UNIVERSIDAD COMPLUTENSE DE MADRID FACULTAD DE CIENCIAS BIOLÓGICAS TESIS DOCTORAL MEMORIA PARA OPTAR AL GRADO DE DOCTOR PRESENTADA POR Laura Domínguez Berzosa DIRECTORES: Nerea, dir Moreno García Jesus María, dir López Redóndo Madrid, 2015 © Laura Domínguez Berzosa, 2012 Organización del hipotálamo en la transición anamnio- amniota : estudio genoarquitectónico y quimioarquitectónico Departamento de Biología Celular , H <3 D o M Universidad Complutense de Madrid o P Facultad de Ciencias Biolôgicas Organizacion del Hipotalamo en la Transicion Anamnio-Amniota: Estudio Genoarquitectonico y Quimioarquitectônico Laura Dominguez Berzosa 2011 J) Universidad Complutense de Madrid Facultad de Ciencias Biolôgicas Organizacion del Hipotalamo en la Transicion Anamnio-Amniota: Estudio Genoarquitectonico y Quimioarquitectônico Trabajo de investigaciôn que présenta Laura Dominguez Berzosa Para optar al grade de Doctor en Ciencias Biolôgicas en la Universidad Complutense de Madrid Fdo. Dna. Laura Dominguez Berzosa. Dirigido por les Doctores Nerea Moreno Garcia Jésus Maria Lôpez Redondo Profesores Titulares del Departamento de Biologia Celular de la Facultad de Ciencias Biolôgicas de la Universidad Complutense de Madrid u Fdo. Dra. Nerea Moreno Garcia 1. Indice 1. Introducciôn general 11 • El prosencéfaio de vertebrados: aspectos evolutivos 13 ❖ Origen embriolôgico y organizacion del prosencéfaio 13 • Organizacion hipotalâmica: antécédentes y perspectiva actual 15 • Morfôgenos y factores de transcripciôn como marcadores prosencefâlicos 17 • Perfîl neuroquimico del hipotalamo 20 • Objetivos y metodologia 22 • Bibliografïa 22 2. Estudios quimioarquitectônicos en el encéfalo adulto 31 • Distribution of thyrotropin-releasing hormone (TRH) immunoreactivity in the brain of urodele amphibians 33 • Immunohistochemical localization of orexins (hypocretins) in the brain of reptiles and its relation to monoaminergic systems 49 3. Estudios genoarquitectonicos en el encéfalo en desarrollo y adulto 65 • Sonic hedgehog expression during Xenopus laevis forebrain development 67 • Ontogenetic distribution of the transcription factor Nkx2.2 in the developing forebrain of Xenopus laevis 83 4. El hipotalamo en la trasiciôn anamnio-amniota: estudios en anuros y reptiles 97 Characterization of the alar hypothalamus of Xenopus laevis during development by molecular marker analysis 99 • Characterization of the basal hypothalamus of Xenopus laevis during development by molecular marker analysis 123 • Subdivisions of the turtle Pseudemys scripta hypothalamus based on the expression of regulatory genes and neuronal markers 141 5. Resumen de los resultados y Discusion general 169 • Resumen de los resultados 171 • Discusion general 172 Consideraciones metodologicas 172 Organizacion del territorio hipotalamico en la evolucion 172 Situacion actual del limite Alar/Basal 180 Hipotesis evolutiva de la organizacion hipotalâmica: existencia de un patron de organizacion comùn en la evolucion y repercusiones de la transicion anamnio-amniota 181 ❖ Bibliografïa 182 6. Conclusiones 189 7. Anexo 193 8. Agradecimientos 196 1. Introducciôn General El prosencéfaio de vertebrados: aspectos evolutivos Origen embriolôgico y organizacion del prosencéfaio Organizacion hipotalâmica: antécédentes y perspectiva actual Morfôgenos y factores de transcripciôn como marcadores prosencefâlicos Perffl neuroquimico del hipotâlamo Objetivos y metodologia Bibliografia El prosencéfaio de vertebrados: aspectos evolutivos El prosencéfaio de vertebrados es probablemente una de las regiones mas complejas del cerebro. Esta compuesto por la union de muchas estructuras heterogéneas que estân implicadas en el control del comportamiento motivado y la modulaciôn de determinados aspectos de la ingesta, reproducciôn, homeostasis, etc. Numerosos estudios a lo largo de estos ùltimos anos han demostrado que las subdivisiones bâsicas del prosencéfaio, asi como diversos aspectos de su organizacion, se encuentran conservados en los diferentes vertebrados. A pesar de esto, existen divergencias evolutivas que han hecho que diferentes grupos de vertebrados adquieran caracteristicas ùnicas en su especie, dada la diferenciaciôn en el grado de complejidad de determinadas estructuras prosencefâlicas (Striedter, 2005; Medina, 2008a), siendo esto una clara consecuencia de los cambios producidos en los mecanismos del desarrollo. Entender los mecanismos por los cuales se produce esta complejidad prosencefâlica, supone un gran paso en la comprension de la historia evolutiva de esta region. Las respuestas a estas preguntas se hacen necesarias para alcanzar una comprension compléta de la organizacion prosencefâlica, y aunque todavia nos encontremos lejos de conocer todos los mecanismos implicados en la especificaciôn prosencefâlica, el présenté estudio nos acerca un poco mâs a la comprension de los procesos evolutivos que rigen la organizacion y regionalizaciôn de esta compleja region. Origen embriologico y organizacion del prosencéfaio El cerebro anterior es una estructura que aparece en la evolucion con los cordados, aunque recibe diferentes nombres entre los procordados y los vertebrados (Holland y Holland, 1999). Los mecanismos responsables de su formaciôn se encuentran muy conservados a lo largo de la evolucion y dan como resultado la formaciôn de compartimentes prosencefâlicos similares a lo largo de los ejes rostrocaudales y dorsoventrales. Este proceso de regionalizaciôn se da gracias a los diferentes mecanismos de inducciôn planar y vertical, que dependen de numerosos factores génicos reguladores, los cuales ejercen influencias ventralizantes y dorsalizantes que se entrecruzan, dando como resultado la distinta compartimentalizaciôn del prosencéfaio. La inducciôn, llevada a cabo por diferentes genes reguladores del desarrollo, sobre la recién formada plaça neural va a tener como consecuencia la adquisiciôn del fenotipo neural, asi como la posterior formaciôn de très vesiculas principales en el tubo neural de los vertebrados; el prosencéfaio, el mesencéfalo y el rombencéfalo (Fig. 1) (Rallu et al., 2002). En todos los vertebrados, el prosencéfaio se desarrolla a partir de la parte anterior de la plaça neural durante la gastrulaciôn (Puelles et al., 2004; Wilson and Houart, 2004). Mâs tarde en el desarrollo, el prosencéfaio se divide para formar las vesiculas telencefâlicas y el diencéfalo. A partir de ese momento, la parte anterior comienza a replegarse sobre la flexura cefâlica y el prosencéfaio comienza a desarrollarse de manera râpida. Este patrôn bâsico de desarrollo aparece en todos los vertebrados, aunque la regionalizaciôn del prosencéfaio ha sido y signe siendo objeto de controversia dada su extrema complejidad. En este sentido, los numerosos estudios acerca de la expresiôn génica en el prosencéfaio llevados a cabo en los ùltimos anos, han ayudado a proponer un patrôn de organizacion prosencefâlico basado en los datos de especificaciôn molecular. De esta manera, la apariciôn del modelo prosomérico ha sido clave en esta nueva concepciôn de la organizaciôn del encéfalo. Dicho modelo fue postulado como un instrumento morfolôgico que divide el prosencéfaio en segmentes transversales (prosômeros) y zonas longitudinales basândose en las caracteristicas moleculares de cada regiôn (Fig. 2A) (Puelles y Rubenstein, 1993; modificado en el 2003). Asi, se establecen compartimentes morfogenéticas, comparables en los distintos grupos de vertebrados, que permiten la comparaciôn de las diferentes estructuras prosencefâlicas asi como la bùsqueda de estructuras homôlogas a lo largo de la filogenia (Fig. 2B). El actual modelo prosomérico basa sus limites en los dominios de expresiôn de diferentes familias de genes reguladores del desarrollo i.e Tbr, Pax, Dix, Nkx, Lhx, Otx, Wnt, Emx, etc, que se encuentran altamente conservados en la evoluciôn. El modelo propone que, durante el desarrollo en vertebrados el prosencéfaio se subdivide en dos vesiculas; la vesicula caudal darâ lugar al diencéfalo propiamente dicho que a su vez estâ subdividido en très prosômeros llamados prosômero 3 (P3), prosômero 2 (P2) y prosômero (PI), mientras que la vesicula mâs rostral darâ lugar al prosencéfaio secundario, el cual estâ formado por el telencéfalo (territorio evaginado) y el hipotâlamo, el cual es considerado actualmente como la porciôn rostral del diencéfalo. La formaciôn de estos segmentos transversales estâ intimamente relacionada con la expresiôn de determinados genes reguladores del desarrollo como Otx2, Gbx2, Wnt, Sonic hedgehog (Shh) por parte de los llamados organizadores primarios y secundarios (Fig. 3 A), que dan lugar a la apariciôn de limites moleculares transversales en la plaça y tubo neural, permitiendo la diferenciaciôn de dominios histogenéticos dentro del territorio prosencefâlico (Fig. 3B). Un ejemplo de ello es la zona limitante intratalâmica, un organizador secundario localizado entre los prosômeros 2 y 3 implicado principalmente en la organizaciôn diencefâlica mediante la expresiôn y secreciôn de proteinas difusibles como Shh entre otras. Ademâs de estos segmentos transversales, el prosencéfaio se encuentra también dividido en segmentos longitudinales que contribuyen a la regionalizaciôn dorsoventtral del tubo neural (Fig. 4). 13 Forebrain Diencephalon Secondary prosencephalon Midbrain / IsO Spinal cordHindbraineye ucr r1+r2 r3 r4 no Somites Figura 1. Representaciôn esquemâtica de la organizaciôn bâsica del cerebro de vertebrados mostrando las très principales vesiculas encefâlicas formadas durante la morfogénesis del tubo neural (Medina, 2008b). De ventral a dorsal esos dominios son el suelo, dominio basai, dominio alar y el techo (ver Puelles y Rubenstein, 2003). La formaciôn las plaças del suelo y basai se llevan a cabo gracias a los procesos de inducciôn vertical ejercidos por determinados genes reguladores expresados en el mesodermo axial, entre los que tiene extrema importancia Shh. Las plaças alar y del techo sin embargo, son el resultado de la acciôn dorsalizante del epitelio extraneural y de la propia plaça del techo una vez que se cierra el tubo neural, estando implicados varios genes reguladores en dicho proceso, taies como noggin, la familia de las BMPs y la familia Wnt, que codifican también para diversas proteinas secretadas difusibles. Las vesiculas telencefâlicas son un territorio exclusivamente alar, mientras que el diencéfalo e hipotâlamo presentan zonas alares y basales. Asi, el dominio basai en el diencéfalo caudal (P1-P3) da lugar al tegmento prerrubal, mientras que la plaça alar forma el pretecho, tâlamo (antiguo tâlamo dorsal) y pretâlamo (antiguo tâlamo ventral) en los respectivos prosômeros 1-3. En los irltimos anos, gran ntimero de estudios comparados han demostrado la existencia de un patrôn comùn de organizaciôn segmentaria en todos los vertebrados estudiados como lamprea (Pombal y Puelles, 1999; Osorio et al., 2005, 2006), pez cebra (Wullimann y Puelles, 1999; Hauptmann y Gester, 2000), pez pulmonado (Gonzâlez y Northcutt, 2009; Moreno y Gonzâlez, 2011), Xenopus (Puelles et al., 1996; Bachy et al., 2001, 2002; Gonzâlez et al., 2002a,b; Brox et al., 2003; 2004), tortuga (Moreno et al., 2010; Moreno y Gonzâlez, 2011), polio (Figdor y Ster, 1993; Abellân y Medina, 2009), ratôn (Puelles y Rubenstein, 1993; 2003; Flames et al., 2007; Abellân et al., 2010), apoyando el modelo prosomérico. De hecho, durante la ultima década el cerebro de los diferentes modelos de vertebrados estâ siendo reevaluado teniendo en consideraciôn los avances en cuanto a su quimioarquitectura, genoarquitectura y hodologia. Cabe destacar la importancia dentro de este anâlisis, del estudio de la organizaciôn del cerebro en la transiciôn anamnio-amniota. En este sentido, son de gran relevancia los estudios comparados llevados a cabo en anfïbios, permitiendo la observaciôn de carcteristicas ancestrales conservadas, y en reptiles, como modelo intermedio hacia mamiferos, los cuales ponen de manifiesto las diferencias evolutivas que se dan en este punto de transiciôn tan relevante de la evoluciôn, permitiendo asi establecer hipôtesis a cerca del proceso evolutivo suffido hasta llegar a los mamiferos, que presentan el mayor grado de complejidad cerebral. Desde un punto de vista evolutivo la transiciôn entre anamnios y amniotas parece haber conllevado multiples cambios en la anatomia neural que fueran necesarios para la conquista y adaptaciôn al nuevo medio terrestre. De esta forma, el anâlisis neuroanatômico evolutivo de las diferentes regiones de cerebro résulta especialmente interesante en estos modelos de transiciôn constituyendo un gran aporte de informaciôn en el establecimiento de las posibles relaciones evolutivas existentes entre los vertebrados (Fig. 5). Este es el caso de los estudios realizados en el complejo amigdalino asi como en los ganglios basales de anfïbios anuros, en los que el anâlisis de la hodologia (Marin et al., 1997a,b; Moreno y Gonzâlez, 2003; 2004, 2005a,b; Moreno et al., 2011), neuroquimica (Marin et al., 1998a,b; Moreno y Gonzâlez, 2006) y perfil molecular (Gonzâlez et al., 2002a,b; Brox et al., 2003; 2004; Moreno et al., 2004; 2008) apoya la idea de un alto grado de conservaciôn en estos sistemas. De la misma forma, existen estudios de hodologia y neuroquimica en reptiles que muestran un alto grado de conservaciôn en estructuras como el complejo amigdalino (Lanuza et al., 1997; Martinez- Marcos et al., 1999; revisado en M artinez-Garcia et al., 2006) y nùcleos talâmicos (Kenigfest et al., 2005). Ademâs, recientemente se ha realizado un estudio en el subpalio de tortuga, en el que la similitud del patrôn molecular y neuroquimico ha permitido la comparaciôn del territorio subpalial con el previamente descrito en otros amniotas y en anuros (Moreno et al, 2010). Todos estos estudios han permitido el establecimiento de homologias entre el prosencéfaio de anuros y reptiles con el del resto de anamniotas y amniotas (Moreno et al., 2009; Moreno y Gonzâlez, 2011), lo cual ayuda en la comprensiôn de los procesos evolutivos que ocurren en esta regiôn. Por lo que el estudio y comprensiôn de las distintas regiones prosencefâlicas atendiendo a éste modelo prosomérico, nos va a facilitar la bùsqueda de relaciones de homologia entre las distintas estructuras, indispensable para conocer la historia evolutiva. De esta 14 □ Ob, I I Nkx2 1 I I Shh MP DP — ch OB VP hemisphenc ST Se Roof Em AEP Bst AHA TH PDA VGSPV PT AlarAHOPv PThEye LG MGSCH AHP .PEP RZl ‘ CZI PH DMH FFVMH D-VTA-SN D-VTA-SNSTh-RM TM SP — RHy prechordal Rosfral dencephakn and telencephalon eptchordal Caudal diencephalon Superior colliculus Inferior colliculus Midbrain CerebellumEmt Th -VTA Pallium PTh Hindbrain Alar Basal Sir Alar hypothalamus Olfactory bulb Basal hypothalamus □ îbr, I I Tbri + SIml I I 01x2/5 I I Dbxl I I Dbxl + Gbx2 I I Dbx1 + Fax? Nkx2.1 NkxS.I Figura 2A. Representaciôn esquemâtica del modelo prosomérico modificado mostrando los diferentes segmentos transversales y zonas longitudinales que dan lugar al establecimiento de los ejes dorsoventral y anteroposterior respectivamente (Puelles y Rubenstein, 2003). B. Representaciôn esquemâtica del los principales compartimentes moleculares observados en el prosencéfaio durante el desarrollo en ratôn. Cada compartimente se encuentra caracterizado por la expresiôn de una combinaciôn especifica de factores de transcripciôn (Puelles y Rubenstein, 2003). esta forma, después de los anâlisis previos de la estructura telencefâlica (Marin et al., 1998b; Moreno y Gonzâlez, 2006; Moreno et al., 2010), ahora ha llegado el tumo del anâlisis de otras regiones prosencefâlicas como el hipotâlamo, en el afân de completar el estudio y profundizaciôn de la organizaciôn prosencefâlica. Organizacion hipotalâmica: antécédentes y perspectiva actual La reciente postulaciôn del modelo prosomérico permitiô una reestructuraciôn del concepto topolôgico del hipotâlamo. La apariciôn de este modelo, que reconoce la existencia de un eje logitudinal prosencefâlico doblado. ha tenido como consecuencia que actualmente se considéré que el hipotâlamo présenta una posiciôn rostral con respecto al diencéfalo (Puelles y Rubenstein, 1993, 2003), reemplazando la idea de la existencia de un eje longitudinal rectilineo que situaba el hipotâlamo topolôgicamente ventral al tâlamo, como previamente habia sido postulado por el modelo columnar de Herrick y Kuhlenbeck (Herrick, 1910; Kuhlenbeck, 1973). Concretamente, el propio término hipotâlamo (hipo viene del griego antiguo ÿpô; debajo) describe literalmente su situaciôn topolôgica bajo el tâlamo. Asi, es frecuente encontrar en la literatura descripciones de la regiôn preôptica (PO) y el hipotâlamo como regiones especiales del diencéfalo ventral implicadas en la regulaciôn del sistema endocrine y del sistema nervioso 15 Midbrain S econdary p rosencephalon Forebrain ; Prechordal Roof plate Notochord ANR Root p ,o ll“ œ ph3on Diencephalon Midbrain i?0 X p3 , p2 pi Dorsal Ventral Pax6 Six3 Tbri, Simi or Dix Pax6 En2 _ Gbx2 Dbxl J Dbxl U ix 1 /5 # L h x 2 m lH x 1 # LHx2/9 S h h + N kxb .l Prechordal pt Rostral B Caudal F ig u ra 3A. Representaciôn esquemâtica de los distintos centros organizadores y senates que controlan la regionalizaciôn prosencefâlica a lo largo de los ejes dorsoventral y anteroposterior (Medina, 2008b). B. Diagrama representative de los centros organizadores y los compartimentos moleculares résultantes de las distintas senates (Medina, 2008b). autônomo (Bruce, 2008; Hodos, 2008). Estas definiciones analizan el cerebro desde el punto de vista columnar sin tener en cuenta ningtin criterio molecular. Numerosos estudios a lo largo de la historia han centrado sus esfuerzos en descifrar la intrincada organizaciôn de la regiôn hipotalâmica considerando diversos aspectos de su neuroquimica, morfogenética y hodologia. No ha supuesto una tarea fâcil debido a la extrema complejidad del estudio de este ârea, en parte provocado por la ausencia de marcadores que caractericen al hipotâlamo de manera exclusiva, asi como por los elaborados procesos migratorios que tienen lugar en el territorio hipotalâmico y las numerosas contribuciones de grupos celulares provenientes de otras regiones prosencefâlicas (Àlvarez-Bolado et al., 2000; Soma et al., 2009; Garcia-Moreno et al., 2010; Bupesh et al., 201 la,b). De esta forma, la organizaciôn anatômica de esta regiôn ha supuesto motivo de debate a lo largo de la historia, no existiendo siempre un acuerdo entre los anatomistas acerca de las diferentes subdivisiones que contribuyen a la formaciôn del territorio hipotalâmico. Asi, la regiôn preôptica ha estado incluida tradicionalmente en el hipotâlamo (Fig. 6a; Butler and Hodos, 2005) en parte debido a la proximidad de ambas regiones en la plaça neural (Garcia-Lôpez et al., 2009; Eagleson y Harris, 1990), aunque actualmente los ùltimos avances en los anâlisis moleculares han revelado que la regiôn preôptica pertenece al territorio subpalial telencefâlico formando el limite dorsal del hipotâlamo (Fig. 6B) (Medina, 2008b; Zhao et al., 2009; Roth et al., 2010), como recientemente se ha mostrado también en anfïbios (Moreno et al., 2008). Actualmente el hipotâlamo estâ considerado como la parte rostral del prosencéfaio secundario que estâ dividido en un dominio alar y otro basai (Fig. 6B). El hipotâlamo alar o protâlamo es rostral al pretâlamo y a las demâs formaciones alares y se extiende dorsalmente hasta las formaciones subpaliales del telencéfalo. El hipotâlamo alar estâ a su vez subdividido en dos subdominios longitudinales. El primero de ellos constituye un dominio adyacente a la regiôn subpalial preôptica caracterizado por la expresiôn de los factores de transcripciôn Sim l y Otp llamado dominio supraoptoparaventricular (SPV) (Medina, 2008b). Esta regiôn darâ lugar entre otros a los nùcleos paraventricular y supraôptico y estâ directamente implicado en la regulaciôn de la funciôn endocrina, conteniendo neuronas magnocelulares encargadas de la producciôn de neuropéptidos como la oxitocina y vasopresina y neuronas parvicelulares encargadas de la regulaciôn de neuronas hipofisiotrôpicas (revisado en Markakis, 2002). El segundo dominio expresa principalmente genes de la familia Dix y producirâ el nùcleo supraquiasmâtico (SC; revisado en Medina, 2008b). El hipotâlamo basai incluye el hipotâlamo tuberal, que darâ lugar entre otros al nùcleo ventromedial y arcuatus, y la regiôn mamilar y retromamilar que incluye el nùcleo subtalâmico (revisado en Medina, 2008b). La divisiôn de la regiôn hipotalâmica en un dominio alar y basai se ha establecido dada la existencia del limite alar-basal, el cual es concordante con el eje longitudinal del encéfalo y es résultante del equilibrio entre las influencias ventralizantes y dorsalizantes. Fue el modelo prosomérico el que postulé la existencia de dicho limite basândose en la existencia de dominios moleculares longitudinales. En este sentido, el morfôgeno Shh y el factor de transcripciôn Nkx2.2 han sido indispensables en el establecimiento de esta separaciôn alar-basal (Puelles y Rubenstein, 1993; Vieira et al., 2005). La organizaciôn del territorio hipotalâmico signe siendo objeto de intenso debate. Un estudio reciente ha propuesto un cambio en el limite alar-basal basândose en la existencia de genes tipicamente alares y tipicamente basales, y restringiendo la regiôn basai del hipotâlamo ùnicamente a la regiôn mamilar, mientras que el dominio tuberal estaria formado por genes que normalmente se encuentran en regiones alares confiriéndole a este territorio un fenotipo alar (Fig. 7; Diez-Roux et al., 2011). Y en este escenario en el que 16 Alar P*®*® Floof plate / ^ plate R oot plate Forebrain ForebrainFloor plate Eye (optic cup) Figura 4. Representaciones esquematicas de las zonas longitudinales que contribuyen a la regionalizaciôn dorsoventral del prosencéfaio (Medina, 2008b). las principales subdivisiones estan siendo cuestionadas, otro estudio reciente ha propuesto la division del hipotalamo en un dominio anterohasal Siml positivo y en un dominio anteroventral Nkx2.1 positivo, los cuales estân separados por una handa llamada intrahipotalâmica diagonal (ID). Esta handa esta caracterizada por la expresiôn de Arx y numerosos genes de la familas Lim- hd, y va paralela al eje neural en el hipotâlamo extendiéndose anteriormente en direcciôn al receso ôptico (Fig. 8; Shimogori et al., 2010). En todo este contexte, la ultima modificaciôn del modelo prosomérico propone otra subdivision en el territorio hipotalâmico separândolo en una regiôn rostral (terminal) y otra caudal (peduncular) que a su vez se encuentran suhdivididas en dominios alares y basales (Fig. 9; ver Allen hrain atlas o f the mouse developing mouse por L. Puelles; Morales-Delgado et al., 2011). En el caso de los anfïbios anuros, el primer prohlema al que nos enfrentamos en el estudio de esta regiôn hipotalâmica es la controversia acumulada durante afios acerca de su organizaciôn y nomenclatura. En particular, el hipotâlamo de anuros fue primero dividido en un hipotâlamo preôptico y otro inftindihular. Mâs tarde, el ârea preôptica, entonces considerada como el actual hipotâlamo alar mâs el ârea preôptica propiamente dicha, fue separada en un dominio anterior y otro posterior, que contenian los nùcleos magnocelular preôptico y el supraquiasmâtico (Neary y Northcutt, 1983; Fig. 10A). La actual panorâmica del hipotâlamo de anfïbios sugiere la existencia de un patrôn de organizaciôn comùn de este territorio en los tetrâpodos (revisado en Moreno y Gonzâlez, 2011; Fig lOB), aunque un anâlisis exhaustive de su organizaciôn en anfïbios anuros se hace necesario para la comprensiôn de la historia evolutiva prosencefâlica. Asi, su comparaciôn con otro grupo de vertebrados tetrâpodos, los reptiles, permite el estudio de la organizaciôn hipotalâmica en la transiciôn anamnio- amniota, lo cual permite la inferencia de posibles relaciones homôlogas para apoyar la idea de un patrôn bâsico de organizaciôn en vertebrados y demostrar la alta conservaciôn de esta regiôn hipotalâmica a lo largo de la evoluciôn. Morfôgenos y factores de transcripciôn como marcadores prosencefâlicos Los estadios iniciales del establecimiento de patrones neuronales estân controlados por proteinas de seùalizaciôn, llamados morfôgenos, que son secretados por los organizadores primarios y secundarios y liberados a distancias variables a lo largo del tejido neural (Lupo et al., 2006). Después, estas senales son también producidas localmente por las plaças del techo y suelo. Estas senales van a inducir, en presencia de receptores especifïcos en el tejido, la expresiôn de factores de transcripciôn determinados, que van a ser los efectores de la especificaciôn y consiguiente formaciôn de los dominios especifïcos neurales de cada regiôn (Wilson y Maden, 2005; Lupo et al., 2006). La especificaciôn molecular estâ llevada a cabo por la expresiôn combinatoria y la acciôn de estos genes reguladores del desarrollo. Estos genes especifican dominios moleculares distintos en el desarrollo, que posteriormente se correlacionan con una determinada poblaciôn o regiôn derivada en el cerebro adulto, como ha sido demostrado en anâlisis de destino celular (Garcia-Lôpez et al., 2009). La expresiôn de genes clave, implicados en procesos de especificaciôn de patrones neuronales, estâ siendo analizada en detalle en los principales modelos de vertebrados, con el fin de establecer tendencias evolutivas en el prosencéfaio de vertebrados. LungAsh M onotrem e m am m als Ray-linned •fish ^ Placenta! Marsupial m mammals Cartilaginous fîsh Turtles Lampreys rs Non-avian ^ * ^ l r d s Drnithlschlan __ dinosaurs Non-avian dinosaurs Figura 5. Esquema filogenético simplificado mostrando la relaciôn evolutiva entre los principales grupos de vertebrados. La flécha roja indica el momento evolutivo de la transiciôn anamnio-amniota. 17 Telencephalon Midbrain / Dorsal \ thalam us Olfactory tract Hindbrain Pituitary Hypothalamus H ypothalam us Subpallium M idbrain PT Th H indbrainPTh Figura 6A. Representaciôn lateral del cerebro de reptil mostrando las regiones tradicionalmente incluidas en la region hipotalâmica, como la region preôptica (Bruce, 2008). B, Representaciôn esquemâtica del cerebro de ratôn que muestra la actual organizaciôn del hipotâlamo, que excluye al ârea preôptica subpalial (Modificado de M edina, 2008b). Como se ha mencionado previamente, el estudio comparado del prosencéfaio en vertebrados es complicado dada la dificultad de establecer relaciones de homologia entre estructuras de vertebrados con diferencias en tamano de areas cerebrales, organizaciôn o conectividad, debidas a los procesos divergentes que ocurren en la evoluciôn (Striedter, 1997). Sin embargo, el estudio de estas familias de genes implicados en la organizaciôn de estructuras, especificaciôn de tejido neural y establecimiento de conexiones estâ siendo muy util en el esclarecimiento de la historia evolutiva del cerebro (Puelles, 2001). Sus dominios de expresiôn segùn el caso, pueden ser exclusives o solapantes, extensos o restringidos a regiones especificas etc, pero en general el estudio de sus fronteras de expresiôn nos proporciona un mapa de las fronteras entre las distintas subdivisiones prosencefâlicas y, es por esto que, dado el alto grado de conservaciôn que suelen tener estas moléculas, son extremadamente utiles en la identificaciôn de estructuras homôlogas en los distintos grupos de vertebrados a lo largo de la evoluciôn. Asi, cambios en la expresiôn regional y en los côdigos de expresiôn en distintos vertebrados podria explicar diferencias en patrones de conectividad y regionalizaciôn (Bachy et al., 2002). En el anfibio Xenopus laevis y en el reptil Pseudemys scripta han sido analizados los patrones de expresiôn de numerosos genes durante los ùltimos anos en pro de la delineaciôn de las diferentes subdivisiones prosencefâlicas y mostrando numerosas homologias con amniotas. De todos estos genes, los marcadores elegidos para el présente estudio se presentan como los mâs conservados, estando implicados en numerosos procesos de especificaciôn hipotalâmica. Sonic Hedgehog (Shh). Sonic es un poderoso morfôgeno (Ingham y Placzek, 2006) implicado en el control de la proliferaciôn de progenitores, establecimiento de patrones de regionalizaciôn y destino celular en el cerebro en desarrollo (revisado en Fuccillo et al., 2006). Es secretado por la linea media ventral de la plaça y tubo neural (Shh neural), asi como por la notocorda y plaça precordal (Shh no neural), dos derivados mesodérmicos con importantes propiedades inductivas (Ericson et al., 1997; Gunhaga et al., 2000; Ingham y McMahon, 2001). El Shh neural es esencial para la coordinaciôn del crecimiento y establecimiento de patrones neuronales a diferentes niveles como en la médula espinal, la uniôn mesencéfalo-rombencéfalo y el cerebelo (Blaess et al., 2006, 2008). En el prosencéfaio Shh se expresa en la plaça basai o del suelo asi como en la zona limitante intratalâmica (Zli), un organizador secundario situado entre el pretâlamo y el tâlamo, donde Shh contrôla la organizaciôn diencefâlica (Kieker y Lumsdem, 2004; Vieira et al., 2005; Zeltser, 2005; Szabô et al., 2009a). Ademâs, Shh se expresa especificamente en el hipotâlamo donde contribuye a su especificaciôn (Chiang et al., 1996; Szabô et al., 2009b). Numerosos estudios en los diferentes grupos de vertebrados corroboran el alto grado de conservaciôn de la expresiôn de este morfôgeno a lo largo de la evoluciôn tanto en amniotas (Vieira et al., 2005; Bardet et al., 2006; Vieira y Martinez, 2006; Garcia-Lôpez et al., 2008) como en anamniotas (Osorio et al., 2005; Menuet et al., 2007; Dominguez et al., 2010). Familia de genes Nkx. Los genes de la familia homeobox Nkx pertenecen a una familia de factores de transcripciôn con un papel importante el la regulaciôn del desarrollo tubo neural basal. En concreto, el factor de transcripciôn Nkx2.1 (T T Fl) se expresa en el pulmôn, glândula tiroidea y dominios especifïcos basales del prosencéfaio (Lazzaro et al., 1991) estando implicado en la regulaciôn del establecimiento de patrones de regionalizaciôn prosencefâlicos (Kimura et al., 1996; Sussel et al., 1999; Marin et al., 2002). Los patrones de expresiôn del prosencéfaio en desarrollo de los genes ortôlogos de Nkx2.1 han demostrado ser similares en otros vertebrados como polio, Xenopus laevis y pez cebra (Puelles et al., 2000; Small et al., 2000; Rohr et al., 2001; Bachy et al., 2002; Gonzâlez et al., 2002a,b) demostrando lo conservado que se encuentra su patrôn de expresiôn a lo largo de la evoluciôn. La expresiôn de Nkx2.1 ha servido para identificar numerosas regiones prosencefâlicas como las regiones preôptica, supraquiasmâtica y mberal durante el desarrollo (Rohr et al., 2001; Gonzâlez et al., 2002a,b; Moreno et al., 2008; van den Akker et al., 18 2008). Ademâs, se ha demostrado mediante la hipofhnciôn de Nkx2.1 en ratôn y rana, que este factor de transcripciôn estâ implicado en la especificaciôn y formaciôn del hipotâlamo basai en diferentes vertebrados (van den Akker et al., 2008). Ademâs, se sabe que la expresiôn de los factores de transcripciôn pertenecientes a esta familia en el tubo neural ventral es inducida por Shh y que en ultimo término son unos de los principales reguladores que ejercen los efectos de Shh en la especificaciôn hipotalâmica. El factor de transcripciôn Nkx2.2 tue originalmente identificado como un gen expresado en las regiones ventrales del sistema nervioso central (Price et al., 1992). Se encuentra implicado en numerosos mecanismos de regionalizaciôn como la especificaciôn temprana de la identidad de las células progenitora y procesos de destino celular en el tubo neural ventral en respuesta a la senalizaciôn graduai de Shh (Briscoe y Ericson, 1999; Briscoe et al., 2000). Nkx2.2 estâ también implicado en el establecimiento del limite alar-basal (Puelles y Rubenstein, 1993; Vieira et al., 2005), en la especificaciôn diencefâlica dada su expresiôn alrededor de la regiôn Shh positiva en Zli (Ericson et al. 1997; Vue et a l , 2007; Kataoka y Shimogori, 2008; Ferrân et a l , 2009) y parece desempanar también un papel activo en la formaciôn hipotalâmica, como se ha demostrado en ratones mutantes para Nkx2.2, en los que se produce transformaciones ventrales hacia destinos dorsales (Briscoe et a l , 1999; Sander et a l , 2000). La expresiôn de Nkx2.2 ha sido analizada en muchos vertebrados (Price et a l , 1992; Barth and Wilson, 1994; Holland et a l , 1998; Schafer et a l , 2005; Vieira y Martinez, 2006; Vue et a l , 2007; Ferrân et a l , 2009; Dominguez et a l , 2011) mostrando un patrôn muy conservado en todos ellos. Familia de genes Dix. Los miembros que componen esta familia estân implicados en el desarrollo del prosencéfaio, formando parte en procesos de especificaciôn neuronal y de diferenciaciôn de las neuronas de proyecciôn e intemeuronas (Eisenstat et a l , 1999). Los genes Dix de vertebrados han incorporado funciones que tienen que ver con el desarrollo de las partes distales de apéndices del cuerpo, cresta neural, placodas sensitivas y el prosencéfaio (Merlo et a l , 2000; Panganiban y Rubenstein, 2002). Mâs especificamente, los genes Dix 1,2,5 y 6 en ratôn son marcadores de neuronas inhibitorias generadas en el subpalio telencefâlico (Puelles et a l , 2000; 2004). De hecho, los factores de transcripciôn pertenecientes a esta familia estân intimamente relacionados con la producciôn de células GABAérgicas en numerosas regiones prosencefâlicas (Price et a l , 1991; Bulfone et a l , 1993; Marin and Rubenstein, 2001), como también se ha sugerido en anfïbios (Brox et a l , 2003). Ademâs, sus patrones de expresiôn defmen los limites de segmentos transversales y longitudinales haciéndoles marcadores muy valiosos para la especificaciôn de estructuras prosencefâlicas dentro de su regionalizaciôn (Bulfone et a l , 1993). Los miembros de esta familia se expresan en el pretâlamo y en la mayor parte del territorio hipotalâmico a lo largo de todos los grupos de vertebrados estudiados POA PV aiar pmt# I basai plate Figura 7. Representaciôn esquemâtica de la reciente propuesta acerca de la situaciôn del limite alar-basal en la que sôlo el territorio mamilar tendria carâcter basai (A) y su comparaciôn con el modelo prosomérico que situa dicho limite delante del territorio tuberal (B). (Diez-Roux et a l, 2011). ■'41V' PM Arc Posterior ID+TT Anterior ID PVN Pth (EmThal) Pth vAH Figura 8. Representaciones esquemâticas del hipotâlamo de ratôn, que muestra la propuesta de organizaciôn hipotalâmica, basada en la existencia de un dominio anterobasal Siml positivo y un dominio anteroventral Nkx2.1 positivo divididos por una banda intrahipotalâmica diagonal (10) y la existencia de una banda tuberomamilar (TT) que sépara el territorio mamilar (M odificado de Shimogori et a l , 2010). (Robinson et a l , 1991; Price et a l , 1991; Bulfone et a l , 1993; Liu et a l , 1997; Eisenstat et al. 1999; Bachy et a l , 2002; Brox et a l , 2003; Dominguez et a l , 2012a,b) siendo requeridos en la histogénesis de dichas âreas (Qiu et al., 1997) y demostrando su alto grado de conservaciôn, lo cual les hace utiles en el estudio de la historia evolutiva hipotalâmica. 19 roof plate ppHrA PPMyB C '/] Peduncu»*» My Q [ PrepvduncuMir Hy { -( Tutoerai area r i Rptroiutiprai «i«a Figura 9. Representaciôn esquemâtica del cerebro de ratôn mostrando la divisiôn del hipotâlamo en un territorio rostral (terminal/prepeduncular) y otro domino caudal (peduncular) (M orales-Delgado et al., 2011). Familia de genes LIM-hd. Los genes LIM homeodominio pertenecen a una familia de factores de transcripciôn implicados con una prominente expresiôn y funciôn en el desarrollo del sistema nervioso (Hobert y Westphal, 2000). Se encuentran implicados en procesos de especificaciôn regional y celular incluyendo procesos de guia axonal y determinaciôn de fenotipo neuronal (Bach, 2000; Rétaux y Bachy, 2002; Zhao et al., 2003). Una particularidad de esta familia es el alto grado de conservaciôn que présenta en la evoluciôn en termines de estructura primaria y funciôn (Hobert y Ruvkun, 1998; Hobert y Westphal, 2000). Los estudios acerca de la expresiôn de estos factores de transcripciôn en el prosencéfaio son numerosos (Bachy et al., 2001, 2002; Nakagawa y O ’Leary, 2001; Sheng et al., 1997; Grigoriou et al., 1998; Rétaux et al., 1999; Bulchand et al., 2003; Zhao et al., 2003; Alunni et al., 2004; Moreno et al., 2004; 2008), y han demostrado que los patrones de expresiôn que présenta esta familia en determinadas regiones cerebrales estân también altamente conservados en la evoluciôn. Su funciôn ha sido particularmente estudiada en las motoneuronas de la médula espinal, donde se ha demostrado que dichos genes codifican para informaciôn posicional, que da como resultado la especificaciôn de determinados grupos neuronales (Tsuchida et al., 1994). Ademâs, se ha observado que los patrones de expresiôn de estos factores de transcripciôn respetan los limites neuroméricos (Rétaux et al., 1999), por lo que sirven de herramienta fundamental para el estudio de las distintas subdivisiones prosencefâlicas. En este sentido, estudios recientes en mamiferos han demostrado que los miembros de la familia LIM-hd son herramientas fundamentales en la identificaciôn de diferentes subdominios dentro del territorio hipotalâmico (Shimogori et al., 2010). Familia de genes Pax. Esta familia contiene numerosos factores de transcripciôn altamente conservados en la evoluciôn, que juegan un papel fundamental en el desarrollo del sistema nervioso central. Asi, median multiples funciones en el desarrollo cerebral, control de la regionalizaciôn dorsoventral del telencéfalo (Stoykova et al., 2000), control de la migraciôn (Chapouton et al., 1999) o la diferenciaciôn de la glia cortical radial (Heins et al., 2002). También estân implicados en la correcta regionalizaciôn dorsoventral del diencéfalo y el correcto desarrollo de las conexiones talamocorticales (Jones et al., 2002). En especial, Pax6 se expresa de forma llamativa en la regiôn palial, en el limite palio-subpalial (W alther y Gruss, 1991; Stoykova y Gruss, 1994; Puelles et al., 2000), tâlamo, pretâlamo y pretecho (Stoykova y Gruss, 1994; Warren y Price, 1997). En cuanto al hipotâlamo, estudios previos describieron su presencia en la regiôn paraventricular de amniotas (Medina, 2008b). La expresiôn de Pax6 ha sido analizada en muchos vertebrados incluyendo humano (Gerard et al., 1995), ratôn (Stoykova y Gruss, 1994; Puelles et al., 2000), polio (Li et al., 1994; Puelles et al., 2000), Xenopus (Bachy et al., 2002) y pez cebra (Hauptmann y Gerster, 2000; Wullimann y Rink, 2001), mostrando un patrôn muy conservado en todas ellas. En cuanto a Pax7, muestra un patrôn de expresiôn altamente conservado (Stoykova and Gruss, 1994; Diez-Roux et al., 2011) siendo especialmente util en la identificaciôn de la plaça basai de P3 (Ferrân et al., 2007; M orona et al., 2011). Perfîl neuroquimico del hipotâlamo Actualmente se considéra que para poder establecer relaciones de homologia entre dos estructuras, se tienen que cumplir una serie de condiciones. La mâs importante y principal es que tengan los mismos progenitores neuroepiteliales en el tubo neural con una misma posiciôn topolôgica, asi como que compartan un côdigo de especificaciôn genética que de lugar a un mismo patrôn histogenético (Puelles et al., 2007). La homologia basada en estas caracteristicas moleculares, viene apoyada por un semejante perfil neuroquimico y un patrôn de conexiones similar, de tal forma que la comparaciôn del patrôn de marcadores de diferenciaciôn neuronal tardia sirve como apoyo para establecer homologias entre estructuras que comparten un mismo perfil molecular, no siendo una condiciôn necesaria, dado que en el curso 20 'i F igu ra 10. Fotomicrografias sobre secciones transversales del cerebro de anuros con la tinciôn de Nissl donde se compara la nomenclatura y organizaciôn hipotalâmica clâsica (A; Neary y Northcutt, 1983) y la nueva propuesta basada en criterios moleculares (B; Dominguez et al., 2012a,b). de la evoluciôn dos estructuras han podido variar sus caracteristicas quimioarquitcctônicas, mantcnicndo cl mismo origen embriolôgico (Puelles et al., 2007). Es por esto que, en el présente trabajo se ha analizado el perfil neuroquimico del hipotâlamo de anuros y reptiles, y su comparaciôn en las distintas subregiones encontradas con el resto de vertebrados ha servido de apoyo al perfil molecular encontrado para establecer homologias en tetrâpodos. El hipotâlamo es una regiôn caracterizada por su implicaciôn en la regulaciôn, coordinaciôn y control del sistema neuroendocrino, por lo que contiene numerosas poblaciones de neuropéptidos y neurohormonas que van a desempenar diferentes funciones relacionadas con este control de la homeostasis y que en su mayoria estarân relacionadas con la hipôfisis. Estas poblaciones neuronales se distribuyen por diferentes centros hipotalâmicos desde el nùcleo paraventricular, supraquiasmâtico, ventromedial y arcuado, donde ejercen su acciôn en el control del comportamiento reproductor, regulaciôn de la ingesta, ciclo sueno-vigilia y comportamiento agresivo entre otros (Nieuwenhuys et al., 2008). De entre todos os neuropéptidos implicados en la funcionalidad del hipotâlamo, estudios previos en anfïbios y reptiles han demostrado que la hormona liberadora de tirotropina (TRH) y las orexinas se encuentran muy conservados a lo largo de la evoluciôn (Lôpez et al., 2008; 2009a,b), de manera que en el présente estudio se ha anlizado el patrôn de distribuciôn en aquellos modelos de anfïbios y reptiles de los que no se disponfan datos, resultando de gran ayuda a la hora de realizar comparaciones entre las principales subdivisiones neuroendocrinas del hipotâlamo de anfïbios y reptiles. Hormona liberadora de tirotropina (TRH). La TRH es un tripéptido localizado en el hipotâlamo y cuya funciôn mâs conocida es la estimulaciôn de la liberaciôn de la hormona estimuladora del tiroides en la hipôfisis (Boler et al., 1969; Schally et al., 1969; Yates et al., 1971; Azizi et al., 1974), aunque se han descrito otras funciones hipofisiotrôpicas como la estimulaciôn de la liberaciôn de prolactina o de la hormona del crecimiento en diferentes especies (Guillemin, 1978; Schally, 1978). Ademâs, el patrôn extrahipotalâmico de distribuciôn de fibras TRH descrito, ha relacionado este neuropéptido con el control de funciones como analgesia, excitaciôn sexual, (Frange, 1974; Horita et al., 1976; Ervin et al., 1981; Horita, 1998), control de 21 ingesta (Ao et al., 2006), del sueno (Reichlin, 1986) y termorregulaciôn (Boschi et al., 1983; Fiedler et al., 2006) entre otros. Su distribuciôn en estructuras hipotalâmicas como el nùcleo paraventricular, se ha descrito en todos los vertebrados estudiados (Del Carmen et al., 2002; Teijido et al., 2002; Diaz et al., 2001; 2002; Geris et al., 1999; Lôpez et al., 2008), mostrando un patrôn inicial muy conservado. Por lo que el estudio de dicho neuropéptido supone un indicador objetivo de las neuronas postmitôticas que van a contribuir a la formaciôn de diferentes estructuras hipotalâmicas, como la regiôn paraventricular. Orexinas (hipocretinas). Las orexinas son neuropéptidos producidos en diferentes regiones hipotalâmicas y que fueron originalmente descritas como reguladores del comportamiento alimenticio (Sakurai et al., 1998). Presentan una amplia distribuciôn a lo largo de todo el cerebro por lo que se las ha asociado con mùltiples funciones fisiolôgicas como el ciclo de sueno-vigilia y patologlas relacionadas como la narcolepsia, alimentaciôn y control de la energia metabôlica (Lubkin y Stricer- Krongrad, 1998; Sakurai et al., 1998; Wolf, 1998; Dube et al., 1999; Haynes et al., 1999; Sweet et a l, 1999; Chemelli et a l , 1999; Volkoff et a l , 2005; Volkoff, 2006; Carter et a l , 2009). Ademâs, las orexinas también estân implicadas en la regulaciôn de funciones hipofisiotrôpicas como la liberaciôn de hormonas adenohipofisiarias (Pu et a l , 1998; Malendowicz et a l , 1999; Mitsuma et a l , 1999; Tamura et a l , 1999; Russell et a l , 2000; Kohsaka et a l, 2001; Seoane et a l , 2004; Barb and Matteri, 2005; Martynska et a l , 2006) y en el control integrado de la regulaciôn del sistema autônomo (Shirasaka et a l , 2002; Ferguson y Samson, 2003; Berthoud et a l , 2005; Yasuda et a l , 2005). Se han realizado numerosos estudios de la distribuciôn de las orexinas en vertebrados mamiferos y no mamiferos observando un patrôn inicial altamente conservado (Kaslin, et a l , 2004; Huesa et a l, 2005; Singletary et a l , 2006; Amiya et a l , 2007; Lôpez et a l, 2009a,b) lo cual apoya el uso de estos neuropéptidos para la identificaciôn de diferentes regiones hipotalâmicas implicadas en la regulaciôn del sistema neuroendocrino. Objetivos y metodologia Como se ha expuesto en la introducciôn previa (capitulo 1), el hipotâlamo de vertebrados es una regiôn de gran complejidad en cuanto a su origen, estructura y regionalizaciôn. En anuros y reptiles el conocimiento de esta regiôn es limitado. Los anfïbios resultan de gran interés en el estudio evolutivo de regiones prosencefâlicas en general y del hipotâlamo en particular dada su posiciôn clave dentro de la escala filogenética, ya que es el ùnico grupo de tetrâpodos anamniota. Este privilegiado enclave en la filogenia permite que los anfïbios posean caracteristicas mâs ancestrales compartidas con otros anamniotas asi como caracteristicas mâs prôximas a otros grupos mâs evolucionados. Por otra parte, la posiciôn filogenética de los reptiles, en especial de las tortugas, les confiere un gran interés en este estudio evolutivo ya que se ha propuesto que este grupo es el pariente mâs cercano de los extintos terâpsidos, grupo a partir del cual los mamiferos evolucionaron (Northcutt, 1970) pero, altemativamente, han sido considerados como un grupo hermano a cocodrilos y aves (Zardoya y Meyer, 2001a,b). Estas razones demuestran el extremo interés de ambos grupos en el estudio de la evoluciôn del cerebro ancestral dado que representan un modelo claro para el anâlisis de la transiciôn anamnio-amniota. Por eso, el uso de dos modelos prôximos en la filogenia como son anfibios y reptiles permite inferir estos cambios evolutivos y, asi, poder aumentar nuestra comprensiôn acerca de la evoluciôn del hipotâlamo en los vertebrados. En un primer anâlisis de la quimioarquitectura del cerebro de estos modelos de transiciôn anamnio- amniota (capitulo 2) identifîcamos el patrôn de distribuciôn de los neuropéptidos TRH y orexinas en los modelos de anfibios y reptiles, que presentaron principalmente una distribuciôn hipotalâmica. Los experimentos realizados en esta investigaciôn se han llevado a cabo en una especie de anfibio anuro, Xenopus laevis, très de anfibios urodelos, Ambystoma tigrinum, Ambystoma mexicanum y Pleurodeles waltl, y dos de reptiles, Pseudemys scripta elegans y Gekko gecko. Para comprender la organizaciôn del cerebro de ambos modelos, y mâs concretamente de la regiôn hipotalâmica, se analizô la distribuciôn de los neuropéptidos TRH y orexinas A y B mediante técnicas inmunohistoquimicas en animales adultos. Tras esta primera aproximaciôn, analizamos la expresiôn del morfôgeno Shh y del factor de transcripciôn Nkx2.2 (capitulo 3), los dos principales factores implicados en el establecimiento del eje alar-basal en vertebrados y fundamentales en la especificaciôn hipotalâmica. Para este anâlisis se llevaron a cabo técnicas de inmunohistoquimica y de hibridaciôn in situ simples y combinadas durante el desarrollo y en individuos adultos. Por ùltimo, llevamos a cabo un estudio detallado de todas las regiones hipotalâmicas, asi como de sus limites con otras regiones prosencefâlicas en base a la expresiôn de factores de transcripciôn claves en el desarrollo mediante técnicas de hibridaciôn in situ simples y combinadas (capitulo 4). El anâlisis conjunto de todos estos datos ha permitido comparar la regionalizaciôn hipotalâmica entre amniotas y anamniotas y la evaluaciôn de una hipôtesis evolutiva de esta regiôn con los datos actuates en mamiferos (capitule 5). Bibliografia Abellân A, Medina L. 2009. Subdivisions and derivatives of the chicken subpallium based on expression o f LIM and other regulatory genes and markers o f neuron subpopulations during development. J Comp Neurol 515(4):465-501. 22 Abellân A, Vernier B, Rétaux S, Medina L. 2010. Similarities and differences in the forebrain expression o f Lhxl and Lhx5 between chicken and mouse: Insights for understanding telencephalic development and evolution. J Comp Neurol 518(17):3512-3528. Alunni A, Blin M, Deschet K, Bourrât F, Vernier P, Retaux S. 2004. Cloning and developmental expression patterns of Dlx2, Lhx7 and Lhx9 in the medaka fish (Oryzias latipes). Mech Dev 121(7- 8):977-983. Àlvarez-Bolado G, Zhou X, Cecconi F, Gruss P. 2000. Expression o f Foxbl reveals two strategies for the formation of nuclei in the developing ventral diencephalon. Dev Neurosci 22(3): 197-206. Amiya N, Amano M, Oka Y, ligo M, Takahashi A, Yamamori K. 2007. Immunohistochemical localization of orexin/hypocretin-like immunoreactive peptides and melanin-concentrating hormone in the brain and pituitary of medaka. Neurosci Lett 427(1): 16-21. Ao Y, Go VL, Toy N, Li T, Wang Y, Song MK, Reeve JR, Jr., Liu Y, Yang H. 2006. Brainstem thyrotropin- releasing hormone regulates food intake through vagal-dependent cholinergic stimulation of ghrelin secretion. Endocrinology 147(12):6004-6010. Azizi F, Vagenakis AG, Bollinger J, Reichlin S, Bush JE, Braverman LE. 1974. The effect of a single large dose of thyrotropin-releasing hormone on various aspects o f thyroid function in the rat. Endocrinology 95(6): 1767-1770. Bach 1. 2000. The LIM domain: regulation by association. Mech Dev 91(l-2):5-17. Bachy 1, Berthon J, Rétaux S. 2002. Defining palliai and subpallial divisions in the developing Xenopus forebrain. Mech Dev 117(1-2): 163-172. Bachy 1, Vernier P, Rétaux S. 2001. The LIM- homeodomain gene family in the developing Xenopus brain: conservation and divergences with the mouse related to the evolution of the forebrain. J Neurosci 21(19):7620-7629. Barb CR, Matteri RL. 2005. Orexin-B modulates luteinizing hormone and growth hormone secretion from porcine pituitary cells in culture. Domest Anim Endocrinol 28(3):331-337. Bardet SM, Cobos 1, Puelles E, Martinez-De-La-Torre M, Puelles L. 2006. Chicken lateral septal organ and other circumventricular organs form in a striatal subdomain abutting the molecular striatopallidal border. J Comp Neurol 499(5):745-767. Barth KA, Wilson SW. 1994. Specification of neuronal identity in the embryonic CNS. Semin Dev Biol 5:349-358. Berthoud HR, Patterson LM, Sutton GM, Morrison C, Zheng H. 2005. Orexin inputs to caudal raphe neurons involved in thermal, cardiovascular, and gastrointestinal regulation. Histochem Cell Biol 123(2):147-156. Blaess S, Corrales JD, Joyner AL. 2006. Sonic hedgehog regulates Gli activator and repressor functions with spatial and temporal precision in the mid/hindbrain region. Development 133(9): 1799-1809. Blaess S, Stephen D, Joyner AL. 2008. Gli3 coordinates three-dimensional patterning and growth of the tectum and cerebellum by integrating Shh and FgfS signaling. Development 135(12):2093-2103. Boler J, Enzmann F, Folkers K, Bowers CY, Schally AV. 1969. The identity o f chemical and hormonal properties of the thyrotropin releasing hormone and pyroglutamyl-histidyl-proline amide. Biochem Biophys Res Commun 37(4):705-710. Boschi G, Nomoto T, Rips R. 1983. Thyrotropin releasing hormone-induced hyperthermia in mice: possible involvement o f adrenal and pituitary glands. Br J Pharmacol 80(2):229-233. Briscoe J, Ericson J. 1999. The specification of neuronal identity by graded Sonic Hedgehog signalling. Semin Cell Dev Biol 10(3):353-362. Briscoe J, Pierani A, Jessell TM, Ericson J. 2000. A homeodomain protein code specifies progenitor cell identity and neuronal fate in the ventral neural tube. Cell 101(4):435-445. Brox A, Puelles L, Ferreiro B, Medina L. 2003. Expression of the genes GAD67 and Distal-less-4 in the forebrain o f Xenopus laevis confirms a common pattern in tetrapods. J Comp Neurol 461(3):370-393. Brox A, Puelles L, Ferreiro B, Medina L. 2004. Expression of the genes Em xl, T bri, and Eomes (Tbr2) in the telencephalon of Xenopus laevis confirms the existence of a ventral palliai division in all tetrapods. J Comp Neurol 474(4):562-577. Bruce L. 2008. Evolution of the hypothalamus in amniotes. "Evolution and Embryological Development of Forebrain" in Enciclopedic Reference of Neuroscience, eds M. D. Binder and N. Hirokawa (Springer-Verlag),. 1363-1367. Bulchand S, Subramanian L, Tole S. 2003. Dynamic spatiotemporal expression of LIM genes and cofactors in the embryonic and postnatal cerebral cortex. Dev Dyn 226(3):460-469. Bulfone A, Puelles L, Porteus MH, Frohman MA, Martin GR, Rubenstein JL. 1993. Spatially restricted expression o f D ix-1, Dlx-2 (Tes-1), Gbx- 2, and Wnt-3 in the embryonic day 12.5 mouse forebrain defines potential transverse and longitudinal segmental boundaries. J Neurosci 13(7):3155-3172. Bupesh M, Legâz 1, Abellân A, Medina L. 2011a. Multiple telencephalic and extratelencephalic embryonic domains contribute neurons to the medial extended amygdala. J Comp Neurol 519:1505-1525. Bupesh M, Abellân A, Medina L. 2011b. Genetic and experimental evidence support the continuum of the central extended amygdala and a mutiple embryonic origin of its principal neurons. J Comp Neurol (in press). Butler A, Hodos W. 2005. Comparative vertebrate neuroanatomy. Sons JW, editor New Jersey: Wiley. 23 1. iin 1 Carter ME, Borg JS, de Lecea L. 2009. The brain hypocretins and their receptors: mediators of allostatic arousal. Curr Opin Pharmacol 9(l):39-45. Chapouton P, Gartner A, Gotz M. 1999. The role o f Pax6 in restricting cell migration between developing cortex and basal ganglia. Development 126(24):5569-5579. Chemelli RM, Willie JT, Sinton CM, Elmquist JK, Scammell T, Lee C, Richardson JA, Williams SC, Xiong Y, Kisanuki Y, Fitch TE, Nakazato M, Hammer RE, Saper CB, Yanagisawa M. 1999. Narcolepsy in orexin knockout mice: molecular genetics of sleep regulation. Cell 98(4):437-451. Chiang C, Litingtung Y, Lee E, Young KE, Corden JL, Westphal H, Beachy PA. 1996. Cyclopia and defective axial patterning in mice lacking Sonic hedgehog gene function. Nature 383(6599):407-413. Del Carmen De Andres M, Anadon R, Manso MJ, Gonzalez MJ. 2002. Distribution of thyrotropin- releasing hormone immunoreactivity in the brain of larval and adult sea lampreys, Petromyzon marinus L. J Comp Neurol 453(4):323-335. Diaz ML, Becerra M, Manso MJ, Anadon R. 2001. Development of thyrotropin-releasing hormone immunoreactivity in the brain of the brown trout Salmo trutta fario. J Comp Neurol 429(2):299-320. Diaz ML, Becerra M, Manso MJ, Anadon R. 2002. Distribution o f thyrotropin-releasing hormone (TRH) immunoreactivity in the brain of the zebrafish (Danio rerio). J Comp Neurol 450(1 ):45-60. Diez-Roux G, Banff S, Sultan M, Geffers L, Anand S, Rozado D, Magen A, Canidio E, Pagani M, Peluso 1, Lin-Marq N, Koch M, Bilio M, Cantiello 1, Verde R, De Masi C, Bianchi SA, Cicchini J, Perroud E, Mehmeti S, Dagand E, Schrinner S, Numberger A, Schmidt K, Metz K, Zwingmann C, Brieske N, Springer C, Hernandez AM, Herzog S, Grabbe F, Sieverding C, Fischer B, Schrader K, Brockmeyer M, Dettmer S, Helbig C, Alunni V, Battaini MA, Mura C, Henrichsen CN, Garcia-Lopez R, Echevarria D, Puelles E, Garcla-Calero E, Kruse S, Uhr M, Kauck C, Feng G, Milyaev N, Ong CK, Kumar L, Lam M, Semple CA, Gyenesei A, Mundlos S, Radelof U, Lehrach H, Sarmientos P, Reymond A, Davidson DR, Dolle P, Antonarakis SE, Yaspo ML, Martinez S, Baldock RA, Eichele G, Ballabio A. 2011. A high- resolution anatomical atlas of the transcriptome in the mouse embryo. PLoS Biol 9(l):el000582. Dominguez L, Gonzalez A, Moreno N. 2010. Sonic hedgehog expression during Xenopus laevis forebrain development. Brain Res 1347:19-32. Dominguez L, Gonzalez A, Moreno N. 2011. Ontogenetic distribution of the transcription factor Nkx2.2 in the developing forebrain of Xenopus laevis. Front. Neuroanat. 5:11. doi: 10.3389/Jhana.2011.00011. Dominguez L, Morona R, Gonzalez A, Moreno N. 2012a. Characterization of the alar hypothalamus of Xenopus laevis during development by molecular markers analysis. J Comp Neurol. (En preparacion). Dominguez L, Morona R, Gonzalez A, Moreno N. 2012b. Characterization of the basal hypothalamus of Xenopus laevis during development by molecular markers analysis. J Comp Neurol. En preparacion. Dube MG, Kalra SP, Kalra PS. 1999. Food intake elicited by central administration of orexins/hypocretins: identification o f hypothalamic sites o f action. Brain Res 842(2) :473-477. Eagleson GW, Harris WA. 1990. Mapping of the presumptive brain regions in the neural plate of Xenopus laevis. J Neurobiol 21(3):427-440. Eisenstat DD, Liu JK, Mione M, Zhong W, Yu G, Anderson SA, Ghattas 1, Puelles L, Rubenstein JL. 1999. DLX-1, DLX-2, and DLX-5 expression define distinct stages of basal forebrain differentiation. J Comp Neurol 414(2):217-237. Ericson J, Rashbass P, Schedl A, Brenner-Morton S, Kawakami A, van Heyningen V, Jessell TM, Briscoe J. 1997. Pax6 controls progenitor cell identity and neuronal fate in response to graded Shh signaling. Cell 90(1): 169-180. Ervin GN, Schmitz SA, Nemeroff CB, Prange AJ, Jr. 1981. Thyrotropin-releasing hormone and amphetamine produce different patterns of behavioral excitation in rats. Eur J Pharmacol 72(l):35-43. Ferguson AV, Samson WK. 2003. The orexin/hypocretin system: a critical regulator of neuroendocrine and autonomic function. Front Neuroendocrino1 24(3): 141-150. Ferrân JL, de Oliveira ED, Merchan P, Sandoval JE, Sânchez-Arrones L, Martinez-De-La-T orre M, Puelles L. 2009. Genoarchitectonic profile of developing nuclear groups in the chicken pretectum. J Comp Neurol 517(4):405-451. Ferrân JL, Sânchez-Arrones L, Sandoval JE, Puelles L. 2007. A model o f early molecular regionalization in the chicken embryonic pretectum. J Comp Neurol 505(4):379-403. Fiedler J, Jara P, Luza S, Dorfman M, Grouselle D, Rage F, Lara HE, Arancibia S. 2006. Cold stress induces metabolic activation o f thyrotrophin- releasing hormone-synthesising neurones in the magnocellular division of the hypothalamic paraventricular nucleus and concomitantly changes ovarian sympathetic activity parameters. J Neuroendocrinol 18(5):367-376. Figdor MC, Stem CD. 1993. Segmental organization of embryonic diencephalon. Nature 363(6430):630- 634. Flames N, Pla R, Gelman DM, Rubenstein JL, Puelles L, Marin O. 2007. Delineation o f multiple subpallial progenitor domains by the combinatorial expression of transcriptional codes. J Neurosci 27(36):9682-9695. Fuccillo M, Rutlin M, Fishell G. 2006. Removal of Pax6 partially rescues the loss o f ventral structures in Shh null mice. Cereb Cortex 16 Suppl 1:196-102. 24 1. l i i  Garcia-Lôpez M, Abellân A, Legâz 1, Rubenstein JL, Puelles L, Medina L. 2008. Histogenetic compartments of the mouse centromedial and extended amygdala based on gene expression patterns during development. J Comp Neurol 506(1 ):46-74. Garcia-Lôpez R, Pombero A, Martinez S. 2009. Fate map of the chick embryo neural tube. Dev Growth Differ 51(3):145-165. Garcia-Moreno F, Pedraza M, Di Giovannantonio LG, Di Salvio M, Lopez-Mascaraque L, Simeone A, De Carlos JA. 2010. A neuronal migratory pathway crossing from diencephalon to telencephalon populates amygdala nuclei. Nat Neurosci 13:680-689. Gerard M, Abitbol M, Delezoide AL, Duller JL, Mallet J, Vekemans M. 1995. PAX-genes expression during human embryonic development, a preliminary report. C R A cadS ci 111 318(l):57-66. Geris KL, D'Hondt E, Kuhn ER, Darras VM. 1999. Thyrotropin-releasing hormone concentrations in different regions of the chicken brain and pituitary: an ontogenetic study. Brain Res 818(2):260-266. Gonzalez A, Northcutt RG. 2009. An immunohistochemical approach to lungfish telencephalic organization. Brain Behav Evol 74:43- 55. Gonzâlez A, Lôpez JM, Marin O. 2002a. Expression pattern of the homeobox protein NKX2-1 in the developing Xenopus forebrain. Brain Res Gene Expr Patterns 1(3-4): 181-185. Gonzâlez A, Lôpez JM, Sânchez-Camacho C, Marin O. 2002b. Regional expression o f the homeobox gene NKX2-1 defines pallidal and intemeuronal populations in the basal ganglia of amphibians. Neuroscience 114(3):567-575. Grigoriou M, Tucker AS, Sharpe PT, Pachnis V. 1998. Expression and regulation o f Lhx6 and Lhx7, a novel subfamily of LIM homeodomain encoding genes, suggests a role in mammalian head development. Development 125(11):2063-2074. Guillemin R. 1978. Control of adenohypophysial functions by peptides of the central nervous system. Harvey Lect 71:71-131. Gunhaga L, Jessell TM, Edlund T. 2000. Sonic hedgehog signaling at gastrula stages specifies ventral telencephalic cells in the chick embryo. Development 127(15):3283-3293. Hauptmann G, Gerster T. 2000. Regulatory gene expression patterns reveal transverse and longitudinal subdivisions of the embryonic zebrafish forebrain. Mech Dev 91 (1-2): 105-118. Haynes AC, Jackson B, Overend P, Buckingham RE, Wilson S, Tadayyon M, Arch JR. 1999. Effects o f single and chronic intracerebroventricular administration of the orexins on feeding in the rat. Peptides 20(9): 1099-1105. Heins N, Malatesta P, Cecconi F, Nakafuku M, Tucker KL, Hack MA, Chapouton P, Barde YA, Gotz M. 2002. Glial cells generate neurons: the role of the transcription factor Pax6. Nat Neurosci 5(4):308-315. Herrick. 1910. The morphology of the forebrain in Amphibian and Reptilia. J Comp Neurol 20:413- 547. Hobert O, Ruvkun G. 1998. A common theme for LIM homeobox gene function across phylogeny? Biol Bull 195(3):377-380. Hobert O, Westphal H. 2000. Functions of LIM- homeobox genes. Trends Genet 16(2):75-83. Hodos W. 2008. Evolution of the hypothalamus in anamniotes. "Evolution and Embryological Development o f Forebrain" in Enciclopedic Reference o f Neuroscience, eds M D Binder and N Hirokawa (Springer-Verlag) : 1361 -1363. Holland LZ, Holland ND. 1999. Chordate origins o f the vertebrate central nervous system. Curr Opin Neurobiol 9(5):596-602. Holland LZ, Venkatesh TV, Gorlin A, Bodmer R, Holland ND. 1998. Characterization and developmental expression of AmphiNk2-2, an NK2 class homeobox gene from Amphioxus. (Phylum Chordata; Subphylum Cephalochordata). Dev Genes Evol 208(2): 100-105. Horita A. 1998. An update on the CNS actions of TRH and its analogs. Life Sci 62(17-18): 1443-1448. Horita A, Carino MA, Smith JR. 1976. Effects of TRH on the central nervous system of the rabbit. Pharmacol Biochem Behav 5(Suppl 1): 111-116. Huesa G, van den Pol AN, Finger TE. 2005. Differential distribution of hypocretin (orexin) and melanin-concentrating hormone in the goldfish brain. J Comp Neurol 488(4):476-491. Ingham PW, McMahon AP. 2001. Hedgehog signaling in animal development: paradigms and principles. Genes Dev 15(23):3059-3087. Ingham PW, Placzek M. 2006. Orchestrating ontogenesis: variations on a theme by sonic hedgehog. Nat Rev Genet 7(11):841-850. Jones L, Lopez-Bendito G, Gruss P, Stoykova A, Molnar Z. 2002. Pax6 is required for the normal development o f the forebrain axonal connections. Development 129(21):5041-5052. Kaslin J, Nystedt JM, Ostergard M, Peitsaro N, Panula P. 2004. The orexin/hypocretin system in zebrafish is connected to the aminergic and cholinergic systems. J Neurosci 24(11):2678-2689. Kataoka A, Shimogori T. 2008. Fgf8 controls regional identity in the developing thalamus. Development 135(17):2873-2881. Kenigfest N, Belekhova M, Repérant J, Rio JP, Ward R, Vesselkin N. 2005. The turtle thalamic anterior entopeduncular nucleus shares connectional and neurochemical characteristics with the mammalian thalamic reticular nucleus. J Chem Neuroanat 30(2- 3): 129-143. Kiecker C, Lumsden A. 2004. Hedgehog signaling from the ZLI regulates diencephalic regional identity. Nat Neurosci 7(11): 1242-1249. Kimura S, Hara Y, Pineau T, Femandez-Salguero P, Fox CH, Ward JM, Gonzalez FJ. 1996. The T/ebp null mouse: thyroid-specific enhancer-binding 25 1. li’N IjrH.l'NBiKAJ-. protein is essential for the organogenesis of the thyroid, lung, ventral forebrain, and pituitary. Genes Dev 10(l):60-69. Kohsaka A, Watanobe H, Kakizaki Y, Suda T, Schioth HB. 2001. A significant participation of orexin-A, a potent orexigenic peptide, in the preovulatory luteinizing hormone and prolactin surges in the rat. Brain Res 898(1): 166-170. Kuhlenbeck H. 1973. The Central Nervous System of Vertebrates (Overall Morphologic Pattern, Vol. 3, Part 11), Karger. Lanuza E, Font C, Martinez-Marcos A, Martinez-Garcia F. 1997. Amygdalo-hypothalamic projections in the lizard Podarcis hispanica: a combined anterograde and retrograde tracing study. J Comp Neurol 384(4):537-555. Lazzaro D, Price M, de Felice M, Di Lauro R. 1991. The transcription factor TTF-1 is expressed at the onset of thyroid and lung morphogenesis and in restricted regions of the foetal brain. Development 113(4): 1093-1104. Li HS, Yang JM, Jacobson RD, Pasko D, Sundin O. 1994. Pax-6 is first expressed in a region of ectoderm anterior to the early neural plate: implications for stepwise determination of the lens. Dev Biol 162(1):181-194. Liu JK, Ghattas 1, Liu S, Chen S, Rubenstein JL. 1997. Dix genes encode DNA-binding proteins that are expressed in an overlapping and sequential pattern during basal ganglia differentiation. Dev Dyn 210(4):498-512. Lopez JM, Dominguez L, Gonzalez A. 2008. Immunohistochemical localization o f thyrotropin- releasing hormone in the brain of reptiles. J Chem Neuroanat 36(3-4):251-263. Lôpez JM, Dominguez L, Moreno N, Morona R, Joven A, Gonzalez A. 2009. Distribution of orexin/hypocretin immunoreactivity in the brain o f the lungfishes Protopterus dolloi and Neoceratodus forsteri. Brain Behav Evol 74(4):302-322. Lubkin M, Stricker-Krongrad A. 1998. Independent feeding and metabolic actions o f orexins in mice. Biochem Biophys Res Commun 253(2):241-245. Lupo G, Harris WA, Lewis KE. 2006. Mechanisms of ventral patterning in the vertebrate nervous system. Nat Rev Neurosci 7(2): 103-114. Malendowicz LK, Tortorella C, Nussdorfer GG. 1999. Orexins stimulate corticosterone secretion o f rat adrenocortical cells, through the activation of the adenylate cyclase-dependent signaling cascade. J Steroid Biochem Mol Biol 70(4-6): 185-188. Marin O, Rubenstein JL. 2001. A long, remarkable journey: tangential migration in the telencephalon. Nat Rev Neurosci 2(11):780-790. Marin O, Baker J, Puelles L, Rubenstein JL. 2002. Patterning of the basal telencephalon and hypothalamus is essential for guidance of cortical projections. Development 129(3):761-773. Marin O, Gonzalez A, Smeets WJ. 1997a. Basal ganglia organization in amphibians: afferent connections to the striatum and the nucleus accumbens. J Comp Neurol 378(1): 16-49. Marin O, Smeets WJ, Gonzalez A. 1997b. Basal ganglia organization in amphibians: development of striatal and nucleus accumbens connections with emphasis on the catecholaminergic inputs. J Comp Neurol 383(3):349-369. Marin O, Smeets WJ, Gonzalez A. 1998a. Basal ganglia organization in amphibians: chemoarchitecture. J Comp Neurol 392(3):285-312. Marin O, Smeets WJ, Gonzalez A. 1998b. Basal ganglia organization in amphibians: evidence for a common pattern in tetrapods. Prog Neurobiol 55(4):363-397. Markakis EA. 2002. Development o f the neuroendocrine hypothalamus. Front Neuroendocrinol 23(3):257-291. Martinez-Garcia F, Novejarque A, Lanuza E. 2006. Evolution of the amygdala in vertebrales. In: Kaas JH, Editor, Evolution of the nervous system. A comprehensive reference, Elsevier Academic Press, Oxford, pp. 255-334. Martinez-Marcos A, Lanuza E, Halpem M. 1999. Organization of the ophidian amygdala: chemosensory pathways to the hypothalamus. J Comp Neurol 412(l):51-68. Martynska L, Polkowska J, Wolinska-Witort E, Chmielowska M, Wasilewska-Dziubinska E, Bik W, Baranowska B. 2006. Orexin A and its role in the regulation of the hypothalamo-pituitary axes in the rat. Reprod Biol 6 Suppl 2:29-35. Medina L. 2008a. Basal ganglia: evolution. In: Squire L et al. (eds). The new encyclopedia o f neuroscience. Elsevier, Oxford. Medina L. 2008b. "Evolution and Embryological Development o f Forebrain" in Enciclopedic Reference of Neuroscience, eds M. D. Binder and N. Hirokawa (Springer-Verlag), 1172-1192. Menuet A, Alunni A, Joly JS, Jeffery WR, Retaux S. 2007. Expanded expression of Sonic Hedgehog in Astyanax cavefish: multiple consequences on forebrain development and evolution. Development 134(5):845-855. Merlo GR, Zerega B, Paleari L, Trombino S, Mantero S, Levi G. 2000. Multiple functions o f Dix genes. Int J Dev Biol 44(6):619-626. Mitsuma T, Hirooka Y, Mori Y, Kayama M, Adachi K, Rhue N, Ping J, Nogimori T. 1999. Effects of orexin A on thyrotropin-releasing hormone and thyrotropin secretion in rats. Horm Metab Res 31(ll):606-609. Morales-Delgado N, Merchan P, Bardet SM, Ferrân JL, Puelles L, Diaz C. 2011. Topography of Somatostatin Gene Expression Relative to Molecular Progenitor Domains during Ontogeny of the Mouse Hypothalamus. Front Neuroanat 5:10. Moreno N, Gonzalez A. 2003. Hodological characterization of the medial amygdala in anuran amphibians. J Comp Neurol 466(3);389-408. 26 Moreno N, Gonzalez A. 2004. Localization and connectivity of the lateral amygdala in anuran amphibians. J Comp Neurol 479(2): 130-148. Moreno N, Gonzalez A. 2005a. Central amygdala in anuran amphibians: neurochemical organization and connectivity. J Comp Neurol 489(1):69-91. Moreno N, Gonzalez A. 2005b. Forebrain projections to the hypothalamus are topographically organized in anurans: conservative traits as compared with amniotes. Eur J Neurosci 21 (7): 1895-1910. Moreno N, Gonzalez A. 2006. The common organization of the amygdaloid complex in tetrapods: new concepts based on developmental, hodological and neurochemical data in anuran amphibians. Prog Neurobiol 78(2):61-90. Moreno N, Gonzalez A. 2011. The non-evaginated secondary prosencephalon of vertebrates. Front Neuroanat 5:12. Moreno N, Bachy I, Rétaux S, Gonzalez A. 2004. LIM- homeodomain genes as developmental and adult genetic markers of Xenopus forebrain functional subdivisions. J Comp Neurol 472(1 ):52-72. Moreno N, Dominguez L, Rétaux S, Gonzalez A. 2008. Islet 1 as a marker o f subdivisions and cell types in the developing forebrain of Xenopus. Neuroscience 154(4): 1423-1439. Moreno N, Gonzalez A, Rétaux S. 2009. Development and evolution of the subpallium. Semin Cell Dev Biol 20(6):735-743. Moreno N, Morona R, Lopez JM, Gonzalez A. 2010. Subdivisions of the turtle Pseudemys scripta subpallium based on the expression of regulatory genes and neuronal markers. J Comp Neurol 518:4877-902. Morona R, Ferrân JL, Puelles L, Gonzalez A. 2011. Embryonic genoarchitecture of the pretectum in Xenopus laevis: a conserved pattern in tetrapods. J Comp Neurol 519(6): 1024-1050. Nakagawa Y, O'Leary DD. 2001. Combinatorial expression patterns o f LlM-homeodomain and other regulatory genes parcellate developing thalamus. J Neurosci 2 1(8):2711-2725. Neary TJ, Northcutt RG. 1983. Nuclear organization of the bullfrog diencephalon. J Comp Neurol 213(3):262-278. Nieuwenhuys R, Voogd J, van Huijzen C. 2008. The Human Central Nervous System. Springer, Germany. Northcutt RG. 1970. The telencephalon of the Western painted turtle (Chrysemys picta bellis). Chicago: University of Illinois Press. Osorio J, Mazan S, Retaux S. 2005. Organization of the lamprey (Lampetra fluviatilis) embryonic brain: insights from LlM-homeodomain, Pax and hedgehog genes. Dev Biol 288(1): 100-112. Osorio J, Megias M, Pombal MA, Retaux S. 2006. Dynamic expression of the LlM-homeodomain gene Lhxl5 through larval brain development of the sea lamprey (Petromyzon marinus). Gene Expr Patterns 6(8):873-878. Panganiban G, Rubenstein JL. 2002. Developmental functions o f the Distal-less/Dlx homeobox genes. Development 129(19):4371-4386. Pombal MA, Puelles L. 1999. Prosomeric map o f the lamprey forebrain based on calretinin immunocytochemistry, Nissl stain, and ancillary markers. J Comp Neurol 414(3):391-422. Prange A, Jr. 1974. Proceedings: Behavioral effects of hypothalamic polypeptides in animals and man. Psychopharmacol Bull 10(4):11-15. Price M, Lazzaro D, Pohl T, Mattei MG, Ruther U, Olivo JC, Duboule D, Di Lauro R. 1992. Regional expression of the homeobox gene Nkx-2.2 in the developing mammalian forebrain. Neuron 8(2):241-255. Price M, Lemaistre M, Pischetola M, Di Lauro R, Duboule D. 1991. A mouse gene related to Distal- less shows a restricted expression in the developing forebrain. Nature 351(6329):748-751. Pu S, Jain MR, Kalra PS, Kalra SP. 1998. Orexins, a novel family of hypothalamic neuropeptides, modulate pituitary luteinizing hormone secretion in an ovarian steroid-dependent manner. Regul Pept 78(1-3):133-136. Puelles L. 2001. Thoughts on the development, structure and evolution of the mammalian and avian telencephalic pallium. Philos Trans R Soc Lond B Biol Sci 356(1414):1583-1598. Puelles L, Rubenstein JL. 1993. Expression patterns of homeobox and other putative regulatory genes in the embryonic mouse forebrain suggest a neuromeric organization. Trends Neurosci 16(ll):472-479. Puelles L, Rubenstein JL. 2003. Forebrain gene expression domains and the evolving prosomeric model. Trends Neurosci 26(9):469-476. Puelles L, Kuwana E, Puelles E, Bulfone A, Shimamura K, Keleher J, Smiga S, Rubenstein JL. 2000. Palliai and subpallial derivatives in the embryonic chick and mouse telencephalon, traced by the expression o f the genes Dlx-2, Emx-1, Nkx-2.1, Pax-6, and Tbr-1. J Comp Neurol 424(3):409-438. Puelles L, Martinez S, Martinez-de-la-Torre M, Rubenstein JL. 2004. The Rat Nervous System . Gene maps and related histogenetic domains in the forebrain and midbrain. In: G. Paxinos E, editor. San Diego: Elsevier. Puelles L, Martinez de la Torre M, Paxinos G, Watson C, Martinez S. 2007. The Chick Brain in Stereotaxic Coordinates: an Atlas featuring Neuromeric Subdivisions and Mammalian Homologies. Academic Press/Elsevier, San Diego. Qiu M, Bulfone A, Ghattas 1, Meneses JJ, Christensen L, Sharpe PT, Presley R, Pedersen RA, Rubenstein JL. 1997. Role of the Dix homeobox genes in proximodistal patterning of the branchial arches: mutations o f Dix-1, Dlx-2, and Dlx-1 and -2 alter morphogenesis o f proximal skeletal and soft tissue structures derived from the first and second arches. Dev Biol 185(2): 165-184. 27 1. LrH.rNJl.KAlj Rallu M, Corbin JG, Fishell G. 2002. Parsing the prosencephalon. Nat Rev Neurosci 3(12);943-951. Reichlin S. 1986. Neural functions of TRH. Acta Endocrinol Suppl (Copenh) 276:21-33. Rétaux S, Bachy 1. 2002. A short history of LIM domains (1993-2002): from protein interaction to degradation. Mol Neurobiol 26(2-3):269-281. Rétaux S, Rogard M, Bach 1, Failli V, Besson MJ. 1999. Lhx9: a novel LlM-homeodomain gene expressed in the developing forebrain. J Neurosci 19(2):783-793. Robinson GW, Wray S, Mahon KA. 1991. Spatially restricted expression o f a member o f a new family of murine Distal-less homeobox genes in the developing forebrain. New Biol 3(12):1183-1194. Rohr KB, Barth KA, Varga ZM, Wilson SW. 2001. The nodal pathway acts upstream of hedgehog signaling to specify ventral telencephalic identity. Neuron 29(2):341-351. Roth M, Bonev B, Lindsay J, Lea R, Panagiotaki N, Houart C, Papalopulu N. 2010. FoxGl and TLE2 act cooperatively to regulate ventral telencephalon formation. Development 137(9): 1553-1562. Russell SH, Kim MS, Small CJ, Abbott CR, Morgan DG, Taheri S, Murphy KG, Todd JF, Ghatei MA, Bloom SR. 2000. Central administration of orexin A suppresses basal and domperidone stimulated plasma prolactin. J Neuroendocrinol 12(12):1213-1218. Sakurai T, Amemiya A, Ishii M, Matsuzaki 1, Chemelli RM, Tanaka H, Williams SC, Richarson JA, Kozlowski GP, Wilson S, Arch JR, Buckingham RE, Haynes AC, Carr SA, Annan RS, McNulty DE, Liu WS, Terrett JA, Elshourbagy NA, Bergsma DJ, Yanagisawa M. 1998. Orexins and orexin receptors: a family o f hypothalamic neuropeptides and G protein- coupled receptors that regulate feeding behavior. Cell 92(5): 1 page following 696. Sander M, Paydar S, Ericson J, Briscoe J, Berber E, German M, Jessell TM, Rubenstein JL. 2000. Ventral neural patterning by Nkx homeobox genes: Nkx6.1 controls somatic motor neuron and ventral intemeuron fates. Genes Dev 14(17):2134-2139. Schafer M, Kinzel D, Neuner C, Schartl M, Volff JN, Winkler C. 2005. Hedgehog and retinoid signalling confines Nkx2.2b expression to the lateral floor plate of the zebrafish trunk. Mech Dev 122(l):43-56. Schally AV. 1978. Aspects o f hypothalamic regulation of the pituitary gland. Science 202(4363): 18-28. Schally AV, Redding TW, Bowers CY, Barrett JF. 1969. Isolation and properties of porcine thyrotropin- releasing hormone. J Biol Chem 244(15):4077-4088. Seoane LM, Tovar SA, Perez D, Mallo F, Lopez M, Senaris R, Casanueva FF, Dieguez C. 2004. Orexin A suppresses in vivo GH secretion. Eur J Endocrinol 150(5):731-736. Sheng HZ, Bertuzzi S, Chiang C, Shawlot W, Taira M, Dawid 1, Westphal H. 1997. Expression of murine Lhx5 suggests a role in specifying the forebrain. Dev Dyn 208(2):266-277. Shimogori T, Lee DA, Miranda-Angulo A, Yang Y, Wang H, Jiang L, Yoshida AC, Kataoka A, Mashiko H, Avetisyan M, Qi L, Qian J, Blackshaw S. 2010. A genomic atlas o f mouse hypothalamic development. Nat Neurosci 13(6):767-775. Shirasaka T, Kunitake T, Takasaki M, Kannan H. 2002. Neuronal effects o f orexins: relevant to sympathetic and cardiovascular fimctions. Regul Pept 104(l-3):91-95. Singletary KG, Deviche P, Strand C, Delville Y. 2006. Distribution of orexin/hypocretin immunoreactivity in the brain of a male songbird, the house finch, Carpodacus mexicanus. J Chem Neuroanat 32(2- 4):81-89. Small EM, Vokes SA, Garriock RJ, Li D, Krieg PA. 2000. Developmental expression of the Xenopus Nkx2-1 and Nkx2-4 genes. Mech Dev 96(2):259- 262. Soma M, Aizawa H, Ito Y, Maekawa M, Osumi N, Nakahira E, Okamoto H, Tanaka K, Yuasa S. 2009. Development o f the mouse amygdala as revealed by enhanced green fluorescent protein gene transfer by means o f in utero electroporation. J Comp Neurol 513(1):113-128 Stoykova A, Gruss P. 1994. Roles of Pax-genes in developing and adult brain as suggested by expression patterns. J Neurosci 14(3 Pt 2): 1395- 1412. Stoykova A, Treichel D, Hallonet M, Gruss P. 2000. Pax6 modulates the dorsoventral patterning o f the mammalian telencephalon. J Neurosci 20(21):8042-8050. Striedter GF. 1997. The telencephalon of tetrapods in evolution. Brain Behav Evol 49(4): 179-213. Striedter GF. 2005. Principles o f brain evolution. Sinauer Associates Inc., editor. Sunderland, MA, USA. Sussel L, Marin O, Kimura S, Rubenstein JL. 1999. Loss o f Nkx2.1 homeobox gene function results in a ventral to dorsal molecular respecification within the basal telencephalon: evidence for a transformation of the pallidum into the striatum. Development 126(15):3359-3370. Sweet DC, Levine AS, Billington CJ, Kotz CM. 1999. Feeding response to central orexins. Brain Res 821(2):535-538. Szabo NE, Zhao T, Cankaya M, Theil T, Zhou X, Alvarez-Bolado G. 2009b. Role of neuroepithelial Sonic hedgehog in hypothalamic patterning. J Neurosci 29(21):6989-7002. Szabo NE, Zhao T, Zhou X, Alvarez-Bolado G. 2009a. The role of Sonic hedgehog o f neural origin in thalamic differentiation in the mouse. J Neurosci 29(8):2453-2466. Tamura T, Irahara M, Tezuka M, Kiyokawa M, Aono T. 1999. Orexins, orexigenic hypothalamic neuropeptides, suppress the pulsatile secretion of luteinizing hormone in ovariectomized female rats. Biochem Biophys Res Commun 264(3):759-762. Teijido O, Manso MJ, Anadon R. 2002. Distribution of thyrotropin-releasing hormone immunoreactivity in 28 the brain o f the dogfish Scyliorhinus canicula. J Comp Neurol 454(1 ):65-81. Tsuchida T, Ensini M, Morton SB, Baldassare M, Edlund T, Jessell TM, Pfaff SL. 1994. Topographic organization of embryonic motor neurons defined by expression of LIM homeobox genes. Cell 79(6):957- 970. van den Akker WM, Brox A, Puelles L, Durston AJ, Medina L. 2008. Comparative functional analysis provides evidence for a crucial role for the homeobox gene Nkx2.1/Titf-1 in forebrain evolution. J Comp Neurol 506(2):211-223. Vieira C, Garda AL, Shimamura K, Martinez S. 2005. Thalamic development induced by Shh in the chick embryo. Dev Biol 284(2):351-363. Vieira C, Martinez S. 2006. Sonic hedgehog from the basal plate and the zona limitans intrathalamica exhibits differential activity on diencephalic molecular regionalization and nuclear structure. Neuroscience 143(1);129-140. Volkoff H. 2006. The role of neuropeptide Y, orexins, cocaine and amphetamine-related transcript, cholecystokinin, amylin and leptin in the regulation of feeding in fish. Comp Biochem Physiol A Mol Integr Physiol 144(3);325-331. Volkoff H, Canosa LF, Unniappan S, Cerda-Reverter JM, Bernier NJ, Kelly SP, Peter RE. 2005. Neuropeptides and the control of food intake in fish. Gen Comp Endocrinol 142(l-2):3-19. Vue TY, Aaker J, Taniguchi A, Kazemzadeh C, Skidmore JM, Martin DM, Martin JF, Treier M, Nakagawa Y. 2007. Characterization of progenitor domains in the developing mouse thalamus. J Comp Neurol 505(1):73-91. Walther C, Gruss P. 1991. Pax-6, a murine paired box gene, is expressed in the developing CNS. Development 113(4):1435-1449. Warren N, Price DJ. 1997. Roles of Pax-6 in murine diencephalic development. Development 124(8):1573-1582. Wilson L, Maden M. 2005. The mechanisms of dorsoventral patterning in the vertebrate neural tube. Dev Biol 282(1): 1-13. Wilson SW, Houart C. 2004. Early steps in the development o f the forebrain. Dev Cell 6(2):167-181. W olf G. 1998. Orexins: a newly discovered family of hypothalamic regulators of food intake. Nutr Rev 56(6): 172-173. Wullimann MF, Puelles L. 1999. Postembryonic neural proliferation in the zebrafish forebrain and its relationship to prosomeric domains. Anat Embryol (Berl) 199(4):329-348. Wullimann MF, Rink E. 2001. Detailed immunohistology o f Pax6 protein and tyrosine hydroxylase in the early zebrafish brain suggests role of Pax6 gene in development of dopaminergic diencephalic neurons. Brain Res Dev Brain Res 131 ( 1 -2): 173-191. Yasuda T, Masaki T, Kakuma T, Hara M, Nawata T, Katsuragi I, Yoshimatsu H. 2005. Dual regulatory effects o f orexins on sympathetic nerve activity innervating brown adipose tissue in rats. Endocrinology 146(6):2744-2748. Yates FE, Russell SM, Maran JW. 1971. Brain- adenohypophysial communication in mammals. Annu Rev Physiol 33:393-444. Zardoya R, Meyer A. 2001a. The evolutionary position o f turtles revised. Naturwissenschaften 88(5): 193- 200. Zardoya R, Meyer A. 2001b. On the origin o f and phylogenetic relationships among living amphibians. Proc Natl Acad Sci U S A 98(13):7380-7383. Zeltser LM. 2005. Shh-dependent formation of the ZLI is opposed by signals from the dorsal diencephalon. Development 132(9):2023-2033. Zhao XF, Suh CS, Prat CR, Ellingsen S, Fjose A. 2009. Distinct expression o f two foxgl paralogues in zebrafish. Gene Expr Patterns 9(5):266-272. Zhao Y, Marin O, Hermesz E, Powell A, Flames N, Palkovits M, Rubenstein JL, Westphal H. 2003. The LIM-homeobox gene Lhx8 is required for the development of many cholinergic neurons in the mouse forebrain. Proc Natl Acad Sci U S A 100(15):9005-9010. 29 30 2. Estudios quimioarquitectônîcos en el encéfalo adulto Distribution of thyrotropin-releasing hormone (TRH) immunoreactivity in the brain of urodele amphibians Brain Behaviour Evolution 71(3):231-246. Immunohistochemical localization of orexins (hypocretins) in the brain of reptiles and its relation to monoaminergic systems Journal of Chemical Neuroanatomy 39(l):20-34 2. ESTUDIOS QUIMIOARQUITECTONICOS EN EL ENCÉFALO ADULTO Original Paper Brain, Belumor andEvolnbon Brain Behav Evol 2008;71:231-246 DOI: 10.1159/000122835 Received: November 19,2007 Returned for revision: December 13,2007 Accepted after revision: January 14,2008 Published online: April 2,2008 Distribution of Thyrotropin-Releasing Hormone (TRH) immunoreactivity in the Brain of Urodele Amphibians Laura Dominguez Jesus M. Lopez Agustfn Gonzalez Departamento de Bioiogia Celular, Facultad de Biologia, Unlversidad Complutense, Madrid, Spain Key Words TRH • Tyrosine hydroxylase • Immunohistochemistry • Preoptic area • Hypothalamus • Hypophysis • Urodeles Evolution found in anurans. Therefore, the important role of skin color adaptation proposed for TRH in anurans on the basis of the direct innervation of the intermediate lobe is not applicable for urodeles. c o p y r ig h t © 2008 s. K arger AG, Basel Abstract To improve knowledge of the peptidergic systems in the brain of amphibians we have conducted a comparative anal­ ysis of the distribution of TRH immunoreactive cell bodies and fibers in three species of urodeles. Fiber labeling was observed in all main brain subdivisions suggesting different control functions for TRH in extrahypothalamic systems. However, as in other vertebrates, TRH neurons were abun­ dant in the hypothalamic nuclei that presumably project to the median eminence and the neural lobe of the hypophysis. Considerable interspecies differences were noted mainly re­ lated to innervation of the olfactory and visual centers (thal­ amus and mesencephalic tectum) and the precise localiza­ tion of immunoreactive cell bodies, which was assessed by double labeling with tyrosine hydroxylase. The comparison of the distribution of TRH immunoreactive neurons and fi­ bers found in urodeles with those reported for other verte­ brates, in particular with anamniotes, reveals a strong resem­ blance but also notable variations not only across vertebrate classes but also within the same class. In this respect, the vir­ tual lack in urodeles of TRH innervation of the intermediate lobe of the hypophysis clearly contrasts with the innervation Introduction Thyrotropin-releasing hormone (TRH) is a tripeptide amide (pGIu-His-Pro-NH2) also known as thyrotropin- releasing factor (TRF) and thyroliberin or protirelin. It was chemically characterized by Schally et al. [1969] and Guillemin [1970] and, isolated from ovine hypothalamic extracts, shown to stimulate the liberation o f thyroid- stimulating hormone (TSH) in the hypophysis o f the rat [Boler et al., 1969; Burgus et al., 1969a, b, 1970]. TRH is synthesized from a large precursor protein that contains multiple copies o f the TRH progenitor tetrapeptide Gln- His-Pro-Gly. The number o f copies varies across species from five [rodents; Lechan et al., 1986; Satoh et al., 1992] to eight [salmonid fish; Ohide et al., 1996]. TRH has been localized in the central nervous system of mammals [Hokfelt et al., 1975; Johansson and Hokfelt, 1980; Johansson et al., 1981; Kreider et al., 1985; Lechan et al., 1986; Tsuruo et al., 1987,1988a, b; Merchenthaler et al., 1988; Sasek et al., 1990; Lynn et a l , 1991]. TRH im ­ munoreactive (TRHir) neurons were primarily found in KARGER Fax+41 6130612 34 E-Mail lcarger@karger.ch www.karger.com © 2008 s. Karger AG, Basel 0006-8977/08/0713-0231*24.50/0 Accessible online at: www.karger.com/bbe Dr. Agustln Gonzalez D epartam ento de Biologia Celular Facultad de Biologia, Universidad Complutense ES-28040 M adrid (Spain) Tel. +34 91 394 4977, Fax +34 91 394 4981, E-Mail agustin@bio.ucm.es 33 mailto:lcarger@karger.ch http://www.karger.com http://www.karger.com/bbe mailto:agustin@bio.ucm.es 2. ESTUDIOS QUIMIOARQUITECTONICOS EN EL ENCEFALO ADULTO the preoptic area, paraventricular nucleus, anterior, dor- somedial and lateral hypothalamic nuclei, and arcuate nucleus. In addition, studies that combined immunohis­ tochemistry and tract tracing techniques revealed projec­ tions from the TRHir neurons o f the supraoptic and para­ ventricular nuclei to the hypophysis o f the rat and high concentration o f TRH was found in the median eminence [Kawano et al., 1991]. According to the distribution o f TRHir neurons and fibers the more clear and common function o f TRH is hypophysiotropic, promoting liberation o f TSH in the adenohypophysis [Boler et al., 1969; Schally et al., 1969; Yates et al., 1971; Azizi et al., 1974]. Moreover, other hy­ pophysiotropic functions for TRH such as stimulation o f secretion of prolactin (PR) [Jackson and Reichlin, 1977] and growth hormone (GH) [Guillemin, 1978; Schally, 1978] were reported in different species. In addition, the extensive extrahypothalamic distribution o f the TRHir fibers and terminals also suggested that this neuropep­ tide could function as neurotransmitter/neuromodulator with implications in processes such as analgesia, sexual excitation [Prange, 1974; Horita et al., 1976a, b; Ervin et al., 1981; Boschi et al., 1983; Horita, 1998], thermoregula­ tion [Fiedler et al., 2006], food intake behavior [Ao et al., 2006], dream control [Reichlin, 1986] and autonomic functions [Helke and Phillips, 1988] among others. These functions were further supported in mammals by the demonstration of a wide distribution of TRH receptors in the brain [Taylor and Burt, 1982; Calza et al., 1992; Wu et al., 1992; Satoh et al., 1997; Heuer et al., 2000]. The structural similarity of TRH across vertebrates led to studies of the brain distribution of TRHir cells and fibers in numerous non-mammalian species from differ­ ent vertebrate classes [agnathans: De Andrés et al., 2002; elasmobranchs: Teijido et al., 2002; teleosts: Batten et al., 1990a, b; Hamano et al., 1990; Matz and Takahashi, 1994; Diaz et al., 2001,2002; birds: Jôzsa et al., 1988,1989; Péc- zely and Kiss, 1988; Jôzsa and Kawano et al., 1991, Geris et al., 1999]. In general, TRH was primarily detected in neurons of the preoptic area and hypothalamus, although the presence o f TRH in neurons o f other brain regions has also been reported [Matz and Takahashi, 1994; Diaz et al., 2001,2002; Teijido et al., 2002]. Studies in amphibians have shown that TRH is the major PR-releasing factor and also stimulates TSH, GH and melanocyte-stimulating hormone (a-MSH) in frogs [Malagon et al., 1989; Gracia-Navarro et al., 1991; Ander­ sen et al., 1992; Nakajima et al., 1993; Gonzâlez de Agui­ lar et al., 1994]. Previous data on the localization o f TRH in the brain of amphibians were reported only for several anurans, with emphasis on the hypothalamohypophysial system [Seki et al., 1983; Mimnagh et al., 1987; Lamacz et a l, 1989; Zoeller and Conway, 1989; Yukata et al., 1990; Miranda and Affanni, 2000]. In these studies some inter­ specific differences were noted, mainly related to TRHir neurons present in various extrahypothalamic areas such as the amygdala, septum or optic tectum. Given the differences observed among anuran species and the total lack of data about the TRH systems in uro­ deles, the aim o f the present comparative study was to assess shared and specific features o f the TRH system in the brain o f amphibians by conducting a detailed analysis of its distribution in the brain of three urodele amphibi­ ans: Ambystoma mexicanum, Ambystoma tigrinum and Pleurodeles waltl. These particular species were chosen because they have been extensively used in neuroana- tomical studies and there is a considerable amount of data available regarding the organization of different neuro- chemically characterized systems and, therefore, the TRH distribution pattern can be precisely interpreted. Immunohistochemistry for tyrosine hydroxylase (TH, the first and rate-limiting enzyme for catecholamine syn­ thesis) has been used in our study in combination with TRH immunohistochemistry because, according to the data in anurans and our previous results in urodeles, both chemicals could coexist in certain brain regions. In addition, the presence o f TRH in dopamine cells o f the rat olfactory bulbs [Tsuruo et al., 1988b] or in noradren­ ergic neurons o f the locus coeruleus in zebrafish [Diaz et al., 2002] suggested the possible coexistence o f these m ol­ ecules in the same neurons. The double stained sections also served to characterize the precise localization of the TRHir elements in relation to the well-established anatomy o f the catecholaminergic neurons in urodeles [Gonzalez and Smeets, 1991,1994,1995]. M aterials and M ethods For the present study, a total of 8 Iberian ribbed newts (Pleuro­ deles waltl), 3 axolotls (Ambystoma mexicanum) and 3 tiger sala­ manders (Ambystoma tigrinum) were used. The animals were ob­ tained from the laboratory stocks of the Department o f Cell Biol­ ogy, University Complutense o f Madrid (P. waltl) and from commercial suppliers (A. tigrinum and A. mexicanum). In the case of P. waltl, the animals were 2-3 year old males and females weighting 12-18 g, whereas the A. mexicanum and A. tigrinum were adult males and females of similar size but their exact age could not be assessed. The original research reported herein was performed under the aninial care guidelines established by European Union (86/609/EEC) and the Spanish Royal Decree 223/1998. 