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

提案摘要 肠神经系统（ENS）是周围神经系统的重要部分，并且负责 控制关键的肠道功能，如消化和胃肠道分泌。发育异常 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。鸡的精液实验表明，两个不同的神经嵴 细胞群支配肠道并产生ENS：迷走神经和骶神经嵴。虽然 迷走神经嵴在ENS中的作用已被广泛研究，但对迷走神经嵴在ENS中的作用知之甚少。 骶神经嵴在后脐肠中的作用。我们的目标是通过使用 现代分子生物学技术，以获得骶骨神经嵴的颗粒状了解- 衍生物在后脐肠道，表征其独特的基因签名，并确定 ENS发育中对骶骨特异性转录调节因子的需求。我们假设 骶神经嵴在脐后肠内形成独特的衍生物，不同于迷走神经源性的 结构，并且这些衍生的细胞是在一种新的基因调控方案的调控下。最后， 这项工作将解决ENS发展中对骶神经嵴缺乏了解的问题，并阐明 ENS衍生的先天性疾病的病因学。 目的1：逆转录病毒介导的鸡骶神经嵴谱系分析：鹌鹑-鸡的前期工作 嵌合体有许多缺点，如创伤性手术和跨物种人工制品。在这里，我们将实现 我们的技术复制缺陷型禽（RIA）逆转录病毒比较细胞谱系分析迷走神经 和骶骨来源的细胞在后脐肠道，可视化两个群体之间的相互作用，并执行 骶骨神经嵴细胞的克隆分析。 目的2：后脐肠中骶骨神经嵴衍生细胞的转录谱：为了 为了鉴定ENS骶骨衍生结构的基因特征及其分化的调节因子，我们 建议在多个时间点对骶骨神经嵴衍生物进行单细胞RNA测序，以表征 骶骨神经嵴进入肠道和神经元分化的转录谱。一旦识别 转录调控因子，我们将进行条件性功能丧失分析，以检查它们在转录调控中的作用。 调节骶神经嵴的神经元分化。

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.