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.

脊椎动物肠神经系统（ENS）是周围神经系统中最大的部分，主要源自在尾声后发生的迷走神经rest，迁移到前肢并沿着肠道的整个长度区分了许多不同的神经元亚型。在ENS神经元发挥作用的同时，在ENS形成中的缺陷会导致Hirschsprung氏病或结肠agligliogenation。在调节胃肠道运动中的关键作用，关于如何或什么控制神经元知之甚少 ENS中的血统规范。鉴定神经元细胞类型和肠神经元分化的分子机制。斑马鱼为解决ENS开发中的重要问题提供了一些优势，因为他们简化的肠神经系统，对基因操纵的可及性和成像的设施。斑马鱼的羊膜均包含神经rest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染色为VIP，物质P和一氧化氮（NO） - 含有神经元。提议在不同发育中执行个体前体和神经元的单个Celle细胞RNA-Seq 发育中的ENS中的阶段（2-6 DPF）。神经元规范将通过斑马鱼和小鸡的CRISPR-CAS9扰动实验进行测试。最后，将使用单个Celle细胞ATAC-SEQ识别和解剖Enterix来构建ENS基因监管网络，我们建议执行以下目的： AIM 1：使用肠神经rest衍生的细胞的转录分析，以其他分辨率使用单细胞RNA-seq和多重荧光原位杂交。肠道前体和神经元被解剖的每时间点（2-6天后）的数千个细胞（2-6天）从斑马鱼胚胎肠道上分类。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。 AIM 2：转录因子在斑马鱼和小鸡中ENS神经元亚型区分区分中的作用。我们将挖掘scrna-seq，以识别其表达与祖细胞相关的转录因子用于功能验证在斑马鱼和小鸡中使用CRISPR-Cas9介导的敲除。 AIM 3：使用单个细胞在ENS中识别与神经元分化相关的主动增强子 atac-seq。神经元前体和发展中的斑马鱼ENS中的神经元。进行测试。