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

脊椎动物肠道神经系统(ENS)是周围神经系统的最大部分， 主要来源于迷走神经脊束，它出现在尾侧后脑，迁移到前脑 沿着肠道的整个长度，分化成许多不同的神经元亚型。在人类身上， ENS形成缺陷会导致先天性巨结肠，或结肠无神经节细胞形成。当ENS神经元在玩的时候 在调节胃肠运动中的关键作用，令人惊讶的是，人们对神经元如何或什么控制知之甚少 ENS中的血统规范。最近出现的单细胞技术有望帮助阐明 肠神经细胞分化的神经细胞类型和分子机制的鉴定。 斑马鱼为解决ENS发展中的重要问题提供了几个优势，这是因为它们的 简化的肠道神经系统，基因操作的可及性和成像的便利性。类似于 羊膜，斑马鱼的肠道含有神经脊源性神经元亚型，从5-羟色胺能， 胆碱能和多巴胺能神经元向血管活性肠肽、P物质和一氧化氮(NO)能神经元转化。在这里，我们 建议对不同发育阶段的单个肠道前体和神经元进行单细胞RNA-SEQ 发育期(2-6个DPF)。候选转录和信号因子在ENS中的作用 神经元的规格将通过CRISPR-Cas9在斑马鱼和雏鸟身上的扰动实验进行测试。 最后，将使用单细胞atac-seq来鉴定并剖析肠道增强子以构建ens基因。 监管网络。我们建议实现以下目标： 目的1：在单个细胞分辨率下对肠神经嵴来源的细胞进行转录图谱分析 单细胞rna-seq和多重荧光原位杂交。我们将进行单细胞RNA-seq 每个时间点(受精后2-6天)解剖数以千计的肠道前体细胞和神经元 从斑马鱼胚胎的内脏中分离出来。我们将特别验证感兴趣基因的表达。 转录因子和信号分子，利用杂交链式反应(HCR)和推断发育 从祖细胞到神经元分化的轨迹。 目的2：转录因子在斑马鱼和雏鸡神经元亚型分化中的作用。 我们将挖掘scrna-seq以确定其表达与祖细胞相关的转录因子。 状态(例如hey1a)和各种神经元亚型标记(例如ebf1a、etv1、Klf6a、Insm1a)用于功能验证 在斑马鱼和雏鸡中使用CRISPR-Cas9介导的基因敲除。 目的3：利用单细胞鉴定与神经元分化相关的活性增强剂 ATAC-SEQ.我们将使用单细胞ATAC-SEQ来识别和测试假定的调控元件在 斑马鱼胚胎发育中的神经元前体和分化神经元。假定的增强区域将 测试它们在斑马鱼中驱动ENS表达的能力，突变斑马鱼，并测试其与羊膜的保存。

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.