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 ident...