Lineage and transcriptional analysis of sacral neural crest contribution to the enteric nervous system

骶神经嵴对肠神经系统贡献的谱系和转录分析

• 批准号: 10679674
• 负责人: JESSICA JACOBS-LI
• 金额: $ 4.77万
• 依托单位: CALIFORNIA INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-01 至 2026-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10679674
• 关键词: Address; Birds; Brain; Cell Lineage; Cells; Chick; Chimera organism; Colon; Communication; Congenital Disorders; Congenital Megacolon; Development; Disadvantaged; Disease; Distal; Enteric Nervous System; Epithelium; Etiology; Fluorescence-Activated Cell Sorting; Foundations; Gene Expression Profile; Gene Expression Profiling; Genetic Transcription; Genus Alpharetrovirus; Immune response; Infection; Knowledge; Label; Life; Mediating; Methods; Modernization; Molecular Biology Techniques; Morphologic artifacts; Myenteric Plexus; Nervous System; Neural Crest; Neural Crest Cell; Neural Tube Closure; Neuroglia; Neuronal Differentiation; Neurons; Obstruction; Operative Surgical Procedures; Peripheral Nervous System; Peristalsis; Population; Proliferating; Quail; Regulation; Regulator Genes; Research; Role; Scheme; Seminal; Skeleton; Skin; Structure; Submucous Plexus; Techniques; Therapeutic; Time; Umbilicus; Visualization; Work; comparative; craniofacial; developmental disease; experimental study; gastrointestinal; gene regulatory network; genetic signature; infection risk; insight; loss of function; melanocyte; nerve supply; nervous system development; novel; nutrient absorption; repaired; retroviral-mediated; single-cell RNA sequencing; stem; stem cell population; trauma surgery; wasting

Proposal Summary The enteric nervous system (ENS) is a vital part of the peripheral nervous system and is responsible for the control of critical gut functions like peristalsis and gastrointestinal secretion. Abnormal development of the ENS can lead to life threatening disorders like Hirschsprung’s disease, characterized by the absence of innervating neurons and glia in the gut. Seminal experiments in chick have shown that two distinct neural crest cell populations innervate the gut and give rise to the ENS: the vagal and the sacral neural crest. Although extensive research has been done on the vagal neural crest’s contribution to the ENS, very little is known about the role of the sacral neural crest in the post-umbilical gut. We aim to address this gap in knowledge by using modern-day molecular biology techniques to gain granular understanding of sacral neural crest- derivatives in the post-umbilical gut, characterize their unique gene signatures, and determine the requirement of sacral-specific transcriptional regulators in ENS development. We hypothesize that the sacral neural crest contributes to unique derivatives within the post-umbilical gut, distinct from vagal-derived structures, and that these derived-cells are under the regulation of a novel gene regulatory scheme. Ultimately, this work will address a lack of understanding of the sacral neural crest in ENS development and shed light on the etiology of ENS-derived congenital disorders. Aim 1: Retroviral mediated lineage analysis of the chick sacral neural crest: Previous work in quail-chick chimeras had multiple disadvantages like traumatic surgery and cross-species artifacts. Here we will implement our technique of replication incompetent avian (RIA) retroviruses for comparative cell lineage analysis of vagal and sacral-derived cells in the post-umbilical gut, visualize interactions between the two populations, and perform clonal analysis of sacral neural crest cells. Aim 2: Transcriptional profiling of sacral neural crest-derived cells in the post-umbilical gut: In order to identify the gene signature of sacral-derived structures of the ENS and regulators of their differentiation, we propose single cell RNA sequencing of sacral neural crest derivatives across multiple time points to characterize the transcriptional profile of sacral neural crest entry into the gut and neuronal differentiation. Upon identification of transcriptional regulators, we will perform conditional loss-of-function analysis to examine their role in regulating the sacral neural crest’s neuronal differentiation.

建议书摘要 肠道神经系统(ENS)是周围神经系统的重要组成部分，负责 控制关键的肠道功能，如蠕动和胃肠道分泌。不正常的发育 ENS可导致危及生命的疾病，如先天性巨结肠，其特征是缺乏 支配肠道中的神经元和神经胶质细胞。在雏鸡身上的开创性实验表明，有两个不同的神经脊 细胞群支配肠道并产生神经核：迷走神经和骶神经脊。虽然 关于迷走神经脊对ENS的贡献已经进行了广泛的研究，但对此知之甚少 脐后肠道中骶神经脊的作用。我们的目标是通过使用以下方法来解决这一知识差距 现代分子生物学技术，以获得对骶骨神经脊的细粒度了解- 在脐带后肠道中的衍生物，表征其独特的基因签名，并确定 在胚胎胚胎发育过程中对骶骨特异转录调控因子的需求。我们假设 与迷走神经来源不同的是，在脐带后的肠道内有独特的衍生物--骶神经脊。 这些衍生细胞受到一种新的基因调控机制的调控。最终， 这项工作将解决在神经内窥镜发育过程中对骶骨神经脊缺乏了解的问题，并揭示 ENS源性先天性疾病的病因学。 目的1：逆转录病毒介导的雏鸡骶神经脊的谱系分析：在鹌鹑-雏鸡中的先前工作 嵌合体有多种缺点，比如创伤性手术和跨物种的文物。在这里，我们将实现 我们的复制失能(RIA)逆转录病毒技术用于迷走神经细胞系的比较分析 和脐带后肠道中的骶骨来源细胞，可视化这两个群体之间的相互作用，并执行 神经脊细胞的克隆分析。 目的2：脐带后肠道中的骶神经脊源性细胞的转录图谱：为了 确定ENS的骶骨来源结构的基因特征以及它们分化的调节因素 提出了多个时间点的骶神经脊衍生物的单细胞RNA测序 骶神经脊进入肠道和神经元分化的转录图谱。一旦被识别 转录调控因子，我们将进行条件性功能丧失分析，以检查它们在 调节骶神经脊的神经元分化。