RNA Seq analysis of E9.5 embryonic vagal regions in Rdh10 mutants of mouse

The enteric nervous system (ENS) is formed from vagal neural crest cells (NCC), which generate the neurons and glia that regulate gastrointestinal function. Defects in the migration, colonization or differentiation of NCC in the gut can result in gastrointestinal disorders such as Hirschsprung disease (HSCR). Although mutations in many genes have been associated with the etiology of HSCR, a significant proportion of affected individuals have an unknown genetic diagnosis. Therefore, it’s important to identify new genes, modifiers and environmental factors that regulate ENS development and HSCR. We discovered that retinol dehydrogenase 10 (Rdh10) loss-of-function mouse embryos exhibit total intestinal aganglionosis, the most severe form of HSCR. Rdh10 catalyzes the first oxidative step in the metabolism of vitamin A to its active metabolite, RA, and is therefore a central regulator of vitamin A metabolism and retinoic acid (RA) synthesis during embryogenesis. Rdh10 is highly expressed in the mesenchyme surrounding the entrance to the foregut and we demonstrate that paracrine retinoid signaling is essential between E7.5-E9.5 for NCC entry into the gut. Vagal NCC form and migrate in Rdh10 mutant embryos but fail to enter the foregut. Comparative RNA-sequencing revealed Ret-Gdnf-Gfra1-signaling which is critical for vagal NCC chemotaxis is downregulated in Rdh10 mutants and furthermore that the composition of the extracellular matrix through which NCC migrate is altered, particularly by increased collagen deposition. Collectively this restricts NCC entry into the gut, demonstrating that Rdh10-mediated vitamin A metabolism and RA signaling pleiotropically regulates the NCC microenvironment during ENS formation and in the pathogenesis of HSCR. The vagal (somite 1-7) regions of Rdh10 mutant or wt E9.5 mouse embryos were isolated in quadruplicate and compared for gene expression using RNA Seq.肠神经系统（enteric nervous system, ENS）由迷走神经嵴细胞（vagal neural crest cells, NCC）发育形成，后者可分化出调控胃肠道功能的神经元与神经胶质细胞。若肠道内神经嵴细胞的迁移、定植或分化出现异常，可引发诸如先天性巨结肠症（Hirschsprung disease, HSCR）这类胃肠道疾病。尽管已有诸多基因的突变被证实与先天性巨结肠症的病因相关，但仍有相当比例的患者无法明确其遗传学诊断。因此，鉴定调控肠神经系统发育与先天性巨结肠症发病的新基因、修饰因子及环境因素具有重要意义。本研究发现，视黄醇脱氢酶10（retinol dehydrogenase 10, Rdh10）功能缺失型小鼠胚胎会表现出完全性肠无神经节症——这是先天性巨结肠症中最为严重的表型。Rdh10可催化维生素A代谢生成活性代谢产物维甲酸（retinoic acid, RA）的第一步氧化反应，因此是胚胎发育过程中维生素A代谢与维甲酸合成的核心调控因子。Rdh10在前肠入口周围的间充质中高表达，本研究证实，在胚胎发育第7.5天至第9.5天（E7.5-E9.5）期间，旁分泌维甲酸信号通路对于神经嵴细胞进入肠道至关重要。迷走神经嵴细胞可在Rdh10突变小鼠胚胎中正常形成并迁移，但无法进入前肠。对比转录组测序（RNA-sequencing, RNA-seq）分析显示，在Rdh10突变体中，对迷走神经嵴细胞趋化迁移至关重要的Ret-Gdnf-Gfra1信号通路表达下调；此外，神经嵴细胞迁移所途经的细胞外基质组成也发生改变，尤以胶原蛋白沉积增加最为显著。综上，上述机制共同限制了神经嵴细胞进入肠道，这表明Rdh10介导的维生素A代谢与维甲酸信号通路可通过多效性调控肠神经系统形成过程中的神经嵴细胞微环境，进而参与先天性巨结肠症的发病机制。本研究将Rdh10突变型或野生型（wild type, wt）小鼠胚胎的迷走（体节1-7）区域进行四次生物学重复取样，通过RNA测序比较其基因表达差异。