Mechanisms of Enteric Neuron Diversification

正常的胃肠道(GI)运动是营养吸收、粪便排出和整体健康的基本前提。近四分之一的美国人口受到肠道疾病的影响，这些疾病会导致胃肠动力异常、慢性便秘和其他功能性肠道疾病。肠神经前体细胞(ENPs)是肠道神经系统(ENS)的神经元和神经胶质细胞，其分化的调节机制需要更深入的了解，才能了解肠道内正常的功能性肠神经补充是如何产生的。SOX10是一种重要的转录因子，在神经脊衍生的前体细胞中发挥作用，产生ENS。患者和小鼠的Sox10基因缺陷会导致远端肠道无神经节细胞增多症，导致巨结肠。最近对Sox10突变小鼠的研究发现，在这些动物的近端神经支配的肠道中，不同类型的肠神经元比例发生了显着的变化，并伴随着肠道运输和运动的异常。虽然Sox10在ENPs中表达，但肠神经细胞中的这些缺陷是意想不到的，因为Sox10随着神经元开始分化而消失，尽管它在肠神经胶质细胞中持续表达。结果表明，Sox10在胚胎胚胎发育中的作用大于在胚胎肠道首次被祖细胞定植的初始阶段简单地促进ENPs的迁移。在拟议的分析中，我们将测试最重要的假设，即Sox10在ENPs中的作用协调转录网络和染色质可及性，从而启动一个协调肠道神经元亚型多样性的调节级联反应。在目标1中，我们将使用单细胞RNA测序(ScRNASeq)来检验假设，即Sox10的突变等位基因通过扰乱ENP中的转录层次来改变肠神经元比率。在目标2中，我们将检验这一假设，即Sox10突变破坏了发育中的ENS谱系中染色质的可及性。整合来自这些研究的信息以及在神经脊培养中验证相互作用的基因效应，将区分产生正常肠道神经元亚型的潜在发育机制，并将促进指导肠道神经元分化治疗胃肠道疾病的努力。

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.