232 Brain Behav Evol 2008;71:231-246 Dominguez/Lôpez/Gonzâlez 34 2. ESTUDIOS QUIMIOARQUITECTONICOS EN EL ENCÉFALO ADULTO AU animais were anesthetized in a 0.3% solution of tricaine methanesulfonate (MS-222, Sandoz; pH 7.3) and perfused trans- cardially with physiological saline followed by 150-200 ml of 4% paraformaldehyde and 1% glutaraldehyde in a 0.1 M phosphate buffer (PB, pH 7.4). Three animals (one male o f each species) re­ ceived an intraperitoneal injection o f colchicine (10 mg for each 30 g of animal weight) dissolved in saline 24-30 h prior the perfu­ sion. The brains were removed and kept in the same fixative for 2-3 h. Subsequently, they were immersed in a solution of 30% su­ crose in PB for 3-5 h at 4°C until they sank, blocked in a solution of 20% gelatin with 30% sucrose in PB, and stored overnight in a solution of 4% formaldehyde and 30% sucrose in PB. Sections were cut on a freezing microtome at 40 jxm thickness in the trans­ verse or sagittal plane and collected in cold PB. TRH Immunohistochemistry The free-floating sections were rinsed twice in PB, treated with 1% H2 O2 in PB for 15 min to reduce endogenous peroxidase activity, rinsed again three times in PB and processed by the per­ oxidase antiperoxidase (PAP) method [Sternberger, 1979]. This included a first incubation o f the sections in rabbit anti-TRH se­ rum (Biogenesis, England; code 8940-0504) diluted 1:100 in PB containing 0.5% Triton X-100 (PBS-T) for 48-72 h at 4 “C. Subse­ quently, they were rinsed in PB for 10 min and incubated in the second antibody swine anti-rabbit (diluted 1:50 in PBS-T; Dako A/S, Glostrup, Denmark), for 60 min at room temperature. After rinsing, the sections were incubated for 90 min in rabbit PAP complex (diluted 1:500 in PBS-T; Dako) and rinsed three times in PB. Finally, the sections were stained in 0.5 mg/ml 3,3'-diamino- benzidine (DAB; Sigma) or in DAB intensified with nickel [Ad­ ams et al., 1981] with 0.01% H 2 O2 in PB for 5-10 min. The sections were then mounted on glass slides from a solution of 0.25% gelatin in 0.05 M Tris-HCl buffer (TB, pH 7.6) and after dehydration the slides were coverslipped with Entellan (Merck, Darmstadt, Ger­ many). Some sections were counterstained with cresyl violet to facilitate analysis o f the results. The TRH antiserum used was raised in rabbit against TRH conjugated to hemocyanin. The specificity of the immunohisto­ chemical reaction was corroborated with controls that included: (1) staining o f some selected sections with preimmune rabbit se­ rum; (2) controls in which either the primary antibody, secondary antibody or the PAP complex was omitted; (3) preabsorptions of the primary antibody with synthetic TRH (Sigma; code P1319; 0.1,1.0 or 10 |x M ). In all these controls, the immunostaining was eliminated, even when the rabbit anti-TRH was preabsorbed with TRH at low concentration (0.1 p ,M ). It should be noted that the sequence of TRH was determined from a frog skin extract and it was found that the primary struc­ ture of TRH is identical in amphibians and in mammals. In fact, the sequence of TRH has been fully conserved across the verte­ brate phylum, indicating that strong evolutionary pressure has acted to preserve the structure of this peptide [Vaudry et al., 1999]. Therefore, the use of antibodies against mammalian TRH seems a valuable tool for unraveling TRH immunoreactive struc­ tures in different vertebrates. Double TRH and TH Immunohistochemistry A procedure based on immunohistofluorescence was used as follows: (1) first incubation was for 72 h at 4 “C in a mixture of rab­ bit anti-TRH (diluted 1:100) and mouse anti-TH (diluted 1:1,000; Immunostar, USA; code P22941); (2) second incubation was for 90 min at room temperature in a mixture of Alexa Fluor 594-conju- gated goat anti-rabbit (red fluorescence, diluted 1:500; Molecular Probes, Denmark) and Alexa Fluor 488-conjugated goat anti­ mouse (green fluorescence, diluted 1:300; Molecular Probes). Af­ ter rinsing, the sections were mounted on glass slides and cover- slipped with Vectashield (Vector, Burlingame, Calif, USA). Prior to all incubations in the second antibody cocktails, the sections were incubated for 1 h at room temperature in normal goat serum. The specificity of the TH antibody was assessed in urodeles and the pattern of immunostaining obtained in this study fully cor­ roborated the distribution of THir cells and fibers reported previ­ ously [Franzoni et al., 1986; Gonzâlez and Smeets, 1991]. Abbreviations used in the figures ac anterior commissure Ip interpeduncular nucleus POp posterior preoptic area Acc nucleus accumbens Is nucleus isthmi Ra raphe nucleus aob accesory olfactory bulb Lp lateral pallium Rf reticular formation Apl amygdala pars lateralis Ls lateral septum Ri inferior reticular nucleus Apm amygdala pars medialis Ifb lateral forebrain bundle Rm median reticular nucleus Cb cerebellum LDT laterodorsal tegmental nucleus Rs superior reticular nucleus cc central canal LF lateral funiculus S septum Dp dorsal pallium Mp medial pallium SC suprachiasmatic nucleus DTh dorsal thalamus Ms medial septum sol solitary tract dh dorsal horn o f spinal cord nPT pretectal nucleus Str striatum DF dorsal funiculus nl neural lobe of hypophysis Tegm mesencephalic tegmentum dl distal lobe of hypophysis Nsol nucleus o f the solitary tract V ventricle glomerular layer ob olfactory bulbs VH ventral hypothalamus Hb habenula oc optic chiasm vh ventral horn of spinal cord igl internal granular layer OT optic tectum VF ventral funiculus U intermediate lobe o f hypophysis POa anterior preoptic area VTh ventral thalamus TRH in the Brain o f Urodeles Brain Behav Evol 2008;71:231-246 233 35 2. ESTUDIOS QUIMIOARQUITECTONICOS EN EL ENCEFALO ADULTO Mp aob i Ms Ms Mo Mp Apm ac .A p ( # # nPT DTh Ifb 234 Brain Behav Evol 2008;71:231-246 Dominguez/Lôpez/Gonzâlez 36 2. ESTUDIOS QUIMIOARQUITECTONICOS EN EL ENCEFALO ADULTO VH %Ra A S Tegm sol Ra DF LF VF Fig. 1. a-p Diagrams of transverse sections through the brain of Ambystoma tigrinum at the levels indicated in the schematic lateral view of the brain. TRHir cell bodies (large dots) and fibers (small dots, wavy lines) are represented in the right half of each section. Scale bars = 500 p,m. Tbe distribution o f TRHir cell bodies and fibers in the brain of the three species used and the pattern o f labeling was charted in representative transverse sections at different brain levels for the case o f Ambystoma tigrinum (fig. 1). Drawings were made by means o f a camera lucida in which the sections counterstained with cresyl violet facilitated the interpretation o f the localization of the [abeled structures. For the double-labeling experiments, the sections were analyzed with an Olympus BX51 fluorescence mi­ croscope with appropriate filter combinations for the identifica­ tion of TRH and TH immunoreactive cells and fibers. Selected sections were photographed using a digital camera (Olympus DP70). Contrast and brightness were adjusted in Adobe Photo­ shop 7.0 (Adobe System, San Jose, Calif., USA). TRH in the Brain of Urodeles Brain Behav Evol 2008;71:231-246 235 37 2. ESTUDIOS QUIMIOARQUITECTONICOS EN EL ENCÉFALO ADULTO Table 1. Comparative localization and relative abundance of TRH-immunoreactive cells and fibers in CNS o f the urodeles am­ phibians Pleurodeles waltl Ambystoma mexicanum Ambystoma tigrinum C F C F C F Telencephalon ob - - - + - + Pallium - + - + - + Acc - +++ - ++ - ++ Str - +++ - +++ - +++ S - +++ - +++ - +++ Amygdala - +++ + +++ + +++ Preoptic area and hypothalamus PO +++ +++ +++ +++ ++ ++ SC - ++ + ++ + ++ VH ++ -♦-++ +++ +++ ->-++ +++ Diencephalon Hb - + - + - + Thalamus - ++ + ++ + ++ Pretectum - ++ - ++ - ++ Mesencephalon and isthmus OT - ++ + ++ ++ ++ Tegm - ++ - ++ - ++ LS - ++ - ++ - ++ Ip - ++ - ++ - ++ Rhombencephalon Rf - + - + - + Nsol - + - + - + Spinal cord - + - + - + C - Immunoreactive cell bodies; F = immunoreactive fibers. + = Low density; ++ = moderate density; +++ = high density; - = no immunoreactive cell bodies or fibers. Results The antibody against TRH used in the present study revealed patterns of immunoreactivity that, for each of the three species examined, were constant from animal to animal. In the cases in which colchicine was used, the pat­ tern o f immunoreactivity obtained was the same although the cell bodies, in general, were more intensely labeled. In addition, no sex differences were observed between ani­ mals o f the same species. Diverse interspecific differences were noted and have been summarized in table 1. In all cases, widespread immunoreactive structures were local­ ized in all major brain subdivisions and will be described from rostral to caudal levels. Selected sections of single labeling experiments are shown in figures 2 and 3, where­ as doubly labeled sections are shown in figure 4. Telencephalon In the main olfactory bulbs of Ambystoma, scattered TRHir fibers were located in the internal granular layer (fig. la) and these fibers were not observed in Pleurodeles. The accessory olfactory bulbs were virtually devoid of immunoreactivity. Also scarce was the innervation o f the pallium in the three species, where only thin varicose fi­ bers and terminal-like structures were dispersed primar­ ily in the superficial portion of the medial, lateral and dorsal palliai subdivisions (fig. Ib-e). Within the subpallium, abundant TRHir fibers were located throughout its rostrocaudal extent. Particularly well innervated were the nucleus accumbens and the stri­ atal neuropil in which TRHir varicose fibers occupied superficial portions (fig. lb, c, 2a, b). In the three species examined, dense terminal-like structures formed con­ spicuous patches in the dorsal and rostral portion o f the striatum (fig. 2b). The rather reduced septal regions of the urodele brain also showed abundant TRHir fibers from rostral to caudal levels, and were more numerous in the lateral septal area than in the medial area (fig. Ic). In the caudal telencephalon, both the medial and lateral portions of the amygdala were densely innervated by TRHir fibers (fig. Id, e, 2c). In these locations, most of the labeling was found in the external fibrous zone although also abundant fibers were labeled among the cell bodies located close to the ventricle (fig. 2c). TRHir cells were consistently observed in the pars medialis o f the amyg­ dala (fig. le), which constituted a rather compact group in A. tigrinum but were dispersed cells throughout the length o f the amygdala in A. mexicanum. Preoptic Area and Hypothalamus Most TRHir elements were located within the preoptic area and the hypothalamus. Starting at the rostral por­ tion of the preoptic area, at the level o f the anterior com­ missure, abundant TRHir cells were observed in relation to the ventricle. They possessed two main cell processes, one long extending laterally, and one short toward the ventricular lining suggesting that they were cerebrospi­ nal fluid (CSF)-contacting neurons (see fig. le, 4a, b). More caudally, two distinct TRHir cell groups in the pos­ terior region o f the preoptic area were distinguished (fig. 2d, e). The first group was made up o f neurons most likely o f the CSF-contacting cell type (arrows in fig. 2d, e, 4c, d), whereas the second TRHir cell group formed a band o f neurons in the external limit between the peri­ ventricular cell layer and the lateral fiber zone (asterisks in fig. 2d, e). The rostrocaudal extent o f these cells reached suprachiasmatic levels only in Ambystoma (fig. Ih), 236 Brain Behav Evol 2008;71:231-246 Dominguez/Lôpez/Gonzâlez 38 2. ESTUDIOS QUIMIOARQUITECTONICOS EN EL ENCÉFALO ADULTO a/oiOG'' R waltl ^ R 0 a l t l f. . i » v A_ / m s - r A.m e mm P O p « Fig. 2. Photomicrographs o f transverse sections through the forebrain illustrating TRHir cell bodies and fibers in the nucleus accumbens (a), striatum (b), amygdala (c), posterior preoptic area (d, e), and ventral hypothala­ mus (f). Arrows in d and e point to CSF-contacting cells and asterisks mark the lateral group o f TRHir cells. Scale bars = 100 p,m. TRH in the Brain o f Urodeles Brain B ehav Evol 2008;71:231-246 237 39 2. ESTUDIOS QUIMIOARQUITECTONICOS EN EL ENCEFALO ADULTO R waltl W b icanum R waltlR w a l t ^ Tegm 5^. S ; V Fig. 3. Photomicrographs o f transverse sections through the forebrain and brainstem illustrating TRHir cell bodies and fibers in the distal lobe o f the hypophysis (a), the thalamus (arrow points to the intensely labeled fiber neuropil in the ventral thalamus) (b), ventral thalamus (c), optic tectum (d), mesencephalic tegmentum (e), and isthm ic region (f). Scale bars = 100 |xm. 238 Brain B ehav Evol 2008;71:231-246 D om inguez/Lopez/G onzâlez 40 2. ESTUDIOS QUIMIOARQUITECTONICOS EN EL ENCEFALO ADULTO whereas in P. waltl they were restricted to more rostral positions. Throughout the preoptic area, primarily in lateral re­ gions, abundant and intense TRHir fibers were located and most frequently corresponded to the lateral dendrit­ ic arborizations of the TRHir cells located more medially in the preoptic area. The labeled fibers formed a disperse pattern and no specific tracts were observed (fig. le -g , 2d, e). The ventral hypothalamus housed a numerous TRHir cell population mainly located in the infundibulum (fig. li-k , 2f). These cells showed perikarya located at dif­ ferent depth within the periventricular cell layer but al­ most all of them possessed a process that reached the periventricular lining, whereas in the opposite side o f the body longer processes directed ventrolaterally reached the lateral portion of the infundibulum and coursed cau­ dally toward the hypophysis (figs Ij, k, 2f). In all three species, numerous TRHir fibers were found in the me­ dian eminence and the neural lobe of the hypophysis. In addition, very scarce fibers also reached the intermediate lobe in Ambystoma. O f note, numerous cells were stained in the pars distalis o f the hypophysis only in P. waltl (fig. 3a). Diencephalon Profuse and disperse TRHir fibers were observed mainly in the dorsal thalamus, habenula and pretectal regions o f the diencephalon (fig. Ig-i). In general, the in­ nervation of the ventral thalamus was lesser than in dor­ sal regions but the visual centers showed a particularly dense innervation in P. waltl (fig. 3b). The presence o f a small group o f TRHir cells located dorsomedially to the lateral forebrain bundle, within the ventral thalamus, was unique to Ambystoma (fig. Ig, 3c). Mesencephalon and Isthmus The optic tectum displayed a layered arrangement o f TRHir fibers that was slightly different in each o f the three species. In the genus Am bystom a, this innervation was m ainly distributed in the efferent fiber layers 4 and 5 and was less abundant in the deep layer 7 (fig. lj-1) [layering according to Roth et a l , 1990]. In contrast, in the tectum o f Pleurodeles layers 7 and 5 were the most conspicuously innervated. A striking difference found in the tectum was the presence o f TRHir cell bodies in A m bystom a but not in Pleurodeles (fig. Ik). They were scattered, pear-shaped neurons located at different lev­ els o f layer 6 and were more abundant in A. tigrinum (fig. 3d). Widely distributed TRHir fibers were also present in the mesencephalic and isthmic tegmentum (fig. Ik-m). In the mesencephalon they formed a disperse field medi­ ally, close to the ventricle, and constituted a dense neuro­ pil laterally (fig. 3e). Caudally in the isthmus, abundant fiber labeling was found in the region o f the isthmic and laterodorsal tegmental nuclei, in the interpeduncular neuropil and in the superior reticular nucleus (fig. Im, 30. Rhombencephalon and Spinal Cord No TRHir cell bodies were found in the medulla and spinal cord o f the three species. However, fiber labeling was found mainly within descending tracts in the medial and lateral aspects o f the rhombencephalon (fig. In, o). Throughout its extent, the fibers seemed to innervate the medial and inferior reticular nuclei, as well as the raphe nuclei and the nucleus of the solitary tract (fig. In, o). A considerable number of fibers reached the spinal cord within the dorsal and lateroventral funiculi (fig. Ip). Only occasionally, some TRHir terminal-like structures were found among the cell bodies of the dorsal and ventral gray spinal fields. Double TRH and TH Immunohistochemistry The codistribution of THir cells with the TRHir neurons was studied by means of double imm unohisto­ fluorescence. With this technique details about fiber dis­ tribution, which are nicely revealed with the DAB pro­ cedure, are not observed because of background fluores­ cence due to the glutaraldehyde included in the fixative mixture. However, the immunoreactive cell bodies were clearly observed and the precise localization o f the TRHir neurons could be assessed in relation to the localization of the THir neurons (fig. 4). In spite o f the extensive co­ distribution o f cells labeled for each case throughout the preoptic area and hypothalamus, no actual colocaliza­ tion o f both chemicals in the same neurons could be dem­ onstrated (fig. 4a-d). In particular, at rostral levels o f the anterior preoptic area both cell populations were largely intermingled (fig. 4a, b), whereas at more caudal levels clear dorsoventral segregation of THir and TRHir cells was found (fig. 4c, d). It should be noted that in the me­ dian eminence and hypophysis a dense innervation of both types o f fibers was found but although THir fibers clearly innervated the intermediate lobe and TRHir fi­ bers reached the neural lobe, some TRHir fibers inter­ mingled with THir fibers were detected in the intermedi­ ate lobe of Ambystoma (fig. 4e). TRH in the Brain of Urodeles Brain Behav Evol 2008;71:231-246 239 41 2. ESTUDIOS QUIMIOARQUITECTONICOS EN EL ENCEFALO ADULTO A.tigrinum I . POa POa P. waltl A.tigrinum i h ■ 240 Brain Behav Evol 2008;71:231-246 D om inguez/Lôpez/G onzâlez 42 2. ESTUDIOS QUIMIOARQUITECTONICOS EN EL ENCÉFALO ADULTO Discussion The aim of the present study was to provide for the first time a detailed description of the organization o f the TRHir cell bodies and fibers in the brain o f urodele am­ phibians. In the following section the general organiza­ tion and variations o f the TRH systems in amphibians (anurans and urodeles) is discussed and compared with those of other vertebrates. In particular, the data of recent studies in different anamniotes obtained with techniques similar to the one used in our study will be discussed [Diaz et al., 2001,2002; De Andrés et al., 2002; Teijido et al., 2002]. In urodeles, as in anurans, most of the TRHir cells were located in the hypothalamus [Andersen et al., 1992]. In addition, interspecies variations were found with re­ spect to the TRHir neurons in extrahypothalamic areas and the widespread distribution of reactive fibers in all the main brain subdivisions. In amniotes variations were reported for the TRH distribution and some o f them were clearly observed between cases in which pretreatment with colchicines was or was not used. Thus, in untreated animals TRHir material was detectable in nerve fibers and terminals, but the cell bodies were only weakly stained or did not stain at all [Tsuruo et al., 1987,1988a, b; Jôzsa et al., 1988]. However, in recent studies in differ­ ent anamniotes wide distribution patterns for TRH were described that included nicely stained cell bodies and in none of these studies colchicines was used [Lamacz et al., 1989; Miranda and Affanni, 2000; Diaz et al., 2001,2002; De Andrés et al., 2002; Teijido et al., 2002]. In our study, we have used one animal o f each species with colchicine pretreatment as controls and have observed that, at least in urodeles, the same immunoreactive cell groups are ob­ served for each species with only a slight improvement of the intensity of the reaction; a result that contrasts with the situation found in amniotes. It should be noted that in a previous study in the an­ uran Xenopus laevis proTRH mRNA was found in nu­ merous places of the brain that could not be revealed with Fig. 4. Photomicrographs showing, in the same sections, staining for TH (green fluorescence) and TRH (red fluorescence). The re­ lationship between the distinct cell populations is illustrated for the anterior (a, b; the framed area in a is shown in higher magni­ fication in b), and posterior preoptic area (c, d). TRH labeling is shown in the neural lobe of the hypophysis whereas TH fibers are entering in the intermediate lobe (e). Scale bars = 100 pum (a, c-e) and 10 |im (b). TRH immunohistochemistry in other anuran species [Bidaud et al., 2004]. Similar discrepancies when com­ paring results o f TRH immunohistochemistry and pro­ TRH mRNA localization were reported for mammals [Tsuruo et al., 1987,1988a]. In addition, we have used glu­ taraldehyde in the fixative mixture because it was found that using glutaraldehyde-containing fixatives in the rat increased the number o f labeled cells and fibers [Tsuruo et al., 1987] and similar results were obtained in urodele amphibians [present results; Anadôn et al., 2002]. The most rostrally located TRHir fibers in the urodele brain were found in the olfactory bulbs, but only in the genus Ambystoma. Species differences were also noted in teleosts where some species o f salmonids possess TRHir cells in the olfactory bulbs [Matz and Takahashi, 1994; Ando et al., 1998; Diaz et al., 2001], whereas in the zebra­ fish and sea bass only scarce fiber labeling was observed [Batten et al., 1990a, b; Diaz et al., 2002]. Lampreys, how­ ever, lack TRHir structures in the olfactory bulbs and it was proposed that this would be the primitive character [De Andrés et al., 2002]. In addition, TRHir cells and fi­ bers have been reported in the olfactory bulbs of an elas- mobranch fish [Teijido et al., 2002]. Therefore, the situa­ tion found in amphibians in which anurans seem to lack TRHir structures in the bulbs [Mimnagh et al., 1987; A n­ dersen et al., 1992; Miranda and Affanni, 2000] which is also the case in the urodele P. waltl (present results) would support the contention that the importance o f a TRH sys­ tem in the olfactory system varies across anamniotes. Available data for mammals confirm the acquisition o f a TRHir system of cells and fibers in the olfactory bulbs [Lechan et al., 1986; Segerson et al., 1987; Merchenthaler et al., 1988; Tsuruo et al., 1988a; Heuer et al., 2000]. W ithin palliai regions, only scarce TRHir fibers are present in urodeles and this feature is shared by anurans [Mimnagh et al., 1987; Andersen et al., 1992; Miranda and Affanni, 2000]. The pallium o f lampreys is almost devoid o f TRHir fibers [De Andrés et al., 2002] but, strik­ ingly different, the dorsal pallium of the dogfish possess­ es TRHir fibers and also abundant cell bodies [Teijido et al., 2002] representing another distinct pattern of neuro­ chemical organization in the pallium of elasmobranchs [Meredith and Smeets, 1987; Anadôn et al., 2000]. Fiber staining distributed over the dorsal and ventral telence­ phalic areas o f teleost fish have been reported for all spe­ cies studied [Batten et al., 1990a, b; Hamano et al., 1990; Matz and Takahashi, 1994; Diaz et al., 2001,2002]. In am­ niotes, only very scarce TRHir fibers have been reported in cortical areas [Parker and Porter, 1983; Jôzsa et al., 1988]. TRH in the Brain of Urodeles Brain Behav Evol 2008;71:231-246 241 43 2. ESTUDIOS QUIMIOARQUITECTONICOS EN EL ENCEFALO ADULTO Concurring with our results in urodeles, most o f the TRHir structures in the telencephalon o f all groups stud­ ied are located in the subpallium. Thus, intense TRHir fibers were found in septal, striatal and amygdaloid areas (or their counterparts) in species of all vertebrate classes. However, the presence of TRHir neurons in telencephal­ ic areas shows marked differences across species. Virtu­ ally no telencephalic cells were found in the lamprey [De Andrés et al., 2002], whereas abundant cells were report­ ed in ventral territories in salmonids teleosts and in the zebrafish [Matz and Takahashi, 1994; Diaz et al., 2001, 2002] that could not be observed in other teleosts [Batten et al., 1990a, b; Hamano et al., 1990]. In anurans, although with some species differences, TRHir cells are located in the diagonal band of Broca and medial amygdaloid ter­ ritories [Seki et al., 1983; Mimnagh et al., 1987; Zoeller and Conway, 1989; Miranda and Affanni, 2000], a situa­ tion also observed in mammals [Lechan et al., 1986; Tsu­ ruo et al., 1987; Merchenthaler et al., 1988; Heuer et al., 2000] but not in birds [Jôzsa et al., 1988]. Our results in urodeles point to the consistent presence o f TRHir neu­ rons in medial territories o f the amygdala in all amphib­ ians but this so-called pars medialis o f the amygdala [Northcutt and Kicliter, 1980] is currently considered (at least in part) as equivalent to the bed nucleus of the stria terminalis (BST) [Moreno and Gonzalez, 2006, 2007, 2008] and, therefore, this TRHir cell population could be compared with the cell population in the BST o f mam­ mals [Heuer et al., 2000]. The general shared pattern of distribution for TRHir cells in all vertebrates includes distinct cell populations in the preoptic area and the hypothalamus. However, the precise localization o f the cells shows differences across species that, in some cases, makes the comparison among cell groups difficult. Our results in urodeles show that the main TRHir cell populations are located in the anterior preoptic area and in the ventral portion of the infundibu­ lar hypothalamus. Although there are differences, this situation is largely comparable to that observed in an­ urans but the abundant cells reported in the dorsal infun­ dibular regions are not found in urodeles [Seki et al., 1983; Mimnagh et al., 1987; Lamacz et al., 1989; Miranda and Affanni, 2000]. In urodeles, numerous TRHir neu­ rons with CSF-contacting processes have been observed in the anterior preoptic area and the ventral hypothala­ mus, similar to observations made in the lamprey and this fact was regarded as a primitive feature [De Andrés et al., 2002]. In fact, only scarce TRHir cells in the hypo­ thalamus of an elasmobranch fish have been described as CSF-contacting cells [Teijido et al., 2002] and these cells were not observed among the relatively numerous cell populations found in the preoptic area and hypothala­ mus of teleosts [Batten et al., 1990a, b; Hamano et al., 1990; Matz and Takahashi, 1994; Diaz et al., 2001,2002]. W ithin amniotes, data available in birds and mammals show that abundant TRHir cells are widely distributed in the preoptic region and hypothalamus but no CSF-con­ tacting cells were reported. In birds, TRHir cells were primarily found in the paraventricular and magnocellu- lar preoptic nuclei [Jôzsa et al., 1988; Péczely and Kiss, 1988]. In the rat, numerous TRHir cells were distributed in the medial preoptic and anterior hypothalamic areas and the paraventricular, dorsomedial and arcuate nuclei [Hokfelt et al., 1975; Lechan et al., 1986; Tsuruo et al., 1987; Merchenthaler et al., 1988]. Both in birds and mam­ mals TRHir cells in the paraventricular nucleus account for the important projections towards the hypophysis [Péczely and Kiss, 1988; Kawano et al., 1991]. The relationship between the TRHir cell system and the hypophysis has received special attention in amphib­ ians [Lamacz et al., 1989; Roubos, 1997; Galas, 1998; Vaudry et al., 1999; Bidaud et al., 2004]. In general, the neurohypophysis of anurans is innervated by a dense net­ work o f TRHir fibers [Andersen et al., 1992] but a lower number o f fibers also reach the intermediate lobe [Mim­ nagh et al., 1987; Verburg-van Kemenade, 1987; Lamacz et al., 1989]. In the neurohypophysis, TRH has been ob­ served in fibers that also contained mesotocin (MST) that most likely arise in the caudal extension of the preoptic nucleus where doubly TRH/MST labeled cells were found [Lamacz et al., 1989]. In addition, it has been demonstrat­ ed that TRH is a potent stimulator o f a-melatonin (a- MSH) secretion in the intermediate lobe of the hypophy­ sis and, therefore, would play a major role in the neuro­ endocrine regulation of skin-color adaptation [Roubos, 1997; Vaudry et al., 1999; Bidaud et al., 2004]. Compara­ tively, our results in urodeles regarding the heavy inner­ vation of the neurohypophysis and the localization of the TRHir cells in the preoptic area would suggest the pos­ sibility of the existence of a TRH/MST hypophysial sys­ tem as in anurans [Gonzâlez and Smeets, 1992; Lowry et al., 1997]. However, in a previous study in Triturus cris- tatus, TRH immunoreactivity was not found in the inter­ mediate lobe and in vitro assays also suggested that TRH does not exert any effect on a-MSH secretion [Danger et al., 1989]. Our results suggested that some TRHir fibers might innervate the intermediate lobe in Ambystoma and they codistribute with TH-containing fibers. In the pres­ ent study o f urodeles, retrograde tracing from the inter­ mediate lobe labeled cells in the suprachiasmatic nucleus 242 Brain Behav Evol 2008;71:231-246 Dominguez/Lôpez/Gonzàlez 44 2. ESTUDIOS QUIMIOARQUITECTONICOS EN EL ENCEFALO ADULTO [Artero et al., 1994] in places where TRHir cells are lo­ cated. However, the suprachiasmatic nucleus also pos­ sesses abundant TH cells, both in anurans and urodeles [Gonzalez and Smeets, 1991,1994] that do project to the intermediate lobe [Tuinhof et al., 1994] and form a sepa­ rate population from the TRHir cells (present results for urodeles and our unpublished results in Xenopus laevis and Rana perezi). In summary, in anurans two important and separated catecholaminergic and TRHergic projec­ tions to the intermediate lobe would control the a-MSH secretion, whereas in urodeles the significance o f the TRH system seems less important. O f note, in teleosts the neurohypophysis receives abundant TRHir fibers [Batten et al., 1990a, b; Hamano et al., 1990; Matz and Takahashi, 1994; Diaz et al., 2001,2002] and some were found close to the a-MSH-producing cells [Batten et al., 1990b; Diaz et al., 2001]. A peculiarity found in P. waltl was the presence of TRHir glandular cells in the posterior lobe of the hypoph­ ysis and the control procedures in our study demonstrat­ ed that this labeling was specific. A similar result has been reported only in the hypophysis o f the dogfish [Teijido et al., 2002]. However, although immunohistochemical studies in anurans have not reported TRHir cells in the posterior lobe, a recent study in Xenopus laevis has shown that proTRH mRNA is expressed in the distal lobe [Bi­ daud et al., 2004], in concordance with results obtained in mammals [Croissandeau et al., 1992; Pagésy et al., 1992; Peters et al., 1997; Bruhn et al., 1998]. Therefore, it appears that TRH synthesis in the posterior lobe of the hypophysis might be a more widespread phenomenon and a para­ crine/autocrine regulation o f hormone secretion in this lobe has been suggested [Bidaud et al., 2004]. Urodeles possess a relatively abundant TRH innerva­ tion of the thalamus and, in addition, some cells were also observed in Ambystoma. Comparatively, anurans lack al­ most completely TRHir structures in the thalamus [An­ dersen et al., 1992] and this correlates well with the scarce localization o f TRH receptors found in Xenopus [Bidaud et al., 2004]. A TRHir cell group was found in the central posterior thalamic nucleus [Diaz et al., 2002] only in ze­ brafish, whereas in other teleosts and birds no TRHir thalamic neurons have been reported [Jôzsa et al., 1988; Péczely and Kiss, 1988; Batten et al., 1990a, b; Hamano et al., 1990; Matz and Takahashi, 1994; Diaz et al., 2001]. Interestingly, immunohistochemical and in situ hybrid­ ization techniques have revealed the presence o f TRHir cells in the reticular thalamic nucleus of the rat [Segerson et al., 1987; Merchenthaler et al., 1988] as well as TRH receptors in some dorsal thalamic nuclei [Heuer et al.. 2000; O'Dowd et al., 2000]. These results suggest impor­ tant functions for TRH in the thalamus o f mammals as recently reported in the ferret, in which regulation o f in­ trinsic thalamocortical activity and function in the pro­ motion o f awakening was demonstrated [Broberger and McCormick, 2005]. The optic tectum o f urodeles shows a layered arrange­ ment o f TRHir fibers, but only in Ambystoma cells also were observed suggesting a role for TRH in the visual system o f these amphibians. Most studies in anurans do not describe TRHir structures in the tectum [Seki et al., 1983; Mimnagh et al., 1987; Miranda and Affanni, 2000], but some exceptions might exist such as in Rana pipiens in which TRHir cells seem to exist [see Andersen et al., 1992]. However, it might be possible that the amount o f TRH in tectal cells is low and not revealed with im m uno­ histochemical techniques; in Xenopus no TRHir cells are observed in the tectum [unpublished results] but abun­ dant proTRH mRNA is contained in tectal cells [Bidaud et al., 2004]. Comparatively, TRHir fibers, but not cell bodies, have been found in the tectum o f all anamniotes studied, whereas amniotes lack TRHir cells and fibers in the optic tectum and superior colliculus [Tsuruo et al., 1987; Jôzsa et al., 1988; Péczely and Kiss, 1988; Heuer et al., 2000]. The mesencephalic and isthmic tegmental regions of urodeles are particularly well innervated by TRHir fibers but no cell bodies were observed. This innervation in­ cluded regions of the laterodorsal tegmental nucleus and the isthmic nucleus, i.e. two cholinergic cell groups in the urodele brain [Marin et al., 1997]. Interestingly, in some fish these cholinergic nuclei possess TRHir cells [Diaz et al., 2001, 2002]. In addition, the locus coeruleus of uro­ deles, whose cells are intermingled with the cholinergic cells o f the laterodorsal tegmental nucleus, are richly in­ nervated by TRHir fibers, as observed in our doubly la­ beled sections for TH and TRH, suggesting a possible role in the noradrenergic function [Gonzâlez and Smeets, 1995]. Notably, in the zebrafish TRH was found in locus coeruleus cells [Diaz et al., 2002]. The presence of TRHir elements in the rhombenceph­ alon and spinal cord of anurans and urodeles is rather scarce and is mainly formed by descending fiber tracts in the ventral and ventrolateral aspects of the medulla that reach the spinal cord in its dorsal and lateral funiculi. Throughout their course, these fibers reach reticular neu­ rons, raphe nuclei and regions of the nucleus of the soli­ tary tract. These main TRHir projections to the reticular regions have been reported for lampreys and teleosts [De Andrés et al., 2002; Diaz et al., 2001, 2002]. However, TRH in the Brain o f Urodeles Brain Behav Evol 2008;71:231-246 243 45 2. ESTUDIOS QUIMIOARQUITECTONICOS EN EL ENCEFALO ADULTO TRHir cells in the vagal motor nucleus o f some teleosts [Ando et al., 1998; Diaz et al., 2001, 2002] and rat [Seger­ son et al., 1987; Tsuruo et al., 1987; Heuer et al., 2000] were not found in amphibians. Finally, the TRHir cells found in the descending trigeminal nucleus of rat and dogfish [Tsuruo et al., 1987; Merchenthaler et al., 1988; Fleming and Todd, 1994; Heuer et al., 2000; Teijido et al., 2002] or within the spinal cord [Fleming and Todd, 1994; Heuer et al., 2000] seem to be unique to these species. Concluding Remarks The present results in urodeles show a widespread sys­ tem o f TRHir fibers in all the main brain regions that would support the notion that this peptide possesses multiple functions in the brain. In particular, TRH could primarily influence the function o f basal telencephalic, thalamic, tectal and tegmental structures. However, our results support the main role for TRH in the hypothala- mo-hypophysial system. In adult urodeles it was shown that TRH is capable of stimulating TSH release [Jacobs et al., 1988] although this stimulation is even more strong­ ly accomplished by corticotrophin-releasing hormone [Licht and Denver, 1990] primarily during the metamor­ phosis [Boorse and Denver, 2002]. The importance of TRH on a-MSH secretion in the intermediate lobe seems to be strikingly different between anurans and urodeles. Thus, TRH is currently considered a potent stimulator o f a-MSH secretion in anurans and this is supported by the strong innervation by TRHir fibers in the intermediate lobe [Lamacz et al., 1989; Roubos, 1997; Vaudry et al., 1999; Bidaud et al., 2004]. However, in urodeles, if this function exists, it would be only supported by the very low amount of TRHir fibers observed in the intermediate lobe o f Ambystoma. A cknow ledgem ents This research was supported by the Spanish Ministry o f Sci­ ence and Technology. Grant number: BFU2006-01014/BFI. R eferences Anadôn R, D iaz ML, Becerra M, M anso MJ (2002) Presence o f thyrotropin-releasing- horm one-im m unoreactive (TRHir) ama- crine cells in the retina o f anuran and uro­ dele am phibians. Brain Res 926:86-93. Anadôn R, M olist P, Rodriguez-M oldes I, Lôpez JM, Q uintela I, Cervino MC, Barja P, G onzâlez A (2000) D istribution o f choline acetyltransferase im m unoreactivity in the brain o f an elasm obranch, the lesser spotted dogfish (Scyliorhinus canicula). J C om p N eu ­ rol 420:139-170. Andersen AC, Tonon MC, Pelletier G, Conlon JM, Fasolo A, Vaudry H (1992) N europep­ tides in the am phibian brain. Int Rev Cytol 138:89-210. Ando H, Ando J, Urano A (1998) Localization o f m RNA encod ing thyrotropin-releasing hor­ m one precursor in the brain o f sockeye salm ­ on. Z ool Sci 15:945-953. Ao Y, G o VL, Toy N , Li T, W ang Y, Song MK, Reeve JR, Jr, Liu Y, Yang H (2006) Brainstem thyrotropin-releasing horm one regulates food intake through vagal-dependent ch o­ linergic stim ulation o f ghrelin secretion. En­ docrinology 147:6004-6010. Artero C, Fasolo A, Franzoni MF (1994) M ultiple sources o f the pituitary pars interm edia in ­ nervation in amphibians: a D il retrograde tract-tracing study. Neurosci Lett 169:163- 166. Azizi F, Vagenakis AG, Bollinger J, Reichlin S, Bush JE, Braverman LE (1974) The effect o f a single large dose o f thyrotropin-releasing horm one on various aspects o f thyroid func­ tion in the rat. E ndocrinology 95:1767-1770. Batten TF, Cambre ML, M oons L, Vandesande F (1990a) Comparative distribution o f neuro- peptide-im m unoreactive system s in the brain o f the green molly, Poecilia latipinna. J Comp N eurol 302:893-919. Batten TF, M oons L, Cambre ML, Vandesande F, Seki T, Suzuki M (1990b) Thyrotropin-re- leasing horm one-im m unoreactive system in the brain and pituitary gland o f the sea bass (Dicentrarchus labrax, Teleostei). Gen Comp Endocrinol 79:385-392. Bidaud I, Galas L, Bulant M, Jenks BG, Ouwens DT, Jegou S, Ladram A, Roubos EW, Tonon MC, Nicolas P, Vaudry H (2004) D istribu­ tion o f the m RNAs encoding the thyrotro­ pin-releasing horm one (TRH) precursor and three TRH receptors in the brain and pitu­ itary o f Xenopus laevis: effect o f background color adaptation on TRH and TRH receptor gene expression. J Comp Neurol 477:11-28. Boler J, Enzm ann F, Folkers K, Bowers CY, Schally AV (1969) The identity o f chem ical and horm onal properties o f the thyrotropin releasing horm one and pyroglutam yl-histi- dyl-proline am ide. Biochem Biophys Res C om m un 37:705-710. Boorse GC, Denver RJ (2002) Acceleration o f A m bystom a tigrinum m etam orphosis by corticotropin-releasing horm one. J Exp Zool 293:94-98. Boschi G, D esiles M, Reny V, Rips R, W riggles- w orth S (1983) Antinociceptive properties o f thyrotropin releasing horm one in mice: com parison w ith morphine. Br J Pharmacol 79:85-92. Broberger C, M cCorm ick DA (2005) Excitatory effects o f thyrotropin-releasing horm one in the thalamus. J N eurosci 25:1664-1673. Bruhn TO, Rondeel JM, Jackson IM (1998) Thy­ rotropin-releasing horm one gene expression in the anterior pituitary. IV. Evidence for paracrine and autocrine regulation. Endo­ crinology 139:3416-3422. Burgus R, D unn TF, D esiderio D , G uillem in R (1969a) M olecular structure o f the hypotha­ lam ic hypophysiotropic TRF factor o f ovine origin: m ass spectrom etry dem onstration o f the PC A -H is-Pro-N H 2 sequence. C R Acad Sci Hebd Seances Acad Sci D 269:1870- 1873. Burgus R, D unn TF, Desiderio D, Vale W, G uil­ lem in R (1969b) Synthetic polypeptide de­ rivatives w ith TRF hypophysiotropic activi­ ty. N ew data. C R Acad Sci Hebd Seances Acad Sci D 269:226-228. Burgus R, D unn TF, D esiderio D, Ward DN , Vale W, G uillem in R (1970) Characterization o f ovine hypothalam ic hypophysiotropic TSH- releasing factor. Nature 226:321-325. Calza L, G iardino L, C eccatelli S, Zanni M, Elde R, Hokfelt T (1992) D istribution o f thyrotro­ pin-releasing horm one receptor messenger RNA in the rat brain: an in situ hybridization study. N euroscience 51:891-909. Croissandeau G, Pagesy P, Grouselle D, Le Daf- niet M, Peillon F, Li JY (1992) Im m unoreac­ tive thyroliberin (TRH) precursor forms in hum an hypothalam us and anterior pituitary tissues. FEBS Lett 298:191-194. 244 Brain Behav Evol 2008;71:231-246 Dominguez/Lôpez/Gonzâlez 46 2. ESTUDIOS QUIMIOARQUITECTONICOS EN EL ENCEFALO ADULTO Danger JM, Perroteau I, Franzoni MF, Saint- Pieire S, Fasolo A, V audry H (1989) Innerva­ tion o f the pars interm edia and control o f al- pha-m elanotropin secretion in the newt. N earoendocrinology 50:543-549. D e Ancrés M C, A nadôn R, M anso MJ, G onzâlez MJ (2002) D istribution o f thyrotropin-re- leaang horm one im m unoreactivity in the brain o f larval and adult sea lampreys, P etro­ m yzon m arinus L. J C om p Neurol 453:323- 335 D iaz NL, Becerra M, M anso MJ, A nadôn R (2001) Developm ent o f thyrotropin-releas­ ing horm one im m unoreactivity in the brain o f the brown trout Salm o tru tta fario . J C om p Neurol 429:299-320. D iaz ML, Becerra M, M anso MJ, Anadôn R (2002) D istribution o f thyrotropin-releasing hormone (TRH) im m unoreactivity in the brain o f the zebrafish (Danio rerio). J Com p Neurol 450:45-60 . Ervin GN, Schm itz SA, N em eroff CB, Prange AJ, Jr ;1981) Thyrotropin-releasing horm one and am phetam ine produce different pat­ terns o f behavioral excitation in rats. Eur J Pharmacol 72:35-43. Fiedler J, Jara P, Luza S, D orfm an M, Grouselle D, Rage F, Lara HE, Arancibia S (2006) Cold stress induces m etabolic activation o f thy- rotrophin-releasing horm one-synthesising neurones in the m agnocellular division o f the hypothalam ic paraventricular nucleus and concom itantly changes ovarian sym pa­ thetic activity parameters. J N euroendocri­ nol 18:367-376. Fleming AA, Todd AJ (1994) Thyrotropin-re- leasing horm one- and GABA-like im m u n o­ reactivity coexist in neurons in the dorsal horn o f the rat spinal cord. Brain Res 638: 347-351. Franzoni MF, Thibault J, Fasolo A, M artinoli MG, Scaranari F, Galas A (1986) O rganiza­ tion o f tyrosine-hydroxylase im m unoposi- tive neurons in the brain o f the crested new t, Triturus cristatus carnifex. J Comp N eurol 251:121-134. Galas L, Lamacz M, G am ier M, Roubos EW, Tonon MC, Vaudry H (1998) Involvem ent o f extracellular and intracellular calcium sources in TR H -induced alpha-MSH secre­ tion from frog m elanotrope cells. M ol Cell Endocrinol 138:25-39. Geris KL, D 'H ond t E, Kuhn ER, Darras VM (1999) Thyrotropin-releasing horm one con­ centrations in different regions o f the chick­ en brain and pituitary: an ontogenetic study. Brain Res 818:260-266. G onzâlez A, Sm eets WJAJ (1991) Comparative analysis o f dopam ine and tyrosine hydroxy­ lase im m unoreactivities in the brain o f tw o am phibians, the anuran Rana ridibunda and the urodele Pleurodeles waltl. J Com p N eurol 303:457-477. G onzâlez A, Sm eets WJAJ (1992) Comparative analysis o f the vasotocinergic and m esoto- cinergic cells and fibers in the brain o f two amphibians, the anuran Rana ridibunda and the urodele Pleurodeles waltl. J Comp N eurol 315:53-73. G onzâlez A, Sm eets WJAJ (1995) Noradrenergic and adrenergic system s in the brain o f the urodele am phibian, Pleurodeles w altl, as re­ vealed by im m unohistochem ical m ethods. C ell Tissue Res 279:619-627. G onzâlez A, Sm eets WJAJ (1994) C atecholam ine system s in the CNS o f am phibians. In: Phy- logeny and D evelopm ent o f C atecholam ine Systems in the CNS o f Vertebrates (Smeets WJAJ, Reiner A, eds), pp 77-102. Cambridge, UK: Cambridge University Press. G onzâlez de A guilar JL, Tonon MC, Ruiz-Na- varro A, Vaudry H, Gracia-Navarro F (1994) M orphological and functional heterogeneity o f frog m elanotrope cells. N euroendocrinol­ og y 59:176-182. Gracia-Navarro F, Castano JP, M alagôn MM, Torronteras R (1991) Subcellular responsive­ ness o f am phibian grow th horm one cells after TSH-releasing horm one stim ulation. G en Com p Endocrinol 84:94-103. G uillem in R (1970) H orm ones secreted by the brain. Isolation, m olecular structure and synthesis o f the first hypophysiotropic hypo­ thalam ic horm one (to be discovered), TRF (thyrotropin-releasing factor). Science 68: 64 -67 . G uillem in R (1978) Peptides in the brain: the new endocrinology o f the neuron. Science 202:390-402. H am ano K, Inoue K, Yanagisawa T (1990) Im­ m unohistochem ical localization o f thyro­ tropin-releasing horm one in the brain o f carp, Cyprinus carpio. Gen Com p Endocri­ nol 80:85-92. H elke CJ, Phillips ET (1988) Thyrotropin-releas­ ing horm one receptor activation in the spi­ nal cord increases blood pressure and sym ­ pathetic tone to the vasculature and the adrenals. J Pharmacol Exp Ther 245:41-46. H euer H, Schafer MK, O ’D onnell D, W alker P, Bauer K (2000) Expression o f thyrotropin- releasing horm one receptor 2 (TRH-R2) in the central nervous system o f rats. J Comp N eurol 428:319-336. H okfelt T, Fuxe K, Johansson O, Jeffcoate S, W hite N (1975) D istribution o f thyrotropin- releasing horm one (TRH) in the central ner­ vous system as revealed w ith im m unoh isto ­ chem istry. Eur J Pharmacol 34:389-392. Horita A (1998) An update on the CNS actions o f TRH and its analogs. Life Sci 62:1443-1448. Horita A, Carino M A, Sm ith JR (1976a) Effects o f TRH on the central nervous system o f the rabbit. Pharm acol Biochem Behav 5:111- 116. H orita A, Carino M A, W eick BG (1976b) Effects o f thyrotropin releasing horm one (TR H , m i­ croinjected into various areas o f conscious and pentobarbital-pretreated rabbits. Proc W est Pharm acol Soc 19:212-213. Jackson IM, Reichlin S (1977) Brain thyrotro- phin-releasing horm one is independent o f the hypothalam us. Nature 267:853-854. Jacobs GF, Kuhn ER (1988) Luteinizing hor­ m one-releasing horm one induces thyroxine release together w ith testosterone in the neo- tenic axolotl A m bystom a m exicanum . Gen C om p Endocrinol 71:502-505. Johansson O, Hokfelt T (1980) Thyrotropin re­ leasing horm one, som atostatin , and enkeph­ alin; distribution studies using im m unohis­ tochem ical techniques. J H istochem Cyto- chem 28:364-366. Johansson O, Hokfelt T, Pernow B, Jeffcoate SL, W hite N , Steinbusch HW, Verhofstad AA, Emson PC, Spindel E (1981) Im m uno­ histochem ical support for three putative transm itters in one neuron: coexistence o f 5- hydroxytryptam ine, substance P and thy­ rotropin releasing horm one-like im m unore­ activity in m edullary neurons projecting to the spinal cord. N euroscience 6:1857-1881. Jôzsa R, K orfHW , Csernus V, M ess B (1988) Thy­ rotropin-releasing horm one (TRH )-im m u- noreactive structures in the brain o f the do­ m estic mallard. C ell T issue Res 251:441- 449. Jôzsa R, M ess B, Csernus V (1989) Ontogenetic developm ent o f thyrotropin-releasing hor­ m one (TRH )-im m unoreactive structures in the brain o f the mallard embryo. Cell Tissue Res 255:657-662. Kawano H, Tsuruo Y, Bando H, Daikoku S (1991) H ypophysiotrophic TRH -producing neu­ rons identified by com bin ing im m unohisto­ chem istry for pro-TRH and retrograde trac­ ing. J Com p Neurol 307:531-538. Kreider MS, Engber TM, Nilaver G, Zim m er­ m an EA, W inokur A (1985) Im m unohisto­ chem ical localization o f TRH in rat CNS: com parison w ith RIA studies. Peptides 6: 997-1000. Lamacz M, H indelang C, Tonon MC, Vaudry H, Stoeckel ME (1989) Three distinct thyrotro­ pin-releasing horm one-im m unoreactive ax­ onal system s project in the m edian em i­ nence-pituitary com plex o f the frog Rana ridibunda. Im m unocytochem ical evidence for co-localization o f thyrotropin-releasing horm one and m esotocin in fibers innervat­ ing pars interm edia cells. N euroscience 32: 451-462. Lechan RM, Wu P, Jackson IM (1986) Im m uno- localization o f the thyrotropin releasing horm one prohormone in the rat central ner­ vous system. Endocrinology 119:1210-1216. Licht P, Denver RJ (1990) Regulation o f pituitary thyrotropin secretion. Prog Clin Biol Res 342:427-432. Lowry CA, Richardson CF, Zoeller TR, M iller LJ, M uske LE, M oore FL (1997) Neuroanatom i- cal distribution o f vasotocin in a urodele am ­ phibian (Taricha granulosa) revealed by im ­ m unohistochem ical and in situ hybridization techniques. J C om p N eurol 385:43-70. Lynn RB, Kreider MS, M iselis RR (1991) Thyro­ tropin-releasing horm one-im m unoreactive projections to the dorsal motor nucleus and the nucleus o f the solitary tract o f the rat. J C om p Neurol 311:271-288. M alagôn M M , G arda-Navarro S, Ruiz-Navarro A, Gracia-Navarro F (1989) M orphometric evaluation o f subcellular changes induced by in vivo TRH treatment in the pituitary gland o f Rana perezi: effects on prolactin and thyrotropic cells. Cell Tissue Res 256:391- 398. TRH in the Brain o f Urodeles Brain Behav Evol 2008;71:231-246 245 47 2. ESTUDIOS QUIMIOARQUITECTONICOS EN EL ENCÉFALO ADULTO M arin O, Sm eets WJAJ, G onzâlez A (1997) D is­ tribution o f choline acetyltransferase im m u­ noreactivity in the brain o f anuran (Rana perezi, Xenopus laevis) and urodele (Pleuro­ deles waltl) am phibians. J Com p N eurol 382: 499-534. Matz SP, Takahashi TT (1994) Im m unohisto­ chem ical localization o f thyrotropin-releas­ ing horm one in the brain o f ch inook salm on (Oncorhynchus tshawytscha). J Com p Neurol 345:214-223. Merchenthaler I, Csernus V, Csontos C, Petrusz P, Mess B (1988) N ew data on the im m u n o­ cytochem ical localization o f thyrotropin-re- leasing horm one in the rat central nervous system. A m J Anat 181:359-376. Meredith GE, Smeets WJAJ (1987) Im m unocy­ tochem ical analysis o f the dopam ine system in the forebrain and m idbrain o f Raja radia- ta: evidence for a substantia nigra and ven­ tral tegm ental area in cartilaginous fish. J Com p N eurol 265:530-548. M im nagh KM, Bolaffi JL, M ontgom ery NM , Kaltenbach JC (1987) Thyrotropin-releasing horm one (TRH): im m unohistochem ical distribution in tadpole and frog brain. Gen Comp Endocrinol 66:394-404. M iranda LA, A ffanni JM (2000) Thyrotropin- releasing horm one im m unoreactivity in the brain and the pituitary during Bufo arena- rum development. Int J D ev N eurosci 18:47- 52. M oreno N , G onzâlez A (2006) The com m on or­ ganization o f the am ygdaloid com plex in tet­ rapods: new concepts based on developm en­ tal. hodological and neurochem ical data in anuran am phibians. Prog Neurobiol 78:61- 90. M oreno N, G onzâlez A (2007) Evolution o f the amygdaloid com plex in vertebrates, with special reference to the anam nio-am niotic transition. J Anat 211:151-163. M oreno N, G onzâlez A (2008) Regionalization o f the telencephalon in urodele amphibians and its bearing on the identification o f the am ygdaloid com plex. Front N eurosci (in press). Nakajima K, Uchida D, Sakai M, Takahashi N , Yanagisawa T, Yam am oto K, Kikuyama S (1993) Thyrotropin-releasing horm one (TRH) is the major prolactin-releasing fac­ tor in the bullfrog hypothalam us. Gen Comp Endocrinol 89:11-16. N orthcutt RG, Kicliter E (1980) O rganization o f the am phibian telencephalon. In: Compara­ tive N eurology o f the Telencephalon (Ebbes- son SOE, ed), pp 203-255. N ew York: Ple­ num. O ’D owd BF, Lee DK, H uang W, Nguyen T, Cheng R, Liu Y, W ang B, Gershengorn MC, George SR (2000) TRH-R2 exhibits sim ilar binding and acute signaling but d istinct regulation and anatom ic distribution com pared with TRH -Rl. M ol Endocrinol 14:183-193. Ohide A, Ando H, Yanagisawa T, Urano A (1996) Hydropathy profiles o f predicted thyrotro­ pin-releasing horm one precursors are highly conserved despite low sim ilarity o f primary structures. J N euroendocrinol 8:695-701. Pagesy P, Croissandeau G, Le D afniet M, Peillon F, Li JY (1992) D etection o f thyrotropin-re­ leasing horm one (TRH) m RNA by the re­ verse transcription-polym erase chain reac­ tion in the hum an normal and tum oral anterior pituitary. Biochem Biophys Res C om m un 182:182-187. Parker CR, Porter JC (1983) Regional localiza­ tion and subcellular com partm entalization o f thyrotropin-releasing horm one in adult hum an brain. J N eurochem 41:1614-1622. Peczely P, Kiss JZ (1988) Im m unoreactivity to vasoactive intestinal polypeptide (VIP) and thyreotropin-releasing horm one (TRH) in hypothalam ic neurons o f the dom esticated pigeon (Colum ba livia). Alterations follow ­ ing lactation and exposure to cold. Cell T is­ sue Res 251:485-494. Peters A, Heuer H, Schom burg L, De Greef WJ, Visser TJ, Bauer K (1997) Thyrotropin-re­ leasing horm one gene expression by anterior pituitary cells in long-term cultures is in flu ­ enced by the culture conditions and cell-to- cell interactions. Endocrinology 138:2807- 2812. Prange A (1974) Proceedings: Behavioral effects o f hypothalam ic polypeptides in anim als and man. Psychopharm acol Bull 10:11-15. Reichlin S (1986) Neural functions o f TRH. Acta Endocrinol Suppl (Copenh) 276:21-33. Roth G, Naujoks-M anteuffel C, Grunwald W (1990) Cytoarchitecture o f the tectum mes- encephali in salamanders: a G olgi and HRP study. J Com p N eurol 291:27-42. Roubos EW (1997) Background adaptation by Xenopus laevis: a m odel for studying neuro­ nal inform ation processing in the pituitary pars interm edia. Com p Biochem Physiol A Physiol 118:533-550. Sasek CA, W essendorf MW, Helke CJ (1990) Ev­ idence for co-existence o f thyrotropin-re- leasing horm one, substance P and serotonin in ventral m edullary neurons that project to the interm ediolateral cell colum n in the rat. Neuroscience 35:105-119. Satoh T, Yamada M, Monden T, lizuka M, Mori M (1992) Cloning o f the m ouse hypothalam ic preprothyrotropin-releasinghorm one(TRH ) cD N A and tissue distribution o f its mRNA. Brain Res M ol Brain Res 14:131-135. Satoh T, Yamada M, Feng P, H ashim oto K, W il­ ber JF, M ori M (1997) Postnatal ontogeny o f the thyrotropin-releasing horm one receptor m essenger ribonucleic acids in the rat fore­ brain. Neuropeptides 31:351-355. Schally AV (1978) A spects o f hypothalam ic reg­ ulation o f the pituitary gland. Science 202: 18-28. Schally AV, Sawano S, Arimura A, Barrett JF, W akabayashi I, Bowers CY (1969) Isolation o f grow th horm one-releasing horm one (GRH) from porcine hypothalam i. Endocri­ nology 84:1493-1506. Segerson TP, H oefler H, Childers H, W olfe HJ, W u P, Jackson IM, Lechan RM (1987) Local­ ization o f thyrotropin-releasing horm one prohorm one m essenger ribonucleic acid in rat brain in situ hybridization. Endocrinol­ o gy 121:98-107. Seki T, Nakai Y, Shioda S, M itsum a T, K ikuyama S (1983) D istribution o f im m unoreactive thyrotropin-releasing horm one in the fore­ brain and hypophysis o f the huUfrog, R ana catesbeiana. Cell T issue Res 233:507-516. Sternberger LA (1979) Im m unocytochem istry. N ew York: Wiley. Taylor RL, Burt DR (1982) Species differences in the brain regional distribution o f receptor binding for thyrotropin-releasing horm one. J Neurochem 38:1649-1656. Teijido O, M anso MJ, A nadôn R (2002) D istribu­ tion o f thyrotropin-releasing horm one im ­ m unoreactivity in th e brain o f the dogfish Scyliorhinus canicula. J Com p N eurol 454: 65-81. Tsuruo Y, Hokfelt T, V isser T (1987) Thyrotropin releasing horm one (TR H )-im m unoreactive cell groups in the rat central nervous system . Exp Brain Res 68:213-217. Tsuruo Y, Hokfelt T, V isser TJ (1988a) Thyrotro­ pin-releasing horm one (T R H )-im m unore- active neuron populations in the rat olfac­ tory bulb. Brain Res 447:183-187. Tsuruo Y, Ceccatelli S, V illar MJ, Hokfelt T, V iss­ er TJ, Terenius L, G oldstein M, Brown JC, Buchan A, W alsh J (1988b) C oexistence o f TRH w ith other neuroactive substances in the rat central nervous system . J Chem N eu­ roanat 1:235-253. T uinhof R, Artero C, Fasolo A, Franzoni MF, Ten Donkelaar HJ, W ism ans PG, Roubos EW (1994) Involvem ent o f retinohypothalam ic input, suprachiasmatic nucleus, m agnocel­ lular nucleus and locus coeruleus in control o f m elanotrope cells o f Xenopus laevis: a ret­ rograde and anterograde tracing study. N eu­ roscience 61:411-420. Vaudry H, Chartrel N , Desrues L, Galas L, Ki­ kuyama S, Mor A , N icolas P, Tonon MC (1999) The pituitary-skin connection in am ­ phibians. Reciprocal regulation o f m elano­ trope cells and derm al m elanocytes. A nn N Y Acad Sci 885:41-56. Verburg-van Kemenade BM, Jenks BG, Visser TJ, Tonon MC, Vaudry H (1987) Assessm ent o f TRH as a potential M SH release stim ulat­ ing factor in Xenopus laevis. Peptides 8 :69- 76. W u W, Elde R, W essendorf MW, Hokfelt T (1992) Identification o f neurons expressing thyro­ tropin releasing-horm one receptor m RNA in spinal cord and lower brainstem o f rat. Neurosci Lett 142:143-146. Yates FE, Russell SM, M aran JW (1971) Brain- adenohypophysial com m unication in m am ­ mals. A nnu Rev Physiol 33:393-444. Yukata T, Tanaka S, K urosum i, K (1990) D istri­ bution o f im m unoreactive thyrotropin-re­ leasing horm one in the brain and hypophy­ sis o f larval bullfrogs w ith special reference to the nerve fibers in the pars distalis. Zool Sci 7:427-433. Zoeller RT, Conway KM (1989) N eurons ex­ pressing thyrotropin-releasing horm one­ like m essenger ribonucleic acid are widely distributed in X enopus laevis brain. Gen Com p Endocrinol 76:139-146. 246 Brain Behav Evol 2008;71:231-246 Dominguez/Lôpez/Gonzâlez 48 2. ESTUDIOS QUIMIOARQUITECTONICOS EN EL ENCEFALO ADULTO Journal of Chemical Neuroanatomy 39 (2010) 2 0 -3 4 C ontents lists available at ScienceDirect Journal of Chemical Neuroanatomy j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / j c h e m n e u (.w^tVairMiCA!. NtURÜANATOMY Immunohistochemical localization of orexins (hypocretins) in the brain of reptiles and its relation to monoaminergic systems Laura Dominguez, Ruth Morona, Alberto Joven, Agustin Gonzalez, Jesûs M. Lopez * Department o f Cell Biology, Faculty o f Biology. University Complutense, 28040 Madrid, Spain A R T I C L E I N F O Keywords: Tyrosine hydroxylase Serotonin Immunohistochemistry Hypothalamus Turtle Lizard Evolution A B S T R A C T Article history: Received 14 May 2009 Received in revised form 30 July 2009 Accepted 30 July 2009 Available online 7 August 2009 W ith th e a im o f g a in in g m ore in sig h t in to th e ev o lu tio n o f th e orex in erg ic sy ste m s in th e brain o f verteb ra tes w e h ave con d u cted a com p arative an a lysis o f th e d istr ib u tion o f o rex in -im m u n o rea ctiv e cell b o d ies and fibers in tw o rep tiles , th e lizard Gekko gecko and th e tu rtle P seudem ys scrip ta elegans. In both sp ec ie s m o st im m u n o r ea ctiv e n eu ron s w e r e fou nd in th e p eriventricu lar h y p oth a lam ic n u cleu s and in th e in fund ibu lar h yp oth a lam u s. O nly in th e geck o, orex in erg ic cell b o d ies w ere p resen t in th e dorso la tera l h y p oth a lam ic n u cleu s and th e p eriventricu lar preop tic n u cleu s. Fiber lab elin g w a s ob served in all m ain brain su b d iv isio n s b u t w a s m ore a b u n d an t in reg ion s su ch as th e sep tu m , p reop tic area, su p rach iasm atic n u cleu s, lateral h yp oth a lam ic area and m ed ian em in en ce . Less co n sp icu o u s w a s the in n ervation o f th e o lfactory bu lbs, palliai reg ions, hab en ula , d o rsom ed ia l and dorso latera l th a lam ic nu clei, torus sem icircu lar is and sp inal cord. D ou b le im m u n o h isto flu o resc en ce tech n iq u es w e re app lied for th e s im u lta n eo u s d e tec tio n o f th e orex in erg ic sy ste m s and th e ca tech o la m in erg ic or sero ton in erg ic sy stem s in th e brain o f rep tiles . A ctual co lo ca liza tio n o f orex in s and ca tech o la m in es or sero to n in in th e sam e n eu ron s w a s n o t o b served . H ow ever , orex in erg ic in n ervation w a s fou nd in d op am inerg ic , noradrenergic and sero ton in erg ic cell groups, su ch as th e su b stan tia nigra and ven tra l teg m en ta l area in th e m idbrain teg m en tu m , th e locu s coeru leu s, th e n u cleu s o f th e so litary tract and th e raphe nu clei. The com p arison o f th e d istr ib u tion o f o rex in -im m u n o rea c tiv e n eu ron s and fibers fou nd in rep tiles w ith th o se rep orted for o th er verteb ra tes revea ls a stron g resem b la n ce bu t a lso n o tab le variation s. In add ition , th e rela tion b e tw e e n th e orex in erg ic and m o n oam in erg ic sy ste m s ob served in th e brain o f rep tiles se e m s to be a shared featu re a m o n g verteb rates. © 2 0 0 9 E lsevier B.V. All r ights reserved . 1. Introduction The orexins, also known as hypocretins, are neuropeptides first found in the human brain as regulators of feeding behavior (Sakurai et al., 1998). Two orexins, A and B (or hypocretins 1 and 2, respectively) w ere described to derive from different proteolitic postraductional processing of the same precursor, prepro-orexin, w hose gene in rat is expressed exclusively in the brain (de Lecea et al., 1998; Sakurai et al., 1998). Immunohistochemical studies in mammals have shown that the neurons producing orexins are localized in particular positions o f the hypothalamus and perifornical region and elaborate networks of immunoreactive Abbreviations: Acc, nucleus accumbens: ADVR, anterior dorsal ventricular ridge; Alh, lateral hypothalamic area; Am, amygdaloid complex; BST, bed nucleus of the stria terminalis: Cb, cerebellum; cc, central canal; Cxd, dorsal cortex; Cxi, lateral cortex; Cxm, medial cortex; dh, dorsal horn of spinal cord; DF, dorsal funiculus: Dl, dorsolateral thalamic nucleus; Dlh, dorsolateral hypothalamic nucleus; Dm, dorsomedial thalamic nucleus; epl, external plexiform layer; flm, fasciculus longitudinalis medialis: Gc, griseum centrale; GP, globus pallidus; Hbl, lateral habenula; Hbm, medial habenula; igl, internal granular layer; inf, infundibulum; Ip, interpeduncular nucleus; ir, infundibular recess; Is, isthmic nucleus; LA, lateral amygdala; Ifb, lateral forebrain bundle; Lc, locus coeruleus; LDT, Laterodorsal tegmental nucleus; LF, lateral funiculus; LM, pretectal lentiform mesencephalic nucleus; MA, medial amygdala; Ma, mamillar region; me, median eminence; MP. medial posterior thalamic nucleus; Mri, medial nucleus of the infundibular recess; Mt, medial thalamic nucleus; NdB, nucleus of the diagonal band of Broca; Nolfa, anterior olfactory nucleus; Nsl, lateral septal nucleus; Nsm, medial septal nucleus; Nsol, nucleus o f the solitary tract; ob, olfactory bulb; oc, optic chiasm; Pb, parabrachial nucleus; pc, posterior commissure; Pd, posterodorsal nucleus; PDVR, posterior dorsal ventricular ridge; Ph, periventricular hypothalamic nucleus; POp, periventricular preoptic nucleus; PV, paraventricular nucleus; Ra, raphe nuclei; Rai, inferior raphe nucleus; Ras, superior raphe nucleus; RA8, reptilian AS nucleus; RF, reticular formation; Ri, inferior reticular nucleus; Rm, median reticular nucleus; Rot, nucleus rotundus; Rs, superior reticular nucleus; SC, suprachiasmatic nucleus; Sn, substantia nigra; SNc, substantia nigra, pars compacta; SNr, substantia nigra, pars reticulata; So, supraoptic nucleus; sol, solitary tract; Str, striatum; tect, mesencephalic tectum; Tegm, mesencephalic tegmentum; to, optic tract; Tor, torus semicircularis: v, ventricle; Vds, nucleus descendens nervi trigemini; Ves, nucleus vestibularis superior; VF, ventral funiculus; vh, ventral horn of spinal cord; Vpr, nucleus sensorius principalis nervi trigemini; VTA, ventral tegmental area; lllv, third ventricle; Xm, nucleus motorius nervi vagi; XI1, nucleus nervi hypoglossi. * Corresponding author. Tel.; +34 91 3944977; fax: +34 91 3944981. E-mail address: agustin@bio.ucm.es (J.M. Lopez). 0891-0618/$ - see front matter © 2009 Elsevier B.V. All rights reserved, doi: 10.1016/j.jchemneu.2009.07.007 49 http://www.elsevier.com/locate/jchemneu mailto:agustin@bio.ucm.es 2. ESTUDIOS QUIMIOARQUITECTONICOS EN EL ENCEFALO ADULTO L Dominguez e t aL/Joumal of Chemical Neuroanatomy 39 (2010) 20-34 21 fibers originated from these neurons are widely distributed in almost all main brain regions (Broberger et al., 1998; Peyron et al., 1998; Cutler et al., 1999; Nambu et al., 1999; Dube et al., 2000; McGranaghan and Piggins, 2001 ; Mintz et al., 2001 ; Zhang et al., 2001, 2004; Nixon and Smale, 2007). Moreover, by means of double immunohistofluorescence it was corroborated that both orexins colocalize in the same neuronal hypothalamic cells and their projections (Zhang and Luo, 2002; Zhang et al., 2004). However, the two orexin receptors 1 and 2 bind the orexins with different affinity and showed distinct patterns of distribution in the brain (Sakurai et al., 1998; Kilduff and de Lecea, 2001 ), raising the possibility that there may be differences in the functional roles for orexins A and B (Nixon and Smale, 2007). In agreement with their widespread distribution in the brain multiple functions for orexins have been demonstrated, such as the control of feeding and energy homeostasis (Lubkin and Stricker-Krongrad, 1998; Sakurai et al., 1998; Wolf. 1998; Dube et al., 1999; Haynes et al., 1999; Sweet et al., 1999; Volkoff et al., 1999,2005; Yamanaka et al.. 1999; Karteris et al., 2005; Volkoff, 2006; Carter et al., 2009) or the regulation of sleep-wake cycle and related pathologies like narcolepsy (Chemelli et al., 1999; Hagan et al., 1999; Lin et al., 1999; Beuckmann and Yanagisawa, 2002; Kukkonen et al., 2002; Baumann and Bassetti, 2005; Matsuki and Sakurai, 2008; Takahashi et al., 2008). In addition, orexins also regulate the release of adenohypophyseal hormones (Pu et al., 1998; Mal- endowicz et al., 1999; Mitsuma et al., 1999; Tamura et al., 1999; Russell et al., 2000; Kohsaka et al., 2001 ; Seoane et al., 2004; Barb and Matteri, 2005; Martynska et al., 2006), the integrated control of autonomic function (Shirasaka et al., 2002; Ferguson and Samson, 2003; Berthoud et al., 2(X)5; Yasuda et al., 2005) and the stimulation of gastrointestinal functions (Okumura and Takaku- saki, 2008). Most investigations about the precise localization of orexins in the brain have been conducted in rodent species (Peyron et al., 1998; Cutler et al., 1999; Nambu et al., 1999; McGranaghan and Piggins, 2001; Mintz et al., 2001; Nixon and Smale, 2007). However, due to the conserved molecular structure of orexins across vertebrates (Sakurai et al.. 1998; Shibahara et al.. 1999; Alvarez and Sutcliffe, 2002; Ohkubo et al., 2002), several studies have used antibodies against mammalian orexins to localize orexin-immunoreactive (orexin-ir) cell bodies and fibers in representatives of fish (Kaslin et al., 2004; Huesa et al., 2005; Amiya et al., 2007), amphibians (Shibahara et al., 1999; Galas et al., 2001 ; Singletary et al., 2005; Suzuki et al., 2008; Lopez et al., 2009) and birds (Ohkubo et al., 2002; Phillips-Singh et al., 2003; Singletary et al., 2006). The results of these studies suggested a highly conserved organization of orexinergic systems in the brain of vertebrates. However, only in a few cases, direct cross-species comparison of the distribution of cell bodies and fibers containing orexins have been made and have shown peculiar differences both among mammals (Nixon and Smale, 2007) and amphibians (Lopez et al.. 2009). Surprisingly, there is no information concerning the specific localization of orexins in the brain of reptiles. To our knowledge, only a brief report has pointed out the presence of orexin-ir cells along the third ventricle within the periventricular nucleus in the green anole lizard (Farrell et al., 2003). Considering the crucial position of reptiles in a phylogenetic perspective and the previously observed differences between lizards and turtles for other neurochemical markers (Smeets et al., 2001, 2003, 2006; Munoz et al., 2008), the present study of the distribution of orexin immunoreactivity in the brain of the turtle Pseudemys scripta elegans and the lizard Gekko gecko has been carried out for a better understanding of primitive and derived traits o f this system in reptiles and, more generally, vertebrates. An additional goal of our study was to establish the relationship between the orexin-ir structures and the aminergic cells and fibers in the reptilian brain. Data in mammals, amphibians and zebrafish are available about the different degree of colocalization and/or codistribution of orexin-ir cells and fibers with catecholaminergic elem ents in the mesencephalic tegmentum, locus coeruleus and caudal rhomben­ cephalon and with serotoninergic cells in the raphe nuclei (Peyron et al., 1998; Baldo et al., 2003; Wang et al., 2003; Kaslin et al., 2004; Zhang et al., 2004; Balcita-Pedicino and Sesack, 2(X)7). Therefore in our study, immunohistochemistry for the detection of orexins was systematically combined with the detection of tyrosine hydro­ xylase (TH, the first and rate-limiting enzyme for catecholamine synthesis) and serotonin. 2. Materials and m ethods For the present study, a total of twelve red-eared turtles, Pseudemys scripta elegans, and seven lizards Gekko gecko were used. The animals were purchased from an authorized commercial supplier and were housed in an air-conditioned room with controlled temperature (25 °C) and natural light conditions. The original research reported herein was performed according to the regulations and laws established by European Union (86/609/EEC) and Spain (Royal Decree 1201/2005) for care and handling of animals in research. The turtles were deeply anesthetized with an intraperitoneal injection of sodium pentobarbital (50-100 mg/kg, Normon Labs, Madrid, Spain). Comeal and pedal withdrawal reflexes disappeared within 10-15 min and the heart was exposed when ventral plastron were removed by two lateral incisions. In turn, the lizards were deeply anesthetized by inhalation of diethyl ether (Panreac, Barcelona, Spain). All animals were perfused transcardially with physiological saline followed by 200 ml of cold 4% paraformaldehyde in a 0.1 M phosphate buffer (PB, pH 7.4). Two animals of each species received an intraperitoneal injection of colchicine (20 mg for each 100 g of animal weight) dissolved in saline, one-day prior the perfusion. The brain and the upper spinal cord were removed and kept in the same fixative for 2-3 h. Subsequently, they were immersed in a solution of 30% sucrose in PB for 4 - 6 h at 4 °C until they sank, embedded in a solution of 20% gelatin with 30% sucrose in PB, and stored for 6 h in a 3.7% formaldehyde solution at 4 °C The brains were cut on a freezing microtome at 40 |xm in the Montai plane and sections were collected in PB. 2.1. Orexin immunohistochemistry The free-floating sections were rinsed twice in PB, treated with 1% HgOz in PB for 20 min to reduce endogenous peroxidase activity, rinsed again three times in PB and processed by the peroxidase antiperoxidase (PAP) method (Sternberger, 1979). This included a first incubation of the sections in a goat anti-orexin-A (Santa Cruz Biotechnology. Santa Cruz, CA USA; code sc-8070) or goat anti-orexin-B serum (Santa Cruz Biotechnology; code sc-8071), diluted 1:500 in PB containing 0.5% Triton X-100, 15% normal rabbit serum, and 2% bovine serum albumin (BSA), for 48 h at 4 X . Subsequently, the sections were rinsed three times in PB for 10 min and incubated for 60 min at room temperature in rabbit anti-goat serum (Chemicon, Temecula, CA) diluted 1:50. After rinsing again three times for 10 min, the sections were incubated for 90 min in goat PAP complex (diluted 1:500; Chemicon). Secondary antiserum and PAP complex were diluted in PB containing 0.5% Triton X- 100,15% NRS and 2% BSA Finally, the sections were rinsed three times for 10 min in PB and subsequently stained in 0.5 mg/ml 3,3'-diaminobenzidine (DAB; Vector SK4100) intensified with nickel (Adams, 1981), with 0.01 % H2 O2 in PB for 5 -1 0 min. The series of sections were mounted on glass slides (mounting medium: 0.25% gelatin) in 0.1 M Tris-HCl buffer (pH 7.6) and, after dehydration, coverslipped with Entellan (Merck, Darmstadt, Germany). Some sections were counterstained with cresyl violet to facilitate analysis o f the results. The specificity o f the immunohistochemical reaction was corroborated with controls that included: ( 1 ) staining o f some selected sections with preimmune goat serum; (2) controls in which either the primary antibody, secondary antibody or the PAP complex was omitted; (3) homologous and heterologous preabsorptions of the primary antibody with synthetic blocking peptides for orexin-A or orexin-B (both of Santa Cruz Biotechnology; code sc-8070P and sc-8071 P respectively; 0.1, 1.0 or 10 p.M). In all these negative controls, the immunostaining was eliminated, even when the goat anti-orexin-A or goat anti-orexin-B was preabsorbed with the synthetic blocking peptides at low concentration (0.1 puM). 2J . Double orexin and TH or serotonin (5-HT) immunohistochemistry A procedure based on immunohistofluorescence was used as follows: (1) first incubation for 72 h at 4 °C in a mixture of goat anti-orexin-A or goat anti-orexin-B (diluted 1:500) and mouse anti-TH (diluted 1:1000; Immunostar, USA; code P22941) or rabbit anti-5-HT (diluted 1:1000; Immunostar, USA; code 20080): (2) second incubation for 90 min at room temperature in a mixture of secondary antisera: donkey anti-goat Alexa 594 (red fluorescence; diluted 1:300; Molecular Probes, Denmark) and chicken anti-mouse Alexa 488 (green fluorescence: diluted 50 2. ESTUDIOS QUIMIOARQUITECTONICOS EN EL ENCEFALO ADULTO 22 L Dominguez e t ai/Journal of Chemical Neuroanatomy 39(2010) 20-34 1:300: Molecular Probes) or FITC-conjugated chicken and-rabbit (green fluores­ cence; diluted 1:100; Chemicon). After rinsing three times in PB, the sections were mounted on glass slides and coverslipped with Vectashield (Vector, Burlingame, CA). The specificity o f the TH and serotonin antibodies was assessed in reptiles and the pattern of immunostaining obtained in this study fully corroborated the distribution of immunoreactive cells and fibers reported previously (Ueda et al., 1983; Wolters et al., 1985; Smeets and Steinbusch, 1988,1990; Kiehn et al., 1992; Medina and Smeets, 1992; Smeets, 1994). 2.3. Evaluation and presentation of the results The distribution o f orexin-ir cell bodies and fibers in the brain of the two species was carefully analyzed and the pattern of labeling was charted in representative transverse sections at different brain levels for the case of Gekko gecko (Fig. 1). Drawings were made by means of camera lucida in which the sections counter­ stained with cresyl violet facilitated the interpretation of the localization of the labeled structures. For the double-labeling experiments, the sections were analyzed with an Olympus BX51 fluorescence microscope with appropriate filter combina­ tions for the identification of orexin and TH/5-HT immunoreactive cells. Selected sections were photographed by using a digital camera (Olympus DP70). Contrast and brightness were adjusted in Adobe Photoshop 7.0 (Adobe System. San Jose. CA). Furthermore, in the double-labeling experiments, the identification of orexin-ir terminal like structures over the cathecolaminergic and serotoninergic cells, the sections were studied with a Leica spectral confocal laser scanning microscope (TCS-SP2). The argon 488-nm and helium/neon lasers were used, respectively, to excite the green or red flourophores. Images series were acquire with steps o f 0.8 or 1 p,m along the z-axis, and collapsed images were obtained from an average of IQ- 12 optical sections. Selected photographs of single stained sections are presented in Figs. 2 -4 and double stained pictures are arranged in Fig. 5. 3. Results The antibodies against orexin-A and orexin-B used in the present study revealed patterns of immunoreactivity that, for each of the tw o species examined, were constant from animal to animal. The labeling observed was restricted to neuronal cell bodies located in the preoptic area/hypothalamus, whereas fibers were distributed in all main divisions of the brain. No differences were observed in the pattern of immunoreactivity when using anti- orexin-A or anti-orexin-B antisera and, therefore, w e will refer to orexin-ir structures. In the cases in which colchicine was used, the Nolfa/ Cxm t x l ADVR ADVR PDVR Nsl • N sm % N sm ; Nsl ^ NdB Cxd CxmCxi Dm PDVR PV scj OC Cxd ,Cxm Cxi PDVR Cv LA Dm V j R ot P h'« A lh Fig. 1. (a-p) Diagrams of transverse sections through the brain of Gekko gecko at the levels indicated in the schematic dorsal view of the brain. Orexin-ir cell bodies (large dots) and fibers (small dots, wavy lines) are represented in the left half of each section. 51 2. ESTUDIOS QUIMIOARQUITECTONICOS EN EL ENCEFALO ADULTO L Dominguez e t aL/Joumal of Chemical Neuroanatomy 39 (2010) 20-34 23 tect tect Pd Dm Ku*- ,>yl LM MR Ph Alh me tect me Cb Cb R s ' \ Ras Ras. (m) tect Tor ■*>>. Tegm VTA Nsol Vds Rai Xnr> fim Rai DF VF Fig. 1. (Continued). 0.5mm pattern of immunoreactivity obtained was the same although the cell bodies, in general, were more intensely labeled. A summary of the results showing the interspecies variations in the distribution of cells and fibers that contained orexins is shown in Table 1. For the description of the results w e will first describe the localization o f labeled cell bodies and, subsequently, the distribu­ tion of labeled fibers from rostral to caudal levels, as presented in the schematic charting for Gekko gecko (Fig. 1). 3.1. Orexin-ir cell bodies The orexin-ir cell population located most rostrally in the brain was found in the periventricular preoptic nucleus of Gekko, especially on the ventrocaudal part of this region (Figs. If; 2a). These small-sized cells possessed pear-shaped perikarya and were primarily located close to the ventricle (Fig. 2a). Only some o f these cells possessed a short process that contacted the cerebrospinal fluid (CSF). These cells were seen in low number in the preoptic area of Pseudemys with the same size and morphology but situated principally in the lateral preoptic area and only som e of them contacted the CSF. The most conspicuous orexineigic cell group was situated in the hypothalamic periventricular nucleus in the two species of reptiles examined (Fig. Ih and i; 2b-d; 5î and b). In Gekko two groups of darkly stained cells were clearly distinguishable on the basis of their morphology. The first population occupied the dorsal portion of the rostral periventricular hypothalamic region, within the dorsolateral hypothalamic nucleus (Fig. Ih; 2c). Large cells 52 2. ESTUDIOS ♦UIMIOARQUITECTONICOS EN EL ENCEF ALO ADULTO 24 L Dominguez e t al./Journal of Chemical Neuroanatomy 39 (2010) 20 -3 4 )lh JÇ Xlh V m e . Fig. 2. Photomicrographs f transverse sections through the forebrain illustrating orexin-ir cell bodies in the rostral periventricular preoptic nucleus of Gekko (a), periventricular hypothalanc nucleus of Pseudemys (b) (enlarged area marked in the insert), dorsolateral hypothalamic nucleus (c) and periventricular hypothalamic nucleus of Gekko (d) (enlarged areamarked in the insert), caudal hypothalamic and infundibular regions o f Gekko (e) and Pseudemys (f). Inset f shows in a higher magnification the CSF-contacting cells in thenfundibulum of Pseudemys. Scale bars = 200 fxm (a-f) and 20 |j,m ( f ). situated away from he ventricle and showing long lateral or ventrolateral processe formed this group. Some of these neurons also possessed a lon^cell process that traversed the ventricular lining and contacted the CSF. The second population showed smaller neurons arraged in a band parallel to the ventricular surface (Fig. 2d). Thœ orexin-ir cells w ere fusiform and lacked CSF-contacting proceses (Fig. li; 2d). Only more caudally in the hypothalamus th e orein-ir cells show ed CSF-contacting processes (Fig. 2e). This distinction vas not recognized in the periventricular hypothalamic nucleu of Pseudemys where a single group of orexin-ir cells was etected forming a row of small neurons parallel to the ventriciar surface (Fig. 2b). These cells in the rostral regions of the hypothlamus show ed laterally or ventrolaterally directed processes an ̂ . • ; ■ . ■ / ' ■ - • cc / V h V f VF Fig. 4. Photomicrographs o f transverse sections through the forebrain and brainstem illustrating orexin-ir fibers in the habenula of Pseudemys (a), the optic tectum o f Gekko (b), the torus semicircularis of Pseudemys (c), the mesencephalic tegmentum of Gekko (d), the caudal part o f the nucleus of the solitary tract (e) and the spinal cord of Pseudemys (f). In all photographs (except for e) lateral is to the left and medial to the right. Scale bars = 100 p.m (d and e) and 200 p.m (a -c and f). (Fig. Ig). whereas the arcuate nucleus of Pseudemys was almost totally devoid of stained fibers. In the tw o reptiles, numerous orexin-ir fibers and terminal-like boutons w ere found preferen­ tially in the external zone of the median em inence ( Figs. 1 i and j ; 2e and f). It should be noted that in the median em inence a dense innervation of orexin-ir and TH-ir fibers was found and both types of fibers were intermingled (Fig. 5d). The orexinergic innervation was moderate in the habenula and was restricted to its lateral zone (Fig. 4a). Conspicuous innervation was also found in the dorsomedial and dorsolateral nuclei of the dorsal thalamus (Fig. Ig -i), formed by fine varicose fibers and terminal-like structures, which clearly contrasted with the virtual absence of im munoreactive structures in the nucleus rotundus (Fig. Ih and i). Ventral to this region, the thalamic medial and posterior nuclei also contained orexin-ir fibers (Fig. Ih and i). The pretectum was another brain region w ith a moderate presence of orexinergic innervation (Fig. l i and j). In the mesencephalon o f reptiles scarce orexin-ir fibers were detected in the tectum (Fig. 1 k). These fibers w ere varicose and were mainly localized in the deep and intermediate zones o f the tectum, lacking a layered arrangement (Fig. 4b). In contrast, the torus semicircularis showed remarkable amounts of orexin-ir varicose fibers and terminal-like structures that were especially abundant in its central nucleus (Figs. Ik; 4c). In the mesencephalic tegmentum orexin-ir fibers were primarily located in the periventricular zone (Fig. Ik; 4d). In addition, as demonstrated in double-labeling experiments, a remarkable amount of orexinergic fibers were found over the dopaminergic cell groups of the ventral tegm ental area, the substantia nigra (Figs. Ik; 5e and f) and the reptilian equivalent of the A8 nucleus (Fig. 5g). Caudally, a considerable am ount of orexin-ir fibers and bouton-like structures was also detected over the noradrenergic cells of the locus coeruleus (Figs. 11 and m; 5h), in the laterodorsal tegmental nucleus (Fig. 11 and m) and in the parabrachial area (Fig. 11). Notably, no orexinergic elem ents were observed in the cerebellum. Only scarce orexinergic innervation was detected in the interpeduncular area. In general, the rhombencephalon of Gekko w as less densely innervated with orexin-ir fibers than that of Pseudemys. The central 55 2. ESTUDIOS QUIMIOARQUITECTONICOS EN EL ENCEFALO ADULTO L Dommguez et al./Joumal o f Chemical Neuroanatomy 39 (2010) 20-34 27 Fig. 5. Photomicrographs showing, in the same sections, staining for orexins (red fluorescence) and TH (a-i) or serotonin (j) (green fluorescence). The relationship between the distinct cell populations is illustrated for the periventricular hypothalamic nucleus of Gekko (a) and Pseudemys (b), and the caudal hypothalamic and infundibular regions of Pseudemys (c) and Gekko (d). The orexin-ir innervation over the TH-ir or serotonin-ir cells is shown for Pseudemys in confocal images of the ventral tegmental area (e), the substantia nigra (f), the RA8 group (g), the locus coeruleus (h), the nucleus of the solitary tract (i) and the medial portion of the superior raphe nuclei (j). Scale bars = 100 p.m (e -i) and 200 p,m (a-d and j). gray in the rostral rhombencephalon showed a moderate amount of orexin-ir fibers and terminal-like boutons, which extended medially into the superior raphe nucleus (Fig. 11 and m). In double labeling experiments a remarkable amount of orexinergic term­ inal-like boutons were found over the serotoninergic cells in the column of the raphe, throughout the rostrocaudal extent of this brain region (Fig. ll-o ) . This orexinergic innervation was profuse over the lateral and medial portions of the superior raphe nuclei (Fig. 5j) and declined gradually at more caudal levels of the inferior raphe nucleus. The superior, middle and inferior nuclei of the reticular formation also showed a small amount of orexin-ir varicose fibers and bouton-like structures. This innervation was more apparent in the caudal rhombencephalon within the area of the solitary tract nucleus (Figs. In and o; 4e). Experiments of double immunohistofluorescence demonstrated the presence of orexin-ir varicose fibers and terminals over the catecholaminergic cells of this nucleus throughout its rostrocaudal extent, these immunoreactive fibers were m ost notable at the level of the obex (Figs. lo ; 4e, 5i). 56 2. ESTUDIOS QUIMIOARQUITECTONICOS EN EL ENCEFALO ADULTO 28 L Dommguez et aL/Joumal of Chemical Neuroanatomy 39 (2010) 20-34 Table 1 Comparative localization and relative abundance o f orexin-immunoreactive cells and fibers in CNS of the reptiles studied. Pseudemys elegans scripta Gekko gecko C F C F Telencephalon ob - - + Cxi - Cxm, Cxd - + - + Acc - Str - + CP - + NdB - Nsl - Nsm - Am - - + Preoptic area and hypothalamus PO + SC - Ph + + + Infundibulum +++ Alh - me - - Diencephalon Hb - Dm - Dl - Pr - Mesencephalon and istmus Tect - Tor - Tegm - VTAISn - Lc - ip + - Rhombencephalon Cc - + Ra - + RF + - + Nsol - - + Spinal cord - - C: immunoreactive cell bodies; F: immunoreactive fibers. +, low density; ++. moderate density; +++. high density; - , no immunoreactive cell bodies or fibers. Within the spinal cord of the two species of reptiles studied a moderate amount of orexin-ir fibers were seen in the lateral funiculus, whereas the ventral and dorsal funiculi showed relatively few fibers (Figs. Ip; 4f). Some orexin-ir bouton-like structures were found among the cell bodies of laminae V, VI and X of the spinal gray and, less abundantly, in laminae IV and VIl-VlII and IX of the ventral horn (Fig. 4f). 4. Discussion In the present study, orexin-like immunoreactivity was investigated in the brain of two reptilian species by means of antibodies against mammalian orexin-A and orexin-B. The specificity of the antibodies used was confirmed with control series immunostained after preabsorption with orexin-A and orexin-B blocking peptides. In these series, no immunostaining was observed in perikarya and fibers. This suggests that the substances recognized are only orexins. It should be noted that the structure of orexins is highly conserved in vertebrates, with only some small variations in the sequence of amino acids (Shibahara et al., 1999; Alvarez and Sutcliffe, 2002). In particular, the last 10- residue peptide in the C-terminus is constant in the orexin peptides of all species examined (Sakurai et al., 1998; Shibahara et al., 1999). The antisera anti-orexin-A and anti-orexin-B (both of Santa Cruz Biotechnology) used in this study, are raised against a peptide mapping at the C-terminus of orexin-A or orexin-B of human origin. This would explain why there were no differences in the pattern of staining that they yield (Singletary et al., 2005,2006; Lopez et al., 2009), being impossible to discern between putative distinct distributions of different orexin peptides and only orexin- immunoreactivity, in general, could be investigated. In addition, the orexins do not have structural homology with other biologically active peptides (Shibahara et al., 1999). The aim of the present study was to provide for the first time a detailed description of the organization of the orexin-ir cell bodies and fibers in the brain of two representative species of reptiles. In the following section w e will discuss the general organization and variations of the orexinergic system in reptiles and will compare our results with those obtained in other vertebrates. 4.1. Localization of orexin-ir neurons in the brain of vertebrates: comparative aspects The most numerous orexin-ir cell group is located in the hypothalamus of the two reptiles studied. Although some small species differences were noted in the localization of the labeled cells, the majority of the orexin-ir neurons are confined within the periventricular hypothalamic nucleus. This observation concurs with one previous report in the green anole lizard Anolis caroiinensis (Farrell et al„ 2003). However, the lizard Gekko has a peculiar group of large orexin-ir cells in the dorsolateral hypothalamic nucleus that is not observed in Pseudemys and Anolis. The presence of orexin-ir cells rostrally in the preoptic area and caudally in the infundibular region seems to be a shared feature of reptiles. In general, the preoptic cells form a scarce neuronal population near the ventricle that is more abundant in Gekko than in Pseudemys. In the infundibulum, many orexin-ir neurons are CSF-contacting cells that appear more numerous in Pseudemys than in Gekko. As in reptiles, orexin-ir cells are consistently found in hypothalamic regions in other vertebrates. In particular, birds possess a similar distribution of orexin-ir cells, in which a single population is localized along the third ventricle and mostly within the paraventricular nucleus extending into the lateral hypothala­ mus (chicken: Ohkubo et al., 2002; quail: Phillips-Singh et al., 2003; house finches: Singletary et al., 2006). Generally in mammals, a single population of orexin-ir cells is described in the caudal part of the lateral hypothalamus (Nambu et al., 1999; McCranaghan and Piggins, 2001 ; Mintz et al., 2001 ) in a region classically recognized as a feeding center (Bernardis and Bellinger, 1993). The precise location of these neurons in the rat extends within the perifomical nucleus, the dorsomedial hypotha­ lamic nucleus and the dorsal and lateral areas of the hypothalamus (Sakurai etal., 1998; Cutler et al., 1999; Date et al., 1999; Nixon and Smale, 2007). The perifomical nucleus and the lateral area of the hypothalamus are implicated in the control of feeding and the energy balance (Winn et al., 1984; Stanley et al., 1996). In the three orders of amphibians (anurans, urodeles and gymnophionans), most orexin-ir cells are localized in the suprachiasmatic nucleus, whereas cells in the preoptic area and the hypothalamic tuberal region are less numerous (Shibahara et al., 1999; Galas et al., 2001 ; Singletary et al., 2005; Suzuki et al., 2008; Lopez et al., 2009). In zebrafish (Danio rerio), cells are distributed in two separated populations, one along the third ventricle within the preoptic area and suprachiasmatic nucleus and the other within the anterior hypothalamus (Kaslin et al., 2004; Faraco et al., 2006). The presence of these cells in the goldfish (Carassius auratus) brain is more restricted and they are located in the anterolateral hypothalamus within the nucleus lateralis tuberis and, more caudally, bordering the third ventricle in the 57 2. ESTUDIOS QUIMIOARQUITECTONICOS EN EL ENCÉFALO ADULTO L Dominguez et aL/Joumal o f Chemical Neuroanatomy 39 (2010) 20-34 29 nucleus posterions periventricularis (Huesa et al., 2005). In this latter nucleus the main group of orexin-ir cells in the brain of the medaka fish Oryzias latipes is also located (Amiya et al., 2007). Therefore, the hypothalamic location of the orexinergic neurons is a shared feature in all vertebrates examined, although each group has some specific differences in their localization. In contrast, in the rat the presence of tw o specific cell groups that contained orexin-B were described within the lateral division of the central nucleus of the amygdala and in the anterior lateral subnucleus of the bed nucleus of stria terminalis (Ciriello et al., 2003b), but in subsequent studies in the rat these cells were not observed (Nixon and Smale, 2007). In addition, cells immunor­ eactive only for orexin-A were observed in the paraventricular nucleus of the lab rat and grass rat, and in the supraoptic nucleus of the lab rat, grass rat and hamster (Nixon and Smale, 2007). These specific variations seem unique to this mammalian group. 