Transcriptional regulation of neuronal cell lineage decisions in the developing enteric nervous system

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The vertebrate enteric nervous system (ENS), the largest portion of the peripheral nervous system, mostly derives from the vagal neural crest which arises in the caudal hindbrain, migrates to the foregut and along the entire length of the gut, differentiating into many different neuronal subtypes. In humans, defects in ENS formation cause Hirschsprung’s Disease, or colonic agangliogenesis. While ENS neurons play critical roles in regulating gastrointestinal motility, surprisingly little is known about how or what controls neuronal lineage specification in the ENS. The recent advent of single-cell technologies promises to help elucidate identification of neuronal cell types and molecular mechanisms underlying enteric neuronal differentiation. Zebrafish offer several advantages for tackling important questions in ENS development due to their simplified enteric nervous system, accessibility to genetic manipulation and facility of imaging. Similar to amniotes, the zebrafish gut contains neural crest-derived neuronal subtypes, ranging from serotonergic, cholinergic and dopaminergic neurons to VIP, Substance P and Nitric Oxide (NO)-containing neurons. Here, we propose to perform single cell RNA-seq of individual enteric precursors and neurons at different developmental stages (2-6 dpf) within the developing ENS. The function of candidate transcription and signaling factors in ENS neuronal specification will be tested by CRISPR-Cas9 perturbation experiments in both zebrafish and chick. Finally, single cell ATAC-seq will be used to identify and then dissect enteric enhancers to build an ENS gene regulatory network. We propose to perform the following aims: Aim 1: Transcriptional profiling of the enteric neural crest-derived cells at individual cell resolution using single cell RNA-seq and multiplex fluorescent in situ hybridization. We will perform single cell RNA-seq on thousands of cells per time point (2-6 days post-fertilization) of enteric precursors and neurons dissected and sorted from the zebrafish embryonic gut. We will validate expression of genes of interest, in particular transcription factors and signaling molecules, using hybridization chain reaction (HCR) and infer developmental trajectories from progenitor to neuronal differentiation. Aim 2: Role of transcription factors in differentiation of ENS neuronal subtypes in zebrafish and chick. We will mine the scRNA-seq to identify transcription factors whose expression correlates with the progenitor state (e.g. hey1a) and various neuronal subtype markers (e.g. ebf1a, etv1, Klf6a, Insm1a) for functional validation using CRISPR-Cas9 mediated knock-out in zebrafish and in chick. Aim 3: Identifying active enhancers associated with neuronal differentiation in the ENS using single cell ATAC-seq. We will use single cell ATAC-seq to identify and test putative regulatory elements functioning in neuronal precursor and differentiating neurons in the developing zebrafish ENS. Putative enhancing regions will be tested for their ability to drive ENS expression in zebrafish, mutated and tested for conservation with amniotes.