Genetic and extrinsic mechanisms governing early enteric nervous system development

驻留在整个胃肠道（GI）道的肌肉壁之间，肠神经系统 (ENS)由一系列相互连接的神经元和神经胶质细胞组成，数量数以亿计。的 ENS控制基本的肠道功能，如消化、水平衡和肠屏障稳态。 ENS来源于肠神经祖细胞（ENPs），其在发育过程中迁移到发育中的肠管中。 胚胎发生并分化成肠神经元或神经胶质。ENS形成的中断导致 先天性条件先天性巨结肠病（HSCR），其中GI的可变区缺乏ENS-最 HSCR的常见形式是沿着远端结肠，也称为结肠无神经节细胞症。的 ENPs用于迁移进入并沿肠管沿着空间定位的潜在细胞机制， 以及它们分化为肠神经元的遗传程序还没有得到很好的研究 在体内，因此限制了我们对ENS如何表现的了解。总体目标是扩大 用于执行ENS所需的复杂机制的基因的基础知识 形成，着眼于为下游翻译治疗研究提供信息。在本提案中，我们 利用斑马鱼胚胎，由于它们与人类的遗传保守，易于观察它们的外部 开发和光学透明度。构建生成的单细胞转录组数据集 从ENP细胞收集在其早期神经发生沿着肠管，目的1将检查一个 新发现的ENP转录亚群的空间排列预测了 未来肠神经元的位置和终末分化沿着肠管。在同意和 通过对哺乳动物模型的进一步观察，我们最近发现维甲酸（RA） 在斑马鱼ENS发育的早期阶段，RA信号在全球范围内是至关重要的;然而， 自主影响ENS在体内的个体发育是没有很好地理解在任何系统的日期。目标2将 研究RA途径自主控制ENP分化状态的假设， 迁移模式沿着肠道管使用尖端单细胞转录组学，光遗传学和 活体成像我们还将测试目标2中候选转录因子功能的机制模型 ENPs中RA的内在下游，以控制ENS的形成，从而扩大了我们对 ENS基因调控网络。目的3将利用基因调控细胞周期，在体内定量 成像和细胞跟踪测试了ENPs将增殖与迁移偶联以 决定了RA通路下游肠道中适当的肠神经元模式。的结果予以 目标将显着增加我们对ENP的遗传、分子和细胞基础的了解 开发和早期ENS创建，它们将为研究这些提供一个新的机制框架。 在体内发育重要的细胞。

Resident between the muscle walls of the entire gastrointestinal (GI) tract, the enteric nervous system (ENS) consists of a series of interconnected neurons and glia, numbered in the hundreds of millions. The ENS controls essential gut functions, such as peristalsis, water balance and intestinal barrier homeostasis. The ENS is derived from enteric neural progenitors (ENPs) that migrate into the developing gut tube during embryogenesis and differentiate into enteric neurons or glia. Disruption in ENS formation results in the congenital condition Hirschsprung disease (HSCR), in which variable regions of the GI lack ENS—the most common form of HSCR presents along the distal colon, also known as colonic aganglionosis. The underlying cellular mechanisms that ENPs utilize to migrate into and spatially position along the gut tube, as well as genetic programs they execute to differentiate into enteric neurons have not been well studied in vivo, therefore limiting our knowledge of how the ENS manifests. The overall goal is to expand foundational knowledge of the genes utilized to execute the complex mechanisms necessary for ENS formation, with an eye for informing downstream translational therapeutic studies. In this proposal, we utilize zebrafish embryos due to their genetic conservation with humans, the ease of viewing their external development and for their optical transparency. Building off of single-cell transcriptomic data sets generated from ENP cells collected during their early neurogenesis along the gut tube, Aim 1 will examine a hypothesis that the spatial arrangement of newly uncovered ENP transcriptional subpopulations predict future enteric neuron placement and terminal differentiation along the gut tube. In agreement with and extending observations in mammalian models, we have recently discovered that Retinoic Acid (RA) signaling is critical globally during early steps of zebrafish ENS development; however, how RA signaling autonomously influences ENS ontogenesis in vivo is not well understood in any system to date. Aim 2 will investigate a hypothesis that the RA pathway autonomously controls ENP differentiation states and migration patterns along the gut tube using cutting edge single-cell transcriptomics, optogenetics and in vivo imaging. We will also test a mechanistic model in Aim 2 that candidate transcription factors function intrinsically downstream of RA in ENPs to govern ENS formation, thereby expanding our understanding of the ENS gene regulatory network. Aim 3 will use genetic modulation of the cell cycle, quantitative in vivo imaging and cell tracking test a cellular mechanistic model that ENPs couple proliferation with migration to dictate proper enteric neuron patterning in the gut downstream of the RA pathway. The results of these aims will significantly increase our knowledge of the genetic, molecular and cellular underpinnings of ENP development and early ENS creation and they will provide a new mechanistic framework for studying these developmentally important cells in vivo.