4.2. Localization of orexin-ir fibers in the brain of vertebrates: comparative aspects The distribution patterns of orexin-ir fibers in the brain also show many similarities among vertebrates. Abundant orexin-ir fibers were observed throughout the septal region, preoptic area and hypothalamus of the two reptiles studied, whereas moderate amounts of fibers were present in the cortex, striatum, thalamus, mesencephalic tectum, torus semicircularis and rhombencepha­ lon. The orexin-ir fibers located most rostrally in the reptilian brain were found in the olfactory bulbs, principally distributed in the internal granular layer, and are more abundant in the turtle. Studies in diverse mammalian species reported that the olfactory bulbs lack orexin-ir fibers (Nixon and Smale, 2007). However, in a recent study in the rat, both orexin-A- and -B-ir fibers were observed, primarily in the granular cell layer and anterior olfactory nucleus (Shibata et al., 2008). Recently, this situation has also been observed in the three amphibian orders (Lopez et al., 2009). Reduced amount of scattered orexin-ir fibers are present in cortical regions of reptiles, especially in the lateral portions, as they are also scarce throughout the cortical areas in mammals and in the palliai regions of amphibians (Nixon and Smale, 2007; Lopez et al., 2009). In contrast, the ventral regions of the reptilian telencephalon, particularly the nucleus accumbens, possess prominent neuropils of orexin-ir fibers and terminal-like struc­ tures, as in amphibians (Lopez et al., 2009). Recently, a role of orexin receptors has been described in the nucleus accumbens shell of rat, playing a modulatory role in turning behavior (Kotani et al., 2008). In our study, intense fiber labeling was noted in the septum of reptiles. Similar observation was made for amphibians and mammals, where GABAergic and cholinergic septohippocam- pal neurons receive excitatory orexinergic innervation (Wu et al., 2002, 2004; Lopez et al., 2009). Orexin cells might also innervate the cholinergic neurons in the basal forebrain of reptiles (Hoogland and Vermeulen-VanderZee, 1990; Powers and Reiner, 1993) and act to modulate cortical acetylcholine release. This activation of the basal forebrain cholinergic system appears to be especially relevant in the context of homeostatic challenges, such as food deprivation in mammals (Fadel and Frederick-Duus, 2008). Moderate amounts of orexin-ir fibers are present in the amygdaloid complex of reptiles, especially within the medial and lateral portions as in amphibians (Galas et al., 2001; Lopez et al., 2009). In mammals, the density of orexin-ir fibers is low to moderate in the amygdaloid complex (Bisetti et al., 2006; Nixon and Smale, 2007). It was shown that in rats orexins can exert an excitatory postsynaptic effect through the amygdala to augment arousal and evoke the autonomic and behavioral responses associated with fear, stress or emotion (Bisetti et al., 2006) and mediate at least a part of the amygdala and bed nucleus o f the stria terminalis-induced cardiorespiratory responses (Zhang et al., 2009). Orexin neuron activation is partly controlled by circadian signals generated in the brain’s main circadian pacemaker, the suprachiasmatic nuclei. The presence of orexin-ir fibers in the suprachiasmatic nucleus of reptiles is conspicuous (present results), in line with results in amphibians (Lopez et al., 2009) and mammals (Date et al., 1999; McCranaghan and Piggins, 2001 ; Mintz et al., 2001 ). Therefore, in non-mammalian species orexins can also alter suprachiasmatic neuronal activity, as in mammals, and may influence the transmission o f information from this nucleus to other brain regions (Brown et aL, 2008). Notable is the presence o f orexin-ir fibers in the dorsal thalamus of reptiles, within the dorsomedial and dorsolateral nuclei. In mammals, orexins have excitatory actions that alter the firing mode of thalamic neurons associated with different states of arousal (Govindaiah and Cox, 2006). In particular, in mammals, all aspects of the anteroposterior paraventricular nucleus of the thalamus are densely innervated by orexin-ir fibers and terminals (Kirouac et al., 2005; Nixon and Smale, 2007). This midline thalamic nucleus projects to limbic forebrain areas, such as the nucleus accumbens, septum and amygdala. Therefore, in reptiles, as in mammals, the basal forebrain and comparable thalamic nuclei receive orexin inputs that have been implicated in arousal (Pasumarthi and Fadel, 2008). The abundant orexin-ir fibers in the lateral hypothalamic area of reptiles suggest that this innervation may act in the regulation o feeding behavior, as is the case in mammals (Sakurai et al., 1998; Sweet et al., 1999). Of note, orexinergic neurons project to the tuberomammillary nucleus in the rat and orexins may release histamine from this nucleus (Yamanaka et al., 2002). This activation of histaminergic neurons by orexins might be important for the modulation of arousal. Similarly, reptiles show a dense innervation by orexin-ir fibers in the hypothalamic area that contains histaminergic cells in the posterior part of the ventral hypothalamus (Inagaki et al., 1990), in line with results reported in amphibians (Airaksinen and Fanula, 1990; Barroso et al., 1993; Lopez et al., 2009) and the teleost zebrafish (Kaslin et al., 2004). Orexin-ir fibers are detected in the median eminence of reptiles. This observation suggests a possible function for these peptides in neuroendocrine regulation. In mammals, the importance of orexins over the neuroendocrine system has been demonstrated (Kukkonen et al., 2002; Ferguson and Samson, 2003). In the rat, orexin-ir fibers have been observed in the median eminence (Peyron et al., 1998; Date et al., 2000b) and in the arcuate nucleus (Peyron et al., 1998; Date et al., 1999; Burdakov et al., 2003; Martynska et al., 2006), which is involved in feeding behavior and in the regulation of the adenohypophysis. RP-HPLC analysis detected orexin-A and orexin-B in rat adenohypophysis, although the amounts were far less than in the median eminence (Date et al., 2000b). On the other hand, intraventricular injections of orexins stimulate the liberation of gonadotropins (Pu et al., 1998) and ACTH (Kuru et al., 2000) and inhibit the secretion o f LH, prolactin (Kohsaka et al., 2001) and GH (Blanco et al., 2001; Seoane et al., 2004; Molik et al., 2008). Recent data suggest that these neuropeptides also increase the production of glucocorticoids in rats and humans (Spinazzi et al., 2006), being implicated in processes of stress produced after the activation of the hypotha­ lamic orexinergic cells by CRF (Winsky-Sommerer et al., 2004). There are contradictory reports describing the presence of orexin-ir cells in the hypophysis of vertebrates. Orexins in specific cells of the adenohypophysis have been reported in human (Blanco et al., 2003), orexin-A in two species o f amphibian anurans (Yamamoto et al., 2004; Suzuki et al., 2007b) and orexin-A and orexin-B in the pituitary o f two species o f teleost fishes (Amiya 58 2. ESTUDIOS QUIMIOARQUITECTONICOS EN EL ENCEFALO ADULTO 30 L Dominguez e t aL/Joumal o f Chemical Neuroanatomy 39 (2010) 20-34 et al., 2007; Suaiki et al., 2007a, 2009). However, none of the other studies in fishe; amphibians, birds or mammals detected orexin-ir cells in the hypphysis (Shibahara et al., 1999; Galas et al., 2001; Phillips-Singh a: al., 2003; Kaslin et al., 2004; Huesa et al., 2005; Singletary et al, 2005, 2006; Nixon and Smale, 2007; Lopez et al., 2009) and the hypophysis is devoid of orexin-ir cells in the two reptiles analyzed in the present study. This discrepancy may be an example of tecinical differences produced by the use of different antibodies. There are daa that indicate interactions between thyrotropin- releasing homone (TRH) and the orexins in the control of wakefulness shte and arousal (Hara et al., 2009) evidenced by the close appaitions observed in mice between TRH-immunor- eactive nerve brminals and orexin-A-immunoreactive cell bodies (Gonzalez et al. 2009). A recent study about the distribution of TRH in the brain of nptiles suggests that a similar relationship between TRH fibers andorexin-ir cells might exist (Lopez et al., 2008). In the midbein of reptiles the torus semicircularis and the optic tectum are moterately innervated by orexin-ir fibers (Farrell et al., 2003; present results). This was also observed in amphibians (Galas et al., 2(01 ; Singletary et al., 2005; Lopez et al., 2009) and teleosts (Kaslinet al., 2004; Huesa et al., 2005; Amiya et al., 2007). In birds, the imervation of these mesencephalic regions is more conspicuous (Sngletary et al., 2006), suggesting an important function for orecins in the auditory and visual system. Notably in mammals, the orexinergic innervation of midbrain structures is moderate to bw (Nixon and Smale, 2007) but conspicuous innervation of the neurons of the mesencephalic trigeminal nucleus was sten in the rat (Stoyanova and Lazarov, 2005). In contrast, both in reptiles (present results) and in amphibians (Lopez et al., 2309) no special relation was observed between orexin-ir fibers and the large cells of this nucleus. Within the mesencephalic tegmentum of reptiles, most orexin-ir fibers are located in relatbn to the dopaminergic cell groups and will be dealt with below. In the isthnus and in the upper rhombencephalon of reptiles particular inner/ation of orexin-ir fibers is located in relation to the cholinergic cels in the pedunculopontine and laterodorsal tegmental nucei (Medina et al., 1993; Powers and Reiner, 1993). Similarh, the homologous cholinergic nuclei in fish and amphibians als) possess abundant orexin-ir fibers (Galas et al., 2001 ; Kaslin et iL, 2004; Lopez et al., 2009). The pedunculopontine nucleus in manmals is also reached by numerous orexinergic fibers (Peyron a al., 1998; Cutler et al., 1999; Nambu et al., 1999; McGranaghan md Piggins, 2001; Mintz et al., 2001) and may regulate the atinia controlled by this nucleus during REM sleep (Takakusaki et al., 2004, 2005) and affect the activity of this nucleus to contiol sleep-wakefulness (Kim et al., 2009). Hypotha­ lamic orexin natrons in rats may affect directly the cholinergic laterodorsal tegnental nucleus neurons and thereby participate in control of sleep (Takahashi et al., 2002). The scarce aexin-ir fibers found in the rhombencephalon of reptiles are manly related to the reticular formation and the griseum centrab, in the rostral rhombencephalon. No particular innervation w * noted on the cranial nerve motor nuclei, in contrast to obstrvations made in mammals (Fung et al., 2001 ; McGregor et al. 2005). Similarly, the dorsal alar portion of the rhombencephalon in reptiles is poorly innervated by orexin-ir fibers and mostoctaval nuclei totally lack innervation. This seems to be a shared feature among vertebrates, although the medial vestibular nucleus of the hamster receives a projection from the orexinergic hypothalamic cells (Horowitz et al., 2005). In the spinal cord of reptiles some orexin-ir terminal-like structures were detected among the cell bodies of laminae V, VI and X of the spiral gray and, less abundantly, in lamina IV and VII- VIII and IX of the ventral horn. Comparatively, in the spinal cord of rats, a high density of orexin-ir fibers was detected in the marginal zone, the lamina 10 and the intermediolateral column and a role for orexins in the modulation of pain sensation and the regulation of the autonomic nervous system was proposed (van den Pol, 1999; Date et al., 2000a; Llewellyn-Smith et al., 2003). In fact, the antinociceptive effect of the orexins has been established in mice (Mobarakeh et al., 2005). Recently, a direct effect of orexin-A on dorsal root ganglion neurons was proposed as a possible mechanism for the orexinergic modulation of spinal nociceptive transmission (Yan et al., 2008). In addition, the orexin-2 receptor seem s to play an inhibitory role in nociceptive transmission in the neonatal rat spinal cord (Shono and Yamamoto, 2008). 4.3. Relationships between orexinergic and monoaminergic systems In the analysis of the relationship between the aminergic and orexinergic systems, the use of double immunohistochemistry reveals in reptiles that both systems can interact in diverse brain regions. In mammals, different studies have revealed that orexinergic neurons are regulated by monoamines and, in addition, there are reciprocal neuronal circuitries between the orexin hypothalamic neurons and several aminergic cell groups. Thus, it was shown that orexin neurons are directly hyperpolarized by noradrenaline, dopamine and serotonin (Yamanaka et al., 2(M)3, 2006; Muraki et al., 2004) and, therefore, inhibited by these monoamines. The anatomical support was provided by immuno­ histochemistry and tract-tracing experiments because orexin-ir cells in the hypothalamus of the rat were embedded in monoaminergic fibers and boutons (Kirchgessner, 2002; Lee et al., 2005). The reciprocal connections were demonstrated when orexinergic innervation was observed in dopaminergic and noradrenergic centers, such as the substantia nigra, ventral tegmental area, locus coeruleus and A l, A2 and A5 cell groups (Kirchgessner, 2002; Baldo et al., 2003; Ferguson and Samson, 2003; Bubseretal.,2005; Balcita-PedicinoandSesack,2007; Nixon and Smale, 2007) and the serotoninergic dorsal raphe nucleus (Lee et al., 2005; Yoshida et al., 2006). The dopaminergic cell groups of the substantia nigra/ventral tegmental area complex of reptiles are likely to be innervated by orexin-ir neurons, because abundant orexin-ir terminal structures were seen in their proximity, as in mammals and birds (Bubser et al., 2005; Singletary et al., 2006; Balcita-Pedicino and Sesack, 2007; present results). This situation is also shared by anamniotes, as demonstrated for several amphibians and teleosts (Kaslin et al., 2004; Lopez et al., 2009). An important role has been attributed to the orexins in the brain system of reward through the innervation of the nucleus accumbens and the ventral tegmental area (Fadel and Deutch, 2002; Korotkova etal., 2003; Harris etal., 2005) and, as w e have observed, a similar situation might exist in reptiles and amphibians since both the nucleus accumbens and the ventral tegmental area contain orexin-ir fibers (Lopez et al., 2009; present results). Recent evidence shows that orexins selectively activate dopamine neurons within the ventral tegmental area that project to the prefrontal cortex and the shell subregion of the nucleus accumbens strengthening the potential role for orexins in cognitive and/or affective processes (Vittoz et al.. 2008). The noradrenergic cell groups in the upper rhombencephalon that constitute the locus coeruleus complex of reptiles (Smeets and Gonzalez, 2000) is richly innervated by orexin-ir fibers, as also observed in mammals (Peyron et al., 1998; Horvath et al., 1999; Baldo et al., 2003; Nixon and Smale, 2007). In addition, in both reptiles and mammals the nucleus of the solitary tract also exhibited moderate innervation by orexin-ir fibers (Kirchgessner, 2002; Baldo et al., 2003; Ferguson and Samson, 2003; Nixon and Smale, 2007; Lopez et al., 2009). Actually, there is evidence that this orexinergic innervation activates the liberation of noradrena- 59 2. ESTUDIOS QUIMIOARQUITECTONICOS EN EL ENCÉFALO ADULTO L Dommguez e t aL/Joumal o f Chemical Neuroanatomy 39 (2010) 20-34 31 line in the locus coeruleus (Hagan et al., 1999; Walling et al., 2004; Chen et al., 2008) and in the nucleus of the solitary tract (Smith et al., 2002a; Ciriello et al., 2003a). In particular, interaction between orexinergic neurons and NMDA receptors in the control of locus coeruleus-cerebrocortical noradrenergic activity of the rat has been demonstrated (Tose et al., 2009). In addition, the hypothalamic orexin neurons that project to the locus coeruleus express a diurnal rhythm of activation that correlates with the neuronal firing frequency of this nucleus, which is important in the modulation of day-night differences of the locus coeruleus impulse activity that promotes wakefulness and behavioral arousal (Gompf and Aston-jones, 2008). The orexin innervation to the nucleus of the solitary tract serves as the anatomical substrate for the involvement of orexins in the regulation of cardiovascular functions (Smith et al.. 2002b; de Oliveira et al., 2003; Shih and Chuang, 2007). Both orexin and serotonin have important roles in the regulation of sleep-wakefulness, as well as in feeding behavior. In the raphe nuclei column, a moderate innervation by orexin-ir fibers is observed in reptiles (present results) and this innervation has also been detected in amphibians (Suzuki et al., 2008; Lopez et al., 2009) and, more abundantly, in teleosts (Kaslin et al., 2004; Huesa et al., 2005). In mammals, the orexin-ir cells project to each subdivision of the raphe nuclei (Wang et al., 2003; Lee et al., 2005) modulating the liberation of serotonin in these nuclei (Takahashi et al., 2005; Wang et al., 2005; Tao et ai., 2006). The orexins depolarize neurons and can increase calcium concentration in the dorsal raphe and laterodorsal tegmental cells, indicating that orexins can function as a neuromodulator in these key serotoni­ nergic and cholinergic neurons of the reticular activating system (Kohlmeier et al., 2008). Thus, orexins might have wake-related influences over a variety of brain functions subject to efferent regulation from the raphe nuclei (Lee et al., 2005). Therefore, on the basis of the neuroanatomical relationships demonstrated, the functions of orexins acting on monoaminergic brain centers would be a primitive shared feature in vertebrates, as seen in reptiles, amphibians and teleosts (Kaslin et al., 2004; Huesa et al., 2005; Suzuki et al., 2008; Lopez et al., 2009; present results). In general, the great similarity in the pattern of brain distribution of orexin-ir fibers and neurons in all species of vertebrate studied indicate a high degree of conservation of these neuropeptides during evolution and also suggests a conservation of the important physiological functions of orexins, which may integrate a variety of interoceptive and homeostatic signals to increase behavioral arousal in response to hunger, stress, circadian signals, and autonomic challenges, via actions on multiple neuromodulatory transmitter systems, such as the monoaminergic systems analyzed here for reptiles. Acknowledgements This research was supported by Santander/Complutense research projects (reference number: PR34/07-15818) and the Spanish Ministry of Science and Technology (Grant number: BFU2006-01014/BF1). References Adams, J.C., 1981. Heavy metal intensification ofDAB-based HRP reaction product. J. Histochem. Cytochem. 29, 775. Airaksinen, M.S., Panula, P., 1990. Comparative neuroanatomy of the histaminergic system in the brain of the frog Xenopus laevis.J. Comp. Neurol. 292, 412-423. Alvarez. CE.. Sutcliffe. J.G.. 2002. Hypocretin is an early member of the incretin gene family. Neurosd. Lett. 324 .169-172 . Amiya. N., Amano, M., Oka, Y., ligo, M., Takahashi, A., Yamamori, K., 2007. Immu- nohistochemical localization of orexin/hypocretin-like immunoreactive pep­ tides and melanin-concentrating hormone in the brain and pituitary of medada. Neurosd. Lett 427 ,16-21 . Baldta-Pedicino, J.J., Sesack, S R.. 2007, Orexin axons in the rat ventral tegmental area synapse infrequently onto dopamine and gamma-aminobutyric acid neu­ rons. J. Comp. Neurol. 503, 668-684. Baldo, BA, Daniel, RA, Berridge, C.W., Kelley, A.E., 2003. Overlapping distributions of orexin/hypocretin- and dopamine-beta-hydroxylase immunoreactive fibers in rat brain regions mediating arousal, motivation, and stress. J. Comp. Neurol. 464, 220-237. Barb, C.R., Matteri, R.L, 2005. Orexin-B modulates luteinizing hormone and growth hormone secretion from porcine pituitary cells in culture. D om est Anim. Endocrinol. 28, 331-337. Barroso, C, Franzoni, M.F., Fasolo, A., Panula, P., 1993. Organization of histamine- containing neurons in the brain of the crested newt. Trituras camifex. Cell Tissue Res. 272 ,147-154 . Baumann, C.R., Bassetti, C.L, 2005. Hypocretins (orexins) and sleep-wake disorders. Lancet Neurol. 4, 673-682. Bemardis, L, Bellinger, LL, 1993. The lateral hypothalamic area revisited: neuroa­ natomy, body weight regulation, neuroendocrinology and metabolism. Neu­ rosd. Biobehav. Rev. 17 .141-193 . Berthoud, H R.. Patterson. LM., Sutton. G.M., Morrison, C, Zheng, H„ 2005. Orexin inputs to caudal raphe neurons involved in thermal, cardiovascular, and gastro­ intestinal regulation. Histochem. Cell Biol. 123 ,147-156 . Beuckmann, C.T., Yanagisawa, M., 2002. Orexins: from neuropeptides to energy homeostasis and sleep/wake regulation. J. Mol. Med. 80, 329-342. Bisetti, A., Cvetkovic, V., Serafin, M., Bayer, L, Machard, D., Jones, B.E., Muhlethaler, M., 2006. Excitatory action of hypocretin/orexin on neurons of the central medial amygdala. Neuroscience 142,999-1004. Blanco, M., Gallego, R., Garcia-Caballero, T., Dieguez, C., Beiras, A, 2003. Cellular localization of orexins in human anterior pituitary. Histochem. Cell Biol. 120, 259-264. Blanco, M., Lopez, M., Garcia-Caballero, T., Gallego, R., Vazquez-Boquete, A, Morel, G., Senarls, R., Casanueva, P., Dieguez, C., Beiras, A., 2001. Cellular localization of orexin receptors in human pituitary.]. Clin. Endocrinol. Metab. 86, 1616-1619. Broberger, C, de Lecea, L, Sutcliffe, J.G., Hokfelt, T., 1998. Hypt^retin/orexin- and melanin-concentrating hormone-expressing cells form distinct populations in the rodent lateral hypothalamus: relationship to the neuropeptide Y and agouti gene-related protein systems. J. Comp. Neurol. 402 ,460-474 . Brown, T.M., Coogan, AN., Cutler, D.J., Hughes, AT., Piggins, H.D„ 2008. Electro- physiological actions of orexins on rat suprachiasmatic neurons in vitro. Neurosci. Lett. 448, 273-278. Bubser, M., Fadel, J R., Jackson, LL, Meador-Woodruff, J.H., Jing, D„ Deutch, A.Y., 2005. Dopaminergic regulation of orexin neurons. Eur. J. Neurosd. 21, 2993- 3001. Burdakov, D., Liss, B., Ashcroft, P.M., 2003. Orexin excites GABAergic neurons of the arcuate nucleus by activating the sodium-calcium exchanger. J. Neurosci. 23, 4951-4957. Carter, M.E., Borg, J.S., de Lecea, L, 2009. The brain hypocretins and their receptors: mediators of allostatic arousal. Curr. Opin. Pharmacol. 9, 39-45. Chemelli, R.M., Willie, J.T., Sinton, CM., Elmquist, J.K., Scammell, T., Lee, C., Richard­ son, JA , Williams, S.C., Xiong, Y., Kisanuki, Y., Fitch. T.E., Nakazato, M., Hammer, R.E., Saper, CB., Yanagisawa, M., 1999. Narcolepsy in orexin knockout mice: molecular genetics o f sleep regulation. Cell 98, 437-451. Chen. X.W., Mu, Y., Huang, H P., Guo, N., Zhang, B., Fan, S.Y., Xiong, JJC, Wang, S R., Xiong, W., Huang, W., Liu, T., Zheng, LH., Zhang, C.X., Li, LH., Yu, Z.P., Hu, ZA, Zhou, Z., 2008. Hypocretin-1 potentiates NMDA receptor-mediated somatoden­ dritic secretion from locus ceruleus neurons. J. Neurosci. 28, 3202-3208. Ciriello, J., McMurray, J.C, Babic, T., de Oliveira, C.V., 2003a. Collateral axonal projections from hypothalamic hypocretin neurons to cardiovascular sites in nucleus ambiguus and nucleus tractus solitarius. Brain Res. 991 ,133-141. Ciriello, J., Rosas-Arellano, M.P., Solano-Flores, LP., de Oliveira, CV„ 2003b. Identi­ fication of neurons containing orexin-B (hypocretin-2) immunoreactivity in limbic structures. Brain Res. 967, 123-131. Cutler, D.J., Morris, R., Sheridhar, V., Wattam, T A , Holmes, S., Patel, S., Arch, J R., Wilson, S., Buckingham, R.E., Evans, M.L, Leslie, RA, Williams, G., 1999. Differential distribution of orexin-A and orexin-B immunoreactivity in the rat brain and spinal cord. Peptides 20 ,1455-1470. Date, Y., Mondai, M.S., Matsukura, S., Nakazato, M., 2000a. Distribution of orexin-A and orexin-B (hypocretins) in the rat spinal cord. Neurosci. Lett 288 ,87-90 . Date, Y., Mondai, M.S., Matsukura, S., Ueta, Y., Yamashita, H„ Kalya, H„ Kangawa, K., Nakazato, M., 2000b. Distribution of orexin/hypocretin in the rat median eminence and pituitary. Brain Res. Mol. Brain Res. 76, 1 -6 . Date, Y., Ueta, Y., Yamashita, H„ Yamaguchi, H., Matsukura, S., Kanawa, K., Sakurai, T., Yanagisawa, M„ Nakazato, M., 1999. Orexins, orexigenic hypothalamic peptides, interact with autonomic, neuroendocrine and neuroregulatory sys­ tems. Proc. Natl. Acad. Sci. U.SA 96, 748-753. de Lecea, L, Kilduff, T.S., Peyron, C, Gao, X., Foye, P.E., Danielson, P.E., Fukuhara, C., Battenberg, E.L, Gautvik, V.T., Bartlett 2nd, F.S., Frankel, W.N., van den Pol, AN., Bloom, F.E., Gautvik, K.M., Sutcliffe, J.G., 1998. The hypocretins: hypothalamus- specific peptides with neuroexcitatory activity. Proc. Natl. Acad. &i. U S A 95, 322-327. de Oliveira, CV., Rosas-Arellano, M.P., Solano-Flores, LP., Ciriello, J„ 2003. Cardi­ ovascular effects of hypocretin-1 in nucleus of the solitary tract Am. J. Physiol. Heart Circ. Physiol. 284, H1369-H1377. Dube, M.G., Horvath, T.L, Kalra, P.S., Kaira, S.P., 2000. Evidence of NPY Y5 receptor involvement in food intake elicited by orexin A in sated rats. Peptides 21 ,1557- 1560. 60 2. ESTUDIOS QUIMIOARQUITECTONICOS EN EL ENCÉFALO ADULTO 32 L Dominguez et aL/Joumal of Chemical Neuroanatomy 39 (2010) 20-34 Dube, M.G., Kalra, S.P., Kalra, PS.. 1999. Food intake elicited by central adminis­ tration of orexins/hypocretins: identification of hypothalamic sites of action. Brain Res. 842 ,473 -477 . Fade!, J„ Deutch, AY., 2002. Anatomical substrates of orexin-dopamine interac­ tions: lateral hypothalamic projections to the ventral tegmental area. Neu­ roscience 111, 379-387. Fadel, j., Frederick-Duus, D., 2008. Orexin/hypocretin modulation of the basal forebrain cholinergic system: insights from in vivo microdialysis studies. Pharmacol. Biochem. Behav. 90 ,156 -162 . Faraco, JH,, Appelbaum, L, Marin, W., Gaus, S.E., Mourrain, P., Mignot, E., 2006. Regulation of hypocretin (orexin) expression in embryonic zebrafish. j. Biol. Chem. 281, 29753-29761. Farrell, W.J., Delville, Y„ Wilczynski, W., 2003. Immunocytochemical localization of orexin in the brain o f the green anole lizard (Anolis caroiinensis). Soc Neur. Abstract 33 (828), 4. Ferguson, AV., Samson, W.K., 2003. The orexin/hypocretin system: a critical reg­ ulator of neuroendocrine and autonomic function. Front. Neuroendocrinol. 24, 141-150. Fung, S.J., Yamuy, j., Sampogna, S., Morales, F.R., Chase, M.H., 2001. Hypocretin (orexin) input to trigeminal and hypoglossal motoneurons in the cat: a double- labeling immunohistochemical study. Brain Res. 903, 257-262. Galas, L, Vaudry, H., Braun, B., van den Pol, A.N., de Lecea, L, Sutcliffe, J.G., Chartrel, N., 2001. Immunohistochemical localization and biochemical characterization of hypocretin/orexin-related peptides in the central nervous system of the frog Rana ridibunda. J. Comp. Neurol. 429. 242-252. Gompf, H.S., Aston-jones, G„ 2008. Role of orexin input in the diurnal rhythm of locus coeruleus impulse activity. Brain Res. 1224,43-52. Gonzalez, JA , Horjales-Araujo, E., Fugger, L, Broberger, C., Burdakov, D., 2009. Stimulation of orexin/hypocretin neurones by thyrotropin-releasing hormona. J. Physiol. 587 ,1179-1186 . Govindaiah, G., Cox, C.L, 2006. Modulation of thalamic neuron excitability by orexins. Neuropharmacology 51 ,414 -425 . Hagan, J.j., Leslie, R.A, Patel, S., Evans, M.L. Wattam, TA , Holmes, S., Benham, C D., Taylor, S.G., Routledge, C., Hemmati. P., Munton, R.P., Ashmeade, T.E., Shah, A.S., Hatcher, J.P., Hatcher, P.D., Jones, D.N., Smith, M.I., Piper, D C., Hunter, A.J., Porter, RA, Upton, N., 1999. Orexin A activates locus coeruleus cell firing and increases arousal in the rat. Proc. Natl. Acad. Sci. U S A 96, 10911-10916. Hara, J., Gerashchenko, D., Wisor, J.P., Sakurai, T., Xie, X., Kilduff, T.S., 2009. Thyrotropin-releasing hormone increases behavioral arousal through modula­ tion of hypocretin/orexin neurons. J. Neurosci. 29, 3705-3714. Harris, G.C., Wimmer, M., Aston-jones, G.. 2005. A role for lateral hypothalamic orexin neurons in reward seeking. Nature 437, 556-559. Haynes, A.C., Jackson, B., Overend, P., Buckingham, R.E., Wilson, S., Tadayyon, M., Arch, J R.. 1999. Effects of single and chronic intracerebroventricular adminis­ tration of the orexins on feeding in the rat. Peptides 20, 1099-1105. Hoogland, P.V., Vermeulen-VanderZee, E., 1990. Distribution of choline acetyltrans- ferase immunoreactivity in the telencephalon of the lizard Cekko gecko. Brain Behav. Evol. 36, 378-390. Horowitz, S.S., Blanchard, J.. Morin, LP., 2005. Medial vestibular connections with the hypocretin (orexin) system. J. Comp. Neurol. 487, 127-146. Horvath, T.L, Peyron, C., Diano, S., Ivanov, A, Aston-jones, C., Kilduff, T.S., van den Pol, A.N., 1999. Hypocretin (orexin) activation and synaptic innervation of the locus coeruleus noradrenergic system. J. Comp. Neurol. 415 ,145-159 . Huesa, G., van den Pol, AN., Finger, T.E., 2005. Differential distribution of hypocretin (orexin) and melanin-concentrating hormone in the goldfish brain. J. Comp. Neurol. 4 8 8 ,476 -491 . Inagaki, N., Panula. P., Yamatodani, A., Wada, H., 1990. Organization of the hista­ minergic system in the brain of the turtle Chinemys revets. J. Comp. Neurol. 297, 132-144. Karteris, E., Machado, R.J., Chen, J., Zervou, S., Hillhouse, E.W., Randeva, H.S., 2005. Food deprivation differentially modulates orexin receptor expression and signaling in rat hypothalamus and adrenal cortex. Am. J. Physiol. Endocrinol. Metab. 288, El 089-E l 100. Kaslin, J., Nystedt, J.M., Ostergard, M., Peitsaro, N., Panula, P., 2004. The orexin/ hypocretin system in zebrafish is connected to the aminergic and cholinergic systems. J. Neurosci. 24, 2678-2689. Kiehn, O., Rostrup, E., Moller, M., 1992. Monoaminergic systems in the brainstem and spinal cord of the turtle Pseudemys scripta elegans as revealed by antibodies against serotonin and tyrosine hydroxylase. J. Comp. Neurol. 325, 527-547. Kilduff, T.S., de Lecea, L, 2001. Mapping of the mRNAs for the hypocretin/orexin and melanin-concentrating hormone receptors: networks of overlapping peptide systems. J. Comp. Neurol. 4 3 5 ,1 -5 . Kim, J., Nakajima, K., Oomura, Y., Wayner, M.J., Sasaki, K., 2009. Electrophysiological effects of orexins/hypocretins on pedunculopontine tegmental neurons in rats: an in vitro study. Peptides 30, 191-209. Kirchgessner. AL. 2002. Orexins in the brain-gut axis. Endocr. Rev. 23 ,1 -1 5 . Kirouac, G.J., Parsons, M.P., Li, S., 2005. Orexin (hypocretin) innervation of the paraventricular nucleus of the thalamus. Brain Res. 1059,179-188. Kohlmeier, KA, Watanabe, S., Tyler, C.J., Burlet, S., Leonard, CS., 2008. Dual orexin actions on dorsal raphe and laterodorsal tegmentum neurons: noisy cation current activation and selective enhancement of Câ * transients mediated by l- type calcium channels. J. Neurophysiol. 100, 2265-2281. Kohsaka, A Watanobe, H., Kakizaki, Y., Suda, T., Schioth, H.B., 2001. A significant participation of orexin-A, a potent orexigenic peptide, in the preovulatory luteinizing hormone and prolactin surges in the rat. Brain Res. 898, 166- 170. Korotkova, T.M., Sergeeva, D A , Eriksson, ICS., Haas, H.L, Brown, R.E., 2003. Excita­ tion o f ventral tegmental area dopaminergic and nondopaminergic neurons by orexins/hypocretins. J. Neurosci. 23, 7-11. Kotani, A , Ikeda, H., Koshikawa, N., Cools, A.R., 2008. Role of orexin receptors in the nucleus accumbens in dopamine-dependent turning behaviour of rats. Neuro­ pharmacology 54, 613-619. Kukkonen. J.P., Holmqvist. T., Ammoun. S., Akerman, K.E., 2002. Functions of the orexinergic/hypocretinergic system. Am. J. Physiol. Cell Physiol. 283, Cl 567- C1591. Kuru, M., Ueta, Y., Serino, R., Nakazato, M., Yamamoto, Y„ Shibuya, 1., Yamashita, H., 2000. Centrally administered orexin/hypocretin activates HPA axis in rats. Neuroreport 11,1977-1980. Lee, H.S., Park, S.H., Song, W.C., Waterhouse, B.D., 2005. Retrograde study of hypocretin-1 (orexin-A) projections to subdivisions of the dorsal raphe nucleus in the rat. Brain Res. 1059, 35-45. Lin, L, Faraco, J., Li, R.. Kadotani, H., Rogers, W., Lin, X., Qiu, X., de Jong, P.J., Nishino, S., Mignot. E., 1999. The sleep disorder canine narcolepsy is caused by a mutation in the hypocretin (orexin) receptor 2 gene. Cell 98, 365-376. Llewellyn-Smith, I.J., Martin, C.L, Marcus, J.N., Yanagisawa, M., Minson, J.B., Scam­ mell, T.E, 2003. Orexin-immunoreactive inputs to rat sympathetic preganglio­ nic neurons. Neurosci. Lett. 351, 115-119. Lopez, J.M., Dominguez, L, Gonzalez, A., 2008. Immunohistochemical localization of thyrotropin-releasing hormone in the brain of reptiles. J. Chem. Neuroanat. 36, 251-263. Lopez, J.M., Dominguez, L, Moreno, N., Gonzalez, A., 2009. Comparative immuno­ histochemical analysis of the distribution o f orexins (hypocretins) in the brain of amphibians. Peptides 30, 873-887. Lubkin, M.. Stricker-Krongrad, A., 1998. Independent feeding and metabolic actions of orexins in mice. Biochem. Biophys. Res. Commun. 253, 241-245. Malendowicz, LK., Tortorella, C, Nussdorfer, G.G., 1999. Orexins stimulate corti­ costerone secretion of rat adrenocortical cells, through the activation of the adenylate cyclase-dependent signaling cascade. J. Steroid. Biochem. Mol. Biol. 70 ,185-188 . Martynska. L, Polkowska, J., Wolinska-Witort. E., Chmielowska, M., Wasilewska- Dziubinska, E., Bik, W., Baranowska, B., 2006. Orexin A and its role in the regulation of the hypothalamo-pituitary axes in the rat. Reprod. Biol. 6 (Suppl. 2), 29-35. Matsuki, T., Sakurai, T., 2008. Orexins and orexin receptors: from molecules to integrative physiology. Results Probl. Cell Differ. 46, 27-55. McGranaghan, PA , Piggins, H.D., 2001. Orexin A like immunoreactivity in the hypothalamus and thalamus of the Syrian hamster (Mesocricetus auratus) and Siberian hamster (Phodopus sungorus), with special reference to circadian structures. Brain Res. 904, 234-244. McGregor, R., Damian, A., Fabbiani, G., Torterolo, P.. Pose, I., Chase, M., Morales, F.R., 2005. Direct hypothalamic innervation of the trigeminal motor nucleus: a retrograde tracer study. Neuroscience 136, 1073-1081. Medina, L, Smeets, W.J., 1992. Cholinergic, monoaminergic and peptidergic inner­ vation of the primary visual centers in the brain of the lizards Cekko gecko and Gallotia galloti. Brain Behav. Evol. 40, 157-181. Medina, L. Smeets, W.J., Hoogland, P.V., Puelles, L, 1993. Distribution of choline acetyltransferase immunoreactivity in the brain of the lizard Callotia galloti. J. Comp. Neurol. 331, 261-285. Mintz, E.M., van den Pol, A.N., Casano, AA, Albers, H E., 2001. Distribution of hypocretin-(orexin) immunoreactivity in the central nervous system of Syrian hamsters (Mesocricetus auratus).]. Chem. Neuroanat. 21, 225-238. Mitsuma, T., Hirooka, Y., Mori, Y., Kayama, M., Adachi, K., Rhue, N„ Ping, J., Nogimori, T., 1999. Effects of orexin A on thyrotropin-releasing hormone and thyrotropin secretion in rats. Horm. Metab. Res. 31, 606-609. Mobarakeh, J.I., Takahashi, K., Sakurada, S., Nishino, S., Watanabe, H., Kato, M., Yanai, K., 2005. Enhanced antinociception by intracerebroventricularly and intrathe- cally-administered orexin A and B (hypocretin-1 and -2) in mice. Peptides 26, 767-777. Molik, E., Zieba, DA, Misztal, T., Romanowicz, K., Wszola, M., Wierzchos, E., Now- akowski, M., 2008. The role of orexin A in the control of prolactin and growth hormone secretions in sheep—in vitro study. J. Physiol. Pharmacol. 59 (Suppl. 9), 91-100. Mufioz, M., Smeets, W.J., Lopez, J.M., Moreno, N., Morona, R., Dominguez, L, Gonzalez, A., 2008. Immunohistochemical localization of neuropeptide FF-like in the brain of the turtle: relation to catecholaminergic structures. Brain Res. Bull. 75, 256-260. Muraki, Y., Yamanaka, A., Tsujino, N., Kilduff, T.S., Goto, K.. Sakurai, T., 2004. Serotonergic regulation of the orexin/hypocretin neurons through the 5- HTIA receptor. J. Neurosci. 24, 7159-7166. Nambu, T., Sakurai, T., Mizukami, K., Hosoya, Y., Yanagisawa, M., Goto, K., 1999. Distribution of orexin neurons in the adult rat brain. Brain Res. 827, 243-260. Nixon, J.P., Smale, L, 2007. A comparative analysis of the distribution of immunor­ eactive orexin A and B in the brains of nocturnal and diurnal rodents. Behav. Brain Funct. 3, 28. Ohkubo, T.. Boswell, T., Lumineau, S., 2002. Molecular cloning o f chicken prepro- orexin cDNA and preferential expression in the chicken hypothalamus. Bio- chim. Biophys. Acta 1577, 476-480. Okumura, T., Takakusaki, K., 2008. Role of orexin in central regulation of gastro­ intestinal functions. J. Gastroenterol. 43, 652-660. Pasumarthi. R.K., Fadel, J., 2008. Activation of orexin/hypocretin projections to basal forebrain and paraventricular thalamus by acute nicotina. Brain Res. Bull. 77, 367-373. 61 2. ESTUDIOS QUIMIOARQUITECTONICOS EN EL ENCÉFALO ADULTO L Dommguez et aL/Joumal of Chemical Neuroanatomy 39 (2010) 20-34 33 Peyron. C. Tighe, O.K.. van den Pol, A.N., de Lecea, L, Heller, H.C., Sutcliffe, J.G., Kilduff, T.S., 1998. Neurons containing hypocretin (orexin) project to multiple neuronal systems, j. Neurosci. 18, 9996-10015. Phillips-Singh, D., Li, Q,, Takeuchi, S., Ohkubo, T., Sharp, P.J., Boswell, T., 2003. Fasting differentially regulates expression of agouti-related peptide, pro-opiomelano- cortin, prepro-orexin, and vasoactive intestinal polypeptide mRNAs in the hypothalamus of Japanese quail. Cell Tissue Res. 313, 217-225. Powers, A.S., Reiner, A., 1993. The distribution of cholinergic neurons in the central nervous system of turtles. Brain Behav. Evol. 41, 326-345. Pu, S., Jain, M.R., Kalra. P.S., Kalra, S.P., 1998. Orexins. a novel family of hypothalamic neuropeptides, modulate pituitary luteinizing hormone secretion in an ovarian steroid-dependent manner. Regul. Pept. 78 ,133-136 . Russell, S.H., Kim, M.S., Small, C.J., Abbott, C.R., Morgan, D.G., Taheri, S., Murphy, K.G., Todd, J.F., Ghatei, M A, Bloom, SR., 2000. Central administration o f orexin A suppresses basal and domperidone stimulated plasma prolactin. J. Neuroen­ docrinol. 12 ,1213-1218. Sakurai, T., Amemiya, A., Ishii, M., Matsuzaki, I., Chemelli, R.M., Tanaka, H., Williams, S.C., Richardson, JA , Kozlowski, G.P., Wilson, S., Arch, J.R., Buckingham, R.E., Haynes, A.C., Carr, SA , Annan, R.S., McNulty, D.E, Liu, W.S., Terrett, JA , Elshourbagy, N A , Bergsma, D.J., Yanagisawa, M., 1998. Orexins and orexin receptors: a family o f hypothalamic neuropeptides and G protein-coupled receptors that regulate feeding behavior. Cell 92, 573-585. Seoane, LM., Tovar, SA , Perez, D., Mallo, F., Lopez, M., Senaris, R., Casanueva, F.F., Dieguez, C., 2004. Orexin A suppresses in vivo GH secretion. Eur. J. Endocrinol. 150, 731-736. Shibahara, M., Sakurai, T., Nambu, T., Takenouchi, T., Iwaasa, H., Egashira, S.I., Ihara, M., Goto, K-, 1999. Structure, tissue distribution, and pharmacological char­ acterization of Xenopus orexins. Peptides 20 ,1169-1176 . Shibata, M., Mondai, M S., Date, Y., Nakazato, M., Suzuki, H., Ueta, Y., 2008. Distribution of orexins-containing fibers and contents of orexins in the rat olfactory bulb. Neurosci. Res. 61, 99-105. Shih, C.D., Chuang, Y.C., 2007. Nitric oxide and G ABA mediate bi-directional cardiovascular effects of orexin in the nucleus tractus solitarii of rats. Neu­ roscience 149, 625-635. Shirasaka, T., Kunitake, T., Takasaki, M., Kannan, H., 2002. Neuronal effects of orexins: relevant to sympathetic and cardiovascular functions. Regul. Pept. 104, 91-95. Shono, K., Yamamoto, T., 2008. Orexin-2 receptors inhibit primary afferent fiber- evoked responses of ventral roots in the neonatal rat isolated spinal cord. Brain Res. 1218, 97-102. Singletary, K.G., Delville, Y., Farrell, W.J., Wilczynski, W., 2005. Distribution of orexin/hypocretin immunoreactivity in the nervous system of the green Tree- frog, Hyla cinerea. Brain Res. 1041, 231-236. Singletary, K.G., Deviche, P., Strand, C., Delville, Y., 2006. Distribution of orexin/ hypocretin immunoreactivity in the brain of a male songbird, the house finch, Carpodacus mexicanus.}. Chem. Neuroanat. 32, 81-89. Smeets, W.J., 1994. Catecholamine systems in the CNS of reptiles: structure and functional correlations. In: Smeets, W.J., Reiner, A. (Eds.), Phylogeny and Devel­ opment of Catecholamine Systems in the CNS of Vertebrates. Cambridge University Press, Cambridge, pp. 103-134. Smeets, W.J., Hoogland, P.V., Voom, P., 1986. The distribution o f dopamine immu­ noreactivity in the forebrain and midbrain of the lizard Gekko gecko: an immunohistochemical study with antibodies against dopamine. J. Comp. Neu­ rol. 253, 46-60. Smeets, W.J., Lopez, J.M., Gonzalez, A., 2001. Immunohistochemical localization of DARPP-32 in the brain of the lizard, Gekko gecko: co-occurrence with tyrosine hydroxylase. J. Comp. Neurol. 435, 194-210. Smeets, W.J., Lopez, J.M., Gonzalez, A , 2003. Immunohistochemical localization of DARPP-32 in the brain of the turtle, Pseudemys scripta elegans: further assess­ ment of its relationship with dopaminergic systems in reptiles. J. Chem. Neuroanat. 25, 83-95. Smeets, W.J., Lopez, J.M., Gonzalez, A., 2006. Distribution of neuropeptide FF-like immunoreactivity in the brain of the lizard Gekko gecko and its relation to catecholaminergic structures. J. Comp. Neurol. 498, 31-45. Smeets, W.J., Gonzalez, A., 2000. Catecholamine systems in the brain of vertebrates: new perspectives through a comparative approach. Brain Res. Rev. 33,308-379. Smeets, W.J., Steinbusch, H.W., 1988. Distribution of serotonin immunoreactivity in the forebrain and midbrain of the lizard Gekko gecko.}. Comp. Neurol. 271 ,419- 434. Smeets, W.J., Steinbusch, H.W., 1990. New insights into the reptilian catecholami­ nergic systems as revealed by antibodies against the neurotransmitters and their synthetic enzymes. J. Chem. Neuroanat 3, 25-43. Smith, B.N., Davis, S.F., van den Pol, AN., Xu, W., 2002a. Selective enhancement of excitatory synaptic activity in the rat nucleus tractus solitarius by hypocretin 2. Neuroscience 115, 707-714. Smith. P.M., Connolly, B.C., Ferguson, AV., 2002b. Microinjection of orexin into the rat nucleus tractus solitarius causes increases in blood pressure. Brain Res. 950, 261-267. Spinazzi, R., Andreis, P.G., Rossi, G.P., Nussdorfer, G.G., 2006. Orexins in the regula­ tion of the hypothalamic-pituitary-adrenal axis. Pharmacol. Rev. 5 8 ,46 -57 . Stanley, B.G., Willett 3rd, V.L, Donias, H.W., Dee 2nd, M.G., Duva, M A, 1996. Lateral hypothalamic NMDA receptors and glutamate as physiological mediators of eating and weight control. Am. J. Physiol. 270, R443-R449. Stemberger, LA, 1979. Immunocytochemistiy. John Wiley & Sons, New York. Stoyanova, LL, Lazarov, N.E, 2005. Localization of orexin-A-immunoreactive fibers in the mesencephalic trigeminal nucleus of the rat Brain Res. 1054, 82-87. Suzuki, H., Kubo, Y., Yamamoto, T., 2008. Orexin-A immunoreactive cells and fibers in the central nervous system of the axolotl brain and their association with tyrosine hydroxylase and serotonin immunoreactive somata. J. Ctiem. Neuroa­ nat. 35, 295-305. Suzuki, H., Matsumoto, A., Yamamoto, T., 2009. Orexin-B-like immunoreactivity localized in both luteinizing hormone- and thyroid-stimulating hormone-con­ taining cells in the Nile tilapia (Oreochromis niloticus) pituitary. Tissue Cell. 41, 75-78. Suzuki, H., Miyoshi, Y., Yamamoto, T., 2007a. Orexin-A (hypocretin l)-like immu­ noreactivity in growth hormone-containing cells o f the Japanese seaperch (Lateolabrax japonicus) pituitary. Gen. Comp. Endocrinol. 150,205-211. Suzuki, H.,Takemoto, Y., Yamamoto, T., 2007b. Differential distribution of orexin-A- like and orexin receptor 1 (0X1R)-Iike immunoreactivities in the Xenopus pituitary. Tissue Cell 39 ,423 -430 . Sweet, D.C., Levine, A.S., Billington, C.J., Kotz, C M., 1999. Feeding response to central orexins. Brain Res. 821, 535-538. Takahashi. K., Koyama, Y., Kayama, Y., Yamamoto, M., 2002. Effects of orexin on the laterodorsal tegmental neurones. Psychiatry Clin. Neurosci. 56, 335-336. Takahashi, K., Lin, J.S., Sakai, K., 2008. Neuronal activi^ o f orexin and non-orexin waking-active neurons during wake-sleep states in the mouse. Neuroscience 153, 860-870. Takahashi, K., Wang, Q,P., Guan, J.L, Kayama, Y., Shioda, S., Koyama, Y., 2005. State- dependent effects of orexins on the serotonergic dorsal raphe neurons in the rat. Regul. Pept. 126, 43-47. Takakusaki, K., Saitoh, K., Harada, H., Okumura, T., Sakamoto, T., 2004. Evidence for a role of basal ganglia in the regulation of rapid eye movement sleep by electrical and chemical stimulation for the pedunculopontine tegmental nucleus and the substantia nigra pars reticulata in decerebrate cats. Neu­ roscience 124, 207-220. Takakusaki, K., Takahashi, K., Saitoh, K., Harada, H., Okumura, T., Kayama, Y., Koyama, Y., 2005. Orexinergic projections to the cat midbrain mediate alter­ nation of emotional behavioural states from locomotion to cataplexy. J. Physiol. 568,1003-1020. Tamura, T, Irahara, M., Tezuka, M., Kiyokawa, M., Aono, T., 1999. Orexins, orexigenic hypothalamic neuropeptides, suppress the pulsatile secretion of luteinizing hormone in ovariectomized female rats. Biochem. Biophys. Res. Commun. 264, 759-762. Tao, R., Ma, Z., McKenna, J.T., Thakkar, M.M., Winston, S., Strecker, R.E, McCarley, R.W., 2006. Differential effect of orexins (hypocretins) on serotonin release in the dorsal and median raphe nuclei of freely behaving rats. Neuroscience 141, 1101-1105. Tose, R., Kushikata, T., Yoshida, H., Kudo, M., Furukawa, K., Ueno, S., Hirota, K., 2009. Interaction between orexinergic neurons and NMDA receptors in the control of locus coeruleus-cerebrocortical noradrenergic activity of the rat Brain Res. 1250, 81-87. Ueda, S., Takeuchi, Y., Sano, Y., 1983. Immunohistochemical demonstration of serotonin neurons in the central nervous system of the turtle (Qemmys Japo- nica). Anat. Embryol. (Berl.) 1 68 ,1 -19 . van den Pol, A.N., 1999. Hypothalamic hypocretin (orexin): robust innervation of the spinal cord. J. Neurosci. 19, 3171-3182. Vittoz, N.M., Schmeichel, B., Berridge, C.W., 2008. Hypocretin/orexin preferentially activates caudomedial ventral tegmental area dopamine neurons. Eur. J. Neu­ rosci. 28, 1629-1640. Volkoff, H., 2006. The role of neuropeptide Y, orexins, cocaine and amphetamine- related transcript, cholecystokinin, amylln and leptin in the regulation of feeding in fish. Comp. Biochem. Physiol. A: Mol. Integr. Physiol. 144,325-331. Volkoff, H., Bjorklund, J.M., Peter, R.E., 1999. Stimulation of feeding behavior and food consumption in the goldfish. Carassius auratus, by orexin-A and orexin-B. Brain Res. 846, 204-209. Volkoff, H., Canosa, LF., Unniappan, S., Cerda-Reverter, J.M., Bernier, N.J., Kelly, S.P., Peter, R.E., 2005. Neuropeptides and the control of food intake in fish. Gen. Comp. Endocrinol. 142, 3-19. Walling, S.G., Nutt, D.J., Lalies, M.D., Harley, C.W., 2004. Orexin-A infusion in the locus ceruleus triggers norepinephrine (NE) release and NE-induced long-term potentiation in the dentate gyrus. J. Neurosci. 24, 7421-7426. Wang, Q,P., Guan, J.L, Matsuoka, T., Hirayana, Y., Shioda, S., 2003. Electron micro­ scopic examination of the orexin immunoreactivity in the dorsal raphe nucleus. Peptides 24, 925-930. Wang, Q,P., Koyama, Y., Guan, J.L, Takahashi, K., Kayama, Y., Shioda, S., 2005. The orexinergic synaptic innervation of serotonin- and orexin 1-receptor-contain­ ing neurons in the dorsal raphe nucleus. Regul. Pept. 126, 35-42. Winn, P., Tarbuck, A., Dunnett, S.B., 1984. Ibotenic acid lesions of the lateral hypothalamus: comparison with the electrolytic lesion syndrome. Neu­ roscience 12, 225-240. Winsky-Sommerer, R., Yamanaka, A., Diano, S., Borok, E, Roberts, AJ„ Sakurai, T., Kilduff, T.S., Horvath, T.L, de Lecea, L, 2004. Interaction between the cortico­ tropin-releasing factor system and hypocretins (orexins): a novel circuit med­ iating stress response. J. Neurosci. 24,11439-11448. Wolf, G., 1998. Orexins: a newly discovered family of hypothalamic regulators of food intake. Nutr. Rev. 56 ,172-173 . Wolters,J.G., ten Donkelaar, H.J., Steinbusch, H.W., Verhofstad, A A , 1985. Distribu­ tion of serotonin in the brain stem and spinal cord o f the lizard Varanus exanthematicus: an immunohistochemical study. Neuroscience 14,169-193. Wu, M., Zaborszky, L, Hajszan, T., van den Pol, AN., Alreja, M , 2004. Hypocretin/ orexin innervation and excitation of identified septohippocampal cholinergic neurons. J. Neurosci. 24, 3527-3536. 62 2. ESTUDIOS QUIMIOARQUITECTONICOS EN EL ENCEFALO ADULTO 34 L Dommguez et aL/Joumal of Chemical Neuroanatomy 39 (2010) 20 -34 Wu, M„ Zhan& Z., Leranth, C, Xu, C, van den Pol, AN., Alreja, M., 2002. Hypocretin increases impulse flow in the septohippocampal GABAergic pathway: implica­ tions for arousal via a mechanism of hippocampal disinhibition, j. Neurosci. 22, 7754-7765. Yamamoto, T., Suzuki, H., Uemura, H., Yamamoto, K., Kikuyama, S., 2004. Localiza­ tion o f orexin-A-like immunoreactivity in prolactin cells in the bullfrog (Rana catesbeiana) pituitary. Gen. Comp. Endocrinol. 135, 186-192. Yamanaka, A , Muraki, Y., Ichiki, K., Tsujino, N., Kilduff, T.S., Goto, K., Sakurai, T., 2006. Orexin neurons are directly and indirectly regulated by catecholamines in a complex manner, j. Neurophysiol. 96, 284-298. Yamanaka, A , Muraki, Y., Tsujino, N., Goto, K., Sakurai, T., 2003. Regulation of orexin neurons by the monoaminergic and cholinergic systems. Biochem. Biophys. Res. Commun. 303 ,120 -129 . Yamanaka, A, Sakurai, T., Katsumoto, T., Yanagisawa, M., Goto, K., 1999. Chronic intracerebroventricular administration of orexin-A to rats increases food intake in daytime, but has no effect on body w eight Brain Res. 849, 248-252. Yamanaka, A , Tsujino, N., Funahashi, H., Honda, K., Guan, J.L, Wang, Q.P., Tominaga, M., Goto, K., Shioda, S., Sakurai, T„ 2002. Orexins activate hista­ minergic neurons via the orexin 2 receptor. Biochem. Biophys. Res. Commun. 290, 1237-1245. Yan, J A , Ce, L, Huang, W., Song, B., Chen, X.W., Yu, Z.P., 2008. Orexin affects dorsal root ganglion neurons: a mechanism for regulating the spinal nociceptive processing. Physiol. Res. 57, 797-800. Yasuda, T., Masaki, T., Kakuma, T., Hara, M., Nawata, T., Katsuragi, I., Yoshimatsu, H., 2005. Dual regulatory effects o f orexins on sympathetic nerve activity inner­ vating brown adipose tissue in rats. Endocrinology 146,2744-2748. Yoshida, K., McCormack, S., Espana, RA, Crocker, A, Scammell, T.E, 2006. Afferents to the orexin neurons o f the rat brain, j. Comp. Neurol. 494, 845-861. Zhang, j., Luo, P., 2002. Orexin B immunoreactive fibers and terminals innervate the sensory and motor neurons of jaw-elevator muscles in the rat. Synapse 4 4 ,106 - 110. Zhang, J.H., Sampogna, S., Morales, F.R., Chase, M.H., 2001. Orexin (hypocretin)-like immunoreactivity in the cat hypothalamus: a light and electron microscopic study. Sleep 24, 67-76. Zhang, J.H., Sampogna, S., Morales, F.R., Chase, M.H., 2004. Distribution of hypo­ cretin (orexin) immunoreactivity in the feline pons and medulla. Brain Res. 995, 205-217. Zhang, W., Zhang, N., Sakurai, T., Kuwaki, T., 2009. Orexin neurons in the hypotha­ lamus mediate cardiorespiratory responses induced by disinhibition of the amygdala and bed nucleus of the stria terminalis. Brain Res. 1262, 25-37. 5/0 lOG' 63 64 3. Estudios genoarquitectonicos en el encéfalo en desarrollo y adulto Sonic hedgehog expression during Xenopus laevis forebrain development. Brain Research 1347:19-32. Ontogenetic distribution of the transcription factor Nkx2.2 in the developing forebrain of Xenopus laevis. Frontiers in Neuroanatomy 5:11. L Not for reproduction, distribution or commercial use. J Brain Research This article appeared In a journal published by Elsevier, The attached copy Is furnished to the author for Internal non-commercial research and education use, Including for Instruction at the au thors Institution and sharing with colleagues. Other uses , Including reproduction and distribution, or selling or licensing copies, or posting to personal, Institutional or third party w ebsites are prohibited. In m ost c a s e s au thors are permitted to post their version of the article (e.g. In Word or Tex form) to their personal website or Institutional repository. Authors requiring further Information regarding Elsevier’s archiving and m anuscript policies are encouraged to visit: http://www.elsevler.com/copyrlght 67 http://www.elsevler.com/copyrlght 3. ESTUDIOS GENOARQUITECTONICOS EN EL ENCEFALO EN DESARROLLO Y ADULTO I R A I N R E S E A R C H 1 3 4 7 ( 2 0 1 0 ) 1 9 - 3 2 a v a i l a b l e a t w w w . s c i e n c e d i r e c t . c o i *•#' ScienceDirect w w w . e l s e v i e r . c o m / l o c a t e / b r a i n r e s BRAIN RESEARCH Research Report Sonic hedgehog expression during Xenopus laevis forebrain development L. Dommguez, A. Gonzalez, N. Moreno* Department of Cell Biology, Faculty of Biology, University Complutense of Madrid, 28040 Madrid, Spain A R T I C L E I N F O Keywords. Prosencephalon Hypothalamus In situ hybridization Evolution Forebrain patterning Thalamus A B S T R A C T Article history; Accepted 2 June 2010 Available online 9 June 2010 We have analyzed the developing expression pattern of x-Shh in the Xenopus forebrain, interpreting the results within the framework of the neuromeric model to assess evolutionary trends and clues. To achieve this goal, we have characterized phenotypically the developing x-Shh expressing forebrain subdivisions and neurons by means of the combination of in situ hybridization for x-Shh and immunohistochemistry for the detection of forebrain essential regulators and markers, such as the homeodomain transcription factors Islet 1, Orthopedia, NKX2.1 and NKX2.2 and tyrosine hydroxylase. Substantial evidence was found for x-Shh expression in the telencephalic commissural preoptic area and this is strongly correlated with the presence of a pallidum and/or a basal telencephalic cholinergic system. In the diencephalon, x-Shh was demonstrated in the zona limitans intrathalam ica and the x-Shh expressing cells were extended into the prethalamus. Throughout development and in the adult hypothalamic x-Shh expression was strong in basal regions but, in addition, in the alar suprachiasmatic region. The findings obtained in the forebrain of Xenopus revealed a largely conserved pattern of Shh expression among tetrapods. However, interesting differences were also noted that could be related to evolutive changes in forebrain organization. © 2010 Elsevier B.V. All rights reserved. 1. Introduction T he forebrain of vertebrates contains the m ost com plex areas of th e brain. It includes th e te lencephalon and th e d ienceph­ alon, w hich develop from the em bryonic prosencephalic vesicle. The m orphology and connectivity betw een forebrain areas vary considerably am ong different vertebrate species (Battler and Hodos, 1996; N ieuw enhuys e t al., 1998). However, m any com m on features are conserved across species regard­ ing th e forebrain developm enta l p rocesses th a t include complex pattern ing , m orphogenetic, m igration and wiring events. Thus, it h as been dem onstrated th a t m any genetic expressions th a t control all aspects of forebrain developm ent are largely shared am ong vertebrates (Rubenstein e t al., 1994; S triedter, 1997; Puelles e t al., 2000; Bachy e t al., 2002; W ullim ann and M ueller, 2004; M urakam i e t al., 2005). In * Corresponding author. Fax; 4-34 913944981. E-mail address: nerea©bio.ucm.e (N. Moreno). Abbreviations: BM, mammillary band; MeA, medial amygdala; oc, optic chiasm; OT, optic tectum; PI, diencephalic prosomere 1; P2, diencephalic prosomere 2; P3, diencephalic prosomere 3; Pa, pallium; PA, pallidum; PO, preoptic area; POC, preoptic commissural area/ commissural septo-preoptic area; PV, paraventricular nucleus; RC, retrochiasmatic nucleus; SC, suprachiasmatic nucleus; SPa, subpallium; SPV, supraoptoparaventricular area; TP, posterior tubercle; Tub, tuberal area; VM, ventromedial nucleus; ZI, zona incerta; Zli, zona limitans intrathalamica 0006-8993/$ - see front m atter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2010.06.007 68 http://www.sciencedirect.coi http://www.elsevier.com/locate/brainres 20 EN DESARROLLO Y ADULTO R A I N R E S E A R C H 1 3 4 7 ( 2 0 1 0 ) 1 9 - 3 2 particular, th e early forebrain develops u n d e r th e in fluence of signaling cen te rs th a t secre te m orphogen m olecu les am ong w hich is th e p ro te in encoded by th e gene Sonic hedgehog (Shh). Shh is a pow erfu l m orphogen (Ingham and Placzek, 2006), w h ich con tro ls p rogen ito r proliferation , regional p a t­ te rn ing an d cell fa te in th e developing b ra in (for review see Fuccillo e t al., 2006). It is secre ted by th e v en tra l m id line of th e n eu ra l p la te an d tube, th e underly ing n o tocho rd and th e p rechordal p la te (non -neu ra l Shh), tw o m eso d erm a l deriva­ tives w ith crucial inductive p roperties (Ericson e t al., 1997; G unhaga e t al., 2000; In g h am an d M cM ahon, 2001). T he n eu ra l Shh is essen tia l for th e coord ination of g row th and p a tte rn at d ifferen t b ra in levels, such as th e sp inal cord, th e m id b ra in - h indb ra in ju n c tio n an d cerebellum (Blaess e t al., 2006, 2008). In th e forebrain , Shh is secre ted from th e ven tra l m idline signaling cen te r in th e te lencepha lon , w h ereas th e develop­ m en t of th e d ien cep h a lo n is con tro lled by Shh p roduced in th e basal or floor p la te an d in th e zona lim itan s in tra th a lam ica (Zli), a secondary o rgan izer located tran sversa lly be tw een th e th a la m u s an d p re th a la m u s (Kiecker an d L um sden, 2004; Vieira e t al., 2005; Zeltser, 2005). In particu lar, Shh secreted from th e Zli specifies th e p re th a lam u s (H ashim oto-Torii e t al., 2003; K iecker an d L um sden , 2004; Vieira e t al., 2005; H irata e t al., 2006; Scholpp e t al., 2006; G uinazu e t al., 2007) and p rom o tes grow th an d d ifferen tia tion of specific subdivisions of th e th a lam u s (Szabo e t al., 2009a). In addition , Shh show s an in trigu ing an d dynam ic p a tte rn of expression in th e h y p o th a ­ lam ic d om ain an d in regions strateg ically s itu a ted to influence th e fo rm ation o f th e d iencepha lic -te lencepha lic boundary (Szabo e t al., 2009b). M ost of th e d a ta abou t Shh expression in th e forebrain w ere ob ta ined in am n io tes , p rim arily m ouse and chick. However, fragm en tary d a ta on Shh expression repo rted for an am n io tes (fish an d am phib ians) u n rave led im p o rtan t differences, m a in ­ ly a t p rosencephalic levels, such as Shh expression in th e alar hy p o th a lam u s, lack o f expression in th e te len cep h a lo n of the lam prey or Shh exp ression in th e zebrafish Zli in absence of th e basa l p la te inductive signal (Barth an d W ilson, 1995; Osorio e t al., 2005; Scholpp e t al., 2006; v an d en Akker e t al., 2008). T he d ev e lopm en t o f th e forebrain in Xenopus h a s recently gained im portance in evolutive stu d ies b ecause th is an u ran am ph ib ian (anam nio te te trapod) show s m an y fea tu res of genetic expression p a tte rn s th a t are readily com parable to th o se in am n io tes (Bachy e t al., 2001, 2002; Brox e t al., 2003, 2004; M oreno e t al., 2004, 2008a,b; v an den Akker e t al., 2008). T hese fea tu res are particu larly h igh ligh ted w h en th e analysis of th e gene expressions is m ad e w ith in th e fram ew ork o f th e p rosom eric m odel (Puelles an d R ubenstein , 2003). Strikingly, in Xenopus d a ta ab o u t th e Shh (x-Shh) expression in th e fo rebrain are n o t available. T he m a in goal o f th is s tu d y w as to provide detailed in fo rm ation on th e localization of x-Shh expression in th e fo rebrain of Xenopus th ro u g h o u t developm ent, analyzing the resu lts in te rm s o f p rosom eric o rgan iza tion to assess fu rth er the d ifferences an d sim ilarities w ith am n io tes . To reach th is goal, w e have charac te rized pheno typ ica lly th e developing x- Shh exp ressing forebrain subdiv isions and n eu ro n s by m ean s of th e com bination o f x-Shh expression w ith th e forebrain e ssen tia l regulators an d m arkers NKX2.1, NKX2.2, ISLETl (ISLl), O rtophedia (OTP) and ty rosine hydroxylase (TH). The resu lts un ravel for th e first tim e in an an am n io te te trap o d th e precise neu ro an a to m ica l d is tribu tion of Shh expression, w hich is strongly usefu l to com pare, in evolutive te rm s, sim ilarities an d differences in th e prosencephalic developing p a tte rn in g am ong am n io tes an d an am nio tes . 2 . Results T he d is tribu tion of th e s ta in in g p a tte rn ob ta ined for x-Shh w as carefully analyzed in th e p ro sencepha lon of Xenopus laeuis from em bryonic s tages th ro u g h th e adult. In th e follow ing sections, w e first describe th e spatio -tem poral sequence of x- Shh expression du rin g forebrain developm ent in series of single s ta ined m ate ria l (Figs. 1-4). Subsequently , th e p a tte rn of x-Shh expression is analyzed in com bination w ith p ro sen ce­ phalic m arkers p rev iously u sed in the analysis of th e Xenopus forebrain deve lopm en t (Figs. 5 an d 6). The n om encla tu re used in th is study follow s th a t p roposed for th e an u ran forebrain, considering th e n ew d iv isions iden tified on th e basis of trac t tracing, im m u n o h is to ch em is try and gene expression p a tte rn s (Puelles e t al., 1996; M arin e t al., 1998; M ilan and Puelles, 2000; G onzalez e t al., 2002a,b; Brox e t al., 2003; M oreno e t al., 2004, 2008a,b; M orona an d G onzalez, 2008). 2 .1 . x-Shh ex p ressio n in th e develop in g prosencephalon In Xenopus, x-Shh w as nev er expressed in th e evaginated te lencephalon , n e ith e r in palliai n o r in subpallial areas (Figs. 1 and 2a), b u t it w as exp ressed in non-evag inated te lencephalic te rrito ries from early developm en ta l stages (Figs. 2b and c) th rough th e ad u lt (Figs. 3a, c, g, 4a, b, and 1). In general, th e x- 8 t1 3 St 14 St 20 hva s t2 4 s t2 8 Zli e % _ s t3 1 Zli f ^ s t 33-34 Zli 1st 35-36 Zli 1 41 h _ Fig. 1 - Photom icrographs o f in toto v iew s illustrating the x-Shh exp ression in early em bryonic stages (from st 13 to st 35-36). Scale bars= 500pm (a,b) and 100pm (c-h). 69 EN DESARROLLO Y ADULTO R A I N R E S E A R C H 1 3 4 7 ( 2 0 1 0 ) 1 9 - 3 2 21 Pa SPa P2 P3 P O C ^ . 4' SPV Zli P2 P3 SPV % o - , Z li, " - r P3 SC P3 P3 P3 TP SC Tub BM Tub BM Tub Tub _ a Zli TP BM Fig. 2 - Photom icrographs o f transverse sectio n s through the develop ing Xenopus forebrain illustrating the x-Shh exp ression in em bryonic sta g es (st38-41). No exp ression is detected in m ost rostral portion o f th e telencephalon (a), although a w eak exp ression starts in th e subpallial POC (b), w h ich is continuous caudally in the forebrain w ith in the ventricular zon e (b-i). At thalam ic lev e ls , x-Shh exp ress ion is restricted to th e Zli in the P2-P3 boundary (b-g). Of note, x-Shh p ositive cells w ere present in th e anterior tuberal region (f-h) w h ereas th ey w ere n o t detected at posterior tuberal lev e ls (see asterisk in g-i). The schem atic draw ings o f sagittal sec tion s illustrate the lev e ls o f the section s a-i, the ex ten t o f th e x-Shh exp ression and the topographic organization o f the im plicated areas. Scale bars = 100 pm . Shh ex p ressin g cells occupied th e v en tr icu la r zone (vz), w ith excep tions in som e areas w h ere th e x-Shh ex p ressin g cells re a c h e d su b v e n tric u la r o r m a n tle zones (svz, m z). From em bryon ic s tages th e m o s t an te r io r x-Shh ex p ress io n de tec ted w as found in th e new ly n a m e d co m m issu ra l sep to -p reop tic a rea (POC) lo ca ted a t th e b ase of th e se p tu m an d in close re la tio n to th e an te r io r co m m issu re (Figs. 2b, 3c, g, 4a, an d a'). In p re m e ta m o rp h ic s tag es a t th is level, s c a t te re d x-Shh exp ressing cells w ere also observed laterally , close to th e la te ra l fo reb ra in b u n d le , in th e reg ion described as pa llidum an d /o r en to p e d u n c u la r a rea (M arin e t al., 1998) (arrow heads in Figs. 4a an d b). A d jacen t to th e POC, x-Shh expression w as ex ten d e d in to th e vz o f th e p reoptic a rea (PO; Figs. 2c, 3a, a ', g, 4b, an d 1). T hus, th e x-Shh exp ression in th e POC and th e PO fo rm ed a c o n tin u o u s ven tricu la r te rrito ry in w h ich th e POC co n stitu ted th e do rsa l tip of th e PO (Figs. 3g an d h). 70 22 EN DESARROLLO Y ADULTO R A I N R E S E A R C H 1 3 4 7 ( 2 0 1 0 ) 1 9 - 3 2 Tub Zli ' P3 SPV “ J . - • , \ OC I i P O in to to la te ra l v ie w to d o r s a l v ie w r ' T P r' SPV P 3 Æ f - d U r Z li- P 3 Tub RC SC SPV PO Fig. 3 - Photom icrographs o f sagittal (a, a', f, g) and horizontal (c, d) sections through the develop ing Xenopus forebrain and in toto lateral and dorsal v iew s illustrating the x-Shh exp ression in prem etam orphic stages (42-50). x-Shh expression is a lm ost continuous from anterior non-evaginated telencephalic areas to the posterior brain covering the ventral ventricular zon e (a, a ). In toto prem etam orphic brains are sh ow n (b, c) before sectioned in horizontal (c, d) and sagittal (f, g) p lanes follow ing the indicated levels. N ote that in th e hypothalam us, x-Shh is observed in the rostral tuberal area, w h ereas the caudal regions lack exp ression (asterisk in f). The schem atic draw ing illustrates, in a sagittal section , the ex ten t o f the x-Shh exp ression . Scale bars = 100 p m (b, c, e-g) and 50 pm (a, a', d). At d iencephalic levels, th e x-Shh ex p ressio n in th e zona lim itan s in tra th a lam ica (Zli) w as s trong th ro u g h th e early d ev e lopm en t (Figs. 2c-g, 3a, a ', f, and 4c-h), b u t d im in ished gradually du ring la te d ev e lo p m en t and in th e ad u lt could n o t be detec ted . T hus, th ro u g h th e ro s tro -cauda l th a lam ic levels a sign ifican t x-Shh exp ression could be observed a t th e b o u n d ­ ary b e tw een th e p ro som eres 2 and 3 (P2 an d P3 o r th a lam u s an d p re th a lam u s, respectively). At an te rio r levels th is ex p res­ sion w as res tric ted to th is b o u n d ary (Figs. 2b, 3a, f, an d 4c-g), w h ereas it ex ten d ed ven tra lly in poste rio r levels, reach ing P3 te rrito ries and fo rm ing an a lm o st co n tin u o u s b an d w ith th e hy po tha lam ic areas (Figs. 2e, f, 3b, f, an d g). T hus, du ring th e p rem etam o rp h o sis , w h en th e b ra in m orpho logy s ta r ts to resem b le th a t of th e adult, x-Shh expression w as observed in P3, ex tend ing caudally in to th e zona incerta (ZI; Figs. 4 f an d g). This x-Shh expression co n tin u ed caudally in th e p oste rio r tubercle (TP; Figs. 3c, 4h, and i) and , m ore caudally, in to th e b ra in s tem (Fig. 4k). W ith in th e h y p o th a lam u s , th e m o s t an te r io r x-Shh ex ­ p ressio n w as fo und in th e su p rach ia sm a tic region (Figs. 2d, e, 3a, g, an d 4c-e). It fo rm ed a co n tin u o u s b an d above th e op tic c h ia s m th a t w as sp ec ia lly e v id e n t in s a g itta l s e c tio n s (Figs. 3a, a ', b, an d g). A t th is region, x-Shh ex p ressio n w as alw ays observed in th e vz o f th e su p rach ia sm a tic (SC) an d re tro ch ia sm a tic (RC) n uc le i b u t, in add ition , x-Shh ex p ress in g cells in th e svz could be d e tec ted la te in d ev e lo p m en t an d th ro u g h th e a d u lt (Fig. 4m). T his ex p ressio n w as co n tin u o u s w ith th a t d e tec ted in th e tu b e ra l reg ion (Tub; Figs. 2 f-h , 3a ', f, g, 4g-i, an d n). H ow ever, from th e early d ev e lo p m en ta l stag es x-Shh ex p ress io n w as n o t d e tec ted in th e m o s t p o s te rio r reg ion of th e tu b e ra l h y p o th a la m u s (Figs. 2i and j). N otably , a sim ila r s i tu a tio n w as found in th e m am m illa ry b an d , in 71 J. J_ / 0 1 C ' X X i-i\^ X Wi > X\_/V.̂ LJ J_jX 1 J_(X_( i-il. ■% V̂X_/X X XJ_(W EN DESARROLLO Y ADULTO - R A I N R E S E A R C H 1 3 4 7 ( 2 0 1 0 ) 1 9 - 3 2 23 'Zlf POC \ P2 P3 U è oc _ b PI pY ' ZjU P3 ( . P3 .S P V PO PI P2 — c 1 "' — VM P3 SC e oc f D 3 1- t P1 P2 a i ZI _ g BM Tub OT PI ÿB M - - 0 J PI OT OT P2 Tub \(TP jB M IffLb _ j t TP BM Tub ___ k à PO sc /Tub m n Fig. 4 - Photomicrographs o f transverse sections through the developing Xenopus forebrain illustrating the x-Shh exp ression in late prem etam orphic larvae (a-k) and in the adult (1-n). The m ost rostral x-Shh exp ression w a s found in a sm all ventricular territory in the com m issural preoptic area (a) that ex ten d s caudally along the ventricle in the preoptic area (b), w here som e scattered cells w ere located laterally (arrowheads). The exp ression continues caudally in the zona lim itan s intrathalam ica and suprachiasm atic region (c-e) and ex ten d s into the prethalam us, m am m illary band and tuberal hypothalam us (f-h). In the caudal diencephalon, x-Shh expression w as located in the posterior tubercle and prerubral tegm ental region (i, j) and continues caudally into the ventral m esencephalon (k). x-Shh exp ression in the adult w a s sh o w n in th e ventricular zone o f the preoptic area (1), the suprachiasm atic region (m) and the tuberal hypothalam us (n). Scale bars = 100 p m (a-k, n) and 50 p m (a', f', 1, m). w h ich x-Shh ex p ressio n w as lack ing a t p o ste rio r levels (BM; Fig-4j). 2.2. x-Shh expression in relation to prosencephalic markers From early p rem etam o rp h ic stages, th e double sta in in g for x- Shh/NKX2.1 clearly confirm ed th e position of x-Shh expressing cells in th e POC, defined by th e expression of x-Shh/NKX2.1 in th e vz, w h ereas th e svz on ly show ed NKX2.1 expression (Fig. 5a). In th e case o f th e PO, th e x-Shh expression occupied th e vz of th e reg ion in w hich TH im m unoreactive cells w ere located in th e svz (Figs. 5b, b ', an d c). In addition , the double lab e lin g w ith th e t r a n sc r ip tio n fac to r OTP allow ed th e iden tifica tion o f th e PO lim its b ecau se OTP w as n o t expressed in th e PO, w h ereas it w as strong ly exp ressed m ore caudally in te rrito ries clearly d is tin c t from th o se th a t expressed x-Shh (Figs. 5a-c). 72 24 EN DESARROLLO Y ADULTO R A I N R E S E A R C H 1 3 4 7 ( 2 0 1 0 ) 1 9 - 3 2 X'ShhnH PO m m m /7/7/ISLix-shhrrH T / N T H Fig. 5 - Photom icrographs of doubly labeled transverse section s through th e Xenopus developing forebrain sh ow in g the x-Shh exp ression at prem etam orphic stages in com bination w ith NKX2.1 (a, e, f, g, j), TH (b-d, k-m ) and ISLETl (n). At preoptic regions, the double x-Shh/NKX2.1 labeling confirm ed the x-Shh telencephalic exp ress ion in the preoptic com m issural area (a), w h ereas the double x-Shh/TH staining d elin eates th e preoptic area (b, c). Sim ilarly, x-Shh/TH (d) and x-Shh/NKX2.1 (g) allow ed the identification o f th e SC, rich in TH and NKX2.1 exp ressin g cells (see arrow head in g). In the d iencephalon, the x-Shh exp ressin g cells in the Zli extend into the prethalam ic (P3) territory, sh ow in g NKX2.1 exp ression (arrowheads in e and f). In posterior diencephalic levels, the x-Shh detected w a s situated in P3 by the double x-Shh/NKX2.1 (h-j), and specifically in the zona incerta, according to the TH expressing cells detected (k). The double labeling for x-Shh/TH (1, m) and x-Shh/ISLETl (n) allow ed the identification of the m am m illary and tuberal boundaries. Scale b ars=100 p m (a-n) and 50 p m (b'). 73 EN DESARROLLO Y ADULTO R A I N R E S E A R C H 1 3 4 7 ( 2 0 1 0 ) 1 9 - 3 2 25 t X;j3hn/N^.X2.2 ^ ' P3 x-StfhIQJP X 'S h h /N K X l : Fig. 6 - Photomicrographs of transverse (a-e) and horizontal (f) sections through the Xenopus developing forebrain showing x-Shh expression in combination to OTP (a-c) and NKX2.2 (d-f). The exclusive expressions of x-Shh, rich in preoptic and preoptic commissural areas, with OTP, rich in the supraoptoparaventricular area, allowed the identification of the boundary (a-c; see asterisk in c). The combination with NKX2.2 confirms the x-Shh expression in the zona limitans intrathalamica (d, e), and the x-Shh and NKX2.2 expression in the suprachiasmatic area (d, f). Scale bars=100 pm. The precise localization in th e d iencephalon of th e areas exp ressing x-Shh w as confirm ed by th e s im u ltan eo u s de tec ­ tion o f TH (Figs. 5k an d 1), NKX2.1 (Figs. 5e-g) an d NKX2.2 (Figs. 6d-f). The double s ta in in g for x-Shh/NKX2.2 allow ed the iden tifica tion of th e Zli b o u n d ary (Figs. 6d and e), and also th e corroboration o f x-Shh exp ressing cells in P3 a reas (Fig. 6e). The la tte r w as also h igh ligh ted by th e x-Shh/NKX2.1 com bination (Figs. 5e-g). In add ition , th e x-Shh expression in th e ZI w as confirm ed by th e double s ta in in g w ith TH (Figs. 5k-m ) and NKX2.1 (Fig. 5j). T he ex ten t of th e x-Shh exp ression in th e h y p o th a lam u s w as analyzed by th e com b in a tio n w ith TH (Fig. 5d) and th e tran sc rip tio n factors NKX2.1 (Figs. 5g an d j), NKX2.2 (Fig. 5d,e) an d ISLl (Fig. 5n). At SC levels, th e x-Shh expression w as clearly d e tec ted in th e a rea w here TH (Fig. 5d), NKX2.1 (see arrow head in Fig. 5g) and NKX2.2 exp ressing cells w ere observed (Figs. 6d a n d e). The x-Shh exp ression in th e m am m illa ry region w as corroborated by th e double s ta in in g x-Shh/TH (Figs. 51 and m). T he x-Shh expression con tin u ed in to th e tubera l area, w here NKX2.1 and ISLl cells w ere loca ted in th e m an tle zone (Figs. 5j a n d n). 3. Discussion Sonic hedgehog is kno w n to be involved in m ultip le actions du ring b ra in developm ent, such as design ing cell fate, cellu lar p ro liferation , d e te rm in a tio n o f specific regions in th e devel­ op ing bra in and d o rso -ven tra l p a tte rn in g o f th e CNS (for rev iew M arti an d B ovolenta, 2002; Fuccillo e t al., 2006). Here w e h av e tho rough ly analyzed th e develop ing expression p a tte rn of x-Shh in th e Xenopus forebrain , in te rp re tin g th e resu lts w ith in th e fram ew ork of th e neu rom eric m odel to assess evo lu tionary tren d s an d functionally clues. To ach ieve th is goal, w e have ch aracterized pheno typ ica lly th e develop ing x- Shh expressing forebrain subdiv isions and n e u ro n s by m ean s o f th e com bination o f in s itu hybrid ization fo r x-Shh and im m u n o h is to ch em is try fo r th e detec tion of th e fo rebrain essen tia l regulators an d m ark ers NKX2.1, NKX2.2, ISLl, OTP an d TH. W e op ted for th e tran sc rip tio n fac to r NKX2.1 b ecau se it is an e ssen tia l regu la to r of th e m ed ia l ganglionic em in en tia (MCE; Sussel e t al., 1999; van den A kker e t al., 2008) an d ac ts in th e specification of th is te lencephalic subdivision th a t gives rise to th e pallidum (PA). In add ition , NKX2.1 expression is h ig h in th e secondary p ro sencepha lon an d served to iden tify th e preoptic, ch iasm atic an d tubera l te rrito ries during dev e lo p m en t (Gon- onzalez e t al., 2002a,b; M oreno e t al., 2008a). M oreover, NKX2.1 is a m ark e r o f th e basa l h y p o th a lam u s in all v e rteb ra te s and m arks th e a lar h y p o th a lam u s in an am n io tes (Rohr e t al., 2001; G onzalez e t al., 2002a,b; M oreno e t al., 2008a; van d en Akker e t al., 2008). W e also u sed ISLl since it h a s been described as an e ssen tia l m ark e r o f several subdiv isions in th e develop ing forebrain o f Xenopus, such as th e p re th a lam u s, th e ch iasm atic an d preo tic region an d th e tu b e ra l an d m am m illa ry p a r ts o f th e basa l h y p o tha lam us. In particu lar, it show s a sim ilar d is trib u ­ tion as th a t o f th e m em b ers o f th e Dix gene fam ily (M oreno e t al., 2008a). To clarify th e p resen ce o f x-Shh in th e p reo p tic area, w e com bined x-Shh exp ression w ith OTP, due to th e fac t th a t it specifically m arks th e sup rao p to p arav en tricu la r reg ion in th e d ifferen t verteb ra tes s tu d ied (Bardet e t al., 2008). W e u sed th e tran sc rip tio n factor NKX2.2 because it h as been described to 74 3. ESTUDIOS GENOARQUITECTONICOS EN EL ENCEFALO EN DESARROLLO Y ADULTO 26 I R A I N R E S E A R C H 1 3 4 7 ( 2 0 1 0 ) 1 9 - 3 2 delineate the alar/basal boundary in m ouse (Shimamura et al., 1995) and consequently served to discriminate the Zli bound­ ary. Moreover, NKX2.2 is strongly expressed in the dienceph­ alon and allowed us the identification of the prethalamus (Kitamura et al., 1997). Finally, we have used TH because in amphibians important dopaminergic populations can be detected in the preoptic area, the suprachiasmatic nucleus, the nucleus of the ZI and in the TP (Gonzalez et al., 1993,1994; Milan and Puelles, 2000; Sm eets and Gonzalez, 2000). 3.1. Shh prosencephalic expression pattern: conservative traits The Shh expression pattern in the brain and its functional significance in differentiating neurons have been analyzed in several amniotes (Vieira et al., 2005; Bardet et al., 2006; Vieira and Martinez, 2006; Garcla-Lopez et al., 2008). Data for anamniotes have also been provided in the last years (Osorio et al., 2005; Menuet et al., 2007; van den Akker et al., 2008). The detailed territorial expression pattern of x-Shh found in the forebrain of Xenopus (anamniote tetrapod) will be discussed in a comparative perspective attending to the main prosence­ phalic regions, only clearly defined in terms of genetic markers for the case of amniotes (Puelles et al., 2000; Puelles and Rubenstein, 2003; Flames et al., 2007; Garcla-Lopez et al., 2008; Abelian and Medina, 2009). 3.1.1. Telencephalon From anterior to posterior levels, the first x-Shh expression detected in Xenopus was localized at the middle line in the telencephalon non-evaginated, just anterior to the preoptic recess, within the vz of the area called POC (Moreno et al., 2008a). In addition, adjacent to the POC, x-Shh expression extends in the vz of the PO and it is also seen in a subset of immigrant mantle layer cells, in the pallidum and/or anterior entopeduncular area (Marin et al., 1998; Gonzalez et al., 2002a, b). The latter cells, as seen as development proceeds, are most likely related to the POC (present results). Previous studies in Xenopus, based on the localization of NKX2.1, have shown that the POC and the ventral portion of the septum (Sv) also constitute a continuous structure in the ventricular lining (Moreno et al., 2008a). According to our present results, x-Shh is not expressed in septal regions. Taking together the expres­ sion of NKX2.1 and x-Shh in this region, it can be considered that within the continuum of PO, POC and Sv two regions exist with distinct specifications, a rostral region x-Shh-/NKX2.1+ (septal territory) and a caudal region x-Shh-h/NKX2.1-i- (preoptic territory). This difference m ost likely is reflected in distinct neuronal specifications. Thus, for example, in the preoptic region, and not in the septal region of Xenopus many cells have been described expressing substances such as enkephalin (Marin et al., 1998), neuropeptide FF (Crespo et al., 2003), orexins (Lopez et al., 2009), NOS (Marin et al., 1998), calretinin (Morona and Gonzalez, 2009) and TH (Gonzalez and Smeets, 1991; Milan and Puelles, 2000). In addition, x-Shh is present in the PO, w hose boundaries were corroborated by the lack of colocalization with the transcription factor OTP that is strongly expressed in the adjacent paraventricular nucleus (Bardet et al., 2008) and does not contain x-Shh+ cells (present results). Therefore, in Xenopus, like in other vertebrates, the PO is defined by the Shh expression (Flames et al., 2007; Menuet et al., 2007; Garda-Lôpez et al., 2008). In the chick forebrain, at least two subdivisions have been described within the preoptic region, a basal area (POB) and a commissural area (POC). Both of them possess c-Shh expres­ sion in the ventricular layer and, exclusively in the POC, scattered cells extend into the mantle layer, likely related to immigrant processes (Abelian and Medina, 2009). This situa­ tion in the chick largely agrees with the results described in the Xenopus preoptic region. The preoptic region is anatomically defined in the m ouse as the region im mediately anterior to the optic recess, at the limit between the telencephalon and the diencephalon, and contains at least two distinct progenitor domains (pPOAl and pP0A2; Flames et al., 2007). It is distinguished from the adjacent subpallial areas by the simultaneous expression of NKX2.1, NKX2.2, and Shh and the lack of detectable levels of Gsh2, Lhx6, Lhx7, or 0Ug2 expression (Flames et al., 2007). In addition, only the POC show s ventricular zone expression of Shh (Garcla-Lopez et al., 2008), in agreement with the present results in Xenopus. It was proposed in the mouse that POC derivatives include at least a subpopulation of Shh expressing cells of the medial amygdala (MeA; Garcla-Lopez et al., 2008), and a similar conclusion has been reached in birds (Abelian and Medina, 2009). In Xenopus, the MeA is defined as the main secondary vomeronasal center in the anuran brain (Moreno and Gonzalez, 2003) and is also a complex area with multiple cell populations (Marin et al., 1998; Brox et al., 2003, 2004; Moreno and Gonzalez, 2003, 2004, 2005, 2006; Moreno et al., 2004, 2008c; Endepols et al., 2006) likely originated from adjacent territories. However, the POC cells that express x- Shh do not belong to the MeA but lie more ventrally in the pallidum (for review see Moreno and Gonzalez, 2006, 2007). Noteworthy, in mam mals POC derivatives appear to include cholinergic cell groups of the basal telencephalon (Garcla- Lopez et al., 2008) and, in the case of Xenopus, the x-Shh migrated cells from the POC largely resemble the population of cholinergic cells found in the sam e location (Marin et al., 1997; Sanchez-Camacho et al., 2006). Therefore, it seem s that the Xenopus amygdala lacks the amniote subdivision derived from the POC, but this area could be the source for the basal cholinergic system present in amphibians. The comparative analysis of the telencephalic Shh expres­ sion in anam niotes reveals very interesting differences. In the lamprey, there is not Shh signaling at the ventral midline (Osorio et al., 2005), suggesting that Shh may be responsible for many of the differences found in the subpallium of agnathans, such as the lack of the pallidum (for review see Osorio and Rétaux, 2008). In the zebrafish (teleost) the double detection of Shh/Dlx2a expression shows that the Shh expression can be localized in the basal telencephalon/preoptic area at late developmental stages (Scholpp et al., 2006). In addition, in the teleost Astyanax mexicanus (closely related to the zebrafish), which possesses several populations of blind cave-living fish that have independently evolved from surface river-dwelling populations in a relatively short period of time, the Shh signal is expanded in the cave fish and includes the basal telencephalon (Menuet et al., 2007). This Shh expression has been related to the fact that a specific GABAergic cell population bom in the 75 3. ESTUDIOS GENOARQUITECTONICOS EN EL ENCEFALO EN DESARROLLO Y ADULTO R A I N R E S E A R C H 1 3 4 7 ( 2 0 1 0 ) 1 9 - 3 2 27 ventral telencephalon was greatly enhanced in developing cavefish embryos (Menuet et al., 2007). No data are available for other fish groups. Our results in Xenopus have shown that in amphibians the recently identified POC region expresses Shh, as in tetrapods (Garcia-Lopez et al., 2008; Abelian and Medina, 2009) and this situation might also be present in teleosts. Interestingly, in the telencephalon of lungfishes, which are considered the closest living relatives of tetrapods (Brinkmann et al., 2004; Takezaki et al., 2004; Hallstrom and Janke, 2009), a pallidum expressing NKX2.1 and a comparable POC region to that of amphibians have been demonstrated (Gonzalez and Northcutt, 2009), although the presence of Shh expression in the telencephalon needs to be investigated. 3.1.2. Diencephalon Based on its morphology and gene expression, the dienceph­ alon has been subdivided into prosomeres 1-3 (P1-P3), which are distinct transverse compartments running along the A/P axis of the forebrain. The only true lineage restriction in the diencephalon occurs at the Zli, which divides P2 and P3 and exhibits Shh expression (Puelles and Rubenstein, 2003). In the last years, fate-mapping experiments in chick have shown that the Zli is cell lineage restricted at its boundaries and is a true developm ental compartment (Zeltser et al., 2001; Garcla-Lopez et al., 2004). During Xenopus development and in the adult, a thin band of x-Lhxl/5 expression just rostral to the x-Lhx2/9-expressing thalamus (P2/P3 boundary) was described as the Zli (Bachy et al., 2001; Moreno et al., 2004). The Shh expression observed in a similar position supports the identification of the Zli. Moreover, in Xenopus x-Shh expression extends to the prethalamus (P3), which has been corroborated by the colocalization of the x-Shh expressing cells in the P3 ISLl+/NKX2.l4-/NKX2.2+ territory (Moreno et al., 2008a; present results). Specifically, x-Shh expressing cells in P3 can be identified in the zona incerta (ZI), where an important TH population is present (Gonzalez et al., 1994; Milan and Puelles, 2000; present results). In the Xenopus developing diencephalon, cells expressing P3 markers, such as x-Lhxl/5 and GAD67, were progressively found in P2 close to the Zli (Brox et al., 2003; Moreno et al., 2004). This suggests that the Zli could either give rise to the subpopulation of cells in P2 expressing P3 markers or that the Zli is not a strict barrier and allows intemeuromeric transit. Both hypotheses could be true. On one hand, it has been shown that acquisition of correct P2 and P3 gene expression is dependent on direct Hh signaling (Scholpp et al., 2006; Szabo et al., 2009a) and, on the other hand, it has been demonstrated that the Zli is not hom ogeneous and the different subzones may account for different permeability properties (Kitamura et al., 1997). It should be noted that the mechanism s that lead to x-Shh expression in the Zli of Xenopus are not known and this aspect would be of interest in evolutive perspective, since differences have been reported between amniotes and ana­ mniotes. Thus, in chick embryos the presence of basal plate Shh is indispensable for the induction of Shh expression in the Zli and, consequently, for the Zli formation and diencephalic specification (Vieira and Martinez, 2006). In contrast, experi­ m ents in zebrafish have shown that there is Shh expression in the Zli even in total absence of basal plate Shh expression (Scholpp et al., 2006). Therefore, it seem s that there are two critical and different pathways to culm inate in th e Shh expression in the Zli, which could have different conse­ quences on the thalamic regionalization. In this context, the study of the Shh expression in the Zli of amphibians under experimental conditions of absence of basal plate Shh would serve to support further the anamniote/am niote differences. In mam mals, on the basis o f the expression o f m any different regulatory genes including Shh, Dlxl, Arx, Brxl, Lhxl, Lhx5 and Nkx2.2, (Kitamura et al., 1997; Nakagawa and O’Leary, 2001; Vieira and Martinez, 2006; Vue e t al., 2007; Szabo et al., 2009a) it was proposed that the Zli is com posed of different cell groups that give rise to different parts o f the prethalamus. In addition, Shh knockout mice presented a significant reduction of the diencephalon (Chiang e t al., 1996) and was demonstrated that Shh m odulates the genetic expression at both sides of the Zli (Braun et al., 2003; Hashimoto-Torii et al., 2003; Kiecker and Lumsden, 2004). Moreover, neural Shh from the Zli and diencephalic tegm en­ tum is essential for prethalamic specification (Kiecker and Lumsden, 2004; Vieira et al., 2005; Zeltser, 2005; Scholpp et al., 2006; Vieira and Martinez, 2006; Delaunay et al., 2009). The area adjacent to the Shh expression of the Zli is induced by Shh to express Nkx2.2, and this area has been proposed to be the source of the subpopulation of GABAergic intem eur- ons observed in the thalam us (Puelles et al., 2004). For the case of particular nuclei, it has been dem onstrated that neurons derived from part of the Zli contribute to GABAergic cells in the ZI (Delaunay et al., 2009). Because thalam ic nuclei contain multiple types of neurons, regulatory genes might exhibit not only nuclei-specific expression but also differen­ tial expression within a nucleus. For example, each subdi­ vision of ZI contains heterogeneous cell populations that express different neurotransmitters and calcium binding proteins (Kolmac and Mitrofanis, 1998). The combinatorial developing analysis in m ice of the LIM-hd genes revealed at least four different subsets of cells; in rostral ZI, ce lls only express ISLl or Lhxl/5, whereas in caudal ZI, m any cells express Isll and Lhxl/5 (Nakagawa and O’Leary, 2001). In Xenopus, the ZI was initially defined by the presence of dopaminergic cells (Gonzalez et al., 1994; Milan and Puelles, 2000) and the analysis o f ISLl and Lhxl/5 confirm ed its position in the posterior portion o f the prethalamus (Moreno et al., 2004, 2008a). Differences along its extension were proposed on the basis o f D1I4 expression (Brox et al., 2003). Thus it seem s that also in anam niotes the ZI h a s rostro­ caudal differences and, like in m am m als, the Shih signal likely plays a direct role in the specification of this nucleus. 3.1.3. Hypothalamus Shh is essential in the specification of the hypothalamus (Chiang et al., 1996) through the action, among others, of the homeobox gene Nkx2.1, which expression is triggered by prechordal Shh signals (Kimura et al., 1996; Puelles et a l., 2004). In Xenopus, x-Shh expression is strong in th e basal hypothalamus, specifically in the mammillary band and the tuberal area. This expression is mainly restricted to the anterior levels, whereas in the m ost posterior basal hypotha­ lam ic levels x-Shh expression was not detected.. It was previously described in the chick that Shh expressioni initially extends in the entire ventral forebrain (basal and floor plates). 76 3. ESTUDIOS GENOARQUITECTONICOS EN EL ENCEFALO EN DESARROLLO Y ADULTO 28 ; R A I N R E S E A R C H 1 3 4 7 ( 2 0 1 0 ) 1 9 - 3 2 but secondarily becom es downregulated in part of the ventral hypothalamus, including the tubero-mammillary primordi- um, but not the retromammillary area (Marti et al., 1995; Shimamura et al., 1995; Crossley et al., 2001; Patten et al., 2003; Manning et al., 2006). In particular, it was described that the ventral tubero-mammillary cells derived from floor plate precursors initially express Shh and later in development suffer a downregulation process by which Shh expression is lost and this confers them the hypothalamic fate (Manning et al., 2006). In Xenopus it might be possible that the posterior tuberal areas have a secondary growth influence by Shh downregulation, however, com plem entary fate-m apping studies are needed to clarify this hypothesis. The colocalization of x-Shh/NKX2.1 expression in the basal hypothalamus suggests that, also in Xenopus, x-Shh plays an essential role in the basal hypothalamic specifica­ tion, likely through the action of NKX2.1. Studies based on the effect of lack of function or hypofunction of NKX2.1 in m ouse and frog showed the relevance of this transcription factor in the specification and form ation of the basal hypothalamus (Marin et al., 2002; Van den Akker et al., 2008). The phylogenetic comparison of NKX2.1 expression in the forebrain showed that it is largely similar in several vertebrates, including chicken, zebrafish and Xenopus (Puelles et al., 2000; Small et al., 2000; Rohr et al., 2001; Bachy et al., 2002; Gonzalez et al., 2002a,b; Moreno et al., 2008a). However, the detailed analysis revealed that this gene show s an additional expression domain in the alar hypothalamus, including the suprachiasmatic nucleus and anterior hypothalamic region, in Xenopus (Gonzalez et al., 2002a; Moreno et al., 2008a) and in zebrafish (Rohr et al., 2001). This situation was corroborated with the combination of x-Shh and specific markers of this area like TH (Moreno et al., 2008a; present results). The alar NKX2.1 expression is consistent with the x-Shh expression detected in the sam e areas (present results) and suggests that in anam niotes the Shh/Nkx2.1 could also activate alar hypothalamic specifica­ tion. In evolutive terms, the comparison of the Nkx2.1 expression in anam niotes, including Xenopus (present results; Moreno et al., 2008a; Van den Akker et al., 2008), zebrafish (Rohr et al., 2001) and lamprey (Osorio et al., 2005) w ith that in am niotes, including turtle (Moreno et al., unpublished data), chicken (Pera and Kessel, 1998; Abelian and Medina, 2009) and m ouse (Shimamura et al., 1995; Puelles et al., 2000), suggests that after the anam niote to amniote transition, the alar hypothalamic expression was greatly reduced. It likely took place gradually during and after the transition being present in som e stem amniotes, like the turtle (Moreno et al., unpublished data), but lacking in mammals. However, information about NKX2.1 expres­ sion in other reptiles is needed and there are not data about the Shh expression in these areas in reptiles, therefore both expressions cannot be related. It seem s interesting the hypothesis that the lack of Shh/NKX2.1 expression in alar hypothalamic areas of mam mals, in contrast to the situation found in anam niotes, could be correlated with the thalamic expansion. In line with this idea is the experimental result obtained in Xenopus in w hich the thalam us is largely increased w hen the NKX2.1 signal is abolished (van den Akker et al., 2008). In m am m als, Shh expression during developm ent is present in the hypothalamic domain and its essential role w as demonstrated in the coordination o f tissue growth and patterning. The neural Shh has a very important and specific role in the developm ent o f the lateral hypothalam us, possibly mediated by regulation of Dlx2, Dbxl, and FoxD. The lack of Shh expression in the hypothalamic neuroe­ pithelium results in a very reduced lateral hypothalamus, in w hich som e of the m ost functionally important and charac­ teristic neuronal subpopulations are either very reduced or com pletely m issing, whereas the preoptic area and the suprachiasmatic nucleus show ed a normal developm ent (Szabo et al., 2009b). 3.2. Conclusions and perspectives All the data obtained in the forebrain of Xenopus reveal a largely conserved pattern o f Shh expression among vertebrates. However, interesting differences were also noted that make this species interesting to test the functional implication of Shh in forebrain development. 1) Amphibians have a well orga­ nized pallidum (for review see Moreno et al., 2009) and this could be related to the telencephalic Shh expression, as observed in Xenopus. 2) The relevance of the Shh signal from the Zli in the diencephalic specification is strongly proved in amniotes and anamniotes (Barth and Wilson, 1995; Vieira and Martinez, 2006). However, it showed two models of specifica­ tion, independent (amniotes; Vieira and Martinez, 2006) or dependent (zebrafish; Barth and Wilson, 1995) on a Shh basal plate signal. The intermediate position of amphibians as anamniote tetrapods make them an interesting model to study the functional implication of Shh. 3) Together with its expression in the basal forebrain territories x-Shh is also expressed in the alar hypothalamus, like NKX2.1 (Moreno et al., 2008a), in contrast to that described in m am mals (Puelles et al., 2000). In evolutive terms this discrepancy could be related to the thalamic expansion in amniotes, even at the expense of reducing alar hypothalamic areas. This evolutive significance of the differences/sim ilarities betw een ana­ mniotes and amniotes could serve to understand the motor that drove the prosencephalic evolution and increase our knowledge about the brain organization and its development. 4. Experimental procedures 4.1. Animals and tissue processing For the present study, adults and tadpoles of X. laeuis were used. Embryos and larvae were classified according to Nieuwkoop and Faber (1967). Embryonic (42-45), premeta- morphic (46-52), prometamorphic (53-58), and metamorphic (59-65) stages were used, minimizing as much as possible the number of animals used and their suffering. All animals were treated according to the regulations and laws of the European Union (86/609/EEC) and Spain (Royal Decree 223/1998) for care and handling of anim als in research, after approval from the University to conduct the experiments described. Adult Xenopus were purchased fi-om commercial suppliers (Xenopus Express; Lyon, France), and the different developing 77 3. ESTUDIOS GENOARQUITECTONICOS EN EL ENCEFALO EN DESARROLLO Y ADULTO R A I N R E S E A R C H 1 3 4 7 ( 2 0 1 0 ) 1 9 - 3 2 29 specim ens were obtained by in vitro fertilization and m ain­ tained in tap water at 20 °C throughout their development. At appropriate times, embryos and larvae were deeply anesthe­ tized in a 0.4 mg/ml solution of tricaine methanesulfonate (MS222, Sigma Chemical Co., St. Louis, MO). The adults and late larvae were perfused transcardially with 0.9% sodium chloride, followed by a phosphate buffered solution (pH 7.4) containing 4% paraformaldehyde. The brains were dissected and post­ fixed in the sam e fixative solution overnight at 4 °C. They were then embedded in gelatin/albumin, and sections were cut on a Vibratome at 30-40 nm thickness in the frontal or sagittal plane. The embryos and premetamorphic larvae were fixed by im m ersion overnight at 4 °C in MEMFA (0.1 M MOPS [4- morpholinopropanesulphonic acid] 2 mM ethyleneglycolte- traacetic acid, 1 mM MgS04, 3.7% formaldehyde) and progres­ sively dehydrated in methanol and stored at 20 °C until use. 4.2. In situ hybridization The cDNA of Xenopus Shh (x-Shh) was provided by Dr. Randal Moon (University of Washington, Seattle; Ekker et al., 1995). For in situ hybridization, antisense digoxigenin (DIG)-labeled riboprobes for x-Shh were synthesized according to the protocol described in Bachy et al. (2001), linearizing the clone in Bluescript KS with Bam HI (Promega, Madison, USA) and transcribing with T3 (Promega). The embryos and premeta­ morphic larvae were processed in toto after progressive re­ hydration and pretreatments (see Bachy et al., 2001), and the adults and late larvae were processed in floating sections (see Moreno et al., 2004). Hybridization step was done with 3 pl/ml of a DIG-labeled RNA probe, in a 50% formamide containing medium overnight at 55 °C. The solution used for prehybridi­ zation (at 60 °C for 1 h) and hybridization contained 50% deionized formamide (Fluka, Steinheim, Germany), 5x stan­ dard saline citrate (Sigma-Aldrich, Steinheim, Germany), 2% blocking reagent (Roche Diagnostics, Mannheim, Germany), 0.1% Tween-20, 0.5% 3-[(3-cholamidopropyl)-dimethylammo- nio]- 1-propanesulfonate (CHAPS; Sigma-Aldrich), 1 mg/ml of yeast tRNA (Sigma-Aldrich), 5 mM of ethylenediaminetetraa- cetic acid (Sigma-Aldrich), and 50 g/ml of heparin (Sigma- Aldrich) in water. Hybridization was detected using an alkaline phosphatase coupled anti-DIG antibody (Roche D iagnostics, dilution 1:1500). Alkaline phosphatase staining was developed with 4-nitroblue tétrazolium chloride/xphosphate/5-brom o-4- chloro-3-indolyl-phosphate solution (NBT/BCIP; Roche Diag­ nostics). After hybridization, the embryos and early larvae were embedded in a solution of 20% gelatin and 30% sucrose in PB, and stored overnight at 4 °C in a solu tion o f 4% formaldehyde and 30% sucrose in PB. Sections were cut at 14-25 nm thickness in the frontal, sagittal and horizontal plane on a freezing microtome. 