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

项目描述 哺乳动物肠神经系统的正常发育需要神经嵴细胞迁移到肠内， 沿着发育中的肠道。ENS发育的扰动可导致肠神经节的变化， 导致患者表型，如胃食管反流、慢性假性梗阻和先天性巨结肠 疾病（HSCR）。HSCR是一种复杂的遗传性发育障碍，其特征在于神经节细胞缺乏症， 沿肠远端沿着无不同长度的肠神经元。HSCR的遗传结构不是 完全理解，在约70%的患者中记录了已鉴定的突变。转录因子Sox 10 是一个可以在HSCR中改变的基因。我们的小组先前已经证明，Sox10Dom小鼠模型， HSCR概括了在患者中观察到的无神经节细胞症的可变表达性和表达率。另外我们 已经表明出生后的Sox10Dom小鼠具有改变的肠神经元类型的比例。这是一个有趣的发现 因为Sox 10在肠神经元中不表达，尽管它在肠神经元祖细胞（ENPs）中表达。 这表明Sox 10可能调节向神经元类型的正常范围的分化过程 通过间接机制。在其他神经嵴来源的谱系中，SOX 10与 控制细胞命运的染色质重塑蛋白。基于Sox10在肠神经元中表达的缺乏， SOX10参与改变其他组织中的染色质，我假设SOX10在改变染色质中起作用。 发育中的ENS中的染色质可及性。将通过以下目的研究这一假设： 目的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ENS神经发生过程中。在本研究中，我将联合收割机结合单核RNA和ATAC测序， ENPs。通过比较野生型和Sox10Dom ENPs之间的这些数据，我将评估染色质可及性 与缺陷性Sox10同种型下游基因表达相关的变化。这些实验将阐明 Sox 10在ENPs向神经元命运分化中的作用。目标2将定义改变Sox 10的影响 结合小鼠的染色质修饰。为了确定Sox 10如何介导其在发育中的间接作用， ENS，我将在野生型和Sox10Dom ENPs中测定全基因组SOX 10结合和组蛋白修饰。这些 研究将SOX10结合的缺陷与染色质结构的改变联系起来，扩展了 基因在ENP基因调控网络中的作用。这些研究还将揭示缺陷SOX 10的影响， 组蛋白修饰，并将指向候选的基因组元件与SOX 10直接结合。 该项目的成功将确定在ENP过程中起作用的Sox10下游的基因组元件 分化成正常神经元亚型比例。识别Sox 10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。这些知识并 利用并应用于人类ENS发育机制的实验，以指导分化 对神经元的命运，这可能会导致细胞疗法的患者胃肠道运动障碍，如HSCR。

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.