Mechanisms of Enteric Neuron Diversification

正常的胃肠运动是营养吸收、粪便消除和整体健康的必要前提。近四分之一的美国人口受到肠道疾病的影响，导致胃肠道运动异常、慢性便秘和其他功能性肠道疾病。肠神经祖细胞（ENPs）形成肠神经系统（ENS）的神经元和胶质细胞，需要更好地了解调节肠神经祖细胞（ENPs）分化的机制，以了解肠内功能性肠神经元的正常补体是如何产生的。Sox10是一种重要的转录因子，在产生ens的神经嵴衍生祖细胞中起作用。患者和小鼠中Sox10的缺陷会引起远端肠神经节病，导致巨结肠。最近对Sox10突变小鼠的研究发现，这些动物的近端神经支配肠中不同肠神经元类型的比例明显改变，并伴有肠道运输和运动异常。虽然Sox10在ENPs中表达，但在肠神经元中这些缺陷是出乎意料的，因为Sox10在神经元开始分化时被消灭，尽管它在肠胶质中持续表达。结果表明，在胚胎肠道首次被祖细胞定殖的初始阶段，Sox10在ENPs的发育中发挥的作用比单纯促进ENPs的迁移更大。在提出的分析中，我们将检验一个总体假设，即ENPs中Sox10的作用协调了转录网络和染色质可及性，启动了一个调节级联，协调了肠神经元亚型的多样性。在Aim 1中，我们将使用单细胞RNA测序（scRNASeq）验证Sox10突变等位基因通过破坏ENPs的转录层次改变肠内神经元比例的假设。在目标2中，我们将检验Sox10突变体在ENS谱系发育中破坏染色质可及性的假设。整合来自这些研究的信息和神经嵴培养中相互作用基因效应的验证将区分产生正常肠道神经元亚型的潜在发育机制，并将促进肠道神经元的直接分化以治疗胃肠道疾病。

Normal gastrointestinal (GI) motility is an essential prerequisite for nutrient absorption, fecal elimination and overall health. Nearly a quarter of the United States population is affected by intestinal disorders that lead to abnormal GI motility, chronic constipation and other functional bowel disorders. Greater understanding of the mechanisms that regulate differentiation of enteric neural progenitors (ENPs), which form the neurons and glia of the enteric nervous system (ENS), are needed to understand how the normal complement of functional enteric neurons within the intestine is generated. Sox10 is an essential transcription factor that functions in the neural crest derived progenitors that generate the ENS. Defects in Sox10 in patients and mice cause aganglionosis of the distal intestine leading to megacolon. Recent studies of Sox10 mutant mice have identified pronounced alterations of the ratios of different enteric neuron types in proximal innervated bowel of these animals that are accompanied by abnormal intestinal transit and motility. Although Sox10 is expressed in ENPs, these deficiencies among enteric neurons were unexpected because Sox10 is extinguished as neurons begin to differentiate, although its expression is sustained in enteric glia. The results suggest that Sox10 has greater roles in ENS development than simply promoting migration of ENPs during initial phases when the fetal gut is first colonized by progenitors. In the proposed analysis we will test the overarching hypothesis that Sox10 action in ENPs orchestrates transcriptional networks and chromatin accessibility setting in motion a regulatory cascade that orchestrates diversity of enteric neuron subtypes. In Aim 1 we will test the hypothesis that the mutant alleles of Sox10 alter enteric neuron ratios by disrupting transcriptional hierarchies in ENPs using single cell RNA sequencing (scRNASeq). In Aim 2 we will examine the hypothesis that Sox10 mutants disrupt chromatin accessibility in developing ENS lineages. Integration of information from these studies and validation of interacting gene effects in neural crest cultures will distinguish between potential developmental mechanisms that generate the normal repertoire of enteric neuron subtypes and will facilitate efforts to direct differentiation of enteric neurons for treatment of GI disease.