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，数量达数亿。的 ENS 控制着重要的肠道功能，例如蠕动、水平衡和肠道屏障稳态。 ENS 源自肠神经祖细胞 (ENP)，它们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神经元或神经胶质细胞。 ENS 形成的破坏导致 先天性先天性先天性巨结肠症 (HSCR)，其中胃肠道的可变区域缺乏 ENS——这是最常见的 HSCR 的常见形式沿远端结肠出现，也称为结肠神经节缺失症。的 ENPs 利用其迁移到肠管并沿肠管进行空间定位的潜在细胞机制， 以及它们执行分化为肠神经元的遗传程序尚未得到充分研究 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，着眼于为下游转化治疗研究提供信息。在这个提案中，我们 利用斑马鱼胚胎，因为它们与人类的遗传保守性，易于观察其外部 开发及其光学透明度。构建生成的单细胞转录组数据集 从沿着肠管的早期神经发生过程中收集的 ENP 细胞中，目标 1 将检查 新发现的 ENP 转录亚群的空间排列预测的假设 未来肠神经元沿肠管的放置和终末分化。同意并 扩展对哺乳动物模型的观察，我们最近发现视黄酸 (RA) 在斑马鱼 ENS 发育的早期阶段，信号传导在全球范围内至关重要；然而，RA信号如何 迄今为止，在任何系统中，对 ENS 体内个体发生的自主影响尚未得到充分了解。目标2将 研究 RA 通路自主控制 ENP 分化状态的假设， 使用尖端的单细胞转录组学、光遗传学和在肠管中的迁移模式 体内成像。我们还将在目标 2 中测试候选转录因子发挥作用的机制模型 ENP 中 RA 的本质下游，控制 ENS 的形成，从而扩大了我们对 ENS 基因调控网络。目标 3 将利用细胞周期的遗传调节，进行体内定量 成像和细胞追踪测试了 ENP 将增殖与迁移结合起来的细胞机制模型 决定了 RA 通路下游肠道中正确的肠神经元模式。这些结果 目标将显着增加我们对 ENP 的遗传、分子和细胞基础的了解 ENS 的开发和早期创建，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.