Temporal Expression of Hox Genes and Phox2b in the Rat Nucleus Ambiguus During Development: Implications on Laryngeal Innervation

Abstract

Objectives Recurrent laryngeal nerve (RLN) injury results in synkinetic reinnervation and vocal fold paralysis. Investigation of cues expressed in the developing brainstem that influence correct selective targeting of intrinsic laryngeal muscles may elucidate post-injury abnormalities contributing to non-functional reinnervation. Primary targets of interest were Hoxb1 and Hoxb2, members of the Hox family that create overlapping gradients in the developing brain, and their target Phox2b, a transcription factor necessary for cranial nerve branchio- and visceromotoneuron survival.

Methods Rat embryos at developmental days E14, E16, E18, and E20 (4 animals/age) were sectioned for RNA in situ hybridization to detect Hoxb1, Hoxb2, and Phox2b mRNA within the brainstem. Slides were costained with Islet1 antibody for identification of the nucleus ambiguus. Results were confirmed using immunohistochemistry. Sections were imaged on a confocal microscope. RNA and protein expressions were quantified using QuPath. Statistical analyses were performed using R.

Results Hoxb1, Hoxb2, and Phox2b expressions varied according to embryologic age. Hoxb1 and Hoxb2 expression peaked at E16, with significant decreases at E18 and E20 (one-way ANOVA p = 0.001 for both). Phox2b expression was highest at E14 and trended downward with increased embryologic age (one-way ANOVA p = 0.005).

Conclusion Peak expression of Hoxb1 and Hoxb2 is observed at time points when the RLN arrives at the larynx and begins to branch toward individual muscles, positioning these gene products to be involved in cueing laryngeal motoneuron identity and target identification. Higher expression of Phox2b earlier in development suggests a role in laryngeal motoneuron formation.