4.3. Double labeling w ith in situ hybridization and immunohistochemistry For double labeling experiments we combined the in situ hybridization for x-Shh with immunohistochemistry with the following antibodies: mouse anti-ISLl (diluted 1:500; Develop­ mental Studies Hybridoma Bank, Iowa City, LA, USA. Clone: 39.4D5), mouse anti-TH (diluted 1:1000; Chemicon International, Inc, USA. Code number P22941), mouse anti-NKX2.2 (diluted 1:500; Developmental Studies Hybridoma Bank, Iowa, USA. Clone: 74.5A5-c), rabbit anti-NKX2.1 (diluted 1:500; Biopat Immu- notechnologies, Italy. Code number PAOlOO) and rabbit anti-OTP (diluted 1:1000; produced by “PinkCell laboratories, Amsterdam, The Netherlands; according to the protocol described in Lin et al., 1999). In the cases in w hich double histofluorescence was used, x-Shh hybridization was performed first and revealed with Fast Red tablets (Roche Diagnostics), followed by im m uno­ histochem istry revealed w ith goat anti-m ouse Alexa 488 (diluted 1:500, Molecular Probes, Denmark) or chicken anti­ rabbit Alexa 488 (diluted 1:500, Molecular Probes). In case of x-Shh/TH double labeling for bright field microscopy, x-Shh hybridization w as performed first and revealed with NBT/ BCIP (Roche Diagnostics) and im m unohistochem istry for TH was revealed w ith biotinylated goat anti-m ouse (diluted 1:100, Calbiochem, Darmstadt, Germany). Visualization was then obtained w ith the ABC kit procedure (Vector, Burlin­ game. CA). 4.4. Imaging The sections were analyzed with an Olympus BX51 microscope that, together with the bright microscopy, was equipped for fluorescence with appropriate filter combinations. Selected sections were photographed by using a digital camera (Olympus DP70). Contrast and brightness of the photomicrographs were adjusted in Adobe Photoshop CS3 (Adobe Systems, San Jose, CA) and figures were mounted in Canvas 11(ACD Systems, Canada). Acknowledgments This work was supported by grants from the Spanish MICINN (grant number: BFU2009-12315). We are grateful to Dr. Jesus M. Lopez for the critical reading of the manuscript. R E F E R E N C E S Abelian, A., Medina, L., 2009. Subdivisions and derivatives of the chicken subpallium based on expression of LIM and other regulatory genes and markers of neuron subpopulations during development. J. Comp. Neurol. 515, 465-501. Bachy, I., Vernier, P., Rétaux, S., 2001. The LIM-homeodomain gene family in the developing Xenopus brain: conservation and divergences with the mouse related to the evolution of the forebrain. J. Neurosci. 21, 7620-7629. Bachy, I., Berthon, J., Rétaux, S., 2002. Defining palliai and subpallial divisions in the developing Xenopus forebrain. Mech. Dev. 117, 163-172. Bardet, S.M., Cobos, I., Puelles, E., Mam'nez-De-La-Torre, M., Puelles, L., 2006. Chicken lateral septal organ and other circumventricular organs form in a striatal subdomain abutting the molecular striatopallidal border. J. Comp. Neurol. 499,745-767. Bardet, S.M., Martinez-de-la-Torre, M., Northcutt, R.G., Rubenstein, J. L., Puelles, L., 2008. Conserved pattern of OTP-positive cells in the paraventricular nucleus and other hypothalamic sites of tetrapods. Brain Res. Bull. 75, 231-235. 78 3. ESTUDIOS GENOARQUITECTONICOS EN EL ENCEFALO EN DESARROLLO Y ADULTO 30 i R A I N R E S E A R C H 1 3 4 7 ( 2 0 1 0 ) 1 9 - 3 2 Barth, K.A., W ilso n , S W ., 1995. E x p ress io n o f z eb ra f ish n k 2 .2 is in f lu e n c e d b y s o n ic h e d g e h o g /v e r te b r a te h e d g e h o g -1 a n d d e m a r c a te s a z o n e o f n e u r o n a l d if fe r e n t ia tio n in th e em b ry o n ic forebrain . D e v e lo p m e n t 1 2 1 ,1 7 5 5 -1 7 6 8 . B la ess , S., C orrales, J.D., Joyner, A.L., 2006. S o n ic h e d g e h o g r eg u la te s Gli a c tiv a to r a n d rep re sso r fu n c t io n s w ith sp a tia l an d tem p o ra l p r e c is io n in th e m id /h in d b r a in reg io n . D e v e lo p m e n t 133, 1799-1809 . B la ess , S., S te p h e n , D ., Joyner, A.L., 2008 . Gli3 c o o r d in a te s th r e e -d im e n s io n a l p a t te r n in g a n d g r o w th o f th e te c tu m an d c e r e b e llu m b y in te g r a t in g S h h a n d Fgf8 s ig n a lin g . D e v e lo p m e n t 135, 2 0 9 3 -2 1 0 3 . B raun, M .M., E th er id ge , A ., B ern ard , A ., R o b ertso n , C.P., R oelink , H., 2003. W n t s ig n a lin g is r eq u ir ed a t d is t in c t s ta g e s o f d e v e lo p m e n t fo r th e in d u c t io n o f th e p o s te r io r foreb ra in . D e v e lo p m e n t 130, 5 5 7 9 -5 5 8 7 . B rin k m an n , H., V e n k a te sh , B., B renn er, S., M eyer, A., 2004. N u c lea r p ro te in -co d in g g e n e s su p p o r t lu n g fis h a n d n o t th e c o e la c a n th a s th e c lo s e s t liv in g re la tiv e s o f la n d v e r teb ra tes . Proc. N atl. A cad. Sci. U. S. A. 101, 4 9 0 0 -4 9 0 5 . Brox, A., P u elles , L., Ferreiro, B., M ed in a , L., 2003. E x p ress io n o f th e g e n e s GAD67 an d D is ta l- le s s -4 in th e foreb ra in o f Xenopus laeuis co n firm s a c o m m o n p a ttern in te tra p o d s. J. C om p. N eu rol. 461, 3 70-393 . Brox, A., P u elles , L., Ferreiro, B., M ed in a , L., 2004. E x p ress io n o f th e g e n e s E m x l, T b rl, a n d E o m e s (Tbr2) in th e te le n c e p h a lo n o f Xenopus laeuis co n firm s th e e x is t e n c e o f a v e n tr a l p a llia i d iv is io n in all te tra p o d s. J. C om p. N eu rol. 474 , 562 -577 . Buttler, A.B., H odos, W ., 1996. C om p a ra tiv e v erteb ra te n eu ro a n a to m y . E vo lu tion a n d A d a p ta tio n . W iley -L iss, N e w York. USA. C hiang, C., L id n gtu n g , Y., Lee, E., Y ou n g , K.E., C orden , J.L., W estp h a l, H., B each y , P.A., 1996. C yclop ia a n d d e fe c tiv e axia l p a tte rn in g in m ic e la c k in g S o n ic h e d g e h o g g e n e fu n c tio n . N atu re 383, 40 7 -4 1 3 . C respo, M., M oreno, N., Lopez, J.M., G on zalez , A., 2003. C om parative a n a ly s is o f n eu ro p ep tid e FF-like im m u n o r e a ct iv ity in th e brain o f an u ran (Rana perezi, Xenopus laeuis) an d u ro d e le (Plcurodcles wait!) am p h ib ia n s. J. C hem . N eu roan at. 25, 53 -71 . C rossley , P.H., M artinez, S., O hkubo, Y., R u b en ste in , J.L., 2001. C oord inate e x p ress io n o f Fgf8, O tx2, B m p4, an d S h h in th e rostral p r o sen ce p h a lo n du rin g d e v e lo p m e n t o f th e te le n c e p h a lic and op tic v e s ic le s . N eu r o sc ie n c e 1 0 8 ,1 8 3 -2 0 6 . D e la u n a y , D ., H eyd on , K., M igu ez, A ., S ch w ab , M., N a v e , K.A., T h o m a s, J.L., S p a ssk y , N., M artin ez , S., Z alc, B., 2009. G en etic tra c in g o f su b p o p u la tio n n e u r o n s in th e p r e th a la m u s o f m ic e (Mus m usculus). J. C om p. N eu ro l. 512, 7 4 -83 . Ekker, S.C., M cG rew, L.L., Lai, C.J., Lee, J.J., v o n K essler , D.P., M oon, R.T., B each y , P.A., 1995. D is tin c t e x p r e s s io n a n d sh a r ed a c tiv it ie s o f m em b er s o f th e h e d g e h o g g e n e fa m ily o f Xenopus laeuis. D e v e lo p m e n t 121, 2 3 3 7 -2 3 4 7 . E n d e p o ls , H., M u h le n b ro c k -L e n ter , S., R oth , G., W a lk o w ia k , W ., 2 0 06 . T h e s e p ta l c o m p le x o f th e f ir e -b e l l ie d to a d Bombina orientaUs: c h e m o a r c h ite c tu r e . J. C h e m . N e u r o a n a t . 31 , 5 9 -7 6 . E ricson, J., R ash b ass, P., S ch ed l, A ., B ren n er-M orton , S., K aw akam i, A., v a n H ey n in g en , V ., J esse ll, T.M ., B riscoe, )., 1997. Pax6 c o n tro ls p ro g en ito r ce ll id e n t ity a n d n e u r o n a l fa te in r e s p o n se to graded S h h s ig n a lin g . C ell 9 0 ,1 6 9 -1 8 0 . F lam es, N ., Pla, R., G elm an , D.M., R u b en ste in , J.L., P u elles , L., M arin, O., 2007. D e lin e a tio n o f m u ltip le su b p a llia l p rogen itor d o m a in s b y th e co m b in a to r ia l e x p r e s s io n o f tra n scr ip tio n a l c o d e s . J. N eu ro sc i. 27, 9 6 82-9695 . F u ccillo , M., R utlin , M., F ish e ll, G., 2006. R em o v a l o f Pax6 p artia lly r e s c u e s th e lo s s o f v e n tr a l s tru c tu res in S h h n u ll m ic e . Cereb. C ortex 16 (Suppl 1), i9 6 - i l0 2 . G arcia-L opez, R., V ieira, C., E ch evarria , D., M artin ez , S., 2004. Fate m a p o f th e d ie n c e p h a lo n a n d th e zo n a lim ita n s a t th e 1 0 -s o m ite s s ta g e in ch ick e m b ry o s . D ev. B iol. 268, 514 -530 . G arda-L ôpez, M., A belian , A ., Legaz, I., R u b en ste in , J.L., P u elles , L., M edina, L., 2008. H isto g en e tic co m p a r tm e n ts o f th e m o u se c en tro m ed ia l an d e x te n d e d a m y g d a la b a se d o n g e n e e x p ress io n p a ttern s d u rin g d e v e lo p m en t. J. C om p. N eurol. 5 0 6 ,4 6 -7 4 . G o n za lez , A ., N o rth cu tt, R.G., 2009. A n im m u n o h is to c h e m ic a l a p p ro a ch to lu n g fis h te le n c e p h a lic o rg a n iza tio n . Brain B eh av. Evol. 74, 4 3 -5 5 . G o n za lez , A ., S m e e ts , W.J., 1991. C o m p arative a n a ly s is o f d o p a m in e a n d ty r o s in e h y d ro x y la se im m u n o r e a c t iv it ie s in th e b rain o f tw o a m p h ib ia n s, th e a n u ra n Rana ridibunda a n d th e u r o d e le Pleurodeles w altlii. J. C om p. N eu ro l. 303, 4 5 7 -477 . G o n za lez , A ., T u in h o f, R., S m e e ts , W.J., 1993. D istr ib u tion o f ty r o s in e h y d ro x y la se an d d o p a m in e im m u n o r e a c t iv it ie s in th e b rain o f th e S o u th A frican c la w e d frog Xenopus laeuis. A nat. E m bryol. (Berl.) 1 8 7 ,1 9 3 -2 0 1 . G o n za lez , A., M arin, O., T u in h o f, R., S m e e ts , W.J., 1994. O n to g en y o f c a te c h o la m in e s y s te m s in th e cen tr a l n e rv o u s s y s te m o f a n u ra n a m p h ib ia n s: a n im m u n o h is to c h e m ic a l s tu d y w ith a n tib o d ie s a g a in s t ty r o s in e h y d r o x y la se an d d o p a m in e . J. C om p. N eu rol. 346, 6 3 -79 . G o n za lez , A ., L opez, J.M., M arin, O., 2002a . E xp ress ion p a ttern o f th e h o m e o b o x p ro te in NKX2-1 in th e d e v e lo p in g Xenopus forebrain . Brain Res. G en e Expr. P a ttern s 1 ,1 8 1 -1 8 5 . G o n za lez , A , L opez, J.M., S a n ch ez -C a m a c h o , C., M arin, O., 2002b. R egion al e x p r e s s io n o f th e h o m e o b o x g e n e NKX2-1 d e fin es p a llid a l a n d in te m e u r o n a l p o p u la t io n s in th e b a sa l gan g lia o f a m p h ib ia n s. N e u r o sc ie n c e 114, 56 7 -5 7 5 . G uinazu , M.F., C h am b ers, D., L u m sd en , A ., K iecker, C., 2007. T issu e in ter a c tio n s in th e d e v e lo p in g c h ick d ie n c e p h a lo n . N eu ral D ev. 2, 25. G u n h aga , L., J e sse ll, T.M., E dlund, T ., 2000. S o n ic h e d g e h o g s ig n a lin g a t g a stru la s ta g e s s p e c if ie s v e n tr a l te le n c e p h a lic c e lls in th e ch ick em b ryo . D e v e lo p m e n t 127, 3283-3293 . H a llstrom , B.M., Janke, A., 2009. G n a th o s to m e p h y lo g en o m ic s u tiliz in g lu n g fis h EST s e q u e n c e s . M ol. Biol. Evol. 2 6 ,4 6 3 -4 7 1 . H ash im oto-T orii, K., M otoyam a, J., H ui, C.C., K uroiwa, A., N akafuku, M., S h im am u ra , K., 2003. D ifferen tia l a c tiv it ie s o f S on ic h e d g e h o g m e d ia te d b y Gli tran scrip tion factors d e fin e d istin ct n eu ro n a l su b ty p es in th e d orsa l th a la m u s. M ech. D ev. 1 2 0 ,1 0 97-1111 . Hirata, T., N a k a za w a , M., M uraoka, O., N a k a y a m a , R., S u da, Y., Hibi, M., 2006. Z in c-fin g er g e n e s Fez a n d F ez-lik e fu n c tio n in th e e s ta b lis h m e n t o f d ie n c e p h a lo n su b d iv is io n s . D e v e lo p m e n t 133, 3 9 9 3 ^ 0 0 4 . In gh am , P.W ., M cM ahon , A.P., 2001. H e d g e h o g s ig n a lin g in a n im a l d e v e lo p m e n t: p a ra d ig m s a n d p r in c ip les . G en es D ev. 15, 3 0 59-3087 . Ingham , P.W., Placzek, M., 2006. O rchestrating o n togen esis: variation s o n a th em e b y so n ic h ed geh og . N at. Rev. G enet. 7 ,8 4 1 -8 5 0 . K iecker, C., L u m sd en , A ., 2004. H e d g e h o g s ig n a lin g from th e ZLI r eg u la te s d ie n c e p h a lic reg io n a l id e n t ity . N at. N eu rosc i. 7, 1242-1249 . K im ura, S., H ara, Y ., P in ea u , T., F e m â n d e z -S a lg u e r o , P., Fox, C.H., W ard, J.M., G o n z a lez , F.J., 1996. T h e T /eb p n u ll m o u se : th y r o id -s p e c if ic e n h a n c e r -b in d in g p r o te in is e s s e n t ia l for th e o r g a n o g e n e s is o f th e th y ro id , lu n g , v e n tr a l foreb ra in , an d p itu ita ry . G e n e s D ev . 10, 6 0 -6 9 . K itam ura, K., M iura, H., Y a n a za w a , M., M iyash ita , T., K ato, K., 1997. E x p ress io n p a tte rn s o f B rx l (R ieg g e n e ). S o n ic h e d g e h o g , N k x2.2 , D lx l a n d A rx d u rin g z o n a lim ita n s in tra th a la m ica an d em b ry o n ic v e n tr a l la tera l g e n ic u la te n u c le a r fo rm a tio n . M ech. D ev. 67, 8 3 -9 6 . K olm ac, C.I., M itrofan is, J., 1998. P a ttern s o f b r a in stem p ro jec tio n to th e th a la m ic reticu lar n u c le u s . J. C om p. N eurol. 3 9 6 ,5 3 1 -5 4 3 . Lin, X., S ta te , M .W ., V accarin o , P.M., G really , J., H ass, M., L eckm an, J.F., 1999. Id en tif ica tio n , c h r o m o s o m a l a s s ig n m e n t, an d e x p r e s s io n a n a ly s is o f th e h u m a n h o m e o d o m a in -c o n ta in in g g e n e O rth op ed ia (OTP). G e n o m ics 60 , 96 -1 0 4 . L opez, J.M., D o m in g u e z , L., M oreno, N ., G o n za lez , A., 2009. C om p a ra tiv e im m u n o h is to c h e m ic a l a n a ly s is o f th e 79 3. ESTUDIOS GENOARQUITECTONICOS EN EL ENCEFALO EN DESARROLLO Y ADULTO B R A I N R E S E A R C H 1 3 4 7 ( 2 0 1 0 ) 1 9 - 3 2 31 d is tr ib u tio n o f o r ex in s (h y p o c re tin s) in th e b ra in o f a m p h ib ia n s. P ep tid es 30, 8 7 3 -8 8 7 . M annin g , L., O h yam a, K., S aeger, B., H ata n o , O., W ilso n , S.A., Logan, M., P laczek , M., 2006. R eg io n a l m o r p h o g e n e s is in th e h y p o th a la m u s: a BM P-Tbx2 p a th w a y co o rd in a te s fa te an d p r o lifera tio n th ro u g h S h h d o w n r e g u la t io n . D ev. C ell 11, 8 7 3-885 . M arin, O., S m e e ts , W.J., G o n za lez , A ., 1997. D istr ib u tion o f c h o lin e a c ety ltr a n s fe ra se im m u n o r e a c t iv ity in th e b ra in o f an u ran (Rana perezi, Xenopus laeuis) a n d u r o d e le (Pleurodeles waltl) a m p h ib ia n s. J. C om p. N eu ro l. 382, 4 9 9 -5 3 4 . Marin, O., S m ee ts , W.J., G onzalez , A., 1998. B asal ganglia organ iza tion in am p h ib ian s: c h e m o a r ch itec tu r e ./. C om p. N eurol. 392 ,285 -312 . M arin, 0 . , Baker, J., P u elles , L , R u b en ste in , J.L., 2002. P a ttern in g o f th e b a sa l te le n c e p h a lo n a n d h y p o th a la m u s is e s s e n t ia l for g u id a n ce o f cortica l p ro jec tio n s . D e v e lo p m e n t 129, 761 -773 . Marti, E., B o v o len ta , P., 2002. S o n ic h e d g e h o g in CNS d ev e lo p m e n t: o n e s ig n a l, m u ltip le o u tp u ts . T ren d s N eu r o sc i. 25, 89 -9 6 . Marti, E., T akada, R., B um crot, D A ., S asak i, H., M cM ahon, A.P., 1995. D istribution o f S on ic h e d g e h o g p e p t id e s in th e d e v e lo p in g ch ick an d m o u s e em bryo. D e v e lo p m e n t 1 2 1 ,2 5 3 7 -2 5 4 7 . M en u et, A ., A lu n n i, A ., Joly, J.S., Jeffery , W .R., R étau x , S., 2007. E xp an d ed e x p r es s io n o f S o n ic H e d g e h o g in A stya n a x cav efish : m u ltip le c o n s e q u e n c e s o n foreb ra in d e v e lo p m e n t an d ev o lu tio n . D e v e lo p m e n t 134, 8 4 5 -8 5 5 . M ilan, F.J., P u elles , L., 2000. P a ttern s o f ca lretin in , ca lb in d in , a n d ty r o s in e -h y d ro x y la se e x p r e s s io n are c o n s is t e n t w ith th e p ro so m er ic m a p o f th e frog d ie n c e p h a lo n . J. C om p. N euro l. 419, 9 6-121 . M oreno, N ., G on za lez , A., 2003. H o d o lo g ica l ch a ra cter iza tio n o f th e m e d ia l a m y g d a la in an u ra n a m p h ib ia n s . J. C om p. N eu rol. 466, 389-408 . M oreno, N ., G on za lez , A ., 2004. L o ca liza tio n an d c o n n e c tiv ity o f th e la tera l a m y g d a la in a n u ra n a m p h ib ia n s . J. C om p. N euro l. 4 7 9 ,1 3 0 -1 4 8 . M oreno, N ., G on za lez , A ., 2005. C en tra l a m y g d a la in an u ra n a m p h ib ia n s: n e u r o c h e m ic a l o rg a n iz a tio n an d c o n n e c tiv ity . J. C om p. N eurol. 489, 6 9 -9 1 . M oreno, N., G o n za lez , A., 2006. T h e c o m m o n o r g a n iza tio n o f th e a m y g d a lo id c o m p le x in te trap od s: n e w c o n c ep ts b a s e d o n d e v e lo p m e n ta l, h o d o lo g ic a l a n d n e u r o c h e m ic a l d a ta in an u ran a m p h ib ia n s. Prog. N eu rob io l. 78, 6 1 -9 0 . M oreno, N ., G on za lez , A., 2007. E v o lu tio n o f th e a m y g d a lo id c o m p le x in v erteb ra tes , w ith s p e c ia l r e fer en ce to th e a n a m n io -a m n io t ic tr a n s itio n . J. A n a t. 2 1 1 ,1 5 1 -1 6 3 . M oreno , N ., B ach y , I., R étau x , S., G o n z a le z , A ., 2004. L IM -h o m eo d o m a in g e n e s a s d e v e lo p m e n t a l a n d a d u lt g e n e t ic m a rk ers o f Xenopus fo reb ra in fu n c t io n a l s u b d iv is io n s . J. C om p. N eu ro l. 4 7 2 , 5 2 -7 2 . M oreno, N ., D o m in g u ez , L., R étau x , S., G o n za lez , A ., 2008a . I s le t l a s a m arker o f su b d iv is io n s a n d c e ll ty p e s in th e d e v e lo p in g forebrain o f Xenopus. N e u r o sc ie n c e 1 5 4 ,1 4 2 3 -1 4 3 9 . M oreno, N., R étaux, S., G o n za lez , A ., 2008b. S p a tio -tem p o r a l e x p r e ss io n o f Pax6 in Xenopus forebra in . Brain R es. 1239, 9 2 -99 . M oreno, N., G on za lez , A., R étaux, S., 2008c. E v id en ces for ta n g en tia l m ig r a tio n s in Xenopus te le n c e p h a lo n : d e v e lo p m e n ta l p a ttern s a n d ce ll track in g e x p e r im e n ts . D ev . N eu rob io l. 68, 504 -520 . M oreno, N ., G on za lez , A., R étau x , S., 2009. D e v e lo p m e n t an d e v o lu tio n o f th e su b p a lliu m . S e m in . C ell D ev. B iol. 20, 735-743 . M orona, R., G on za lez , A., 2008. C a lb in d in -D 28k a n d ca lr e tin in e x p r es s io n in th e foreb ra in o f a n u ra n a n d u ro d e le a m p h ib ia n s: fu rther su p p o rt for n e w ly id e n t if ie d su b d iv is io n s . J. C om p. N eurol. 5 1 1 ,1 8 7 -2 2 0 . M orona, R., G on za lez , A ., 2009. I m m u n o h is to c h e m ic a l lo c a liz a tio n o f ca lb in d in -D 28k an d c a lr e t in in in th e b r a in s te m o f an u ra n an d u ro d e le a m p h ib ia n s. J. C om p. N eu ro l. 515, 50 3 -5 3 7 . M urakam i, Y., U ch id a , K., Rijli, P.M., K uratan i, S., 2005. E vo lu tion o f th e brain d e v e lo p m e n ta l p lan : in s ig h t s from a g n a th a n s . D ev. Biol. 280, 249 -259 . N a k agaw a , Y., O’Leary, D.D., 2001 . C om b in ator ia l e x p r e s s io n p a tte rn s o f L IM -h o m eo d o m a in an d o th e r regu la tory g e n e s p a rce lla te d e v e lo p in g th a la m u s . J. N eu ro sc i. 21, 2711-2725 . N ie u w e n h u y s , R., T en D on k elaar , H.J., N ic h o lso n , C., 1998. T h e C entral N erv o u s S y s te m o f V erteb ra tes. Sp rin ger-V erlag Berlin H eid elb erg , G erm any. N ieu w k o o p , P.D., Faber, J., 1967. N o rm a l ta b le o f Xenopus laeuis (D audin). O sorio, J., R étaux, S., 2008. T h e la m p re y in e v o lu tio n a r y s tu d ies . D ev. G en es Evol. 218, 2 2 1 -2 3 5 . O sorio, J., M azan , S., R étaux, S., 2005. O rgan isa tion o f th e la m p rey (Lampetra flu via tilis) em b r y o n ic brain: in s ig h ts from L IM -h om eod om ain , Pax a n d h e d g e h o g g e n e s . D ev. Biol. 288, 100- 112. P atten , L, K ulesa , P., S h en , M.M., Fraser, S., P laczek , M., 2003. D is tin c t m o d e s o f floor p la te in d u c tio n in th e ch ick em b ryo . D e v e lo p m e n t 130, 4 8 0 9 -4 8 2 1 . Pera, E.M., K esse l, M., 1998. D e m a rca tio n o f v e n tr a l territor ies b y th e h o m e o b o x g e n e NKX2.1 d u rin g early ch ick d e v e lo p m e n t . D ev. G en es Evol. 2 0 8 ,1 6 8 -1 7 1 . P u elles , L., R u b en ste in , J.L., 2003. Forebrain g e n e e x p r e s s io n d o m a in s an d th e e v o lv in g p ro so m e r ic m o d e l. T ren d s N eu rosc i. 26, 46 9 -4 7 6 . P u elles, L., M ilan, F., M artinez-de-la -T orre, M., 1996. A seg m en ta l m a p o f arch itecton ic su b d iv is io n s in th e d ie n ce p h a lo n o f th e frog Rana perezi: a ce ty lc h o lin e s te ra se -h is to c h e m ic a l ob serva tion s . Brain B ehav. Evol. 4 7 ,2 7 9 -3 1 0 . P u elles , L., K uw an a, E., P u elles , £., B u lfon e, A., S h im a m u ra , K., K eleher, J., Sm iga , S., R u b en ste in , J.L., 2000. Palliai an d su b p a llia l d er iv a tiv e s in th e em b r y o n ic ch ick a n d m o u s e te le n c e p h a lo n , traced b y th e e x p r e s s io n o f th e g e n e s D ix -2, E m x-1, N kx-2 .1 , Pax-6, an d T b r-l.J . C om p. N eurol. 4 2 4 ,4 0 9 -4 3 8 . P u elles , E., A n n in o , A ., T u orto , F., U sie llo , A., A cam p ora , D., C zerny, T., Brodski, C., A ng, S.L., W u rst, W ., S im eo n e , A., 2004. O tx2 reg u la te s th e e x te n t, id e n t ity a n d fa te o f n eu ro n a l p ro g en ito r d o m a in s in th e v en tra l m id b ra in . D e v e lo p m e n t 131, 2037-2048 . Rohr, K.B., Barth, K.A., V arga, Z.M., W ilso n , S.W ., 2001. T h e n o d a l p a th w a y acts u p str e a m o f h e d g e h o g s ig n a lin g to s p e c ify v en tra l te le n c e p h a lic id e n t ity . N eu ro n 29, 341-351 . R u b en ste in , J.L., M artinez, S., S h im a m u ra , K., 1994. T h e em b ry o n ic v erteb ra te forebrain: th e p r o so m e r ic m o d e l. S c ie n c e 266, 578-580 . S a n ch ez -C a m a ch o , C., L opez, J.M., G on zalez , A., 2006. B asal forebrain ch o lin erg ic s y s te m o f th e an u ran a m p h ib ia n Rana perezi: e v id e n c e for a sh a r ed o rg a n iza tio n p a tte rn w ith a m n io te s . J. C om p. N eu ro l. 494, 9 6 1 -975 . S ch o lp p , S., W olf, O., Brand, M., L u m sd en , A., 2006. H ed g eh o g s ig n a llin g from th e zo n a lim ita n s in tra th a la m ica o rch estr a te s p a tte rn in g o f th e zeb ra fish d ie n c e p h a lo n . D e v e lo p m e n t 133, 8 5 5-864 . S h im a m u ra , K., H artigan, D.J., M artin ez, S., P u elles , L., R u b en ste in , J.L., 1995. L on g itu d in a l o r g a n iz a tio n o f th e an ter ior n eu ra l p la te a n d n eu ra l tu be. D e v e lo p m e n t 121, 3923-3933 . S m all, E.M., V ok es, S.A., Garriock, R.J., Li, D., Krieg, P.A., 2000. D e v e lo p m e n ta l e x p r e s s io n o f th e Xenopus N k x2-1 a n d N k x2-4 g e n e s . M ech . D ev. 96, 25 9 -2 6 2 . S m ee ts , W.J., G on za lez , A ., 2000. C a tec h o la m in e s y s te m s in th e brain o f verteb rates: n e w p e r sp e c t iv e s th ro u g h a co m p a ra tiv e ap p roach . Brain Res. Brain R es. Rev. 33, 308 -379 . S triedter, G.F., 1997. T h e te le n c e p h a lo n o f te tr a p o d s in e v o lu tio n . Brain B eh av . Evol. 4 9 ,1 7 9 -2 1 3 . S u sse l , L., M arin, O., K im ura, S., R u b en ste in , J.L., 1999. L oss o f N kx2.1 h o m e o b o x g e n e fu n c tio n r e su lts in a v en tra l to d orsa l m o lecu la r r esp e c ific a tio n w ith in th e b a sa l te le n c e p h a lo n : e v id e n c e for a tr a n sfo rm a tio n o f th e p a llid u m in to th e s tr ia tu m . D e v e lo p m e n t 126, 3 3 59-3370 . Szabo, N.E., Z hao, T., C ankaya, M., T heil, T., Zhou, X.,  lvarez-B olado, G., 2009a. Role o f n e u ro e p ith e lia l S on ic h e d g e h o g in h y p o th a la m ic pattern in g . J. N eu rosc i. 29, 6989-7002. 80 3. ESTUDIOS GENOARQUITECTONICOS EN EL ENCEFALO EN DESARROLLO Y ADULTO 32 R A I N R E S E A R C H 1 3 4 7 ( 2 0 1 0 ) 1 9 - 3 2 S zabo, N.B., Z hao, T., Z hou , X., A lvarez-B o lad o , G., 2009b. T h e role o f S o n ic h e d g e h o g o f n e u r a l orig in in th a la m ic d iffe ren tia tio n in th e m o u s e . J. N eu ro sc i. 29, 2453-2466 . T a k e za k i, N ., F ig u ero a , F., Z a le sk a -R u tc z y n sk a , Z., T a k a h a ta , N ., K le in , J., 2 0 04 . T h e p h y lo g e n e t ic r e la t io n s h ip o f te tr a p o d , c o e la c a n th , a n d lu n g f is h r e v e a le d b y th e s e q u e n c e s o f fo r ty - fo u r n u c le a r g e n e s . M ol. B iol. E vol. 21 , 1 5 1 2 -1 5 2 4 . v a n d e n A kker, W .M ., Brox, A ., P u elles , L., D u rston , A.J., M ed in a , L., 2008. C om p a ra tiv e fu n c tio n a l a n a ly s is p ro v id es e v id e n c e for a cru cia l ro le for th e h o m e o b o x g e n e N k x2 .1 /T itf-1 in forebrain e v o lu tio n . J. C om p. N eu ro l. 506, 211 -223 . V ieira, C., M artin ez, S., 2006. S o n ic h e d g e h o g from th e b a sa l p la te a n d th e z o n a lim ita n s in tra th a la m ica e x h ib its d ifferen tia l a c tiv ity o n d ie n c e p h a lic m o lecu la r r e g io n a liza tio n a n d n u c lea r stru ctu re . N e u r o sc ie n c e 1 4 3 ,1 2 9 -1 4 0 . V ieira , C., G arda, A.L., S h im a m u ra , K., M artinez, S., 2005. T h a la m ic d e v e lo p m e n t in d u c e d b y S h h in th e ch ick em b ryo . D ev . Biol. 284, 3 5 1 -3 6 3 . V u e, T.Y., A aker, J., T a n ig u c h i, A , K a zem za d e h , C., Sk id m ore , J.M., M artin, D.M ., M artin, J.F., T reier, M., N a k agaw a , Y., 2007. C h a ra cter iza tio n o f p ro g en ito r d o m a in s in th e d e v e lo p in g m o u s e th a la m u s . J. C om p . N eu ro l. 505, 7 3 -9 1 . W u llim a n n , M.F., M ueller, T., 2004. T e le o s te a n a n d m a m m a lia n foreb ra in s c o n tra sted ; e v id e n c e from g e n e s to b eh a v io r . J. C om p. N eu ro l. 475, 1 4 3 -162 . Z eltser , L.M., 2005. S h h -d e p e n d e n t fo r m a tio n o f th e ZLI is o p p o se d b y s ig n a ls fro m th e d o rsa l d ie n c e p h a lo n . D e v e lo p m e n t 132, 2 02 3 -2 0 3 3 . Z eltser, L.M., Larsen, C.W., L u m sd en , A., 2001. A n e w d e v e lo p m e n ta l c o m p a r tm en t in th e forebrain reg u la ted b y L unatic fringe. N at. N eu rosc i. 4 ,6 8 3 -6 8 4 . 81 82 NEUROAIM ATOM Y 3. ESTUDIOS GENOARQUITECTONICOS EN EL ENCEFALO EN DESARROLLO Y ADULTO doi: 10.3389/fnana,2011.00011 Ontogenetic distribution of the transcription factor Nkx2.2 in the developing forebrain of Xenopus laevis Laura Dominguez, Agustin Gonzalez and Nerea Moreno* Faculty of Biology, Department of Cell Biology, University Complutense of Madrid, Madrid, Spain Edited by: Fernando Martinez-Garcia, Universidad de Valencia, Spain Reviewed by: Loreta Medina, Universidad de Lleida, Spain Isabel Rodriguez-Moldes, University of Santiago de Compostela, Spain *Conespondence: Nerea Moreno, Faculty of Biology, Department o f Cell Biology, University Complutense of Madrid, C/José Antonio Novais 2, Madrid E-28040, Spain. e-mail: nerea@bio.ucm.es The expression of the Nkx2.2 gene is involved in the organization of the alar-basal boundary in the forebrain of vertebrates. Its expression in different diencephalic and telencephalic regions, helped to define distinct progenitor donnains in mouse and chick. Here we investigated the pattern of Nkx2.2 protein distribution throughout the development of the forebrain of the anuran amphibian, Xenopus laevis. We used immunohistochemical and in situ hybridization techniques for its detection in combination with other essential territorial markers in the forebrain. No expression was observed in the telencephalon. In the alar hypothalamus, Nkx2.2 positive cells were scattered in the suprachiasmatic territory, but also in the supraopto-paraventricular area, as defined by the expression of the transcription factor Orthopedia (Otp) and the lack of xDII4. In the basal hypothalamus Nkx2.2 expressing cells were localized in the tuberal region, with the exception of the arcuate nucleus, rich in Otp expressing cells. In the diencephalon it was expressed in all three prosomeres (P1-P3) and not in the zona limitans intrathalamica. The presence of Nkx2.2 expressing cells in P3 was restricted to the alar portion, as well as in prosomere P2, whereas in PI the Nkx2.2 expressing cells were located in the basal plate and identified the alar/basal boundary. These results showed that Nkx2,2 and Sonic hedgehog are expressed in parallel adjacent stripes along the anterior-posterior axis.The results of this study showed a conserved distribution pattern of Nkx2.2 among vertebrates, crucial to recognize subdivisions that are otherwise indistinct, and supported the relevance of this transcription factor in the organization of the forebrain, particularly in the delineation of the alar/basal boundary of the forebrain. K eyw ords: p ro s e n c e p h a lo n , h y p o th a la m u s , in situ h y b rid iza tio n , ev o lu tio n , fo reb ra in p a t te rn in g , th a la m u s INTRODUCTION During the last 10 years our understanding o f the organization o f the developing forebrain has dramatically changed, in part as a consequence o f the impressive number o f morphological, chem- oarchitectonic, embryological, and, primarily, genoarchitectonic data. In most o f the recent studies, the columnar conception o f the brain organization (Herrick, 1910) has been frequently challenged by the interpretation o f the data in a current prosomeric model ( Puelles and Rubenstein, 1993,2003), which is emerging as the most useful tool in the evolutionary genoarchitectonic analysis. In this scheme, a prosomere, as all neural segments, is composed o f four longitudinal zones: roof, alar plate, basal plate, and floor. Their boundaries are defined by molecular patterns and cellular fates commonly established by dorso-ventral patterning o f the forebrain wall (Puelles and Rubenstein, 1993,2003) that show an impressive grade o f conservation across vertebrates in morphological and gene expression terms (Puelles et al., 2000; Bachy et al., 2001,2002; Brox et al., 2003, 2004; Moreno et al., 2003, 2004, 2005, 2008a,b, 2010; Osorio et al., 2005,2006; Flames et al., 2007; Bardet et al. 2008,2010; Garcia-Lopez et al., 2008; Abellân and Medina, 2009; Ferran et al., 2009; Dominguez et al., 2010; Morona et al., 2011). In this scenario, the expressions o f key patterning genes involved both in regional and cellular specification processes are currently being analyzed in detail in the main vertebrate models, in order to establish precise traits of the forebrain evolution. Among these developmental regulators is Nkx2.2, a member o f the vertebrate homeodomain transcription factor gene family homologous to the Drosophila NK2/ventral nervous system defective (vnd) gene (Kim and Nirenberg, 1989; Price et al., 1992; Jimenez et al., 1995). It was originally identified as a gene that is expressed in ventral regions of the developing vertebrate central nervous system (Price et al., 1992), and is closely related to Sonic hedgehog (Shh), a powerful mor- phogen that controls progenitor proliferation, regional patterning, and cell fate in the developing brain (for review see Fuccillo et al., 2006). It is involved in a wide range o f regionalization mechanisms including the early specification o f progenitor cell identity and cell fate processes in the ventral neural tube, in response to graded Shh signaling (Briscoe and Ericson, 1999; Briscoe et al., 2000). It is also implicated in the establishment o f the alar-basal boundary ( Puelles and Rubenstein, 1993; Vieira et al., 2005) and in the specification o f the diencephalic patterning, helping to define distinct progeni­ tor domains (Ericson et al., 1997; Vue et al., 2007; Kataoka and Shimogori, 2008; Ferran et al., 2009). Most o f the data about Nkx2.2 expression in the forebrain were obtained in amniotes, primarily mouse, and chick, and only frag­ mentary data on Nkx2.2 expression have been reported in anamni­ otes, especially fishes and amphioxus (Holland et al., 1998; Schafer et al., 2005). Surprisingly, the expression o f Nkx2.2 has not been analyzed in the forebrain o f amphibians, which constitute the only anamniote group o f tetrapods. O f interest, in numerous recent Frontiers in Neuroanatomy www.frontiersin.org M arch 2011 | Volume 5 | Article 11 | 1 83 mailto:nerea@bio.ucm.es http://www.frontiersin.org D om inguez e t al. 3. ESTUDIOS GENOARQUITECTONICOS EN EL ENCEFALO EN DESARROLLO Y ADULTO Nkx2.2 In developing X enopusforebrain studies it is shown that the forebrain organization in amphibians shares many key features with amniotes, mainly in terms o f genetic specification, as revealed when the prosomeric paradigm is used in the interpretation o f many territorial gene expression patterns (Bachy et al., 2001,2002; Brox et al., 2003,2004; Moreno et al., 2004, 2008a,b; van den Akker et al., 2008; Dominguez et al., 2010; Morona et al., 2011 ). In particular, we have recently reported the distribution of xShh in the forebrain o f the anuran amphibian Xenopus laevis during development (Dominguez et al., 2010) and, following this line o f research, herein we have analyzed the pattern o f distribution of Nkx2.2, a functionally and anatomically related transcription factor in vertebrates. The comparative analysis, following the proso­ meric model, serves to assess evolutionary trends. We have charac­ terized phenotypically the developing Nkx2.2 expressing forebrain subdivisions and neurons by means o f the combination o f Nkx2.2 expression with forebrain essential regulators and markers, such as Nkx2.1, Tbrl, Pax6, G ABA, Pax7, Orthopedia (Otp), xDll4, xShh, tyrosine hydroxylase (TH), mesotocin (MST), and somatostatin (SOM). The results of this study showed an extremely conserved distribution pattern o f Nkx2.2 among vertebrates, crucial to delin­ eate subdivisions that were otherwise indistinct, and supported the relevance o f this transcription factor in the establishment and organization o f the forebrain. MATERIALS AND METHODS ANIMALS AND TISSUE PROCESSING For the present study, adults, juveniles, and tadpoles of X. laevisv/ere used. Embryos and larvae were classified according to Nieuwkoop and Faber (1967). Embryonic (42-45), premetamorphic (46-52), prometamorphic (53-58), and metamorphic (59-65) stages were used, minimizing as much as possible the number o f animals used. All animals were treated according to the regulations and laws of the European Union (86/609/EEC) and Spain (Royal Decrees 1201/2005) for care and handling o f animals in research, after approval from the University to conduct the experiments described. Adult Xenopus were purchased from commercial suppliers (Xenopus Express; France), and the different developing specimens were obtained by in vitro fertilization and maintained in tap water at 20°C throughout their development. At appropriate times, embryos and larvae were deeply anesthetized in a 0.4-m g/ml solution of tricaine methanesulfonate (MS222, Sigma-Aldrich, Steinheim, Germany). The adults, juveniles, and late larvae were perfused tran­ scardially with 0.9Ü sodium chloride, followed by cold 4Ü parafor­ maldehyde in a 0.1-M phosphate buffer (PB, pH 7.4). The brains were removed and kept in the same fixative for 2-3 h. Subsequently, they were immersed in a solution o f 30ii sucrose in PB for 4 -6 h at 4°C until they sank, embedded in a solution o f 20Ü gelatin with 30Ü sucrose in PB, and stored for 6 h in a 3.7ii formalde­ hyde solution at 4°C. The brains were cut on a freezing microtome at 40 pm (adults) or 20-30 pm (juveniles and late larvae) in the transverse or sagittal plane and sections were collected and rinsed in cold PB. The embryos and premetamorphic larvae were fixed by immersion overnight at 4°C in MEMFA [0.1 M MOPS (4-mor- pholinopropanesulfonic acid) 2 mM ethyleneglycoltetraacetic acid, 1 mM MgSO^, 3.7Ü formaldehyde], then they were processed in toto and finally sectioned at 14-16 pm thickness in the transverse, horizontal, or sagittal plane on a freezing microtome. IMMÜNOCHEMISTRY Single immunohistochemistry for Nkxl2 A immunohistofluorescence procedure was conducted with the primary antibody on the free-floating sections that, in all cases, was diluted in 5-1 Oil normal serum o f the species in which the second­ ary antibody was raised in PB vrith 0.1 ii Triton X-100 (Sigma) and 2Ü bovine serum albumin (BSA, Sigma).The protocol included two steps, as follows: ( 1 ) Incubation for 72 h at 4°C in the dilution o f the primary antibody mouse anti-Nkx2.2 ( 1:500; Developmental Studies Hybridoma Bank, DSHB, Iowa City, USA. Clone: 74.5A5) and (2) the second incubation was conducted for 90 min at room temperature with the labeled secondary antibody Alexa 488-conjugated goat anti­ mouse (1:500; Molecular Probes; catalog reference: A21042). After being rinsed, the sections were mounted on glass slides and cover- slipped with Vectashield mounting medium (Vector Laboratories, Burlingame, CA, USA; catalog number: HIOOO). Double immunohistochemistry for Nkx2^0tp, Nkx2.Z/MST, Nkx22/ BONI. Nkx22/NkxZ 1, Nkx22/Thrl. NkxZ2/TH, Nkx2^axB. and NkxZ2/GABA The cocktails o f primary antibodies were diluted in PB with O.lü Triton X -100 and used for 60 h at 4°C. They always included mouse anti-Nkx2.2 (1:500; DSHB) in combination with: rabbit anti-Otp (1:1000; produced by “PickCell laboratories” Amsterdam, The Netherlands; according to the protocol described in Lin et al., 1999), rabbit anti-MST (diluted 1:2000; donated by Dr. J. M. Guerné Université de Strasbourg, France), rabbit anti-SOM (1:1000; Incstar, Wisconsin, USA, Code number: 20067), rabbit anti-Nkx2.1 (1:500; Biopat Immunotechnologies, Italy, Code number: PAOlOO), rab­ bit anti-Tbrl (1:250; Santa Cruz Biotechnology, Inc., USA, Code number: sc-48816), rabbit anti-TH (diluted 1:1000; Chemicon International, Inc., USA, Code number: P22941), rabbit anti-Pax6 (1:200; Covance, California, USA, Code number: PBR-278P), and rabbit anti-GABA (1:3000; Sigma-Aldrich, Steinheim, Germany, Code number: A2052). The secondary antibodies were used in appropriated combinations and were: Alexa 488-conjugated goat anti-mouse (1:500, Molecular Probes) and Alexa 594-conjugated goat anti-rabbit (1:500, Molecular Probes; catalog number: A11012). In all cases, secondary antibodies were diluted in PB with O.lü Triton X-100 for 90 min at room temperature. After rinsing, the sections were mounted on glass slides and coverslipped with Vectashield. IN SITU HYBRIDIZATION Double labeling with in situ hybridization and immunohistochemistry: Nkx22fi(Shh and NkxZZ/xDII4 For double histofluorescence labeling experiments, we combined the immunohistochemistry for Nkx2.2 with in situ hybridization for the following markers: xShh (provided by Dr. Randal Moon. University of Washington; Ekker et al., 1995) and xDll4 (provided by Dr. Nancy Papalopulu. University o f Manchester; Papalopulu and Kintner, 1993). For in situ hybridization, which was performed first, antisense dig­ oxigenin (DIG)-labeled riboprobes for these markers were synthesized according to the protocol described in Bachy et al. (2001), linearizing the clones in Bluescript KS with Bam HI (Promega, Madison, USA) and transcribing with T3 (Promega) for xShh, with N otl (Promega, Madison, USA) and T3 (Promega) for xDll4. The embryos and Frontiers in Neuroanatomy www.frontiersin.org M arch 2011 | Volume 5 | Article 11 | 2 84 http://www.frontiersin.org D om inguez e t al. 3. ESTUDIOS GENOARQUITECTONICOS EN EL ENCEFALO EN DESARROLLO Y ADULTO NkxZ.Z In developing X enopusfo rebrain premetamorphic larvae were processed in toto after progressive re­ hydration and pretreatments (see Bachy et al., 2001), and the late larvae were processed in floating sections (see Moreno et al., 2004). Hybridization step was done with 3 pl/ml o f a DIG-labeled RNA probe, in a 50ii formamide containing medium overnight at 55°C. The solution used for prehybridization (at 60°C for 1 h) and hybridization contained 50ü deionized formamide (Fluka, Steinheim, Germany), 5x standard saline citrate (Sigma-Aldrich, Steinheim, Germany), 2ü blocking reagent (Roche Diagnostics, Mannheim, Germany), O.lü Tween-20, 0.5Ü 3-[(3-cholamidopropyl)-dimethylammonio]-l- propanesulfonate (CHAPS; Sigma-Aldrich), I mg/ml o f yeast tRNA (Sigma-Aldrich), 5 mM of ethylenediaminetetraacetic acid (Sigma- Aldrich), and 50 g/ml o f heparin (Sigma-Aldrich) in water. Hybridization was detected using an alkaline phosphatase coupled anti-DIG antibody (Roche Diagnostics, dilution 1:1500). Alkaline phosphatase staining was developed with Fast red tablets (Roche Diagnostics). The in situ hybridization was followed by the immunohistochemistry for mouse anti-Nkx2.2 ( 1:500; DSHB) revealed with Alexa 488-conjugated goat anti-mouse (diluted 1:500, Molecular Probes). Subsequently, embryos and early larvae were immersed in a solution o f 30ü sucrose in PB until they sank, embedded in a solution o f 20ü gelatin and 30ü sucrose in PB, and stored overnight at 4°C in a solution o f 4ü formaldehyde and 30Ü sucrose in PB. Sections were cut at 14-25 pm thickness in the frontal, sagittal, and horizontal plane on a freezing microtome. IMAGING The sections were analyzed with an Olympus BX51 microscope that was equipped for fluorescence with appropriate filter com­ binations. Selected sections were photographed by using a digital camera (Olympus DP72). Contrast and brightness of the phot­ omicrographs were adjusted in Adobe PhotoShop CS3 (Adobe Systems, San Jose, CA) and figures were mounted in Canvas 11 (ACD Systems, Canada). RESULTS The distribution of the Nkx2.2 protein has been analyzed in the prosencephalon o f X. laevis from embryonic stages through the adult. In the following sections we analyze, using both immunohis­ tochemistry and in situ hybridization procedures, the spatio-tem­ poral sequence of Nkx2.2 expression during forebrain development in single (Figure 1) and double (Figures 2-5) stained material. All o f the markers used in combination with Nkx2.2 have been previously used in the analysis o f the Xenopus forebrain devel­ opment, and the nomenclature used in the present study largely follows that used in our preceding mapping studies o f the anuran forebrain (Moreno et al., 2004, 2008a,b; Morona and Gonzalez, 2008; Dominguez et al., 2010; Morona et al., 2011). A schematic representation of the Nkx2.2 distribution in the case o f the early (premetamorphic) forebrain is shown in Figure 6. NkxZ2 DISTRIBUTION IN THE DEVELOPING PROSENCEPHALON In Xenopus, Nkx2.2 immunoreactive (Nkx2.2-ir) cells were not observed in telencephalic areas, neither evaginated nor non- evaginated territories, from early developmental stages through the juvenile, when the brain morphology is close to that observed in adults (Figure 1). The most anterior expression detected was observed in the hypothalamic territory, helping to the identification o f the alar and basal domains (Figures lA-D). Form early (Figures 1A,B) to late (Figures 1C,D) developmental stages, Nkx2.2-ir cells were observed in the supraopto-paraventricular (SPV) region o f the alar hypothalamus (Figures lE-H). At embryonic developmental stages (Figure IE), virtually all the cells observed in the ventricular (vz) and subventricular layers (svz) o f this zone were Nkx2.2-ir, whereas from early larval (Figures 1F,G) through juvenile stages the cells decreased notably occupying a band in the svz (Figure IH). In addition, in the alar hypothalamus scattered Nkx2.2 cells were observed in the suprachiasmatic (SC) territory that became more numerous from late developmental stages and through the adult­ hood (Figures 1A-D,G,H,P). These cells formed a continuous band above the optic chiasm (oc) that was especially evident in sagittal sections (Figures 1C,D). In the basal hypothalamus (BH), scarce Nkx2.2-ir cells were found in the svz o f the medial part o f the tuberal region, whereas the most posterior portion o f this basal region was devoid o f Nkx2.2 positive cells (Figures II-L). Caudally, Nkx2.2 expressing cells were detected in the diencephalon, defining the midbrain-diencephalic boundary (MDB; Figures lA-D) with the exception o f the zona limitans intrathalamica (Zli), which was devoid o f Nkx2.2 expression (Figures 1A-D,M-P). In prosomeres 2 (P2) and 3 (P3) Nkx2.2 positive cells were restricted to the alar portion (Figures IM P). At early stages o f development, these cells were observed in the vz and svz o f P2 and P3, respectively (Figures 1M,N), whereas from prometamorphic stages Nkx2.2-ir cells were only detected in the svz and mz (Figures 10,P). In P I, the Nkx2.2-ir cells were located in the alar/basal boundary, representing the ventral limit o f the pretectum (Figures II-L). NkxZZ DISTRIBUTION IN RELATION TO PROSENCEPHAUC MARKERS In order to further characterize the localization o f Nkx2.2-ir cells within the forebrain, we carried out double labeling experiments throughout development, using different prosencephalic markers (Figures 2-5). To analyze the distribution o f the Nkx2.2-ir cells in the alar hypothalamus we combined Nkx2.2 with the transcription factor Orthopedia (Otp; Figures 2A,B,E), the neuropeptides mesotocin (MST; Figures 2C,F) and somatostatin (SOM; Figure 2D), the dopaminergic marker tyrosine hydroxylase (TH; Figures 2G,H), and the transcription factor xDll4 (Figure 21). From early premeta­ morphic stages, the double staining for Nkx2.2/Otp (Figure 2A) defined the position o f a restricted population o f Nkx2.2 expressing cells in the svz o f the SPV. Whereas almost all the SPV cells expressed Otp (Figure 2B), only a subpopulation o f the SPV cells coexpressed Nkx2.2 and Otp (see arrowhead in Figure 2B), just in the most posterior portion o f the SPV, defining the limit between the SPV and the SC (Figures 2E,E'). This was also confirmed by the double labeling Nkx2.2/MST (Figure 2C) and Nkx2.2/SOM (Figure2D). At SC levels, the double staining for Nkx2.2/MST (Figure 2F), Nkx2.2/TH (Figures 2G,H), and Nkx2.2/xDll4 (Figure 21) con­ firmed the position o f Nkx2.2-ir cells in this territory, anterior to the oc and posterior to the SPV (Figure 2F'). In this area, the Nkx2.2-ir cells were found in the catecholaminergic (Figures 2G,H) and xDll4-expressing region (Figure 21). During the development the Nkx2.2 expressing cells were numerous and from the early larvae Frontiers in Neuroanatomy www.frontiersin.org M arch 2011 I Volume 5 I Article 11 I 3 85 http://www.frontiersin.org D om inguez e t al. 3. ESTUDIOS CjENOAKguriEClUJNlOUS EM E E E M E E P A L U EN DESARROLLO Y ADULTO Nkx2.2 in developing X en o p u s forebrain Embryonic Prem etam orphic Prom etam orphic Juvenile FIG U RE 1 I P h o to m ic r o g r a p h s o f s a g i t t a l (A -D ) a n d t r a n s v e r s e |E -P ) s e c t io n s th r o u g h t h e X e n o p u s fo r e b ra in s u b d iv is io n s a lo n g t h e d if fe re n t r e p r e s e n t a t i v e d e v e lo p m e n ta l s t a g e s . T he sag itta l s e c t io n s s h o w th e a lm o s t c o n tin u o u s N kx2.2 e x p re s s io n from an te rio r h y p o th a lam ic a re a s to th e m o s t caudal reg io n s of th e fo reb ra in (A -D ). N kx2.2 is n o t e x p re s s e d in te le n c e p h a lic a re a s , from em b ry o n ic s ta g e s th ro u g h th e adu lt. N kx2.2 e x p re s s io n s ta r t s in th e SPV te rrito ry of th e alar h y p o th a la m u s (E -H ). In th e b asa l h y p o th a lam u s N kx2.2 e x p re s s io n is re s tr ic te d to th e tu b e ra l h y p o th a la m u s (l-L ). In th e d ie n c e p h a lo n , N kx2.2 is o b s e rv e d in th e th re e p ro s o m e re s (P 1 -P 3 ) an d th e Zli lacks N kx2.2 e x p re s s in g ce lls ( l-P ). A bbrev ia tions: Arc, n u c le u s a rc u a tu s ; BH, b asa l h y p o th a lam u s; MB, m am m illa ry band ; M DB, m id b ra in -d ien cep h a lic boun d ary ; OB, o lfa c to ry bulb; oc, op tic ch iasm ; 01, o p tic te c tu m ; P1-P3, d ie n cep h a lic p ro s o m e re s 1 -3 ; Pa, pallium ; PO C, p reo p tic c o m m issu ra l a re a /c o m m is s u ra l s e p to -p re o p tic a rea ; PO, p reo p tic a rea ; PI, p re te c tu m ; PTh, p re th a la m u s; PThE, p re th a lam ic e m in e n c e ; PV, p a rav e n tric u la r n u c leu s ; SC, su p ra c h ia sm a tic n u c le u s ; SPa, su bpallium ; SPV, su p rao p to -p a ra v e n tr ic u la r a rea ; Tel, te le n c e p h a lo n ; Th, th a la m u s;T R p o s te r io r tu b e rc le ;T u b , tu b e ra l a rea ; Zl, zona in certa ; Zli, zona lim itans in tra tha lam ica . S ca le b ars : 5 0 0 pm (D,L), 2 0 0 pm (C ,G ,H ,K ,0 ,P ), 100 pm (A,B), and 50 pm (E ,F ,I,J,M ,N ). stages these cells showed a clear patterning, however it is from late developmental stages when these cells clearly formed an independ­ ent and exclusive subpopulation in the SC, situated in the ventral portion avoiding the anterior region (see asterisk in Figure 2F'). From early premetamorphic stages, the double staining for Nkx2.2/ Nkx2.1 (Figure 3A) confirmed the Nkx2.2 expression in the svz of the BH. The double staining Nkx2.2/Otp confirmed the position of Nkx2.2 positive cells in the tuberal area of the BH, but avoiding the nucleus arcuatus (Arc), and the mammillary band (MB), both rich in Otp expressing cells (Figures 3B,C). In addition, the lack o f Nkx2.2 expression in the mammillary region was confirmed by the Nkx2.2/ TH (Figure 3D) and Nkx2.2/xDll4 double staining (Figure 3E). Caudally in the diencephalon, the banded staining pattern obtained for the pair N kx2.2/Tbrl confirmed the lack o f Nkx2.2 expressing cells in the prethalam ic eminence (PThE), rich in T b rl expression (Figure 4A), but allowed the identification of Nkx2.2-ir cells in the thalam us and prethalam us. The expres­ sion observed in the alar portion o f P3 and P2 was confirmed by the double staining Nkx2.2/Pax6 (Figure 4B), Nkx2.2/xDll4 (Figure 4C), and Nkx2.2/GABA (Figure 4D). In addition, spe­ cifically the Nkx2.2-ir cells in P3 were noted in the zona incerta (ZI; Figures 4E,F), where Nkx2.1 expression is present (M oreno et al., 2008a) and a conserved catecholaminergic group is located (Smeets and Gonzalez, 2000). The com bination of Nkx2.2/xShh confirm ed the lack o f Nkx2.2 expressing cells in the zona limitans intrathalamica (Zli; Figure 4G) defined by the xShh-expression (Dom inguez et al., 2010). Finally, Nkx2.2 has been functionally related to the m orpho- gen Shh implied in the alar-basal boundary establishment (for review see Fuccillo et al., 2006). We have recently reported the Frontiers in Neuroanatomy www.frontiersin.org M arch 2011 I V olume 5 I A rticle 11 I 4 86 http://www.frontiersin.org D om inguez e t al. 3. ESTUDIOS GENOARQUITECTONICOS EN EL ENCEFALO EN DESARROLLO Y ADULTO Nkx2.2 In developing Xenopus forebrain s t 46 MB Tub Arc \ S t 46 S t 48 Adult d ; d SPV s t 57 I Adult d ■ I • I \ PTh S t 57 s t 48 d Tub oc ' \ SC> - " s c 19 o c ' ' . _ ■ , T . L o L ' 1 oc . V > I I < I FIGU RE 2 I P h o to m ic r o g r a p h s o f s a g i t t a l (A ,D -G ,I) a n d t r a n s v e r s e (B ,C ,E ',H ) s e c t io n s t h r o u g h t h e a la r h y p o th a la m u s i l lu s t r a t in g t h e N kx2 .2 e x p r e s s io n in c o m b in a t io n w i th s e v e ra l h y p o th a la m ic m a rk e r s . T he m o s t a n te rio r N kx2.2 e x p re ss in g ce lls a re o b s e rv e d in th e SPV (the O tp e x p re ss in g territory) during d e v e lo p m e n t (A ,B ,E ,E '), in th e reg ion w h e re it co localizes w ith m e so to c in {C,F,F') an d s o m a to s ta tin (D), b u t only a su b p o p u la tio n of th e SPV ce lls c o e x p re s s N kx2.2 and O tp [s e e a rro w in (B,E,E')1. A t s u p rach ia sm a tic levels, th e N kx2.2 positive ce lls a re lo c a ted in th e ca tech o lam in e rg ic |G ,G ',H ) an d xDII4 (I) e x p re s s in g reg ion , co n s titu tin g an in d e p e n d e n t te rrito ry w ith in th is dom a in (G -l) .T h e yellow lines in (A ,E,G ) ind ica te th e level of th e s e c t io n s of (B ,E ',H ), respective ly . Yellow b o x es in (EG ) ind ica te th e h igher m ag n ifica tio n s sh o w n in (F ',G '), respective ly . A bbrev ia tions a s in F ig u re 1. S cale b ars : 50 0 pm (F), 2 0 0 pm (E ,E ',G ), 100 pm (D,H) and 50 pm (A ,B ,C ,P ,G ',I) precise distribution of xShh in the Xenopus developing forebrain (Dominguez et al., 2010) and in the present study we have ana­ lyzed the extent o f the Nkx2.2 expressing cells along the entire forebrain in com bination with xShh, in transverse (Figures 5A,B) and horizontal sections (Figure 5C) to analyze their relation with the Xenopus forebrain axis. O f note, this double labeling shows that the pattern of distribution of Nkx2.2-ir cells formed a series of bands that extended along the anterior-posterior axis in parallel to adjacent stripes labeled for xShh (Figure 5). All the results obtained from the double labeling analysis confirmed the localization of the Nkx2.2-ir cells in the regions described above and summarized in Figure 6. DISCUSSION Nkx2.2 was the first gene of the NK2 hom eobox class to be dem ­ onstrated in all the deuterostomes, showing hom ology in all the models analyzed. Even possible homologies were suggested in the nervous system between invertebrates and vertebrates given the high conservations in the expression and function of this gene (Holland et al., 1998). The presence o f two paralogs, Nkx2.2a and Nkx2.2b, was dem onstrated in zebrafish and phylogenetic and expression analysis suggests that these genes arose by a fish-specific gene duplication and acquired differential transcriptional control. Subsequently one paralog was lost and only the Nkx2.2a is con­ sidered ortholog o f the mammal gene (Schafer et al., 2005) and, in addition, its expression dom ain is very conserved. However, both paralogs are regulated by Shh (Barth and Wilson, 1995; Schafer et al., 2005). In general terms, in the vertebrate central nervous system Nkx2.2 has been implicated in the ventral neuronal patterning, at early developmental stages (Briscoe and Ericson, 1999), defining the alar/ basal boundary along to Shh-expression (Puelles and Rubenstein, 1993; Vieira et al., 2005). This is found even in amphioxus, in which there is not obvious anatomical boundary separating alar and basal regions along the dorso-ventral axis o f the cerebral vesicle (Holland et al., 1998). Frontiers in Neuroanatomy www.frontiersin.org M arcfi2011 I V olume 5 j Article 11 I 5 87 http://www.frontiersin.org D om inguez e t al. 3. ESTUDIOS GENOARQUITECTONICOS EN EL ENCEFALO EN DESARROLLO Y ADULTO Nkx2.2 In deve lop ing Xenopus forebrain St 45 PTh St 57 Th, - -Zli FIG U R E 3 I P h o to m i c r o g r a p h s o f t r a n s v e r s e (A ,C ,D ) a n d s a g i t t a l (B,E) s e c t i o n s th r o u g h t h e b a s a l h y p o th a la m u s i l lu s t r a t in g t h e N k x 2 .2 e x p r e s s io n in c o m b in a t io n w i th s e v e r a l h y p o th a la m ic m a rk e r s . T he Nkx2.1 e x p re s s io n co n firm s th a t th e lim ited N kx2.2 e x p re s s io n fo u n d in th e b asa l h y p o th a lam ic te rr ito ry is s itu a te d in th e tu b e ra l reg ion (A), re s tr ic te d to th e an te r io r p a r t [a s te risk in (B)[ a n d avoid ing th e n u c le u s a rc u a tu s , la b e led fo r O tp (B,C), a n d th e m am m illa ry ban d , d e f in e d byTH (D) an d xDII4 (E ,E '). Yellow box in e in d ic a te s th e a re a s h o w n in h ig h e r m agn ifica tio n in e '. A b b rev ia tio n s a s in F ig u re 1. S ca le bars : 100 pm (B.E), 50 pm (A ,C ,D ,E '). s t 48 \P T h E PTh S t 46 Th PTh Tub ' I S t 48 z t i ' J h I St 46 T h , . - ' / , Zli ' A PTh L _ r . s c ; PO I ' . . ! o c • ' D E FIGU RE 4 1 P h o to m ic r o g r a p h s o f t r a n s v e r s e (A,E,F,G) a n d s a g i t ta l (B,C,D) s e c t io n s t h r o u g h t h e d ie n c e p h a lo n i l lu s tr a t in g t h e N k x 2 .2 e x p r e s s io n in c o m b in a t io n w i th d iv e r s e d ie n c e p h a i ic m a rk e rs . The p re th a lam ic e m in e n c e , rich inT brI (A), w a s devoid of N kx2.2 ex p re s s io n , in c o n tra s t to th e p re th a la m u s , d e f in ed by th e Pax6 (B) and xDII4 e x p re s s io n s (C ).T he yellow box in d s h o w s a h ighe r m agn ifica tion of th e Nkx2.2/GABA d o u b le labeled ce lls found in th e th a la m u s [ s e e a rro w h e a d s in (D ,D ')l.T he d o u b le labeling Nkx2.2/N kx2.1 (E) an d N kx2.2/TH (F) d efin e th e position o f th e N kx2.2 ce lls in th e zona incerta o f th e p re th a lam u s. The xShh d ie n cep h a lic e x p re s s io n d e fin e s th e lack of N kx2.2 ex p re s s io n in th e Zli {zona linnitans intrathalamica) |G ). A bbrev ia tions a s in F ig u re 1. S ca le bars: 100 pm (B,C,D), 50 pm (A,E,F,G), 25 pm (D ) Frontiers in Neuroanatomy www.frontiersin.org M arch 2011 | V olume 5 I Article 11 j 6 88 http://www.frontiersin.org D om inguez e t al. 3. ESTUDIOS GENOARQUITECTONICOS EN EL ENCEFALO EN DESARROLLO Y ADULTO Nkx2.2 In developing Xenopus forebrain S t 46 Z lir \ SPV , ' PTh ; FIGURE 5 I P h o to m ic ro g ra p h s o f t r a n s v e r s e (A,B) a n d h o r iz o n ta l (C) s e c t io n s t h r o u g h t h e fo re b ra in i l lu s tra t in g N kx2 .2 e x p re s s io n in c o m b in a t io n w i th x S h h . The e x p re ss io n s of N kx2.2 and xShh ex ten d along th e am erio r-p o ste rio r axis in parallel ad jacen t s tr ip e s , form ing longitudinal co lum ns along th e forebrain. A bbreviations a s in F ig u re 1. S cale bars: 50 pm . N kx2 .2 EXPRESSION IN THE FOREBRAIN AND COM PARISON W ITH OTHER VERTEBRATES: CONSERVATIVE TRAITS AND EVOLUTIONARY IMPORTANCE In all vertebrates analyzed Nkx2.2 is expressed in the prosen­ cephalon where it is known to be involved in the specification of progenitor domains and in establishing regionalization patterns. Moreover, it contributes to the differentiation of distinct areas and their compartmentalization, as well as to the acquisition of cel­ lular identity and the regulation of the distribution of the earliest neurors in the brain (Wilson et al., 1993; Barth and Wilson, 1994; Vieira ind Martinez, 2006; Vue et al., 2007). Thus, the analysis in anamn ote and amniote vertebrates of the spatio-temporal distri­ bution of this transcription factor is considered o f relevance in the unders'.anding of the establishment of the different subdivisions within the forebrain and in evaluating the degree of conserva­ tion across vertebrates (Price et al., 1992; Barth and Wilson, 1995; Holland et al., 1998; Schafer et al., 2005; Vieira and Martinez, 2006; Vue et al., 2007; Ferran et al., 2009). In the present study we have provided for the first tim e in amphibians a detailed analysis of its distribution in X laevis, a tetrapod anamniote with a forebrain organization that in genoarchitectonic terms shares many features with its counterpart in amniotes (Bachy et al., 2001, 2002; Brox et al., 2003, 2004; M oreno et al., 2004, 2008a,b; van den Akker et al., 2008; Dominguez et al., 2010; M orona et al., 2011). To fully understand the precise topological distribution of Nkx2.2 expres­ sion, its com bination with the respective expression of different forebrain markers has been shown to be extremely useful. Thus, we have analyzed the distribution of Nkx2.2 in combination with the localization o f Nkx2.1, Otp, Pax6, GABA, T brl, TH, MST, and SOM, and in situ hybridization for the detection of xShh and xDll4. The expression o f Nkx2.1 has been deeply analyzed in the pros­ encephalon of many vertebrates and it has served to identify the preoptic (PO), SC, and tuberal territories (Tub) during develop­ m ent (Rohr et al., 2001; Gonzalez et al., 2002a,b; M oreno et al., 2008a; van den Akker et al., 2008), like the developmental regulatory gene xDll4 (Papalopulu and Kintner, 1993; Puelles and Rubenstein, 1993; Shimam ura and Rubenstein, 1997; Puelles et al., 2000; Bachy et al., 2002; M arin et al., 2002; Brox et al., 2003). In addition, the localization of TH highlighted the regionalization of the SC and the ZI in P3 (Gonzalez et al., 1993,1994; Milan and Puelles, 2000; Smeets and Gonzalez, 2000). Specifically in the hypothalamus, O tp expression served in the identification of the SPV region and it also defines the Arc and the MB within the BH (Bardet et al., 2008; Dominguez et al., 2010). The presence of MST (or its homolog oxytocin) and SOM in the magnocellular neurons of the SPV is also a shared feature between amniotes and anamniotes (Blasher and Heinrichs, 1982; Gonzalez et al., 1995,2003; Petkô and Orosz, 1996; Lopez et al., 2007). In the diencephalon, the morphogen xShh was crucial to delimit the Zli, as well as to define the alar-basal bound­ ary and the longitudinal columns that establish the dorso-ventral patterning (Puelles and Rubenstein, 2003; Dominguez et al., 2010). The homeobox gene T brl was extremely useful in the recognition of the PThE (Puelles et al., 2000; Brox et al., 2004) and the relative expression of xDll4, Pax6, and GABA served to identify the bounda­ ries of the three diencephalic prosomeres (Bachy et al., 2002; Brox et al., 2003; M oreno et al., 2008b; M orona et al., 2011). Telencephalon In mammals, the m ost anterior Nkx2.2 expression was observed in the medial ganglionic eminence (MGE), also called pallidal domain, which consists of, at least, five progenitor domains glo­ bally defined by the strong expression of Nkx2.1, weak expression of Nkx2.2, and lack of Pax6 and Shh-expressions (Flames et al., 2007). In the case of the chicken, three subdivisions were recently identified in the pallidal domain, comparable to those described for mammals (Abelian and Medina, 2009). In chicken, the Nkx2.2 expression was not described in detail but it seems to be Weakly expressed in the subpallium (see Figures 4F,G in Vieira et al., 2005 and see Figures 1 K,Q in Bardet et al., 2010) in contrast to the strong expression found in mammals (Flames et al., 2007). Differently, in Frontiers in Neuroanatomy www.frontiersin.org M arch 2011 I V olum e 5 I Article 11 I 7 89 http://www.frontiersin.org D om inguez e t al. j . U U l U i b U t i N U / V K V ^ u i i i i X i U i N i L ^ w ; ^ n iN c .l . r . i N ^ j 3 r / \ i ^ v j EN DESARROLLO Y ADULTO Nkx2.2 In d eve lop ing Xrnopius forebrain S c h e m a tic r e p r e s e n ta t io n s , pr e m e t a m o r ph ic s t a g e s a ico en b asa PTh ' FIGURE 6 I S c h e m a t ic d ra w in g s o f s a g i t ta l a n d c o ro n a l s e c t io n s t h r o u g h a p r e m e ta m o r p h ic b ra in o f Xenopus laevis il lu s tr a t in g t h e d is tr ib u tio n o f N kx2 .2 e x p re s s in g z o n e s (g re e n re g io n s in t h e s a g i t ta l v ie w ) a n d c e lls (g re e n d o t s in t h e c o ro n a l v ie w ) a lo n g t h e fo re b ra in . T he app ro p ria ted levels of the coronal s e c t io n s a re ind ica ted in th e sagittal view . A bbreviations a s in F ig u re 1. Xenopus (present results) and in the turtle (Moreno et al., 2010) Nkx2.2 expression has not been observed in the svz of the MGE. This interesting result might be in correlation with the presence of Shh-expression in the subventricular zone o f the mouse MGE (Garcia-Lopez et al., 2008) and chicken (Abelian and Medina, 2009) and its absence in the pallidal region of amphibians (Dominguez et al., 2010). However, Shh and Nkx2.2 are not always expressed in the same regions. Also in the telencephalon, the PO region in mammals contains at least two distinct progenitor dom ains (Flames et al., 2007). The ventricular zone of the PO is uniquely defined by the simultaneous expression o f Nkx2.1, Nkx2.2, and Shh, and m ost prom inently by the lack o f detectable levels of Gsh2, Lhx6, Lhx7, or 01ig2 expression (Flames et al., 2007). Like in the case of the MGE, in Xenopus (present results) bu t also in turtle (M oreno et al., 2010), there is no t Nkx2.2 expression in the POA. However, in this case it is not correlated with the lack of the Shh-expression, because Shh-expression is observed in the preoptocom m issural area (Dom inguez et al., 2010). The boundary between the telencephalon and the hypothalamus has been recently defined, first in chicken (Bardet et al., 2006) and later in mouse (Flames et al., 2007), as the preoptohypothalamic zone (POH). It constitutes the border of the subpallium and was described by its characteristic and specific expression of Nkx2.2 as a narrow territory separating longitudinally the PO firom the magnocellular hypothalamus (Bardet et al., 2006). This region contains progenitor cells that express Dlx2, Dlx5, Pax6, Qlig2, and Gsh2, lacks expression of Nkx2.1, Shh, Nkx6.2, and Dbxl and can be further subdivided into two areas because the POH 1, but not the POH2, expresses high levels of Nkx2.2 in mammals (Flames et al., 2007; Bardet et al., 2010). In Xenopus, Nkx2.2 expression is not detected in a comparable region, given that the most anterior expression found coincides with the Otp SPV expressing zone within the hypothalamus (see below). Hypothalamus The hypothalam us constitutes a very com plex structure, as regards its functionality, cell complexity, hodology, develop­ m ent, and m orphology. Therefore, it is no t surprising that the Frontiers in Neuroanatomy www.frontiersin.org M arch 2011 | V olum e 5 | Article 11 | 8 90 http://www.frontiersin.org D om inguez e t al. 3. ESTUDIOS GENOARQUITECTONICOS EN EL ENCEFALO EN DESARROLLO Y ADULTO Nkx2.2 In developing Xenopus forebrain elucidation o f its genoarchitecture is particularly im portant to understand how is generated and maintained the diversity in the hypothalamus. That is one of the reasons that makes fas­ cinating the hypothalamus and, thereby, the focus of analysis in many comparative studies. This has as its counterparts the numerous studies in which the nomenclature and interpreta­ tion of the nuclei and boundaries are constantly under debate (Szabô et al., 2009b; Bardet et al., 2010; Dominguez et al., 2010; Shimogori et al., 2010). On the basis of combined expression analysis, at least two dif­ ferent longitudinal alar domains have been proposed in the chick hypothalamus: the Dix- and Shh-negative SPV area, which lies under the border of the FoxGl-positive telencephalic field, and the subparaventricular area, which lies under it and is adjacent to the Shh-positive basal plate, and expresses Dlx5 and Nkx2.2 (Bardet et ah, 2010). In Xenopus, the most anterior Nkx2.2 posi­ tive cells were localized in a Shh-/Nkx2. l-/xDll4—/Otp-t- territory forming a thin strip of cells that delimit the region just anterior to the SC (present results) and their localization within this distinct region is highlighted because some of these cells contain both Otp and Nkx2.2. The SC region is complex in terms of nuclei organization that most likely reflects a complex genetic specification. In general, the SC is defined as a Dlx-expressing zone in all vertebrates analyzed (Bachy et ah, 2002; Brox et ah, 2003; Puelles and Rubenstein, 2003; Flames et ah, 2007; present results). In mammals, this region does not express Nkx2.1 and Shh, in contrast to the situation in other amniotes (Medina, 2008) and anamniotes (Medina, 2008; Moreno et ah, 2008a; Dominguez et ah, 2010). In particular, although these two markers were not specifically described in the SC region of the chick, their presence can be inferred from the published map­ ping studies (compare Figures 61,1 from Puelles et ah, 2000 and Figures 1D,P,F,J from Bardet et ah, 2010) suggesting possible evo­ lutionary differences between birds and mammals in this area and/ or the existence of different progenitor domains. The SC of X. laevis has been carefully studied in the last years, mapping the distribution of numerous markers that identified different regions within this territory. In general terms, mainly on the basis of calcium binding proteins expression, rostral, and caudal SC zones were defined (Milan and Puelles, 2000; Morona and Gonzalez, 2008). In addition, based on the expression of neuropeptide Y and TH (Kramer et ah, 2001) three differ­ ent nuclei were identified, the ventrolateral, dorsomedial, and caudal nuclei. All of them differ from each other in location, shape, number of cells, and function (Kramer et ah, 2001) and, therefore, it is logic to think that also in genetic specification. In terms of genoarchitecture, the expression of several genes of the LIM-homeodomain family appears to define distinct territories within this SC area (Moreno et ah, 2004, 2008a). Among others, Isletl is expressed in a cell population that forms a curved band surrounding externally the Nkx2.1/Shh vz expression, i.e., the only area where Nkx2.2, TH, and xGAD67/xDll4 populations seem to be intermingled (present results; Brox et ah, 2003; Moreno et ah, 2008a; Dominguez et ah, 2010). Noteworthy, it is known that Nkx2.2 and Shh are involved in the control of the development of midbrain dopaminergic neurons (Prakash and Wurst, 2006) and, given the close spatial relationship between Shh, Nkx2.2, and TH expressing neurons in the SC of Xenopus, a possible implication of Shh/Nkx2.2 may exist for the acquisition of the dopaminergic phenotype in this region. Finally in the BH, like in the rest of the forebrain regions, the elucidation of genetic combinatorial expression patterns has served to characterize and define regions, and their comparative analysis in different vertebrates has been used as a useful tool to establish their grade of conservation. Thus, it was defined that Nkx2.1 expression in the BH is required to maintain molecular characteristics o f the developing hypothalamus (Kimura et al., 1996; Takuma et al., 1998; Sussel et al., 1999; Marin et al., 2002; van den Akker et al., 2008), but other members of this gene family also seem to have roles in hypothalamic formation because, for example, Nkx2.2 or Nkx6.1 m utant mice have ventral to dorsal transformations (Briscoe and Ericson, 1999; Sander et al., 2000). In addition, in all vertebrates studied the transcription factor Otp is expressed in the arcuate nucleus and the oblique perimammil- lary band of the BH (Simeone et al., 1994; Puelles and Rubenstein, 2003; Del Giacco et al., 2006; Bardet et al., 2008), where con­ tributes to progenitor cell proliferation, survival, and migration (Goshu et al., 2004), and operates in the proper differentiation of several neurohormone-secreting nuclei (Acampora et al., 1999, 2000; Wang and Lufkin, 2000; Michaud, 2001; Blechman et al., 2007; Eaton and Glasgow, 2007; Ryu et al., 2007; Del Giacco et al., 2008; Eaton et al., 2008). Additionally, the activity of Otp is essential for the induction of the dopaminergic phenotype in the hypothalamus of vertebrates (Del Giacco et al., 2008). In Xenopus, the mammillary area has been defined by TH and dopamine or histamine immunohistochemistry (Airaksinen and Panula, 1990; Gonzalez et al., 1993,1994; Milan and Puelles, 2000) and further identified by the expression of Otp (Dominguez et al., 2010) and the lack of Nkx2.2 (present results). In contrast, the Nkx2.2 com ­ binatorial expression patterns identified different zones within the Nkx2.1 expressing tuberal hypothalamus in Xenopus: an inter­ mediate Nkx2.2-i-/Otp- zone and the Nkx2.2-/Otp-l- zone that constitutes the arcuate nucleus (Bardet et al., 2008; Dominguez et al., 2010; present results). Of note, the lack of Nkx2.2 expressing cells in the most posterior region of the tuberal area, coincides with a total absence of xShh-expression in the same territory (Dominguez et al., 2010). Diencephalon In the last 10 years an impressive number of morphological, chemo- and genoarchitectural data has helped to the interpreta­ tion in the diencephalon of the boundaries, extent of the areas and identification of distinct nuclei and subnuclei. These data have often been gathered not only for mammals but also in a number of non-mammalian species and the evolutionary perspec­ tive shows the impressive degree of conservation of this forebrain region, not only regarding the expression of different genes but also their functions. In all vertebrates analyzed, the alar diencephalon early develops into three major neuroepithelial domains along the anterior-posterior (A/P) axis, known as the prethalamus (P3), the thalamus (P2), and the pretectum (PI), and it is limited by two boundaries, the most anterior one that lies along the supraopto- mammillary region and the posterior one that lies between the pretectum and the mesencephalon (Puelles and Rubenstein, 2003; Frontiers in Neuroanatomy www.frontiersin.org M arch 2011 I Volume 5 I Article 11 I 9 91 http://www.frontiersin.org Dom inguez e t al. 3. ESTUDIOS GENOARQUITECTONICOS EN EL ENCEFALO EN DESARROLLO Y ADULTO Nkx2.2 In developing Xenopus forebrain Moreno et al., 2004, 2008b; Vieira et al., 2005; Ferran et al., 2009; Morona et al., 2011 ). Also in all the vertebrates exists the Zli as a secondary morphogenetic organizer in diencephalic histogenesis that appears as a transverse ventricular ridge between the pre­ thalamus and the thalamus from neural tube stages (Echevarria et al., 2003; Puelles and Rubenstein, 2003). The final position, boundaries, organizations, and functions are in the end defined by signaling molecules such as Shh or FgfB (Echevarria et al., 2003; Vieira et al., 2005, 2010; Kataoka and Shimogori, 2008), which act regulating the expression of developmental genes that will specify compartmentalization and cell fate in the diencephalon (Echevarria et al., 2003; Hashimoto-Torii et al., 2003; Kiecker and Lumsden, 2004). In mammals, from early embryonic stages Nkx2.2 is expressed in a distinct pattern in all three diencephalic prosomeres (Price et al., 1992; Puelles and Rubenstein, 2003; Vue et al., 2007; Kataoka and Shimogori, 2008) and is induced by Shh, which diffuses from the ventral territory and from the Zli, and is directly or indirectly repressed by Pax6 (Pratt et al., 2000; Kiecker and Lumsden, 2004). However, the interaction Pax6-Nkx2.2 it is not simple and recipro­ cal given that the downregulation of Pax6 in the posterior thalamus is not followed by the upregulation of Nkx2.2 (Pratt et al., 2000). In many studies, the influence of Shh over Nkx2.2 has been dem­ onstrated (Barth and Wilson, 1995; Kiecker and Lumsden, 2004; Vieira and Martinez, 2006), however there are also evidences about their independence in different models. This is the case of the mam­ malian Nkx2.2 expression in the posterior region of the Zli, which is under the control of Fgf8 and not Shh (Kataoka and Shimogori, 2008), but also in the cave-living form of the teleost fish Astyanax mexicnnus, which shows a clear expansion of Shh-expression at the ventral midline but is not correlated with a larger Nkx2.2 expression domain; in contrast to other transcription factors expressed in the forebrain such us Nkx2.1 (Menuet et al., 2007). In the diencephalon of mouse and chicken, the gene Nkx2.2 is expressed during development in a rostroventral band of the tha­ lamus (Puelles and Rubenstein, 1993; Martinez-de-la-Torre et al., 2002). On the basis of the Nkx2.2 expression, a distinct progenitor domain has been characterized in the developing thalamus (also marked by Mash 1 ; Vue et al., 2007; Kataoka and Shimogori, 2008), which is controlled by FGF signaling, with independence of the Shh activity (Kataoka and Shimogori, 2008). Later in the thalamic development, the expressions o f Nkx2.2 and Gad67 were detected in different nuclei (Vue et al., 2007; Kataoka and Shimogori, 2008), specifically in the posterior ventral lateral geniculate nucleus (vLGN), concluding that at least a portion of the vLGN, classically a considered a prethalamic nuclei (Kitamura et al., 1997), belongs to the set of retinorecipient thalamic nuclei that do not project to the cortex (Paxinos, 1994) and arises from the thalamic domain (Vue et al., 2007; Kataoka and Shimogori, 2008). In the chick, the combined expression of Gbx2, Nkx2.2, and Pax6 (Martinez-de-la-Torre et al., 2002), the cadherin expression (Redies, 2000), and fate maps analysis (Garcia-Lopez et al., 2004) allowed the identification in the thalamus of four subdivisions, the anter- oventral, dorsal, intermediate, and ventral regions. Interestingly, Nkx2.2 expression was only detected into the anteroventral region (adjacent to the Zli) and its derivatives, among which are the set retinorecipient thalamic nuclei (Martinez-de-la-Torre et al., 2002) that express Nkx2.2 and are proposed as thalamic dérivâtes (Vue et al., 2007). In Xenopus, we have found an extended Nkx2.2 positive region along the developing thalamus that might define a distinct progeni­ tor domain whose cells would contribute to the formation o f other diencephalic nuclei. The Nkx2.2 thalamic expression is observed in different nuclei expressing diverse transcription factors such as x-Lhx2/9 (Moreno et al., 2004) and scattered GAD67 (Brox et al., 2003). In addition, it is suggested that in Xenopus the Nkx2.2 gene is a good candidate involved in the acquisition of the GABAergic phenotype in the thalamus, the pretectum, and the basal plate of the caudal diencephalon, where there is not Dll expression (Brox et al., 2003; present results). In mammals and chick it has been proposed that the area adjacent to the Shh-expression of the Zli is induced by Shh to express Nkx2.2, and this area has been proposed to be the source of the subpopulation of GABAergic interneurons observed in the thalamus (Fode et al., 2000; Martinez-de-la-Torre et al., 2002; Puelles et al., 2004). In this context, we have observed Nkx2.2/GABA double labeled cells in the thalamus (present results), a region devoid of D114 expression (Brox et al., 2003; present results), suggesting that Nkx2.2 could be implicated in the acquisition of the GABAergic phenotype, like in amniotes. Interestingly, also in Xenopus, Nkx2.2 expression is observed in the prethalamus, and its precise localization is corroborated by the colocalization of the Nkx2.2 expressing cells in the territory of P3 that is xDll4-t-/Nkx2. l+/Pax6-l-/TH-(-/xShh-l- (present results; Bachy et al., 2002; Brox et al., 2003; Moreno et al., 2008a,b; Dominguez et al., 2010). As regards the Zli, it expresses Shh in ail vertebrates analyzed and is involved in the correct acquisition of the P2 and P3 gene expres­ sion and regionalization pattern (Braun et al., 2003; Hashimoto- Torii et al., 2003; Kiecker and Lumsden, 2004; Scholpp et al., 2006; Szabô et al., 2009a). In this context, Nkx2.2 expression in the thalamus and prethalamus is induced by Shh secreted by Zli cells (Kiecker and Lumsden, 2004; Vieira et al., 2005; Vieira and Martinez, 2006). Thus, the xShh-expression in the Zli found in Xenopus suggests that also in amphibians Shh is likely involved in the specification of the diencephalic territory (Dominguez et al., 2010), and that xShh-expression in the Zli could lead to establish the Nkx2.2 expression pattern in the P2/P3 territory of amphib­ ians, like in amniotes. Finally, the Nkx2.2 expression observed in the thalamus of the mouse and chicken continues into PI (Puelles and Rubenstein, 1993; Martlnez-de-la-Torre et al., 2002). Studies about the pretectal molecular regionalization in chick and Xenopus have described that the precise Nkx2.2 expression within PI marks the alar/basal boundary, representing a tentative ventral limit of the pretectum (Ferran et al., 2009; Morona et al., 2011; present results). ACKNOWLEDGMENTS This work supported by grants from Spanish MICINN and the UCM (Grant numbers: BFU2009-12315 and BSCH-UCM GR58/08). We are grateful to Dr. Ruth Morona for the fruitful discussions about the diencephalic regionalization and to Dr. Jesus M. Lôpez for the critical reading of the manuscript. Frontiers in Neuroanatomy www.frontiersin.org M arch 2011 | Volume 5 | Article 11 | 10 92 http://www.frontiersin.org D om inguez e t al. 3. ESTUDIOS GENOARQUITECTONICOS EN EL ENCEFALO EN DESARROLLO Y ADULTO developing x e n o p u s forebrain REFERENCES A bellén , A., a n d M ed in a , L. (2009). S ubdivisions a n d derivatives o f the chicken subpallium based on expres­ sion o f LIM and o ther regulatory genes an d m arkers o f n e u ro n subpopu la­ tio n s du rin g developm ent. /. Comp. NeuroL 515 ,465-501. A c a m p o ra , D ., P o s t ig lio n e , M . P., Avantaggiato, V., Di Bonito, M., and Sim eone, A. (2000). T he role o f O tx and O tp genes in b ra in development. Int. J. Dev. Biol. 44 ,669-677. A c a m p o ra , D ., P o s t ig lio n e , M . P., A van tagg ia to , V., D i B on ito , M ., V accarino, F. M ., M ichaud , J., and S im e o n e , A. (1 9 9 9 ). P rog ressive im pairm en t o f developing neuroen­ docrine cell lineages in the hypo tha­ lam us o f mice lacking the orthopedia gene. Genes Dev. 13,2787-2800. Airaksinen, M. S., and Panula, P. (1990). C om parative n eu ro an a to m y o f the h is tam ine rg ic system in the b ra in o f th e frog Xenopus laevis. J. Comp. N eurol 292,412-423. Bachy, I., B erthon , J., a n d R étaux, S. (2002). Defining palliai and subpallial divisions in the developing Xenopus forebrain. Mech. Dev. 117, 163-172. Bachy, I., V ernier, P., a n d R étaux , S. (2001). The L IM -hom eodom ain gene fam ily in the dev e lo p in g Xenopus brain : conservation and divergences w ith the m ouse related to the evolu­ tion o f the forebrain . /. Neurosci. 21, 7620-7629. B arde t, S. M ., C obos, I., P uelles, E., M a r tin e z -D e -L a -T o rre , M ,, a n d Puelles, L. (2006). Chicken lateral sep­ tal organ and o ther circum ventricular o rgans form in a striatal subdom ain abu tting the m olecular striatopallidal border./. Comp. Neurol. 499,745-767. B arde t, S. M „ F erran , J. L., Sanchez- A rrones, L , and Puelles, L. (2010). O n to g e n e tic e x p re ss io n o f son ic hedgehog in the chicken subpaUium. Front. Neuroanat. 4:28. doi: 10.3389/ fiiana.2010.00028 Bardet, S. M., M artinez-de-la-Torre, M., N orthcutt, R. G., Rubenstein, J. L., and Puelles, L. (2008). Conserved pattern o f OTP-positive cells in the paraven­ tricu la r nucleus and o ther hypo tha­ lamic sites o f tetrapods. Brain Res. Bull 75,231-235. B arth , K. A., an d W ilson, S. W. (1994). Specification o f neuronal identity in the em bryonic CNS. Semin. Dev. Biol. 5 ,349-358. B arth , K. A., a n d W ilson, S. W. (1995). Expression o f zebrafish nk2.2 is influ­ enced by sonic hedgehog/vertebrate h edgehog -1 an d dem arcates a zone o f n eu ro n a l d iffe re n tia tio n in th e em bryon ic fo reb ra in . Development 121,1755-1768. Blasher, S., an d H ein richs, M . (1982). Im m unoreactive neu ro p ep tid e sys­ tem s in avian em bryos (dom estic m al­ lard, dom estic fowl, Japanese quail). Cell Tissue Res. 223,287-303. Blechman, J., Borodovsky, N ., Eisenberg, M ., Nabel-Rosen, H ., G rim m , J., and Levkowitz, G. (2007). Specification o f h y p o th a lam ic n e u ro n s by d ua l regulation o f the hom eodom ain p ro ­ te in o r th o p e d ia . Development 134, 4417-4426. B raun , M . M ., E theridge, A., B ernard , A., R obertson, C. P., and Roelink, H. (2003). W nt signaling is requ ired at d istinct stages o f developm ent for the induction o f the posterior forebrain. Development 130,5579-5587. Briscoe, J., an d Ericson, J. (1999). T he specification o f neuronal identity by g raded son ic hedgehog signalling . Semin. Cell Dev. Biol. 10,353—362. Briscoe, J., Pierani, A., Jessell, T. M ., and Ericson, J. (2000). A hom eodom ain protein code specifies progenitor cell identity and neuronal fate in the ven­ tral neural tube. Cell 101,435—445. Brox, A., Puelles, L., F erreiro , B., an d M edina, L. (2003). Expression o f the genes GAD67 and distal-less-4 in the forebrain o f Xenopus laevis confirm s a co m m o n p a tte rn in te trap o d s. J. Comp. Neurol. 461, 370-393. Brox, A., Puelles, L„ Ferreiro , B„ and M edina, L. (2004). Expression o f the genes E m x l,T b r l ,a n d Eom es (Tbr2) in the telencephalon o f Xenopus laevis confirm s the existence o f a ventral pal­ liai division in all te trapods. /. Comp. Neurol 474,562-577. Del G iacco, L., P istocch i, A., C ote lli, F., F ortunato , A. E., an d Sordino , P. (2008). A peek inside the neurosecre­ to ry brain through orthoped ia lenses. Dev Dyn. 237,2295-2303. Del G iacco, L., S ord ino , P., P istocchi, A., A ndreak is , N ., T arallo , R., Di Benedetto, B., and Cotelli, F. (2006). D i f f e r e n t ia l r e g u la t i o n o f th e zebrafish o rth o p e d ia 1 gene du rin g fate d e te rm in a tio n o f d iencephalic neu rons . BMC Dev. B iol 6, 50. doi: 10.1186/1471-213X-6-50 D om inguez, L , Gonzalez, A., and M oreno, N. (2010). Sonic hedgehog expression during Xenopus laevis forebrain devel­ opm ent. Brain Res. 1347,19-32. E a ton , J. L., an d G lasgow , E. (2007). Zebrafish orthopedia (o tp) is required for iso tocin cell developm ent. Dev. Genes Evol 217,149-158. Eaton, I. L., H olm qvist, B., and Glasgow, E. (2008). O n togeny o f vaso toc in - expressing cells in zebrafish: selective requ irem en t for th e tran sc rip tiona l reg u la to rs o r th o p e d ia a n d single- m inded 1 in the p reop tic area. Dev Dyn. 237, 995-1005. Echevarria, D., V ieira, C., G im eno, L , and M artinez, S. (2003). N euroepithelial seco n d ary o rgan izers an d cell fate specification in the developing brain . BrainRes. BrainRes. Rev. 43 ,179-191 . Ekker, S. C., M cGrew, L. L., Lai, C. J., Lee, J. J., von Kessler, D. P., M oon , R. T., an d Beachy, P. A. (1995). D is tin c t expression a n d sh ared ac tiv ities o f m em bers o f the hedgehog gene fam ­ ily o f Xenopus laevis. Development 121, 2337-2347. E ricson , J., R ashbass , P., S ched l, A., B renne r-M orton , S., K aw akam i, A., van H eyningen, V , Jessell, T. M ., an d Briscoe, J. (1997). Pax6 contro ls p ro ­ genitor cell identity an d neuronal fate in response to graded Shh signaling. Cell 9 0 ,169-180. Ferran, J. L., de Oliveira, E. D., M erchân, P., Sandoval, J. E., S ânchez-A rrones, L., M artinez-D e-L a-T orre , M ., a n d Puelles, L. (2009). G enoarchitectonic profile o f developing nuclear g roups in the chicken p re tec tu m . /. Comp. Neurol 517,405—451. F lam es, N ., P la, R., G e lm an , D. M ., R u b en ste in , J. L., P uelles, L., a n d M arin, O. (2007). D elineation o f m u l­ tip le subpallia l p ro g en ito r d o m a in s by the com b in a to ria l expression o f transcrip tional codes. ]. Neurosci. 27, 9682-9695. Fode, C., M a, Q ., C asarosa, S., A ng, S. L., A nderson, D. J., and G uillem ot, F. (2000). A role for neural de te rm in a­ tion genes in specifying the dorsoven- tral identity o f telencephalic neurons. Genes Dev. 14 ,67-80 . Fuccillo, M ., R utlin, M ., an d Fishell, G. (2006). Removal o f Pax6 partially res­ cues the loss o f ventral s truc tu res in Shh null mice. Cereb. Cortex 16( Suppl. 1), 96-102. G arcia-L opez, M ., A belian , A., Legaz, I., R ubenste in , J. L., Puelles, L., an d M edina, L. (2008). H istogenetic com ­ partm ents o f th e m ouse centrom edial and extended am ygdala based o n gene expression pattern s du rin g develop­ m ent. / Comp. NeuroL 506 ,46-74 . Garcia-Lopez, R„ V ieira, C., Echevarria, D., and M artinez, S. (2004). Fate m ap o f the diencephalon an d the zona lim i­ tans at the 10-som ites stage in chick em bryos. Dev. Biol 268 ,514-530 . G onzalez, A., Lôpez, J. M ., a n d M arin , O. (2002a). Expression pattern o f the ho m eo b o x p ro te in NKX 2-1 in th e developing Xenopus forebrain. Brain Res. Gene Expr. Patterns 1 ,181-185. G onzalez , A ., L ôpez, J. M ., S anchez- C am acho, C., and M arin, O. (2002b). Regional expression o f the hom eobox gene NKX2-1 defines pallida l a n d interneuronal populations in the basal ganglia o f am phib ians. Neuroscience 114, 567-575. Gonzâlez, A., M arin, O., Tuinhof, R., and Sm eets, W. J. (1994). O n to g en y o f catecholam ine systems in the central nervous system o f anu ran am phibians: an im m unohistochem ical study w ith antibodies against tyrosine hydroxy­ lase a n d dopam ine. J. Comp. Neurol 346 ,63-79 . Gonzâlez, A., M oreno, N ., M orona, R., and Lôpez, J. M . (2003). Som atostatin-like im m unoreactiv ity in the brain o f the u rodele am ph ib ian Pleurodeles waltl C olocalization w ith ca techolam ines a n d n i t r ic ox id e . Brain Res. 965, 246-258. G onzâlez, A., M unôz, A., M unôz, M ., M arin , O ., an d Sm eets, W. J. (1995). O ntogeny o f vasotocinergic and meso- tocinergic systems in th e brain o f the S outh A frican claw ed frog Xenopus laevis.}. Chem. Neuroanat. 9 ,27-40 . G onzâlez, A., T uinhof, R., and Smeets, W. J. (1993). D istribu tion o f tyrosine hydroxylase an d dopam ine im m u n o ­ reactivities in the b ra in o f the South A frican claw ed frog Xenopus laevis. Anat. Embryol (Berl) 187,193-201. G oshu, E., Jin, H., Lovejoy, J., M arion, J. E, M ichaud, J. L., and Fan, C. M. (2004). Sim2 con tribu tes to neuroendocrine horm one gene expression in the ante­ rio r hypo thalam us. M ol Endocrinol 18,1251-1262. H ashim oto-T orii, K., M otoyam a, J., Hui, C. C., Kuroiwa, A., N akafiiku, M ., and S h im am ura , K. (2003). D ifferential ac tiv ities o f son ic hedgehog m e d i­ a ted by G li tr a n s c r ip t io n fac to rs define d istinct neu ronal subtypes in the dorsal tha lam us. Mech. Dev. 120, 1097-1111. H errick, C. J. (1910). T he M orphology o f the forebrain in am phibia and reptilia. J. Comp. Neurol 20 ,413-547. H olland, L. Z., Venkatesh, T. V , G orlin, A., Bodmer, R .,and H olland, N. D. ( 1998). C haracterization an d developm ental expression o f A m phiN k2-2 , an NK2 class hom eobox gene from Amphioxus. (phylum chordata; subphylum cepha- lo c h o rd a ta ). Dev. Genes Evol. 208, 100-105. Jiménez, E , M artin -M orris, L. E., Velasco, L , C hu, H ., Sierra, J., Rosen, D. R., and W hite, K. ( 1995). vnd, a gene required for early neurogenesis o f Drosophila, encodes a h o m e o d o m a in p ro te in . £M B O /. 14 ,3487-3495. K ataoka, A., a n d Shim ogori, T. (2008). FgfS contro ls regional identity in the deve lop ing th a lam u s. Development 135,2873-2881. K iecker, € . , a n d L u m sd en , A. (2004). H edgehog sign a lin g fro m th e ZLI regulates diencephalic regional iden­ tity. Nat. Neurosci. 7 ,1242-1249. K im , Y., a n d N ire n b e rg , M . (1989). D ro so p h ila N K -h o m e o b o x genes. Frontiers in Neuroanatomy www.frontiersin.org M arch 2011 I V olum e 5 I Article 11 I 11 93 http://www.frontiersin.org D om inguez e t al. 3. ESTUDIOS GENOARQUITECTONICOS EN EL ENCEFALO EN DESARROLLO Y ADULTO Nkx2.2 In developing Xenppusforebrain Proc. Natl. Acad. Sci. U.S.A. 86, 7716-7720. Kimura, S., H ara, Y., Pineau, T., Femândez- Salguero, P., Fox, C. H ., W ard, J. M ., a n d G onzâlez, F. J. (1996). T he T / e b p n u ll m o u se : th y ro id -sp e c if ic enhancer-b inding pro te in is essential for the organogenesis o f the thyroid, lung, ventral forebrain, and pituitary. Genes Dev. 10 ,60-69. K itam ura, K., M iura, H ., Yanazawa, M ., M iyashita, T., and Kato, K. (1997). E xpression p a tte rn s o f B rx l (R ieg g e n e ), so n ic h ed g e h o g , N k x 2 .2 , D lx l an d A rx d u rin g zona lim itans in trathalam ica and em bryonic ventral lateral geniculate nuclear form ation . Mech. Dev. 67 ,83-96 . Kramer, B. M ., W elting, ]., Berghs, C. A., Jenks, B.G., an d Roubos, E. W. (2001 ). Functional organization o f the sup ra ­ chiasm atic nucleus o f Xenopus laevis in relation to background adaptation. /. Comp. Neurol. 432,346-355. Lin, X., State, M. W., V accarino, F. M ., Greally, J., Hass, M., and Leckman, J. P. (1999). Identification, chrom osom al assignm ent, and expression analysis o f the hum an hom eodom ain containing gene O rthopedia (OTP). GenomicsSO, 96-104. Lôpez, J. M ., M oreno, N ., M orona, R., M u n ô z , M ., D o m in g u e z , L., a n d Gonzâlez, A. (20071. D istribu tion o f som atostatin-like im m unoreactiv ity in the brain o f the caecüian Dermophis mexicanusi am phibia: gym nophiona) : com parative aspects in am phibians. /. Comp. Neurol. 501,413-340. M arin , O ., Baker, J., P uelles, L., a n d Rubenstein, J. L. (2002). Patterning o f the basal telencephalon and h ypo tha­ lam us is essential for guidance o f cor­ tical p ro jections. Development 129, 761-773. M artinez-de-la-Torre, M ., G arda, A. L., Puelles, E., an d Puelles, L. (2002). Gbx2 expression in the late em bryonic chick dorsal thalam us. Brain Res. Bull. 57,435-438. M edina, L. (2008).“Evolution and em bry­ ological developm ent o f forebrain,” in Enciclopedic Reference ofNeuroscience, eds M . D. B inder and N . H irokaw a (Springer-Verlag), 1172-1192. M enuet, A., A lunni, A., Joly, J. S., Jeffery, W.R., and Rétaux, S. (2007). Expanded exp ress ion o f son ic h ed g e h o g in Astyanax cavefish: m u ltip le conse­ quences on forebrain deve lopm en t a n d ev o lu tio n . Development 134, 845-855. M ichaud, J. L. (2001 ). The developm ental p rogram o f the hypothalam us and its disorders. Clin. Genet. 60 ,255-263. M ilân,F. J.,and Puelles, L. (2000). Patterns o f calretinin, calbindin, and tyrosine- hydroxylase expression are consistent w ith the prosom eric m ap o f the frog d iencephalon . J. Comp. Neurol. 419, 96-121. M oreno , N ., Bachy, I., R étaux , S., an d G onzâlez, A. (2003). Palliai origin o f m itral cells in the o lfactory bulbs o f Xenopus. Neuroreport 14,2355-2358. M o re n o , N ., B achy, I., R é ta u x , S., a n d G o n z â le z , A. (2 0 0 4 ) . L IM - h o m e o d o m a in genes as d ev e lo p ­ m ental an d adu lt genetic m arkers o f Xenopus forebrain functional subd i­ visions. J. Comp. Neurol. 472 ,52-72 . M o re n o , N ., Bachy, I., R é ta u x , S., a n d G o n z â le z , A. (2 0 0 5 ) . L IM - h o m e o d o m a in genes as te r r i to ry m arkers in the brainstem o f adult and developing Xenopus laevis. J. Comp. Neurol. 485,240-254. M oreno, N ., D om inguez, L., Rétaux, S., and Gonzâlez, A. (2008a). Isletl as a m arker o f subdivisions an d cell types in the developing forebrain o f Xenopus. Neuroscience 154,1423-1439. M oreno, N ., Rétaux, S., and G onzâlez, A. (2008b). Spatio-tem poral expression o f Pax6 in Xenopus forebrain . Brain Res. 1239,92-99. M oreno, N., M orona, R., Lôpez, J.M ., and G onzâlez, A. (2010). Subdivisions of the tu rtle Pseudemys scripta subpal­ lium based on the expression o f regu­ latory genes and neuronal m arkers. /. Comp. NeuroL 518,4877—4902. M orona , R., F erran , J. L., Puelles, L., and Gonzâlez, A. (2011). Em bryonic g en o a rc h ite c tu re o f p re te c tu m in Xenopus laevis: a conserved pattern in te trap o d s. /. Comp. Neurol. 519, 1024-1050. M orona, R., a n d G onzâlez, A. (2008). C a lb in d in -D 2 8 k a n d c a lr e t in in expression in the forebrain o f anuran and urodele am phibians: fu rthe r sup ­ p o rt for newly identified subdivisions. /. Comp. Neurol. 511 ,187-220. N ie u w k o o p , P. D ., a n d F a rb e r , J. (1967). Norm al Table o f Xenopus laev is (D a u d in ) . A m s te rd a m : N orth-H olland . O so rio , J., M azan , S., a n d R étaux , S. (2005). O rganiza tion o f the lam prey ( Lampetra fluviatilis) em bryonic brain: in sigh ts from L IM -h o m eo d o m ain , Pax an d hedgehog genes. Dev Biol. 288,100-112. O sorio, J., Megfas, M., Pom bal, M . A., and R étaux, S. (2006). D ynam ic expres­ sion o f the L IM -hom eodom ain gene L hxl5 th rough larval b ra in develop­ m en t o f the sea lam prey (Petromyzon m arinus). Gene Expr. Patterns 6, 873-878. Papalopulu, N ., an d K intner, C. (1993). Xenopus distal-less related hom eobox genes are expressed in the developing forebrain an d are induced by planar signals. Development 117,961-975. Paxinos, G. ( 1994). Atlas o f the Developing R at Nervous System. N ew York: A cadem y press. P e tk ô , M ., a n d O ro sz , V. (1 9 9 6 ) . D istribution o f som atostatin-im m u- noreactive s truc tu res in the cen tra l ne rv o u s system o f the frog, Rana esculenta.}. Himforsch. 37,109-120. Prakash, N ., and Wurst, W. (2006). Genetic networks controlling the developm ent o f m idbrain dopam inergic neurons. J. Physiof. 