Assessing Sox 10's effect on chromatin accessibility in enteric neuron lineage diversification

项目说明 哺乳动物肠道神经系统的正常发育需要神经脊细胞迁移到 沿着发育中的肠道。ENS发育的扰动可导致肠神经节的变化， 导致患者的表型，如胃食道反流、慢性假性梗阻和先天性巨结肠 疾病(HSCR)。HSCR是一种复杂的遗传发育障碍，以无神经节细胞缺乏症为特征， 沿远端肠管可见不同长度的肠神经元。HSCR的遗传结构不是 完全理解，在约70%的患者中记录了已识别的突变。转录因子Sox10 是一个可以在HSCR中改变的基因。我们的小组之前已经证明，Sox10Dom鼠标模型 HSCR重述了在患者中所见的无神经节细胞增多症的不同表现和外显率。此外，我们 已经表明，出生后的Sox10Dom小鼠改变了肠道神经元类型的比例。这是一个耐人寻味的发现 由于Sox10在肠神经元中不表达，但在肠神经前体细胞(ENPs)中表达。 这表明Sox10可能正在调节分化为正常神经元类型的过程。 通过一种间接机制进行比例调整。在其他神经脊派生的谱系中，Sox10与 染色质重塑蛋白控制细胞命运。基于肠神经细胞中缺乏Sox10的表达和 S参与改变其他组织染色质，我推测SOX10在改变染色质中有作用 显影ENS中染色质的可及性。这一假设将通过以下目标进行调查： 目标1将定义Sox10突变等位基因对全基因组染色质可及性和基因表达的影响 在小鼠神经发生的过程中。在这项研究中，我将结合单核RNA和ATAC-测序 ENPs。通过比较野生型和Sox10Dom ENPs的这些数据，我将评估染色质的可及性 与缺陷Sox10亚型下游基因表达相关的变化。这些实验将澄清 Sox10‘S在ENPs向神经元命运分化中的作用目标2将定义更改后的Sox10的效果 与小鼠染色质修饰的结合。确定Sox10如何在发育过程中调节其间接影响 我将在野生型和Sox10Dom ENPs中分析全基因组的Sox10结合和组蛋白修饰。这些 研究将把Sox10结合缺陷与染色质结构的变化联系起来，扩大 发展中的ENP基因调控网络中的基因。这些研究还将揭示SOX10缺陷的影响 在组蛋白修饰上，并将指向Sox10直接结合的候选基因组元件。 该项目的成功将确定Sox10下游在ENP期间发挥作用的基因组元件 分化成正常的神经元亚型比例。辨析SOX10的S对监管的影响 ENPs中的基因组区域将识别正常ENS发育所需的基因。这一知识可能 被利用并应用于人类发育机制的实验以指导分化 走向神经元的命运，这可能导致对HSCR等胃肠动力障碍患者的细胞治疗。

Project Description Normal development of the mammalian enteric nervous system requires migration of neural crest cells into and along the developing intestine. Perturbations of ENS development can result in changes in enteric ganglia that result in patient phenotypes such as gastroesophageal reflux, chronic pseudo-obstruction, and Hirschsprung’s disease (HSCR). HSCR is a complex genetic developmental disorder characterized by aganglionosis, the absence of enteric neurons in varying lengths, along the distal bowel. The genetic architecture of HSCR is not completely understood, with identified mutations documented in ~70% of patients. The transcription factor Sox10 is one gene that can be altered in HSCR. Our group has previously shown that the Sox10Dom mouse model of HSCR recapitulates the variable expressivity and penetrance of aganglionosis seen in patients. In addition, we have shown that postnatal Sox10Dom mice have altered ratios of enteric neuron types. This is an intriguing finding since Sox10 is not expressed in enteric neurons, although it is expressed in enteric neuronal progenitors (ENPs). This suggests that Sox10 could be regulating the differentiation process into the normal range of neuronal type proportions through an indirect mechanism. In other neural crest derived lineages, SOX10 interacts with chromatin remodeler proteins to control cell fate. Based on the lack of Sox10 expression in enteric neurons and SOX10’s participation in altering chromatin in other tissues, I hypothesize that Sox10 has a role in altering chromatin accessibility in the developing ENS. This hypothesis will be investigated through the following aims: Aim 1 will define effects of a Sox10 mutant allele on genome-wide chromatin accessibility and gene expression during mouse ENS neurogenesis. In this study, I will combine single nucleus RNA and ATAC-sequencing in ENPs. By comparing these data between wild-type and Sox10Dom ENPs, I will evaluate chromatin accessibility changes linked with gene expression downstream of a defective Sox10 isoform. These experiments will clarify Sox10’s role in the differentiation of ENPs towards neuronal fates. Aim 2 will define effects of altered Sox10 binding on chromatin modifications in mice. To determine how Sox10 mediates its indirect effect in the developing ENS, I will assay genome-wide SOX10 binding and histone modifications in wild type and Sox10Dom ENPs. These studies will link deficits in SOX10 binding to alternations in chromatin architecture, expanding the framework of genes in the developing ENP gene regulatory network. These studies will also reveal effects of defective SOX10 on histone modifications and will point towards candidate genomic elements to which SOX10 directly binds. Success of this project would identify genomic elements downstream of Sox10 that function during ENP differentiation into normal neuronal subtype proportions. Discernment of Sox10’s influence on regulatory genomic regions in ENPs will identify genes that are required for normal ENS development. This knowledge may be leveraged and applied to experiments in human ENS developmental mechanisms to direct differentiation towards neuronal fates, which may lead to cell therapies for patients with GI motility disorders such as HSCR.