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

发育中的肠神经系统神经细胞谱系决定的转录调控

基本信息

批准号：
10646306
负责人：
Marianne Bronner
金额：
$ 52.18万
依托单位：
CALIFORNIA INSTITUTE OF TECHNOLOGY
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-06-15 至 2027-04-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10646306
关键词：
ATAC-seq Autonomic nervous system Birth CRISPR/Cas technology Cell Lineage Cells Chick Childhood Colon Complex Congenital Abnormality Congenital Megacolon Data Set Defect Development Disease Distal Embryo Enhancers Enteral Enteric Nervous System Event Fertilization Fluorescent in Situ Hybridization Ganglia Gastrointestinal Motility Gene Expression Gene Expression Profiling Genetic Genetic Transcription Human Individual Intestines Jaw Knock-out Lead Left Length Life Mediating Mining Molecular Mus Mutate Mutation Nervous System control Neural Crest Neural Crest Cell Neuronal Differentiation Neurons Neurotransmitters Nitric Oxide Pattern Peripheral Nervous System Play Population Primitive foregut structure Proteins Reaction Regulatory Element Reporter Resolution Role Side Signal Pathway Signal Transduction Signaling Molecule Sorting Specific qualifier value Stream Substance P Testing Time Transcriptional Regulation Transgenic Organisms Undifferentiated Validation Vertebrates Zebrafish cell motility cell type cholinergic neuron dopaminergic neuron experimental study gene regulatory network genetic manipulation hindbrain imaging facilities interest migration nervous system development progenitor public health relevance single cell technology single molecule single-cell RNA sequencing teleost tool transcription factor transcriptome

项目摘要

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.

脊椎动物的肠神经系统（ENS）是周围神经系统中最大的一部分，