575,403-410. P ratt, T., V italis, T., W arren, N ., Edgar, J. M ., M ason, J. O., an d Price, D. J. (2000). A role for Pax6 in the norm al developm ent o f dorsal thalam us and its cortical connections. Development 127,5167-5178. Price, M., Lazzaro, D., Pohl, T., M attei, M. G., Riither, U., Olivo, J. C., D uboule, D., and Di Lauro, R. (1992). Regional expression o f th e ho m eo b o x gene N kx-2.2 in the developing m am m a­ lian forebrain. Neuron 8 ,241-255. P uelles, L., K uw ana, E., P uelles, E., Bulfone, A., Shimamura, K., Keleher, J., Smiga, S., and Rubenstein, J. L. (2000). Palliai and subpallial derivatives in the em bryonic chick and m ouse te l­ encephalon, traced by the expression o f the genes Dlx-2, Emx-1, Nkx-2.1, Pax-6, an d T br-1 . J. Comp. Neurol. 424, 409-438. Puelles, L., M artinez, S., M artinez-de-la- Torre, M ., and Rubenstein, J. L. (2004). “G ene m aps and related histogenetic dom ains in the forebrain and m id ­ b ra in ,” in The Rat Nervous System, 3rd Edn, ed. G. Paxinos (San Diego: Elsevier), 3-25. Puelles, L., and Rubenstein, J. L. (1993). Expression patterns o f hom eobox and o ther putative regulatory genes in the em bryonic m ouse forebrain suggest a neu ro m eric o rgan iza tion . Trends Neurosci. 1 6 ,4 7 2 ^ 7 9 . Puelles, L., and Rubenstein, J. L. (2003). F orebrain gene expression dom ains an d the evolving prosom eric m odel. Trends Neurosci. 26 ,469-476. Redies, C. (2000). Cadherins in the central nervous system. Prog. Neurobiol. 61, 611-648. Rohr, K. B., Barth, K. A., Varga, Z. M ., and W ilson, S. W. (2001 ). The nodal p a th ­ way acts upstream o f hedgehog sign­ aling to specify ventral telencephalic identity. Neuron 29 ,341-351. R yu, S., M ah le r, J., A cam p o ra , D ., H olzschuh, J., Erhardt, S., O m odei, D„ Sim eone, A., and Driever, W. (2007). O rthoped ia hom eodom ain pro te in is essential for diencephalic d o p am in ­ ergic neuron developm ent. Curr. Biol. 17,873-880. Sander, M ., Paydar, S., Ericson, J., Briscoe, J., Berber, E., G erm an, M., Jessell, T. M ., and Rubenstein, J. L. (2000). Ventral neural patterning by N kx hom eobox genes: N kx6.1 cœitrois som atic m otor neu ron and ventral in tem euron fetes. Genes Dev. 14,2134-2139. Schafer, M „ Kinzd, D., Neimer, C., Schartl, M ., VolfF, J. N .,and W inkler, C . (2005). H edgehog and retinoid signalling con­ fines nkx2.2b expression to th e lateral flo o r p la te o f d ie zebra fish tru n k . Mech. Dev. 122,43-56. S cholpp , S., W olf, O ., B rand , M ., an d Lum sden, A. (2006). H edgehog signal­ ling from the zona lim itans in tra th a­ lamica orchestrates pa ttern ing o f the zebrafish diencephalon. Development 133,855-864. S h im a m u ra , K., a n d R u b e n s te in , J. L. (1 9 9 7 ). Induc tive in te ra c t io n s d ire c t early reg ionaliza tion o f the m ouse forebrain. Development 124, 2709-2718. S h im o g o ri, T , Lee, D. A ., M ira n d a - Angulo, A., Yang,Y., Wang, H., Jiang, L , Yoshida, A. C., Kataoka, A., M ashiko, H ., Avetisyan, M., Q i, L., Q ian, J., and Blackshaw, S. (2010). A genom ic atlas o f m ouse hypothalam ic developm ent. Nat. Neurosci. 13,767-775. Sim eone, A., D’Apice, M . R., N igro, V., Casanova, J., Graziani, E , A cam pora, D., a n d A van tagg iato , V. (1994 ). O r th o p e d ia , a n o v e l h o m e o b o x - c o n ta in in g gene exp ressed in th e developing CNS o f b o th m ouse and Drosophila. Neuron 13,83—101. Smeets, W. J., and G onzâlez, A. (2000). Catecholam ine systems in the brain o f vertebrates: new perspectives th rough a com parative approach. Brain Res. Brain Res. Rev. 33,308-379. Sussel, L., M arin , O., K im ura , S., and R u b en s te in , J. L. (1 9 9 9 ). Loss o f N kx2.1 h o m eobox gene fu n c tio n results in a ventral to dorsal m olecular respecification irith in the basal te len­ cephalon: evidence for a transfo rm a­ tion o f the pallidum in to the striatum . Development 126,3359-3370. Szabô, N . E., Zhao, T., Z hou , X ., an d Alvarez-Bolado, G. (2009a). T he role o f sonic hedgehog o f neural origin in thalam ic differentiation in the m ouse. /. Neurosci. 29,2453-2466. Szabô, N. E., Zhao, X, Cankaya, M ., Theil, T., Z hou, X., and Alvarez-Bolado, G. (2009b). Role ofneuroepithelial sonic hedgehog in hypothalamic patterning. /. Neurosci. 29 ,6989-7002. T akum a, N ., Sheng, H . Z ., F u ru ta , Y., W ard, J. M ., Shaima, K., H ogan, B. L., Pfaff, S. L., Westphal, H ., K im ura, S., and M ahon, K. A. (1998). Form ation o f R a th k e ’s p ouch req u ire s d u a l in d u c tio n from th e d iencephalon . Development 125,4835-4840. van den Akker, W. M , Brox, A., Puelles, L , D urston , A. J., and M edina, L (2008). C om parative functional analysis pro Frontiers in Neuroanatomy www.frontiersin.org March 2011 | Volume 5 | Article 11 | 12 94 http://www.frontiersin.org Dominguez e t al. 3. ESTUDIOS GENOARQUITECTONICOS EN EL ENCEFALO EN DESARROLLO Y ADULTO Nkx2.2 In developing Xenopus forebrain vides evidence fr a crucial role for the hom eobox gae Nkx2.1/Titf-1 in forebrain evolutin. /. Comp. Neurol. 506,211-223. Vieira, C , G arda, AL., S h im am ura, K., an d M artin ez , S (2005). T ha lam ic d e v e lo p m e n t id u c e d by Shh in th e ch ick em b ro . Dev. Biol. 284, 351-363. V ie ira , C ., a n d M rtln e z , S. (2006 ). Sonic hedgehog om the basal plate and the zona lim ans in trathalam ica exhibits differenal ac tiv ity on d ie n ­ cephalic m o lec ia r reg ionaliza tion an d nuclear s tru tu re . Neuroscience 143,129-140. V ieira, C., P om bero , A., G arcia-Lopez, R., G im eno , L., E chevarria , D., a n d M artinez, S. (2010). M olecular m echa­ nism s controlling brain developm ent: an overview o f neuroep ithelia l sec­ o n d a ry organizers. Int. J. Dev. Biol. 54 ,7 -20 . V ue, T. Y., A aker, J., T a n ig u ch i, A ., K azem zadeh, C ., S k idm ore , J. M ., M artin , D. M ., M artin , J. P., Treier, M ., a n d N ak ag aw a , Y. (2 0 0 7 ) . C h a r a c te r iz a t io n o f p ro g e n i to r d o m a in s in th e deve lop ing m ouse thalam us. J. Comp. Neurol. 505,73-91. W ang, W., an d Lufkin, T. (2000). T he m urine O tp hom eobox gene plays an essential ro le in the specification o f neuronal cell linet^es in the developing hypothalam us. Dev. B io llT l,432-449. W ilson, S. W , Placzek, M ., an d Furley, A. (1993). Border disputes: do b ounda­ ries play a role in grow th-cone gu id ­ ance? Trends Neurosci. 16,316-322. C o n flic t o f In te r e s t S ta te m e n t: T he authors declare tha t the research was con­ ducted in the absence o f any com mercial o r financial re lationships th a t could be construed as a potential conflict o f interest. Received: 05 November 2010; paper pend­ ing published: 29 December 2010; accepted: 16 February 2011; published online: 02 March 2011. Citation: Dominguez L, Gonzâlez A and Moreno N (2011 ) Ontogenetic distribu­ tion o f the transcription factor Nkx2.2 in the developing forebrain o f Xenopus lae­ vis. Front. Neuroanat. 5:11. doi: 10.3389/ fnana.2011.00011 Copyright © 2011 Dominguez, Gonzâlez and Moreno. This is an open-access article subject to an exclusive license agreement between the authors and Frontiers Media SA, which permits unrestricted use, distri­ bution, and reproduction in any medium, provided the original authors and source are credited. Frontiers In Neuroinatomy www.frontiersin.org M arch 2011 I Volume 5 I Article 11 I 13 95 http://www.frontiersin.org 96 4. El hipotâlamo en la transîcion anamnio-amnîota: estudîos en anuros y reptiles Characterization of the alar hypothalamus of Xenopus laevis during development by molecular marker analysis. Journal of Comparative Neurology (En preparacion) Characterization of the basal hypothalamus of Xenopus laevis during development by molecular marker analysis. Journal of Comparative Neurology (En preparacion) Subdivisions of the turtle Pseudemys scripta hypothalamus based on the expression of regulatory genes and neuronal markers Journal of Comparative Neurology. DOI: 11-0165.22762 J. n,L, nirvj 1 la A iA vimiw- AMNIOTA: ESTUDIOS EN ANUROS Y REPTILES THE JOURNAL OF COMPARATIVE NEUROLOGY 000: 00-00 (2012) Characterization of the alar hypothalamus of Xenopus laevis during development by molecular marker analysis LAURA DOMINGUEZ, RUTH MORONA, AGUSTIN GONZALEZ, NEREA MORENO* Departamento de Biologia Celular, Facultad de Biologia, Universidad Complutense, 28040, Madrid, Spain A BSTRA CT The patterns of expression of a set o f conserved developmental regulatory transcription factors and neuronal markers were analyzed in the alar hypothalamus o f Xenopus laevis throughout development Combined immunohistochemical and in situ hybridization techniques were used for the identification of subdivisions and their boundaries. The alar hypothalamus was located rostral to the diencephalon in the secondary prosencephalon and represents the rostral continuation of the alar territories of the brainstem and diencephalon, according to the prosomeric model. It is composed by the supraoptoparaventricular (dorsal) and the suprachiasmatic (ventral) regions, and limits dorsally with the preoptic region, caudally with the prethalamic eminence and the prethalamus, and ventrally with the basal hypothalamus. The whole supraopto-paraventricular area was defined by the Otp expression and it could be subdivided into rostral and caudal portions, on the basis o f the distinct Nkx2.2 expression only in the rostral portion. This region is the source of many neuroendocrine cell populations that were primarily found in the rostral subdivision. The suprachiasmatic region was characterized by the D114/Isll expression and, it was also subdivided into rostral and caudal portions, based on the expression of Nkx2.1/Nkx2.2 and Lhxl/7 exclusively in the rostral portion. All these data support that the topology and molecular specification of the unraveled subdivisions of the anuran alar hypothalamus possess many features shared with their counterpart in amniotes, likely controlling similar reflexes, responses, and behaviors. Keywords: Development, preoptic, supraoptoparaventricular, suprachiasmatic, tetrapods, homology, in situ hybridization, evolution, forebrain patterning The hypothalamus is a fundamental component of the brain of all vertebrates that is critically involved in the regulation of endocrine functions, control o f autonomic reactions, and generation o f basic behavioral patterns (Nieuwenhuys et al., 1998; Bruce, 2008; Hodos, 2008). Within the morphological organization of the forebrain analyzed in comparative anatomical studies (Herrick, 1910; Kuhlenbeck, 1954), and attending to mere topographical positions, the hypothalamus was considered to be the ventralmost part of the diencephalon. Actually, the diencephalon was traditionally subdivided into four dorsoventrally arranged zones (as observed in conventional transverse brain sections): the epithalamus, the dorsal thalamus, the ventral thalamus, and the hypothalamus. However, the current notion in neuromorphological research focus on topological rather than topographical relations (Puelles, 2001; Puelles and Medina, 2002) and topological relations of forebrain subdivisions become clear if the strong curvature o f the neural tube at the junction o f the midbrain and forebrain is taken into consideration. Thus, modem synthetic models such as the prosomeric one (Puelles and Rubenstein, 1993, 2003; Puelles, 1995, 2001) that take into account topological relations and the combinatorial analysis o f expression patterns in the developing forebrain have excluded the hypothalamus from the diencephalon, which is formed by three neuromeres (prosomeres 1-3). The rostralmost (prechordal) forebrain is designated as the secondary prosencephalon and it possibly represents a single prosomere that contains the hypothalamus (rostral to the diencephalic P3), the telencepalon impar and the telencephalic hemispheres (Puelles and Rubenstein, 2003). The interpretation of the parts o f the secondary 99 3 . E L m r U l A L A M U EJN L A iK A N S iC lU N A N A M N IU - AMNIOTA: ESTUDIOS EN ANUROS Y REPTILES prosencephalon is fraught with difficulties mainly derived from the early optic and hemispheric évaginations and the different degree of development across vertebrates that disturb the primary pattern of this region (Nieuwenhuys et al., 2008). However, morphological and molecular results have progressively contributed to highlight the organization o f the main parts o f the secondary prosencephalon and its subdivisions (Bulfone et al., 1993; Puelles, 1995, 2001, Puelles and Medina, 2002; Puelles and Rubenstein 1993, 2003; Rubenstein and Beachy, 1998; Rubenstein et al, 1998; Nieuwenhuys et al., 2008; Medina, 2008; Moreno and Gonzalez, 2011). Most important information is derived from the fact that genes involved in CNS development are often expressed in morphologically restricted patterns and the boundaries of their expression frequently coincide with those of the morphological units. In recent years, considerable progress has been made in identifying these morphological units in representatives of different vertebrate classes on the basis o f shared patterns of combinatorial gene expressions, which are highly conserved in evolution. Thus the analysis o f the molecular neural regionalization in the developing diencephalon mainly agrees with the proposed molecular maps and seems to be strictly conserved across vertebrates (Figdor and Stem, 1993; Rubenstein et al., 1994; Puelles and Rubenstein, 2003; Medina, 2008; Moreno and Gonzalez, 2011). Current concepts on hypothalamic organization consider it as being topologically ventral to the telencephalic preoptic area and rostral to P3, the rostral diencephalic prosomere that contains the prethalamic eminence and the prethalamus (formerly named ventral thalamus). The longitudinal domains o f the alar and basal plates, which extend along the neuraxis, also extend in the hypothalamus and the alar-basal boundary was considered to end rostrally just behind the optic chiasm in all vertebrates (Puelles, 1995), although this notion has recently been challenged by the combinatorial analysis of expression pattems during development (Diez-Roux et al., 2011). In the highly complex anatomy of the hypothalamus, distinct regions are considered that can be summarized into two main alar components, the supraoptoparaventricular area (SPV) and the suprachiasmatic area (SC), and two basal components, the tuberal hypothalamus and the mammillary band (reviewed in Medina, 2008, Moreno and Gonzalez, 2011). Most data about the organization and molecular specification o f the hypothalamic regions have been obtained in amniotes, particularly in mice where controversial models have been postulated for the regionalization and topology (Figdor and Stem, 1993; Puelles and Rubenstein, 2003; Shimogori et al., 2010). Only in a few studies similar data have been provided for the hypothalamus of nonmammalian amniotes (Abelian and Medina, 2009; Bardet et al., 2010; Moreno et al., 2010, 2011b) and sparse and fragmentary data are available for anamniotes (Blechman et al., 2007; Machluf et al., 201 IDel Giacco et al., 2006; 2008). Notably, recent studies in amphibians have revealed strikingly conserved pattems of combinatorial gene expression in morphological units of the prosencephalon, as compared to amniotes (Brox et al., 2003; Moreno et al., 2004; 2008a; Dominguez et al., 2010; 2011). In accordance with Puelles et al. (2007), we consider that brain development pattems studied comparatively, essentially molecular regionalization and brain histogenesis, provide fundamental cues for setting analysis o f any vertebrate brain into proper evolutionary perspective. In addition, chemoarchitecture and hodology represent essential complementary approaches for defining homologies across vertebrates. In this context, the present study aims to clarify the main genoarchitectonic and neurochemical features o f the alar hypothalamic regions in anuran amphibians, in an attempt to support their homology with their counterparts in amniotes. Together with the SPV and the SC, we have included in our analysis the preoptic area (PO), which flanks the rostral end of the third ventricle, topologically dorsal to the SPV. Although this region is o f telencephalic origin (Flames et al., 2007; Garcia-Lopez et al., 2008; Medina, 2008; Sânchez-Arrones et a l, 2009; Moreno and Gonzâlez, 2011), we have analyzed its main developmental properties because it is stmcturally and functionally tied in with the hypothalamus and has classically been considered a “rostral” hypothalamic region. The approaches used in our study include the analysis in Xenopus laevis during development and in the adult o f the pattem of distribution o f main regulatory transcription factors and proteins involved in neural patteming and that are also expressed after development (Moreno et a l, 2011a). Toward this aim, we have analyzed the expression pattem of different transcription factors (Isletl, xLhxl, xLhx5, xLhx7,xLhx9, Nkx2.1, Nkx2.2, Otp, xShh, and Tbrl), and different active substances used as neuroanatomical markers, such as y-aminobutyric acid (GABA), mesotocin (MST), somatostatin (SOM) and tyrosine hydroxylase (TH). The markers used have been selected because of their known specific expression in distinct developing hypothalamic portions, and the combination of all markers highlighted boundaries, nuclei, and morphogenetic domains and was extremely useful for assessing shared features and differences with other vertebrates. In our accompanying paper, we have conducted a similar analysis o f the basal subdivisions of the hypothalamus (Dominguez et a l , 2012b). MATERIALS AND METHODS Animals and tissue processing For the present study, series o f tadpoles o f Xenopus laevis, staged according to Nieuwkoop and Faber (1967) and sorted into embryonic (4 2 ^ 5 ) and premetamorphic (46-52), prometamorphic (53-58), and 100 J. LL mrWlALAiVlVJ nil'll LA 1 JVAî aiLlUi'̂ Al̂ AlViHll̂ - AMNIOTA: ESTUDIOS EN ANUROS Y REPTILES Abbreviations AH alar hypothalamus SC suprachiasmatic region BH basal hypothalamus SCc caudal suprachiasmatic region MeA medial amygdale SCr rostral suprachiasmatic region MGE medial ganglionic eminence SPa subpallium oc optic chiasm SPV supraoptoparaventricular region Pa pallium SPVc caudal supraoptoparaventricular region PO preoptic region SPVr rostral supraoptoparaventricular region POC commissural preoptic area Th thalamus PTh prethalamus Zli zona limitans intrathalamic PThE prethalamic eminence metamorphic (59-65), stages were used. In addition, postmetamorphic juveniles specimens were used. All animals were treated according to the regulations and laws of the European Union (86/609/EEC) and Spain (Royal Decree 1201/2005) for care and handling of animals in research, after approval from the University to conduct the experiments described. All animals were obtained from the laboratory stocks of the Department of Cell Biology, University Complutense of Madrid, and the different developing specimens were obtained by Pregnyl- induced (Organon) breeding and maintained in tap water at 20°C throughout their development. At appropriate times, embryos, larvae, and juveniles were deeply anesthetized by immersion in a 0.3% solution of tricaine methanesulfonate (MS222, Sigma-Aldrich, Steinheim, Germany), pH 7.4, and used for the different sets of experiments. The number of animals used in the present study was the minimum to guarantee the correct interpretation of the results and to minimize their suffering. Immunohistochemistry To characterize the PO, SPV and SC chemically, immunohistochemistry for the detection of y- aminobutyric acid (GABA), Islet-1 (Isll), mesotocin (MST), Nkx2.1, Nkx2.2, orthopedia (Otp), somatostatin (SOM), Tbrl and tyrosine hydroxylase (TH) was conducted (Table 1). Juveniles and late larvae were perfused transcardially with 0.9% NaCl solution, followed by 100-200 ml o f 4% paraformaldehyde in 0.1 M phosphate buffer (PB; pH 7.4). The brains were removed and kept in the same fixative overnight at 4°C. Subsequently, they were immersed in a solution of 30% sucrose in PB for 5 hours at 4°C until they sank, embedded in a solution o f 20% gelatin with 30% sucrose in PB, and then immersed in a 3.7% formaldehyde solution at 4°C for 8-10 hours. The brains were cut on a freezing microtome at 25-30 pm in the transverse or sagittal plane and collected and rinsed in cold PB. The embryos and premetamorphic larvae were fixed by immersion overnight at 4”C in MEMFA (0.1 M MOPS [4-morpholinopropanesulphonic acid], 2 mM ethylene glycol tetraacetic acid, 1 mM MgS04, 3.7% formaldehyde) and then they were processed in toto. Finally, the brains were gelatin blocked (as detailed above) and cut on a freezing microtome at 14-16 pm in the transverse, horizontal, or sagittal plane (see Dominguez et al., 2010,2011). Immunohistofluorescence procedures were carried out on the free-floating sections (or in toto for embryos and young larvae) as follows: 1). First, incubation was conducted for 60 hours at 4°C in the dilution of each primary serum (see Table 1): rabbit anti-GABA, mouse anti-lsll, rabbit anti-MST, rabbit anti-Nkx2.1, mouse anti-Nkx2.2, rabbit anti-Otp, rabbit anti-SOM, rabbit anti-Tbrl, mouse anti-TH, rabbit anti-TH. 2). According to the species in which the primary antibody was raised, the second incubations were conducted with the appropriately labeled secondary antibody diluted 1:500 for 90 minutes at room temperature: Alexa 594- conjugated goat anti-rabbit (Molecular Probes, Eugene, OR; catalog reference A11037) or Alexa 488- conjugated goat anti-mouse (Molecular Probes; catalog reference A21042). In all cases, the antibodies were diluted in PB and containing 0.5-1% Triton X-100. After being rinsed, the sections were mounted on glass slides and coverslipped with Vectashield mounting medium (Vector, Burlingame, CA; catalog No. HIOOO). To study the relative distribution o f two markers in the same sections, the two-step protocol for immunohistofluorecence was used with cocktails of pairs o f primary antibodies (one developed in rabbit and one in mouse), at the same dilutions and conditions specified above, and secondary cocktail o f Alexa 594- and Alexa 488-conjugated antibodies (as above). In all cases, after being rinsed, the sections were mounted on glass slides and coverslipped with Vectashield Western blotting analysis Two juveniles were anesthetized in MS222, and the brains were quickly removed and mechanically homogenized in an equal volume o f cold buffer (5 mM EDTA, 20 mM Tris, pH 7.4, 150 mM NaCl, 10% glycerol, 1% Nonidet P40; Roche) supplemented with protease and phosphatase inhibitors (50 pg/ml phenylmethylsulfonyl fluoride, 10 pg/ml aprotinin, 25 jig /ml leupeptin, and 100 nM ortho vanadate; all from 101 3. E L H IF U I A L A M U LA 1KAJ>S1L1UJ> AJ^AMPNIU- AMNIOTA: ESTUDIOS EN ANUROS Y REPTILES Sigma). Samples of the supernatants containing in each case 50 pg of protein were applied in each lane of a 12% polyacrylamide gel (161-0801; Bio-Rad, Hercules, CA) and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with a Mini-Protean system (Bio-Rad). The samples of rat brain and molecular weight standards (Precision Plus Protein Dual Color Standards; Bio-Rad) were run in other lanes. The separated samples in the gel were transferred to a nitrocellulose membrane (Bio-Rad). Nonspecific binding sites were blocked by incubation overnight in Tris-HCl buffer (TBS) containing 0.1% Tween-20 (TBST) and 5% nonfat milk, at 4°C. The blots were then incubated for 24 hours at 4°C in primary antibody dilution (as for immunohistochemistry). After rinsing in TBS, the blots were incubated in horseradish peroxidase-coupled secondary goat anti-mouse or goat anti-rabbit antisera (Jackson Immunoresearch, West Grove, PA; diluted 1:15,000) for 2 hours at room temperature. Immunoreactive bands were detected by using an enhanced chemiluminescence system (Super Signal West Pico Chemiluminiscent Substrate; Pierce, Thermo Scientific, Rockford, IL). Photographs were taken after applying an autoradiographic film to the membrane, in darkness, for 1-4 minutes. Controls and specificity of the antibodies All the antibodies had been previously tested in Xenopus', many of them were used as territory markers in the forebrain, and the patterns o f staining showed the same distribution as that observed in the present study (Gonzalez and Smeets, 1992; Gonzalez et al., 1993; Marin et al., 1998a,b; Gonzalez et al., 2002a,b; Bachy and Rétaux, 2006; Moreno et al., 2008a, b; Morona and Gonzalez, 2008; Maier et al., 2010; Dominguez et al., 2011; Morona et al., 2011). Controls for the immunohistochemical experiments included: 1) Western blot (see the previous section and Fig. 1), 2) incubation of some selected sections with preimmune mouse serum (1:1,000 for Isll, Nkx2.2 and TH) or rabbit serum (1:1,000 for Nkx2.1, Otp, Tbrl, GAB A, MST, SOM, and TH) instead of the primary antibody, 3) controls in which either the primary or the secondary antibody was omitted, and 4) preadsorption of the primary antibodies with synthetic peptides. The latter included adsorption with Isll peptide (Abeam, Cambridge, MA; see Moreno et al., 2008a), synthetic NKX2.1 peptide (Biopat; 0100-P; see Moreno et al., 2008a), synthetic Tbrl blocking peptide (amino acids 50 to 150 o f the murine TBR-1; Abeam ab25853;), synthetic isotocin (MyBiosource, San Diego, CA: MBS230170), and synthetic somatostatin S14 and S28 (Bachem Bioscience Inc., formerly Peninsula, King of Prussia, PA; codes H I490 and H4955; see Gonzalez et al., 2007). In all the controls, the immunostaining was eliminated. The specificity of the antibodies used has been assessed by the commercial suppliers (Table 1); in addition, immunoblotting was conducted. The Western blots o f brains extract o f Xenopus laevis showed that all antibodies used labeled a single band, which with small variations corresponded well with the bands labeled in the rat lanes (Fig. 1). Many o f the antibodies and sera used here (rabbit anti-GABA, mouse anti-Isll, rabbit anti-Nkx2.1, mouse anti-TH) were also used in our previous study o f the Xenopus bed nucleus of the stria terminalis and their specificity was then detailed (Moreno et al., 2011a). Only in the case of TH, monoclonal and polyclonal antibodies were used (Table 1) with fully comparable results in the obtained pattern of immunostaining. [Ser'*,Ile*]oxytocin or isotocin is a nonapeptide that is present in many bony fish in place of oxytocin, whereas [Ile^joxytocin or MST is the homologous peptide present in amphibians. For the detection of MST we have used a rabbit-derived antiserum to isotocin (donated by Dr. J.M. Guemé, Université de Strasbourg, France) because it cross-reacts with MST and, to a lesser extent, with arginine vasotocin, which is the amphibian homologous to vasopressin (Thepen et al., 1987; Gonzalez and Smeets, 1992). The latter cross­ reaction, however, was lowered to a non-immune serum level by absorption of the antibody with vasotocin beads (Gonzalez et al., 1995). In our study, the use of this staining was chosen because it reveals the neurosecretory region of the SPV and has served as territory marker for the location of this region in the combination with other antibodies. The distribution of MST in the forebrain of Xenopus detected by this anti- isotocin antibody fully agrees with that described with specific anti-MST antibodies (Conway and Gainer, 1987). The anti-Nkx2.2 monoclonal antibody was developed by Dr. Jessell (Columbia University; New York). This antibody was raised against a sequence of 219aa (published in GenBank: AAD04630) of the chick Nkx2.2 protein (the antigen used was a recombinant protein made in E. coli, containing the amino acid range cited). The Nkx2.2 antibody has been tested in mouse, rat, chick and human (see datasheet). The results of brain distribution of this protein coincide with those obtained by the expression of the RNA of the same protein using in situ hybridization techniques (Viera and Martinez, 2006). It has been used previously in other studies in Xenopus (Dominguez et al., 2010) and mouse (Vue et al., 2007) with similar results, supporting the conservation o f this transcription factor in the evolution. The Western blot analysis labeled a single band in the Xenopus lane at the same molecular weight o f that o f the major product detected in rat brain extract (Fig. Ic). Otp is a homeodomain containing factor that is expressed in alternating and highly conserved hypothalamic domains in vertebrates (Simeone et al., 1994; Del Giacco et al., 2006; Bardet et al., 2008). The Otp polyclonal anti serum is generated from the highly conserved sequence 69-bp region at the C-terminal domain and is directed against the C-terminal sequence of the OTP predicted protein product. This antibody 1 0 2 j. Kj L j n irij 1 Ai-iAivivj Ai'̂ Aivii’Niu- AMNIOTA: ESTUDIOS EN ANUROS Y REPTILES Table 1. Antibodies used in the present study Name Immunogen Commercial Supplier MW(KDa) Dilution GABA GABA-BSA Polyclonal rabbit anti-y- aminobutyric acid Sigma; Catalogue reference: A2052 0.0103 1:3000 I s l l Amino acids 247-349 at the C-terminus of rat Isll Monoclonal mouse anti-Isl 1 Developmetal Studies Hybridoma Bank. Catalogue reference: 39.4D5 39 1 :500 MST Isotocin (Peptide Sequence: CYISNCPIG-NH2 Polyclonal rabbit anti-MST Dr. Guemé, Univ. Strasbourg, France 0.996 1:2000 N kxl.l Amino acids 110-122 from the amino terminus Polyclonal rabbit anti-TTF Biopatlmmunotechnologies, Caserta, Italy; catalogue reference: PA 0100 37-42 1 :500 Nkx2.2 E. co//-derived recombinant chick NKX2.2 Monoclonal mouse anti-Nkx2.2 Developmental Studies Hybridoma Bank. Catalogue reference: 74.5A5 28-30 1 :500 Otp Amino acid sequence: RKALEHTVSMSFT of the C-terminal OTP Polyclonal rabbit anti-Otp Pikcell Laboratories, Kruislaan, Amsterdam, The Netherlands 34 1 :1000 SOM Amino acid sequence: AGCKNFFWKTSC Polyclonal rabbit anti-SOM ImmunoStar, Wisconsin, USA. Catalogue reference: P20067 13 1:1000 Tbrl Amino acids 1 -200 at the N-terminus of mouse TBR-1 Polyclonal rabbit anti-Tbr-1 Santa Cruz Biotechnology; Catalogue reference: sc-48816 73 1:1000 TH Catalitic core of TH molecule Protein purified from rat pheochromocytoma Monoclonal mouse anti-TH ImmunoStar; Cataloque reference: 22941 Policlonal rabbit anti-TH Millipore (Chemicon). Catalogue reference: AB152 62 1:1000 was raised against a 13-amino-acid sequence, which is completely conserved in the species studied by BLAST (Lin et al., 1999). Rabbits were immunized against this peptide linked to keyhole limpet hemocyanin. Sera were screened by Western blot and immunocytochemistry, using cell lines transfected with an expression vector encoding the full-length mouse Otp and appropriate controls. Sera were affinity-purified against the Otp C- terminal peptide coupled to cyanogen bromide-activated Sepharose 4b according to standard protocols (Lin et al., 1999). The Western blot detected a single band that corresponded to that detected in the rat brain extract (Fig. Id). This band corresponded to the Otp homolog protein in Xenopus laevis and rat because it coincides with the calculated molecular weight (about 34KDa) in relation to the published nucleotide sequence for rat orthopedia homolog (NCBl accession number XM_215445). The polyclonal anti-SOM was purchased from Immunostar, Inc. (formerly Incstar, Hudson, WI) and was generated in rabbit against somatostatin conjugated to Keyhole limpet hemocyanin with carbodiimide (see manufaturer’s data sheet). The specificity of the SOM antibody was tested by the manufacturers, and the cross-reactivity data provided state that, when present, SOM-14, SOM-25, and SOM-28 are revealed by the antiserum, whereas cross-reaction does not exist with prosomatostatin or other neuropeptides, such as SP, NPY, VIP, insulin, glucagon, and amylin. We used the staining in the developing brain as reference in combination with other immunostainings since the pattern of SOM-like immunoreactivity in the forebrain o f Xenopus has been fully detailed (Olivereau et al., 1987; Moreno and Gonzalez, 2007). Our results in this study fully agree with the previous descriptions of the SOM-staining. Furthermore, the staining with this antibody colocalizes with the mRNA distribution of the same protein (Morales-Delgado et al., 2011). Tbr-1 is a rabbit polyclonal antibody raised against a part o f the protein highly conserved in vertebrates (see Santa Cruz Inc. data sheet), as revealed 103 3. EL HIPOTALAMO EN LA TRANSICION ANAMNIO- AMNIOTA: ESTUDIOS EN ANUROS Y REPTIIES Table 2. List o f the gene markers used, gene bank account, origin of the plasmid and enzymes/polymerases used in the probe synthesis GENE GENEBANK ACC. N" ORIGIN PRIMERS LINEARIZATION ENZIME/ POLIMERASI xShh NMOO1088313 Dr. Randal Moon. University of Washington, USA F:CGCAAATGGGCGG TAGGCGTG RrCAGGAAACAGCT ATGAC BamHl/T3 xD114 NMOO1090563 Dr. Nancy Papalopulu. University of Manchester,UK F:AG(GA)AA(GA)CC( CAT)CG(CT)AC(CAT) AT(CA)TA; R:CA(GA)GT(AGCT)A A(GA) AT(TC) TGG TTCCAG AA Notl/T3 xLhxl NMOO 1090659 Dr. Sylvie Rétaux. CNRS. Paris, France FXl iTGCCTTCTATTC TCCTAATCCGCCC; RXLCAGCTTAGGCT ACCACACTGCCG Ncol/SP6 xLhx5 NMOO 1090569.1 Dr. Sylvie Rétaux. CNRS. Paris, France FXl: TGCCTTCTATTCTCC TAATCCGCCC; RXl: CAGCTTAGGCTACC ACACTGCCG; FX5: GGATTTCACTGGAC TTGGCTTCTGC and RX5: GTTGGAATCAGGCG TACAAGCACC Ndel/T7 xLhxV NMOO 1086489 Dr. Sylvie Rétaux. CNRS. Paris, France Fdeg7:AARGTIAAYG AYYTITGYTGGCAY GT; Rdeg7:TGICKIGCICK RCARTTYTGRAACC A Notl/T7 xLhx9 NMOO 1094058.1 Dr. Sylvie Rétaux. CNRS. Paris, France Fdeg9:TIGCIGTIGAY AARCARTGGCAY- (ACT)Tand 32, MAYTTIGCYCTIGCR TTYTGRAACCA NC0I/SP6 by comparing sequences by BLAST analysis. The Western blot shows a single band that coincides with the band o f about 50 KDa obtained from the rat brain extract (Fig. le). This band coincides with the expected molecular weight o f this protein in relation to the published nucleotide sequence for rat Tbrl (NCBI accession number XM OO1056898). In situ hybridization The cDNA o f Xenopus xD114, xShh, xLhxl, xLhx5, xLhx7 and xLhx9 (Table!) has been previously used (Bachy et al., 2001; 2002; Moreno et al., 2004; Dominguez et al., 2010). Fo in situ hybridization, which was performed first, antisense digoxigenin (DIG)-labeled riboprobes for thes markers were synthesized according to the protocol dscribed in Bachy et al (2001; see table 2). The emlryos and premetamorphic larvae were processed in oto after progressive re-hydration and pretreatments (ee Bachy et al., 2001), and the late larvae were prcessed in floating sections (see Moreno et al., 2004; 2011). Hybridization step was done with 3 |l/ml of a DIG-labeled RNA probe, in a 50% firmamide containing medium overnight at 55°C. The solution used for prehybridization (at 60°C for 1 hour) ind 104 AMNIOTA: ESTUDIOS EN ANUROS Y REPTILES lslet1(39KDa) X.I Rat 50 37 Nkx2.1 (42KDa) Nkx2.2(30KDa) X.I Rat 50 X I Rat 37 25 Otp(34KDa) X.I Rat 29 Tbr1(50KDa) X I Rat mTH(62KDa) X I Rat rTH(62KDa) X I Rat 97 50 e f g Fig. 1. Identification by Western blots o f protein bands recognized in Xenopus brain extract stained for the mouse anti-Isll (a), rabbit anti-Nkx2.1 (b), mouse anti-Nkx2.2 (c), rabbit anti-Otp (d), rabbit anti-Tbr 1 (e), mouse anti-TH (f) and rabbit anti-TH (g) antibodies. A single band is seen in each of the lanes corresponding to the Xenopus brain extracts that are compared with the band stained in each case for rat brain extracts. The expected molecular weight is indicated for each transcription factor or enzyme and the molecular weight standard is represented at right in each photograph. hybridization contained 50% deionized formamide (Fluka, Steinheim, Germany), 5x standard saline citrate (Sigma-Aldrich, Steinheim, Germany), 2% blocking reagent (Roche Diagnostics, Mannheim, Germany), 0.1% Tween-20, 0.5% 3-[(3-cholamidopropyl)- dimethylammonio]-l-propanesulfonate (CHAPS; Sigma- Aldrich), 1 mg/ml o f yeast tRNA (Sigma-Aldrich), 5 mM o f ethylenediaminetetraacetic acid (Sigma-Aldrich), and 50 g/ml of heparin (Sigma-Aldrich) in water. Hybridization was detected using an alkaline phosphatase coupled anti-DlG antibody (Roche Diagnostics, dilution 1:1500). Alkaline phosphatase staining was developed with 4-nitroblue tetrazolium/5- bromo-4-chloro-3-indolyl-phosphate solution (NBT/BCIP; Roche Diagnostics). In cases o f double in situ hybridization labeling, one o f the probes was combined with DIG and the other with fluorescein, and both were revealed with NBT/BCIP (Roche Diagnostics) and INT/BCIP (Roche Diagnostics), respectively. In the cases in which hybridization was detected by fluorescence, it was revealed with Fast Red tablets (Roche Diagnostics). When the in situ hybridization was combined with immunohistochemistry, in situ hybridization was revealed with Fast Red tablets followed by the incubation in the primary antibodies (see table 1) and a second incubation with goat anti-mouse Alexa 488 (diluted 1:500, Molecular Probes), for the cases o f mouse developed primary antibodies, or chicken anti-rabbit Alexa 488 (diluted 1:500, Molecular Probes) for the case o f prim an antisera developed in rabbit. Subsequently, embryos aid early larvae were embedded in a solution o f 20% gelatin and 30% sucrose in PB, and stored overnight at 4 °C ii a solution of 4% formaldehyde and 30% sucrose in PB. Sections were cut on a freezing microtome at 14-25 pm i n the transverse, sagittal or horizontal plane Im agin g The sections were analyzed with an Olympus BX51 microscope that was equipped for fluorescence with appropriate filter combinations. Some o f the sections were also analyzed with a Leica SP2 confocal microscope to corroborate the coexpression o f two different markers. Selected sections were photographed by using a digital camera (Olympus DP72). Contrast and brightness o f the photomicrographs were adjusted in Adobe Photo Shop CS3 (Adobe Systems, San Jose, CA) and figures were mounted in Canvas 11 (ACD Systems, International). RESULTS D ev elo p m en ta l p a ttern in g o f the alar h yp oth a lam u s The analysis throughout development o f Xenopus laevis o f the different markers used in the present study showed that they have distinct distribution patterns in the forebrain and, specifically, in the hypothalamic and adjacent regions. The combination o f the expression o f these markers was used as a tool for the identification o f the precise boundaries and subdivisions o f the alar hypothalamus, a rather complicate region with scarce cell migration from the ventricle and poorly individualized nuclei during development and also in adult amphibians, as observed in Nissl-stained sections (Fig. 2). The specific distribution patterns observed during development for the markers used in this study (xD114, GABA, Isll, xL hxl, xLhx5, xLhx7, xLhx9, Nkx2.1, Nkx2.2, MST, Otp, xShh, SOM, T b rl, and TH) in the ventricular (vz). 105 DORSAL VIEW i d a m j m u i a : L î s i u m u î » l i n a t n u k u » y LATERAL VIEW i d h Fig. 2. Photomicrographs of Nissl-stained transverse and sagittal sections through the hypothalamus of a prometamorphic Xenopus from medial to lateral (a-c) and rostral to caudal (d-i) at the approximate levels indieated on the dorsal and lateral views of the brains. These photomierographs illustrate the poorly segregated regions roughly recognized in the hypothalamus. The scale bar in a is valid for b and c = 200pm. The scale bar in d is valid for e-i = 200pm The scale bar in a’ is valid for b ’ and c’ = 100pm. subventricular (svz) and/or mantle zones (mz) allowed us to define the extent o f the alar hypothalamus and its subdivisions and boundaries (Fig. 3) as well as the progenitor and histogenetic domains in the PO (Fig. 4), the SPV (Fig. 5), and the SC (Figs. 6, 7). The results about the combinatorial expression o f the markers used have been summarized in the schemes o f Fig. 8. Boundaries. The expression o f xShh and Nkx2.1 in the PO, as opposed to the lack o f expression in the dorsal hypothalamus, was useful to highlight the limit topologically dorsal o f the alar territory o f the SPV (Fig. 3a,b). In addition, the boundary between the PO and the SPV was clearly identified by the expression o f Otp (Fig. 3 c-f) and, to a lesser extent, Nkx2.2 (Fig. 3g,h) in the SPV. Moreover, the expression during development in the PO o f XD114 (Fig. 3c,g), Isll (Fig. 3d,e), xShh (Fig. 3f), and Nkx2.1 (Fig. 3h) in combination with the SPV markers (Otp and Nkx2.2) allowed the identification of the dorsal boundary between the SPV in the alar hypothalamus and the PO at any stage, including the juveniles. Ventral to the Otp expressing SPV and dorsal to the optic chiasm, the SC region was identified in the alar hypothalamus by the expression o f xD114 (Fig. 3g, k). xShh (Fig. 3i) and xLhxl (Fig. 3j). The caudal boundary between the alar hypothalamus and the diencephalic p3 was also distinctily noticeable by the combinatorial expression patterns o f several markers. Thus, at dorsal levels, the caudal boundaiy o f the SPV was highlighted by the expression o f xLhx9 and Tbrl in the prethalamic eminence (PThE) o f the dorsal P3 (Fig. 31, m). In turn, the caudal boundary between the ventral part o f SPV and the SC with the prethalamus (P3) was seen by the expression o f xShh, xLhxl, Nkx2.1 and xD114 (Fig. 3a-c, j). All these boundaries have been summarized in the bottom scheme shown in Fig. 3n. Preoptic region. The PO anatomically belongs to the non-evaginated telencephalon located topologically dorsal to the alar hypothalamus (Fig. 3). It has been traditionally considered as part o f the hypothalamus, thus in the present analysis we will pay special attention to the identification o f the boundaries between the PO and the hypothalamus. We combined immunohistochemistry for the markers Nkx2.1, Nkx2.2, Is ll, and TH with in situ hybridization for xShh and xD114 to characterize the PO (Fig. 4). The xShh expression defined the PO vz, whereas Nkx2.1 106 st45 / S P V , , . Sc \ '^ O SPV/ (VIGE S C \ ,'PO _ b n B H c ® F ''P O r ' PO I ' I ' s c B H ^ :^P,V:1 r sc , — 9 _ h X SPV SPV' PO _ j SC ' , , SPa — k PThE PT h^PThE,-SPV XDII4 Nkx2.1 PTh xLhx1 xShh PThE PTh-SC XDII4 xLhx1 PTh-SPV I I xLhx9 Tbr1 Otp Nkx2.2 Isl1 SC Shh Nkx2.1 8 P V 8 C Otp SPV Nkx2.2 P O 8 P V 8hh XDII4 PO Isl1 Fig. 3. Photomicrographs of sagittal (a-c,e,g,h,j,m) and transverse (d,f,i,k,l) sections through the main alar hypothalamic subdivisions of Xenopus. The boundaries are depicted on the basis o f the single expression for xShh, Nkx2.1 and xD114 (a,b,k) and the combined expressions of xD114 and Otp (c), Isll and Otp (d,e), xShh and Otp (f,i), xD114 and Nkx2.2 (g), Nkx2.1 and Nkx2.1 (h), xLhxl and Otp (j), xLhx9 and Otp (1) and Trbrl and Isll (m). The boundaries identified are the PO-SPV (c-f), SPV-SC (g-i), SPV-PTh (c-f,i,j), SC-PTh (k), and SPV-PThE (l,m). The schematic representation at the bottom summarizes the boundaries between the different alar hypothalamic subdivisions and the combinatorial code of markers of each region. The yellow dashed lines indicate the limit between two adjacent regions revealed by the different markers. Scale bars: 200pm (e), 100pm (k,l) and 50pm (a-d, f-j,m). expression was detected in the vz and svz, particularly starting at late embryonic stages (Fig. 4a-c). The combination o f Nkx2.1 and Isll from embryonic (Fig. 4d- f) through larval stages (Fig. 4g,h) showed that the Isll expression occupied a subventricular band adjacent laterally to the Nkx2.1 expressing cells in the PO (Fig. 4e,g). The staining with TH confirmed the location o f the previous markers within the PO, where abundant dopaminergic cells are distributed (Gonzalez et ah, 1994). This TH positive cell population was always revealed close to the vz within the Nkx2.1 positive territory (Fig. 4i), deep to the Isll positive territory (Fig. 4j). GABAergic cells were also observed in the PO intermingled with the Isll expressing cells (Fig. 4k,l), and some cells co-expressed both markers (see arrowhead in Fig. 4k). Finally, from embryonic to metamorphic stages, the ventral boundary o f the telencephalic PO was discernible by the combination 107 st46 I / st4î PTh SC SPa — \ b st45 / r ' c st45 I / PTh V /pQ SPa e f IMCs^D/ st45 I / — g _ d st48 / . PTh ° \ s P V s c h °" st45 / s e t : / P O st46 / rd \ S P V , ' : ; SC ' p st451 / PO ?PV . PTh \ SPV PO PO ' SPV TRANSVERSAL VIEWSAGITTAL VIEW \ SPV A/B boundary Nkx2.1 ■ I xShh+Nkx2.1 ^ XDII4 Isleti TH E Ï 3 GABA Otp Figure 4. Pliotomicrographs of sagittal (a,b,f,h,l,p,q) and transverse (c-e,g,i-k,m-o,r-t) through the developing Xenopus PO region showing the single expressions of Nkx2.1 (a) and xShh (b) and the combined expressions of xShh and Nkx2.1 (c), Isll md Nkx2.1 (d-h), Nkx2.1 and TH (i), Isll and TH (j), Isll and GABA (k,l), Isll and Otp (m-p), xD114 and Otp (q), xShh and Otp (r), Nkx2.1 and Nkx2.2 (s) and xShh and Nkx2.2 (t). The schematic representation at the bottom summarizes the combinatorial code of markers present in the PO and its adjacent domains (u,v). The arrowhead in k indicates double labeled Isll and GABA cells. The photomicrograph o shows a higher magnification of the area shown in n by confocal microscropy to illustrate the limit between the Isll+ PO and the Otp+ SPV. Scale bars: 200pm (p), 50pm (a-n, q-t), 25pm (o). 108 st33/34 N k::2 2 / st37/38 . / i . I— d < r V ▼ T h F / st46 b N ky? / _ f I st48 Th , ' y SPVr ' PTh ' 'SPVc — J / st48 PTh SPVr SC I/ ' ' ' ' ',o c . 1 ̂ - ;- sp v c C \ , J h ’/• SPVr ■ ■ H Q SPVc SPVr. / Adult I Adult SPVr sc , . ' -SPVc \S P V r SC . / st48 ' - SPVc ' SPVr PTh SPVr s c ' / >0 - SPV" SC ' t . ' SPVr - ; /■ sc ' , I— d SPVCj, , i >^SPVc SAGITTAL VIEW TRANSVERSAL VIEW S P V c S P V r F 7 1 Nkx2.2 Otp V 7 ^ xLhxS EZ3 MST |ooo| Som ■ I Nkx2.1 xShh+Nkx2.1 ŸZÀ XDII4 Figure 5. Photomicrographs of transverse (a,c,e,e’,f,h,j,l,p,q) and sagittal (b,d,g,i,k,m-o,r-t) sections through the developing Xenopu5 SPV region showing the expressions of Otp (a), Nkx2.2 and Otp (b-g), Nkx2.2 and MST (h-k), Nkx2.2 and SOM (1-n), xLhx5 (0), xShh and Otp (p), Nkx2.2 and Nkx2.1 (q), xD114 and Otp (r), xD114 and Nkx2.2 (s), Otp and Isll (t). The schematic represen ation at the bottom summarizes the combinatorial code of markers present in the SPV and its adjacent domains (u, u ’, v). The arrowhead in a indicates the Otp+ ventricular cells. The yellow line in b indicates the level of the section shown in c. The arrowheads in e’ and f point to the ventricular double labeling for Nkx2.2 and Otp in the SPVr portion, whereas the upper double arrowhead in f indicates the unique ventricular expression of Otp in the SPVc domain. The yellow box in m indicates the higher magnification shown in n and the arrowhead in n shows the coexpression of Nkx2.2 and SOM in SPVr. Scale bars; 250pm (g), 200pm (h,t), 100pm (b,f), 50pm (a,c-e,h,i,k-m,o-s), 25pm (e’), 12,5pm (n). 109 jcjIy «jfvvyo x ivcjx xxxjxi.j PTh S P V ' X xLhx7/xDII4 st45xLhx7 SPV, . I ' S P a» % :“ ¥ . . P o 4 PTh .'S P V SCr scr ' : ;po S t 46 . S t 48 PTh^^'- ■' S t 46 SPV - SC c X , . , PO SCr^ TRANSVERSAL VIEWSAG TTAL VIEW A/B boundary Nkx2.1 V/̂ A Nkx2.2 xLhx7 EZ3 GA3A xShh+Nkx2.1 ŸZÀ xDII4+lsl1 xLhx1 TH Figure 6. Photomicrographs of transverse (a-e,i,n-p) and sagittal (f-h,j-m) sections through the developing Xenopus SC region showing the single expressions of xShh (d), Nkx2.1 (e), xLhxV (f), xLhxl (h) and the combined expressions of xD114 and fell (a), Isll and GABA (b), Isll and Nkx2.1 (c), xLhx7/xD114 (g), Nkx2.1 and Nkx2.2 (i-k), xLhxl and Nkx2.2 (1), xShh and TB(m,n), Nkx2.1 and TH (o) and Nkx2.2 and TH (p). The schematic representation at the bottom summarizes the combinatorial lode of markers present in the SC (q, q', r). The arrowhead in b indicates a cell double labeled for Isll and GABA. Scale bars: 200im (k), 50pm (a-j,l-p). 110 J . ÏÏL é lj IL i^ I j A A i ’N A iV li'M lV J- AMNIOTA: ESTUDIOS EN ANUROS Y REPTILES of Isll/Otp because Isll labels the PO and Otp is specifically expressed in the SPV (Fig. 4m-p). Thus, from early developmental stages, a clear boundary between both territories was observed (Fig. 4m,n) and no double labeled cells were observed (Fig. 4o). This boundary was confirmed with other markers of the PO, such as xD114 (Fig. 4q), xShh (Fig. 4r) and of the SPV, such as Nkx2.2 (Fig. 4s,t). All these results have been summarized in the bottom schematic representation in Fig. 4u,v. Supraoptoparaventricular region. From early embryonic stages, the dorsalmost hypothalamic domain was defined by the expression of Otp in the vz/svz and, within this expression zone, a low number of cells also expressed Nkx2.2 (Fig. 5a-c). Later, from premetamorphic to metamorphic larval stages, Otp and Nkx2.2 were more clearly observed in the SPV and double labeled cells were detected (Fig. 5d, e; see arrowhead in Fig. 5e' and 5f). The combination of both markers allowed the distinction of a rostral portion (SPVr) rich in Otp/Nkx2.2 expressing cells and a caudal part (SPVc) that only expressed Otp (Fig. 5f,g). In addition, from early larval to adult stages, in this hypothalamic territory important cell populations were identified by immunohistochemistry to several neuropeptides constituting the endocrine hypothalamus (see ten Donkelaar, 1998). Thus, the combination of Nkx2.2 with MST (Fig. 5h-k) and SOM (Fig. 51-n) showed that these neuropeptidergic cell populations occupied the SPVc and, in addition, double labeled cells were detected (see arrowhead in Fig. 5n). Another differential marker in the SPV was xLhx5 that was observed only in the SPVr and continued ventrally into the SC (Fig. 5o). The ventral boundary of the SPV with the SC was identified form embryonic to metamorphic stages by the combination of Otp or Nkx2.2 with markers o f the SC in Xenopus that avoided the SPV region. It is the case of xShh/Otp (Fig. 5p), Nkx2.1/Nkx2.2 (Fig. 5q), xD114/0tp (Fig. 5r), xD114/Nkx2.2 (Fig. 5s) and Isll/Otp (Fig. 5t). All these results have been summarized in the bottom schematic representation in Fig. 5u,v. Suprachiasmatic region. From embryonic stages, the ventral portion of the alar hypothalamus, the SC domain, was distinct by the expression o f xD114 and Isll, as well as by the presence of GABA expressing cells (Fig. 6a-c), some of which were double labeled for Isll (see arrowhead in Fig. 6b). Additionally, the analysis of the distribution patterns for Nkx2.1 (Fig. 6c,e), xShh (Fig. 6d), xLhx7 (Fig. 6f) and xLhxl (Fig. 6g) allowed the identification o f a rostral subdivision (SCr), rich in all these markers, in contrast to a caudal portion (SCc) where only xD114/Isll expressing cells were detected (Fig. 6h). The combination o f Nkx2.1/Nkx2.2 through development (Fig. 6i-k) showed that the caudal portion lacked Nkx2.1 and the number o f cells expressing Nkx2.2 was low (compare Fig. 6j and Fig. 6k). In addition, some markers of the SCr such as xLhxl were almost absent in the SCc (Fig. 61). The combinations of markers of the SC with TH (Fig. 6m-p), allowed the identification of the catecholaminergic population mainly within the SCr, as confirmed by the distribution o f xShh (Fig. 6m,n), Nkx2.1 (Fig. 6o) and Nkx2.2 (Fig. 6p) in this region. All these results have been summarized in the bottom schematic representation in Fig. 6q,r. The distinct extents o f the subdivisions in the SC were clearly observed from prometamorphic larval stages to the juveniles. This was particularly illustrated by the combination o f Nkx2.1 and Isll that allowed the identification o f the boundary between the rostral and caudal SC regions, in transverse and sagittal sections (Fig. 7a-f). Isll was expressed in the whole SC, but the cell density was markedly higher in the caudal portion (Fig. 7a, c, d), whereas Nkx2.1 was restricted to the rostral portion (Fig. 7b,d,e), from anterior (Fig. 7a) to posterior (Fig. 7f) levels. As in earlier developmental stages, xLhxl (Fig. 7g) and xLhx7 (Fig. 7h) are distinctly expressed in the rostral portion. At these late developmental stages, the SC boundaries with the SPV were better highlighted by the combination of Isll with Otp (Fig. 7i). Finally, at late developmental stages the staining for GABA and TH showed the distribution of both markers in the SC, as defined by the expression of Isll (Fig. 7j, k) and in both cases double labeled cells were observed (see arrowheads in Fig. 7k). All the results of expression pattern domains have been summarized in Fig. 8. DISCUSSION The present study represents a first analysis o f the development o f a portion of the hypothalamus in anuran amphibians, namely the alar hypothalamus. This dorsal portion of the hypothalamus has been characterized during development and in juveniles by the combinatorial expression patterns of a set of molecular markers known to be involved in the specification of hypothalamic territories in amniotes (Puelles and Rubenstein, 2003; Bardet et al., 2008; Shimogori et al., 2010; Morales-Delgado et al., 2011; Moreno et al., 2010; 2011b). Within the poorly segregated neuronal populations in the amphibian brain, this approach has been most valuable for identifying hypothalamic subregions and heir boundaries with adjacent territories (Dominguez et al., 2010, 2011; Moreno and Gonzalez, 2011). We have followed the current conception o f brain subdivisions considered in the prosomeric model and, thus, we have identified the alar hypothalamic regions as rostrally located to the diencephalon, ventral to the telencephalic PO, and dorsal to the alar-basal boundary just beneath the optic chiasm (Puelles and Rubenstein, 1993, 2003; Puelles, 1995, 2001). Although other criteria have served to consider alternative hypothalamic subdivisions (le Gros Clark, 1938; Crosby and Woodbume, 1940; Swanson, 1987; Simerly, 2004), we have preferred to attend to topological relationships among the distinct prosencephalic territories that can be individualized by 111 /A.iTXlNlW 1 . X u rxm^xxv^kj m. xv^x x xx-/x l̂j ' POC S C r ' S P V S C c \S C c SPV 0 0 , S P V — c _ d xLhx1 Juvenile xLhx7 Th ' S C c S to b SC / SPV Fig. 7. Photomicrographs o f transverse (a,e-k) and sagittal (b-d) sections through the SC territory :n late prometamorphic and juvenile Xenopus showing the single expressions o f Nkx2.1 (b), Isll (b), xLhxl (g) and xLhx7 (h) and the combined expressions o f Isll and Nkx2.1 (a,d-f), Isll and Otp (i), Isll and GABA (j) and Isll and '7H (k). Scale bars: 100 pm. the differential expression o f developmental genes that codify, primarily, transcription factor (Puelles, 2001; Puelles and Medina, 2002). In concordance with several previous studies in different amniotes, the territories here considered within the alar hypothalamus are similarly subdivided into supraoptoparaventricular (SPV) and suprachiasmatic (SC) regions (Puelles and Rubenstein, 2003; Bardet et al., 2008; Shimogori et al., 2010; Morales-Delgado et al., 2011; Moreno et al., 2010; 201 Ib). However, it should be noted that recent studies in the mouse have challenged this interpretation because a different alar-basal boundary has been proposed that leaves most o f the current basal hypothalamus within the alar domain (Diez-Roux et al., 2011). It should be also strengthened that in the present study, we have abandoned the classical interpretation o f the hypothalamus o f anurans as part o f the diencephalon (Herrick, 1910, 1917), roughly subdivided into preoptic and infundibular parts. O f note, the preoptic hypothalamus, including the PO proper plus the current alar hypothalamus, was further subdivided into anterior and posterior areas that contained the magnocellular and the suprachiasmatic nuclei (Neary and Northcutt, 1983). However, the classically termed PO hæ been demonstrated to be a telencephalic territory (Zhao et al., 2009; Roth et al., 2010; for review Medina, 2008 . P relim in ary con sid eration s: experim ental approach All the markers used in the present study ha\e been demonstrated to have highly conserved expression patterns during evolution. Among the transcription factors whose expression could help ii the identification o f the alar hypothalamus we have opted for Nkx2.1 and D114 because their expression; have been thoroughly analyzed in the prosencephaon o f many vertebrates and both have been used to identify the subpallial preoptic region, dorsally adjacent to the alar hypothalamus (Gonzalez et al., 2002a,b; ?uelles and Rubenstein, 2003; Puelles et al., 2000; Bach) et al., 2002; Brox et al., 2003; Moreno et al., 2008a; vm den 112 ô . EiJ-i Xlirvj 1 AĴ AiVlU 1-.A AMNIOTA: ESTUDIOS EN ANUROS Y REPTILES Akker et a l , 2008). In all vertebrates studied, the transcription factor Otp has been demonstrated to be a good marker of the SPV (Puelles and Rubenstein, 2003; Bardet et a l , 2008) and the analysis of the Otp expression in combination with the expression of Nkx2.1/D114 highlighted the boundaries of this alar territory (Brox et a l , 2003; Puelles and Rubenstein, 2003; Bardet et a l, 2010; Dominguez et a l , 2010; Moreno et a l, 2010; 201 la; Martinez de la Torre et a l, 2011). The homeobox transcription factor Nkx2.2 resulted useful in the identification o f the alar-basal boundary, also evidenced by the expression o f xShh (Puelles and Rubenstein, 2003; Bardet et a l, 2010; Dominguez et a l , 2010, 2011). In addition, both markers served to the identification of the SC, as in other tetrapods (Bardet et a l , 2010; Medina, 2008; Dominguez et a l, 2010, 2011; Moreno et a l, 2011b). We have also used transcription factors of the LIM-homeodomain family, such as xLhxl, xLhx5, xLhx7 and Isll, which have been demonstrated to encode positional information and resulted useful to determine the SC boundaries (Moreno et a l, 2004, 2008a; Shimogori et a l , 2010). Finally, Tbrl and xLhx9 showed a specific expression in the prethalamic eminence (PThE) (Puelles and Rubenstein, 2003; Moreno et a l, 2004; Medina, 2008), which have helped in the delineation of the alar hypothalamic boundary with the diencephalic P3. In addition, the localization of TH, a catecholaminergic marker, highlighted the regionalization o f the SC (Gonzalez et a l, 1993, 1994; Smeets and Gonzalez, 2000) and the presence of MST (the oxytocin homologous peptide present in amphibians) and SOM in the neuroendocrine neurons of the SPV is also a shared feature between amniotes and anamniotes (Blasher and Heinrichs, 1982; Gonzalez et a l, 1995; 2003; Petkô and Grosz, 1996; Lopez et a l, 2007). Finally, the GABA has been used as a marker to identify the Dix positive territories including the PO and the SC (Brox et a l, 2003). The preoptic region The PO is defined anatomically as the region immediately in front of the preoptic recess and has classically been considered to be a part o f the hypothalamus as the rostralmost part o f the diencephalon (for review Hodos, 2008). However, the current knowledge about the morphogenetic patterning o f the forebrain has changed this view and the PO is considered as a non-evaginated part of the telencephalon, on the basis o f its topological position in the neural plate, uimaveled by fate mapping studies, and its genetic specification (Flames et a l , 2007; Garcfa-Lôpez et a l, 2009; Sânchez-Arrones et a l, 2009). The present topological and molecular analysis o f the PO in anurans supports its telencephalic nature, as in amniotes, and it has been included in the description because it has classically been left out in the studies o f the subpallium and because it ventrally borders on the actual alar hypothalamus. Patterning and neuronal specification. In contrast to the hypothalamus, the PO is currently regarded as derived from the FoxGl-positive telencephalic neuroepithelium. FoxGl is a member o f the Fox/Forkhead family o f winged helix transcription factors, which is involved in the specification, proliferation and differentiation of the telencephalon, being expressed in dividing progenitors o f the ventricular zone and in early postmitotic neurons in all vertebrates analyzed (Tao and Lai, 1992; Murphy et a l, 1994; Bourguignon et a l , 1998; Zhao et a l, 2009; Roth et a l, 2010). Thus, the PO is part o f the Dix- expressing subpallium and is also characterized by the expression of Nkx2.1 (Puelles et a l , 2000; Brox et a l , 2003; Garcia-Lôpez et a l , 2008; Abellân and Medina, 2009; Moreno et a l , 2009; Martinez de la Torre et a l , 2011). O f note, in anurans as in amniotes, Shh expression is expressed in PO within the subpallium (Flames et a l, 2007; Bardet et a l, 2010; Dominguez et a l , 2010). The preoptic region of Xenopus is composed by the commissural preoptic area (POC) and the preoptic area proper (PO) (Moreno et a l, 2008a; 2009; Dominguez et a l , 2010; present results); subdivisions that were previously described in anuiiotes (Garcia- Lopez et a l, 2008; Abellân and Medina, 2009). The results of our present study in Xenopus showed that the vz of the PO is uniquely defined by the expression of xShh and Nkx2.1, whereas in the svz a stripe of Nkx2.1 cells is embraced laterally by Isll and D114 expressing cells (see Figure 4v). This pattern of distinct bands arranged mediolaterally was early observed in amphibians although its significance was not explained (Neary and Northcutt, 1983). In line with these observations it should be noted that in mouse and chick, neurons of the hypothalamus are generated in an “outside-in” pattern with neurons bom earlier occupying a more lateral position in the mantle zone (Altman and Bayer, 1986; Markakis, 2002; Caqueret et a l, 2006). Moreover, some of these studies found a correlation between these waves of neurogenesis and the layered expression of typical markers in the some hypothalamic areas. There are no data about the developing pattern of neurogenesis in the amphibian hypothalamus, however, we have found a close relationship between the neurons bom in the ventricular proliferative zone and the migrated populations that form in the mantle zone suggesting that this outside-in developing pattem is likely to occur also in amphibians. In terms of chemoarchitecture, the GABAergic cell population in the PO was located within the xD114 and Isll expression domains and some cells coexpressed both markers (present results), suggesting that these transcription factors could be involved in the GABAergic phenotype establishment in the PO of Xenopus, as has been described in the forebrain o f other vertebrates (Price et a l, 1991; Bulfone et a l , 1993; Marin and Rubenstein, 2001). 113 A M f N lU l a : E S I u m u s EJ> A JSU K U S y K E E l l E E S A/B boundary xShh+Nkx2.1 VZA XDII4, Isl1 Otp YU Nkx2.2 xLhx1 [=1 xLhx7 O xLhx9 VZA xLhxS ■ i Tbrl PThE % S P V c Figure 8. Schematic sagittal representation o f the alar hypothalamus of a premetamorphic Xenopus summarizing the combinatorial code of transcription factors used in the present study, following the color code indicated in the box. Boundaries. Our results have highlighted that the PO is distinct from the hypothalamic SPV territory by their differential molecular gene expressions. The PO is a Shh/Nkx2.1/xD114 subpallial positive territory, in contrast to the lack o f these transcription factors in the SPV, rich in Otp expression (present results; Figure 8). This situation seems to be very conserved in all tetrapods (Puelles and Rubenstein, 2003; Bardet et a l , 2008; Moreno et a l , 2008a; 201 la). In addition, it has been described in amniotes the existence of a thin territory called the preopto-hypothalamic region (POH; Bardet et a l , 2006; Flames et a l , 2007; Moreno et a l , 201 lb). This territory represents the molecular boundary separating longitudinally the PO from the magnocellular hypothalamus and it is characterized in mammals by the ventricular expression o f Dlx2, Glig2, Gsh2, Pax6 and Nkx2.2, whereas it lacks expression of Nkx2.1, Shh and Dbx (Flames et a l , 2007). Comparable results were obtained for other amniotes in which the POH could be distinguished (Bardet et a l , 2006; Moreno et a l , 2011b). The results in Xenopus along development showed that there are not Nkx2.2+ /Otp-/Nkx2.1- ventricular cells in an anatomically similar territory. The first Nkx2.2 expressing cells along the A-P axis were located in the SPV region, as revealed its coexpression with Otp (Dominguez et a l , 2011; present results). A similar situation has been observed in different groups o f fishes (unpublished results) suggesting that the existence o f this evident boundary seems a feature only o f amniotes. The supraoptoparaventricular area The SPV is the dorsal part o f the alar hypothalamic territory and contains multiple neuroendocrine cell populations being an essential part o f the hypothalamo- hypophyseal system. Characteristically and specifically, the SPV can be individualized by the expression o f the gene Orthopedia (Otp), which serves for its distinct identification from adjacent territories, both in amniotes and in anurans (Bardet et a l , 2008; Moreno et al. 2011b). Patterning and neuronal specification. The SPV in amniotes is a Dix negative subdomain in the most dorsal region of the alar hypothalamus, adjacent to the PO, which expresses the transcription factors Otp and Siml (Bulfone et a l , 1993; Puelles and Rubenstein, 2003; Medina, 2008). In Xenopus, this area was previously called optoeminential band, only on the basis o f the lack o f Dix genes (Brox et a l , 2003), following the terminology used in amniotes at that moment (Puelles and Rubenstein, 2003). However, the current term supraoptoparaventricular area was later coined (see Medina, 2008) because it, at least in mammals, includes the supraoptic and the paraventricular nuclei. This term has been rapidly adopted and used to identify similar regions across vertebrates (see Moreno and Gonzalez, 2011). In fact, Otp was seen to be expressed in the SPV o f the mouse. 114 ô . m l ij m ru 1 aj-iAivuj t L ï y L jA ai-naiviinu -̂ AMNIOTA: ESTUDIOS EN ANUROS Y REPTILES chicken, turtle, amphibians, zebrafish and lungfishes (Acampora et a., 1999; Lin et al., 1999; Caqueret et al., 2006; Del Giacco et al., 2006; Blechman et al., 2007; Bardet et al., 2008; Gonzalez and Nothcutt, 2009; Machluf et al., 2011; Moreno and Gonzalez 201 l;Moreno et al., 2011b; present results). In addition, the lack of Shh, Nkx2.1 and Dix expression in this region, as opposed to its neighboring areas such as the PO, SC and PTH, is also highly conserved (Brox et al., 2003; Puelles and Rubenstein, 2003; Bardet et al., 2008; Dominguez et al., 2010, 2011; Moreno et al 2010; 2011b; Martinez de la Torre et al., 2011). We have also analyzed the expression pattem of members o f the LIM homeodomain family, and xLhx5 was specifically expressed in a band corresponding to the SPV region, in concordance with results in the chicken (Abellân and Medina, 2010). Previous studies in the mouse revealed that Lhx5 was expressed in a Dlx5- negative gap that was defined as the “optoeminential zone” and that most likely corresponds to the SPV (Bulfone et al., 1993). This SPV can be further subdivided in amniotes and anurans (Bardet et al., 2008; Moreno et al., 2011b; present results). The distinction in the SPV of rostral and caudal portions was initially suggested by the distinct organization o f two cell groups within the Otp+ conglomerate and the relative amount of cells, higher in the caudal portion (Bardet et ah, 2008). In the present study, we have defined two distinct parts along the rostro- caudal extent o f the SPV in Xenopus based on additional molecular criteria. The rostral part (SPVr) is characterized by the ventricular and subventricular expression of Otp, whereas only the caudal part (SPVc) also expresses Nkx2.2 in vz and svz cells. The expression o f Nkx2.2 in only one part o f the SPV of Xenopus is a feature observed also in the SPV o f the turtle, where comparable rostral and caudal parts were identified (Moreno et al., 2011b). Studies in the developing mouse showed that the Nkx2.2 expression is found in the POH and in the subparaventricular area (SPa), which lies under it and is adjacent to the Shh-positive basal plate (Bardet et al., 2010; Morales-Garcia et al., 2011). However, the presence of Nkx2.2+ cells in the anterior hypothalamic region o f mouse, which partially overlaps the Siml expression domain, has also been reported (Caqueret et al., 2006). Regardless this conserved pattem o f Otp and Nkx2.2 expression, interspecific differences within this region were noted. This is the case o f the lack of Pax6 and Tbrl expressions in Xenopus (Moreno et al., 2008b; present results), both present in amniotes (Medina, 2008; Moreno et al., 201 lb). However this lack is also shared by other anamniotes such as the lamprey (Murakami et al., 2001) and lungfishes (Moreno and Gonzalez, 2011). The lack o f a distinct POH (marked by Nkx2.2) and Pax6 expressing cells in the SPV of Xenopus (present results), contrast with the results reported in amniotes (Flames et al., 2007; Medina, 2008). This situation might reflect differences in the specification of this area between amniotes and anamniotes (Moreno and Gonzalez, 2011). In fact, it has been demonstrated in mammals that Nkx2.2 expression in the ventral neural tube is restricted to progenitors that do not express Pax6, but it is Pax6 who regulates the expression o f Nkx2.2 (Ericsson et al., 1997). This relation has also been observed in the diencephalon, and in mice Pax6 mutants abnormal Nkx2.2 expression is observed in the thalamus (Pratt et ah, 2000). Interestingly, prosencephalic migratory pathways from and to the hypothalamus are being currently evaluated, specially in mouse (Bupesh et ah, 201 la,b), but also in other tetrapods (Moreno et ah, 2008c) and a particular contribution of neurons generated in the SPV to the medial amygdala has been demonstrated (Garcla-Moreno et ah, 2010; Bupesh et ah, 2011a). Our present observations in Xenopus strongly support the participation of Otp expressing cells generated in the SPV in the composition of the region identified as the anuran medial amygdala during development (Moreno and Gonzalez, 2007, 2011). In regard to the neuronal specification, the differential expression along the ontogeny of the transcription factor Otp, together with the pattem of some posttranscriptional markers, has allowed to correlate the emergence of differentiated cellular types with the presence of this transcription factor, highlighting the role o f Otp in the differentiation of postmitotic cells in the SPV. Thus, the Otp mutant mice failed to develop properly the paraventricular (PV) and supraoptic (SO) nuclei and these animals die soon after birth (Acampora et ah, 1999; Wang and Lufkin, 2000). Particularly, Otp acts in a complicated cascade of transcription factors for the differentiation of several neuroendocrine cells o f the SPV, including the tirotropin releasing hormone (TRH), corticotropin releasing hormone (CRH), oxytocin (OT), vasopressin (VSP) and somatostatine (SOM) expressing cell types (Acampora et ah, 1999; Wang and Lufkin, 2000; Goshu et ah, 2004; Caqueret et ah, 2005; Morales-Delgado et ah, 2011) what contributes to the differentiation of independent nuclei within this territory. The present results showed that the Otp expression domain in the SPV within the alar hypothalamus of Xenopus correlates with the emergence o f distinct neuroendocrine cells types such as SOM and MST (present results). The finding of MST and SOM positive cells in the Otp expression domain, and the presence of Otp/SOM coexpressing cells, suggest that this transcriptional regulator controls the differentiation of these SOM+ and MST+ populations in Xenopus, like in amniotes, and that likely the nuclei organization could also be comparable (present results). In addition, the presence o f TRH immunoreactive neurons in this region is also a conserved feature along evolution. Thus, TRH-containing cells have been described in the paraventricular nucleus of amniotes (Vandenbome et ah, 2005; Aoki et ah, 2007; Lopez et ah, 2008; Radar et ah, 2010) and also in amphibians (Dominguez et ah, 2008). Furthermore, numerous data in anamniotes support the strongly conserved role o f Otp in the differentiation of the neurohormone- 115 3. E L H lP O 1 ALA M O EN LA 1RA NSICIU N ANAM NIO- AMNIOTA: ESTUDIOS EN ANUROS Y REPTILES secreting cells in other vertebrates. Specifically, an Otp isoform (Otpl) is required for the development o f the zebrafish vasotocin-neurophysin and isotocin- neurophysin (Tessmar-Raible et al., 2007; Eaton and Glasgow, 2007; Blechman et al., 2007; Eaton et al., 2008). Boundaries. In Xenopus, the Otp positive SPV territory borders caudally on the PThE, which expresses Tbrl/xLhx9, and on the PTh, rich in Isll and xD114 expression (present results; see figure 8). This situation agrees with that reported in mouse, where the boundary between both regions was discernible by the expression of Sim-1, Otp and Bm-2 in the SPV, in contrast to the expression o f Arx, Dix and Pax6 in the PTh (Puelles and Rubenstein, 2003). In the mouse, the dorsal end of this border (SPV-PTh) surrounds the PThE along the stria terminalis into the vicinity o f the amygdala, apparently to end at the fissura choroidea, in the caudomedial wall of the telencephalon (Puelles and Rubenstein, 2003). In addition, the Otp positive expression domain also marks the ventral limit o f the SPV with the Dlx+ SC territory in all vertebrates studied (Puelles and Rubenstein, 2003; Bardet et al., 2008; Morales-Delgado et al., 2011; present results). Notably, in a recent study o f the turtle hypothalamus, a similar situation has been described in which the SPV borders dorsally on the PO, through the POH Nkx2.2 expressing zone, ventrally on the SC, and caudally on the PThE and the PTh; all these limits being confirmed on the basis of the combinatorial expression patterns for the same markers used in the present study (Moreno et al., 201 lb). The suprachiasmatic area The molecular and neurochemical data collected in the present study have allowed us to perform a genoarchitectonic and chemoarchitectonic map of the SC territory. Thus, the combinatorial expression of xD114, Islet, Nkx2.1, Nkx2.2, and xLhxl/7 allowed the analysis o f the SC territory. The chemiarchitectonic of this region was also analyzed by the distribution of TH and GABA, which provided information about the postmitotic young cells that will contribute to the individualization of specific subnuclei in the adult. Patterning and neuronal specification. In Xenopus, we have defined two distinct subdivisions along the rostro-caudal axis o f the SC, the SCr and SCc. The SCr is characterized by the ventricular expression o f the developmental regulators xShh and Nkx2.1, whereas Nkx2.2 is expressed subventricularly. In turn, SCc lacks the expression of these regulators, both in vz and in svz, and expresses xD114/Isll (present results; see Figure 8). A similar rostro-caudal subdivision has also been described recently in the turtle, mainly on the basis o f the Nkx2.1, Nkx2.2 and Isll combinational expressions (Moreno et al., 2011b). Nkx2.1 expression in the SC has only been detected in non-mammalian vertebrates including zebrafish (Rohr et al., 2001), several amphibians (van den Akker et al., 2008; Dominguez et al., 2010; present results), turtle (Moreno et al., 2011b) and chicken (Abellân and Medina, 2009). In particular, in the chicken, Nkx2.1 expression has been described in the subparaventricular area (SPa), which belongs to the suprachiasmatic domain (Abellân and Medina, 2009). The SPa has also been defined as a Nkx2.2 positive territory (Bardet et al., 2010), suggesting that this region could be equivalent to the Nkx2.1/Nkx2.2 positive SCr of Xenopus (present results) and turtle (Moreno et al., 2011b). In the evolutionary context, it seems that the Nkx2.1 expression is being progressively restricted in the SC territory. Thus, in the zebrafish the Nkx2.1/Shh expression occupies the whole the SC (Rohr et al., 2001), whereas in Xenopus, an anamniote tetrapod, and sauropsids (birds and reptiles) this expression is partial and restricted to subregions (present results; van der Akker et al., 2008; Medina; 2008; Abellân and Medina, 2009; Moreno et al., 2011b), in contrast to mammals that lack Nkx2.1 expression in the SC (Puelles and Rubenstein, 2003; Bardet et al., 2010). This lack in mammals is supported by the fact that mutant mice with absence of Shh in the hypothalamic neuroepithelium showed a normal development of the SC zone (Szabo et al., 2009). These changes in the SC Shh/Nkx2.1 expression have been related to the reduction of the alar hypothalamus in amniotes in contrast to anamniotes and the correlative expansion o f the thalamus (van den Akker et al., 2008; for review see Medina, 2008), The present data about the gradual restriction of Shh/Nkx2.1 expression found in mammals in comparison with non­ mammalian vertebrates such as Xenopus (present results) and turtle (Moreno et al., 2011b) support this hypothesis. Thus the Isll+/Nkx2.1- regions in the SC would be the most conserved regions in all vertebrates in contrast to the xShh/Nkx2.1+ region, totally lacking in mammals. Previous studies in Xenopus described Isll, xLhxl and xLhx7 expression in the SC (Moreno et al., 2004; Moreno et al., 2008a; present results). Recently, a detailed molecular atlas o f mouse hypothalamic development has been presented in which several members o f the LlM-hd family were used as essential tools in the identification o f distinct hypothalamic subdomains (Shimogori et al., 2010). A particular zone was described containing Arx- and 7-positive cells that was termed the intrahypothalamic diagonal, also defined by the expression o f Lhx6, Lhx8 and Lhxl (Shimogori et al., 2010). This domain appeared to give rise later in development to the inhibitory interaeurons of the suprachiasmatic nucleus, dorsomedial hypothalamic nucleus and posterior hypothalamus. This situation resembles that described in the chicken, in which a territory o f the SC was defined by the expression of Lhx6/7/8 and also by the presence of cGAD67 and the lack of cVGlut2 (Abellân and Medina, 2009). In the medaka fish, Dlx2 and Lhx7 expressions showed comparable patterns to those detected in other vertebrates, defining a hypotalamic expression domain 116 J. üilj nirvi 1 AljAiVlW î 'XAiVllXIHJ- AMNIOTA: ESTUDIOS EN ANUROS Y REPTILES that likely corresponds to the intrahypothalamic diagonal region (Alunni et al., 2004). Therefore, the LIMhd expression pattem seems to be conserved in this area across vertebrates. The comparison of the Isll and Dix expressions suggested that their expression domains are largely overlapped in all prosencephalic regions (Moreno et al., 2008a). The SC has been shown to be a Dlx-positive territory in all vertebrates analyzed (Bachy et al., 2002; Brox et al., 2003; Flames et al., 2007; Medina, 2008; Moreno et al., 2011b; present results) and the results obtained in this study corroborate that both Isll and xD114 expression domains overlap in the SC domain. We have analyzed the expression pattern of xD114 (that shows 64% sequence identity with mouse Dlx2) in combination with other hypothalamic markers to identify different domains within the alar hypothalamus, and have detected xD114 expression in the entire SC, occupying the svz, as was also seen for the Isll expression (see Figure 8). In mammals, Dix 1/2 are expressed in the least mature cells passing from the vz to the svz, whereas Dlx5/6 are expressed in svz and mz (Liu et al., 1997). Even in the larval lamprey forebrain, the expression o f Dll genes was described in the subparaventricular area, which is the primordium of the suprachiasmatic nucleus (Martinez de la Torre et al., 2011). Regarding neuronal specification in the SC region, recent data have indicated that the production of GABAergic neurons in the forebrain is restricted to the Dix expressing territories (Price et al., 1991; Bulfone et al., 1993; Marin and Rubenstein, 2001). Previous studies \nXenopus (Brox et al., 2003) and the present results have showed that in the SC territory the GABAergic positive neurons were located within the xD114/lsl 1 expressing area, suggesting that also in Xenopus members of the Dix genes family are involved in the specification of the GABAergic phenotype. O f note, it was reported in mice that Dix transcription factors regulate dopaminergic differentiation in the prethalamus (Andrews et al., 2003). The close spatial relationship between the postmitotic marker TH with Isll and xD114 in the SC of Xenopus suggest that these genes could also be controlling the dopaminergic specification. In terms of chemoarchitecture, previous studies in amphibians have divided the SC region into rostral and caudal portions, mainly on the basis o f calcium binding proteins expression (Milan and Puelles, 2000; Morona and Gonzalez, 2008). In those studies, the expression of calbindin-D28k was found in almost the entire SC, whereas calretinin was restricted to the rostral portion (Morona and Gonzalez, 2008). Moreover, three distinct nuclei within the SC region have been identified in Xenopus following neurochemical criteria. These nuclei are the ventrolateral, dorsomedial and caudal, which are differentiated from each other by the expression of neuropeptide Y and TH (Kramer et al., 2001). These neurochemical differences observed between the distinct subdivisions described within the SC are probably the result o f the different genetic specification that underlies the regionalization o f this area. Boundaries. In anurans, the SC region is flanked dorsally and ventrally by the Otp expression domains of these SPV and the tuberal hypothalamus, respectively (present results). In addition, the combination of Otp/lsll observed through development allowed the identification of the caudal boundary of SC. Thus, at dorsal levels the SC borders on the posterior tip of the SPV, whereas ventrally forms a boundary with the PTh (present results, see Figure 8). This situation agrees with that recently reported in the turtle Pseudemys scripta, where the boundary between the SC and the dorsal PO and the ventral Tub is discernible by the expression o f Otp (Moreno et al., 2011b). In addition, the SPV Otp positive expression domain also marks the limit o f the Dlx+ SC and PTH territory in all vertebrates studied (Morales-Delgado et al., 2011; Puelles and Rubenstein, 2003; Bardet et al., 2008; present results). In contrast to the observations in Pseudemys, the SC region in Xenopus limits caudally with the PTh, as observed by the continuous xDdl4 expression observed in these regions (present results), whereas in the turtle a strip of the SPV separates the SC and the PTh (Moreno et al., 201 lb). Hypothalamic formation: evolutionary considerations All the data obtained in the present study point to a largely conserved pattem of organization among vertebrates, thus the main conclusion of this study in a evolutionary comparative context is that major histogenetic processes in the alar hypothalamus, thought to be a hallmark of mammals, actually existed early in vertebrate phylogeny. Therefore, similar regions most likely control similar reflexes, responses, and behaviors in vertebrates. In particular, the current view of the anuran hypothalamus suggests the existence of a basic plan in the organization of this territory shared by all tetrapods (for review see Moreno and Gonzalez, 2011). The analyzed alar hypothalamus in the secondary prosencephalon is constituted by the SPV and the SC. Its boundaries are dorsally with the PO, caudally with the prethalamic eminence and the prethalamus, and ventrally with the basal hypothalamus (present results). It shows in all the vertebrates studied a banded exelusive pattem for the Otp/D114 gene expressions in the SPV/SC respectively, (present results, Bardet et al., 2008). The main exception is found in the Nkx2.1/Shh expression observed in the SC region (Medina, 2008; Moreno et al., 2008a; present results), in contrast to that described in mammals (Puelles et al., 2000). In evolutionary terms, this discrepancy could be related to the thalamic expansion that oecurs in amniotes, even at the expense of reducing alar hypothalamic areas. The significance o f the differences/similarities observed between anamniotes and amniotes could serve to understand the motor that drove the hypothalamic evolution and increase our knowledge about the brain organization and its development. 117 3. EL H i r U l ALAM U EN EA IK A N M LIU N ANAMJNIU- AMNIOTA: ESTUDIOS EN ANUROS Y REPTILES LITERATURE CITED Abellân A, Medina L. 2009. Subdivisions and derivatives o f the chicken subpallium based on expression of LIM and other regulatory genes and markers of neuron subpopulations during development. J Comp Neurol 515:465-501. Abellân A, Vernier B, Rétaux S, Medina L. 2010. Similarities and differences in the forebrain expression of Lhxl and Lhx5 between chicken and mouse: Insights for understanding telencephalic development and evolution. J Comp Neurol 518:3512-3528. Acampora D, Postiglione MP, Avantaggiato V, Di Bonito M, Vaccarino FM, Michaud J, Simeone A. 1999. Progressive impairment of developing neuroendocrine cell lineages in the hypothalamus of mice lacking the Orthopedia gene. Genes Dev 13:2787-2800. Altman J, Bayer SA. 1986. The development of the rat hypothalamus. Adv Anat Embryol Cell Biol 100:1- 178. Alunni A, Blin M, Desehet K, Bourrât F, Vernier P, Rétaux S. 2004. Cloning and developmental expression patterns of Dlx2, Lhx7 and Lhx9 in the medaka fish (Oryzias latipes). Mech Dev 121:977- 983. Andrews GL, Yun K, Rubenstein JL, Mastick OS. 2003. Dix transcription factors regulate differentiation of dopaminergic neurons of the ventral thalamus. Mol Cell Neurosci 23:107-120. Aoki Y, Ono H, Yasuo S, Masuda T, Yoshimura T, Ebihara S, ligo M, Yanagisawa T. 2007. Molecular evolution of prepro-thyrotropin-releasing hormone in the chicken (Gallus gallus) and its expression in the brain. Zoolog Sci 24:686-692. Bachy 1, Berthon J, Rétaux S. 2002. Defining palliai and subpallial divisions in the developing Xenopus forebrain. Mech Dev 117:163-172. Bachy 1, Rétaux S. 2006. GABAergic specification in the basal forebrain is controlled by the LlM-hd factor Lhx7. Dev Biol 291:218-226. Bachy 1, Vernier P, Rétaux S. 2001. The LIM- homeodomain gene family in the developing Xenopus brain: conservation and divergences with the mouse related to the evolution o f the forebrain. J Neurosci 21:7620-7629. Bardet SM, Cobos 1, Puelles E, Martinez-De-La-Torre M, Puelles L. 2006. Chicken lateral septal organ and other circumventricular organs form in a striatal subdomain abutting the molecular striatopallidal border. J Comp Neurol 499:745-767. Bardet SM, Martinez-de-la-Torre M, Northcutt RG, Rubenstein JL, Puelles L. 2008. Conserved pattem o f OTP-positive cells in the paraventricular nucleus and other hypothalamic sites of tetrapods. Brain Res Bull 75:231-235. Bardet SM, Ferrân JL, Sânchez-Arrones L, Puelles L. 2010. Ontogenetic expression of sonic hedgehog in the chicken subpallium. Front Neuroanat. 4:28. doi: 10.3389/fiiana.2010.00028 Blasher S, Heinrichs M. 1982. Immunoreactive neuropeptide systems in avian embryos (domestic mallard, domestic fowl, Japanese quail). Cell Tissue Res 223:287-303. Blechman J, Borodovsky N, Eisenberg M, Nabel-Rosen H, Grimm J, Levkowitz G. 2007. Specification of hypothalamic neurons by dual regulation o f the homeodomain protein Orthopedia. Development 134:4417-4426. Bourguignon C, Li J, Papalopulu N. 1998. XBF-1, a winged helix transcription factor with dual activity, has a role in positioning neurogenesis in Xenopus competent ectoderm. Development 125:4889-4900. Brox A, Puelles L, Ferreiro B, Medina L. 2003. Expression of the genes GAD67 and Distal-less-4 in the forebrain o f Xenopus laevis eonfirms a common pattem in tetrapods. J Comp Neurol 461:370-393. Bmce L. 2008. Evolution if the hypothalamus in amniotes. In: Encyclopedia of Neuroscience, Binder, M.D., Hirokawa, N., Windhorst, U. (Eds). Springer, Berlin, pp 1363-1367. Bulfone A, Puelles L, Porteus MH, Frohman MA, Martin GR, Rubenstein JL. 1993. Spatially restricted expression of Dix-1, Dlx-2 (Tes-1), Gbx- 2, and Wnt-3 in the embryonic day 12.5 mouse forebrain defines potential transverse and longitudinal segmental boundaries. J Neurosci 13:3155-3172. Bupesh M, Legâz 1, Abellân A, Medina L. 2011a. Multiple telencephalic and extratelencephalic embryonic domains contribute neurons to the medial extended amygdala. J Comp Neurol 519:1505-1525. Bupesh M, Abellân A, Medina L. 2011b. Genetie and experimental evidence supports the continuum of the central extended amygdala and a mutiple embryonic origin of its principal neurons. J Comp Neurol 519:3507-3531. Caqueret A, Coumailleau P, Michaud JL. 2005. Regionalization of the anterior hypothalamus in the chick embryo. Dev Dyn 233:652-658. Caqueret A, Boucher F, Michaud JL. 2006. Laminar organization of the early developing anterior hypothalamus. Dev Biol 298:95-106. Conway KM, Gainer H. 1987. Immunocytochemical studies o f vasotocin, mesotocin, and neurophysins in the Xenopus hypothalamo-neurohypophysial system. J Comp Neurol 264:494-508. Crosby EC, Woodbume RT. 1940. The comparative anatomy o f the preoptic area and the hypothalamus. Res Publ Assoc Res Nerv Ment Dis 20:52-169. Del Giacco L, Sordino P, Pistocchi A, Andreakis N, Tarallo R, Di Benedetto B, Cotelli F. 2006. Differential regulation o f the zebrafish orthopedia 1 gene during fate determination of diencephalic neurons. BMC Dev Biol 6:50. 118 ô . E E m r U lA E A iV U J El'N E A 1 KAT>l»lEHJi>l ATN AiVli'NlU- AMNIOTA: ESTUDIOS EN ANUROS Y REPTILES Del Giacco L, Pistocchi A, Cotelli F, Fortunate AE, Sordino P. 2008. A peek inside the neurosecretory brain through Orthopedia lenses. Dev Dyn 237:2295- 2303. Diez-Roux G, Banfi S, Sultan M, Geffers L, Anand S, Rozado D, Magen A, Canidio E, Pagani M, Peluso 1, Lin-Marq N, Koch M, Bilio M, Cantiello 1, Verde R, De Masi C, Bianchi SA, Cicchini J, Perroud E, Mehmeti S, Dagand E, Schrinner S, Numberger A, Schmidt K, Metz K, Zwingmann C, Brieske N, Springer C, Hernandez AM, Herzog S, Grabbe F, Sieverding C, Fischer B, Schrader K, Brockmeyer M, Dettmer S, Helbig C, Alunni V, Battaini MA, Mura C, Henrichsen CN, Garcia-Lopez R, Echevarria D, Puelles E, Garcia-Calero E, Kruse S, Uhr M, Kauck C, Feng G, Milyaev N, Ong CK, Kumar L, Lam M, Semple CA, Gyenesei A, Mundlos S, Radelof U, Lehrach H, Sarmientos P, Reymond A, Davidson DR, Dolle P, Antonarakis SE, Yaspo ML, Martinez S, Baldock RA, Eichele G, Ballabio A. 2010. A high- resolution anatomical atlas of the transeriptome in the mouse embryo. PLoS Biol 9 :e l000582. Dominguez L, Lopez JM, Gonzalez A. 2008. Distribution of thyrotropin-releasing hormone (TRH) immunoreactivity in the brain of urodele amphibians. Brain Behav Evol 71(3):231-246. Dominguez L, Gonzalez A, Moreno N. 2010. Sonic hedgehog expression during Xenopus laevis forebrain development. Brain Res 1347:19-32. Dominguez L, Gonzalez A, Moreno N. 2011. Ontogenetic distribution of the transcription factor Nkx2.2 in the developing forebrain of Xenopus laevis. Front Neuroanat 5:11. Eaton JL, Glasgow E. 2007. Zebrafish orthopedia (otp) is required for isotocin cell development. Dev Genes Evol 217:149-158. Eaton JL, Holmqvist B, Glasgow E. 2008. Ontogeny of vasotocin-expressing cells in zebrafish: selective requirement for the transcriptional regulators orthopedia and single-minded 1 in the preoptic area. Dev Dyn 237:995-1005. Ericson J, Rashbass P, Schedl A, Brenner-Morton S, Kawakami A, van Heyningen V, Jessell TM, Briscoe J. 1997. Pax6 controls progenitor cell identity and neuronal fate in response to graded Shh signaling. Cell 90:169-180. Figdor MC, Stem CD. 1993. Segmental organization of embryonic diencephalon. Nature 363(6430):630-634. Flames N, Pla R, Gelman DM, Rubenstein JL, Puelles L, Marin O. 2007. Delineation o f multiple subpallial progenitor domains by the combinatorial expression of transcriptional codes. J Neurosci 27:9682-9695. Garcia-Lopez M, Abellân A, Legâz 1, Rubenstein JL, Puelles L, Medina L. 2008. Histogenetic compartments of the mouse centromedial and extended amygdala based on gene expression pattems during development. J Comp Neurol 506:46- 74. Garcia-Lopez R, Pombero A, Martinez S. 2009. Fate map of the chick embryo neural tube. Dev Growth Differ 51:145-165. Garcia-Moreno F, Pedraza M, Di Giovannantonio LG, Di Salvio M, Lopez-Mascaraque L, Simeone A, De Carlos JA. 2010. A neuronal migratory pathway crossing from diencephalon to telencephalon populates amygdala nuclei. Nat Neurosci 13:680- 689. Gonzâlez A, Smeets WJ. 1992. Distribution of vasotocin- and mesotocin-like immunoreactivities in the brain of the South African clawed frog Xenopus-laevis. J Chem Neuroanat 5:465-479. Gonzâlez A, Tuinhof R, Smeets WJ. 1993. Distribution of tyrosine hydroxylase and dopamine immunoreactivities in the brain of the South African clawed frog Xenopus laevis. Anat Embryol (Berl) 187:193-201. Gonzâlez A, Marin O, Tuinhof R, Smeets WJ. 1994. Ontogeny of catecholamine systems in the central nervous system o f anuran amphibians: an immunohistochemical study with antibodies against tyrosine hydroxylase and dopamine. J Comp Neurol 346:63-79. Gonzâlez A, Munoz A, Munoz M, Marin O, Smeets WJ. 1995. Ontogeny of vasotocinergic and mesotocinergic systems in the brain o f the South African clawed frog Xenopus laevis. J Chem Neuroanat 9:27-40. Gonzâlez A, Lopez JM, Marin O. 2002a. Expression pattem of the homeobox protein NKX2-1 in the developing Xenopus forebrain. Brain Res Gene Expr Pattems 1:181-185. Gonzâlez A, Lopez JM, Sânchez-Camacho C, Marin O. 2002b. Regional expression of the homeobox gene NKX2-1 defines pallidal and intemeuronal populations in the basal ganglia of amphibians. Neuroscience 114:567-575. Gonzâlez A, Moreno N, Morona R, Lopez JM. 2003. Somatostatin-like immunoreactivity in the brain of the urodele amphibian Pleurodeles waltl. Colocalization with catecholamines and nitric oxide. Brain Res 965:246-258. Gonzâlez A, Northcutt RG. 2009. An immunohistochemical approach to lungfish telencephalic organization. Brain Behav Evol 74:43-55. Goshu E, Jin H, Lovejoy J, Marion JF, Michaud JL, Fan CM. 2004. Sim2 contributes to neuroendocrine hormone gene expression in the anterior hypothalamus. Mol Endocrinol 18:1251-1262. Herrick. 1910. The morphology o f the forebrain in Amphibian and Reptilia. J Comp Neurol 20:413- 547. Herriek. 