Gene Regulatory Analysis of the Developing Enteric Nervous System of Zebrafish (Danio rerio)

Citation

Li, Can (2025) Gene Regulatory Analysis of the Developing Enteric Nervous System of Zebrafish (Danio rerio). Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/mc6z-3k03. https://resolver.caltech.edu/CaltechTHESIS:02172025-204256436

Abstract

Neural crest cells give rise to the neurons of the enteric nervous system (ENS) that innervate the gastrointestinal tract to regulate gut motility. The immense size and distinct subregions of the gut present a challenge to understanding the spatial organization and sequential differentiation of different neuronal subtypes. To tackle this, we profile enteric neurons and progenitors at single cell resolution during zebrafish embryonic and larval development to provide a near complete picture of transcriptional changes that accompany emergence of ENS neurons throughout the gastrointestinal tract. Multiplex spatial RNA transcript analysis was then used to reveal the temporal order and distinct localization patterns of neuronal subtypes along the length of the gut. Next, we show that functional perturbation of select transcription factors Ebf1a, Gata3 and Satb2 alters the cell fate choice, respectively, of inhibitory, excitatory and serotonergic neuronal subtypes in the developing ENS. To decipher the molecular mechanism underlying the development of ENS, we further performed single cell ATAC-seq to profile the epigenetic landscape of the developing ENS. Together with CUT&RUN results, we found the master regulator Phox2bb harbors extensive binding sites throughout the genome and plays versatile roles in neuronal differentiation, including regulating progenitor gene Sox10, activating transcription factors Phox2a and Insm1b for early neural development and regulating genes Etv1 and Hmx3a for neuronal differentiation. Integrated with single cell RNA-seq analysis, we further reconstruct a putative gene regulatory circuit involving in the specification and maturation of ENS neurons.

Thesis Files