Genetic and extrinsic mechanisms governing early enteric nervous system development

驻留在整个胃肠道(GI)的肌壁之间，即肠道神经系统 (ENS)由一系列相互连接的神经元和胶质细胞组成，数量以亿计。这个 ENS控制基本的肠道功能，如蠕动、水分平衡和肠道屏障稳态。 ENS来源于肠神经前体细胞，在发育过程中迁移到发育中的肠管中。 胚胎发生并分化为肠道神经元或神经胶质细胞。ENS形成的中断导致 先天性先天性巨结肠(HSCR)，其中GI的可变区缺乏ENS-最多 常见的HSCR表现为远端结肠，也称为结肠无神经节细胞增多症。这个 ENPs用来迁移到肠管中并沿肠道管空间定位的潜在细胞机制， 除了它们执行的分化为肠道神经元的遗传程序外，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基础知识 形成，着眼于为下游的转化治疗研究提供信息。在这项提案中，我们 利用斑马鱼胚胎，因为它们与人类的基因保守，易于查看它们的外部 它们的发展和光学透明性都很重要。建立在生成的单细胞转录数据集基础上 从肠管内早期神经发生过程中收集的ENP细胞中，Aim 1将检查一个 新发现的ENP转录亚群的空间排列可预测的假设 未来肠神经元沿肠管的放置和终末分化。与和达成协议 在哺乳动物模型中的扩展观察，我们最近发现维甲酸(RA) 在斑马鱼胚胎发育的早期阶段，信号在全球范围内是至关重要的；然而，RA信号如何 到目前为止，任何系统都不能很好地理解在体内自主影响Es个体发生的机制。目标2将 研究一种假说，即RA通路自主控制ENP分化状态和 应用尖端单细胞转录学、光遗传学和生物信息学的肠管迁移模式 活体成像。我们还将在目标2中测试候选转录因子起作用的机制模型 在ENPs中内在地位于RA下游以管理ENS的形成，从而扩展了我们对 ENS基因调控网络。目标3将使用遗传调节细胞周期，在体内定量 成像和细胞跟踪测试了ENPs将增殖与迁移联系在一起的细胞机制模型 在RA通路下游的肠道中决定适当的肠道神经元模式。这些研究的结果 AIMS将显著增加我们对ENP的遗传、分子和细胞基础的了解 开发和早期的ENS创建，它们将为研究这些提供一个新的机制框架 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.