1917. The intemal stmcture of the midbrain and thalamus of Necturus. J Comp Neurol 28; 215- 348. Hodos W. 2008. Evolution of the hypothalamus in anamniotes. In: Encyclopedia of Neuroscience, 119 j . EL H IEU l A L A M U EN LA 1 KAN SICIUN A N A M N lü- AMNIOTA: ESTUDIOS EN ANUROS Y REPTILES Binder, M.D., Hirokawa, N., Windhorst, U. (Eds). Springer, Berlin, pp 1361-1363. Kâdar A, Sanchez E, Wittmann G, Singru PS, Fuzesi T, Marsili A, Larsen PR, Liposits Z, Lechan RM, Fekete C. 2010. Distribution of hypophysiotropic thyrotropin-releasing hormone (TRH)-synthesizing neurons in the hypothalamic paraventricular nucleus o f the mouse. J Comp Neurol 518:3948-3961. Kramer BM, Welting J, Berghs CA, Jenks BG, Roubos EW. 2001. Functional organization of the suprachiasmatic nucleus of Xenopus laevis in relation to background adaptation. J Comp Neurol 432:346- 355. Kuhlenbeck H. 1954. The human diencephalon; a summary of development, structure, funetion, and pathology. Confin Neurol 14: 1-230. Le Gross Clark WE. 1938. Morphological aspects of the hypothalamus. In: Le Gross Clark WE, Beattie J, Riddoch G, Dott NM (eds). The hypothalamus. Oliver and Boyd, Edinburgh, pp 1-68. Lin X, State MW, Vaccarino FM, Greally J, Hass M, Leckman JF. 1999. Identification, chromosomal assignment, and expression analysis of the human homeodomain-containing gene Orthopedia (OTP). Genomics 60:96-104. Liu JK, Ghattas 1, Liu S, Chen S, Rubenstein JL. 1997. Dix genes encode DNA-binding proteins that are expressed in an overlapping and sequential pattem during basal ganglia differentiation. Dev Dyn 210:498-512. Lopez JM, Dominguez L, Gonzalez A. 2008. Immunohistochemical localization of thyrotropin- releasing hormone in the brain of reptiles. J Chem Neuroanat 36:251-263. Lopez JM, Moreno N, Morona R, Munoz M, Dominguez L, Gonzalez A. 2007. Distribution o f somatostatin­ like immunoreactivity in the brain of the caecilian Dermophis mexicanus (Amphibia: Gymnophiona): comparative aspects in amphibians. J Comp Neurol 501(3):413-430. Machluf Y, Gutnick A, Levkowitz G. 2011. Development of the zebrafish hypothalamus. Ann N Y Acad Sci 1220:93-105. Maier S, Walkowiak W, Luksch H, Endepols H. 2010. An indirect basal ganglia pathway in anuran amphibians? J Chem Neuroanat 40:21-35. Marin 0 , Rubenstein JL. 2001. A long, remarkable joumey: tangential migration in the telencephalon. Nat Rev Neurosci 2:780-790. Marin O, Smeets WJ, Gonzalez A. 1998. Basal ganglia organization in amphibians: chemoarchitecture. J Comp Neurol 392:285-312. Marin O, Smeets WJ, Gonzalez A. 1998. Basal ganglia organization in amphibians: evidence for a common pattem in tetrapods. Prog Neurobiol 55:363-397. Markakis EA. 2002. Development of the neuroendocrine hypothalamus. Front Neuroendocrinol 23:257-291. Martinez-de-la-T orre M, Pombal MA, Puelles L. 2011. Distal-less-like protein distribution in the larval lamprey forebrain. Neuroscience 178:270-284. Medina L. 2008. Evolution and embryo logical development o f forebrain. In: Encyclopedia of Neuroscience, Binder, M.D., Hirokawa, N., Windhorst, U. (Eds). Springer, Berlin, pp 1172- 1192. Milan FJ, Puelles L. 2000. Pattems o f calretinin, calbindin, and tyrosine-hydroxylase expression are consistent with the prosomeric map o f the fi-og diencephalon. J Comp Neurol 419:96-121. Morales-Delgado N, Merchan P, Bardet SM, Ferrân JL, Puelles L, Diaz C. 2011. Topography of Somatostatin Gene Expression Relative to Molecular Progenitor Domains during Ontogeny of the Mouse Hypothalamus. Front Neuroanat 5:10. doi:. 10.3289/friana.2011.00012. Moreno N, Gonzâlez A. 2007. Evolution of the amygdaloid complex in vertebrates, with special reference to the anamnio-amniotic transition. J Anat 211:151-163. Moreno N, Gonzâlez A. 2011. The non-evaginated secondary prosencephalon of vertebrates. Front Neuroanat 5:12. doi: 10.3389/fiiana.2011.00012. Moreno N, Bachy 1, Rétaux S, Gonzâlez A. 2004. LIM- homeodomain genes as developmental and adult genetic markers of Xenopus forebrain functional subdivisions. J Comp Neurol 472:52-72. Moreno N, Dominguez L, Rétaux S, Gonzâlez A. 2008a. Islet 1 as a marker of subdivisions and cell types in the developing forebrain o f Xenopus. Neuroscience 154:1423-1439. Moreno N, Retaux S, Gonzâlez A. 2008b. Spatio- temporal expression o f Pax6 in Xenopus forebrain. Brain Res 1239:92-99. Moreno N, Gonzâlez A, Rétaux S. 2008c. Evidences for tangential migrations in Xenopus telencephalon: developmental pattems and cell tracking experiments. Dev Neurobiol 68:504-520. Moreno N, Gonzâlez A, Rétaux S. 2009. Development and evolution of the subpallium. Semin Cell Dev Biol 20:735-743. Moreno N, Morona R, Lopez JM, Gonzâlez A. 2010. Subdivisions of the Turtle Pseudemys scripta Subpallium Based on the Expression of Regulatory Genes and Neuronal Markers. J Comp Neurol. 518:4877-902. Moreno N, Morona R, Lopez JM, Dominguez L, Joven A, Bandin S, A G . 2011 a. Characterization of the bed nucleus of the stria terminalis (BST) in anuran amphibians. J Comp Neurol. doi: 10.1002/cne.22694. Moreno N, Dominguez L, Morona R, Gonzâlez A. 2011. Subdivisions o f the turtle Pseudemys scripta hypothalamus based on the expression of regulatory genes and neuronal markers. J Comp Neurol, doi: 11-0165.22762 Morona R, Ferrân JL, Puelles L, Gonzâlez A. 2011. Embryonic genoarchitecture of the pretectum in Xenopus laevis: a conserved pattem in tetrapods. J Comp Neurol 519:1024-1050. 1 2 0 J. EE nirV l̂AEAiVHJ En EA Al̂ AiViniW- AMNIOTA: ESTUDIOS EN ANUROS Y REPTILES Morona R, Gonzalez A. 2008. Calbindin-D28k and calretinin expression in the forebrain o f anuran and urodele amphibians: further support for newly identified subdivisions. J Comp Neurol 511:187-220. Murakami Y, Ogasawara M, Sugahara F, Hirano S, Satoh N, Kuratani S. 2001. Identification and expression of the lamprey Pax6 gene: evolutionary origin of the segmented brain of vertebrates. Development 128:3521-3531. Murphy DB, Wiese S, Burfeind P, Schmundt D, Mattei MG, Schulz-Schaeffer W, Thies U. 1994. Human brain factor 1, a new member of the fork head gene family. Genomics 21:551-557. Neary TJ, Northcutt RG. 1983. Nuclear organization of the bullfrog diencephalon. J Comp Neurol 213:262- 278. Nieuwenhuys R. ten Donkelaar H, Nicholson C (Eds) 1998. The Central Nervous System of Vertebrates. London: Springer. Nieuwenhuys R, Voogd J, van Huijzen C. 2008. The Human Central Nervous System. Springer, Germany. Nieuwkoop PD, and Faber J. 1967. Normal table of Xenopus laevis (Daudin). Amsterdam: Noth Holland. Olivereau M, Vandesande F, Boucique E, Ollevier F, Olivereau JM. 1987. Immunocytochemieal localization and spatial relation to the adenohypophysis o f a somatostatin-like and a corticotropin-releasing factor-like peptide in the brain o f four amphibian species. Cell Tissue Res 247:317- 324. Petkô M, Orosz V. 1996. Distribution of somatostatin- immunoreactive structures in the central nervous system o f the frog, Rana esculenta. J Himforsch 37:109-120. Prati T, Vitalis T, Warren N, Edgar JM, Mason JO, Price DJ. 2000. A role for Pax6 in the normal development of dorsal thalamus and its cortical connections. Development 127:5167-5178. Price M, Lemaistre M, Pischetola M, Di Lauro R, Duboule D. 1991. A mouse gene related to Distal- less shows a restricted expression in the developing forebrain. Nature 351:748-751. Puelles L. 1995. A segmental morphological paradigm for understanding vertebrate forebrains. Brain Behav Evol 46:319-337. Puelles L. 2001. Brain segmentation and forebrain development in amniotes. Brain Res Bull 55:695- 710. Puelles L, Rubenstein JL. 1993. Expression pattems of homeobox and other putative regulatory genes in the embryonic mouse forebrain suggest a neuromeric organization. Trends Neurosci 16:472-479. Puelles L, Medina L. 2002. Field homology as a way to reconcile genetic and developmental variability with adult homology. Brain Res Bull 57:243-255. Puelles L, Rubenstein JL. 2003. Forebrain gene expression domains and the evolving prosomeric model. Trends Neurosci 26:469-476. Puelles L, Kuwana E, Puelles E, Bulfone A, Shimamura K, Keleher J, Smiga S, Rubenstein JL. 2000. Palliai and subpallial derivatives in the embryonic chick and mouse telencephalon, traced by the expression of the genes Dlx-2, Emx-1, Nkx-2.1, Pax-6, and Tbr-1. J Comp Neurol 424:409-438. Puelles L, Martinez de la Torre M, Paxinos G, Watson C, Martinez S. 2007. The Chick Brain in Stereotaxic Coordinates: an Atlas featuring Neuromeric Subdivisions and Mammalian Homologies. Academic Press/Elsevier, San Diego. Rohr KB, Barth KA, Varga ZM, Wilson SW. 2001. The nodal pathway acts upstream of hedgehog signaling to specify ventral telencephalic identity. Neuron 29:341-351. Roth M, Bonev B, Lindsay J, Lea R, Panagiotaki N, Houart C, Papalopulu N. 2010. FoxGl and TLE2 act cooperatively to regulate ventral telencephalon formation. Development 137:1553-1562. Rubenstein JL, Beachy PA. 1998. Patterning of the embryonic forebrain. Curr Opin Neurobiol 8:18-26. Rubenstein JL, Martinez S, Shimamura K, Puelles L. 1994. The embryonic vertebrate forebrain: the prosomeric model. Science 266: 578-580. Rubenstein JL, Shimamura K, Martinez S, Puelles L. 1998. Regionalization of the prosencephalic neural plate. Annu Rev Neurosci 21:445-477. Sânchez-Arrones L, Ferrân JL, Rodriguez-Gallardo L, Puelles L. 2009. Incipient forebrain boundaries traced by differential gene expression and fate mapping in the chick neural plate. Dev Biol 335:43-65. Shimogori T, Lee DA, Miranda-Angulo A, Yang Y, Wang H, Jiang L, Yoshida AC, Kataoka A, Mashiko H, Avetisyan M, Qi L, Qian J, Blackshaw S. 2010. A genomic atlas of mouse hypothalamic development. Nat Neurosci 13:767-775. Simeone A, D'Apice MR, Nigro V, Casanova J, Graziani F, Acampora D, Avantaggiato V. 1994. Orthopedia, a novel homeobox-containing gene expressed in the developing CNS of both mouse and Drosophila. Neuron 13:83-101. Simerly C, Navara C, Hyun SH, Lee BC, Kang SK, Capuano S, Gosman G, Dominko T, Chong KY, Compton D, Hwang WS, Schatten G. 2004. Embryogenesis and blastocyst development after somatic cell nuclear transfer in nonhuman primates: overcoming defects caused by meiotic spindle extraction. Dev Biol 276:237-252. Smeets WJ, Gonzalez A. 2000. Catecholamine systems in the brain of vertebrates: new perspectives through a comparative approach. Brain Res Brain Res Rev 33:308-379. Swanson LW. 1987. The hypothalamus. In: Bjorklund A, Swanson LW (eds). Integrated systems of the CNS, part 1. Elsevier, Amsterdam, pp 125-277 (Handbook of chemical neuroanatomy, vol 5). Szabo NE, Zhao T, Cankaya M, Theil T, Zhou X, Alvarez-Bolado G. 2009. Role of neuroepithelial Sonic hedgehog in hypothalamic patterning. J Neurosci 29:6989-7002. 121 3. EL H lF O lALA M U EN LA IK A N SIC IU N AJNAMPIIU- AMNIOTA: ESTUDIOS EN ANUROS Y REPTILES Tao W, Lai E. 1992. Telencephalon-restricted expression of BF-1, a new member of the HNF-3/fork head gene family, in the developing rat brain. Neuron 8(5):957- 966. Ten Donkelaar HJ. 1998. Anurans. In: Nieuwenhuys R, ten Donkelaar HJ, Nicholson C, editors. The central nervous system of vertebrates. London; Springer. P 1151-1314. Tessmar-Raible K, Raible F, Christodoulou F, Guy K, Rembold M, Hausen H, Arendt D. 2007. Conserved sensory-neurosecretory cell types in annelid and fish forebrain: insights into hypothalamus evolution. Cell 129:1389-1400. Thepen T, Voom P, Stoll CJ, Sluiter AA, Pool CW, Lohman AH. 1987. Mesotocin and vasotocin in the brain of the lizard Gekko gecko. An immunocytochemical study. Cell Tissue Res 250:649-656. van den Akker WM, Brox A, Puelles L, Durston AJ, Medina L. 2008. Comparative functional analysis provides evidence for a crucial role for the homeobox gene Nkx2.1/Titf-1 in forebrain evolution. J Comp Neurol 506:211-223. Vandenbome K, Roelens SA, Darras VM, Kuhn ER, Van der Geyten S. 2005. Cloning and hypothalamic distribution of the chicken thyrotropin-releasing hormone precursor cDNA. J Endocrinol 186:387- 396. Vieira C, Martinez S. 2006. Sonic hedgehog from the basal plate and the zona limitans intrathalamica exhibits differential activity on diencephalic molecular regionalization and nuclear structure. Neuroscience 143:129-140. Vue TY, Aaker J, Taniguchi A, Kazemzadeh C, Skidmore JM, Martin DM, Martin JF, Treier M, Nakagawa Y. 2007. Characterization of progenitor domains in the developing mouse thalamus. J Comp Neurol 505:73- 91. Wang W, Lufkin T. 2000. The murine Otp homeobox gene plays an essential role in the specification of neuronal cell lineages in the developing hypothalamus. Dev Biol 227:432-449. Zhao XF, Suh CS, Prat CR, Ellingsen S, Fjose A. 2009. Distinct expression o f two foxgl paralogues in zebrafish. Gene Expr Pattems 9:266-272. 122 J . E E n i r w i AEAiVlYi Ei>l E A Ai^ AiVli'>IVi- AMNIOTA: ESTUDIOS EN ANUROS Y REPTILES THE JOURNAL OF COMPARATIVE NEUROLOGY 000: 00-00 (2012) Characterization of the basal hypothalamus of Xenopus laevis during development by molecular marker analysis LAURA DOMINGUEZ, AGUSTIN GONZALEZ, NEREA M ORENO Departamento de Biologia Celular, Facultad de Biologia, Universidad Complutense, 28040, Madrid, Spain ABSTRACT The pattems of expression o f a set o f conserved developmental regulatory transcription factors and neuronal markers were analyzed in the basal hypothalamus of Xenopus laevis throughout development. Combined immunohistochemical and in situ hybridization techniques were used for the identification of subdivisions and their boundaries. The basal region, rostral to the hypothalamo-diencephalic boundary, produced the ventral hypothalamus, including the tuberal, and mammillary hypothalamic regions, according to the prosomeric model. It limited dorsally with the optic chiasm and the alar hypothalamus, and caudally with the diencephalic prosomere P3. The tuberal hypothalamus was defined by the expression of Nkx2.1, xShh and Isll and it was further subdivided into rostral and caudal portions, on the basis o f the distinct Otp expression only in the rostral portion and the expression o f Nkx2.2 restricted to the caudal subdivision. In the mammillary region the combination of xShh and Nkx2.1 defined the rostral mammillary region, expressing Nkx2.1, and the caudal retromammillary area expressing xShh. The boundary between both basal hypothalamic territories was observed by the combination of xLhxl, xD114 and Otp, expressed in the mammillary region, with Isll, absent in this region and only expressed in the tuberal hypothalamus. Finally, in the mammillary region, Otp was expressed by the catecholaminergie cell population detected in this area. All these data illustrate that the basal hypothalamus shows a very conserved organization pattem, as illustrated by the comparison o f the features observed in anurans to those reported in amniotes, suggesting the existence of a basic bauplan in the organization of this prosencephalic region in all tetrapods. Keywords: Development, tuberal, mammillar, tetrapods, homology, in situ hybridization, evolution, forebrain patterning. The hypothalamus is a specialized region of the forebrain o f vertebrates that is involved in regulation of the endocrine system, the autonomic nervous system, and related brain systems. Thus, it is concerned with various visceral functions and behavioral processes such as reproductive and parental behavior, temperature regulation, territory management, and biological rhythms (Swanson, 1987; Rink and Wullimann, 1998; Shimizu et al., 1999; Butler and Hodos, 2005; Yamamoto and Ito, 2005). The hypothalamus is formed, as the rest of the forebrain, from the anterior neural plate trough complex processes o f morphogenesis. As a result, this brain region in the mature brain is highly distorted, mainly by the sharp flexure of the longitudinal brain axis and by differential degree o f development of its components. These phenomena make difficult to identify the basic units/subdivisions in the mature hypothalamus and to understand the topological relationships between units. Moreover, the different degree of development of the hypothalamus in the different vertebrates obscures the interpretation of anatomical data and the comparison across vertebrates and greatly complicates forebrain evolution studies (Nieuwenhuys et al., 1998; Butler and Hodos, 2005; Hodos, 2008, Bruce, 2008). Current concepts o f brain anatomy agree in considering the hypothalamus as a component of the secondary prosencephalon located rostral to the diencephalon and ventral to the telencephalon (Puelles and Rubenstein, 2003; Puelles et al. 2004; Medina 2008; Shimogori et al., 2010), therefore abandoning the classical neuroanatomical view of the hypothalamus as a specialized region of the ventral part diencephalon (Hodos, 2008, Bruce, 2008). The hypothalamus is not embryologically unitary and the longitudinal units developed in the dorsoventral axis o f the neural tube are also present in the 123 3. EL H IE U 1 ALAM O EN LA I KANSICION ANAM NIO- AMNIOTA: ESTUDIOS EN ANUROS Y REPTILES hypothalamus. Thus, the alar territories of the brainstem and diencephalon are rostrally continued in the alar portion o f the hypothalamus, whereas the basal parts of the eaudal brain continue rostrally and form the basal hypothalamus (Puelles and Rubenstein, 2003). The mechanisms involved in dorsoventral patterning are highly similar across vertebrates, from fish to mammals (Lupo et al., 2006), and so are the resulting dorsoventral brain domains. The latter are better studied by the expression of regulatory genes that constitute a great help for correctly identifying major subdivisions at forebrain levels (Puelles et al., 2004). Thus, each molecular subdivision is characterized by the expression of a specific combination o f developmental regulatory genes whose action will produce a specific set o f brain derivatives (Medina 2008). From a comparative perspective, it is important to note that many data have been reported about the highly conserved molecular/genetic specification features shared by different vertebrates, and these data were based on the restricted, combinatorial expression and action of genes that regulate important aspects o f development. Thus, the expression pattems o f orthologous regulatory genes are highly conserved throughout vertebrate phylogeny (with a few exceptions), and so are the brain subdivisions/units and their molecular profile (Brox et al., 2003; Puelles et al., 2004; Wullimaim and Mueller, 2004; Medina, 2006; Moreno et al., 2010; Moreno and Gonzalez, 2011), which facilitates one-to-one comparison of the molecular brain subdivisions/units across vertebrates. In particular, the precise organization o f the hypothalamic subdivisions is currently being investigated in amniotes, particularly in amniotes (Shimogori et al., 2010; Diez-Roux et al., 2011; Moreno et al., 2011). O f note, the anamniote hypothalamus has many similarities to that o f amniotes, but it also has some important differences and it is a relatively large and well developed region in many groups, such as amphibians (Neary and Northcutt, 1983; Puelles et al., 1996; ten Donkelaar 1998a, b; Hodos 2008). As part of a research program that aims to construct a model o f hypothalamic histogenetic fields and evaluate topological and homologous relationships between anurans (anamniotes) and amniotes, in particular mammals, we have analyzed in a previous study the development of the alar hypothalamus in Xenopus laevis (Dominguez et al., 2012a). It was revealed that it is also subdivided into smaller subdomains, which show distinct molecular features and produce different cell groups, and these observations were readily comparable to those reported in amniotes (Shigomory et al., 2010; Diez-Roux et al., 2011; Moreno et al., 2011). In the present study, we examined the organization o f the basal (ventral) territories o f the hypothalamus. In amniotes, the basal region rostral to the p3/hypothalamic boundary was demonstrated to be primarily under the influence of prechordal plate/rostral mesendoderm signals, to expresses Nkx2.1, and to produce the ventral hypothalamus (Puelles and Rubenstein, 2003; Puelles et al., 2004; Medina et al., 2008). Furthermore, the expression o f Nkx2.1 in this ventral region is induced by prechordal Shh signals and this transcription factor appears to play a key role in the specification and formation o f the ventral hypothalamus in different vertebrates (Marin and Rubenstein, 2002; van den Akker et al., 2008; Lupo et al., 2006). Therefore, the basal hypothalamus forms the prechordal or hypothalamic tegmentum, rostrally located to the tegmental zone of p3, and it is constituted by the retromammillary/mammillary portion (caudal) and the tuberaFinfundar portion (rostral) (Bulfone et al., 1993; Puelles and Rubenstein, 1993; Rubenstein and Puelles, 1994; Eisenstat et al., 1999; Puelles and Rubenstein, 2003, Puelles et al., 2004; Shimogori et al., 2010). These major subdomains within the basal hypothalamus were primarily identified in the murine hypothalamus on the basis o f their distinct combinatorial expression pattems. Therefore, in the present study we have investigated molecular subdivisions or compartments within the developing basal hypothalamus and have compared expression domains of hypothalamic markers in Xenopus laevis to those reported in similar studies in amniotes (Puelles and Rubenstein, 2003; Shimogori et al., 2010; Diez-Roux et al., 2011; Moreno et al., 2011). In particular we have analyzed the expression pattems of makers implied in the amniote hypothalamic specification, such as Islet 1 (Isll), xLhxl, Nkx2.1, Nkx2.2, Orthopedia (Otp), Pax7, Sonic hegdehog (xShh), somatostatin (SOM) and tyrosine hydroxylase (TH). Several o f these markers were previously demonstrated to be expressed in distinct hypothalamic regions of Xenopus (Gonzalez et al., 2002; Moreno et al., 2008; Dominguez et al., 2010, 2011). In general, we showed that the topology and main molecular features of the subdivisions in the basal hypothalamus have been highly conserved from amphibians to mammals. MATERIALS AND METHODS Animals and tissue processing For the present study, adults, juvenile and tadpoles of Xenopus laevis were used. Embryos and larvae were staged according to Nieuwkoop and Faber (1967) into embryonic (35-45), premetamorphic (46-52), prometamorphic (53-58), and metamorphic (59-65) stages. All animals were treated according to the regulations and laws of the European Union (86/609/EEC) and Spain (Royal Decrees 1201/2005) for care and handling of animals in research, after approval from the University to conduct the experiments described. Adult Xenopus were purchased from commercial suppliers (XenopusOne, Michigan, USA), and the different developing specimens were obtained by Prcgnyl- induced (Organon) breeding and maintained in tap water at 20°C throughout their development. At appropriate times, embryos, larvae, and juveniles were deeply anesthetized by 124 3 . E E m r u 1 A E A iV lU E N E A 1 K A N » 1 E 1 U N A N A IV IN IU - AMNIOTA; ESTUDIOS EN ANUROS Y REPTILES Abbreviations CT caudal tuberal region Hyp hypophysis Ma mammilar region Mab mammillar band oc optic chiasm PI prosomere 1 P2 prosomere 2 P3 prosomere 3 P3b basal plate of P3 PO preoptic region PT pretectum PTh prethalamus RM retromammillar region SC suprachiasmatic region SPV supraoptoparaventricular region Th thalamus TP posterior tuberculum Tub tuberal region RT rostral tuberal region Zli zona limitans intrathalamic immersion in a 0.3% solution o f tricaine methanesulfonate (MS222, Sigma-Aldrich, Steinheim, Germany), pH 7.4, and used for the different sets o f experiments. The number of animals used in the present study was the minimum to guarantee the correct interpretation of the results and to minimize their suffering. Immunohistochemistry To chemically characterize the subdivisions of the basal hypothalamus, immunohistochemistry for the detection of Islet 1 (Isll), Nkx2.1, Nkx2.2, Orthopedia (Otp), Pax7, somatostatin (SOM) and tyrosine hydroxylase (TH), was carried out (Table 1). Adults, juveniles, and late larvae were perfused transcardially with 0.9% NaCl solution, followed by 100-200 ml of 4% paraformaldehyde in 0.1 M phosphate buffer (PB; pH 7.4). The brains were removed and kept in the same fixative overnight at 4°C. Subsequently, they were immersed in a solution o f 30% sucrose in PB for 5 hours at 4°C until they sank, embedded in a solution o f 20% gelatin with 30% sucrose in PB, and then immersed in a 3.7% formaldehyde solution at 4°C for 8-10 hours. The brains were cut on a freezing microtome at 25-30 pm in the transverse or sagittal plane and collected and rinsed in cold PB. The embryos and premetamorphic larvae were fixed by immersion overnight at 4“C in MEMFA (0.1 M MOPS [4-morpholinopropanesulphonic acid], 2 mM ethylene glycol tetraacetic acid, 1 mM M gS04, 3.7% formaldehyde) and then they were processed in toto. Finally, the brains were gelatin blocked (as detailed above) and cut on a freezing microtome at 14—16 pm in the transverse, horizontal, or sagittal plane (see Dominguez et al., 2010,2011). Immunohistofluorescence procedures were carried out on the free-floating sections (or in toto for embryos and young larvae) as follows: 1). First, incubation was conducted for 60 hours at 4"C in the dilution of each primary serum (see Table 1): 1) mouse anti-Isll, rabbit anti-Nkx2.1, mouse anti-Nkx2.2, rabbit anti-Otp, mouse anti-Pax7, rabbit anti-SOM, mouse anti-TH, rabbit anti- TH. 2) According to the species in which the primary antibody was raised, the second incubations were conducted with the appropriately labeled secondary antibody diluted 1:500 fbr 90 minutes at room temperature: Alexa 594-conjugated goat anti-rabbit (Molecular Probes, Eugene, OR; catalog reference A11037) or Alexa 488-conjugated goat anti-mouse (Molecular Probes; catalog reference A21042). In all cases, the antibodies were diluted in PB and containing 0.5-1% Triton X-100. After being rinsed, the sections were mounted on glass slides and coverslipped with Vectashield mounting medium (Vector, Burlingame, CA; catalog No. H I000). To study the relative distribution of two markers in the same sections, the two-step protocol for immunohistofluorecence was used with cocktails of pairs o f primary antibodies (one developed in rabbit and one in mouse), at the same dilutions and conditions specified above, and secondary cocktail of Alexa 594- and Alexa 488-conjugated antibodies (as above). In all cases, after being rinsed, the sections were mounted on glass slides and coverslipped with Vectashield. Controls and specificity of the antibodies Controls for the immunohistochemical procedures were conducted as in the companion study of the alar hypothalamus and their specificity was then detailed (Dominguez et al., 2012a). The controls included: 1) Western blot analysis, 2) incubation of some selected sections with preimmune mouse or rabbit sera instead of the primary antibody, 3) controls in which either the primary or the secondary antibody was omitted, and 4) predsorption o f the primary antibodies with synthetic peptides. Only the Pax7 antibody was not used in our previous paper but was fully characterized in similar studies in Xenopus (Morona et al., 2011). In situ hybridization Double labeling with in situ hybridization and immunohistochemistry fo r xShh/Otp, xShh/Isll, xShh/TH, xDll4/0tp, xD lW Isll, xDll4/Nkx2.2, xLhxl/Otp, xLhxl/Nkx2.2, and xLhxl/TH an single labeling fo r xS h h . The gene markers used in the present study are summarized in the table 2 with the gene bank account, origin o f the plasmid and enzymes/polymerases used in the probe synthesis. For double histofluorescence labeling experiments 125 3. E L H lF O l ALA M U EN LA I KANSICIUN ANAM NIU- AMNIOTA: ESTUDIOS EN ANUROS Y REPTILES Table 1. Antibodies used in the present study Name Immunogen Commercial Supplier MW (KDa) Dilution GABA GABA-BSA Polyclonal rabbit anti-y- aminobutyric acid Sigma; Catalogue reference: A2052 0.0103 1:3000 I s l l aa 247-349 at the C-terminus o f rat Isll Monoclonal mouse anti-Isl 1 Developmetal Studies Hybridoma Bank. Catalogue reference: 39.4D5 39 1 :500 N kxl.l aa 110-122 from the amino terminus Polyclonal rabbit anti-TTF Biopatlmmunotechnologies, Caserta, Italy; catalogue reference: PA 0100 37-42 1 :500 N k xl.l E. co/f-derived recombinant chick NKX2.2 Monoclonal mouse anti-Nkx2.2 Developmental Studies Hybridoma Bank. Catalogue reference: 74.5A5 28-30 1 :500 Otp aa sequence: RKALEHTVS of the C-terminal OTP Polyclonal rabbit anti-Otp Pikcell Laboratories, Kruislaan, Amsterdam, The Netherlands 34 1 :1000 Fax? E.coli-derived recombinant chick PAX7. aa 352-523 of chick Pax7 Monoclonal mouse anti-Pax7 Developmental Studies Hybridoma Bank. Catalogue reference: PAX7 55 1:500 SOM Aa sequence: Ala-Gly- Cys-Lys-Asn-Phe-Phe- T rp-Lys-Thr-Ser-Cys Polyclonal rabbit anti-SOM ImmunoStar, Wisconsin, USA. Catalogue reference: 20067 13 1:1000 TH Catalitic core of TH molecule A sequence of the N- terminus TH Monoclonal mouse anti-TH ImmunoStar; Cataloque reference: 22941 Policlonal rabbit anti-TH Millipore (Chemicon). Catalogue reference: AB152 62 1:1000 we combined the immunohistochemistry for Isll, Nkx2.2, Otp, and TH with in situ hybridization for the following markers: xD114 (provided by Dr. Nancy Papalopulu. University of Manchester; Papalopulu and Kintner, 1993), xLhxl (provided by Dr. Sylvie Rétaux. CNRS. Paris, France; Bachy et al., 2001) and xShh (provided by Dr. Randal Moon. University of Washington; Ekker et al., 1995). For in situ hybridization, which was performed first, antisense digoxigenin (DIG)-labeled riboprobes for these markers were synthesized according to the protocol described in Bachy et al (2001), linearizing the clones in Bluescript KS with Bam HI (Promega, Madison, USA) and transcribing with T3 (Promega) for xShh, with Notl (Promega) and T3 (Promega) for xD114 and with Xbal (Promega) and T3 (Promega) in the case o f xLhxl. The embryos and premetamorphic larvae were processed in toto after pretreatments (see Bachy et al., 2001), and the late larvae were processed in floating sections (see Moreno et al., 2004). Hybridization step was done with 3 pFml of a DIG- labeled RNA probe, in a 50% formamide containing medium overnight at 55°C. The solution used for prehybridization (at 60°C for 1 hour) and hybridization contained 50% deionized formamide (Fluka, Steinheim, Germany), 5x standard saline citrate (Sigma-Aldrich, Steinheim, Germany), 2% blocking reagent (Roche Diagnostics, Mannheim, Germany), 0.1% Tween-20, 0.5% 3-[(3-cholamidopropyl)-dimethylammonio]-l - propanesulfonate (CHAPS; Sigma-Aldrich), 1 mg/ml of yeast tRNA (Sigma-Aldrich), 5 mM of ethylenediaminetetraacetic acid (Sigma-Aldrich), and 50 g/ml of heparin (Sigma-Aldrich) in water. Hybridization was detected using an alkaline phosphatase coupled anti-DIG antibody (Roche Diagnostics, dilution 1:1500). Alkaline phosphatase staining was developed with Fast red tablets (Roche Diagnostics). The in situ hybridization was followed by the immunohistochemistry for the primary antibodies mouse anti-Isll, rabbit anti-Nkx2.1, mouse anti-Nkx2.2, rabbit anti-Otp and mouse anti-TH, used with the same dilution as described above and revealed with chicken anti-rabbit Alexa 488 (diluted 1:500, Molecular Probes) for the case of primary antibodies developed in rabbit 126 ô . JLL- m r V J lA ljA iV U J ILiX L , A I AT^AiVli'^lU- AMNIOTA: ESTUDIOS EN ANUROS Y REPTILES Table 2. List of the gene markers used, gene bank account, origin of the plasmid and enzymes/polymerases used in the probe synthesis. GENE GENEBANK ACC. N" ORIGIN PRIMERS LINEARIZATIO N ENZIME/POLIM ERASE xShh NMOO1088313 Dr. Randal Moon. University of Washington, USA F:CGCAAATGGGCGGT AGGCGTG R:CAGGAAACAGCTA TGAC BamHl/T3 xDU4 NMOO1090563 Dr. Nancy Papalopulu. University of Manchester,UK F:AG(GA)AA(GA)CC(C AT)CG(CT)AC(CAT)AT (CA)TA; R:CA(GA)GT(AGCT)AA (GA) AT(TC) TGG TTCCAG AA Notl/T3 xLhxl NMOO 1090659 Dr. Sylvie Rétaux. CNRS. Paris, France FXl iTGCCTTCTATTCT CCTAATCCGCCC; RXl iCAGCTTAGGCTA CCACACTGCCG Xbal/T3 and goat anti-mouse Alexa 488 (diluted 1:500, Molecular Probes) for the case of the other ones developed in mouse. Subsequently, embryos and early larvae were embedded in a solution o f 20% gelatin and 30% sucrose in PB, and stored overnight at 4 °C in a solution o f 4% formaldehyde and 30% sucrose in PB. Sections were cut on a freezing microtome at 14-25 pm in the transverse, sagittal or horizontal plane. Imaging The sections were analyzed with an Olympus BX51 microscope that was equipped for fluorescence with appropriate filter combinations. Selected sections were photographed by using a digital camera (Olympus DP72). Contrast and brightness of the photomicrographs were adjusted in Adobe PhotoShop CS3 (Adobe Systems, San Jose, CA) and figures were mounted in Canvas 11 (ACD Systems, Canada). RESULTS Developmental patterning of the basal hypothalamus In the present study we have analyzed the anatomical organization of the developing basal hypothalamus in Xenopus laevis based on the differential expression patterns of prosencephalic markers, which are expressed through the juvenile and adult specimens. The markers used, including the transcription factors, in the ventricular (vz), subventricular (svz), and mantle (mz) zones, allowing the analysis o f progenitor domains. First, Nissl staining (Fig. 1) allowed the identification of the main different areas classically defined by the anatomical landmarks. The combination of the markers xD114, Isll, xLhxl, Nkx2.1, Nkx2.2, Otp, Pax7, xShh, SOM and TH allowed the observation of the boundaries that delimit the different subdivisions (Fig. 2), and the identification o f the two main portions of the basal hypothalamus throughout development, i.e. the tuberal (Tub, Fig. 3) and mammillary band (Mab; Figs. 4,5) regions. Finally, all the results on the combinatorial expression in the basal hypothalamus of the markers used have been summarized in Figure 6 to show schematically the subdivisions and their boundaries identified on the basis o f their chemo- and genoarchitecture. Boundaries.The basal hypothalamus was characterized by the ventricular expression of xShh (Fig. 2a) and Nkx2.1 (Fig. 2b). In addition, at embryonic stages, Nkx2.1 was also expressed in the basal part of the rostral diencephalic neuromere (P3 in Fig. 2b). Distinctly, Isll was exclusively observed in the Tub region, primarily in its posterior part (Fig. 2c), and the simultaneous detection of Nkx2.1 and Isll allowed the identification o f the limit between the Tub and the Mab regions since the latter lacked Isll expression (Fig. 2d). This boundary was better observed at larval stages and in sagittal sections (Fig. 2e). Another tool for visualizing the Tub-Mab boundary was the combination of Isll and Otp, because Otp was expressed in the Mab but not in the posterior part of Tub (Fig. 2f). At dorsal levels, the tuberal region limits with the optic chiasm rostrally, and the alar suprachiasmatic (SC) domain caudally. In particular. 127 3. EL H IF O 1ALAM U EN LA IK A N SILIU N AJNAMNIO- AMNIOTA; ESTUDIOS EN ANUROS Y REPTILES DORSAL VIEW d b LATERAL VIEW d b Figure 1. Photomicrographs of Nissl-stained sagittal (a) and transverse (b-d) sections through the basal hypothalamus of a prometamorphic Xenopus at the approximate levels indicated on the dorsal and lateral views of the brains. These photomicrographs illustrate the poorly segregated regions roughly recognized in the hypothalamus. Scale bars: 200pm the boundary between the caudal part o f Tub and the SC in the alar hypothalamus could be highlighted by the combination of the markers Nkx2.1 and Nkx2.2 (Fig. 2g). Thus, Nkx2.1 labeled both the Tub and the dorsal aspect of the SC region, whereas Nkx2.2 was not expressed in the SC but showed distinct expression in the dorsal part of Tub (Fig. 2g). Finally, the caudal boundary of the basal hypothalamus is formed with the basal (tegmental) part of P3. The border between the two regions was evident by the combination of Nkx2.1, which was expressed in the basal hypothalamus and in P3, and Pax7 that, from early developmental stages labelled the basal part of P3 (Fig. 2h). All these boundaries have been summarized in the bottom scheme shown in Fig. 2. The tuberal hypothalamus. The Tub was characterized by the expression of Nkx2.1 in the vz and svz (Fig. 3a) and by the expression of Isll in the svz (Fig. 3b), with the exception of a caudal portion in which Nkx2.1 was restricted, in late embryonic stages, to the vz (see asterisk in Fig. 3a, c). The vz of the tuberal hypothalamus is also characterized by the expression of xShh (Fig. 3d). From embryonic stages to late larvae, the combination of Isll, Otp and Nkx2.2 allowed the identification of subdivisions within this territory (Fig. 3 e-i). The developing expression of Otp was restricted to the rostral portion (Fig. 3e,f), whereas Nkx2.2 was expressed in the caudal regions (Fig. 3h,i), occupying the region devoid of Nkx2.1 svz expression (see asterisk in Fig. 3g). In addition, SOM expressing cells were observed only in the rostral tuberal region, as observed by the combination with Nkx2.2 expressed in the CT (Fig. 3i,j). Finally, scarce and disperse Lhxl expressing cells were observed in the CT, confirmed by the combination with Nkx2.2 (Fig. 3k). All these results have been summarized in the bottom schematic representation in Fig 31,m. The mammillary band. The Mab was characterized by the Nkx2.1 expression in the mammillary portion (Ma) and the lack o f Isll (Fig. 4a). This combination allowed the identification of the boundary between the Tub, rich in Isll/Nkx2.1 expressions and the Ma, only expressing Nkx2.1 (Fig. 4b). In addition, the distinction between the Ma and retromammillary (RM) subdivisions was observed by the combination of Nkx2.1, rich in Ma, and xShh expressed in the RM portion (Fig. 4c). From embryonic to late larval stages, Otp expression was detected in the Ma portion, form anterior (Fig. 4d,e) to posterior levels (Fig. 4f), forming a banded pattern in which the Isll expression was detected in the whole Tub, whereas Otp was restricted to the rostral portion of the Tub and to the Ma (Fig. 4d-f). At early embryonic stages, the combination o f Otp and Nkx2.2 (Fig. 4g) allowed the identification of the boundary between the Ma, expressing Otp, and P3 and the alar hypothalamus both rich in Nkx2.2 and lacking Otp (Fig. 4g). The early region expressing Nkx2.2 extended in later stages of development into the adjacent Ma region and the CT, evidenced by the expression of Nkx2.1 in both regions (Fig. 4h). The cell population in Ma was heterogeneous and, most likely had different embryologie origin. Thus, two clear populations of Nkx2.2 and Otp intermingled expressing cells in the Ma were detected, from early larva stages (Fig. 4iJ). Similarly, the combination of 128 1/v; tio 1 n,iYi ufVL̂ a i KJLriiLiJLa st46 • r ' r ' - st46 / st46 st46 st43 j W h / P 3 ' / SC •X Mab' ' Tub ' T u b / o c ' , _ f g * h PThPax7P3Nkx2.1 SC Nkx2.2 Nkx2.1Mab Otp Nkx2.1 xDil4 Nkx2.2xLhx1 OtpxShh Nkx2.1 Isl1XDII4 ocxLhx1xShh Tub Figure 2. Photomicrographs of sagittal (a,e,g) and transverse (b-d,f,i) sections through the mammillary and tuberal hypothalamus of Xenopus, showing the expressions of xShh (a), Nkx2.1 (b), and Isll (c) and the combined expressions of Isll and Nkx2.1 (d,e), Isll and Otp (f), Nkx2.1 and Nkx2.2 (g) and Fax? and Nkx2.1 (h). The schematic representation at the bottom summarizes the distribution of the main markers along the basal hypothalamic subdivisions and the boundaries between them. Scales bars: 100pm (a, f-h), 50pm (e). The scale bar in b is valid for c and d = 100pm. Nkx2.2 and Nkx2.1 allowed the identification subpopulations o f cells in the Ma that expressed one of the markers and, later in development, double labeled cells were also detected (Fig. 4k-m). Another molecular marker o f Ma cells was xD114 (Fig. 4n,o) and its presence in this region was confirmed by the combination with Isll, lacking in the Ma (Fig. 4n), and Otp, rich in the Ma (Fig. 4o). In addition, xLhxl was observed in the Ma by the combination with Otp (Fig. 4p). This xLhxl expression (Fig. 4q) confirmed the expression o f Nkx2.2 restricted to the dorsalmost portion o f the Ma (Fig. 4r), and evidenced the extent o f the RM lacking both Nkx2.1 and xLhxl (Fig. 4s). The combination o f Nkx2.1 and Pax7 from embryonic to late larvae stages (Fig. 5a-e) allowed the identification o f the basal portion o f P3 in which both Pax7 and Nkx2.1 were expressed and numerous double labeled cells were observed in the vz and the svz from early stages (Fig. 5a), in contrast to the Tub that lacked Pax7 expression. Later in larval development, this expression was expanded to ventral levels and double labelled cells along P3 were detected, avoiding the RM in both cases (Fig. 5b-d), but reaching in the case o f Pax7 the Ma zone, earlier devoid o f Pax7 expression (see arrowhead in Fig. 5d). This population o f Pax7 expressing cells, which was detected in the Ma from premetamorphic larval stages, was different from the Otp expressing cells detected more laterally in this region o f the Ma (Fig. 5e). 129 A i v i i 'N i u i / v : \ t u L r 1 l i j iL a 2.1 st42 Isl1 P3b Mab *T ub b st46 st42 I lsl1/^; :% .st42 " M a b {■ • '*• > rvj st57 Nkx2,2/Nkx st45 st48 I 5xL M / : RT' o c TRANSVERSAL VIEWSAGITTAL VIEW A/B boundary xShh+Nkx2.1 xShh Nkx2.1 Isl1 Otp P Z ^ Nkx2.2 1***1 Som Figure 3. Photomicrographs of transverse (a-e, g,h) and sagittal (f,i-k) sections through the tuberal hypothalamus of Xempus showing the expressions of Nkx2.1 (a) and Isll (b), and the combined expressions of Isll and Nkx2.1 (c,), xShh and Isll (d), Isll and Otp (e,f), Nkx2.2 and Nkx2.1 (g), Nkx2.2 and otp (h,i), Nkx2.2 and SOM (j), and xLhxl and Nkx2.2 (k). The schematic sagttal and transverse representations at the bottom summarize the combinatorial codes of the markers in the tuberal hypothalamus. S:ale bars: 100pm. The scale bar in a is valid for b and c. 130 / st42 st46 R M ^ ; M a - - PTh, P 3 ' ' Ma Tgb \ , ' SPV,' s c '^ i Tub /'oc st46 St 66 b I c St 56 , ' C T d » 4 t st35 Zli V SPV A sc ' . Tub st46 st46 ,Ma c R : R T n h _ i RT' oc', j RT RM ' / st48 / ' r r • MS - RT Rfvr 'iVla Tub / 0 0 _RM/ Ma__ Tub' ' o c y Figure 4. Photomicrographs of transverse (a,c-e,h„l-nj) and sagittal (b,g,i,k,o-s) sections through the mammillary hypothalamus of Xenopus showing the combined expression of Isll and Nkx2.1 (a,b), xShh and Nkx2.1 (c), Isll and Otp (d), Isll and Otp (e,f), Nkx2.2 and Otp (g), Nkx2.2 and Nl«2.1 (l,m) xD114 and Isll (n), xD114 and Otp (o), xLhxl and Otp (p). xLhxl (q) and Nkx2.2 (r) expression in the same section and its merge image (s). The level o f the transverse section showed in j is indicated by the vertical yellow line drawn in i. Scale bars; 100pm. The scale bar in q is valid for r and s. 131 st33/34 A .p ; st46 Pax7/Nks„^,^% ' st54 RM, # ' ' \ : I M a * . '. '- r^ R M r- , ' • ■ M a r b Tub c ■ st48 sxfLteD/ st48 I P 1 S t 5 7 T h St57 RM — _ 4 Ma , -P3 Ma TP ^ , _ j A/B boundary xShh+Nkx2.1 xShh V 7 ^ Nkx2.2 Nkx2.1 V X A xLhx1 SC SPV SAGITTAL VIEW Basal i TRANSVERSAL VIEW P3 Tub Otp loo of t h Pax7 Figure 5. Photomicrographs of transverse (a,b,d-k) and sagittal (c,l) sections through the mammillary hypothalamus of Xenopus showing the combined expressions of Fax? and Nkx2.1 (a,-d) and Pax7 and Otp (f). The arrowheads in a and d show double labeled cells. In addition, imagines are shown of the combined labelling for TH with Nkc2.1 (f), xShh (g), Isll (h), Otp (i), xLhxI (j) and Nkx2.2 (k,l),. The schematic sagittal and transverse representations at the bottom summarize the combinatorial codes of the markers in the mammillary hypothalamus. Scale bars: 100pm (a,b,d-l), 50pm (c). The scale bar in e is valid for d. 132 ô . Üj L j n ir U lA L iA iV U J L i A IKAi'M Slt.lUrN ATNAiVmiU- AMNIOTA: ESTUDIOS EN ANUROS Y REPTILES Finally, the detection in the same sections of the transcription factors used to define the Ma and RM and the enzyme TH (fig. 5 f-1) resulted very useful to characterize these regions, given the important dopaminergic cell population (revealed for TH) that is very conserved in this portion of the hypothalamus across vertebrates (Smeets and Gonzalez, 2000). This was the case o f Nkx2.1 rich in the Ma where TH immunoreactive cells were detected (Fig. 5f). The presence of TH positive cells in the Ma also identified the lack o f xShh expression in the vz of the Ma (Fig. 5g). The combination of TH and Isll confirmed the lack of Isll in the Ma (Fig. 5h), in contrast to the colocalization observed in the case of Otp/TH (arrowhead in Fig. 5i). Finally, xLhxl (Fig. 5j), and Nkx2.2 (Fig. 5k,l) were also detected in the TH positive Ma region. All these results about the characterization of the mammillary band have been summarized in the bottom schematic representation in Fig. 5. The summary of the expression patterns detected in the different subdivisions of the basal hypothalamus in our study is presented in Fig. 6. DISCUSSION In the present study we have focused our analysis on the basal hypothalamus. This hypothalamic region largely corresponds to the posteroventral hypothalamus, recently described in the mouse as the Nkx2.1 positive hypothalamic region (Shimogori et al., 2010). This territory contains the basal parts o f the terminal (rostral) and peduncular (caudal) hypothalamus; terms recently coined by L. Puelles (Allen developmental mouse brain atlas). O f note, in a recent study the alar/basal boundary in the murine hypothalamus has been differently interpreted and the tuberal region considered in our study (the basal terminal region o f Puelles) is regarded as alar (Diez-Roux et al., 2011). All the disparities that exist in the current terminology/interpretation between the different authors have resulted from very recent studies about molecular specifications. Moreover, in chemical, hodological or anatomical studies the complexity of the terminology used is even more varied making very difficult to understand, in a comparative perspective, which part of the hypothalamus the authors refer to. Therefore, in our study we have tried to simplify the comparative analysis and we have opted, in this and in our previous studies of the hypothalamus (Moreno et al., 2011; Dominguez et al., 2011b), to analyze the main hypothalamic subdivisions proposed in the prosomeric model (Puelles and Rubenstein, 2003). We felt impelled to use this model because, independently of the alar/basal subdivisions, the topologically identified hypothalamic regions that are specified by a combinatorial code of gene expressions have been demonstrated to be very conserved across vertebrates (Brox et al., 2003; Puelles and Rubenstein, 2003; Abelian and Medina, 2009; Moreno et al., 2004; 2008a;b; 2011, Bardet et al., 2008; Dominguez et al., 2010; 2011, Morona et a l, 2011). In this context, the use of the conserved developmental regulatory genes as tools in the genoarchitectonic analysis o f the hypothalamus has been proven to be specially efficient to highlight conserved patterns, but also differences that likely reflect divergences in the evolution. The selection o f the different transcription factors used in our study was prompted by previous results obtained in similar studies in other vertebrates that showed a high degree of conservation in most o f the expression patterns in hypothalamic regions (Puelles and Rubenstein, 2003; Bardet et a l , 2008; Dominguez et a l, 2010; 2011; Shimogori et a l, 2010; Morales- Delgado et a l , 2011; Moreno et a l , 2011). Thus, it was described that the basal region rostral to the P3/hypothalamic boundary is primarily under the influence o f prechordal plate/rostral mesoderm signals (Garcia-Calero et a l , 2008), and produces the ventral hypothalamus (including neurohypophysis and the ventromedial/tuberal, mammillary and tuberomammillary hypothalamic regions), and the expression of Nkx2.1 in this ventral region is induced by prechordal Shh signals (reviewed in Medina, 2008). In line with those observations, in Xenopus xShh and Nkx2.1 expression define in the embryo the vz of the tuberal hypothalamus (Tub) and, later in the development, also the svz, in which Isll is also is expressed. Moreover, analysis of Nkx2.1 hypofunction in mouse and frog demonstrated that this transcription factor plays a key role in the specification and formation of the basal hypothalamus in both classes of vertebrates (van den Akker et a l, 2008). The combination of Otp and Nkx2.2 allowed the distinction in Xenopus o f rostral (RT) and caudal (CT) regions in the Tub (present results). The use of these two markers was based on previous studies that demonstrated the expression of Otp in the tuberal hypothalamus of different vertebrates, with a very restricted pattern of expression (Bardet et al,. 2008; Del Giacco et a l , 2008; Morales-Garcia et a l, 2011), whereas Nkx2.2 was seen to be a good marker of the basal plate (Puelles et a l, 2004; Garcia-Lôpez et a l, 2004). In the case of the analysis of the mammillary band (Mab), the combiantion of xShh and Nkx2.1 defined in Xenopus the rostral mammillary region (Ma) as a Nkx2.1+/xShh- territory and the caudal retromammillary area (RM) as a Nkx2.1-/xShh+ region (present results). This pattern of expression in similar regions seems to be highly conserved (Garcia-Calero et a l, 2008; Medina, 2008). In addition, the combination o f xLhxl, xD114 and Otp, expressed in the Ma, with Isll, absent in the whole Mab, allowed the identification of this region and its boundaries in Xenopus, also in line with previous studies in different vertebrates (Brox et a l, 2003; Moreno et a l, 2004; 2008a; Bardet et a l, 2008). In the Ma region Otp/TH double labeled cells have been demonstrated in Xenopus suggesting in amphibians molecular relationship described in other vertebrates (DelGiacco 133 A/B b o u n d ary xShh+Nkx2.1 xShh Nkx2.1 V 7 ^ Nkx2.2 Ÿ /À xLhx1 O tp H I Isl1 TH Pax7 ! • • • ! S o m Figure 6. Schematic sagittal and transverse (a,b) representations of the basal hypothalamus of Xenopus summarizing the combinatorial code of expression for the transcription factors used in the present study ( following the indicated colour code). et al., 2006). Finally, from early development, Pax7 (Stoykova and Gruss, 1994) and Nkx2.1 (Medina, 2008) were described in the murine basal plate o f prosomere 3 (P3), in striking similarity with our results in Xenopus, and following the pattern of expression for these two markers, double labeled cells are located in the Ma, likely migrated from the adjacent P3 region. 134 J. CjLj n iru 1 AJ-iAiVHJ U.I’N Al̂ A VlHiU- AMNIOTA: ESTUDIOS EN ANUROS Y REPTILES T uberal hypothalam us The Tub has traditionally been included in the basal hypothalamic territory, constituting the most rostral portion o f the hypothalamus, which expresses in all vertebrates the developmental regulators Shh and Nkx2.1 both essential for the hypothalamic organization and specification (reviewed in Medina, 2008; Moreno and Gonzalez, 2011). In mammals, the mature tuberal hypothalamus contains different nuclei, such us the ventromedial and arcuate nuclei, which are bilateral cell groups at the base of the hypothalamus that are organized through the aggregation of neurons bom along the third ventricle and then migrate laterally (Altman and Bayer, 1986). In amphibians the classical studies divided the infundibulun (term frequently used to refer to the current tuberal hypothalamus) into two periventricular nuclei, the dorsal and ventral hypothalamic nuclei and a migrated nucleus, the lateral hypothalamic nuclei (Neary and Northcutt, 1983). Subsequent studies based on chemoarchitecture in adult anurans have considered the tuberal hypothalamus as it is currently regarded, and caudal, intermedite and medial tuberal nuclei were described in the periventricular cell layer (Puelles et al., 1996, Milan and Puelles, 2000; Morona and Gonzalez, 2008). The medial and, at least part of, the intermediate nuclei would correspond to the here identified as rostral tuberal (RT) region on the basis of gene expression during development, whereas the caudal nucleus practically corresponds to the caudal tuberal (CT) region. Patterning and neuronal specification. Recently, Shimogori and collaborators defined the Nkx2.1-positive posteroventral hypothalamic neuroepithelium, which contains the primordia of the ventromedial hypothalamus and arcuate nucleus, and the premammillary and mammillary regions (Shimogori et al., 2010). However, a subsequent study has proposed a new conception of the alar-basal boundary, and the Tub has been identified as an belonging to the alar plate, in contrast to basal Mab, supported by the analysis of several regional gene expression patterns, but with the exception of Shh and Nkx2.1 (Diez-Roux et al., 2011). Nevertheless, the discussion is still open since experiments in chick, in which the prechordal plate was ablated, showed that it is essential to obtain a correctly ventralized and regionalized tuberal and mammillary regions, thus both regions would be under similar inductive signals (Garcia-Calero et al., 2008). In Xenopus, the vz of the Tub is characterized by the expression of xShh and Nkx2.1, which extends into the svz (present results; see Figure 6). Amniote vertebrates share this expression pattern and it was related to the dorsoventral patterning along the rostrocaudal axis (reviewed in Medina, 2008). In particular, the morphogene Shh plays a key role in the organization of the basal hypothalamus through the action of Nkx2.1, whose expression is triggered by prechordal signals (Kimura et al., 1996; Puelles et al., 2004). In addition, Nkx2.1has been involved in the nuclear specification of the ventromedial and arcuate nuclei, as it was investigated in relation to the neurons in these locations synthesize several neurotransmitters such as G ABA, NPY and dopamine (McClellan et al., 2006; Yee et al., 2009). In mammals, Isll was importantly implied in the development o f the ventromedial and arcuate nuclei o f the tuberal hypothalamus, suggesting a role in the reproductive behaviour through an interaction with estrogen receptor a (Davis et al., 2004). In Xenopus, Isll was also observed in the Tub (Moreno et al., 2008a) and its expression allowed the identification of its boundary with the Mab, which is devoid of Isll but expressed Nkx2.1 and Shh (present results, Dominguez et al., 2010). Additionally, a similar pattern has been described in the turtle Pseudemys scripta (Moreno et al., 2011) showing the important grade of conservation of this region in all tetrapods. In the Tub of Xenopus, marked by the expression of xShh/Nkx2.1/Isll, we have identified two subdomains on the basis o f the differential Otp/Nkx2.2 expression. The transcription factor Otp is exclusively located at the most rostral portion (RT), that likely give rise to counterpart in anurans of the arcuate nucleus, as in all the vertebrates examined (Acampora et al., 1999; Bardet et al., 2008; Morales-Delgado et al., 2011; Moreno et al., 2011; present results). In addition, in mammals Otp has been implicated in the specification of SOM+ neuronal type (Acampora et a l , 1999; Wang and Lufkin, 2000) and, recently, in the developing hypothalamus of the mouse the presence o f SO/ Otp cells in the Nkx2.1/Shh expressing tuberal hypothalamus has been reported (Morales-Delgado et a l, 2011). In Xenopus, we have also identified an Otp/SOM positive territory in a comparable position within the RT, just beneath the optic chiasm. In addition, in the caudal domain (CT) the lack of Otp and the Nkx2.2 expression during the larval stages defined its boundaries. Nkx2.2 is a typical marker o f the basal plate, essential for the maintenance of the ventral phenotype (Briscoe et a l, 1999; Sander et a l , 2000; Puelles et a l , 2004; Garcia-Lôpez et a l, 2004) and Otp has served to identify similar subregions in the tutle and mouse (Morales-Delgado et a l, 2011; Moreno et a l, 2011). O f note, in the basal hypothalamus o f lungfish, currently considered the closet living relatives of tetrapods, subdivisions observed in the tuberal hypothalamus using the same markers are largely comparable to those of anurans and amniotes (Moreno et a l, 2011). In Xenopus, the Nkx2.2 positive cell population found in the Tub was located in the CT, within the negative gap for Nkx2.1 expression in the svz. In mammals, the neurons o f the ventromedial (VM) nucleus of the tuberal hypothalamus distinctly express Nkx2.1 and Nkx2.2 during development (Tran et a l, 2003; Kurrasch et a l, 2007). However, the VM neurons bom earlier, which occupy the ventrolateral part o f the nucleus (McCellan et a l, 2006), downregulate Nkx2.1 as the VM precursors differentiate into a morphologically distinct nucleus (Tran et a l, 2003). On 135 i . EL H lF U l A LA M U EN LA IK A N M L IU N AJNAMJNIU- AMNIOTA: ESTUDIOS EN ANUROS Y REPTILES the basis o f the expression pattern observed in our study it is possible to speculate a functional regulation of both transcription factors in which the expression o f Nkx2.2 could downregulate the Nkx2.1 expression in CT. However, following the development of the Nkx2.2 expression in the Tub from early embryonic stages it seems that the expression found in the CT is derived from the Ma/ P3-Nkx2.2 expressing territory (see Figure 6 of Dominguez et al., 2011; present results). Previous studies had reported xD114 expression in the tuberal territory of Xenopus, (Brox et al., 2003). Specifically xD114 expressing population is located in the most caudal portion of the tuberal hypothalamus, whereas the rostral domain has no detectable expression (present results). Furthermore, there is a close spatial relationship between this xD114 expression and the GABAergic positive cell population in CT (unpublished observations), suggesting a role of xD114 in the specification o f the GABAergic phenotype in Tub. In fact, it was demonstrated that the production of GABAergic neurons in the forebrain is largely restricted to specific histogenetic territories that express developmental regulatory genes of the Dix family (Price et al., 1991; Bulfone et al., 1993; Marin and Rubenstein, 2001). In addition, it was also demonstrated in mammals the existence of DLX positive cells in the arcuate nucleus of the tuberal hypothalamus that specifically marked the GABA-containing cells (Yee et al., 2009). Interestingly, studies in agnathan anamniotes such as the lamprey, revealed a substantia) number of Dll-expressing cells in the tuberal hypothalamic nucleus and the tuberomammillary region (Martinez de la Torre et al., 2011). The mammillar hypothalamus The mammillary complex, formed in anurans by the mammillary and the retromammillary areas, is a caudoventral hypothalamic specialization located between the diencephalic tegmentum of P3 and the tuberal hypothalamic area (Puelles and Rubenstein, 2003; Puelles et al., 2004, 2007). Similar to the rest o f the hypothalamic regions, the terminology, boundaries, and interpretation of this area differs between the different authors. Puelles and cols (Morales-Delgado et al., 2011; and see: Allen brain developmental mouse brain atlas) proposed that this area in the mouse is caudal to the tuberal hypothalamus and is composed by the perimammillary and mammillary regions in the terminal (rostral) hypothalamus, and the periretromammillary and retromammillar regions in the peduncular (caudal) hypothalamus. In contrast, Shimogori and cols composed a genomic atlas o f mouse hypothalamic development in which this region is divided into perimammilary (PM), mammillary (MM) and supramammi 1 lary (SMM) subdivisions and defines an additional intermediate zone termed tuberomammillary terminal (TT) that separates the mammillary and premammillary neuroepithelium (Shimogori et al., 2010). In classical anatomical studies in anurans, there were not special references to comparable regions and the hypothalamic regions that are currently identified as mammillary (Puelles et al., 1996; Brox et al., 2003; Moreno et al., 2004) were previously included in the posterior tubercle (Neary and Northcutt, 1983). Neural patterning and nuclear specification. By means of the analysis o f the combinatorial expression of Shh, Nkx2.1, Otp, Lhxl, Isll and Pax7 we have tentatively identified distinct rostral cauda subdivisions in the Mab of Xenopus. The combination of xShh and Nkx2.1 defined the mammillary region proper (Ma) being Nkx2.1+/xShh-, whereas the caudal retromammillar area (RM) was Nkx2.1-/xShh+, and this pattern is very conserved (Garcia-Calero et al., 2008; Medina, 2008). This subdivision is supported by the additional expression in the Ma o f xLhxl, Nkx2.2, and Otp and also by the lack of Isll in this subdivision. Interestingly, in the chick it was described that Shh expression initially extends in the entire ventral forebrain (basal and floor plates) but secondarily becomes downregulated in part o f the ventral hypothalamus, including the tuberomammillary primordium but not the retromammillary area (Marti et al., 1995; Shimamura et al., 1995; Crossley et al., 2001; Patten et al., 2003; Manning et al., 2006) and this particular loss of Shh expression is what conferred the hypothalamic fate to these cells (Manning et al., 2006). The detailed subdivision proposed by Shimogori and coworkers on the basis o f the combinatorial code of many different genes, also included members of the LIM-hd family. Thus a tuberomammillary terminal (TT) zone was defined by the Lhx6 expression, the supramammillary nucleus (SMM) by the expression of Lhx5, and the anterior portion of the premammillary nucleus (PM) expresses Lhx9 (Shimnogori et al., 2010). In Xenopus, the existence of a region comparable to the TT was proposed as a ventral continuation of the Lhx7 expressing territory o f the alar hypothalamus (Dominguez et al., 2012a) that, along development, extends into the anterior portion o f the mammillary complex (see Figs. 8e and 9d of Moreno et al., 2004). Also in Xenopus, it has been described Lhxl/5 expression in the Ma, but not Lxxh9 (present results; Moreno et al., 2004), which suggests that also in this case this region is molecularly distinct in anamniotes but with some specific differences. A distinct feature o f the Ma in Xenopus is the Dll expression (Brox et al., 2003; present results), which contrasts with the results reported in vertebrates as distant as lampreys and mammals where the Dll expression is restricted to the tuberal hypothalamus (Puelles and Rubenstein, 2003; Martinez de la Torre et al., 2011). In this Ma region we have observed Otp/TH double labeled cells and in zebrafish it was demonstrated that Otp is necessary to induce the dopaminergic phenotype in the hypothalamus and posterior tuberculum (Blechman et al., 2007; Del Giacco et al., 2006; Ryu et al., 2007; Lôhr et al., 2009). Thus, a similar role in the fate specification o f this cell population might be present in Xenopus. In addition, we 136 ô . Ih i-I n iru 1 AL«AiVUJ IjA AiHAiViî iW- AMNIOTA: ESTUDIOS EN ANUROS Y REPTILES found that some of the TH positive cells also coexpressed Nkx2.1 in line with previous studies that suggested an implication o f Nkx2.1 in the dopaminergic specification (kawano et al., 2003). In addition to this molecular neuronal specification, in mammals the existence of SOM expressing cells in the Otp positive Ma region was reported (Morales-Delgado et al., 2011), in contrast there are not SOM expressing cells in this region in Xenopus. In terms of nuclear specification, in mammals and birds this region gives rise to the subthalamic nucleus which arises from the retromammillary region and migrates to the deep surface o f the peduncle (Altman and Bayer, 1986, Puelles and Rubenstein, 2003; Jiao et al., 2000). About the actual origin and molecular specification o f this nucleus there are discrepancies in the literature. In mammals, it is a caudoventral hypothalamic specialization located between the diencephalic tegmentum (P3) and the tuberal hypothalamic area (Puelles and Rubenstein, 2003; Puelles et al., 2004, 2007) and recent fate map studies in birds focused on the prethalamic basal plate have described that the basal plate of P3 generates the retromammillary tegmentum and the subthalamic nucleus (Garcia-Lôpez et al., 2009). In amphibians the existence of an equivalent o f the subthalamic nucleus has been suggested (Marin et al., 1998). Neary and Nothcutt (1983) in their analysis o f the diencephalic cell masses tentatively considered that the called porsterior entupenduclar nucleus, close to the lateral forebrain bundle, could be part o f the hypothalamus (Neary and Nothcutt, 1983) and in a recent study in anurans based on hodological and neurochemical data a comparable subthalamic region was proposed to comprise the dorsocaudal suprachiasmatic nucleus, the posterior entopeduncular nucleus (after Northcutt and Neary, 1983), and the ventral part of the prethalamus (Maier et al., 2010). In Xenopus, from very early stages of development the basal plate o f P3 is characterized by the ventricular expression of Pax7 and Nkx2.1 and along development the cells characterized by this expressions are progressively located in the Ma (present results), likely migrating from the adjacent P3 region. Noteworthy, some years ago Stoykova and Gruss postulating the roles of Pax-genes suggested by their expression patterns, identified the subthalamic nucleus of mammals on the basis on the Pax7 expression (Stoykova and Gruss, 1994). However, we have not found ftirther specific references in the literature, but Pax7 is expressed during the development and in postnatal life in the mouse subthalamic nucleus (see mouse developmental Allen Brain Atlas). Therefore, only attending to the expression of Pax7 the counterpart of the subthalamic nucleus in anurans would be dispersed in the Mab region. LITER A TU R E CITED Abelian A, Medina L. 2009. Subdivisions and derivatives of the chicken subpallium based on expression of LIM and other regulatory genes and markers of neuron subpopulations during development. J Comp Neurol 515:465-501. Acampora D, Postiglione MP, Avantaggiato V, Di Bonito M, Vaccarino FM, Michaud J, Simeone A. 1999. Progressive impairment of developing neuroendocrine cell lineages in the hypothalamus of mice lacking the Orthopedia gene. Genes Dev 13:2787-2800. Altman J, Bayer SA. 1986. The development o f the rat hypothalamus. Adv Anat Embryol Cell Biol 100:1- 178. Alvarez-Bolado G, Zhou X, Cecconi F, Gruss P. 2000. Expression of Foxbl reveals two strategies for the formation of nuclei in the developing ventral diencephalon. Dev Neurosci 22:197-206. Bachy I, Vernier P, Rétaux S. 2001. The LIM- homeodomain gene family in the developing Xenopus brain: conservation and divergences with the mouse related to the evolution of the forebrain. J Neurosci 21:7620-7629. Bardet SM, Martinez-de-la-Torre M, Northcutt RG, Rubenstein JL, Puelles L. 2008. Conserved pattern of OTP-positive cells in the paraventricular nucleus and other hypothalamic sites of tetrapods. Brain Res Bull 75:231-235. Blechman J, Borodovsky N, Eisenberg M, Nabel-Rosen H, Grimm J, Levkowitz G. 2007. Specification of hypothalamic neurons by dual regulation of the homeodomain protein Orthopedia. Development 134:4417-4426. Briscoe J, Sussel L, Serup P, Hartigan-O'Connor D, Jessell TM, Rubenstein JL, Ericson J. 1999. Homeobox gene Nkx2.2 and specification of neuronal identity by graded Sonic hedgehog signalling. Nature 398:622-627. Brox A, Puelles L, Ferreiro B, Medina L. 2003. Expression of the genes GAD67 and Distal-less-4 in the forebrain of Xenopus laevis confirms a common pattern in tetrapods. J Comp Neurol 461:370-393. Bruce, L. 2008. Evolution of the hypothalamus in amniotes. In: Encyclopedia of Neuroscience, Binder, M.D., Hirokawa, N., Windhorst, U. (Eds.). Springer, Berlin, pp 1363-1367. Bulfone A, Puelles L, Porteus MH, Frohman MA, Martin GR, Rubenstein JL. 1993. Spatially restricted expression o f Dix-1, Dlx-2 (Tes-1), Gbx- 2, and Wnt-3 in the embryonic day 12.5 mouse forebrain defines potential transverse and longitudinal segmental boundaries. J Neurosci 13:3155-3172. Butler AB, Hodos W (2005) Comparative vertebrate neuroanatomy. Evolution and Adaptation, 2nd edn. John Wiley & Sons, Hoboken, NJ, USA. Crossley PH, Martinez S, Ohkubo Y, Rubenstein JL. 2001. Coordinate expression o f Fgf8, Otx2, Bmp4, and Shh in the rostral prosencephalon during development of the telencephalic and optic vesicles. Neuroscience 108:183-206. 137 3. EL H lE U l ALA M U EN LA IK A N M LIU N ANAMNIU- AMNIOTA: ESTUDIOS EN ANUROS Y REPTILES Davis AM, Seney ML, Walker HJ, Tobet SA. 2004. Differential colocalization of Islet-1 and estrogen receptor alpha in the murine preoptic area and hypothalamus during development. Endocrinology 145:360-366. Del Giacco L, Sordino P, Pistocchi A, Andreakis N, Tarallo R, Di Benedetto B, Cotelli F. 2006. Differential regulation of the zebrafish orthopedia 1 gene during fate determination o f diencephalic neurons. BMC Dev Biol 6:50. Del Giacco L, Pistocchi A, Cotelli F, Fortunato AE, Sordino P. 2008. A peek inside the neurosecretory brain through Orthopedia lenses. Dev Dyn 237:2295- 2303. Diez-Roux G, Banfi S, Sultan M, Geffers L, Anand S, Rozado D, Magen A, Canidio E, Pagani M, Peluso I, Lin-Marq N, Koch M, Bilio M, Cantiello I, Verde R, De Masi C, Bianchi SA, Cicchini J, Perroud E, Mehmeti S, Dagand E, Schrinner S, Numberger A, Schmidt K, Metz K, Zwingmann C, Brieske N, Springer C, Hernandez AM, Herzog S, Grabbe F, Sieverding C, Fischer B, Schrader K, Brockmeyer M, Dettmer S, Helbig C, Alunni V, Battaini MA, Mura C, Henrichsen CN, Garcia-Lopez R, Echevarria D, Puelles E, Garcia-Calero E, Kruse S, Uhr M, Kauck C, Feng G, Milyaev N, Ong CK, Kumar L, Lam M, Semple CA, Gyenesei A, Mundlos S, Radelof U, Lehrach H, Sarmientos P, Reymond A, Davidson DR, Dolle P, Antonarakis SE, Yaspo ML, Martinez S, Baldock RA, Eichele G, Ballabio A. 2011. A high- resolution anatomical atlas o f the transcriptome in the mouse embryo. PLoS Biol 9:e 1000582. Dominguez L, Morona R, Gonzalez A, Moreno N. 2012a. Characterization of the alar hypothalamuc of Xenopus laevis during development by molecular markers analysis. J Comp Neurol. Submitted. Eisenstat DD, Liu JK, Mione M, Zhong W, Yu G, Anderson SA, Ghattas I, Puelles L, Rubenstein JL. 1999. DLX-1, DLX-2, and DLX-5 expression define distinct stages of basal forebrain differentiation. J Comp Neurol 414:217-237. Ekker SC, Ungar AR, Greenstein P, von Kessler DP, Porter JA, Moon RT, Beachy PA. 1995. Patterning activities o f vertebrate hedgehog proteins in the developing eye and brain. Curr Biol 5:944-955. Garcia-Calero E, Femandez-Garre P, Martinez S, Puelles L. 2008. Early mammillary pouch specification in the course of prechordal ventralization of the forebrain tegmentum. Dev Biol 320:366-377. Garcia-Lopez R, Vieira C, Echevarria D, Martinez S. 2004. Fate map o f the diencephalon and the zona limitans at the 10-somites stage in chick embryos. Dev Biol 268:514-530. Garcia-Lopez R, Pombero A, Martinez S. 2009. Fate map of the chick embryo neural tube. Dev Growth Differ. 51: 145-165. Gonzalez A, Lopez JM, Marin O. 2002a. Expression pattern o f the homeobox protein NKX2-1 in the developing Xenopus forebrain. Brain Res Gene Expr Patterns 1:181-185. Hodos, W. 2008. Evolution of the hypothalamus in anamniotes. In: Encyclopedia of Neuroscience, Binder, M.D., Hirokawa, N., Windhorst, U. (Eds.). Springer, Berlin, pp 1361-1363. Jiao Y, Medina L, Veenman CL, Toledo C, Puelles L, Reiner A. 2000. Identification of the anterior nucleus of the ansa lenticularis in birds as the homolog of the mammalian subthalamic nucleus. J Neurosci 20:6998-7010. Kawano H, Horie M, Honma S, Kawamura K, Takeuchi K, Kimura S. 2003. Aberrant trajectory of ascending dopaminergic pathway in mice lacking Nkx2.1. Exp Neurol 182:103-112. Kimura S, Hara Y, Pineau T, Femandez-Salguero P, Fox CH, Ward JM, Gonzalez FJ. 1996. The T/ebp null mouse: thyroid-specific enhancer-binding protein is essential for the organogenesis of the thyroid, lung, ventral forebrain, and pituitary. Genes Dev 10:60-69. Kurrasch DM, Cheung CC, Lee FY, Tran PV, Hata K, Ingraham HA. 2007. The neonatal ventromedial hypothalamus transcriptome reveals novel markers with spatially distinct patterning. J Neurosci 27:13624-13634. Lopez JM, Dominguez L, Moreno N, Gonzalez A. 2009. Comparative immunohistochemical analysis o f the distribution of orexins (hypocretins) in the brain of amphibians. Peptides 30:873-887. Lohr H, Ryu S, Driever W. 2009. Zebrafish diencephalic A 11-related dopaminergic neurons share a conserved transcriptional network with neuroendocrine cell lineages. Development 136:1007-1017. Lupo G, Harris WA, Lewis KE. 2006. Mechanisms of ventral patterning in the vertebrate nervous system. Nat Rev Neurosci 7:103-114. Maier S, Walkowiak W, Luksch H, Endepols H 2010. An indirect basal ganglia pathway in anuran amphibians? J Chem Neuroanat 40:21-35. Manning L, Ohyama K, Saeger B, Hatano O, Wilson SA, Logan M, Placzek M. 2006. Regional morphogenesis in the hypothalamus: a BMP-Tbx2 pathway coordinates fate and proliferation through Shh downregulation. Dev Cell 11:873-885. Marin O, Smeets WJ, Gonzalez A. 1998. Evoludon of the basal ganglia in tetrapods: a new perspective based on recent studies in amphibians. Trends Neurosci 21:487-494. Marin O, Rubenstein JL. 2001. A long, remarkable journey: tangential migration in the telencephalon. Nat Rev Neurosci 2:780-790. Marin O, Rubenstein JLR. 2002. Patterning, regionalization, and cell differentiation in the forebrain. In: Rossant J, Tam PPL (eds) Vlouse development. Patterning, morphogenesis, and organogenesis. Academic Press, San Diegc, CA, pp 75-106 Marti E, Takada R, Bumcrot DA, Sasaki H, McMahon AP. 1995. Distribution of Sonic hedgehog pq)tides 138 Ô . ILL, mrUlALAinU en l a lKAl>l»lLlUrN ANAiVlNlU- AMNIOTA: ESTUDIOS EN ANUROS Y REPTILES in the developing chick and mouse embryo. Development 121:2537-2547. Martinez-de-la-T orre M, Pombal MA, Puelles L. 2011. Distal-less-like protein distribution in the larval lamprey forebrain. Neuroscience 178:270-284. McClellan KM, Parker KL, Tobet S. 2006. Development o f the ventromedial nucleus o f the hypothalamus. Front Neuroendocrinol 27:193-209. Medina L. 2006. Field homologies. In: Kaas J, Striedter G, Rubenstein, JLR (eds). Encyclopedia on evolution o f nervous systems, vol 1: History of ideas, basic Concepts and developmental mechanisms. Elsevier, Oxford, UK. Medina L. 2008. Evolution and embryo logical development o f forebrain. In: Encyclopedia of Neuroscience, Binder, M.D., Hirokawa, N., Windhorst, U. (Eds ). Springer, Berlin, pp 1172- 1192. Morales-Delgado N, Merchan P, Bardet SM, Ferran JL, Puelles L, Diaz C. 2011. Topography o f somatostatin gene expression relative to molecular progenitor domains during ontogeny of the mouse hypothalamus. Front Neuroanat 5:10, doi: 10.3389/jhana.2011.00012. Moreno N, Gonzalez A. 2011. The non-evaginated secondary prosencephalon of vertebrates. Front Neuroanat 5:12, doi: 10.3389/jhana.2011.00012. Moreno N, Bachy I, Rétaux S, Gonzalez A. 2004. LIM- homeodomain genes as developmental and adult genetic markers of Xenopus forebrain functional subdivisions. J Comp Neurol 472:52-72. Moreno N, Dominguez L, Rétaux S, Gonzalez A. 2008a. Islet I as a marker o f subdivisions and cell types in the developing forebrain of Xenopus. Neuroscience 154:1423-1439. Moreno N, Rétaux S, Gonzalez A. 2008b. Spatio- temporal expression o f Pax6 in Xenopus forebrain. Brain Res 1239:92-99. Moreno N, Morona R, Lopez JM, Gonzalez A. 2010. Subdivisions o f the Turtle Pseudemys scripta Subpallium Based on the Expression o f Regulatory Genes and Neuronal Markers. J CompNeurol 518:4877-902. Moreno N, Dominguez L, Morona R, Gonzalez A. 2011. Subdivisions o f the turtle Pseudemys scripta hypothalamus based on the expression o f regulatory genes and neuronal markers. Journal o f Comparative Neurology (in press). Papalopulu N, Kintner C. 1993. Xenopus Distal-less related homeobox genes are expressed in the developing forebrain and are induced by planar signals. Development 117:961-975. Patten I, Kulesa P, Shen MM, Fraser S, Placzek M. 2003. Distinct modes o f floor plate induction in the chick embryo. Development 130:4809-4821. Price M, Lemaistre M, Pischetola M, Di Lauro R, Duboule D. 1991. A mouse gene related to Distal- less shows a restricted expression in the developing forebrain. Nature 351:748-751. Puelles L, Rubenstein JL. 1993. Expression patterns of homeobox and other putative regulatory genes in the embryonic mouse forebrain suggest a neuromeric organization. Trends Neurosci 16:472- 479. Puelles L, Rubenstein JL. 2003. Forebrain gene expression domains and the evolving prosomeric model. Trends Neurosci 2:469-476. Puelles L, Javier Milan F, Martinez-de-la-T orre M. 1996. A segmental map of architectonic subdivisions in the diencephalon of the frog Rana perezi: acetylcholinesterase-histochemical observations. Brain Behav Evol 47(6):279-310. Puelles L, Martinez S, Martinez-de-la-T orre M„ Rubenstein JLR (2004) Gene maps and related histogenetic domains in the forebrain and midbrain. In: Paxinos G (ed) The rat nervous system, 3rd edn. Elsevier, Amsterdam, pp 3-25. Puelles L, Martinez de la Torre M, Paxinos G, Watson C, Martinez S. 2007. The Chick Brain in Stereotaxic Coordinates: an Atlas featuring Neuromeric Subdivisions and Mammalian Homologies. Academic Press/Elsevier, San Diego. Rink E, Wullimann MF. 2004. Connections of the ventral telencephalon (subpallium) in the zebrafish (Danio rerio). Brain Res 1011:206-220. Rubenstein JL, Puelles L. 1994. Homeobox gene expression during development of the vertebrate brain. Curr Top Dev Biol 29:1-63. Ryu S, Mahler J, Acampora D, Holzschuh J, Erhardt S, Omodei D, Simeone A, Driever W. 2007. Orthopedia homeodomain protein is essential for diencephalic dopaminergic neuron development. Curr Biol 17:873-880. Sander M, Paydar S, Ericson J, Briscoe J, Berber E, German M, Jessell TM, Rubenstein JL. 2000. Ventral neural patterning by Nkx homeobox genes: Nkx6.1 controls somatic motor neuron and ventral Morona R, Ferran JL, Puelles L, Gonzalez A. 2011. Embryonic intemeuron fates. Genes Dev 14:2134-2139. genoarchitecture of the pretectum in Xenopus laevis: Shimamura K, Hartigan DJ, Martinez S, Puelles L, conserved pattern in tetrapods. J Comp Neurol 519:1024- Rubenstein JL. 1995. Longitudinal organization of 1050. Neary TJ, Northcutt RG. 1983. Nuclear organization of the bullfrog diencephalon. J Comp Neurol 213:262- 278. Nieuwenhuys R. ten Donkelaar H, Nicholson C (Eds) 1998. The Central Nervous System of Vertebrates. London: Springer. the anterior neural plate and neural tube. Development 121:3923-3933. Shimizu K, Okada M, Takano A, Nagai K. 1999. SCOP, a novel gene product expressed in a circadian manner in rat suprachiasmatic nucleus. FEBS Lett 458(3):363-369. Shimogori T, Lee DA, Miranda-Angulo A, Yang Y, Wang H, Jiang L, Yoshida AC, Kataoka A, 139 3. EL H lP U l ALA M U EN LA IK A N SIC IU N ANAM NIU- AMNIOTA: ESTUDIOS EN ANUROS Y REPTILES Mashiko H, Avetisyan M, Qi L, Qian J, Blackshaw S. 2010. A genomic atlas of mouse hypothalamic development. Nat Neurosci 13:767-775. Stoykova A, Gruss P. 1994. Roles o f Pax-genes in developing and adult brain as suggested by expression patterns. J Neurosci 14:1395-1412. Swanson LW (1987) The hypothalamus. In: Bjorklund A, Hokfelt T, Swanson LW (eds) Handbook of chemical neuroanatomy, vol 5. Integrated systems of the CNS, Part I. Elsevier, the Netherlands, pp 1-124. Ten Donkelaar HJ. 1998a. Anurans. In: Nieuwenhuys R, ten Donkelaar HJ, Nicholson C, editors. The central nervous system of vertebrates. London: Springer, p 1I5I-1314. Ten Donkelaar HJb. 1998. Urodeles. In: Nieuwenhuys R, ten Donkelaar HJ, Nicholson C, editors. The central nervous system of vertebrates. London: Springer, p Tran PV, Lee MB, Marin O, Xu B, Jones KR, Reichardt LF, Rubenstein JR, Ingraham HA. 2003. Requirement o f the orphan nuclear receptor SF-1 in terminal differentiation of ventromedial hypothalamic neurons. Mol Cell Neurosci 22:441- 453. van den Akker WM, Brox A, Puelles L, Durston AJ, Medina L. 2008. Comparative functional analysis provides evidence for a crucial role for the homeobox gene Nkx2.1/Titf-1 in forebrain evolution. J Comp Neurol 506:211-223. Wang W, Lufkin T. 2000. The murine Otp homeobox gene plays an essential role in the specification of neuronal cell lineages in the developing hypothalamus. Dev Biol 227:432-449. Wullimann MF, Mueller T. 2004. Teleostean and mammalian forebrains contrasted: Evidence from genes to behavior. J Comp Neurol 475:143-162. Yamamoto N, Ito H. 2005. Fiber connections of the anterior preglomerular nucleus in cyprinids with notes on telencephalic connections o f the preglomerular complex. J Comp Neurol 491:212-233. Yee CL, Wang Y, Anderson S, Ekker M, Rubenstein JL. 2009. Arcuate nucleus expression o f NKX2.1 and DLX and lineages expressing these transcription factors in neuropeptide Y(+), proopiomelanocortin(+), and tyrosine hydroxylase(+) neurons in neonatal and adult mice. J Comp Neurol 517:37-50. Zhao XF, Suh CS, Prat CR, Ellingsen S, Fjose A. 2009. Distinct expression of two foxgl paralogues in zebrafish. Gene Expr Patterns 9:266-272. 140 3. ILL tlirUlALAiVlU LN LA ItCANaiLlUN ANAiVlNlU- AMNIOTA: ESTUDIOS EN ANUROS Y REPTILES THE JOURNAL OF COMPARATIVE NEUROLOGY 000: 00-00 (2011) DOI: 11-0165,22762 Subdivisions of the turtle Pseudemys scripta hypothalamus based on the expression of regulatory genes and neuronal markers LAURA DOMINGUEZ , NEREA MORENO , RUTH MORONA AND AGUSTIN GONZALEZ Departamento de Biologia Celular, Facultad de Biologia, Universidad Complutense, 28040, Madrid, Spain 'Both authors have contributed equally to this work A B STR A C T The patterns o f distribution of a set o f conserved brain developmental regulatory transcription factors and neuronal markers were analyzed in the hypothalamus of the juvenile turtle, Pseudemys scripta. Combined immunohistochemical techniques were used for the identification of the main boundaries and subdivisions in the optic, paraventricular, tuberal and mammillary hypothalamic regions. The combination o f Tbrl and Pax6 with Nkx2.1 allowed the identification of the boundary between the telencephalic preoptic area, rich in Nkx2.1 expression, and the prethalamic eminence, rich in Tbrl expression. In addition, at this level Nkx2.2 expression defined the boundary between the telencephalon and the hypothalamus. The dorsalmost hypothalamic domain was the supraoptoparaventricular region that was defined by the expression of Otp/Pax6 and the lack of Nkx2.1/Isll. It is subdivided into rostral, rich in Otp and Nkx2.2, and caudal, only Otp positive, portions. Ventrally, the suprachiasmatic area identified by its catecholaminergic groups and the lack of Otp, and could be further divided into a rostral portion, rich in Nkx2.1 and Nkx2.2, and a caudal portion, rich in Isll and devoid of Nkx2.1 expression. The expressions of Nkx2.1 and Isll defined the tuberal hypothalamus, whereas only the rostral portion expressed Otp. Its caudal boundary was evident by the lack of Isll in the adjacent mammillary area, which expressed Nkx2.1 and Otp. All these results provided an important set of data on the interpretation of the hypothalamic organization in a reptile and hence make a useful contribution to the understanding of hypothalamic evolution. Keywords: supraoptoparaventricular, suprachiasmatic, tuberal, mammillar, reptile, evolution. The term hypothalamus (hypo from the old Greek ÿpô: under) literally describes the topographical position of the hypothalamus beneath the thalamus and it has been traditionally considered as a special region of the ventral diencephalon, involved in regulation of the endocrine system, the autonomic nervous system and related brain systems (Nieuwenhuys, 1998; Bruce, 2008; Hodos, 2008). However, taking into account molecular and developmental data gathered in recent years, mainly based on the detailed comparison of diverse developmental gene expression patterns, Puelles and Rubenstein postulated the currently accepted prosomeric model of the forebrain (Puelles and Rubenstein, 1993, 2003). Within this model, the hypothalamus is clearly rostral to the diencephalon and is distinctly specified during development (Shimogori et al., 2010). It corresponds to the extratelencephalic portion of the secondary prosencephalon and does not contain the preoptic area, classically included in the hypothalamus but currently identified as a telencephalic territory (Flames et al., 2007; Garcia-Lôpez et al., 2008; Medina, 2008; Sanchez-Arrones et al., 2009; Moreno and Gonzalez, 2011). In the prosomeric model, each division is characterized by the expression of a unique combination o f developmental regulatory genes, and each appears to represent a self-regulated and topologically constant histogenetic brain compartment that gives rise to specific cell groups. In the current view of the highly complex anatomy o f the hypothalamus, distinct regions are considered that can 141 3. EL H lF U l A LA M O EN LA IK A N SIC IO N AN A M N IO AMNIOTA: ESTUDIOS EN ANUROS Y REPTILES Abbreviations Pa paraventricular nucleus PDVR posterior dorsal ventricular ridge Ph nucleus periventricularis hypothalami PC preoptic area POC commissural preoptic area POH preoptohypothalamic boundary Ppo preiventricular preoptic area PTh prethalamus PThE prethalamic eminence Rot nucleus rotundus RT rostral tuberal hypothalamus SC suprachiasmatic region SCc caudal suprachiasmatic region SCr rostral suprachiasmatic region SPa subpallium SPV supraoptoparaventricular region SPYc caudal supraoptoparaventricular region SPVr rostral supraoptoparaventricular region Sv ventral septum Th thalamus Tub tuberal hypothalamus Vmh nucleus ventromedialis hypothalami be summarized into four main components: the supraoptoparaventricular area, the suprachiasmatic area, the tuberal hypothalamus and the mammillary area (reviewed in Medina, 2008, Moreno and Gonzalez, 2011). Most interestingly, the comparative analysis o f numerous data conducted in the last years has highlighted the segmental organization of the forebrain and has shown conserved patterns in all vertebrates studied, in terms of embryological origin, cell types, neurochemical organization, hodology, and functional implications of the subregions in the prosencephalon, suggesting that the molecular specifications unravel the morphogenetic field formation with a very conserved pattern (Puelles and Rubenstein, 1993; 2003; Bachy et al., 2001; 2002; Gonzalez et al., 2002a,b; Brox et al., 2003; 2004; Moreno et al., 2004; 2008a,b; 2010; Osorio et al., 2005; 2006; Flames et al., 2007; Garcia-Lôpez et al., 2008; Abellân and Medina, 2009; Ferrân et al., 2009; Gonzalez and Northcutt, 2009; Dominguez et al., 2010; 2011; Morona et al., 2011). Among amniotes, most data on the regionalization of the prosencephalon have been obtained for mammals and birds. In contrast, only scarce and fragmentary data have been reported concerning the location of markers involved in the formation of the forebrain in reptiles (Smith-Femandez et al., 1998; Métin et al., 2007; Pritz and Ruan, 2009; Moreno et al., 2010). This is surprising, considering the crucial position of reptiles in a phylogenetic perspective specially turtles, which were reported to be the most closely related to the extinct therapsids from which mammals arose (Northcutt, 1970) although, alternatively, they have been considered the sister group to crocodiles and birds (Zardoya and Meyer, 2001a,b). Given the strategic evolutionary position of reptiles and the fact that important differences may also exist among vertebrates, which may reflect divergent evolution in the different lineages and also secondary adaptations of each group, we herein have studied in the hypothalamus of the turtle Pseudemys scripta the pattern of distribution of main regulatory transcription factors and proteins involved in neural patterning and that are also expressed after development (Moreno et al., 2010). The markers used have been selected because they are forebrain essential regulators and markers, such as Islet 1 (Isll), Nkx2.1, Nkx2.2, Orthopedia (Otp), Pax6, Pax7, Tbrl, and tyrosine hydroxylase (TH). The combination of all these markers highlighted boundaries, nuclei, and morphogenetic domains and provided a gene expression atlas in the turtle hypothalamus that facilitates the understanding of the nuclear organization and appears extremely useful for assessing shared features and differences with other vertebrates. MATERIALS AND METHODS For the present study, 8 prehatching (1-2 weeks prehatching and lesser than 5 cm of length), 6 hatching (just hatching and 5 cm of length) and 20 posthatching (1-2 weeks posthatching and 5-7 cm o f length) Pseudemys scripta were used. The animals were obtained from the laboratory stock of the Department o f Cell Biology, University Complutense, Madrid, or from authorized commercial suppliers (Aquarium, Madrid, Spain). They were kept in a room with controlled temperature (25°C) and natural light/dark conditions. The original research reported here was performed according to the regulations and laws o f the European Union (86/609/EEC) and Spain (Royal Decree 1201/2005) for care and handling of animals in research. Immunohistochemistry The turtles were deeply anesthetized with an intraperitoneal injection of sodium pentobarbital (50- 100 mg/kg, Normon Labs, Madrid, Spain) and perfused transcardially with physiological saline followed hy 200 ml of cold 4% paraformaldehyde in a 0.1 M phosphate buffer (PB, pH 7.4). The brains were removed and kept in the same fixative for 2-3 hours. Subsequently, they were immersed in a solution o f 30% sucrose in PB for 4-6 hours at 4 °C until they sank, embedded in a solution of 20% gelatin with 30% sucrose in PB, and stored for 6 hours in a 3.7% formaldehyde solution at 4 “C. The brains were cut on a freezing microtome at 30 pm in the transverse or sagittal plane, and sections were collected and rinsed in cold PB. Immunohistofluorescence procedures were conducted for different primary antibodies that, in all cases, were diluted in 5-10% normal goat serum in PB with 0.1% Triton X-100 (Sigma, St. Louis, MO) and 142 3 . L L m r U lA L A iV lO EN L A IK A i'N a iL lU N AI'NAiVlNlU- AMNIOTA: ESTUDIOS EN ANUROS Y REPTILES Table 1. Antibodies used in the present study Name Immunogen Commercial supplier MW (KDa) Dilution Isll amino acids 247-349 at the c- terminus of rat Islet I Monoclonal Mouse-anti-Isl Developmental studies hybridoma bank; catalogue reference: 40.2D6 39 1:500 Nkx2.1 amino acids 110-122 from the amino terminus Polyclonal Rabbit -anti-TTF Biopat Immunotechnologies, Caserta, Italy, catalogue reference: PA 0100 42-37 1:500 Nkx2.2 E. coli-derived recombinant chick NKX2.2 NK2 transcription factor related Monoclonal Mouse-anti-Pax6. Developmental Studies Hybridoma Bank; catalogue reference: 74.5A5 30 1:500 Otp amino-acid sequence: RKALEHTVS of the C- terminal OTP Polyclonal Rabbit-anti-Otp Pikcell Laboratories, Kruislaan, Amsterdam, The Netherlands 34 1:1000 Pax6 E. coli-derived recombinant chick PAX6. aa 1-223 of chick Pax6 Monoclonal Mouse-anti-Pax6. Developmental Studies Hybridoma Bank; catalogue reference: PAX6 46 1:500 Pax6 peptide sequence: QVPGSEPDMSQYWPRLQ of the C-terminus of the mouse Pax6 protein Polyclonal Rabbit-anti- Pax6 Covance, California, USA. Code number: PBR-278 46 1:250 Pax7 E. coli-derived recombinant chick PAX7. aa 352-523 of chick Pax7 Monoclonal Mouse-anti-Pax7. Developmental Studies Hybridoma Bank; catalogue reference: PAX7 55 1:500 Tbrl amino acids 1-200 at the N-terminus of mouse TBR-1 Polyclonal Rabbit-anti-Tbrl Santa Cruz, Inc Catalogue reference: SC-48816 73 1:1000 TH TH purified from rat PC 12 cells Monoclonal Mouse-anti-TH ImmunoStar, Inc. Catalogue reference: 22941 62 1:1000 TH Protein purified from rat pheochromocytoma Polyclonal Rabbit-anti-TH Chemicon International, Inc, USA. Code number: AB152 62 1:1000 2% bovine serum albumin (BSA, Sigma). Immunohistochemical protocols were carried out on the free-floating sections as follows: 1) Incubation for 72 hours at 4°C in the dilution of each primary serum (see Table 1) in PB with 0.1% Triton X-100. 2) According to the species in which the primary antibody was raised, the second incubations were conducted with the appropriately labeled secondary antibody diluted 1:500 for 90 minutes at room temperature: Alexa 594-conjugated goat anti­ rabbit (Molecular Probes, Eugene, OR, USA; catalogue reference: A11037), Alexa 488-conjugated goat anti­ mouse (Molecular Probes; catalogue reference: A21042). To study the relative distribution of two proteins in the same sections, the two-step protocol for immunohistofluorecence was used with cocktails o f pairs of primary antibodies (one developed in rabbit and the other in mouse), at the same dilutions and conditions specified above, and secondary cocktail o f Alexa 594- and Alexa 488-conjugated antibodies (as above). In all cases, after being rinsed, the sections were mounted on glass slides and coverslipped with Vectashield mounting medium (Vector Laboratories, Burlingame, CA, USA; catalogue number: H I000). Western blotting analysis Two animals were cold anesthetized, and the brains were quickly removed and mechanically homogenized in an equal volume of buffer (5 mM EDTA, 20 mM Tris, pH 7.4, 150 mM NaCl, 10% glycerol, 1% Nonidet P40; Roche, Mannheim, Germany) supplemented with protease and phosphatase inhibitors (50 |Lig/ml phenylmethylsulfonyl fluoride, 10 pg/ml aprotinin, 25 |Xg/ml leupeptin, and 100 nM orthovanadate; all from Sigma). Samples of the supernatants containing in each case 50 |ig of protein were applied in each lane of a 12% polyacrylamide gel (161-0801; Bio-Rad, Hercules, CA) and separated by sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) with a Mini-Protean system (Bio-Rad). The samples of rat brain and molecular weight standards (Precision Plus 143 Isletl (39KDa) Ps Rat 50 37 Nkx2.1 (42KDa) Ps Rat 50 37 Nkx2.2 (30KDa) Ps Rat Otp (34KDa) Ps Rat 37 29 mPax6 (46KDa) Ps Rat 50 37 rPax6 (46KDa) Ps Rat Pax7 (55KDa) Ps Paf Tbr1 (73KDa) Ps Paf 97 50 rTH (62KDa) Ps Paf mTH 62 (KDa) Ps Paf 97 50 37 Figure 1. Identification by Western blots o f protein bands recognized in Pseudemys scripta for the mouse anti-Isll antibody (a), rabbit anti-Nkx2.1 antiserum (b), mouse anti-Nkx2.2 antibody (c), rabbit anti-Otp antiserum (d), mouse anti-Pax6 antibody (e), rabbit anti-Pax6 antiserum (f), mouse anti-Pax7 antibody (g), rabbit anti-Tbrl antiserum (h), rabbit anti-TH antiserum (i), and mouse anti-TH antibody (j). A single band is seen in each o f the lanes corresponding to the turtle brain extracts that are compared with the band stained in each case for rat brain extracts. The expected molecular weight is indicated for each transcription factor or enzyme and the molecular weight standard is represented on the right o f each photograph. Protein Kaleidoscope Standards, Bio-Rad) were run in other lanes. The separated samples in the gel were transferred to nitrocellulose membrane (Bio-Rad). Nonspecific binding sites were blocked by incubation overnight in Tris-HCl buffer (TBS) containing 0.1% Tween-20 (TBST) and 5% nonfat milk, at 4°C. The blots were then incubated for 24 hours at 4°C in primary antibody dilution (as for immunohistochemistry). After rinsing in TBS, the blots were incubated in horseradish peroxidase-coupled secondary goat anti-mouse or goat anti-rabbit IgG (Jackson ImmunoResearch, West Grove, PA; diluted 1:15,000) for 2 hours at room temperature. Immunoreactive bands were detected by using an enhanced chemiluminescence system (Super Signal West Pico Chemiluminiscent Substrate; Pierce, Thermo Scientific, Rockford, IL). Photographs were taken after applying an autoradiographic film to the membrane, in darkness, for 1 -4 minutes. Controls and specificity of the antibodies General eontrols for the immunohistochemical reaction included: 1) Western blot (see the previous section), 2) staining some selected sections with preimmune rabbit serum; 3) controls in which either the primary and/or the secondary antibody was omitted. In all these negative controls, the immunostaining was eliminated. In addition, all the antibodies used have been tested, under identical conditions, in tissues devoid o f antigen (mouse brain slices at levels devoid o f expression), as negative control, and in tissues positive for the antigen (mouse brain slices at levels expressing the antigen). In all the cases, the controls have been satisfactory. The specificity o f the antibodies used has been assessed by the commercial companies (Table 1), and, in addition, immunoblotting was conducted (see above). The Western blots o f brain extract o f Pseudemys scripta showed that all antibodies used labeled a single band, which with small variations corresponded well to the bands labeled in the rat lanes (Fig. 1). Many o f the antibodies used here (Isll, Nkx2.1, Otp, mouse anti-Pax6, mouse anti-TH and T brl) were also used in our previous study o f the Pseudemys scripta forebrain and their specificity was then detailed (Moreno et al., 2010). However, it should be noted that the antisera were generated against non­ turtle sequences and, unfortunately, there is not information available regarding sequence similarity between these molecules in turtle and other vertebrates, and we cannot analyze the degree o f conservation of each epitope recognized by the antibodies. Nevertheless, given the similarities observed in the expression patterns for each antibody across species, it is strongly suggested similarities in the sequences. In the cases o f TH and Pax6 , monoclonal and polyclonal antibodies were used (table 1) with fully comparable results in the obtained pattern of immunostaining. The anti-Nkx2.2 monoclonal antibody was developed by Dr. Jessell (Columbia University; New York). The DNA region o f NK2 transcription factor of chick was cloned by polymerase chain reaction into the E. coli expression vector. Recombinant protein was expressed and purified. The monoclonal antibody was generated by immunization o f mice with the recombinant protein. It has been tested in mice, rat, chick and human (see Developmental Studies Hybridoma Bank Data sheet). The specificity o f the Nkx2.2 antibody has been confirmed by an absence of labeling in Nkx2.2-/- mice (Cai et al., 2010). Western blot analysis with the anti-Nkx2.2 antibody detected a single band at the same molecular weight as that o f the major product detected in rat brain extract (Fig. Ic). 