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

脊椎动物肠神经系统（ENS），周围神经系统的最大部分， 主要来源于迷走神经嵴，起源于后脑尾部，迁移到前肠 并且沿着整个肠道，分化成许多不同的神经元亚型。在人类中， ENS形成的缺陷导致先天性巨结肠或结肠无神经节细胞生成。当ENS神经元 在调节胃肠道运动中起着关键作用，令人惊讶的是，人们对如何或什么控制神经元运动知之甚少。 最近出现的单细胞技术有望帮助阐明 神经元细胞类型的鉴定和肠神经元分化的分子机制。 斑马鱼在解决ENS开发中的重要问题方面提供了几个优势， 简化的肠神经系统，遗传操作的可及性和成像的便利性。类似于 斑马鱼肠道含有神经嵴衍生的神经元亚型，范围从多巴胺能， 胆碱能和多巴胺能神经元向VIP、P物质和一氧化氮（NO）能神经元转化。这里我们 建议在不同发育阶段对单个肠前体和神经元进行单细胞RNA-seq， 在发育中的ENS中，转录和信号传导因子的功能 神经元特化将通过斑马鱼和鸡中的CRISPR-Cas9扰动实验来测试。 最后，单细胞ATAC-seq将用于鉴定并随后解剖肠增强子以构建ENS基因。 监管网络。我们建议实现以下目标： 目的1：使用单个细胞分辨率的肠神经嵴衍生细胞的转录谱分析 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）解剖数千个肠前体细胞和神经元， 是从斑马鱼胚胎肠道中分离出来的我们将验证感兴趣的基因的表达，特别是 转录因子和信号分子，使用杂交链反应（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鸡ENS神经元亚型分化中的作用。 我们将挖掘scRNA-seq以鉴定其表达与祖细胞相关的转录因子。 状态（例如hey 1a）和各种神经元亚型标志物（例如ebf 1a、etv 1、Klf 6a、Insm 1a）用于功能验证 在斑马鱼和鸡中使用CRISPR-Cas9介导的敲除。 目的3：利用单细胞技术鉴定ENS中与神经元分化相关的活性增强子 ATAC-seq.我们将使用单细胞ATAC-seq来鉴定和测试推定的调控元件， 神经元前体和分化的神经元在发育中的斑马鱼ENS。推定的增强区域将 测试它们在斑马鱼中驱动ENS表达的能力，突变并测试它们与斑马鱼的保守性。

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.