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

脊椎动物肠神经系统（ENS）是周围神经系统的最大部分， 主要源自尾侧后脑的迷走神经嵴，迁移到前肠 并沿着整个肠道长度，分化成许多不同的神经元亚型。在人类中， ENS 形成缺陷会导致先天性巨结肠症或结肠无神经节生成。当 ENS 神经元玩耍时 令人惊讶的是，对于调节胃肠运动的关键作用，人们对如何或什么控制神经元知之甚少。 ENS 中的谱系规范。最近出现的单细胞技术有望帮助阐明 鉴定神经元细胞类型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具有多种优势，因为它们 简化的肠神经系统、基因操作的可及性和成像设施。类似于 羊膜动物中，斑马鱼肠道含有神经嵴衍生的神经元亚型，范围从血清素能、 胆碱能和多巴胺能神经元到 VIP、P 物质和含一氧化氮 (NO) 的神经元。在这里，我们 提议对不同发育阶段的个体肠前体和神经元进行单细胞 RNA-seq ENS 发育阶段（2-6 dpf）。 ENS 中候选转录和信号转导因子的功能 神经元规范将通过 CRISPR-Cas9 扰动实验在斑马鱼和小鸡身上进行测试。 最后，单细胞 ATAC-seq 将用于识别并解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： 目标 1：使用单细胞分辨率对肠神经嵴衍生细胞进行转录分析 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）解剖和分析数千个肠前体细胞和神经元细胞 从斑马鱼胚胎肠道中分选出来。我们将验证感兴趣基因的表达，特别是 转录因子和信号分子，使用杂交链式反应（HCR）并推断发育 从祖细胞到神经元分化的轨迹。 目标 2：转录因子在斑马鱼和雏鸡 ENS 神经元亚型分化中的作用。 我们将挖掘 scRNA-seq 来识别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 使用 CRISPR-Cas9 介导的斑马鱼和雏鸡基因敲除。 目标 3：使用单细胞识别与 ENS 神经元分化相关的活性增强子 ATAC 序列。我们将使用单细胞 ATAC-seq 来识别和测试在 发育中的斑马鱼 ENS 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，并进行突变和羊膜动物保护测试。

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.