144 d c b a I I I I Th PTh, r ' S t r T v ^ Xy ;ll- This same antibody has also been tested by Western blot with chicken brain extracts and two bands were obtained o f 43 kDa and 28 kDa and two isoforms were suggested (Ferran et al., 2009). The band observed in Pseudemys corresponds well to the band o f 28 kDa observed in chick. The Pax6 serum was generated against a sequence that is highly conserved (see Table 1). The antibody was subsequently purified on a Protein A column and is useful in studying brain, neuronal and olfactory development in eukaryotes (see Covance Data sheet). The Western blot for the brain extract o f Pseudemys shows a band that corresponded well to that in the rat lane and coincides with the calculated molecular weight (Fig. If), and is similar to that obtained with the monoclonal anti-Pax6 (Fig. le). The Pax6 antibody detects two major and two minor products in Western blots o f chicken brain, suggesting the existence o f Pax6 isoforms (Kawakami et. al., 1997) and the results o f the immunoreaction produced essentially the same topographic localization as the mRNA expression pattern in the brain (Ferran et al., 2009). The anti-Pax7 antibody (Kawakami et al., 1997) was developed by Dr. Kawakami (Division o f Biological Science, Nagoya University Graduate School of Science, Nagoya, Japan). The DNA region corresponding to amino acids 352-523 o f chick Pax7 was cloned by polymerase chain reaction into the E. coli expression vector. Recombinant protein was expressed and purified. The monoclonal antibody was generated by immunization of mice with the recombinant protein. It has been tested in chicken, mouse, zebrafish, rat, human, and axolotl (see Developmental Studies Hybridoma Bank data sheet). In Western blots o f chicken brain tissue the Pax7 antibody detects three bands but the spatiotemporal distribution pattern obtained by immunohistochemistry corresponded with the mRNA expression patterns (Ferran et al., 2009). In Pseudemys, the Western blot analysis with the Pax7 antibody detected a single band at the same molecular weight as the major product detected in rat brain extract (about 55 kDa; see Fig. Ig) and also coincides with the band observed in Xenopus brain extracts (Morona et al., 2011). The immunogen used to produce the TH serum was denatured tyrosine hydroxylase from rat pheochromocytoma (denatured by sodium dodecyl Figure 2. Photomicrographs of Nissl-stained transverse sections through the hypothalamus of juvenile Pseudemys scripta, from rostral (a,b) to caudal (c,d) levels showing in the left side of the sections the nomenclature proposed by Smeets and cols (1987) and in the right side the major subdivisions considered in our study. The approximate levels of the sections are indicated in the upper scheme of the lateral view of the brain in which the preoptic area and hypothalamus are highlighted in grey. Note the change in the longitudinal axis due to the pronounced brain flexure and the orientation of the actual location of dorsal and rostral portions of the hypothalamus. Scale bar = 500 pm. 145 J. r-m.# xiJkj x x̂ x-rxv-rk_7 x̂ i ̂ r-xi. ̂ %_/x-vv-̂ k̂ x x'vx.jx x xj Nkx2.1 P ax b Nkx2.2 P ax 7 Figure 3. Photomicrographs of transverse sections through the telencephalon (a-e) and hypothalamus (f-t) of Pseudemys scrota, showing Nkx2.1 (a,f,k,p), Isll(b,g,l,q), Otp (c,h,m,r), Pax6 (d,i,n,s), Tbrl (e,j), Nkx2.2 (o) and Pax7 (t) immunostaining. Scale bar = 200 pm (valid for all photomicrographs). A magenta-green version of this figure is provided as Supporting Information Figure 1. sulfate). W estern blot analysis using mouse and rat brain tissue demonstrated a single band at 62kDa and no band was detected in rat liver used as negative eontrol tissue (Yee et al., 2009). Similarly, the polyclonal anti-TH antibody detected a similar single band in W estern blots o f chick (Bardet et al., 2006) and Xenopus (Morona and Gonzalez, 2008) brain tissue. The Western blot analysis in Pseudemys shows a single band at the expected molecular weight and similar o f that o f the major product detected in rat brain extract (about 62kDa) (Fig. li). This result is comparable to that observed with the monoclonal antibody (Fig. Ij). Finally, the antigen used to raise the antibody Tbrl was a recombinant protein corresponding to the amino acid region 1-200 o f the murine TBR-1. Thus, the antigen corresponds to the entire N-terminus o f the protein and within this protein different immunoglobulines bind. Preadsorption experiments were also done as a further control o f the specificity o f the antibody in the turtle brain. Preadsorption was done mixing the primary antibody with a Tbrl blocking peptide that consisted in amino acids 50 to 150 o f the murine TBR-1 (Abeam ab25853; 0.1, 1.0 or 10 mM). The preadsorbed antibody was then used for immunohistochemistry instead the primary antibody, following the procedure indicated above and in these controls, the immunostaining vas eliminated, even when the rabbit anti-Tbrl vas preadsorbed with the peptide at low concentration 0.1 mM). Imaging The sections were analyzed with an Olympus BI51 microscope equipped for fluorescence with appropiate filter combinations. Selected sections vere photographed using a digital camera (Olympus DP'O). Contrast and brightness were adjusted in Acbbe Photoshop CS3 (Adobe systems, San Jose, CA), ind photographs were mounted on plates using Canvas 11 (ACS systems. International Inc). RESULTS At the prehatching and posthatching stages usee in the present study, the brain maintains the expressioi o f the different markers used, including the transcripion factors, in the ventricular (vz), subventricular (fvz), ind mantle zones {mz), allowing the analysis o f progentor domains. The results obtained were comparable in tie 146 A iV liX lV JlA : Ï KJLrUJLiJLa Tbr1/lsl1 PDVR PThE Nkx2.1/Pax6 ,. i ' -:■■ Nkx2.1/Pax6 PDVR P T h E \ ‘X :'\ I : \ j\ ! po ' ! " SPV Nkx2.1 /rax6 P ih E ▲ > PTh % POC Nk: 2. :/Pax6 PThE PO i Tbr1/Pax6 f PDVR P T h ' P T h E p/lsl1 S P a S C ; : P O g Tji:1/Pax6' PDVR # # & • . m ' P Q - ç , a SPV PThE 147 3. EL H lF U l A LA M O EN LA I KANSICIUN ANAMJNIU- AMNIOTA: ESTUDIOS EN ANUROS Y REPTILES Figure 4. Photomicrographs o f transverse (a, b, e, h-jj) and sagittal (c,d,f and g) sections through the telencephalon o f Pseudemys scripta, showing the combined expressions o f Tbrl and Isll (a), Nkx2.1 and Pax6 (b- e), Tbrl and Pax6 (f,h) and Otp and Isll (g,i and j). The arrowheads in b, d and e indicate the Pax6 vz expression in the prethalamic eminence. The yellow lines in the sagittal sections presented in c, and g point to the level o f the transverse sections indicated in each figure. Scale bar in a (valid for b,f,g and h) = 500pm, in c (valid for e, i and j) = 200 pm and in d = 100 pm. A magenta-green version o f this figure is provided as Supporting Information Figure 2. different developmental stages used. Thus the results will be described with independence of the stage used. First, Nissl staining (Fig. 2) allowed the identification of the different hypothalamic and adjacent areas classically recognized and distinctly highlighted by the expression of the markers Isll, Nkx2.1, Nkx2.2, Otp, Pax6, Pax7, and Tbrl (Fig. 3). The classical conception of the brain described the hypothalamus under, thus ventral, to the thalamus . However, currently following the prosomeric model the prosencephalon is subdivided in longitudinal prosomeres following the brain flexure. Thus now the hypothalamus constitutes the most rostral prosencephalic region, with the telencephalon at its dorsal border and the prethalamus constitutes the caudal boundary (Puelles and Rubenstein, 2003). These orientation clues are followed in the description o f the results. The combination of these markers helped to the identification of the boundaries of the hypothalamus with the telencephalon and diencephalon (Figs. 4, 5), and the distinct nuclei and territories within the dorsal (Figs. 6-8) and ventral hypothalamic regions (Figs. 9-11). The results on the combinatorial expression in the hypothalamus o f the markers used have been summarized in the schemes of Fig. 12. General identification of the main hypothalamic regions in the turtle The preoptic area (PO) has been traditionally considered as part of the hypothalamus and, thus in the present analysis special attention was paid to the identification of the boundaries between the PO and the hypothalamus. At the level o f the preoptic recess of the third ventricle, Nkx2.I (Fig. 3a) was expressed in the vz and the svz o f the PO, including the PO proper and a particular zone close to the anterior commissure, recently termed the commissural preoptic area (POC; Moreno et al., 2010). The Isll expression was also noticeable in the svz of the PO (Fig. 3b). At this level, the expression pattern shown by Otp was useful for identifying the dorsal tip of the supraoptoparaventricular region (SPV; Fig. 3c), whereas Pax6, expressed in the SPV, also served to identify the caudal extent of the septum, defined as the ventral septum (Sv; Fig. 3d). Also at these levels, the Tbrl expression helped to the recognition of the most anterior extent o f the dorsal part o f the diencephalic prosomere 3 (p3), which is identified as the prethalamic eminence (PThE; Fig. 3e). At the level o f the optic chiasm, Nkx2.1 was distinctly expressed in the vz of the suprachiasmatic area (SC), whereas Isll labeled cells in this area in the svz (Fig. 3f,g). This zone was adjacent to the SPV, defined by the Otp expression (Fig. 3h). At this level, the labeling in the vz for Pax6 (Fig. 3i) and in the svz for Tbrl (Fig. 3j) highlighted the caudal and ventral boundaries of the PThE above the prethalamus (PTh). In the tuberal hypothalamus (Tub), Nkx2.1 and Isll were expressed from anterior through posterior levels (Fig. 31,p,q), whereas the Otp expression only labeled one portion (Fig. 3m,r). The Tub was seen to be adjacent to the posterior extent o f the SPV, which is at this level directly rostral to the PTh, distinctly labeled for Pax6 (Fig. 3n). These regions were also highlighted by the expression of Nkx2.2 (Fig. 3o). At posterior (caudal) levels o f the hypothalamus, the mammillary area (Ma) was defined by the expression o f Nkx2.1 (Fig. 3p) and Otp (Fig. 3r), and by the lack of Isll expressing cells (Fig. 3q); the adjacent territory in P3 at this level was visualized by the expression of Pax7 (Fig. 3t). Analysis of the hypothalamo-telencephalic and hypothalamo-diencephalic boundaries At telencephalic levels (Fig. 4) the combination of Isll and Tbrl (Fig. 4a) showed that the PO, rich in Isll svz expression directly limits with the PThE defined by the svz Tbrl expression (Fig. 4a). This boundary was also confirmed by the expression of Nkx2.1 in the vz of PO and Pax6 in the vz of PThE (Fig. 4b-e), both observed from anterior (Fig. 4b) to posterior (Fig. 4e) levels. The expression of these two markers, Pax6 and Tbrl, in the vz defined the PThE (Fig. 4f,h) but only Tbrl was subventricularly expressed. In addition, Pax6 was expressed in the svz o f the PTh (Fig. 4f). The combination of Isll and Otp (Fig. 4g) showed the dorsal tip of the SPV defined by the Otp expression (Fig. 4i j) . Once analyzed the boundary between the PO and the PThE at caudo-dorsal levels (Fig. 4), the ventral extent of the telencephalic PO (Fig. 5) was evidenced by the Nkx2.1 vz and svz expression, and its limits with the hypothalamic SPV were defined by the Otp vz and svz expression (Fig. 5a). In this location, a gap between the PO and the SPV was observed (see astericks in Fig. 5). This domain was defined by the Nkx2.2 vz expression (Fig. 5 b-e). Thus, the combination o f Nkx2.2 with Otp (Fig. 5b,c) and Nkx2.1 (Fig. 5d,e) defined this narrow territory between the telencephalic PO and the hypothalamic SPV (Fig. 5e). We have used the term preoptohypothalamic boundary (POH) already used in other amniotes to define a similar border area (Bardet et al., 2006; Flames et al., 2007). 148 Oip/Nkx2.2 Figure 5. Photomicrographs o f transverse sections through the limit between the telencephalon and the hypothalamus o f Pseudem ys scrip ta , showing the combined expressions o f Otp and Nkx2.1 (a), Otp and Nkx2.2 (b,c) and Nkx2.1 and Nkx2.2 (d,e). The asterisks in the panel show the preopto hypothalamic boundary. The dashed boxes in b and e show the magnified figure o f c and e respectively. Scale bar in a and b = 200pm, in c and e =100 pm and in d = 500 pm. A magenta-green version o f this figure is provided as Supporting Information Figure 3. Analysis of the supraoptoparaventricular region In the hypothalamus, the dorsalmost region identified was the SPV, which was defined by the Otp expression and the lack of Isll and Nkx2.I (Figs. 6, 7). The SPV showed Otp expressing cells in the vz and svz from anterior (Fig. 6a) through posterior levels (Fig. 6i). It was seen to limit dorsally with the POH, close to the PO identified by the Nkx2.1/Isll expressions (Fig. 6a). Ventrally, the SPV formed a direct boundary with the SC region (Fig. 6b-f) in which a conspicuous TH positive cell group is located in all vertebrates (Smeets and Gonzalez, 2000). In addition, the paraventricular nucleus (Pa), which is also TH positive (Smeets et al., 1987), was identified in the SPV (Fig. 6f,g; Otp positive). These catecholaminergic cell groups allowed the distinction of the SPV from the adjacent regions, from anterior (Fig. 6d,e) through posterior levels (Fig. 6g,h). The SPV region was also defined by the expression in the vz o f Nkx2.2 (Fig. 6h) and Pax6 (Fig. 6j,k) and its boundary with the caudally located PTh was thus observed (Fig. 6j-l). The detailed analysis o f the Nkx2.2 expression in this SPV region allowed the identification o f subregions (Fig. 7). The whole SPV was characterized by the vz expressions o f Otp, Pax6 and Nkx2.2 (Fig. 7a-d). However, Nkx2.2 was only expressed in the svz at the rostral portion (SPVr; Fig. 7e). Thus, the SPVr expressed Otp and Nkx2.2 in the vz and svz, therefore many cells were doubly labeled in our experiments, whereas in the caudal portion (SPVc) practically only Otp was expressed in the svz (Fig. 7f,g). Analysis of the suprachiasmatic region The SC region o f all the vertebrates analyzed contains important catecholaminergic cell groups in relation to the optic chiasm (Smeets and Gonzalez, 2000; Fig. 8a-e). In addition, rostro/caudal subregions within the SC could be distinguished mainly on the basis o f the expression pattern o f Nkx2.2 (Fig. 8). Thus, the combination o f Otp (marker o f the SPV) and TH helped to the identification o f the SPV-SC boundary(Fig. 8a). The TH positive cell group was situated in the region close to the optic chiasm and. 149 fiai uwiwa i iiijfLa TH/Nfex2 2 Pa Æ 3 \ V \ sc RT k |S P V . . 's i Tub •kx2,1/THOtp/isH PDVR N k x 2 , 1 / m C oc Otp/TH SPV | s c . ' \ . l l ' . 8 0 : ' Tub ‘ otp/isn - - # SC ■ Otp/Pax(jTbr1/Pi,xo Figure 6. Photomicrographs o f transverse sections through the hypothalamus o f Pseudem ys scrip ta , showing the combned expressions o f Otp and Isll (a and i), Nkx2.1 and TH (b and c), Otp and TH (d-g), TH and Nkx2.2 (h), Tbrl and Pax6 (j), O tpind Pax6 (k) andTbrl and Isll (1). The dashed boxes in b and g show the magnified figure o f c and h respectively. The yellow lins in the sagittal sections presented i point to the level o f the transverse sections indicated. Scale bar in a (valid for b,d,f-l) = 500pm, n c (valid for e) = 200 pm. A magenta-green version o f this figure is provided as Supporting Information Figure 4. 150 /ViVli’SlkFl/V; UfVkfa Ï fULr 1 PTh SPV I tTub /Pax6 PTh SPV Pc^xn/Nkx2., PTh sc ' Tub SPV Tub / njK x 2 . 2 /Nt-: 2̂.2 # 7 ' PQH' SPVc S F » V 3 l 'sc m /S C c Tub SCr Figure 7. Photomicrographs of transverse sections through the hypothalamus of Pseudemys scripta, showing the combined expressions of Otp and Isll (a), Otp and TH (b), Otp and Pax6 (c), Pax6 and Nkx2.2 (d) and Otp and Nkx2.2 (e-g). The dashed box in f shows the magnified figure of g. Scale bar in a (valid for a-f) = 500pm, in g = 200 pm. A magenta-green version of this figure is provided as Supporting Information Figure 5. according to the longitudinal brain axis, was called rostral SC (SCr; Fig. 8b, c). In this rostral portion o f the SC, Nkx2.2 expressing cells in the svz (Fig. 8d) and Nkx2.1 expressing cells in the vz and svz (Fig. 8e) were observed. In contrast, the Isll expression allowed the identification o f the caudal portion (SCc; Fig. 81), which was also devoid o f Nkx2.1 expression (Fig. 8g). The eombination of Nkx2.1 and Nkx2.2 allowed the distinction o f the boundary between the SCr, rich in vz and svz cells expressing both transcription factors (Fig. 8h), and the SCc that only expressed Nkx2.2 in vz eells and limited caudally with the SPVr (Fig. 8h,i). In addition, the SCr limited rostrally with the tuberal hypothalamus, whieh lacks Nkx2.2 expression in the vz (Fig. 8j). T he tu b eral and m am m illary regions o f the h yp othalam us The rest o f the hypothalamus corresponds to the basal hypothalamus and is distinctly and totally defined by the Nkx2.1 expression (Figs. 9 and 10). It is composed rostrally by the tuberal part o f the hypothalamus (Tub; Fig. 9) and caudally by the mammillary region (Ma; Figs. 10, 11). The Tub showed expression o f Nkx2.1 in the vz and svz (Fig. 9a), whereas Isll was expressed only in the svz (Fig. 9c). Strikingly, the Otp expression distinctly defined a subregion in the rostral portion that we called rostral tuberal hypothalamus (RT; Fig. 9b). Thus, the RT, in contrast to the eaudal tuberal region (CT), can be identified by the Otp expression within the N kx2.I/Isll tuberal hypothalamus (Fig. 9d,e). The mammillary region could be distinguished from the tuberal region by the eombination o f Nkx2.1, Isll and Otp (Fig. 9f-j). Thus, the limit with the tuberal hypothalamus was highlighted because Nkx2.1 labeled both regions (Fig. 9f, g) but Isll was mainly located in the svz o f the tuberal region, whereas Otp labeled the svz o f the mammillary region (Fig. 9h-j). More in detail, the Ma was delineated by the combination o f Nkx2.1, which defined all this ventral territory (Figs. 10, 11), and Isll that was expressed only in the Tub (Figs. 10a,d, 11 c,d). In addition, the combination o f both allowed the identification o f the Ma vz (Nkx2.1+/Isll-), and the svz Ma population 151 Pa, SPV ? V T H , . SPVc S P V r 'fi. SCr Figure 8. Photomicrographs of transverse (c,-e, g-j) and sagittal (a,b,f) sections through the hypothalamus of Pseudemys scrota, showing the combined expressions of Otp and TH (a-c), TH and Nkx2.2 (d), Nkx2.1 and TH (e), Otp and Isll (f), Nkx2.1 and [si 1 (g), Nkx2.1 and Nkx2.2 (h,i) and Otp and Nkx2.2 (j). The yellow line in the sagittal sections presented in a point to the level olthe transverse sections indicated. The dashed box in a shows the magnified figure of b. Scale bar in a = 500pm, in e (valid for c,dg,h and j) = 200 pm and in e (valid for f and i) = 100 pm. A magenta-green version of this figure is provided as Supporting Informaion Figure 6. 152 1 I is j i ,r 1 iLiiLcs r)tp/Pax6 ,'PTh N k x 2 .1 /P s î Nkx2.1/rax6 Nkx2 1/ls!1 Nkx2.1 Figure 9. Photomicrographs of transverse sections through the hypothalamus of Pseudemys scripta, showing the combined expressions of Nkx2.1 and Pax6 (a, f), Otp and Pax6 (b), Nkx2.1 and Isll (c-e) and Nkx2.1 (g). The figures h-j presented the same section with Isll (h), Otp (i) and the combination of both (j). The dashed box in d shows the magnified figure of i. Scale bar in a (valid for b,c,d and I) = 500pm, in e (valid for g-j) = 100 pm. A magenta-green version of this figure is provided as Supporting Information Figure 7. (Nkx2.1+), which occupied a lateral position separated from the ventricle (Fig. 10d,e). In these locations, the combination o f Otp (rich in the svz of Ma) and Isll (marker of Tub), revealed an Nkx2.1+ thin territory between the Otp+ and the Isll+ zones (see asterisks in Figs. 9d and I lf) . To highlight this particular boundary, the combination with TH was useful, because a particular catecholaminergic cell group was described in that area (Smeets et al., 1987). It was possible to identify the Nkx2.1 expressing cells intermingled with the TH positive cells (Fig. 10b,e). In addition, Otp was also expressed in the Ma svz (Figs. lOf, l lg ) . The Otp expression in the Ma occupied two parallel bands (Fig. 11 h) and only in the caudal one the labeled Otp cells were intermingled with the TH expressing cells (Fig. H i), where a subpopulation o f cells coexpressed both markers (Fig. ll j) . The combination o f markers o f the Ma such as Nkx2.1 (Fig. 10c) or Otp (Fig. lOf) with Pax6 showed the caudal boundary of the Ma with the basal part o f p3, where can be found scattered Pax6 expressing cells. Also the combinations o f Nkx2.1 or Otp with Nkx2.2 and Pax7 served to characterize the caudal boundary o f Ma with p3, and also some subdivisions within Ma. Thus, Nkx2.1 and Nkx2.2 153 IXW 1. JL JLL/l 1 i 1.1 ̂ ̂ X XVJLJX 1. J.XJXJLJ Nkxk l/lsH . ', Ma I i ' ' - M.Tub Nkx2//TH J Vfa ’■ i * CT RT Nkxz /Pax6 -P3 : i , . < x 2 . 1 / » T f a x 7 - - : # # # 3 X .-7Pax7 i.\>' ■ % t f t •• : Û i ü , - m t . Tub ; • . 1. ’ n* 0 ' • ' . ■ ■ f5v •♦I ■ i t ' . ■ ' Ma Figure 10. Photomicrographs of transverse sections through the hypothalamus of Pseudemys scripta, showing the combned expressions of Nkx2.1 and Isll (a, d), Nkx2.I and TH (b, e), Nkx2.1 and Pax6 (c), Otp and Pax6 (f), Nkx2.1 and Nkx2.2 (g,i)ind Nkx2.1 and Pax7 (h,j). Scale bar in a (valid for b,c,f,g) and = 500pm, in d (valid for i and j) = 200 pm and in e = 100 pn. A magenta-green version of this figure is provided as Supporting Information Figure 8. 154 Nkx2.1 Otp Ma PO Tub o c Tub ' SPV Ma , ' , it ' 2.1/isn Th h,i Pa A SPV Nkx2.2 ^ Ma m o c Figure 11. Photomicrographs of sagittal sections through the hypothalamus of Pseudemys scripta, showing the expressions of Nkx2.1 (a), Otp (b,h) and Nkx2.2 (o) and the combined expressions of Nkx2.I/Isll (c,d), Otp/Isl 1 (e,f), Otp/TH (g,i,j), Otp/Pax7 (k,l), TH/Nkx2.2 (m,n). The dashed box in d, e, i and k show the magnified figure indicated in each figure. Scale bar in a (valid for b,c,e,g,k and m) = 500pm, in h (valid for d,f,i,n,o,l) = 200 pm and in j = 100 pm. A magenta-green version of this figure is provided as Supporting Information Figure 9. showed overlapping expression in the Ma, and an important group o f cells coexpressed both markers, whereas only Nkx2.2 continued into p3 and Nkx2.1 into the Tub (Fig. 10g,i). In turn, Pax7 was intensely expressed in the basal part o f p3 and the combination with Nkx2.1 marked the boundary with the rostrally located Ma (Fig. 10h,j). The latter boundary between the Ma and p3 could also be analyzed by the combination o f Otp with Pax? (Fig. llk ,l). The use o f TH, as marker o f a catecholaminergic cell population in the Ma, in combination with Nkx2.2 showed that within this hypothalamic region only the anterior part expressed Nkx2.2 whereas the TH cells were located more caudally (Fig. 11m, n). DISCUSSION The main concepts established during many years in the study o f the forebrain topology have been dramatically changed in recent years, mainly by the assimilation o f the prosomeric model (Puelles and Rubenstein, 1993; 2003) that contrasts with the classically used columnar model o f the brain (Herrick, 1910). According to the prosomeric model, the rostral forebrain, called secondary prosencephalon, comprises the telencephalon, including the evaginated vesicles and the preoptic area, and the hypothalamus, which lies rostral to the diencephalon (Puelles and Rubenstein, 2003). This paradigm, supported by molecular and fate map analysis (Sanchez-Arrones et al., 2009; Garcia- Lopez et al., 2009; reviewed in Medina, 2008) is constantly under revision, given the high complexity o f the region, the technical advances, and the vast number o f newly generated data. In particular, the complex anatomy o f the hypothalamus is currently being unraveled as new molecular and developmental data are gathered. In general, we have considered the hypothalamus as formed by the supraoptoparaventricular (SPV), the suprachiasmatic (SC), the tuberal (Tub) and the mammillary (Ma) regions (reviewed in Medina, 2008; Moreno and 155 3. EL H lP O l A LAM O EN LA 1RA NSICION ANAM NIU- AMNIOTA: ESTUDIOS EN ANUROS Y REPTILES Gonzalez, 2011), which include, among others, the suprachiasmatic, supraoptic, paraventricular, ventromedial, arcuate, and subthalamic nuclei o f former studies (Altman and Bayer, 1986; Smeets et al., 1986). However, recent reports have favored different subdivisions of the hypothalamus and different terminologies have been coined. Thus, a new division was proposed attending to “terminal” (rostral) and “peduncular” (caudal) regions (Morales-Delgado et al., 2011 in reference to “Allen development mouse brain atlas” done by L. Puelles). In addition, in a recent study Shimogori and collaborators have subdivided the hypothalamus into anterodorsal (Siml positive) and posteroventral (Nkx2.1 positive) hypothalamic regions, with an additional intermediate band termed “intrahypothalamic diagonal” (Shimogori et al., 2010). Furthermore, another current subdivision of the hypothalamus attends to alar (dorsal) territories (containing the SPV and SC) and basal (ventral) components (containing the Tub and Ma), in accordance with the prosomeric model (Puelles and Rubenstein, 2003). However, also this interpretation of the hypothalamus has been recently questioned because a high-resolution anatomical atlas of the transcriptome in mouse embryos (Diez-Roux et al., 2011) changed this subdivision based on the new concept o f “alar and basal genes”, proposing that only the Ma is a basal territory. Thus in this really complex scenario in which the interpretation o f the hypothalamic territories and boundaries are constantly being challenged, we have opted for the strategy of interpreting the hypothalamic region of the turtle following the main subdivisions considered in the prosomeric model, independently of their alar/basal identification. Prelim inary considerations: selection o f the m arkers used As mentioned above, the deep change in the conception of the brain organization that has occurred in the last years has led to reanalysis o f the newly recognized regions under a new perspective. One o f the main tools used consists o f the genoarchitectonic analysis, in which each brain domain is characterized by the expression of a unique combination of developmental regulatory genes, which demonstrates the crucial importance of this histogenetic code in the understanding of the brain (Puelles and Rubenstein, 1993, 2003; Moreno et al., 2004; 2008a,b; 2010; Ferran et al., 2009; Morona et al., 2011). Moreover, results in different vertebrates have provided substantial evidence to support the validity of the prosomeric model across the different classes. This knowledge is crucial to evaluate the differences and similarities in the different groups, and shed more light on the molecular brain specification during the vertebrate evolution.The hypothalamus o f mammals has been analyzed under this new perspective o f territory specification by molecular cues grouped in distinct subregions (Puelles and Rubenstein, 2003; Flames et al., 2007; Shimogori et a., 2010). In addition, some data have also been provided for birds (Bardet et al., 2006; 2008) and amphibians (Dominguez et a l, 2010; 2011) but similar molecular analysis were totally lacking for reptiles. Abundant immunohistochemical studies have described distinct distributions of many neuropeptides/neurotransmitters in the reptilian hypothalamus (Femandez-Llebrez et a l , 1988; Sherwood and Whittier, 1988; Inagaki et a l , 1990; Jimenez et a l , 1994; D'Aniello et a l , 1999; Wang et a l , 1999; Smeets et a l , 2003; Lôpez et a l , 2008; Munoz et a l , 2008; Dominguez et a l, 2009) and its hodology has also been analyzed (Bruce and Neary, 1995a,b; Lanuza et a l , 1997). In addition, a few studies have reported important molecular data on telencephalic (Smith-Femandez, 1998; Métin et a l, 2007; Moreno et a l , 2010) and diencephalic (Pritz and Ruan, 2009) specification in reptiles. In the present study, we have analyzed in the turtle hypothalamus the distribution patterns of many transcription factors expressed in the hypothalamus of other vertebrates, by means of single and double immunohistochemistry. The expression of Nkx2.1 has been deeply analyzed in the prosencephalon of many vertebrates and it has served to identify the preoptic, suprachiasmatic and tuberal territories during development (Gonzalez et a l , 2002a,b; Moreno et a l, 2008a; van den Akker et a l, 2008; Moreno et a l, 2010). The pattern of expression of Islet 1 (Isll) resembles those described for the main members o f the Dix family (Moreno et a l , 2008b; 2010), which help to the identification of main prosencephalic boundaries. Orthopedia (Otp) is a highly conserved homeodomain- containing factor that is transcribed during murine embryonic development in a segment-like expression pattern including the anterior hypothalamus, supraoptoparaventricular region, retrochiasmatic, and ventral tuberal areas (Puelles and Rubenstein, 2003; Bardet et a l, 2008; Dominguez et a l, 2010). The homeobox gene Tbrl was extremely useful in the recognition of the prethalamic eminence (Puelles et a l , 2000; Brox et a l, 2004). Pax6 and Pax7 transcription factors show conserved expression patterns in all vertebrates studied, including reptiles (Stoykova and Gruss, 1994; Smith-Femandez et a l , 1998; Puelles et a l, 2000; Bachy et a l , 2002; Moreno et a l , 2008a; 2010; Pritz and Ruan, 2009). In addition the expression of Pax6 served to identify the boundaries o f the diencephalic prosomeres (Moreno et a l, 2008a; Ferran et a l , 2009; Morona et a l , 2011). Finally, these gene expression domains have been systematically analyzed in combination with TH, a catecholaminergic neuronal marker of specific cell populations carefully described in turtle (Smeets et a l , 1987) and very conserved in the evolution (Smeets and Gonzalez, 2000). The combination of all these markers highlighted boundaries, nuclei, and morphogenetic domains and was extremely useful for assessing shared features and differences with other vertebrates. In general, strikingly conserved patterns of expression of the markers selected are present in the hypothalamus 156 # # PThE SPVc SPVr w MeA SPVr P3 Ma CT RT isl1 I Pax6 I Nkx2.2 | Otp | Nkx2.1 Q Pax7 | Tbrl TH 157 3. EL H IPO TA LA M O EIN LA TRAJNSICIOIN ANAM NIO- AMNIOTA: ESTUDIOS EN ANUROS Y REPTILES Figure 12. Schematic representations of different coronal levels through the hypothalamus of Pseudemys scripta summarizing the combinatorial code of transcription factors used in the present study, following the color code indicated in the box. The levels of the transverse sections (a-d) are indicated in the schematic sagittal representation in Figure 13. The gray dashed boxes illustrate amplified drawings showing the composition of different areas analyzed in the present study. When the expression of the transcription factor was detected in the vz it has been illustrated in the schemes in this position (in gray lines). When they are expressed in the svz it has been illustrated by a corresponding color frame or by filling in the region with the color to simplify the scheme (for example, a blue region surrounded by an orange frame means that both factors are expressed in the region, in that case Nkx2.1 and Nkx2.2). The dots in the schemes in the svz position indicated expressing cells detected in the present study, whose origin is likely from adjacent histogenetic domains. The arrows indicate the most likely migratory pathway followed by these cells, based on the migratory movements identified in other vertebrates their most probable origin, given the histogenetic domains identified in the present study. of the turtle that allow the identification of putative homological relationships across vertebrates. The main results obtained in our study can be summarized: 1) The combination of Tbrl and Pax6 with Nkx2.1 allows the identification of the boundary between the telencephalic PO, rich in vz Nkx2.1 expression, and the PThE, rich in Tbrl expression. In addition, 2) at this level Nkx2.2 expression defines an intermediate territory that marks the boundary between the telencephalon and the hypothalamus, the POH boundary. 3) The dorsalmost hypothalamic domain is the SPV that is defined by the vz/svz Otp and vz Pax6 expressions and by the lack of Nkx2.1/Isll expressions and includes the catecholaminergic paraventricular nucleus. 4) The caudal boundary of the SPY is highlighted by the Pax6/Isll expression in the prethalamus and its dorsal boundary is marked by the Nkx2.1/Isll expression in the PO. 5) A subdivision o f SPV can be considered since the rostral portion is rich in Otp and Nkx2.2 in the vz and svz, whereas the caudal portion only showed Otp. 6) Ventral to the SPV, the SC is identified by its catecholaminergic groups and the lack of Otp and can be further divided into a rostral portion, rich in Nkx2.1 and Nkx2.2, and a caudal portion, rich in Isll and devoid o f Nkx2.1 expression. 7) The tuberal hypothalamus is defined by the expressions of Nkx2.1 and Isll and is subdivided into a rostral portion rich in Otp expression in the vz/svz and a caudal portion lacking Otp. 8) The caudal boundary of the tuberal hypothalamus is evident by the lack o f Isll in the adjacent mammillary area, which expresses Nkx2.1 and Otp. Finally, 10) the mammillary area is defined by Nkx2.1 expression in the vz/svz and Otp expression in the svz, wheras Nkx2.2 is only expressed in its dorsal portion. Hypothalamic subdivisions; comparative aspects Most data about the organization o f the hypothalamus have been obtained in mice, and in particular developmental data. Hypothalamic neurogenesis occurs between embryonic day 10 (EIO) and E l6, with individual hypothalamic nuclei typically being generated in an outside-inside sequence (Ifft, 1972; Altman and Bayer, 1986). Apparently, a lateromedial gradient exists along the whole hypothalamus, and possibly a dorsoventral gradient (Ifft, 1972; Wyss and Sripanidkulchai, 1985; Caqueret et al., 2005; 2006; McClellan et al., 2006; 2008), and this neurogenetic activity is still observed in the adult (Kokoeva et al., 2005). In the chick, two phases in the neurogenesis o f the hypothalamus have been defined. First, there is a phase with intense radial migrations in which a clonal expansion of the ventricle occurs by proliferative activity. Subsequently, a second phase of development is characterized by tangential migratory movements through which the cells occupy their final destinations, forming nuclei and finally synthesizing the neurotransmitters that are used to communicate with other cells, once the hypothalamus is developed (Caqueret et al., 2005). There are not data about the hypothalamic neurogenesis in reptiles, however, given that it appears very conserved in the evolution of amniotes, and that the genoarchitecture is also very conserved (see below), it seems reasonable to think that the neurogenetic process could be similar. In the case of the developing hypothalamus in the turtle, the nuclear organization is simpler than described in other amniotes, especially in mammals. Close relationship of the hypothalamic populations and their proliferative ventricular origin can be established (present results). This latter situation resembles that observed in amphibians in which the hypothalamic populations are in close relation to the ventricle (Moreno et al., 2008b; Dominguez et al., 2010; 2011). As previously mentioned, recent data on the genetic specification of the mouse hypothalamus has led to a change in the interpretation of this brain region. Thus, it was early recognized that Shh is essential to specify the hypothalamus (Chiang et al., 1996), specially the lateral hypothalamus (Szabo et al., 2009). In addition, based on the Siml/Nkx2.I expressions, an anterodorsal Siml expressing hypothalamic neuroepithelium (containing the primordium of the paraventricular nucleus) and a postero ventral Nkx2.1-positive hypothalamic neuroepithelium (containing the primordia o f the ventromedial and arcuate nuclei, and the premammillary and mammillary neuroepithelia) were described (Shimogori et al., 2010). Furôiermore, in a recent study (Diez-Roux et al., 2011) it has been reported that genes mainly expressed in the diencephalic alar plate and/or in the telencephalon are also expressed into the tuberomammillary hypothalamus and/or anterior hypothalamic and suprachiasmatic nucleus (12 genes were identified with 158 1 JL a • M-J Æ -J± l 1 ^ ■ V V ^ L_y X X'VX^ x X XX^XJk_7 A. Turtle hypothalam ic combinatorial ex p ress ion IsM Pax6 ■ Nkx2.2 g Otp ■ Nkx2.1 U Pa%7 ■ Tbrl d| c| b f a | PThE POH S C r S C O s p V ® P ' ' " B. Com parison of hypothalam ic gene ex p re ss io n patterns in am nio tes M am m als Birds Turtle PO Nkx2.1 47 Dlx5 ̂* Nkx2.1 01x5^®* Shh2* IsM ®* Nkx2.1 Isll POH Nkx2.2 4 Dlx5 Pax6 4* (- Nkx2.1 4) Nkx2.2 2 01x5 >̂3 (- Nkx2.1 ®) Nkx2.2 (- Nkx2.1 ) SPV Otp ‘' ’® Pax6 4 (-01x5 1>®*) (- Nkx2.1 4.6) Otp ̂ Pax6« (-01x5 1*) (- Nkx2.1 3) (-Isll ®*) Otp Pax6 (- Nkx2.1 ) (-Isll ) SC 01x5 ’̂4,6,7* (- Nkx2.1 01x5 i'3.7* Nkx2.1 3.5* Nkx2.2 3* IsM ®* (-Shh 3*) Nkx2.1 Nkx2.2 Isll Tub Nkx2.1 ®'7 01x5 Otp® Nkx2.1 7 01x5 ̂ Otp ^ Nkx2.1 IsM* Otp Ma Nkx2.1 Otp Nkx2.1 Otp Nkx2.1 Otp 159 3. EL H IF U 1A LA M U EN LA 1KANSILIUN ANAM NIO- AMNIOTA: ESTUDIOS EN ANUROS Y REPTILES Figure 13. A. Schematic representations of a sagittal view of the forebrain of Pseudemys scripta summarizing the combinatorial code of transcription factors used in the present study, following the color code indicated in the box. The levels of the coronal sections drawn in the figure 12 are indicated (a-d). The combinatorial expression in the different histogenetic domains has been illustrated in the frame or in the center to simplify the scheme (as in Figure 12). B. Comparison of the different gene expression patterns detected in amniotes. * Indicates the lack of information in the literature for one or two of the amniote groups. + Indicates actual differences in the expression patterns among the three groups. Given the lack of detailed information in the literature about the expression patterns in distinct subregions as those established in our study, only the complete histogenetic domains are compared in this table. The indicated main reports considered to make this table are: 1: Bardet et al., 2008. 2: Bardet et al., 2006. 3: Bardet et al., 2010. 4: Flames et al., 2007. 5: Abellân and Medina, 2009. 6: Morales-Delgado et al., 2011.7: Medina, 2008. this pattern: Lhx2, Lhx6, Lhx9, D lxl, Dlx2, Dlx5, Unc4, Cited, Rorb, Arx, Foxa2, and Otx2), giving to these regions an alar plate character. In contrast, genes mainly expressed in the basal plate domains o f the diencephalon, including the hypothalamus, were exclusively expressed in the caudal hypothalamic regions: mammillary and retromammillary areas (13 genes were identified with this pattern: Foxal, M xla, Lm xlb, Barhll, Dbxl, Pax7, 01ig2, Rarb, Dfp3, Lhxl, Lhx5, Irx l, and Irx3). Most interestingly, this new interpretation identifies primary sensorial hypothalamic areas as alar plate derivatives and this agrees with the hypothesis o f “ functional columns” in the vertebrate brain, where sensorial information is primarily processed by alar derivatives (reviewed in Nieuwenhuys, 1999). In the following sections, the molecular data obtained in the turtle are discussed separately for each of the main regions analyzed in our study. Preoptic area. The PO has been recently analyzed in a study about the subpallium o f the turtle Pseudemys scripta (Moreno et al., 2010) because current data considering its topological position in the neural plate and its genetic specification support the PO as a distinct telencephalic region (Flames et al., 2007; Garcia-Lôpez et al., 2008; Sânchez-Arrones et al., 2009). However, we have included information about the PO for two main reasons, first because in most previous studies has the PO been considered as part o f the hypothalamus (Butler and Hodos, 2005) and second because it constitutes the dorsal border of the hypothalamus and their boundaries are not cytoarchitectonically clear. The combination of the expressions of Tbrl and Pax6 with Nkx2.1 in the PO allows the identification of the boundary between the telencephalic PO, rich in vz Nkx2.I expression, and the PThE, rich in Pax6 and Tbrl expression in the vz and svz, respectively. In mammals, the embryonic PO is defined anatomically as the region immediately in front of the preoptic recess of the third ventricle, just between the caudal hemispheres and the diencephalon, as part o f the non-evaginated telencephalon (reviewed in Moreno et al., 2009; Moreno and Gonzalez, 2011). In contrast to the hypothalamic regions, it expresses Foxgl, a conserved transcriptional repressor that plays a key role in the specification, proliferation and differentiation of the telencephalon (Zhao et al., 2009; Manuel et al., 2010; Roth et al., 2010). It is adjacent to the medial ganglionic eminence and, like it, expresses Nkx2.I (Flames et al., 2007) and Shh, Nkx5.1 and D bxl, but does not express Siml or Nkx6.2 (Gelman et al., 2009). Most o f these molecular expressions resemble those found in other amniotes like the chicken (Bardet et al., 2010) and turtle (Moreno et al., 2010; present results), but also in the amphibian Xenopus (Moreno et al., 2008b; Dominguez et al., 2010, 2011). Therefore, the specification of this region seems to have been conserved, at least, in tetrapods. Preoptohypothalamic boundary. This particular region has been recognized as a narrow territory that expresses Nkx2.2 between the telencephalic vz expressing Nkx2.1 and the hypothalamic vz expressing Otp. In other amniotes has a similar boundary been defined, just adjacent to the subpallium that was identified as the POH. Thus, in mammals, the POH is identified as the outermost structure o f the Dlx2- positive telencephalic stalk (Flames et al., 2007). From a molecular perspective, the POH vz contains progenitor cells that express Dlx2, Nkx2.2, Pax6, 01ig2, and Gsh2, and lack expression of Nkx2.1, Shh, Nkx6.2, and Dbxl (Flames et al., 2007). In the chick, the POH has been defined as a thin territory separating longitudinally the PO from the magnocellular hypothalamus (Bardet et al., 2006). Thus, with the current data it seems that this is a conserved region in amniotes (present results), since there are no data about this region in anamiotes. Interestingly, in Xenopus Nkx2.2 expressing cells have been described in the most anterior border of the Otp hypothalamic territory but a counterpart o f the POH of amniotes could not be distinguished (Dominguez et al., 2011). Supraoptoparaventricular area. The dorsal hypothalamic domain is defined by the expressions of Otp, Siml, and Pax6 and the lack of Nkx2.I/DIx3 (reviewed in Markakis, 2002; Medina, 2008 and Moreno and Gonzalez, 2011). In mammals, this subdomain produces the paraventricular, supraoptic and anterior hypothalamic nuclei, and for that is currently called supraoptoparaventricular region (SPV). The terms optoeminential band and optopeduncular were previously used by Rubenstein and Puelles (1993) in mammals, and later also in Xenopus (Brox et al., 2003) to refer the Dix negative alar hypothalamic territory, the current SPV. This new term was later coined (see Medina, 2008) because, at least in mammals, it includes the supraoptic the paraventricular nuclei. In the turtle, this pattern is conserved (present results), and the SPV is directly continued caudally by the prethalamus, which expresses Pax6/Isll intensively. In the turtle SPV, the Nkx2.2 expression allows the 1 6 0 J . ILL t l i r U l A L A i V i U ILi>l L A IK A T N a iL lU rN Ai>l A iV li'N iU - AMNIOTA: ESTUDIOS EN ANUROS Y REPTILES identification of two regions, a rostral region rich in Otp and Nkx2.2 vz and svz expressions and a caudal region, which in the svz only expresses Otp (see Fig. 12). The SPV in the mouse has also been divided into two portions called rostral paraventricular and caudal paraventricular areas, respectively, based on the Otp expression (Morales- Delgado et al., 2011). In mammals, the SPV is considered the neuroendocrine hypothalamic region, which integrates central and peripheral signals to modulate secretion by the pituitary of peptidic hormones involved in homeostasis (Markakis, 2002). The paraventricular and supraoptic nuclei o f the hypothalamus contain five major types of magnocelluar and parvocellular neurons secreting oxytocin, vasopressin, CRH, somatostatin, and TRH (Caqueret et al., 2005). The transcription factors Siml, Amt2, and Otp, are coexpressed in this anterior hypothalamic portion, and are essential for terminal differentiation of the neurosecretory neurons (Michaud et al., 1998, 2000, Hosoya et al., 2001, Acampora et al., 1999; Wang and Lufkin, 2000). One of their common downstream genes, Bm2, is necessary for the differentiation o f the cells producing oxytocin, vasopressin, and CRH, whereas Sim2, a paralog of Siml, contributes to the expression of TRH and somatostatin genes (Caqueret et al., 2006). Inactivation of any o f these genes results in the elimination of the paraventricular and supraoptic structures and neuroendocrine hormone expression (reviewed in Markakis, 2002). In particular, Otp was shown to participate in cell proliferation and migration, whereas the other genes were suggested to participate in the differentiation program leading to hormone gene expression (Caqueret et al., 2006). The homeodomain transcription factor Otp is distinctly expressed in the SPV in mice, chicks, turtle, amphibians, zebrafish and lungfishes (Acampora et al., 1999; Del Giacco et al., 2006; Bardet et al., 2008; Gonzalez and Northcutt, 2009; Dominguez et al., 2010; Moreno et al., 2010; reviewed in Moreno and Gonzalez, 2011), allowing the SPV to be distinguished from the adjacent domains expressing Shh, Nkx2.1, and Dix (Bardet et al., 2008; Dominguez et al., 2010; Moreno et al., 2010). Other shared feature by the SPV o f amniotes is the Pax6 vz expression (Medina, 2008; present results). In contrast, this hypothalamic region in Xenopus does not include Pax6 expressing cells (Moreno et al., 2008a; Dominguez et al., 2010; 2011), which seems to be a shared characteristic feature of anamniotes because both in lampreys and in zebrafish the hypothalamic distribution of Otp seems similar (Del Giacco et al., 2006; Joly et al., 2007), but Pax6 is not expressed in the hypothalamus (Murakami et al., 2001). Thus, in the evolution o f this area the Pax6 expression seems a difference between amniotes and anamniotes and could be related to the specific size and functionality of this area and the implication o f Pax6 in the dorsoventral brain organization as has been demonstrated in the telencephalon (Torreson et al., 2000). In the reptilian region of the SPV, different cell groups producing many neuropeptides have been described, such as arginine vasotocin, FRMFa, CRH, galanin, TRH, and orexins (Smeets et al., 1990; Propper et al., 1992; D'Aniello et al., 1999; Lôpez et al., 2008; Munoz et al., 2008; Dominguez et al., 2009). AU these data about the neuropeptidic cell populations, along with the present results, suggest that in the turtle this region also constitutes the neuroendocrine hypothalamus, which is very conserved in amniotes. Furthermore, recent studies in the zebrafish have mapped the expression o f the ortholog genes of many known hypothalamic peptides (including OXT, A VP, CRH, TRH and SST) and demonstrated that Siml and Otp are core components of a conserved transcriptional network, which specifies neuroendocrine in the fish hypothalamus and posterior tuberculum (Lohr et al., 2009). In addition, it has been recently described a link between a conserved molecular address (Nk2.1+, otp+) and a conserved cell type (vasotocinergic extraocular photoreceptors), proposing that the vertebrate hypothalamus and the insect pars intercerebralis trace back to a simple brain with neurosecretory cells that existed in the common bilaterian ancestors (De Velasco et al., 2007; Hartenstein, 2006). This kind of evidences suggest that sensory-neurosecretory cell types have been present at the starting point of bilaterian brain evolution, extending the “ protoneuron concept” (Vigh et a l, 2004), which had postulated that surface-contacting sensory-neurosecretory neurons are ancestral for deuterostomes (Tessmar-Raible, 2007; Tessmar-Raible et a l, 2007). The hypothalamus in mice is generated in an “outside-in” fashion, with neurons bom earlier occupying a more lateral position in the mantle zone (Altman and Bayer, 1978; Markakis, 2002) and the developing hypothalamic region can be divided into two main domains along the mediolateral axis o f the mantle layer. The medial domain, identifiable by the coexpression o f Siml and Bm2 at E l2.5, gives rise to the paraventricular and supraoptic nuclei, whereas the lateral domain, defined by the expression of Rgs4, gives rise to a heterogeneous population of neurons (Michaud et a l , 1998; Goshu et a l , 2004). In addition to this radial development, tangential migratory pathways have also been described in the hypothalamus. In particular, a portion of the amygdaloid complex has been described to arise from extratelencephalic regions (Soma et a l, 2009; Bupesh et a l, 2011), likely from hypothalamic regions (Garcia- Lôpez et a l , 2008; Bupesh et a l, 2011) that express Otp (Garcfa-Moreno et a l, 2010). In the chick, lineage studies with retroviral markers have also revealed this two phases of cell migration in the developing hypothalamus (Amold- Aldea and Cepko, 1996), related to radial migration leading to laminar organization and tangential cell migration resulting in the final nuclear organization (Caqueret et a l , 2005). It is interesting to note that not only in the turtle similar migratory movements have been described (Métin et a l , 2007) but also in amphibians (for review see Moreno et a l , 2009). In particular, it is likely that the Otp expressing cells described in the medial amygdala of the turtle (Moreno 161 3. EL H lP U l A LA M O EN LA TRAJNSICTON AJNAMJNIO- AMNIOTA: ESTUDIOS EN ANUROS Y REPTILES et al., 2010) have an extratelencephalic origin in the SPV (present results; see Figure 12b) and likely reach their final telencephalic location through tangential migration at earlier developmental stages to those analyzed here (data not shown), comparable to the situation described in mammals (Soma et al., 2009; Garcia-Moreno et al., 2010). In addition, it can also be postulated, based on the pattern o f Otp expression observed, that cells from the SPV expanded laterally and dorsally reaching the SC (present results). Suprachiasmatic area. In the turtle, ventral to the SPV the suprachiasmatic region (SC) is identified by its catecholaminergic cell groups (Lopez et al., 2008; present results), and is caudally continuous with the PTh. The SC is divided into a rostral portion rich in Nkx2.I and a caudal portion rich in Isll, both lacking Otp (present results). In all vertebrates examined, the SC was demonstrated to be a Dlx-positive territory (Bachy et al., 2002; Brox et al., 2003; Flames et al., 2007; Dominguez et al., 2010; Martinez de la Torre et al., 2011). In contrast, the expression of Nkx2.1 in the SC has been described only in non-mammalian vertebrates (Rorh et al., 2001; Bachy et al., 2002; Moreno et al., 2008a; van den Akker et al., 2008; Abellân and Medina, 2009; Dominguez et al., 2010; Moreno et al., 2010). In fact, in the Nkx2.1 knockout mice this hypothalamic zone is almost unaffected (Marin et al., 2002), whereas it is very reduced in Nkx2.1 morphant Xenopus (van der Akker et al., 2008). The pattern of Nkx2.1 expression in the SC of the turtle seems closer to that o f anamniotes than to that of mammals. However, only a subdivision of the SC expresses Nkx2.1 in contrast to zebrafish (Rohr et al., 2001) and Xenopus (Gonzalez et al., 2002a,b; Moreno et al., 2008b) and it might indicate that the Nkx2.1 expression is gradually reduced during evolution, specially at the anamniote-amniote transition. Therefore, summarizing the SPV, rich in Otp, dorsally limits with the PO (present results) and both are separated by the POH in amniotes. Ventral to the SPV is located the SC that expressed Dlx/Isll genes and lacks Otp. The lack of Otp was the first evidence to identify the boundary between the SPV-SC. The Isll expression in the SCc defined it as non-SPV region, and the lack of Pax6 svz expressing cells distinguished it as non-PTh. The SCr is ventrally limiting with the Tub, being distinct by the Nkx2.2 expression and the lack of Isll, which is characteristic of the Tub regions (see Figures 12 and 13). Tuberal hypothalamus. It constitutes the most ventral portion o f the hypothalamus, defined in all vertebrates by its Shh/Nkx2.1 expression (reviewed in Medina, 2008; Moreno and Gonzalez, 2011). In addition, in the mouse a notable novel compartment, termed the intra­ hypothalamic diagonal, has been recently described and consists o f a zone o f Arx-positive, Gad67-positive cells that separates the anterodorsal and posteroventral portions of the hypothalamus and expresses multiple genes of the Lhx family and may give rise to the bulk of hypothalamic intemeurons (Shimogori et al., 2010). The mammalian tuberal hypothalamus contains different nuclei, such us the ventromedial and arcuate nuclei, which are bilateral cell groups at the base of the hypothalamus that are organized through the aggregation o f neurons bom along the third ventricle that migrate laterally (Altman and Bayer, 1986). The ventromedial nucleus o f the hypothalamus in mice, develops under the specification of Nkx2.1 and SF-I, appears as a bilateral cell group at the base of the diencephalon around embryonic day 16/17, and follows an outside-in developing pattern (McClellan et al., 2006). Islet-1 is another transcription factor localized in this nucleus important in its development (Davis et al., 2004). G ABA is also likely to be involved in determining the boundaries of the nucleus by influencing the movement characteristics of migrating neurons (McClellan et al., 2006). In turn, Nkx2.1+ cells are broadly distributed in the arcuate nucleus and synthesize many neurotransmitters such as G ABA, NPY, POMC, and dopamine, whereas Dlx+ cells mark only GABAergic and dopaminergic neurons (Yee et al., 2009). In addition, the arcuate nucleus characteristically expresses the Otp transcription factor (Acampora et al., 1999; Bardet et al., 2008). In the case of the turtle, the tuberal hypothalamus shows gene expression pattern largely similar to those of mammals (present results), but also common to birds, in which the ventral tubero-mammillary cells in chick embryogenesis arise from a set o f floor plate-like precursors that initially express Shh (Manning et al., 2006). Interestingly, in the anamniote Xenopus the Nkx2 1 /Shh/D Ix/Isl 1 expression resulted largely comparable to that observed in the tuberal hypothalamus of amniotes (Brox et al., 2003; Moreno et al., 2008b; Dominguez et al., 2010). Of note, in turtle the tuberal hypothalamus expresses Nkx2.1/Isll, but only the rostral portion, which likely gives rise to the arcuate nucleus, expresses Otp and this situation is similar to that described in mammals (Morales-Delgado et al., 2011). Mammillary region. Studies o f this region have reported different results in terms of its subdivisions and organization, and the terminology used was also different. On the basis o f fate maps analysis in chicken (Garcfa-Lopez et al., 2009), it was described that the basal plate of p3 generates the retromammillary tegmentum and the subthalamic nucleus, which then migrates ventrodorsally close to the telencephalic peduncle. In a genomic atlas o f mouse hypothalamic development, Shimogori and collaborators described in the posteroventral hypothalamic region the supramammillary, mammillary, tuberomammillary, and premammillary zones (Shimogori et al., 2010). In addition, four distinct regions have been recently considered (Puelles in; Allen Brain Atlas; 2011. http://developingmouse.brain-map.org): perimammillary and mammillary region in the terminal hypothalamus and periretromammillary and retromammillary regions in the peduncular hypothalamus. Independently o f their origin and components, the mammillary pouch is formed just rostral to the postulated boundary between the so-called 1 6 2 http://developingmouse.brain-map.org j . ILL tlirU lA L A JV lU LN LA 1 K A N aiLlU N ATNAIVINIU- AMNIOTA: ESTUDIOS EN ANUROS Y REPTILES epichordal (diencephalic) and prechordal (hypothalamic) territories o f the forebrain (Puelles, 2001; Garcia-Calero et al., 2008). These areas are assumed to receive vertical inductive signals from the notochord and the prechordal plate, respectively, due to their close topographic relationship (Puelles and Rubenstein, 1993; 2003). In terms o f genetic specification, Nkx2.1 and Shh have partly complementary patterns, Nkx2.1 being expressed in the mammillary body but not in the retromammillary area, whereas the opposite is true for Shh (Morales-Delgado et al., 2011). In mammals, the retromammillary area, is a well known caudoventral hypothalamic specialization located between the prethalamic tegmentum (basal p3) and the tuberal hypothalamic area (Puelles and Rubenstein, 2003). Shh expression is present initially in the entire ventral forebrain (basal and floor plates), but secondarily becomes downregulated in part o f the ventral hypothalamus, including the mammillary primordium but not the retromammillary area (Crossley et al., 2001; Manning et al., 2006; Shimamura et al., 1995). In the turtle, the caudal boundary of the tuberal hypothalamus is evident by the lack o f Isll in the adjacent mammillary area, rich in Nkx2.1 and Otp. In addition, the combination of Nkx2.I, Otp and Isll allowed the identification o f a distinct portion (premammillary) between the tuberal hypothalamus (Nkx2.1/Isll) and the proper mammillary region (Otp+/Isll-) in which only Nkx2.1 is expressed (present results). Furthermore, the Nkx2.2 expression also defines a distinct dorsal portion within the mammillary region. In the hypothalamus and posterior tuberculum of the mouse, it has been demonstrated that development of specific groups of dopaminergic neurons is dependent on the Otp function (Blechman et al., 2007; Del Giacco et al., 2006; Ryu et al., 2007; Lohr et al., 2009). In the case of the turtle, the Otp expression found in the dopaminergic cells o f the posterior hypothalamus would suggest a similar functional implication. As regard the nuclei formation in the mammillary region, it was described in mice that a subpopulation of progenitor cells in the diencephalic basal plate and posterior hypothalamus appears to generate glutamatergic neurons in the subthalamic nucleus and GABAergic neurons in the mammillary and retromammillary derivatives (Delaunay et al., 2009). The subthalamic nucleus is an almond-shaped structure that occupies the lateral wall o f the rodent “ventral thalamus” (Altman and Bayer, 1986; Marchand, 1987), and it provides an important linkage between the direct and indirect output pathways o f the basal ganglia (Albin et al., 1989; Gerfen, 1992). In rat, neurons of the subthalamic nucleus exit the cell cycle at El 2 .5-E l 5.5 in the medial mammillary recess and migrate to occupy the subthalamic nucleus (Altman and Bayer, 1986; Marchand, 1987). It is a glutamatergic diencephalic cell group that requires PITX2 for its normal development (Martin et al., 2004). In birds, it was postulated that the nucleus of the ansa lenticularis constitutes the counterpart o f the subthalamic nucleus (Jiao et al., 2000). It is a glutamatergic nucleus that belongs to the basal ganglia circuits and develops within the mammillary hypothalamic area and migrates to a position adjacent to the cerebral peduncle. In the case of reptiles, it was postulated that the anterior entopeduncular nucleus (Brauth and Kitt, 1980; Brauth, 1988) could be the reptilian counterpart o f the mammalian subthalamic nucleus, due to its hodology and topographical position at the border of the hypothalamus and its glutamatergic nature (Puelles and Medina, 1994). The present results are based on late developmental stages and juveniles and do not allow the identification of this nucleus in the turtle hypothalamus, but it is clear that the genetic pathways that specify this region are very conserved in the evolution. LITER A TU R E CITED Abelian A, Medina L. 2009. Subdivisions and derivatives of the chicken subpallium based on expression of LIM and other regulatory genes and markers of neuron subpopulations during development. J Comp Neurol 515:465-501. Acampora D, Avantaggiato V, Tuorto F, Barone P, Perera M, Choo D, Wu D, Corte G, Simeone A. 1999. Differential transcriptional control as the major molecular event in generating Otxl-/- and Otx2-/- divergent phenotypes. Development 126:1417-1426. Albin RL, Young AB, Penney JB. 1989. The functional anatomy of basal ganglia disorders. Trends Neurosci 12:366-375. Altman J, Bayer SA. 1978. Development of the diencephalon in the rat. II. Correlation of the embryonic development of the hypothalamus with the time of origin of its neurons. J Comp Neurol 182:973-993. Altman J, Bayer SA. 1986. The development o f the rat hypothalamus. Adv Anat Embryol Cell Biol 100:1- 178. Arnold-Aldea SA, Cepko CL. 1996. Dispersion patterns o f clonally related cells during development o f the hypothalamus. Dev Biol 173:148-161. Bachy I, Failli V, Rétaux S. 2002. A LIM- homeodomain code for development and evolution o f forebrain connectivity. Neuroreport 13:23-27. Bachy I, Vernier P, Rétaux S. 2001. The LIM- homeodomain gene family in the developing Xenopus brain: conservation and divergences with the mouse related to the evolution of the forebrain. J Neurosci 21:7620-7629. Bardet SM, Cobos I, Puelles E, Martinez-De-La-Torre M, Puelles L. 2006. Chicken lateral septal organ and other circumventricular organs form in a striatal subdomain abutting the molecular striatopallidal border. J Comp Neurol 499:745-767. Bardet SM, Ferran JL, Sânchez-Arrones L, Puelles L. 2010. Ontogenetic expression o f sonic hedgehog in the chicken subpallium. Front Neuroanat 4. doi: 10.3389/fhana.2010.00028. 163 3. EL HIPOTALAMO EN LA 1 RANSICION ANAMNIO- AMNIOTA: ESTUDIOS EN ANUROS Y REPTILES Bardet SM, Martînez-de-la-Torre M, Northcutt RG, Rubenstein JL, Puelles L. 2008. Conserved pattern of OTP-positive cells in the paraventricular nucleus and other hypothalamic sites o f tetrapods. Brain Res Bull 75:231-235. Blechman J, Borodovsky N, Eisenberg M, Nabel-Rosen H, Grimm J, Levkowitz G. 2007. Specification of hypothalamic neurons by dual regulation of the homeodomain protein Orthopedia. Development 134:4417-4426. Brauth SE. 1988. Catecholamine neurons in the brainstem of the reptile Caiman crocodilus. J Comp Neurol 270:313-326. Brauth SE, Kitt CA. 1980. The paleostriatal system of Caiman crocodilus. J Comp Neurol 189:437-465. Brox A, Puelles L, Ferreiro B, Medina L. 2003. Expression of the genes GAD67 and Distal-less-4 in the forebrain of Xenopus laevis confirms a common pattern in tetrapods. J Comp Neurol 461:370-393. Brox A, Puelles L, Ferreiro B, Medina L. 2004. Expression of the genes Emxl, Tbrl, and Eomes (Tbr2) in the telencephalon of Xenopus laevis confirms the existence of a ventral palliai division in all tetrapods. J Comp Neurol 474:562-577. Bruce LL. 2008. Evolution of the Hypothalamus in Amniotes. In: M.D. Binder NH, editor. Enciclopedic Reference of Neuroscience: Springer-Verlag. pp 1363- 1367. Bruce LL, Neary TJ. 1995a. Afferent projections to the lateral and dorsomedial hypothalamus in a lizard, Gekko gecko. Brain Behav Evol. 46:30-42. Bruce LL, Neary TJ. 1995b. Afferent projections to the ventromedial hypothalamic nucleus in a lizard, Gekko gecko. Brain Behav Evol. 46:14-29. Bupesh M, Legaz I, Abellân A, Medina L.2011. Multiple telencephalic and extratelencephalic embryonic domains contribute neurons to the medial extended amygdala. J Comp Neurol. 519:1505-25. Butler A, Hodos W. 2005. Comparative vertebrate neuroanatomy. Sons JW, editor. New Jersey: Wiley. Cai J, Zhu Q, Zheng K, Li H, Qi Y, Cao Q, Qiu M. 2010. Co-localization of Nkx6.2 and Nkx2.2 homeodomain proteins in differentiated myelinating oligodendrocytes. Glia 58:458-468. Caqueret A, Boucher F, Michaud JL. 2006. Laminar organization of the early developing anterior hypothalamus. Dev Biol 298:95-106. Caqueret A, Coumailleau P, Michaud JL. 2005. Regionalization of the anterior hypothalamus in the chick embryo. Dev Dyn 233:652-658. Chiang C, Litingtung Y, Lee E, Young KE, Corden JL, Westphal H, Beachy PA. 1996. Cyclopia and defective axial patterning in mice lacking Sonic hedgehog gene function. Nature 383:407-413. Crossley PH, Martinez S, Ohkubo Y, Rubenstein JL. 2001. Coordinate expression of Fgf8, Otx2, Bmp4, and Shh in the rostral prosencephalon during development of the telencephalic and optic vesicles. Neuroscience 108:183-206. D’Aniello B, Pinelli C, Jadhao AG, Rastogi RK, Meyer DL. 1999. Comparative analysis o f FMRFamide- like immunoreactivity in caiman {Caiman crocodilus) and turtle (Trachemys scripta elegans) brains. Cell Tissue Res 298:549-559. Davis AM, Seney ML, Walker HJ, Tobet SA. 2004. Differential colocalization of Islet-1 and estrogen receptor alpha in the murine preoptic area and hypothalamus during development. Endocrinology 145:360-366. De Velasco B, Erclik T, Shy D, Sclafani J, Lipshitz H, Mclnnes R, Hartenstein V. 2007. Specification and development o f the pars intercerebralis and pars lateralis, neuroendocrine command centers in the Drosophila brain. Dev Biol 302:309-323. Del Giacco L, Sordino P, Pistocchi A, Andreakis N, Tarallo R, Di Benedetto B, Cotelli F. 2006. Differential regulation o f the zebrafish orthopedia 1 gene during fate determination o f diencephalic neurons. BMC Dev Biol 6:50. Delaunay D, Heydon K, Miguez A, Schwab M, Nave KA, Thomas JL, Spassky N, Martinez S, Zalc B. 2009. Genetic tracing of subpopulation neurons in the prethalamus o f mice {Mus musculus). J Comp Neurol 512:74-83. Diez-Roux G, Banfi S, Sultan M, Geffers L, Anand S, Rozado D, Magen A, Canidio E, Pagani M, Peluso I, Lin-Marq N, Koch M, Bilio M, Cantiello I, Verde R, De Masi C, Bianchi SA, Cicchini J, Perroud E, Mehmeti S, Dagand E, Schrinner S, Numberger A, Schmidt K, Metz K, Zwingmann C, Brieske N, Springer C, Hemândez AM, Herzog S, Grabbe F, Sieverding C, Fischer B, Schrader K, Brockmeyer M, Dettmer S, Helbig C, Alunni V, Battaini MA, Mura C, Henrichsen CN, Garcia-Lopez R, Echevarria D, Puelles E, Garcia-Calero E, Kruse S, Uhr M, Kauck C, Feng G, Milyaev N, Ong CK, Kumar L, Lam M, Semple CA, Gyenesei A, Mundlos S, Radelof U, Lehrach H, Sarmientos P, Reymond A, Davidson DR, Dolle P, Antonarakis SE, Yaspo ML, Martinez S, Baldock RA, Eichele G, Ballabio A. 2011. A high-resolution anatomical atlas of the transcriptome in the mouse embryo. PLoS Biol 9 :el000582. Dominguez L, Gonzâlez A, Moreno N. 2010. Sonic hedgehog expression during Xenopus laevis forebrain development. Brain Res 1347:19-32. Dominguez L, Gonzâlez A, Moreno N. 2011. Ontogenetic distribution of the transcription factor Nkx2.2 in the developing forebrain of Xenopus laevis. Front Neuroanat 5:11. Doi: 10.3389/fhana.2011.00011. Dominguez L, Morona R, Joven A, Gonzâlez A, Lôpez JM. 2009. Immunohistochemical localization of orexins (hypocretinsj in the brain of reptiles and its relation to monoaminergic systems. J Chem Neuroanat 39:20-34. Femândez-Llebrez P, Perez J, Nadales AE, Cifuentes M, Grondona JM, Mancera JM, Rodriguez EM. 1988. Immunocytochemical study o f the 164 J . ILL t l i r u 1 ALAiYlU LfN L A lK A T N »lL lU r\ ANAJYINIU- AMNIOTA: ESTUDIOS EN ANUROS Y REPTILES hypothalamic magnocellular neurosecretory nuclei o f the snake Natrix maura and the turtle Mauremys caspica. Cell Tissue Res 253:435-445. Ferran JL, de Oliveira ED, Merchan P, Sandoval JE, Sanchez-Arrones L, Martinez-De-La-T orre M, Puelles L. 2009. Genoarchitectonic profile o f developing nuclear groups in the chicken pretectum. J Comp Neurol 517:405-451. Flames N, Pla R, Gelman DM, Rubenstein JL, Puelles L, Marin O. 2007. Delineation of multiple subpallial progenitor domains by the combinatorial expression of transcriptional codes. J Neurosci 27:9682-9695. Garcia-Calero E, Femandez-Garre P, Martinez S, Puelles L. 2008. Early mammillary pouch specification in the course o f prechordal ventralization of the forebrain tegmentum. Dev Biol 320:366-377. Garcia-Lôpez M, Abelian A, Legaz I, Rubenstein JL, Puelles L, Medina L. 2008. Histogenetie compartments o f the mouse centromedial and extended amygdala based on gene expression patterns during development. J Comp Neurol 506:46-74. Garcia-Lôpez R, Pombero A, Martinez S. 2009. Fate map of the chick embryo neural tube. Dev Growth Differ 51:145-165. Garcia-Moreno F, Pedraza M, Di Giovannantonio LG, Di Salvio M, Lôpez-Mascaraque L, Simeone A, De Carlos JA. 2010. A neuronal migratory pathway crossing from diencephalon to teleneephalon populates amygdala nuclei. Nat Neurosci 13:680-689. Gelman DM, Martini FJ, Nobrega-Pereira S, Pierani A, Kessaris N, Marin O. 2009. The embryonic preoptic area is a novel source o f cortical GABAergic intemeurons. J Neurosci 29:9380-9389. Gerfen CR. 1992. The neostriatal mosaie: multiple levels of compartmental organization in the basal ganglia. Annu Rev Neurosci 15:285-320. Gonzalez A, Lôpez JM, Marin O. 2002a. Expression pattern o f the homeobox protein NKX2-1 in the developing Xenopus forebrain. Brain Res Gene Expr Patterns 1:181-185. Gonzâlez A, Lôpez JM, Sânehez-Camaeho C, Marin O. 2002b. Regional expression of the homeobox gene NKX2-1 defines pallidal and intemeuronal populations in the basal ganglia of amphibians. Neuroscienee 114:567-575. Gonzâlez A, Northcutt RG. 2009. An immunohistochemical approach to lungfish telencephalic organization. Brain Behav Evol 74:43- 55. Goshu E, Jin H, Lovejoy J, Marion JF, Michaud JL, Fan CM. 2004. Sim2 contributes to neuroendocrine hormone gene expression in the anterior hypothalamus. Mol Endocrinol 18:1251-1262. Hartenstein V. 2006. The neuroendocrine system of invertebrates: a developmental and evolutionary perspective. J Endocrinol 190:555-70. Herrick CJ. 1910. The morphology of the forebrain in amphibia and reptilia. J Comp Neurol 20:413-547. Hodos W. 2008. Evolution of the Hypothalamus in Anamniotes. In: M.D. Binder NH, editor. Eneiclopedie Referenee of Neuroscience: Springer- Verlag. p 1361-1363. Hosoya T, Oda Y, Takahashi S, Morita M, Kawauchi S, Ema M, Yamamoto M, Fujii-Kuriyama Y. 2001. Defective development of secretory neurones in the hypothalamus of Amt2-knockout mice. Genes Cells 6:361-374. Ifft JD. 1972. An autoradiographic study of the time of final division o f neurons in rat hypothalamic nuclei. J Comp Neurol 144:193-204. Inagaki N, Panula P, Yamatodani A, Wada H.1990. Organization of the histaminergic system in the brain of the turtle Chinemys reevesii. J Comp Neurol.297:132-44. Jiao Y, Medina L, Veenman CL, Toledo C, Puelles L, Reiner A. 2000. Identification of the anterior nucleus of the ansa lenticularis in birds as the homolog of the mammalian subthalamic nucleus. J Neurosci 20:6998-7010. Jimenez AJ, Mancera JM, Pérez-Figares JM, Femândez-Llébrez P. 1994. Distribution of galanin- like immunoreactivity in the brain of the turtle Mauremys caspica. J Comp Neurol 349:73-84. Joly JS, Osorio J, Alunni A, Auger H, Kano S, Retaux S. 2007. Windows of the brain: towards a developmental biology of eircumventricular and other neurohemal organs. Semin Cell Dev Biol 18:512-524. Kawakami A, Kimura-Kawakami M, Nomura T, Fujisawa H. 1997. Distributions of PAX6 and PAX7 proteins suggest their involvement in both early and late phases o f chiek brain development. Mech Dev 66:119-130. Kokoeva MV, Yin H, Flier JS. 2005. Neurogenesis in the hypothalamus o f adult mice: potential role in energy balance. Science 310:679-683. Lanuza E, Font C, Martfnez-Marcos A, Martinez- Garcia F. 1997. Amygdalo-hypothalamic projections in the lizard Podarcis hispanica: a combined anterograde and retrograde tracing study. J Comp Neurol. 38:537-55. Lohr H, Ryu S, Driever W. 2009. Zebrafish diencephalic A ll-related dopaminergic neurons share a conserved transcriptional network with neuroendocrine cell lineages. Development 136:1007-1017. Lôpez JM, Dominguez L, Gonzâlez A. 2008. Immunohistochemical localization of thyrotropin- releasing hormone in the brain of reptiles. J Chem Neuroanat 36:251-263. Manning L, Ohyama K, Saeger B, Hatano O, Wilson SA, Logan M, Placzek M. 2006. Regional morphogenesis in the hypothalamus: a BMP-Tbx2 pathway coordinates fate and proliferation through Shh downregulation. Dev Cell 11:873-885. Manuel MN, Martynoga B, Molinek MD, Quinn JC, Kroemmer C, Mason JO, Price DJ. 2010. The transcription factor Foxgl regulates telencephalic progenitor proliferation cell autonomously, in part 165 3. EL H lF U l ALA M O EN LA IK A N SILIU N A N A M N lü- AMNIOTA: ESTUDIOS EN ANUROS Y REPTILES by controlling Pax6 expression levels. Neural Dev 6:9. Marchand R. 1987. Histogenesis o f the subthalamic nucleus. Neuroscience 21:183-195. Marin O, Baker J, Puelles L, Rubenstein JL. 2002. Patterning of the basal telencephalon and hypothalamus is essential for guidance of cortical projections. Development 129:761-773. Markakis EA. 2002. Development o f the neuroendocrine hypothalamus. Front Neuroendocrinol 23:257-291. Martin DM, Skidmore JM, Philips ST, Vieira C, Gage PJ, Condie BG, Raphael Y, Martinez S, Camper SA. 2004. PITX2 is required for normal development of neurons in the mouse subthalamic nucleus and midbrain. Dev Biol 267:93-108. Martinez-de-la-Torre M, Pombal MA, Puelles L. 2011. Distal-less-like protein distribution in the larval lamprey forebrain. Neuroscience 178:270-284. McClellan KM, Calver AR, Tobet SA. 2008. G ABA B receptors role in cell migration and positioning within the ventromedial nucleus of the hypothalamus. Neuroscience 151:1119-1131. McClellan KM, Parker KL, Tobet S. 2006. Development of the ventromedial nucleus of the hypothalamus. Front Neuroendocrinol 27:193-209. Medina L. 2008. Evolution and Embryological Development of Forebrain. In: M.D. Binder NH, editor. Eneiclopedie Reference of Neuroscience: Springer-Verlag. p 1172-1192. Métin C, Alvarez C, Moudoux D, Vitalis T, Pieau C, Molnar Z. 2007. Conserved pattern of tangential neuronal migration during forebrain development. Development 134:2815-2827. Michaud JL, DeRossi C, May NR, Holdener BC, Fan CM. 2000. ARNT2 acts as the dimerization partner of SIMl for the development of the hypothalamus. Mech Dev 90:253-261. Michaud JL, Rosenquist T, May NR, Fan CM. 1998. Development o f neuroendocrine lineages requires the bHLH-PAS transcription factor SIM l. Genes Dev 12:3264-3275. Morales-Delgado N, Merchan P, Bardet SM, Ferran JL, Puelles L, Diaz C. 2011. Topography of Somatostatin Gene Expression Relative to Molecular Progenitor Domains during Ontogeny of the Mouse Hypothalamus. Front Neuroanat 5:10. Doi: 10.3389/fhana.2011.00010. Moreno N, Bachy I, Rétaux S, Gonzalez A. 2004. LIM- homeodomain genes as developmental and adult genetic markers of Xenopus forebrain functional subdivisions. J Comp Neurol 472:52-72. Moreno N, Dominguez L, Rétaux S, Gonzalez A. 2008b. Islet 1 as a marker o f subdivisions and cell types in the developing forebrain of Xenopus. Neuroscience 154:1423-1439. Moreno N, Gonzalez A. 2011. The non-evaginated secondary prosencephalon of vertebrates. Front Neuroanat 5:12. Doi: 10.3389/fhana.2011.00012. Moreno N, Gonzâlez A, Rétaux S. 2009. Development and evolution of the subpallium. Semin Cell Dev Biol 20:735-743. Moreno N, Morona R, Lôpez JM, Gonzâlez A. 2010. Subdivisions of the turtle Pseudemys scripta subpallium based on the expression o f regulatory genes and neuronal markers. J Comp Neuol 518:4877-4902. Moreno N, Retâux S, Gonzâlez A. 2008a. Spano- temporal expression of Pax6 in Xenopus forebrxin. Brain Res 1239:92-99. Morona R, Ferran JL, Puelles L, Gonzâlez A. 2011. Embryonic genoarchitecture of the pretectum in Xenopus laevis: A conserved pattern in tetrapodj. J Comp Neurol 519:1024-1050. Morona R, Gonzâlez A. 2008. Calbindin-D28k md calretinin expression in the forebrain of anuran md urodele amphibians: further support for nevly identified subdivisions. J Comp Neurol 511:187- 220 . Munoz M, Smeets WJAJ, Lôpez JM, Moreno N, Morona R, Dominguez L, Gonzâlez A. 2008. Immunohistochemical localization o f neuropeptide FF-like in the brain of the turtle: relation to eatecholaminergie structures. Brain Res Bull 75:256-260. Murakami Y, Ogasawara M, Sugahara F, Hirano S, Satoh N, Kuratani S. 2001. Identification ind expression of the lamprey Pax6 gene: evolutiorary origin of the segmented brain of vertebrates. Development 128:3521-3531. Nieuwenhuys R. 1998. The Central Nervous Systen of Vertebrates. Nieuwenhuys R, ten Donkelaar HI, Nicholson C, editors. London: Springer. Nieuwenhuys R. 1999. The morphological pattern of the vertebrate brain. Eur J Morphol 37:81-84. Northcutt R. 1970. The telencephalon of the western painted turtle (Chrysemys picta bellis). Universiy of Illinois Press, Chicago. Osorio J, Mazan S, Rétaux S. 2005. Organisation of the lamprey {Lampetra Jluviatilis) embryonic bnin: insights from LIM-homeodomain, Pax ind hedgehog genes. Dev Biol 288:100-112. Osorio J, Megias M, Pombal MA, Rétaux S. 2006. Dynamic expression of the LIM-homeodomain gme Lhx 15 through larval brain development of the sea lamprey (Petromyzon marinus). Gene Expr Pattons 6:873-878. Pritz MB, Ruan YW. 2009. PAX6 immunoreactivity in the diencephalon and midbrain o f alligator duiing early development. Brain Behav Evol 73:1-15. Propper CR, Jones RE, Lôpez KH. 1992. Distributon of arginine vasotocin in the brain of the Hard Anolis carolinensis. Cell Tissue Res 267:391-398 Puelles L. 2001. Brain segmentation and forebnin development in amniotes. Brain Res Bull 55:6)5- 710. Puelles L, Kuwana E, Puelles E, Bulfone A, Shimamira K, Keleher J, Smiga S, Rubenstein JL. 2000. Palial and subpallial derivatives in the embryonic chck and mouse telencephalon, traced by the express on of the genes Dlx-2, Emx-1, Nkx-2.1, Pax-6, md Tbr-1. J Comp Neurol 424:409-438. 136 3 . L L m r U lA L A iV lL » L N L A I K A N a iL lU N A N A iV ilN lU - AMNIOTA: ESTUDIOS EN ANUROS Y REPTILES Puelles L, Medina L. 1994. Development of neurons expressing tyrosine hydroxylase and dopamine in the chicken brain: a comparative segmental analysis. In: Smeets WJAJ , Reiner A, editors. Phylogeny and development of catecholamine systems in the CNS o f vertebrates Cambridge, UK: Cambridge UP. p 381- 404. Puelles L, Rubenstein JL. 1993. Expression patterns of homeobox and other putative regulatory genes in the embryonic mouse forebrain suggest a neuromeric organization. Trends Neurosei 16:472-479. Puelles L, Rubenstein JL. 2003. Forebrain gene expression domains and the evolving prosomeric model. Trends Neurosci 26:469-476. Rohr KB, Barth KA, Varga ZM, Wilson SW. 2001. The nodal pathway acts upstream of hedgehog signaling to specify ventral telencephalic identity. Neuron 29:341- 351. Roth M, Bonev B, Lindsay J, Lea R, Panagiotaki N, Houart C, Papalopulu N. 2010. FoxGl and TLE2 act cooperatively to regulate ventral telencephalon formation. Development 137:1553-1562. Ryu S, Mahler J, Acampora D, Holzschuh J, Erhardt S, Omodei D, Simeone A, Driever W. 2007. Orthopedia homeodomain protein is essential for diencephalic dopaminergic neuron development. Curr Biol 17:873- 880. Sanchez-Arrones L, Ferran JL, Rodriguez-Gallardo L, Puelles L. 2009. Incipient forebrain boundaries traced by differential gene expression and fate mapping in the chick neural plate. Dev Biol 335:43-65. Sherwood NM, Whittier JM. 1988. Gonadotropin- releasing hormone from brains of reptiles: turtles {Pseudemys scripta) and snakes {Thamnophis sirtalis parietalis). Gen Comp Endocrinol 69:319-327. Shimamura K, Hartigan DJ, Martinez S, Puelles L, Rubenstein JL. 1995. Longitudinal organization o f the anterior neural plate and neural tube. Development 121:3923-3933. Shimogori T, Lee DA, Miranda-Angulo A, Yang Y, Wang H, Jiang L, Yoshida AC, Kataoka A, Mashiko H, Avetisyan M, Qi L, Qian J, Blackshaw S. 2010. A genomic atlas of mouse hypothalamic development. Nat Neurosci 13:767-775. Smeets WJAJ, Gonzalez A. 2000. Catecholamine systems in the brain of vertebrates: new perspectives through a comparative approach. Brain Res Brain Res Rev 33:308-379. Smeets WJAJ, Jonker AJ, Hoogland PV. 1987. Distribution of dopamine in the forebrain and midbrain of the red-eared turtle, Pseudemys scripta elegans, reinvestigated using antibodies against dopamine. Brain Behav Evol 30:121-142. Smeets WJAJ, Lopez JM, Gonzalez A. 2003. Immunohistochemical localization of DARPP-32 in the brain of the turtle, Pseudemys scripta elegans: further assessment of its relationship with dopaminergic systems in reptiles. J Chem Neuroanat 25:83-95. Smeets WJAJ, Sevensma JJ, Jonker AJ. 1990. Comparative analysis o f vasotocin-like immunoreactivity in the brain of the turtle Pseudemys scripta elegans and the snake Python regius. Brain Behav Evol 35:65-84. Smith-Femandez A, Pieau C, Repérant J, Boncinelli E, W assef M. 1998. Expression of the Emx-1 and Dlx- 1 homeobox genes define three molecularly distinct domains in the telencephalon of mouse, chick, turtle and frog embryos: implications for the evolution of telencephalic subdivisions in amniotes. Development 125:2099-2 111. Soma M, Aizawa H, Ito Y, Maekawa M, Osumi N, Nakahira E, Okamoto H, Tanaka K, Yuasa S. 2009. Development of the mouse amygdala as revealed by enhanced green fluorescent protein gene transfer by means of in utero electroporation. J Comp Neurol 513:113-128. Stoykova A, Gruss P. 1994. Roles of Pax-genes in developing and adult brain as suggested by expression patterns. J Neurosei 14(3 Pt 2): 1395- 1412. Szabo NE, Zhao T, Cankaya M, Theil T, Zhou X, Àlvarez-Bolado G. 2009. Role of neuroepithelial Sonic hedgehog in hypothalamic patterning. J Neurosei 29:6989-7002. Tessmar-Raible K. 2007. The evolution of neurosecretory centers in bilaterian forebrains: insights from protostomes. Semin Cell Dev Biol 18:492-501. Tessmar-Raible K, Raible F, Christodoulou F, Guy K, Rembold M, Hausen H, Arendt D. 2007. Conserved sensory-neurosecretory cell types in annelid and fish forebrain: insights into hypothalamus evolution. Cell 129:1389-1400. Toresson H, Potter SS, Campbell K. 2000. Genetic control o f dorsal-ventral identity in the telencephalon: opposing roles for Pax6 and Gsh2. Development 127:4361-4371. van den Akker WM, Brox A, Puelles L, Durston AJ, Medina L. 2008. Comparative functional analysis provides evidence for a crucial role for the homeobox gene Nkx2.1/Titf-1 in forebrain evolution. J Comp Neurol 506:211-223. Vigh B, Manzano e Silva MJ, Frank CL, Vineze C, Czirok SJ, Szabo A, Lukats A, Szel A. 2004. The system of cerebrospinal fluid-contacting neurons. Its supposed role in the nonsynaptic signal transmission o f the brain. Histol Histopathol 19:607-628. Wang W, Lufkin T. 2000. The murine Otp homeobox gene plays an essential role in the specification of neuronal cell lineages in the developing hypothalamus. Dev Biol 227:432-449. Wang Y, Lance VA, Nielsen PF, Conlon JM. 1999. Neuroendocrine peptides (insulin, panereatic polypeptide, neuropeptide Y, galanin, somatostatin, substance P, and neuropeptide gamma) from the desert tortoise, Gopherus agassizii. Peptides 20:713-722. 167 3. EL H IPO TA LA M O EN LA TRANSICTON ANAM NIO- AMNIOTA: ESTUDIOS EN ANUROS Y REPTILES Wyss JM, Sripanidkulchai B. 1985. An autoradiographic analysis o f the time of origin o f neurons in the hypothalamus of the cat. Brain Res 353:89-98. Yee CL, Wang Y, Anderson S, Ekker M, Rubenstein JL. 2009. Arcuate nucleus expression of NKX2.1 and DLX and lineages expressing these transcription factors in neuropeptide Y(+), proopiomelanoeortin(+), and tyrosine hydroxylase(+) neurons in neonatal and adult mice. J Comp Neurol 517:37-50. Zardoya R, Meyer A. 2001a. The evolutionary position of turtles revised. Naturwissensehaften 88:193-200. Zardoya R, Meyer A. 2001b. On the origin of and phylogenetic relationships among living amphibians. Proc Natl Acad Sci U S A 98:7380-7383. Zhao XF, Suh CS, Prat CR, Ellingsen S, Fjose A. 2009. Distinct expression o f two foxgl paralogues in zebrafish. Gene Expr Patterns 9:266-272. 168 5. Resumen de los resultados y Discusîon General Resumen de los resultados Discusion general Consideraciones metodologicas Organizacion del territorio hipotalàmico en la evoluciôn Situaciôn actual del limite Alar/Basal Hipôtesis evolutiva de la organizacion hipotalàmica: existencia de un patron comûn en la evoluciôn y repercusiones en la transiciôn anamnio- amniota Bibliogrq/la 3. KLaUlVlLN UtL L U » K L »U L 1 A U U » Y U ia L L M U N U L N L K A L Resumen de los resultados Como hemos detallado en la introducciôn, el hipotâlamo en vertebrados es una region extremadamente compleja en cuanto a su origen, estructura y regionalizaciôn, y el estudio de su organizacion en anfibios y reptiles es menor que en otros vertebrados. Por ello, en el présente trabajo de investigaciôn se ha llevado a cabo un anâlisis detallado de la organizacion hipotalàmica en base a la expresiôn de determinados genes reguladores del desarrollo y el anâlisis de su perfil neuroquimico. Si tenemos en cuenta la privilegiada posiciôn filogenética que ocupan los anfibios, dada su condiciôn de ùnicos tetrâpodos anamniotas, y los reptiles, siendo un grupo muy semejante al grupo ancestral del que derivan los mamiferos, su estudio comparado se hace extremadamente relevante a la hora de establecer posibles homologias entre anamniotas y amniotas, ya que representan un modelo claro en la transiciôn anamnio- amniota. En este sentido, los resultados del présente estudio nos aportan nuevos conocimientos acerca del patrôn bâsico de organizaciôn de la regiôn prosencefâlica en los vertebrados y su conservaciôn filogenética. En el cap itu lo 2, siguiendo estudios previos en otros modelos de la transiciôn anamnio-amniota (Lôpez et al., 2008; 2009a) se analizô la distribuciôn de la hormona liberadora de tirotropina (TRH) y de las orexinas (hipocretinas) en el cerebro adulto de très especies de anfibios urodelos, Ambystoma tigrinum, Ambystoma mexicanum y Pleurodeles waltl, y dos especies de reptiles, Pseudemys scripta elegans y Gekko gecko, respeetivamente, mediante el uso de técnicas inmunohistoquimicas. En todos los casos se encontraron numerosas estructuras inmunorreactivas dispersas a lo largo del eje rostrocaudal de la mayoria de las subdivisiones encefâlicas. En cuanto a la TRH, se observaron fibras inmunoreactivas para TRH en numerosas regiones distribuidas a lo largo de todo el sistema nervioso central (SNC) de las très especies de urodelos analizadas. Eue en regiones subpaliales e hipotalâmicas donde se observô mayor concentraciôn de fibras TRH positivas, concretamente invervando los nùcleos aecumbens, estriado, septo, amigdala, ârea preôptica, supraquiasmâtico e hipotâlamo tuberal, asi como en la eminencia media y el lôbulo neural de la hipôfisis. Las células inmunoreactivas para TRH se localizaron principalmente en regiones hipotalâmicas, aunque se observaron ciertas diferencias entre especies. En el caso de las dos especies de urodelos del género Ambystoma, se observaron células TRH positivas en la amigdala medial que, sin embargo no ftieron detectadas en regiones similares del urodelo Pleurodeles waltl, asi como una numerosa poblaciôn de células inmnuorreactivas en el ârea preôptica de las très especies analizadas. Con respecto al territorio hipotalàmico, se localizaron células positivas en el nùcleo supraquiasmâtico ùnicamente en el género Ambystoma, mientras que en el hipotâlamo tuberal se detectô un grupo celular localizado en el infimdibulo de las très especies de urodelos y cuyas células exhibfan procesos contactantes eon el liquide cefalorraquideo. También se detectaron células en otras regiones encefâlicas fuera del subpalium e hipotâlamo como en el diencéfalo y techo ôptieo en las especies del género Ambystoma. Ademâs, el anâlisis de la expresiôn de TRH junto con el de la enzima tirosina hidroXilasa (TH), mediante técnicas de doble inmunohistofiuorescencia, permitiô la observaciôn de un alto grado de codistribuciôn de ambos sistemas en la regiôn preôptica y supraquiasmâtica, aunque no se observaron células doblemente marcadas. En cuanto a la inmunodetecciôn de las orexinas en los reptiles Pseudemys scripta elegans y Gekko gecko, se observaron numerosas fibras inmunorreactivas distribuidas a lo largo de todas las subdivisiones del cerebro de ambos reptiles, aunque la regiôn hipotalàmica y la eminencia media destacaron por tener un mayor numéro de fibras positivas para orexinas. Los grupos celulares inmunorreativos mâs numerosos se localizaron en el territorio hipotalàmico de las dos especies estudiadas, aunque también se encontraron células positivas para orexina en el ârea preôptica subpalial, donde la mayoria de las eélulas se situaban periventricularmente contactando con el ventriculo. Dentro de la regiôn hipotalàmica, la ausencia de células inmunorreactivas en el nùeleo supraquiasmâtieo fue una caracteristica comûn a las dos especies de reptiles analizadas. Sin embargo, se observô un numeroso grupo celular orexinérgico en el infiindibulo y nùcleo periventricular hipotalàmico tanto en tortuga como en Gekko. Ademâs, se analizaron los patrones de distribuciôn de la enzima TH y la serotonina en conjunciôn con el de las orexinas observando codistribueiôn con células TH positivas en el hipotâlamo tuberal asi como la existencia de fibras inmunorreactivas para orexina sobre células serotoninérgicas de la columna del rate. En el c ap itu lo 3, se analizaron los patrones de expresiôn del morfôgeno Shh y del factor de transcripciôn Nkx2.2, mediante técnicas de hibridaciôn in situ e inmnohistoquimica en el prosencéfalo en desarrollo y adulto del anuro Xenopus laevis. En base al alto grado de conservaciôn de ambos genes reguladores y a que sus patrones de expresiôn recapitulan las ffonteras de ciertas subdivisiones encefâlicas, su estudio en anuros nos da una idea del origen de diferentes regiones encefâlicas en base a su organizaciôn. Es por esto que, podemos eoneluir que los patrones de expresiôn de ambos marcadores se centran en la regiôn prosencefâlica de vertebrados, destacando su preseneia en el territorio hipotalàmico principalmente basai. A nivel telencefâlico, no se encontrô expresiôn del factor de transcripciôn Nkx2.2, sin embargo, cabe destacar la expresiôn de Shh en la regiôn preôptica del telencéfalo no evaginado, concretamente en el ârea preoptocomisural (POC) y en el ârea preôptica propiamente dicha (PO). En el diencéfalo, se detectô una fiierte expresiôn de Shh en la zona limitante intratalâmica (Zli) asi como en la regiôn pretalâmica. Por el contrario, Nkx2.2 no se localizô en 171 5. RESUMEN DE LUS RESULTADOS Y DISCUSION GENERAL Zli aunque si se encontraron células Nkx2.2 positivas en la zona ventricular (vz) y subventricular (svz) del tâlamo, pretâlamo y pretecho. Dentro del hipotâlamo, en la region supraoptoparaventricular (SPV) del hipotâlamo alar no se detectô expresiôn de Shh mientras que si se localizaron células Nkx2.2 positivas en una porciôn de este territorio. La segunda regiôn con carâcter alar del hipotâlamo, la regiôn supraquiasmâtica (SC), se caracterizô por la expresiôn de Shh y Nkx2.2 aunque ùnicamente en un dominio ventral dentro de la regiôn supraquiasmâtica. En el hipotâlamo basai, se detectô expresiôn de Shh y Nkx2.2 el hipotâlamo tuberal y mamilar. Por otra parte, los resultados de estos estudios demostraron que las expresiones de ambos marcadores se acompanan delineando el limite alar-basal a lo largo del eje longitudinal de cerebro en anfibios. La importante preseneia de estos dos reguladores en el hipotâlamo de anfibios, trente a otras regiones proseneefâlicas, sugiere que ambos desempenan un relevante papel en el control y regulaciôn de la especificaciôn hipotalàmica y por tanto, apoya su implicaciôn en la organizaciôn hipotalàmica. Una vez analizado el perfil neuroquimico y molecular del territorio hipotalàmico identificando su complejidad, en el capitu lo 4 se procediô al estudio detallado de la regionalizaciôn hipotalàmica analizando cada uno de los subdominios que forman esta regiôn prosencefâlica en el cerebro en desarrollo e individuos adultos del anfibio Xenopus laevis y el reptil Pseudemys scripta elegans utilizando técnicas inmunohistoquimicas y de hibridaciôn in situ. En este estudio se utilizaron numerosos marcadores implicados en la organizaciôn prosencefâlica durante el desarrollo, asi como marcadores postmitôticos que ayudaron a la identificaeiôn de los derivados de dichas regiones de estudio en el cerebro adulto. También se analizaron los patrones de expresiôn moleculares de la regiôn preôptica, tradicionalmente considerada hipotalàmica, asi como los limites rostrocaudales y dorsoventrales del territorio hipotalàmico. Los resultados del estudio del hipotâlamo alar pusieron de manifesto la existencia de dos subregiones mayoritarias, el ârea supraoptoparaventricular (SPV) y la regiôn supraquiasmâtica (SC), diferenciadas claramente por una combinatoria de los patrones de expresiôn de los principales marcadores prosencefâlicos, que a su vez se encontraron divididas en diferentes subdominios. De esta forma, el SPV estâ caracterizado por la expresiôn del factor de transcripciôn Otp y se encuentra subdividido en una regiôn caudal y en otra rostral caracterizada por la expresiôn de Nkx2.2, formando dos compartimentos citogenéticos molecularmente diferenciados. La regiôn supraquiasmâtica se encuentra caracterizada por la expresiôn de los factores de transcripciôn Isll y xD114 pero el posterior anâlisis de los marcadores Nkx2.1, Nkx2.2, xShh xLhx7 y xLhxl permitieron la identificaeiôn de dos subdominios. El subdominio caudal se encuentra caracterizado por la exclusiva expresiôn de xD114 e Isll, mientras que la porciôn mâs rostral se caracteriza por la expresiôn de xShh, Nkx2.1, Nkx2.2 xLhxl y xLhx7. En el hipotâlamo basai se definieron dos subregiones principales, el hipotâlamo tuberal y mamilar gracias a la combinaciôn de los patrones de expresiôn de los marcadores prosencefâlicos utilizados. Asi, la regiôn tuberal, en la que encontramos expresiôn de xShh y Nkx2.1 y Isll, se encuentra dividida en una porciôn rostral caracterizada por la expresiôn de Otp, y otra porciôn caudal caracterizada por la expresiôn de xD114 y Nkx2.2. De la misma forma, dentro del territorio mamilar xD114 y xLhxl positivo, diferenciamos dos subregiones, el ârea retromamilar (RM) y el ârea mamilar propiamente dieha (Ma). El ârea mamilar oeupa la regiôn mâs rostral y se encuentra caracterizada por la expresiôn de Nkx2.1 y es negativa para xShh, mientras que la regiôn retromamilar expresa xShh y es negativa para Nkx2.1. Discusion general Consideraciones metodologicas En el présente estudio se han realizado técnicas inmunohistoquimicas, de hibridaciôn in situ, asi como técnicas combinadas. Con respecto a las técnicas inmunohistoquimicas, la especificidad de los anticuerpos utilizados tue rigurosamente probada en cada caso. En todos los expérimentes se realizaron contrôles en paralelo en ausencia de anticuerpo y no se observô marcaje residual. Ademâs, se realizaron contrôles incubando el tejido en preseneia de suero hecho en las especies en las que los anticuerpos secundarios fueron obtenidos. Por otra parte, se realizaron contrôles en los que se prebloquearon los anticuerpos con la proteina correspondiente que reconocen, y al usarlo en el tejido no se observô ningùn marcaje. La especificidad de los anticuerpos en las distintas especies fue también identificada mediante anâlisis de Western blot, en los que los extractos de cerebro de Xenopus y tortuga mostraron que los anticuerpos utilizados marcaban una ùnica banda, que se correspondia con la banda marcada en los extractos de cerebro de rata. De la misma forma, en todos los casos el marcaje producido por el anticuerpo era similar al obtenido en experimentos de hibridaciôn in situ, quedando demostrada la especificidad de los anticuerpos utilizados en el présente estudio. Organizaciôn del territorio hipotalàmico en la evoluciôn El territorio hipotalâmieo debe su nombre a su posiciôn topogrâfica debajo del tâlamo descrita en los estudios clâsicos que no atendian a la flexura encefâlica. La comparaciôn de este territorio ha resultado dificil a lo largo de la eseala filogenética dadas las diferencias existentes entre los distintos grupos de vertebrados en cuanto a tamaflo. 