Retinoic Acid Contribution to Embryonic Gastrointestinal Tract Development

Hirsch sprung病（HSCR）是由于神经细胞（NCC）定植或发育失败而导致的肠道部分先天性神经元缺失。这种异常发生在1/5000活产婴儿中，通常需要手术切除a神经节肠。只有一半的HSCR病例可归因于已知的遗传缺陷，因此有必要探索这种疾病的其他途径和潜在的修饰因子。本研究拟采用新的类视黄醇缺乏症动物模型，探讨维生素A代谢在NCC和胃肠道发育中的作用。我将验证RDH10对器官发生所需的RA合成有重大影响的假设，特别是在肠神经系统（ENS）形成过程中对NCC增殖、分化或迁移的影响。具体来说，该项目解决了类视黄醇缺乏对等位基因系列小鼠的影响，并研究了HSCR的原因和潜在的拯救。目的1将定义肠道发育过程中类维生素a信号的时空要求，验证我们的假设，即RA信号是正确形成ENS所需的时空条件。这是相关的，因为维生素A缺乏可能发生在怀孕前和怀孕期间，并可能影响NCC的发展，导致先天性异常，可能包括HSCR。因此，了解在ENS形成和肠道发育过程中NCC发育的哪个阶段需要类维生素a信号是至关重要的。RDH10是维生素a氧化为维甲酸（RA）的第一步所需的视黄醇脱氢酶。我们在Aim 2中假设RDH10是神经嵴细胞初始迁移所必需的，如果没有适当的RA信号，NCCs就无法接收到定殖肠道所需的适当引导信号并形成正常的encs。我们在培养和子宫灌胃中使用类维生素a补充剂，并通过条件敲除小鼠来确定RA信号的时间需求。我们的研究结果将作为理解rdh10缺陷小鼠ENS发育缺陷的细胞和分子机制的跳板，并确定RA对已知肠道NCC调节途径和肠道微环境的相互作用。我们将使用免疫组织化学和原位杂交对整个胚胎、肠道、培养肠道和切片进行检测，以检测肠道NCC的增殖、凋亡、迁移和分化，以及已知对这些发育过程有贡献的基因，同时结合候选基因和RNA测序方法。这些研究结果将为研究HSCR建立一个新的模型，提供有关胚胎肠道发育过程中RA的时空需求、RDH10缺陷动物的致病细胞机制以及RA与正常ENS NCC定殖途径的相互作用等信息。这些知识可能会导致创新的非手术治疗，以减少这种常见的先天性疾病的发病率和死亡率。

DESCRIPTION (provided by applicant): Project Summary Hirsch sprung disease (HSCR) is a congenital absence of neurons in a portion of the intestinal tract, due to a failure of neural cres cell (NCC) colonization or development. This anomaly occurs in 1/5000 live births, and typically requires surgical resection of the a ganglionic bowel. Only half of the HSCR cases are attributable to known genetic defects, and it is therefore necessary to explore other pathways and potential modifiers of this disease. The proposed studies investigate the role of vitamin A metabolism in NCC and gastrointestinal tract development using new animal models of retinoid deficiency. I will test the hypothesis that RDH10 has a significant impact on the RA synthesis necessary for organogenesis, specifically on NCC proliferation, differentiation or migration during formation of the enteric nervous system (ENS). Specifically, this project addresses the effects of retinoid deficiency in an allelic series of mice as well as investigates the cause and potential rescue of HSCR. Aim 1 will define the temporal and spatial requirements for retinoid signaling during gut development, testing our hypothesis that RA signaling is spatiotemporally required for proper ENS formation. This is relevant, as Vitamin A deficiency can occur prior to and during pregnancy and can affect NCC development, resulting in congenital anomalies that may include HSCR. Hence it is critical to understand which stage of NCC development requires retinoid signaling during ENS formation and gut development. RDH10 is a retinol dehydrogenase enzyme required for the first oxidation step of vitamin A to retinoic acid (RA). We hypothesize in Aim 2 that RDH10 is required for the initial migration of neural crest cells, and without proper RA signaling, NCCs do not receive the appropriate guidance signals required to colonize the gut and form a normal ENS. We utilize retinoid supplementation both in culture and via in utero gavage supplementation, along with conditional knockout mice to define the temporal requirement for RA signaling. Our results will act as a springboard for understanding the cellular and molecular mechanisms underlying defective ENS development in RDH10-deficient mice, as well as determine the interactions of RA on known enteric NCC regulatory pathways and gut microenvironment. We will use immunohistochemistry and in situ hybridization on whole embryos, guts, cultured guts, and sections to assay for enteric NCC proliferation, apoptosis, migration, and differentiation, as well as for genes known to contribute to these developmental processes, alongside a combination of candidate gene and RNA sequencing approaches. Results from these studies will establish a new model for studying HSCR, providing information regarding the spatiotemporal requirements for RA during embryonic intestinal development, the pathogenic cellular mechanisms of RDH10 deficient animals, as well as the interactions of RA with pathways that govern normal ENS NCC colonization. This knowledge may lead to innovative non-surgical treatments to reduce the morbidity and mortality of this common congenital disease.