172 3. KLMJMLfN UL LU » K L »L L iA U U » Y U1»LL»1UN G EN ERA L organizaciôn, migraciones, tipos celulares, etc. Sin embargo, cada vez son mâs los estudios que abogan por la existencia de homologias entre anamniotas y amniotas en diferentes regiones hipotalâmicas y que por tanto apoyan la idea de im patrôn de organizaeiôn comûn en el hipotâlamo de vertebrados. La mayoria de los datos acerca de la organizaciôn y especificaciôn molecular del hipotâlamo han sido obtenidos en amniotas, especialmente en ratôn donde se han propuesto diferentes modelos (Figdor and Stem, 1993; Puelles and Rubenstein, 2003; Shimogori et al., 2010; Diez-Roux et al., 2011). Es por ello que, el présente estudio se ha centrado en el anâlisis de la organizaciôn hipotalàmica en el anamniota y anfibio anuro Xenopus laevis asi como en un amniota no mamifero, el reptil Pseudemys scripta elegans, pudiendo de esta forma completar la historia evolutiva del territorio hipotalàmico dentro de la escala filogenética. El estudio de ambos vertebrados ha permitido el anâlisis de la regionalizaciôn hipotalàmica en la transiciôn anamnio- amniota observando la existencia de un patrôn bâsico altamente conservado en la evoluciôn. De esta forma, en el présente estudio se han ido analizando comparativamente cada una de los caracteristicas bâsicas que el territorio hipotalàmico présenta en otros vertebrados, con el fin de discutir la posibilidad de existeneia de un patrôn de organizaeiôn eomùn en los vertebrados (Fig, 1). Asi, de manera general, los resultados obtenidos en el présente estudio y su comparaciôn con otros vertebrados nos permiten eoneluir que, a grandes rasgos, el territorio hipotalàmico comparte en todos los vertebrados analizados diferentes aspectos a tener en cuenta a la hora de establecer homologias, como son: 1 ) su situaciôn topogrâfica es rostral al diencéfalo; 2) se encuentra dividido en regiones alares y basales, a su vez subdivididas rostrocaudalmente en distintas porciones segùn su especificaciôn molecular, que en todos los casos coinciden con territorios ôpticos y neuroendocrinos; 3) présenta un patrôn quimioarquitectônico conservado, dada la preseneia de poblaciones neuronales postmitôticas, principalmente neuropeptidicas, ubicadas en regiones similares en la evoluciôn. Ademâs, todos los datos obtenidos nos han permitido eoneluir que en la transiciôn anamnio-amniota se dan caracteristicas moleculares y neuroquimicas que influyen en la organizaciôn del hipotâlamo, representando un relevante momento evolutivo. Région preôptica Actualmente, la regiôn preôptica estâ incluida en el telencéfalo impar no evaginado y ha sido anatômicamente definida durante el desarrollo como la regiôn inmediatamente en frente del receso preôptico, en el limite entre el telencéfalo y el diencéfalo (Flames et al., 2007). Sin embargo, tradicionalmente la regiôn preôptiea ha sido considerada como parte del hipotâlamo (revisado en Butler y Hodos, 2005). El avance en el conocimiento acerca de los patrones morfogenéticos del prosencéfalo permitiô la separaciôn de la regiôn preôptica del hipotâlamo y su aceptaciôn como un territorio subpalial. En concreto, el estudio de la expresiôn del gen FoxGl fiie imprescindible, ya que se sabe que dicho gen estâ implicado en la especificaciôn, proliferaciôn y diferenciaciôn del territorio telencefâlico y, con la demostraciôn de la expresiôn de FoxGl en la regiôn preôptica se afianzô su origen telencefâlico (Zhao et al., 2009; Roth et al., 2010). De esta forma, actualmente se considéra el ârea preôptica como parte del telencéfalo no evaginado dada su posiciôn topolôgica en la plaça neural, desvelada gracias a los experimentos de destino celular, y dada su especificaciôn genética (Flames et al., 2007; Garcia-Lôpez et al., 2009; Sânchez-Arrones et al., 2009). En el présente estudio se ha considerado el anâlisis de la regiôn preôptica debido a su tradicional concepciôn como parte del territorio hipotalàmico, dado que se encuentra estructural y funcionalmente ligada a él, y ademâs porque représenta el limite dorsal con dicho territorio. De hecho, hay que tener en consideraciôn que los anâlisis de especificaciôn molecular del subpalio han demostrado que la regiôn preôptica présenta un perfil molecular diferente al resto de los componentes subpaliales (Flames et al., 2007), por lo que el ârea preôptica podria suponer un territorio intermedio de transiciôn de perfiles moleculares entre el telencéfalo y el hipotâlamo propiamente dicho. Amniotas. En mamiferos la regiôn preôptica se encuentra compartimentada en el ârea preôptica comisural (POC) y en el ârea preôptica propiamente dicha (PO) (Garcia-Lôpez et al., 2008). El POC, recientemente propuesta, estâ localizada en la base del septo y relacionada con la comisura anterior. Estâ principalmente caracterizada por la expresiôn ventricular de Shh, aportando células a la amigdala medial contribuyendo al origen heterogéneo de ésta (Garcia-Lôpez et al., 2008). El ârea preôptica, como parte del territorio subpalial, se caracteriza por la expresiôn de genes de la familia Dix (Flames et al., 2007), y comparte la expresiôn ventricular del factor de transcripciôn Nkx2.1 con la eminencia gangliônica medial (MGE), que es el territorio prospectivo del pâlido (Nkx2.1+) (Puelles et al., 2000; Garcia-Lôpez et al., 2008; revisado en Moreno et al., 2009). Desde un punto de vista molecular, el PO de ratôn se encuentra definida por la expresiôn ventricular de Nkx2.1 y Shh, asi como por la falta de niveles détectables de Gsh2, Lhx6, Lhx7 u Glig2 (Flames et a., 2007). Ademâs, se ha identificado recientemente en ratôn un limite entre la regiôn preôptica y el hipotâlamo llamado limite preoptohipotalâmico (POH), el cual se caracteriza por la expresiôn de Nkx2.2 (Flames et al., 2007). Con respecto al perfil neuroquimico, la regiôn preôptica de mamiferos se caracteriza por la preseneia de células GABAérgicas en la zona subventricular, cuya especificaciôn viene determinada por la expresiôn de genes de la familia Dix (Price et al., 1991; Bulfone et al., 1993; Marin y Rubenstein, 2001). Ademâs, en rata se ha detectado en la regiôn preôptica, una poblaciôn de células positivas para el neuropéptido TRH que, concretamente, se ubican en el nùcleo preôptico medial 173 Raton E x p a n s i o n p a l ia l EPT h Pol o A u s e n c ia d e N kx2 .1 e n S C AM NIOTAS Tortuga P O H N k x 2 .2 + Xenopus P O C S h h + »Cr SCc SPVI R e s t r i c c io n N kx2 .1 e n S C Fez pulmonado P à l id o N kx2 .1 +Lamprea A p o r ta c io n c é lu la s O tp + a M eA Tetrâpodos h b i Limite Alar/Basal g g Shh+Nkx2.1 Shh ■ I Nkx2.1 n Nkx2.2 V7X Dix2 01x5 Isi1 Lhxl r ~ l Lhx7 ^ 3 Lhx5 ■ I Lhx9 Otp a T brl Pax6 Figura 1. Representaciôn de la evoluciôn del hipotâlamo en base a patrones de expresiôn moleculares. El côdigo de color utilizado se indica en la parte superior izquierda. VER AMPLIACIÔN EN EL DESPLEGABLE ANEXO 1. Las principales diferencias encontradas en la evoluciôn de la regiôn hipotalàmica en base a sus patrones moleculares utilizados son, la ausencia de expresiôn de Shh y Lhxl en el ârea preôptica de lamprea (Osorio et al., 2005). La ausencia de expresiôn de Pax6 en la regiôn SPV de anamnios (Murakami et al., 2001; Dominguez et al., 201 le; Moreno y Gonzâlez, 2011) con respecto a amniotas. En la regiôn supraquiasmâtica en mamiferos no se ha descrito expresiôn de Nkx2.1 ni Shh (Puelles y Rubenstein, 2003) a diferencia de amniotas no mamiferos y anamniotas. Finalmente, en la regiôn mamilar de Xenopus se détecta expresiôn de genes Dix a diferencia de mamiferos y lamprea (Puelles y Rubenstein, 2003; Martinez de la Torre et al., 2011). El resto de las diferencias de expresiôn de los marcadores présentés en el esquema se deben a la ausencia de datos en la bibliografia. (Hôkfelt et al., 1975; Lechan et al., 1986; Tusuruo et al., 1987; Merchenthaler et al., 1988), el cual ha sido directamente relacionado con la regulaciôn del comportamiento sexual (Bruce et al., 2008). Ademâs, la regiôn preôptica de mamiferos se encuentra también caracterizada por la preseneia de la enzima tirosina hidroxilasa (Hôkfelt et al., 1984; Simmons y Yahr, 2011). En la regiôn preôptica de aves también han sido descritas dos subdivisiones (Abellân y Medina, 2009). El ârea POC, comparable a la regiôn homônima en ratôn (POC; en Garcia-Lôpez et al., 2008), caracterizada por la expresiôn ventricular de Shh y Lhx7 y su relaciôn con la comisura anterior, y el ârea preôptica basai (POB) comparable al PO y que expresa también Shh en vz pero no Lhx7 (PO; en Garcia-Lôpez et al., 2008). Ademâs, se ha observado que no toda la regiôn preôptica es positiva para Shh, si no que existe un dominio ventral que es dorsal al pedùnculo ôptieo, que es negativo para Shh pero expresa marcadores subpaliales como FoxG l, Dlx5 y Nkx2.1 (Bardet et al., 2010). En cuanto a su patrôn de especificaciôn molecular, la regiôn preôptica en aves se encuentra principalmente caracterizada por la expresiôn de Shh y Nkx2.1, y se distingue de la divisiôn palidal adyacente (MGE) gracias a la expresiôn del factor de transcripciôn Islet 1 en la regiôn preôptica (Abellân y Medina, 2009). Similar a lo descrito en mamiferos, se caracteriza por la expresiôn de genes de la familia Dix los cuales estân directamente implicados en la especificaciôn del territorio subpalial (Puelles et al., 2000). Ademâs, la regiôn peôptica de polio se caracteriza por la expresiôn de FoxGl (Bardet et al., 2010), y también se ha descrito el limite POH caracterizado por la expresiôn de Dlx5 y Nkx2.2 (Bardet et al., 2006). Desde el punto de vista neuroquimico, en aves ha sido defmido un grupo TRH positivo en el nùcleo magnocelular preôptico 174 3. KE»LiYlEN LIE E U » K E»EE 1 ALIU» ï U1»EE»1UN G EN ERA L comparable al descrito en otros vertebrados amniotas y anamiotas (Jôzsa et al., 1988; Péczely y Kiss, 1988). Se ha descrito la preseneia de poblaciones celulares positivas para TH (Baillien et al., 1999) y GABA, las cuales se han relacionado con la regulaciôn de la temperatura y del ciclo sueno-vigilia en PO de paloma (Ekimova y Pastukhov, 2005). En reptiles, estudios recientes en tortuga han demostrado una organizaciôn subpalial comparable a la descrita en otros amniotas (Métin et al., 2007; Moreno et al., 2010). En concreto, en base a la expresiôn de diferentes genes reguladores del desarrollo y marcadores neuronales, se han establecido diferentes compartimentos histogenéticos en el subpalio (Moreno et al., 2010). De esta forma, se ha observado que toda la regiôn preôptica expresa Nkx2.1, y es la expresiôn diferencial de Isll la que define dos regiones comparables a las descritas en el resto de amniotas. Asi, se distingue una porciôn negativa para Isll situada en la porciôn mâs medial y caudal del telencéfalo ventral, ventral al septo, medial al nùcleo del lecho de la estria terminal (BST) y dorsal a la comisura anterior, comparable al POC de mamiferos, y una segunda regiôn Isll positiva comparable a PO (Moreno et al., 2010). Ademâs, en tortuga se ha identificado una regiôn que expresa Nkx2.2 situada entre la regiôn preôptica y el ârea supraoptoparaventricular, que es comparable al POH descrito en amniotas (Dominguez et al., 201 Ib). Desde el punto de vista neuroquimico, el présente estudio ha revelado la existencia de un grupo celular orexinérgico en la regiôn preôptica de reptiles localizado periventricularmente y con células contactantes con el ventriculo, el cual se encuentra muy conservado a lo largo de la evoluciôn (Dominguez et al., 2010a). También se han encontrado células positivas para TH que caracterizan la regiôn preôptica de tortuga (Moreno et al., 2010). Anamniotas. La regiôn preôptica de anuros ha sido analizada en el présente estudio observando un patrôn de organizaciôn altamente conservado. Estudios previos en Xenopus propusieron la existencia de una regiôn comparable al POC descrito en mamiferos, basândose en la existencia de expresiôn ventricular de Nkx2.1 en una regiôn comparable en la base del septo, relacionada con la comisura anterior y negativa para Isletl (Moreno et al., 2008a). Posteriormente, se analizô la expresiôn de Shh en Xenopus confirmando la existencia de una regiôn preôptica precomisural (POC) que expresa ventricularmente Shh y Nkx2.1 (Dominguez et al., 2010b). De la misma forma se ha identificado otra divisiôn llamada ârea preôptica propiamente dicha (PO) comparable a la también descrita en anmiotas, y que se caracteriza por la expresiôn de Shh, Nkx2.1, Lhx7, Isletl y genes de la familia Dix (Brox et al., 2003; Moreno et al., 2004; 2008a; Moreno y Gonzâlez, 2011; Dominguez et al., 2010b; 2012a). A diferencia de lo descrito en amniotas, en el caso de Xenopus no se ha descrito una regiôn Nkx2.2 independiente que separase la regiôn preôptica del hipotâlamo (Dominguez et al., 2011a; 2012a). Desde un punto de vista quimioarquitectônico, se ha observado una poblaciôn de células GABAérgicas localizadas dentro del dominio de expresiôn Dix y Isletl positivo, lo cual sugiere una posible implicaciôn de ambos factores de transcripciôn en la especificaciôn de fenotipo GABAérgico en la regiôn preôptica de anuros (Dominguez et al., 2012a), al igual que ha sido previamente descrito en mamiferos. Ademâs se han encontrado grupos celulares postmitôticos positives para TRH y orexinas, dos neuropeptides con una localizaciôn tipicamente hipotalàmica y directamente implicados en la regulaciôn del sistema neuroendocrino que comparten un patrôn de distribuciôn muy similar a amniotas (Dominguez et al., 2008; Lôpez et al., 2009a). La regiôn preôptica de anuros también se caracteriza por la expresiôn de TH alrededor del ventriculo (Gonzâlez et al., 1993; Dominguez et al., 2012a), caracteristica muy conservada a lo largo de la evoluciôn. Con respecto a la regiôn preôptica de peces, en teleôsteos se observa la expresiôn de Shh y Dix en la regiôn del telencéfalo basal/preôptica en el desarrollo tardio (Scholpp et al., 2006; Menuet et al., 2007) aunque todavia no se distinguen diferentes subdominios dentro de esta ârea. Sin embargo, es en el telencéfalo de pez pulmonado, considerado como el grupo vivo mâs cercano a los tetrâpodos (Brinkmann et al., 2004; Takezaki et al., 2004; Hallstrom y Janke, 2009), donde se observa por primera vez una regiôn palidal como tal que expresa Nkx2.1 asi como una regiôn Nkx2.1 vz positiva comparable al POC y PO descritos en amniotas (Gonzâlez y Northcutt, 2009; revisado en Moreno et al., 2009) a falta de un anâlisis de la expresiôn de Shh en dicha regiôn. Los estudios realizados en lamprea, correlacionan la falta de expresiôn de Nkx2.1 y Shh en el telencéfalo basai Dix positivo (Martinez de la Torre et al., 2011), con la falta de un palido y regiôn preôptica propiamente dichas en el subpalio (Murakami et al., 2001; Osorio et al., 2005). Atendiendo a su patrôn neuroquimico, en lamprea también se han observado células TRH positivas localizadas en el ârea preôptica anterior, lo cual refieja su condiciôn de caracteristica ancestral (Del Carmen et al., 2002). De la misma forma, estas células fueron observadas alrededor del ârea preôptica de teleôsteos (Batten et al., 1990a,b; Hamano et al., 1990; Matz y Takahashi, 1994; Diaz et al., 2001, 2002), donde también se han detectado numerosas células orexinérgicas en pez cebra (Kaslin et al., 2004; Faraco et al., 2006) y en pez pulmonado (Lôpez et al., 2009b). Todos estos datos han llevado a homologar el POC y PO de anuros y reptiles con sus équivalentes de la regiôn preôptica descritos en mamiferos, ya que comparten un patrôn de especificaciôn molecular comùn, que révéla un mismo origen, y ademâs, presentan numerosas similitudes en cuando a la hodologia (datos no mostrados) y el patrôn neuroquimico, lo que hace inferir que esta regiôn preôptica pueda estar relacionada en funciones comparables en los distintos vertebrados. Asi, la regiôn preôptica de anuros y reptiles estaria probablemente 175 5. RESUMEN DE LOS RESULTADOS Y DISCUSION GENERAL implicada en la regulaciôn de determinadas funciones del sistema neuroendocrino como por ejemplo el control del comportamiento sexual, a través de su estrecha relaciôn con determinados centros subpaliales, al igual que ocurre en amniotas (Bruce et al., 2008). Ârea Supraoptoparaventricular El ârea supraoptoparaventricular (SPV) es una regiôn directamente implicada en el sistema neuroendocrino, constituyendo una parte esencial del sistema hipotâlamo- hipofisiario. Anatômicamente se encuentra entre la regiôn preôptica y el territorio supraquiasmâtico (SC), y estâ formado principalmente por dos nùcleos que le dan el nombre: nùcleo supraôptico y nùcleo paraventricular (Medina, 2008). Esta regiôn del hipotâlamo alar consta de numerosas poblaciones de células neuroendocrinas que vienen especificadas por el gen orthopedia (Otp) y que finalmente van a liberar al torrente circulatorio o a la hipôfisis numerosas neurohormonas encargadas del control y la regulaciôn de diferentes funciones homeostâticas como respuesta al estrés, comportamientos reproductivos y sociales, etc (revisado en Markakis, 2002). Amniotas. El SPY de mamiferos y aves se define como un territorio Dix negativo adyacente a la regiôn preôptica y que expresa los factores de transcripciôn Otp, Siml, Pax6 y Tbrl (Bulfone et al., 1993; Acampora et a., 1999; Lin et al., 1999; Puelles and Rubenstein, 2003; Del Giacco et al., 2006; Flames et al., 2007; Bardet et al,. 2008; Medina, 2008). Este territorio también se caracteriza por la expresiôn del gen de la familia LlM-hd Lhx5, cuya expresiôn se ha observado en el SPV tanto en ratôn como en polio (Bulfone et a l , 1993; Abellân et a l, 2010). Otra caracteristica muy conservada de esta regiôn es la falta de expresiôn de Shh, Dix y Nkx2.1, aunque estâ flanqueado dorsalmente por PO, ventralmente por SC y caudalmente por el pretâlamo (PTh), donde se expresan todos esos genes reguladores del desarrollo (Puelles and Rubenstein, 2003). En cuanto a su organizaciôn general, el territorio SPV de mamiferos y aves se dividiô inicialmente en una regiôn rostral y otra caudal, en base a la aglomeraciôn celular y a la cantidad de células Otp positivas (Bardet et a l, 2008), aunque actualmente no se ha confirmado una subdivisiôn de este territorio atendiendo estrictamente a criterios moleculares. Ademâs de esta complejidad molecular en cuanto a su especificaciôn, estudios de migraciones dentro del prosencéfalo han descrito que, algunas de estas células que expresan Otp migran hacia la amigdala medial contribuyendo a su origen heterogéneo (Garcia-Moreno et a l, 2010). Numerosos estudios han demostrado la implicaciôn de Otp en la especificaciôn neural y diferenciaciôn de un gran grupo de células neuroendocrinas como son las células que expresan la TRH, hormona liberadora de corticotropina, oxitocina, vasopresina y somatostatina (SOM) (Acampora et a l , 1999; Wang and Lufkin, 2000; Goshu et a l , 2004), siendo imprescindible para la correcta formaciôn de los diferentes nùcleos para los eue contribuyen dichas poblaciones dentro del territorio SPV. De esta forma, en mamiferos y aves se han descrito très nùcleos principales dentro del SPV, el nùcleo paraventricular, formado entre otras por células que sintetizan TRH (Aoki et a l , 2007; Vandenbome et a l , 2005; Kâdâr et a l, 2010), el nùcleo supraôptico, que contiene células oxitocina positivas, y el nùceo periventricular (aPV), que se encuentra adyacente a ?V y contiene células que expresan SOM y proyecta a la eminencia media (Wang and Lufkin, 2000; Caquerel et a l , 2005; Medina, 2008; Morales-Delgado et a l, 2011). En reptiles, los datos obtenidos en el campo de la neuroquimica, de trazado neuronal y de expresiôn de genes, muestran un alto grado de conservaciôn en esta regiôn SPV. La regiôn que corresponde al SPV en reptiles, es un territorio caracterizado por la preseneia de Otp y Pax6, y la ausencia de Nkx2.1 (Domingue? et a l , 2011b). La expresiôn del factor de transcripcôn Nkx2.2 ha permitido la distinciôn de dos subdivisioies dentro del SPV en reptiles. De esta forma en el présenté trabajo se ha descrito una porciôn rostral rica en Otj» y Nkx2.2 localizados en la zona ventricular y subventricular (SPVr) y una porciôn caudal caracterizada por la ùnica expresiôn de Otp (SPVc) (Dominguez et a l, 2011b). En el cerebro de reptiles,en este ârea se ha descrito la preseneia de mùltiples neuropéptidos, como orexinas (Dominguez et i l , 2010a), o TRH (Lôpez et a l , 2008). La TRH se encuentra localizada dentro del territorio Otp positi/o, lo que sugiere que la poblaciôn neuronal TRH positva estâ especificada por la acciôn de Otp, al igual que ocurre en el resto de amniotas (revisado en Del Giacco et al, 2008). Todos estos datos acerca de la distribueôn de estas poblaciones neuropeptidicas en el S^V sugieren que esta regiôn de reptiles también constitiye el hipotâlamo neuroendocrino y que se encuertra altamente conservado en amniotas. Ademâs, de igial forma que ocurria en mamiferos, se han observtdo células que expresan Otp en la amigdala medial (MeA) de tortuga (Moreno et a l , 2010). De modo qie, teniendo en cuenta los conocidos movimiertos migratorios que se han descubierto en los ùltimos aios en este grupo de amniotas (Métin et a l, 2007), ©ta preseneia de células Otp positivas en MeA de tortiga sugiere que pueda tener un origen hipotalàmico y tue provengan del SPV como previamente se ha descrito en mamiferos (Soma et a l , 2009; Garcia-Moreno et il, 2010). Esta regiôn de reptiles también se caracteriza jor la preseneia de un grupo catecolaminérgico descrito jor la preseneia de la enzima TH (Smeets et a l, 1917; Dominguez et a l, 201 Ib). Anamniotas. El territorio SPV de anuros se encuentra caracterizado por la expresiôn de Otp y Lbc5 asi como por la falta de expresiôn de Shh, Nkx2.I y genes de la famila Dix, los cuales se expresan en territorios adyacentes formando los limites del S>V (Brox et a l, 2003; Dominguez et a l , 2010b; 2012a). De manera similar a lo descrito en tortuga (Dominguez et 1T6 5. KlLSLMILrN U I L LUS KLSUL1AUUS Y UlSLUSlUrN ULfNlLKAL al., 2011b), en Xenopus se ha propuesto una regionalizaciôn rostro-caudal de SPV en base al patron de expresiôn molecular identificado (Dominguez et al., 2012a). De esta forma se ha descrito un dominio rostral que expresa Otp y Nkx2.2 (SPVr) y un dominio caudal que ùnicamente expresa Otp (SPVc). Sin embargo, una de las caracteristicas tipicas de anuros es la falta de expresiôn de los factures de transcripeiôn Pax6 y Tbrl, a diferencia de lo que ocurre en amniotas donde si se ha observado su expresiôn (Medina 2008; Dominguez et al., 2011b). Desde el punto de vista neuroquimico, se ban observado numerosas poblaciones de neuropéptidos y neurohormonas présentes en el SPV de anuros que también han sido detectadas en regiones similares en amniotas. De esta forma, el SPV de anuros présenta poblaciones de células que sintetizan TRH (Dominguez et al., 2008) y que se encuentran en el territorio que expresa Otp (Dominguez et al., 2012a), sugiriendo que también en anuros Otp se encargaria de la diferenciaciôn celular y la adquisiciôn del destino celular hacia células TRH positivas, contribuyendo a la formaciôn del nùcleo paraventricular, al igual que se ha descrito en amniotas (Goshu et al., 2004; Del Giacco et al., 2008). En anuros también se han observado células postmitôticas positivas para somatostatina (SOM) en una regiôn concreta del SPV (Dominguez et al., 2012a), que podria corresponderse al territorio prospectivo del nùcleo aPV ya descrito en amniotas (Wang and Lufkin, 2000; Caqueret et al., 2005; Medina, 2008). Dichas células SOM+ probablemente son especificadas por Otp como ocurre en amniotas (Morales-Delgado et al., 2011) ya que se ha observado una correlaciôn entre la apariciôn de expresiôn de Otp y la emergencia de poblaciones celulares neuroendocrinas SOM y mesotocina positivas (Dominguez et al., 2012a). Ademâs, también se ha puesto de manifïesto la existencia de células que expresan Otp situadas en la MeA de Xenopus (Dominguez et al., 2012a) sugiriendo la posibilidad de una ruta migratoria tangencial similar por la que la MeA reciba aportaciôn de células hipotalâmicas. La regiôn SPV de peces también se eneuentra principalmente caracterizada por la expresiôn de Otp, lo cual como hemos visto es un rasgo comùn en la evoluciôn (Del Giaceo et al., 2006; Blechman et al., 2007; Machluf et al., 2011), asi como también lo es la ausencia de expresiôn de genes de la familia Dix como se ha observado en lamprea (Martinez de la Torre et al., 2011). La falta de expresiôn de Tbrl y Pax6 que se observô en anuros parece ser una caracteristica comùn en anamniotas, ya que estudios realizados en lamprea (Murakami et al., 2001) y pez pulmonado (Moreno y Gonzalez, 2011) han revelado su ausencia en el territorio équivalente al SPV. Estudios realizados en peces también apoyan el papel conservado de Otp en la especificaciôn de poblaciones neuronales neurosecretoras, como es el caso de pez cebra, en el que Otp esta implicado en la diferenciaciôn de la vasotocina-neurofisina e isotocina-neruofisina, homôlogos de la oxitocina y mesotocina en mamiferos (Tessmar-Raible et al., 2007; Eaton y Glasgow, 2007; Blechman et al., 2007; Eaton et al., 2008). Los datos recopilados aeerca de la neuroquimiea y expresiôn génica muestran que en tetrâpodos el ârea supraoptoparaventricular aparece como un centro perteneeiente al sistema neuroendoerino hipotalâmieo altamente conservado en la filogenia. Este patrôn de organizaciôn molecular y neuroquimico similar apoya la hipôtesis de un origen comùn. Ârea Supraquiasmâtica La regiôn supraquiasmâtica constituye una parte del sistema neuroendoerino hipotalâmieo siendo uno de los mayores centros coordinadores de diferentes comportamientos, estados fisiolôgicos y ritmos circadianos. Dada su condiciôn de intégrante del sistema endocrino, se caracteriza por contener numerosas poblaciones celulares que expresan diferentes neuropéptidos como vasopresina, neuropéptido Y, somatostatina, preproencefalina, etc. Amniotas. La regiôn supraquiasmâtica (SC) de mamiferos y aves se caracteriza por la expresiôn de genes de la famila Dix, a los que se ha relacionado con procesos de especificaciôn de células GABAérgicas (Price et al., 1991; Bulfone et al., 1993; Marin y Rubenstein, 2001). La expresiôn de Nkx2.1 se ha observado solamente en vertebrados no mamiferos (van den Akker et al., 2008). Asi, en polio Nkx2.1 se expresa ùnicamente en una porciôn llamada ârea subparaventricular que pertenece al SC y que también expresa el factor de transcipciôn Nkx2.2 y los miembos Lhx6/7 y 8 de la familia LIM-hd (Abellân y Medina, 2009; Bardet et al., 2010). En mamiferos, el territorio supraquiasmâtico también expresa numerosos genes de la famila LIM-hd, que se sabe estân implicados en numerosos procesos de especificaciôn y determinaciôn celular que tienen lugar durante el desarrollo. Concretamente, en un estudio reciente realizado en ratôn en base a las expresiones de Lhxl, Lhx6, Lhx7 y Lhx8, entre otros genes, se propone la existencia de un territorio denominado banda intradiagonal (ID), que constituiria el origen de las poblaciones de intemeuronas supraquiasmâticas (Shimogori et al., 2010). En términos neuroquimicos, esta regiôn supraquiasmâtica también se caracteriza por la existencia de poblaciones neuronales positivas para los neuropéptidos TRH (Jôzsa et al., 1988; Péczely y Kiss, 1988; Merchenthaler et al., 1988), asi como por la existencia de numerosas células TH positivas (Hôkfelt et al., 1984; Smeets y Gonzâlez, 2000) y la expresiôn de GAD67 (Abellân y Medina, 2009) que caracterizan la regiôn supraquiasmâtica de amniotas. La regiôn supraquiasmâtica de tortuga se caracteriza por la expresiôn de Isl 1 en todo el territorio (Dominguez et al., 2011b), siendo la expresiôn diferencial de Nkx2.1 y Nkx2.2 lo que permitiô la distinciôn de dos subregiones (Dominguez et al., 2011b). Una porciôn rostral llamada SCr que expresa ventricularmente Nkx2.1, Nkx2.2, y otra porciôn caudal llamada SCc, negativa para estos marcadores y que 177 5. RESLMEIN DE LOS RES LET ADOS Y DISCUSION GENERAL ùnicamente expresa Isll (Dominguez et al., 2011b). El analisis del patron neuroquimico en tortuga y Gecko desvelo la ausencia de poblaciones orexinérgicas y TRH positivas en el SC de reptiles, patron similar a mamiferos y aves y contrario al existente en anamniotas donde existe un numeroso grupo celular positivo para orexinas y TRH en el SC (Kaslin et al., 2004; Dominguez et al., 2008; 2010a; Lôpez et al, 2008; 2009a). Ademâs, se ha observado la presencia de poblaciones positivas para TH localizadas en la regiôn mâs rostral (SCr) (Dominguez et al., 2011b). Anamniotas. El ârea supraquiasmâtica de anuros es un territorio que expresa genes de la famila Dix (Brox et al., 2003; Dominguez et al., 2012a). Ademâs, esté caracterizada por la expresiôn de xD114 y Isll en todo el territorio (Dominguez et al., 2012a) siendo los patrones de expresiôn de Nkx2.1, Nkx2.2 y xShh los que muestran la presencia de dos subregiones a lo largo del eje rostrocaudal diferenciadas claramente por su patrôn molecular (Dominguez et al., 2010b; 201 la; 2012a). La poreiôn mâs rostral, SCr, estâ caracterizada por la presencia de xD114, Isll, Nkx2.1, Nkx2.2, xShh, xLhxl y xLhx7, mientras que la porciôn mâs caudal, SCc, expresa ùnicamente xD114 y Isll (Dominguez et al., 2012a). La existencia en anuros de dos porciones en el territorio supraquiasmâtico en base a la expresiôn diferencial de Nbe2.1, es una situaciôn comparable a la previamente descrita en reptiles (Dominguez et al., 201 Ib). Ademâs, la porciôn SCr Nkx2.1/Nkx2.2 positiva podria ser comparable a la subregiôn de SC descrita en polio caracterizada también por la expresiôn de Nkx2.2 y Lhx6/7/8 (Abellân y Medina, 2009; Bardet et al., 2010; Dominguez et al., 2012a). En cuanto a su perfil neuroquimico, la presencia de células GABAérgicas dentro del dominio de expresiôn de xD114 de anuros sugiere la implicaciôn de Dix en la especificaciôn celular hacia el fenotipo GABAérgico en Xenopus, al igual que ocurre en mamiferos (Price et al., 1991; Bulfone et al., 1993; Marin y Rubenstein, 2001). Ademâs de constituir una caracteristica muy conservada en la evoluciôn, el SC de anuros présenta numerosas poblaciones neuropeptidérgicas, como células TRH y orexina positivas (Dominguez et al., 2008; Lôpez et al., 2009a) que no aparecen en reptiles (Lôpez et al., 2008; Dominguez et al., 2010a), sugiriendo que dichas poblaciones postmitôticas desaparecen en la transiciôn anamnio-amniota. Ademâs, la regiôn supraquiasmâtica de anuros présenta una importante poblaciôn de células TH positivas (Gonzâlez et al., 1993; Dominguez et al., 2012a), como se ha descrito an amniotas (Hôkfelt et al,. 1984; Smeets y Gonzâlez, 2000). En cuanto al patrôn de expresiôn génico, estudios en pez cebra han descrito expresiôn de Shh y Nkx2.1 en toda la regiôn supraquiasmâtica sin observar diferentes subdominios (Rohr et al., 2001). En medaka, se ha observado expresiôn de Dlx2 y Lhx7 en regiones similares al resto de los vertebrados, que probablemente corresponda con esta regiôn supraquiasmâtica (Alunni et al., 2004). Ademâs, en lamprea se ha observado expresiôn de genes de la familia Dix en el primordio de la regiôn supraquiasmâtica (Martinez de la Torre et al., 2011). Con respecto a su patrôn neuroquimico, el SC de peces présenta un grupo orexina positivo aunque no se observa en todas las especies, apareciendo en pez cebra pero no en goldfish (Kaslin et al., 2004; Huesa et al., 2005; Faraco et al., 2006). Ademâs, estudios en pez pulmonado han identificado células orexinérgicas en el SC, al igual que en tetrâpodos (Lôpez et al., 2009b), apoyando el alto grado de conservaciôn de dicho sistema peptidérgieo en el hipotâlamo de anamniotas. También se ha observado un grupo celular TRH positivo en el nùcleo supraquiasmâtico de teleôsteos (Matz y Takahasi, 1994, Diaz et al., 2002). Regiôn tuberal La regiôn tuberal actualmente estâ eonsiderada como una parte del hipotâlamo basai que expresa principalmente Shh y Nkx2.1, ambos esenciales para el correcto desarrollo hipotalâmieo (revisado en Medina, 2008). Sin embargo un estudio reciente en ratôn ha propuesto que el territorio tuberal tiene un carâcter alar, basândose en el anâlisis de expresiôn de numerosos genes régionales (Diez-Roux et al., 2010). El anâlisis de esta regiôn ha puesto de manifiesto su patrôn de organizaciôn altamente conservado entre anamniotas y amniotas. Amniotas. La regiôn tuberal de amniotas estâ constituida por diferentes nùcleos como son el nùcleo ventromedial (VMN), dorsomedial (DMN) y el nùcleo arcuado (Arc). Estân formados por células que nacen en el tercer ventriculo y migran lateralmente (Atlman y Bayer, 1986). Recientemente Puelles y colaboradores han propuesto una divisiôn rostrocaudal del hipotâlamo en una regiôn peduncular caudal y otra terminal rostral, la cual incluye los nùcleos VMN y arcuado (Arc) (Morales-Delgado et al., 2011; en referenda a “Allen development mouse brain atlas” por L.Puelles). Esta regiôn se caracteriza principalmente por la expresiôn de Shh, el cual estâ directamente implicado en la organizaciôn del hipotâlamo basai a través de la acciôn de Nkx2.1 (Kimura et al., 1996; Puelles et al., 2004). En mamiferos, Isll se expresa en los nùcleos ventromedial y arcuado, revelando un papel en el desarrollo hipotalâmieo y actuando en el control del comportamiento reproductive a través del receptor de estrôgeno a (Davis et al., 2004). El nùcleo arcuado de ratôn y polio expresan el factor de transcripeiôn Otp (Acampora et al., 1999; Morales-Delgado et al., 2011), implicado en la especificaciôn de células SOM positivas (Acampora et al., 1999; Wang y Lufkin, 2000), y expresa Dix, encargado de la especificaciôn GABAérgica (Yee et al., 2009). Ademâs, en la regiôn tuberal de ratôn, se ha descrito expresiôn de Otp en una regiôn llamada anterobasal (Morales-Delgado et al., 2011) localizada justo detrâs del quiasma ôptico, comparable al denominado nùcleo retroquiasmâtico de aves (Puelles et al., 1987). Por otra parte, el nùcleo 178 5. KESUMEN DE LUS K E SLLIA D U S Y UISLLSIUN GENERAL ventromedial expresa Nkx2.2 el cual estâ implicado en la adquisiciôn de fenotipo basai y en la especificaciôn de células con destino ventromedial (Kurrasch et al., 2007). En mamiferos, en cuanto a su neuroquimiea se ha encontrado un numeroso grupo de células orexinérgicas en el ârea lateral hipotalâmica y el DMN (de Lecea et al., 1998; Sakurai et al., 1998; Peyron et al., 1998; van den Pol., 1998) que se sabe estân implicadas en el control de funciones homeostâticas como la regulaciôn de la alimentaciôn, el ciclo del sueno-vigilia y de la temperatura (Powley and Keesey, 1970; van den Pol, 1982; De Lecea et a l , 1998; Sakurai et a l , 1998; Peyron et a l , 1998; Sutcliffe and de Lecea, 2000). De hecho, la mayoria de los neuropéptidos encargados del control de la ingesta se encuentran en la regiôn tuberal. El neuropéptido TRH también se eneuentra distribuido en esta regiôn tuberal, concretamente en el DMN y Arc (Hôkfelt et a l , 1975; Lechan et a l , 1986; Tusuruo et a l, 1987; Merchenthaler et a l, 1988). El hecho de que ambos neuropéptidos estén présentes en la regiôn tuberal sugiere una posible interacciôn entre ellos. Ademâs, estudios en ratas han demostrado que expiantes hipotalâmicos con orexinas inhiben la liberaciôn de TRH (Mitsuma et a l, 1999). En reptiles, esta zona tuberal se caracteriza por la expresiôn de Nkx2.1 y Isll (Dominguez et a l , 2011b). Ademâs, se han identificado dos subregiones dentro del ârea tuberal de tortuga en base a su patrôn molecular diferencial De esta forma, présenta una regiôn rostral (RT) earacterizada por la expresiôn de Nkx2.1, Isll y Otp, por lo que probablemente constituya el territorio prospectivo del Arc y comparable a dicha regiôn de mamiferos, y una porciôn caudal que expresa Nkx2.1 y Isll, de la que podrian derivar el resto de nùcleos tuberales (Dominguez et a l, 2011b). Con respecto a su patrôn neuroquimico, existe una poblaciôn de células orexina positivas en el nùcleo periventricular hipotalâmieo localizado en la regiôn tuberal infundibular de tortuga y Gecko (Dominguez et a l, 2010a), el cual podria ser comparable al descrito en la regiôn tuberal de otros amniotas (de Lecea et a l , 1998; Sakurai et a l, 1998; Peyron et a l , 1998; van den P o l, 1998) ejerciendo una fimciôn similar en el control de ciertos sistemas comportamentales y funciones neuroendocrinas, como el control de la ingesta, sueno-vigilia, etc. Ademâs también se han observado células TRH positivas en este nùcleo periventricular (Lôpez et a l , 2008), lo cual indica un patrôn neuroquimico conservado en amniotas. Anamniotas. La regiôn tuberal de anuros muestra un patrôn de organizaciôn comparable al observado en amniotas. Se trata de una regiôn caracterizada por la expresiôn de Nkx2.1, Shh y Isll (Dominguez et a l, 2010b; 2012b). Al igual que ocurre en reptiles, esta regiôn se divide en dos dominios en base a un patrôn de expresiôn diferencial de Otp y Nkx2.2. De esta forma, se divide en una porciôn rostral (RT), que expresa Otp, y otra caudal (CT), que expresa Nkx2.2 (Dominguez et a l, 2011a; 2012b). El marcador Isll define el limite de la regiôn tuberal con el territorio mamilar al igual que en amniotas, y la similar situaciôn de Otp con respecto a las células positivas para SOM dentro de RT lo hacen comparable con el Arc de mamiferos y aves (Acampora et a l , 1999; Morales-Delgado et a l , 2011). La regiôn tuberal de anuros présenta numerosas células orexinérgicas y TRH positivas localizadas a lo largo del hipotâlamo tuberal (Dominguez et a l , 2008; Lôpez et a l, 2009a), en regiones comparables a amniotas, por lo que estos péptidos en anuros podrian mantener sus funciones neuroendocrinas descritas en mamiferos. Banda mamilar La regiôn mamilar es una especializaciôn ventral del hipotâlamo basai que yace entre la parte epicordal y la precordal del prosencéfalo (Puelles y Rubenstein, 2003). La organizaciôn del territorio mamilar se eneuentra en los ùltimos anos bajo revisiôn debido a los continuos datos obtenidos en el campo de la morfogenética. De esta forma, recientemente se ha subdividido basândose en un perfil molecular. En el présenté estudio se ha analizado eomparativamente dicha regiôn en anuros y reptiles observando un alto nivel de conservaciôn. Amniotas. La regiôn mamilar de mamiferos se ha subdividido recientemente en base a su patrôn de expresiôn molecular en euatro regiones segùn Puelles y cols: perimamilar, mamilar, periretromamilar y retromamilar (Developing Allan Brain Atlas; 2011) y en très regiones segùn Shimogori: supramamilar, mamilar y premamilar (Shimogori et a l, 2010). Ademâs, en base a la expresiôn diferencial de Nkx2.1 y Shh en mamiferos y aves se ha descrito una regiôn mamilar Nkx2.1+/Shh- y una regiôn retromamilar Nkx2.1-/Shh+ (Garcia-Calero et a l , 2008; Morales- Delgado et a l , 2011). El faetor de transcripeiôn Otp también se expresa en la regiôn mamilar siendo una caracteristica muy conservada observada en todos los vertebrados (Bardet et a l , 2008; Morales-Delgado et a l, 2011). Sin embargo, en cuanto a la especificaciôn nuclear, el origen de algunas de las regiones de la banda mamilar no estâ claro, y résulta muy controvertido. En aves se ha propuesto recientemente que la regiôn retromamilar y el nùcleo subtalâmico provienen de la plaça basa de P3 (Garcia-Lôpez et a l, 2009). De esta forma, en ratôn se ha descrito que una poblaciôn de células de la plaça basai de P3 podrian generar neuronas glutamatérgicas en el nùcleo subtalâmico y GABAérgicas en la regiôn mamilar (Delaunay et a l, 2009). Finalmente, con respecto a su perfil neuroquimico, el complejo mamilar de mamiferos se ha asociado con la regulaciôn de la temperatura, memoria y reproducciôn, llevando a cabo dichas acciones a través de GABA y numerosos neuropéptidos como NPY, substancia P, VIP, galanina y somatostatina (Swaab, 2003). La presencia de Nkx2.1 en la regiôn mamilar ha sido relacionada con la especificaciôn del destino catecolaminérgico, como se observô en mutantes de Nkx2.1 en los que se desarrollaba una via 179 5. RESUMEN DE LOS RESULTADOS Y DISCLSION GENERAL dopaminérgica ascendente aberrante (Kawano et al., 2003). Ademâs, la presencia de células positivas para SOM en el dominio de expresiôn de Otp, ha relacionado este faetor de transcripeiôn con la especificaciôn de células somatostatinérgicas (Morales-Delgado et al., 2011). En cuanto a su especificaciôn génica, el ârea mamilar de tortuga se caracteriza principalmente por la expresiôn de Nkx2.1 en vz, de Nkx2.2, Otp en svz y por la presencia de células Pax7 positivas dispersas en svz (Dominguez et al., 2011b) por lo que esta regiôn podria eompararse al patrôn de expresiôn molecular de la banda mamilar de mamiferos y aves. Las células Pax7 positivas probablemente provengan de la poblaeiôn que expresa Pax7 en la plaça basai de P3, apoyando la idea de que parte de la regiôn mamilar présenta su origen en el tegmento pretalâmico, como se ha descrito en mamiferos (Garcia-Lôpez et al., 2009). Por otra parte, en anamniotas se ha sugerido la implicaciôn de Otp en la especificaciôn dopaminérgica (Blechman et a l, 2007; Del Giacco et a l, 2006; Ryu et a l, 2007; Lôhr et a l, 2009), de tal forma que la existencia en tortuga de células TH positivas en el dominio de expresiôn mamilar de Otp (Dominguez et a l, 2011b) sugiere el alto grado de conservaciôn en la evoluciôn de esta implicaciôn funcional Anamniotas. En anuros la banda mamilar présenta dos subdivisiones diferentes con respecto a su patrôn de expresiôn molecular. El ârea mamilar propiamente dicha (Ma) expresa Nkx2.1 en vz pero es negativa para xShh, mientras que el ârea retromamilar (RM) présenta el patrôn contrario (Dominguez et a l , 2012b), situaciôn comparable a la previamente descrita en amniotas (Garcia-Calero et a l, 2008; Morales-Delgado et a l, 2011). Ademâs, en Xenopus la regiôn mamilar se eneuentra caracterizada por la expresiôn de Nkx2.2 y Otp (Dominguez et a l , 2011a; 2012b). La presencia de expresiôn de Otp en la regiôn mamilar es una caracteristica muy conservada a lo largo de la evoluciôn, asi como su implicaciôn en la especificaciôn del fenotipo dopaminérgico, como se ha sugerido en Xenopus dada la coexpresiôn de la enzima TH y Otp en numerosas células de Ma (Dominguez et a l, 2012b). Ademâs, en la banda mamilar de Xenopus se han observado células Pax7 positivas que parecen tener un origen en la plaça basai de P3, situaciôn comparable a la observada en amniotas (Dominguez et a l , 201 Ib) y la cual apoya la idea de que parte del territorio mamilar podria presentar un origen diencefâlico también en anuros, como se ha propuesto en amniotas (Delaunay et a l , 2009; Garcia-Lôpez et a l, 2009). Situaciôn actual del limite Alar/Basal La interpretaciôn de las diferentes regiones del prosencéfalo secundario, entre ellas el hipotâlamo, ha sido tradicionalmente controvertido debido a las evaginaciones hemisféricas y ôpticas que tienen lugar y a los diferentes grados de desarrollo a lo largo de los distintos grupos de vertebrados, que distorsionan el patrôn primario de esta regiôn (Niewenhuys et a l, 2008). Sin embargo, los avances realizados en el campo de la morfogenética en los ùltimos anos han contribuido a définir las diferentes subregiones prosencefâlicas atendiendo a sus relaciones topolôgicas y no topogrâficas (Puelles, 2001; Puelles y Medina, 2002; Puelles y Rubenstein, 2003; Niewenhuys et a l , 2008). De esta forma, las relaciones topolôgicas de las divisiones prosencefâlicas nos proporciona su origen embriolôgico y la posibilidad de establecer relaciones de homologia entre vertebrados. Asi, estas relaciones topolôgicas solamente se pueden desvelar si se tiene en cuenta la curvatura del tubo neural y sus fiexuras. En este sentido, Puelles y Rubenstein propusieron el modelo prosomérico, el cual tiene en cuenta la enorme flexura del eje longitudinal del cerebro que proporcionaba una nueva posiciôn topogrâfica al hipotâlamo, situândolo rostral al diencéfalo (Puelles y Rubenstein, 1993; modificado en Puelles y Rubenstein, 2003) en vez de bajo él, como habia sido propuesto en el modelo columnar de Herrick and Kuhlenbeck (Herrick, 1910; Kuhlenbeck, 1973). Este eje longitudinal divide al cerebro en diferentes regiones longitudinales como son las plaças del techo, alar, basai y del suelo, cada una con un perfil molecular especifico que define diferentes compartimentos moleculares comparables entre los distintos vertebrados (Puelles y Rubenstein, 2003). El establecimiento del limite alar/basal en mamiferos se ha hecho en base al eje longitudinal del cerebro, de tal forma que se define como un territorio a lo largo del eje longitudinal que se caracteriza por la expresiôn de determinados marcadores genéticos como la expresiôn concreta del morfôgeno Shh y del faetor de transcripeiôn Nkx2.2 en todos los vertebrados estudiados (Puelles and Rubenstein, 1993; Shimamura et a l , 1995; Vieira et a l , 2005) y que divide el cerebro en regiones alares y basales (Puelles y Rubenstein, 1993, 2003). Asi, este limite ha permitido la separaciôn del territorio hipotalâmieo en zonas alares, que incluyen las regiones clâsicas ôpticas, y basales, que ineluye el hipotâlamo tuberal y mamilar (Puelles y Rubenstein, 1993; modificado en Puelles y Rubenstein, 2003). Teniendo en cuenta que el modelo prosomérico se ha establecido como un paradigma que establece unas propiedades morfogenéticas concretas para cada regiôn del cerebro haciéndolas comparables entre los distintos grupos de vertebrados, el establecimiento del limite alar-basal en otros vertebrados no mamiferos debe cumplir las mismas caracteristicas moleculares. De esta forma, nuestro estudio ha analizado la expresiôn de los marcadores xShh y Nkx2.2 permitiendo el establecimiento del limite alar-basal en anfibios siguiendo los mismos criterios morfogenéticos que en mamiferos (Dominguez et a l, 2010b; 2011a) y demostrando que también en anfibios dicho limite no es rectilineo sino que termina justamente detrâs del quiasma ôptico, como se habia descrito previamente en mamiferos (Puelles, 1995; Puelles y Rubenstein, 1993; 2003). Asi, el présenté estudio ha identificado por 180 5. KESUM EN DE EU S K E SU E IA U U S Y D iSE E SlU N G EN ERA L primera vez el limite alar-basal en anfibios, apoyando el uso del modelo prosomérico como un instrumento morfolôgico comparable entre vertebrados y demostrando el alto grado de eonservaciôn de dieho limite en la evoluciôn. Por otra parte, bay que tener en consideraciôn que el concepto del limite alar-basal estâ siendo actualmente objeto de controversia. De esta forma, recientemente se ha propuesto un cambio en el limite alar/basal, lo cual implicaria una modificaciôn de las regiones hipotalâmieas. Asi, Diez-Roux y eolaboradores han analizado los patrones de expresiôn de multiples genes tipicamente alares como Lhx2, Lhx6, Lhx9, Dlxl Dlx2 y Dlx5 entre otros, y tipicamente basales, entre lo que se encuentran Pax7, 01ig2, Lhxl, Lhx5, Irxl e Irx3. El resultado de dicho anâlisis revelô que segùn esta discriminaciôn alar-basal, el territorio tuberal tendria un origen alar mientras que el territorio mamilar séria el ùnico dentro del hipotâlamo con carâcter basai (Diez- Roux et al., 2010). Esta aproximaciôn tiene en contra los propios patrones de expresiôn de Shh y Nkx2.1, los cuales se eonsideran tipicamente basales y se eneuentran expresados especificamente en el territorio tuberal. Aùn asi, es cierto que estudios de transplantes en polio han sugerido una diferente naturaleza alar o basai dentro de la expresiôn de Shh en el dieneéfalo e hipotâlamo (Vieira y Martinez et al., 2006), por lo que se necesitaria un anâlisis exhaustivo del posible origen alar o basai del Shh expresado en la regiôn tuberal para confirmar dicha hipôtesis. Los resultados de nuestro estudio en anuros ponen de manifiesto la existencia de expresiôn de Shh y Nkx2.1 en regiones hasta el momento consideradas como tipicamente alares, como es el caso de la regiôn preôptiea, lo que podria sugerir también una posible modificaciôn de este limite alar-basal (Dominguez et al., 2010b; 201 la). Hipôtesis evolutiva de la organizaciôn hipotalâmica: existencia de un patrôn de organizaciôn comùn en la evoluciôn y repercusiones de la transiciôn anamnio- amniota En el présente estudio las distintas aproximaciones expérimentales nos han permitido obtener abondante informaciôn sobre la organizaciôn hipotalâmica en los reptiles y anuros. La realizaciôn de este estudio en dos grupos de vertebrados représentantes de la transiciôn anamnio-aminota ha contribuido a la identificaciôn de las caracteristicas primitivas en la organizaciôn de este sistema, asi como a inferir algo mâs acerca de su evoluciôn en los vertebrados, haciendo un seguimiento de los eambios evolutivos mâs importantes sucedidos entre anfibios y reptiles. De forma que, hasta el momento, los datos existentes en amniotas, principalmente obtenidos en mamiferos, proponian la existencia de un patrôn conservado de organizaciôn con unas caracteristicas bâsicas (Puelles y Rubenstein, 2003). Los resultados de nuestro estudio en reptiles y anuros y su eomparaciôn con otros vertebrados sugieren la existencia de un plan bâsico de organizaciôn hipotalâmica en tetrâpodos, dado que las diferentes subdivisiones que lo eomponen presentan el mismo origen embriolôgico y un patrôn neuroquimico comparable en todos los vertebrados analizados. En anuros y reptiles hemos observado expresiôn de Shh y Nkx2.1 en la regiôn preôptiea (Dominguez et al., 2010b, 2011b). Esta expresiôn se mantiene en todos los vertebrados estudiados exeepto en la lamprea (pertenece al primitive grupo de vertebrados agnatos), donde el subpalio parece no estar dividido y no se ha identificado un palido como tal, lo cual se piensa que estâ relacionado con esta ausencia de expresiôn de Shh y Nkx2.1 (Osorio et al., 2005). Esto sugiere que el paso evolutive a vertebrados con mandibula implica la formaciôn de una regiôn subpalial con un dominio pâlido-PO que probablemente sea consecuencia de la expresiôn de Shh y Nkx2.1 en el telencéfalo basai. De tal forma que la existencia de Shh y Nkx2.1 en la regiôn preôptiea en anuros y reptiles apoya la idea de la existencia de un pâlido como tal en el subpalio de estos grupos de vertebrados y la implicaciôn de dichos marcadores en este fenômeno. Ademâs, el présente estudio ha puesto de manifiesto la existencia de una regiôn preoptocomisural (POC) en anuros y reptiles similar a la descrita en mamiferos, de tal forma que los anfibios anuros, junto con pez pulmonado (Moreno y Gonzâlez, 2011), se revelan como los primeros anamniotas en los que aparece un POC con las mismas caracteristicas moleculares que en mamiferos, demostrando que la organizaciôn preôptiea mantiene una patrôn de estructura comùn en tetrâpodos (Gonzâlez y Northcutt, 2009), aunque se necesitaria un anâlisis exhaustivo de la expresiôn de Shh que pudiera homologar dichos territorios. Los datos obtenidos en el présenté estudio han puesto de manifiesto que en la transiciôn a amniotas se producen eambios en la organizaciôn hipotalâmica asi como en los propios limites de esta regiôn que definen su extensiôn dentro del prosencéfalo. De esta forma, en mamiferos se ha establecido recientemente una regiôn llamada preoptohipotalâmica (POH) caracterizada por la expresiôn de Nkx2.2 y que représenta el limite entre la regiôn preôptiea y el hipotâlamo (Flames et al., 2007; Bardet et al., 2006). Esta regiôn se ha identificado también en tortuga gracias a la existencia de expresiôn de Nkx2.2 en una regiôn comparable (Dominguez et al., 2011b) mientras que en Xenopus no se ha observado dicha expresiôn (Dominguez et al., 2012a), por lo que se sugiere que la existencia de este limite entre la regiôn preôptiea y el hipotâlamo es una caracteristica que aparece en tetrâpodos amniotas, evidenciando la posiciôn de Xenopus como un estado de transiciôn entre anamniotas y amniotas. Con respecto a la regiôn supraoptoparaventricular, como hemos visto en apartados anteriores, présenta un patrôn muy conservado a lo largo de la evoluciôn, manteniendo la expresiôn de Otp. Sin embargo, la 181 5. RESUMEN DE LOS RESULTADOS Y DISCUSION GENERAL transiciôn de anamniotas hacia amniotas ha tenido sus repercusiones en el patrôn de especificaciôn molecular de esta regiôn. En Xenopus, este territorio no présenta expresiôn de Pax6, mientras que en amniotas como reptiles y mamiferos la expresiôn de este faetor de transcripeiôn es una caracteristica compartida (Flames et al., 2007; Moreno et al., 2008b; Dominguez et al., 201 Ib; revisado en Moreno y Gonzalez, 2011). Ademâs, la conservaciôn de la expresiôn de Otp en esta regiôn se ha relacionado en todas las especies de vertebrados con la aportaciôn de eélulas Otp+ hipotalâmieas al eomplejo amigdalino (Garcia-Moreno et al., 2010; Moreno et al., 2010; Dominguez et al., 2012a). La expresiôn de Nkx2.1 en el territorio supraquiasmâtico va siendo cada vez mâs restringida a lo largo de la evoluciôn hasta su total desapariciôn. Asi, en anamniotas no tetrâpodos como pez cebra, Nkx2.1 y Shh ocupan la totalidad de la regiôn supraquiasmâtica (Rohr et al., 2001), y en Xenopus, un anamniota tetrâpodo, asi como en los amniotas reptiles y aves, esta expresiôn estâ restringida a un ùnico subdominio (van der Akker et al., 2008; Medina; 2008; Abellân y Medina, 2009; Dominguez et al., 2012a; Dominguez et al., 2012b) mientras que en mamiferos no existe expresiôn de Shh ni Nkx2.1 en el supraquiasmâtico (Puelles y Rubenstein, 2003; Bardet et al., 2010). Estos resultados muestran que la tendencia evolutiva de desapariciôn de la expresiôn de estos dos marcadores en la evoluciôn comienza en anuros, es decir, eon la apariciôn de tetrâpodos, y sugieren que esta graduai desapariciôn de Shh/Nkx2.1 en la regiôn supraquiasmâtica en la evoluciôn se correlaciona con la expansiôn palial producida en amniotas (Bruce y Neary, 1995; Striedter, 1997) asi como con la reducciôn del hipotâlamo alar en amniotas con respecto a anamniotas en pro de la expansiôn del tâlamo (van den Akker et al., 2008; revisado en Medina et al., 2008). Finalmente, el mantenimiento de la expresiôn parcial de Shh y Nkx2.1 en anuros, reptiles y aves indica que la restricciôn de su expresiôn en el supraquiasmâtico constituye un importante cambio que se produce de una manera graduai en la evoluciôn dependiendo de las necesidades adaptativas de cada grupo. De tal forma que el dominio supraquiasmâtico que expresa Shh y Nkx2.1 representaria la porciôn mâs conservada, preservando caracteristicas moleculares similares a anamniotas, mientras que el dominio Shh/Nkx2.1 negativo representaria la porciôn mâs evolucionada y que darâ origen al territorio supraquiasmâtico de mamiferos. Por otra parte, el anâlisis del perfil neuroquimico de las poblaciones TRH positivas y orexinérgicas en el supraquiasmâtico revelô una expresiôn diferencial de estos marcadores entre anuros, donde se detectaron, y en reptiles, en los que no se detectaron poblaciones positivas para ambos marcadores (Dominguez et al., 2008; 2010a; Lôpez et al., 2008, 2009a). Estas diferencias en la expresiôn de poblaciones celulares postmitôticas implican una diferencia en la individualizaciôn de los distintos nùcleos que forman el territorio supraquiasmâtico en reptiles. La desapariciôn de estas poblaciones en la transiciôn anamnio-amniota sugiere una diferente implicaciôn en reptiles en la regulaciôn de los diferentes procesos homeostâticos y neuroendocrinos llevados a cabo por la regiôn supraquiasmâtica. Con respecto al hipotâlamo basai, se ha revelado como la regiôn hipotalâmica con un patrôn de organizaciôn mâs conservado en la evoluciôn. Los datos recopilados en el présenté estudio han demostrado la existencia de un perfil molecular comùn tanto en anuros y reptiles como con el resto de tetrâpodos, formado por la expresiôn de Nkx2.1 en todo en territorio tuberal y mamilar, a excepciôn de la regiôn retromamilar. Mientras Shh, présenta un patrôn de expresiôn complementario a Nkx2.1, ocupando la regiôn tuberal y retromamilar y estando ausente en la mamilar propiamente dicha (Garcia-Calero et al., 2008; Morales-Delgado et al., 2011; Dominguez et al., 201 Ib; Dominguez et al., 2012b). La expresiôn de ambos marcadores es tipica de esta regiôn hipotalâmica basai en todos los vertebrados, donde se han revelado como piezas fundamentales en la inducciôn de la organizaciôn hipotalâmica (revisado en Medina, 2008). Los procesos esenciales de especificaciôn molecular de las distintas subdivisiones hipotalâmicas, se producen tempranamente en la evoluciôn. Los procesos histogenéticos que defmen el territorio hipotalâmieo estân muy conservados en los tetrâpodos, observando un patrôn de organizaciôn molecular y neuroquimico comùn entre anuros y reptiles, asi como caracteristicas especificas de la transiciôn anamnio- amniota, que revelan los diferentes procesos de divergencia que se dan en la evoluciôn. Bibliografîa Abellân A, Medina L. 2009. Subdivisions and derivatives of the chicken subpallium based on expression of LIM and other regulatory genes and markers of neuron subpopulations during development. J Comp Neurol 515(4):465-501. Abellân A, Vernier B, Rétaux S, Medina L. 2010. Similarities and differences in the forebrain expression of Lhxl and Lhx5 between chicken and mouse: Insights for understanding telencephalic development and evolution. J Comp Neurol 5 18(17):3512-3528. Acampora D, Postiglione MP, Avantaggiato V, Di Bonito M, Vaccarino FM, Michaud J, Simeone A. 1999. Progressive impairment of developing neuroendocrine cell lineages in the hypothalamus of mice lacking the Orthopedia gene. Genes Dev 13(21):2787-2800. Altman J, Bayer SA. 1986. The development o f the rat hypothalamus. Adv Anat Embryol Cell Biol 100:1- 178. Alunni A, Blin M, Deschet K, Bourrât F, Vernier P, Retaux S. 2004. Cloning and developmental expression patterns of Dlx2, Lhx7 and Lhx9 in the 182 KESUM EN DE EU S K E S L E l ALIUS Y U ISE ESIU N G EN ERA L medaka fish (Oryzias latipes). Mech Dev 121(7- 8):977-983. Aoki Y, Ono H, Yasuo S, Masuda T, Yoshimura T, Ebihara S, ligo M, Yanagisawa T. 2007. Molecular evolution of prepro-thyrotropin-releasing hormone in the chicken (Gallus gallus) and its expression in the brain. Zoolog Sci 24(7):686-692. Baillien M, Foidart A, Balthazart J. 1999. Regional distribution and control o f tyrosine hydroxylase activity in the quail brain. Brain Res Bull 48(1):31- 37. Bardet SM, Cobos I, Puelles E, Martinez-De-La-Torre M, Puelles L. 2006. Chicken lateral septal organ and other circumventricular organs form in a striatal subdomain abutting the molecular striatopallidal border. J Comp Neurol 499(5);745-767. Bardet SM, Martinez-de-la-Torre M, Northcutt RG, Rubenstein JL, Puelles L. 2008. Conserved pattern of OTP-positive cells in the paraventricular nucleus and other hypothalamic sites of tetrapods. Brain Res Bull 75(2-4):231-235. Bardet SM, Ferrân JL, Sanchez-Arrones L, Puelles L. 2010. Ontogenetic expression of sonic hedgehog in the chicken subpallium. Front Neuroanat 4. Batten TF, Cambre ML, Moons L, Vandesande F. 1990. Comparative distribution of neuropeptide- immunoreactive systems in the brain of the green molly, Poecilia latipinna. J Comp Neurol 302(4):893- 919. Batten TF, Moons L, Cambre ML, Vandesande F, Seki T, Suzuki M. 1990. Thyrotropin-releasing hormone- immunoreactive system in the brain and pituitary gland of the sea bass (Dicentrarchus labrax, Teleostei). Gen Comp Endocrinol 79(3):385-392. Blechman J, Borodovsky N, Eisenberg M, Nabel-Rosen H, Grimm J, Levkowitz G. 2007. Specification of hypothalamic neurons by dual regulation o f the homeodomain protein Orthopedia. Development 134(24)4417-4426. Brinkmann H, Venkatesh B, Brenner S, Meyer A. 2004. Nuclear protein-coding genes support lungflsh and not the coelacanth as the closest living relatives of land vertebrates. Proc Natl Acad Sci U S A 101(14):4900-4905. Brox A, Puelles L, Ferreiro B, Medina L. 2003. Expression of the genes GAD67 and Distal-less-4 in the forebrain of Xenopus laevis confirms a common pattern in tetrapods. J Comp Neurol 461(3):370-393. Bruce L. 2008. Evolution of the hypothalamus in amniotes. "Evolution and Embryological Development of Forebrain" in Encielopedic Reference of Neuroscience, eds M. D. Binder and N. Hirokawa (Springer-Verlag),. 1363-1367. Bruce LL, Neary TJ. 1995. The limbic system of tetrapods: a comparative analysis o f cortical and amygdalar populations. Brain Behav Evol 46(4- 5):224-234. Bulfone A, Puelles L, Porteus MH, Frohman MA, Martin GR, Rubenstein JL. 1993. Spatially restricted expression o f Dix-1, Dlx-2 (Tes-1), Gbx-2, and Wnt- 3 in the embryonic day 12.5 mouse forebrain defines potential transverse and longitudinal segmental boundaries. J Neurosci 13(7):3155- 3172. Butler A, Hodos W. 2005. Comparative vertebrate neuroanatomy. Sons JW, editor New Jersey: Wiley. Caqueret A, Coumailleau P, Michaud JL. 2005. Regionalization of the anterior hypothalamus in the chick embryo. Dev Dyn 233(2):652-658. Davis AM, Seney ML, Stallings NR, Zhao L, Parker KL, Tobet SA. 2004. Loss of steroidogenic faetor 1 alters cellular topography in the mouse ventromedial nucleus of the hypothalamus. J Neurobiol 60(4):424-436. de Leeea L, Kilduff TS, Peyron C, Gao X, Foye PE, Danielson PE, Fukuhara C, Battenberg EL, Gautvik VT, Bartlett FS, 2nd, Frankel WN, van den Pol AN, Bloom FE, Gautvik KM, Sutcliffe JG. 1998. The hypocretins: hypothalamus-specific peptides with neuroexcitatory activity. Proc Natl Acad Sci U S A 95(l):322-327. Del Carmen De Andres M, Anadon R, Manso MJ, Gonzalez MJ. 2002. Distribution of thyrotropin- releasing hormone immunoreactivity in the brain of larval and adult sea lampreys, Petromyzon marinus L. J Comp Neurol 453(4):323-335. Del Giacco L, Sordino P, Pistocchi A, Andreakis N, Tarallo R, Di Benedetto B, Cotelli F. 2006. Differential regulation of the zebrafish orthopedia 1 gene during fate determination of diencephalic neurons. BMC Dev Biol 6:50. Del Giacco L, Pistocchi A, Cotelli F, Fortunate AE, Sordino P. 2008. A peek inside the neurosecretory brain through Orthopedia lenses. Dev Dyn 237:2295-2303. Delaunay D, Heydon K, Miguez A, Schwab M, Nave KA, Thomas JL, Spassky N, Martinez S, Zale B. 2009. Genetic tracing of subpopulation neurons in the prethalamus of mice (Mus musculus). J Comp Neurol 512(l):74-83. Diaz ML, Becerra M, Manso MJ, Anadon R. 2001. Development of thyrotropin-releasing hormone immunoreactivity in the brain of the brown trout Salmo trutta fario. J Comp Neurol 429(2):299-320. Diaz ML, Becerra M, Manso MJ, Anadon R. 2002. Distribution of thyrotropin-releasing hormone (TRH) immunoreactivity in the brain of the zebrafish (Danio rerio). J Comp Neurol 450(1):45- 60. Diez-Roux G, Banfi S, Sultan M, Geffers L, Anand S, Rozado D, Magen A, Canidio E, Pagani M, Peluso 1, Lin-Marq N, Koch M, Bilio M, Cantiello 1, Verde R, De Masi C, Bianchi SA, Cicchini J, Perroud E, Mehmeti S, Dagand E, Schrinner S, Numberger A, Schmidt K, Metz K, Zwingmann C, Brieske N, Springer C, Hernandez AM, Herzog S, Grabbe F, Sieverding C, Fischer B, Schrader K, Brockmeyer M, Dettmer S, Helbig C, Alunni V, Battaini MA, Mura C, Henrichsen CN, Garcia- Lopez R, Echevarria D, Puelles E, Garcia-Calero 183 5. RESUMEN DE LOS RESULTADOS Y DISCUSION GENERAU E, Kruse S, Uhr M, Kauck C, Feng G, Milyaev N, Ong CK, Kumar L, Lam M, Semple CA, Gyenesei A, Mundlos S, Radelof U, Lehrach H, Sarmientos P, Reymond A, Davidson DR, Dolle P, Antonarakis SE, Yaspo ML, Martinez S, Baldock RA, Eichele G, Ballabio A. 2011. A high-resolution anatomical atlas of the transcriptome in the mouse embryo. PLoS Biol 9(l):el000582. Dominguez L, Lopez JM, Gonzalez A. 2008. Distribution of thyrotropin-releasing hormone (TRH) immunoreactivity in the brain of urodele amphibians. Brain Behav Evol 71(3):231-246. Dominguez L, Morona R, Joven A, Gonzâlez A, Lopez JM. 2010a. Immunohistochemical localization of orexins (hypocretins) in the brain of reptiles and its relation to monoaminergic systems. J Chem Neuroanat 39(l);20-34. Dominguez L, Gonzâlez A, Moreno N. 2010b. Sonic hedgehog expression during Xenopus laevis forebrain development. Brain Res 1347:19-32. Dominguez L, Gonzâlez A, Moreno N. 2011a. Ontogenetic distribution of the transcription factor nkx2.2 in the developing forebrain of Xenopus laevis. Front Neuroanat 5:11. Dominguez L, Moreno N, Morona R, Gonzâlez A. 2011b. Subdivisions of the turtle Pseudemys scripta hypothalamus based on the expression of regulatory genes and neuronal markers. Journal o f Comparative Neurology. DOl: 11-0165.22762 Dominguez L, Morona R, Gonzalez A, Moreno N. 2012a. Characterization of the alar hypothalamus of Xenopus laevis during development by molecular marker analysis. Journal of Comparative Neurology. (En preparacion) Dominguez L, Gonzâlez A, Moreno N. 2012b. Characterization of the basal hypothalamus of Xenopus laevis during development by molecular marker analysis. Journal o f Comparative Neurology. (En preparacion) Eaton JL, Glasgow E. 2007. Zebrafish orthopedia (otp) is required for isotocin cell development. Dev Genes Evol 217(2): 149-158. Eaton JL, Holmqvist B, Glasgow E. 2008. Ontogeny of vasotocin-expressing cells in zebrafish: selective requirement for the transcriptional regulators orthopedia and single-minded 1 in the preoptic area. Dev Dyn 237(4):995-1005. Ekimova IV, Pastukhov lu F. 2005. [GABA-ergic mechanisms o f the ventrolateral preoptic area of the hypothalamus in regulation of sleep and wakefulness and temperature homeostasis in pigeon Columba livia]. Zh Evol Biokhim Fiziol 41(4):356-363. Faraco JH, Appelbaum L, Marin W, Gaus SE, Mourrain P, Mignot E. 2006. Regulation of hypocretin (orexin) expression in embryonic zebrafish. J Biol Chem 281(40):29753-29761. Figdor MC, Stem CD. 1993. Segmental organization of embryonic diencephalon. Nature 363(6430):630-634. Flames N, Pla R, Gelman DM, Rubenstein JL, Puelles L, Marin O. 2007. Delineation of multiple subpallial progenitor domains by the combinatorial expression of transcriptional codes. J Neurosci 27(36):9682-9695. Gareia-Calero E, Femândez-Garre P, Martinez S, Puelles L. 2008. Early mammillary pouch specification in the course of prechordal ventralization of the forebrain tegmentum. Dev Biol 320(2):366-377. Garcia-Lôpez M, Abellân A, Legâz 1, Rubenstein JL, Puelles L, Medina L. 2008. Histogenetic compartments of the mouse centromedial and extended amygdala based on gene expression patterns during development. J Comp Neurol 506(1 ):46-74. Garcia-Lôpez R, Pombero A, Martinez S. 2009. Fate map of the chick embryo neural tube. Dev Growth Differ 51(3): 145-165. Garcia-Moreno F, Pedraza M, Di Giovannantonio LG, Di Salvio M, Lôpez-Mascaraque L, Simeone A, De Carlos JA. 2010. A neuronal migratory pathway crossing from diencephalon to telencephalon populates amygdala nuclei. Nat Neurosci 13(6):680-689. Gonzâlez A, Tuinhof R, Smeets WJ. 1993. Distribution of tyrosine hydroxylase and dopamine immunoreactivities in the brain of the South African clawed frog Xenopus laevis. Anat Embryol (Berl) 187(2): 193-201. Gonzâlez . A, Northcutt RG. 2009. An immunohistochemical approach to lungflsh telencephalic organization. Brain Behav Evol 74(l):43-55. Goshu E, Jin H, Lovejoy J, Marion JF, Michaud JL, Fan CM. 2004. Sim2 contributes to neuroendocrine hormone gene expression in the anterior hypothalamus. Mol Endocrinol 18(5):1251-1262. Hallstrom BM, Janke A. 2009. Gnathostome phylogenomics utilizing lungflsh EST sequences. Mol Biol Evol 26(2):463-471. Hamano K, Inoue K, Yanagisawa T. 1990. Immunohistochemical localization of thyrotropin- releasing hormone in the brain o f carp, Cyprinus carpio. Gen Comp Endocrinol 80(l):85-92. Herrick. 1910. The morphology of the forebrain in Amphibian and Reptilia. J Comp Neurol 20:413- 547. Hodos W. 2008. Evolution o f the hypothalamus in anamniotes. "Evolution and Embryological Development o f Forebrain" in Encielopedic Reference of Neuroscience, eds M D Binder and N Hirokawa (Springer-Verlag): 1361-1363. Hôkfelt T, Fuxe K, Johansson O, Jeffcoate S, White N. 1975. Distribution of thyrotropin-releasing hormone (TRH) in the central nervous system as revealed with i mmunohi stochemistry. Eur J Pharmacol 34(2):389-392. Hôkfelt T, Martensson R, Bjôrklund A, Kleinau S, Goldstein M. 1984. Distributional maps of tirosine hydroxilase immunoreactive neurons in the rat brain. En: Bjôrklund, A. Hôkfelt, T. (eds): 184 5. RESUM EN UE LUS R E S U L l AUUS Y UISLUSIU N U EN ERA L Classical Transmitters in the CNS. I. Handbook of Chemical Neuroanatomy. Amsterdam: Elsevier, pp 277-386. Huesa G, van den Pol AN, Finger TE. 2005. Differential distribution of hypocretin (orexin) and melanin- concentrating hormone in the goldfish brain. J Comp Neurol 488(4):476-491. Jôzsa R, Korf HW, Csemus V, Mess B. 1988. Thyrotropin-releasing hormone (TRH)- immunoreactive structures in the brain of the domestic mallard. Cell Tissue Res 251(2):441-449. Kâdâr A, Sanchez E, Wittmann G, Singru PS, Fuzesi T, Marsili A, Larsen PR, Liposits Z, Lechan RM, Fekete C. 2010. Distribution of hypophysiotropic thyrotropin-releasing hormone (TRH)-synthesizing neurons in the hypothalamic paraventricular nucleus of the mouse. J Comp Neurol 518(19):3948-3961. Kaslin J, Nystedt JM, Ostergard M, Peitsaro N, Panula P. 2004. The orexin/hypocretin system in zebrafish is connected to the aminergic and cholinergic systems. J Neurosci 24(11):2678-2689. Kawano H, Horie M, Honma S, Kawamura K, Takeuchi K, Kimura S. 2003. Aberrant trajectory of ascending dopaminergic pathway in mice lacking Nkx2.1. Exp Neurol 182(1):103-112. Kimura S, Hara Y, Pineau T, Femandez-Salguero P, Fox CH, Ward JM, Gonzalez FJ. 1996. The T/ebp null mouse: thyroid-specific enhancer-binding protein is essential for the organogenesis of the thyroid, lung, ventral forebrain, and pituitary. Genes Dev 10(1):60- 69. Kuhlenbeck H. 1973. The Central Nervous System of Vertebrates (Overall Morphologic Pattern, Vol. 3, Part 11), Karger. Kurrasch DM, Cheung CC, Lee FY, Tran PV, Hata K, Ingraham HA. 2007. The neonatal ventromedial hypothalamus transcriptome reveals novel markers with spatially distinct patterning. J Neurosci 27(50): 13624-13634. Lechan RM, Wu P, Jackson IM. 1986. Immunolocalization of the thyrotropin-releasing hormone prohormone in the rat central nervous system. Endocrinology 119(3):1210-1216. Lin X, State MW, Vaccarino FM, Greally J, Hass M, Leckman JF. 1999. Identification, chromosomal assignment, and expression analysis o f the human homeodomain-containing gene Orthopedia (OTP). Genomics 60(1):96-104. Lohr H, Ryu S, Driever W. 2009. Zebrafish diencephalic A 11-related dopaminergic neurons share a conserved transcriptional network with neuroendocrine cell lineages. Development 136(6): 1007-1017. Lôpez JM, Dominguez L, Gonzalez A. 2008. Immunohistochemical localization of thyrotropin- releasing hormone in the brain o f reptiles. J Chem Neuroanat 36(3-4):251-263. Lôpez JM, Dominguez L, Moreno N, Gonzâlez A. 2009a. Comparative immunohistochemical analysis of the distribution of orexins (hypocretins) in the brain of amphibians. Peptides 30(5):873-887. Lopez JM, Dominguez L, Moreno N, Morona R, Jôven A, Gonzâlez A. 2009b. Distribution of orexin/hypocretin immunoreactivity in the brain of the lungfishes Protoptenis dolloi and Neoceratodus forsteri. Brain Behav Evol 74(4):302-322. Machluf Y, Gutnick A, Levkowitz G. 2011. Development of the zebrafish hypothalamus. Ann N Y Acad Sci 1220:93-105. Marin O, Rubenstein JL. 2001. A long, remarkable journey: tangential migration in the telencephalon. Nat Rev Neurosci 2(11):780-790. Markakis EA. 2002. Development of the neuroendocrine hypothalamus. Front Neuroendocrinol 23(3):257-291. Martinez-de-la-Torre M, Pombal MA, Puelles L. 2011. Distal-less-like protein distribution in the larval lamprey forebrain. Neuroscience 178:270-284. Matz SP, Takahashi TT. 1994. Immunohistochemical localization of thyrotropin-releasing hormone in the brain of chinook salmon (Oncorhynchus tshawytscha). J Comp Neurol 345(2):214-223. Medina L. 2008. "Evolution and Embryological Development o f Forebrain" in Encielopedic Reference of Neuroscience, eds M. D. Binder and N. Hirokawa (Springer-Verlag), 1172-1192. Menuet A, Alunni A, Joly JS, Jeffery WR, Retaux S. 2007. Expanded expression of Sonic Hedgehog in Astyanax cavefish: multiple consequences on forebrain development and evolution. Development 134(5):845-855. Merchenthaler 1, Csemus V, Csontos C, Petrusz P, Mess B. 1988. New data on the immunocytochemical localization of thyrotropin- releasing hormone in the rat central nervous system. Am J Anat 181(4):359-376. Métin C, Alvarez C, Moudoux D, Vitalis T, Pieau C, Molnar Z. 2007. Conserved pattern of tangential neuronal migration during forebrain development. Development 134(15):2815-2827. Mitsuma T, Hirooka Y, Mori Y, Kayama M, Adachi K, Rhue N, Ping J, Nogimori T. 1999. Effects of orexin A on thyrotropin-releasing hormone and thyrotropin secretion in rats. Horm Metab Res 31(ll):606-609. Morales-Delgado N, Merchân P, Bardet SM, Ferrân JL, Puelles L, Diaz C. 2011. Topography of Somatostatin Gene Expression Relative to Molecular Progenitor Domains during Ontogeny of the Mouse Hypothalamus. Front Neuroanat 5:10. Moreno N, Bachy 1, Rétaux S, Gonzâlez A. 2004. LIM- homeodomain genes as developmental and adult genetic markers of Xenopus forebrain functional subdivisions. J Comp Neurol 472(1 ):52-72. Moreno N, Dominguez L, Rétaux S, Gonzâlez A. 2008a. Islet 1 as a marker of subdivisions and cell types in the developing forebrain of Xenopus. Neuroscience 154(4): 1423-1439. Moreno N, Rétaux S, Gonzâlez A. 2008b. Spatio- temporal expression o f Pax6 in Xenopus forebrain. Brain Res 1239:92-99. 185 5. RESUMEN DE LOS RESULTADOS Y DISCUSION GENERAL Moreno N, Gonzalez A, Retaux S. 2009. Development and evolution of the subpallium. Semin Cell Dev Biol 20(6):735-743. Moreno N, Morona R, Lôpez JM, Gonzalez A. 2010. Subdivisions of the Turtle Pseudemys scripta Subpallium Based on the Expression of Regulatory Genes and Neuronal Markers. JCompNeurol. Moreno N, Gonzalez A. 2011. The non-evaginated secondary prosencephalon of vertebrates. Front Neuroanat 5:12. Murakami Y, Ogasawara M, Sugahara F, Hirano S, Satoh N, Kuratani S. 2001. Identification and expression of the lamprey Pax6 gene: evolutionary origin of the segmented brain of vertebrates. Development 128(18):3521-3531. Nieuwenhuys R, Voogd J, van Huijzen C. 2008. The Human Central Nervous System. Springer, Germany. Osorio J, Mazan S, Retaux S. 2005. Organisation of the lamprey (Lampetra fluviatilis) embryonic brain: insights from LIM-homeodomain, Pax and hedgehog genes. Dev Biol 288(1): 100-112. Péczely P, Kiss JZ. 1988. Immunoreactivity to vasoactive intestinal polypeptide (VIP) and thyreotropin- releasing hormone (TRH) in hypothalamic neurons of the domesticated pigeon (Columba livia). Alterations following lactation and exposure to cold. Cell Tissue Res251(2):485-494. Peyron C, Tighe DK, van den Pol AN, de Lecea L, Heller HC, Sutcliffe JG, Kilduff TS. 1998. Neurons containing hypocretin (orexin) project to multiple neuronal systems. J Neurosci 18(23):9996-10015. Powley TL, Keesey RE 1970. Relationship of body weight to the lateral hypothalamic feeding syndrome. J Comp Physiol Psychol 70(l):25-36. Price M, Lemaistre M, Pischetola M, Di Lauro R, Duboule D. 1991. A mouse gene related to Distal- less shows a restricted expression in the developing forebrain. Nature 351(6329):748-751. Puelles L. 1995. A segmental morphological paradigm for understanding vertebrate forebrains. Brain Behav Evol 46(4-5):319-337. Puelles L. 2001. Brain segmentation and forebrain development in amniotes. Brain Res Bull 55(6):695- 710. Puelles L, Medina L. 2002. Field homology as a way to reconcile genetic and developmental variability with adult homology. Brain Res Bull 57(3-4):243-255. Puelles L, Rubenstein JL. 1993. Expression patterns of homeobox and other putative regulatory genes in the embryonic mouse forebrain suggest a neuromeric organization. Trends Neurosci 16(11):472-479. Puelles L, Rubenstein JL. 2003. Forebrain gene expression domains and the evolving prosomeric model. Trends Neurosci 26(9):469-476. Puelles L, Amat JA, Martinez-de-la-Torre M. 1987. Segment-related, mosaic neurogenetic pattern in the forebrain and mesencephalon of early chick embryos: 1. Topography o f AChE-positive neuroblasts up to stage HH18. J Comp Neurol 266(2):247-268. Puelles L, Kuwana E, Puelles E, Bulfone A, Shimamura K, Keleher J, Smiga S, Rubenstein JL. 2000. Palliai and subpallial derivatives in the embryonic chick and mouse telencephalon, traced by the expression of the genes Dlx-2, Emx-1, Nkx-2.1, Pax-6, and Tbr-1. J Comp Neurol 424(3):409-438. Puelles L, Martinez S, Martinez-de-la-Torre M, Rubenstein JL. 2004. The Rat Nervous System . Gene maps and related histogenetic domains in the forebrain and midbrain. In: G. Paxinos E, editor. San Diego: Elsevier. Rohr KB, Barth KA, Varga ZM, Wilson SW. 2001. The nodal pathway acts upstream of hedgehog signaling to specify ventral telencephalic identity. Neuron 29(2):341-351. Roth M, Bonev B, Lindsay J, Lea R, Panagiotaki N, Houart C, Papalopulu N. 2010. FoxGl and TLE2 act cooperatively to regulate ventral telencephalon formation. Development 137(9): 1553-1562. Ryu S, Mahler J, Acampora D, Holzschuh J, Erhardt S, Omodei D, Simeone A, Driever W. 2007. Orthopedia homeodomain protein is essential for diencephalic dopaminergic neuron development. Curr Biol 17(10):873-880. Sakurai T, Amemiya A, Ishii M, Matsuzaki 1, Chemelli RM, Tanaka H, Williams SC, Richarson JA, Kozlowski GP, Wilson S, Arch JR, Buckingham RE, Haynes AC, Carr SA, Annan RS, McNulty DE, Liu WS, Terrett JA, Elshourbagy NA, Bergsnm DJ, Yanagisawa M. 1998. Orexins and orexin receptors: a family of hypothalamic neuropeptides and G protein-coupled receptors that regulate feeding behavior. Cell 92(5): 1 page following 696. Sanchez-Arrones L, Ferrân JL, Rodriguez-Gallardo L, Puelles L. 2009. Incipient forebrain boundaries traced by differential gene expression and fate mapping in the chick neural plate. Dev Biol 335(1 ):43-65. Scholpp S, Wolf O, Brand M, Lumsden A. 2006. Hedgehog signalling from the zona limitans intrathalamica orchestrates patterning of the zebrafish diencephalon. Development 133(5):855- 864. Shimamura K, Hartigan DJ, Martinez S, Puelles L, Rubenstein JL. 1995. Longitudinal organization of the anterior neural plate and neural tube. Development 121(12):3923-3933. Shimogori T, Lee DA, Miranda-Angulo A, Yang Y, Wang H, Jiang L, Yoshida AC, Kataoka A, Mashiko H, Avetisyan M, Qi L, Qian J, Blackshaw S. 2010. A genomic atlas of mouse hypothalamic development. Nat Neurosci 13(6):767-775. Simmons DA, Yahr P. 2011. Distribution of catecholaminergic and peptidergic cells in the gerbil medial amygdala, caudal preoptic area and caudal bed nuclei o f the stria terminalis with a focus on areas activated at ejaculation. J Chem Neuroanat 41(1):13-19. 186 3. KESUM EN LIE EU!> K ESU E1A U U 5 Y LIl^LUMUrN UENEKAE Smeets WJAJ, Gonzalez A. 2000. Catecholamine systems in the brain o f vertebrates: new perspectives through a comparative approach. Brain Res Brain Rev. 33(2- 3): 308-79. Smeets WJAJ, Jonker AJ, Hoogland PV. 1987. Distribution of dopamine in the forebrain and midbrain of the red-eared turtle, Pseudemys scripta elegans, reinvestigated using antibodies against dopamine. Brain Behav Evol 30:121-142. Soma M, Aizawa H, Ito Y, Maekawa M, Osumi N, Nakahira E, Okamoto H, Tanaka K, Yuasa S. 2009. Development of the mouse amygdala as revealed by enhanced green fluorescent protein gene transfer by means of in utero electroporation. J Comp Neurol 513(1):113-128. Striedter GF. 1997. The teleneephalon of tetrapods in evolution. Brain Behav Evol 49(4):179-213. Sutcliffe JG, de Lecea L. 2000. The hypocretins: excitatory neuromodulatory peptides for multiple homeostatic systems, including sleep and feeding. J Neurosci Res 62(2):161-168. Swaab DF. 2003. The human hypothalamus: Basic and clinical aspects. Part 1: Nuclei o f the human hypothalamus. Amsterdam: Elsevier. Takezaki N, Figueroa F, Zaleska-Rutczynska Z, Takahata N, Klein J. 2004. The phylogenetic relationship of tetrapod, coelacanth, and lungflsh revealed by the sequences of forty-four nuclear genes. Mol Biol Evol 21(8):1512-1524. Tessmar-Raible K, Raible F, Christodoulou F, Guy K, Rembold M, Hausen H, Arendt D. 2007. Conserved sensory-neurosecretory cell types in annelid and fish forebrain: insights into hypothalamus evolution. Cell 129(7): 1389-1400. Tsuruo Y, Hokfelt T, Visser T. 1987. Thyrotropin releasing hormone (TRH)-immunoreactive cell groups in the rat central nervous system. Exp Brain Res 68(1):213-217. van den Akker WM, Brox A, Puelles L, Durston AJ, Medina L. 2008. Comparative functional analysis provides evidence for a crucial role for the homeobox gene Nkx2.1/Titf-1 in forebrain evolution. J Comp Neurol 506(2):211-223. van den Pol AN. 1982. Lateral hypothalamic damage and body weight regulation: role of gender, diet, and lesion placement. Am J Physiol 242(3):R265-274. van den Pol AN, Gao XB, Obrietan K, Kilduff TS, Belousov AB. 1998. Presynaptic and postsynaptic actions and modulation of neuroendocrine neurons by a new hypothalamic peptide, hypocretin/orexin. J Neurosci 18(19):7962-7971. Vandenbome K, Roelens SA, Darras VM, Kuhn ER, Van der Geyten S. 2005. Cloning and hypothalamic distribution of the chicken thyrotropin-releasing hormone precursor cDNA. J Endocrinol 186(2):387- 396. Vieira C, Garda AL, Shimamura K, Martinez S. 2005. Thalamic development induced by Shh in the chick embryo. Dev Biol 284(2):351-363. Vieira C, Martinez S. 2006. Sonic hedgehog from the basal plate and the zona limitans intrathalamica exhibits differential activity on diencephalic molecular regionalization and nuclear structure. Neuroscience 143(1): 129-140. Wang W, Lufkin T. 2000. The murine Otp homeobox gene plays an essential role in the specification of neuronal cell lineages in the developing hypothalamus. Dev Biol 227(2):432-449. Yee CL, Wang Y, Anderson S, Ekker M, Rubenstein JL. 2009. Arcuate nucleus expression of NKX2.1 and DLX and lineages expressing these transcription factors in neuropeptide Y(+), proopiomelanocortin(+), and tyrosine hydroxylase(-t-) neurons in neonatal and adult mice. J Comp Neurol 517(l):37-50. Zhao XF, Suh CS, Prat CR, Ellingsen S, Fjose A. 2009. Distinct expression of two foxgl paralogues in zebrafish. Gene Expr Patterns 9(5):266-272. 187 188 6. Conclusiones t». CUNLUJMUNES El hipotâlamo se eneuentra dividido dorso-ventralmente en euatro grandes regiones, supraoptoparaventrieular y supraquiasmâtiea, en el llamado hipotâlamo alar y tuberal y mamilar, en el hipotâlamo basai. Estas a su vez se eneuentran divididas rostro-eaudalmente en dos poreiones difereneiadas por su patron de espeeifieaeiôn génieo. Frente a regiones altamente eonservadas, prineipalmente en el hipotâlamo basai, el hipotâlamo alar présenta un mayor numéro de difereneias que apuntan a una mayor divergeneia evolutiva en esta region en la transieiôn anamnio-amniota. Los estudios quimioarquiteetônieos basados en la loealizaeiôn de la hormona liberadora de tirotropina y orexinas, en los modelos de anfibios y reptiles, demostraron patrones de distribuciôn muy similares entre estos grupos, que representan la transiciôn anamnio-amniota. En concreto en el hipotâlamo se identifïcaron poblaciones neuronales fâcilmente homologables en los vertebrados, sugiriendo una organizaciôn muy conservada neuroanatômicamente. La regiôn preôptiea, clâsicamente eonsiderada hipotalâmica, estâ compuesta por dos subregiones, preôptiea y comisural, ambas defînidas por la expresiôn del gen Nkx2.1 y la ausencia de Isll en la regiôn comisural. Este patrôn de organizaciôn apoya su origen telencefâlico, como ha sido demostrado en aves y mamiferos. Entre la regiôn preôptiea y el hipotâlamo se ha identificado en Pseudemys el limite preoptohipotalâmico en base a la expresiôn de Nkx2.2, como en otros amniotas. Este limite no se ha encontrado en Xenopus, lo que sugiere diferencias en la transiciôn anamnio-amniota en el establecimiento de los limites telencéfalo-hipotâlamicos. La regiôn supraoptoparaventricular présenta un patrôn de especificaciôn molecular muy conservado, con la expresiôn del faetor de transcripeiôn Otp en ambos modelos. Asi como su regionalizaciôn rostrocaudal en base a la expresiôn diferencial de Nkx2.2 en la porciôn rostral. Sin embargo, existen discrepancias en la expresiôn del gen Pax6, sôlo identificado en Pseudemys, como en otros amniotas, pero no en Xenopus, aportando nue vas diferencias entre anamnios y amniotas. El hipotâlamo supraoptoparaventricular constituye la principal porciôn neuroendocrina del hipotâlamo y contiene numerosas poblaciones neuronales neuropeptidicas, como las que poseen la hormona liberadora de tirotropina, mesotocina y somatostatina, muy eonservadas en la evoluciôn. La expresiôn en ambos modelos de Otp en la regiôn de la amfgdala medial, tradicionalmente eonsiderada de origen exclusivamente telencefâlico, sugiere que estas células proceden en el desarrollo de regiones hipotalâmicas supraoptoparaventriculares 191 que emigrarian hasta su posiciôn final, como ha sido descrito en mamiferos, constituyendo un sistema muy conservado en la evoluciôn. La regiôn supraquiasmâtica, definida por la expresiôn de Isll, présenta una regionalizaciôn rostrocaudal en base a la expresiôn de Nkx2.1 en la porciôn rostral, como ha sido descrito en aves, pero a diferencia de lo observado en mamiferos. Esta discrepancia sugiere la pérdida de esta subregiôn en la evoluciôn entre amniotas mamiferos y el resto de los tetrâpodos. Este hecho ha sido relacionado con la expansiôn talâmica y palial en detrimento de la regiôn supraquiasmâtica que tiene lugar en mamiferos y no asi en otros amniotas y anamniotas. La regiôn tuberal, definida por la expresiôn de Nkx2.1 y Isll, présenta una regionalizaciôn rostrocaudal en base a la expresiôn de Otp en la porciôn rostral. Esto se ha relacionado con el origen del nùcleo arcuado en todos los vertebrados, con un patrôn de especificaciôn y organizaciôn muy conservado en la escala evolutiva. La banda mamilar estâ definida por la expresiôn de Nkx2.1 en la porciôn rostral mamilar y xShh en la regiôn caudal retromamilar. La expresiôn de Otp en la porciôn rostral apoya su identificaciôn como mamilar, asi como la presencia de poblaciones catecolaminérgicas en esta misma regiôn con un patrôn de expresiôn muy conservado. Con los marcadores genéticos y neuroquimicos empleados se ha comprobado la existencia de una organizaciôn hipotalâmica conservada en los modelos de anfibio y reptil utilizados, comparable a lo descrito en aves y mamiferos, lo que sugiere la existencia de un patrôn de organizaciôn hipotalâmieo comùn en todos los tetrâpodos. 192 7. Anexo 0) CM \ .^1 %■a S3 a « F - I C O C O Z Z Q Q i 2 _j I Dll DO 8. Agradecimientos No se si a lo largo de estos anos he conseguido trasmitir lo agradecida que he estado a todas las personas que han participado en esta travesla, de modo que es hora de al menos intentarlo. En primer lugar, debo agradecer al Dr Agustin Gonzalez la oportunidad de trabajar en su laboratorio. Gracias por el apoyo y confianza. En el recuerdo quedan sus tarareos al entrar en el laboratorio.. .Laurigena!. A la Dra Margarita Munoz quiero agradecerle sus constantes palabras amables y su enorme empatia durante estos anos. A mi directora la Dr Nerea Moreno, por ser referenda absoluta de tenacidad e inquietud. Espero que el haber trabajado este tiempo a su lado baya servido para que se me peguen algunas de sus cualidades. Gracias por tu espiritu critico constructivo y tu objetividad, por no poner barreras, y por haberme recogido cuando mâs lo necesitaba. A mi director el Dr Jésus Lôpez, gracias por haberme iniciado en el mundo de la investigaciôn, por ensenarme un buen método de trabajo y a ser constante en ello. En el recuerdo me quedan largas conversaciones de ânimo y confianza en mi trabajo. A mis companeros, diria que no tengo palabras.. pero si que las tengo. A Ruth, por haber tenido tanta paciencia con esta doctoranda tan preguntona...por estar siempre dispuesta y disponible para resolverme problemas, por las risas y tantos buenos momentos y por haberme ensenado tantas cosas. A Albertito Joven, gracias por todas las risas del mundo, fuiste un soplo de aire fresco para mi, y lo sigues siendo, por acompanarme en todos los momentos tanto buenos como malos y por ser tan increfblemente auténtico. A Sandra, por sus palabras de carino y ânimo y su comprensiôn, se que dejo en muy buenas manos la ciclopamina...A Jorge, por su etema sonrisa y su continua disposiciôn. Imposible olvidarme de los companeros del Dpto de Biologfa Celular. Al primero que tengo que agradecerle es al Dr Benjamin Femândez, fuente inagotable de ânimo y palabras amables desde el corazôn. En el recuerdo siempre quedarân sus palabras de aliento desde el marco de la puerta, gracias por contagiarme su alegria. Tengo que destacar al Dr Inigo Azcoitia, gracias creo que.. por todo, desde que le conozco creo que he mejorado considerablemente mi manera de expresarme...Gracias por transmitir constantemente sabiduria, en mi opinion esa es una de las caracteristicas de un gran cientifico.Gracias al resto de companeros del Departamento, desde todos los miembros del grupo VIP, pasando por los chicos Zapata y por supuesto las técnicos Isabel y Mar, y la secretaria Teresa, gracias por vuestra constante preocupaciôn por mi y por haberme ayudado todos estos anos, creo que os debo unos eppys... Ahora viene un GRACIAS en mayùsculas para todos y cada uno de los miembros del laboratorio del Dr Salvador Martinez en el Institute de Neurociencias de Alicante, primero por haberme acogido con los brazos abiertos, por haberme ayudado incondicionalmente en absolutamente todo lo que necesitara y ademâs, por hacerlo con una sonrisa. En especial tengo que destacar al “zulo team”, con quién mâs estrechamente comparti mi estancia, dirigido por el Dr Diego Echevarria Aza, al que le tengo que agradecer simplemente el ser como es, asi que gracias por haberme contagiado tu espiritu investigador y tu curiosidad. A mis amigos, por estar ahi siempre. Gracias por ayudarme en todo y por hacerme feliz dia a dia. Todo esto empezô un 2 de octubre de 2001 jy de ahi al Nobel!...alguno lo ganarâ ^no?; sois los mejores. Lauri, Rous, Javi, Mery, José...gracias por sacarme siempre una sonrisa, por creer que no hay limites, por confiar en mi y por demostrar que tanto el éxito como los fracasos no valen de nada si no tienes con quién compartirlos. A mi hermana Carol, por estar SIEMPRE, por ser mi principal apoyo, por ensenarme, por haber sido y seguir siendo el mejor referente a seguir, porque por muy mal que vayan las cosas y por muy negro que esté el panorama.. .siempre estâ ella en forma de luz para guiarme. Gracias por tu fortaleza, por hacer que mi vida sea mejor y por no dejarme caer. A Juan Pablo, mi hermano en funciones, por confiar en que yo puedo y por contagiarme felicidad y seguridad, por estar siempre ahi y tener siempre las palabras correctas que me hagan seguir adelante. A mis padres, mis queridos padres, porque a ellos les debo TODO. Por impulsarme siempre a ser mejor, por ensenarme constantemente a vivir, por confiar en mi y creer que si me propongo algo soy capaz de conseguirlo. Gracias a los dos por vuestra fuerza y valor, por vuestro espiritu de superaciôn y por haber sacrifïcado todo por y para nosotras. 198