Development of the enteric nervous system

肠神经系统的发育

• 批准号: RGPIN-2014-06351
• 负责人: Pilon, Nicolas
• 金额: $ 3.42万
• 依托单位: Université du Québec à Montréal
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2018
• 资助国家: 加拿大
• 起止时间: 2018-01-01 至 2019-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=662200
• 关键词: Development; enteric; nervous; system

BACKGROUND: The enteric nervous system (ENS) is the largest and most complex part of the peripheral nervous system. It is found throughout the length of the intestines and is organized into two interconnected networks of neural ganglia that are notably in charge of gut motility. The ENS is formed during embryonic development, being derived from neural crest cells (NCC) that migrate from the dorsal neural tube. The foregut is initially colonized and then, enteric (e) NCC migrate in an anterior to posterior direction to colonize the midgut and hindgut. Incomplete colonization leads to lack of neural ganglia over varying lengths of the distal gut, resulting in lethal constipation soon after birth (aganglionic megacolon). Multiple processes such as cell proliferation, survival and differentiation are also critically required for completion of ENS formation. At the molecular level, two signaling pathways have emerged as key players of ENS development: one controlled by the Glial cell Derived Neurotrophic Factor (GDNF) and the other by Endothelin-3 (ET3). Nevertheless, based on genetic studies in human and model organisms, it is clear that supplemental players important for ENS development have yet to be identified. **In order to identify such new genes/loci, we have performed an insertional mutation screen in transgenic mice. Due to random insertion of transgenic DNA in the genome, this screen allowed the identification of three lines of mice, named "TashT", "Spot" and "Holstein" that display an aganglionic megacolon phenotype. Deep sequencing of genomic DNA revealed that, for every line, the transgene insertion site is located in a gene/locus that is either totally novel (TashT and Spot) or that has not been previously associated with ENS formation (Holstein). Thus, this unique collection of mouse mutants represents an extremely rare opportunity for making major breakthroughs in the field.**MAIN OBJECTIVE: This research program is aimed at identifying and characterizing the causative gene for each of our mutant lines. **SPECIFIC OBJECTIVES: Renewal of NSERC funding is now requested for working on the Spot and Holstein lines, with a special focus on Spot in accordance with the following specific aims:*1) Characterize the developmental defect leading to the Spot megacolon phenotype: we will identify which cellular process (migration, proliferation, survival or differentiation) is deregulated in Spot eNCC. We will also determine the hierarchical position of the Spot candidate gene (A830082K12Rik) in relation to the critical GDNF and ET3 signaling pathways. Moreover, the complete set of genes affected by the Spot mutation in eNCC will be revealed via RNAseq, comparing the transcriptome profile between wild-type and mutant embryos. *2) Confirm that A830082K12Rik is the Spot causative gene: a novel mouse model with targeted mutation of A830082K12Rik will be generated and used to test allelism with the Spot locus. *3) Characterize the novel long non-coding RNA gene A830082K12Rik: Gene expression patterns will be evaluated via in situ hybridization while subcellular localization will be analyzed using an RNA tagging system coupled with fluorescent labeling. A screen for protein interactants in eNCC will be performed using pull-down experiments coupled with mass spectrometry.**CONCLUSION: This work is an excellent opportunity for increasing our general understanding of an exciting novel class of regulatory molecules (i.e. lncRNA). Together with the fact that other NCC-derived structures (e.g. melanocytes and inner ear sensory epithelia) are also severely affected in Spot animals, our studies are thus expected to have a very significant and long-lasting impact not only on the ENS field but also on the wider field of developmental biology.

背景：肠神经系统是周围神经系统中最大、最复杂的部分。它存在于整个肠道的长度，并组织成两个相互连接的神经节网络，主要负责肠道运动。ENS在胚胎发育期间形成，源自从背侧神经管迁移的神经嵴细胞（NCC）。前肠最初被定殖，然后肠（e）NCC以从前到后的方向迁移以定殖中肠和后肠。不完全的定植导致在不同长度的远端肠道上缺乏神经节，导致出生后不久的致命性便秘（无神经节巨结肠）。完成ENS形成也需要多个过程，如细胞增殖、存活和分化。在分子水平上，两种信号通路已经成为ENS发育的关键参与者：一种由胶质细胞源性神经营养因子（GDNF）控制，另一种由内皮素-3（ET 3）控制。然而，根据对人类和模式生物的遗传研究，很明显，对ENS开发重要的补充参与者尚未确定。** 为了鉴定这些新基因/基因座，我们在转基因小鼠中进行了插入突变筛选。由于转基因DNA在基因组中的随机插入，该筛选允许鉴定三个品系的小鼠，命名为“TashT”、“Spot”和“Holstein”，其显示无神经节巨结肠表型。基因组DNA的深度测序显示，对于每个品系，转基因插入位点位于完全新的基因/基因座（TashT和Spot）或先前未与ENS形成相关的基因/基因座（Holstein）。因此，这种独特的小鼠突变体集合代表了在该领域取得重大突破的极其难得的机会。主要目的：本研究计划旨在确定和表征我们的每一个突变株系的致病基因。** 具体要求：现在需要更新NSERC的资金用于Spot和Holstein系的研究，特别关注Spot，具体目标如下：*1）表征导致Spot巨结肠表型的发育缺陷：我们将确定Spot eNCC中哪些细胞过程（迁移、增殖、存活或分化）被解除调控。我们还将确定Spot候选基因（A830082 K12 Rik）与关键GDNF和ET 3信号通路的关系的层次位置。此外，eNCC中受Spot突变影响的全套基因将通过RNAseq揭示，比较野生型和突变胚胎之间的转录组谱。*2）确认A830082 K12 Rik是Spot致病基因：将产生具有A830082 K12 Rik靶向突变的新型小鼠模型，并用于测试与Spot基因座的等位性。*3）表征新的长非编码RNA基因A830082 K12 Rik：将通过原位杂交评估基因表达模式，同时将使用与荧光标记结合的RNA标签系统分析亚细胞定位。将使用下拉实验结合质谱法进行eNCC中蛋白质相互作用物的筛选。结论：这项工作是一个很好的机会，增加我们的一般性理解的一个令人兴奋的新型调节分子（即lncRNA）。加上其他NCC衍生结构（例如黑色素细胞和内耳感觉上皮细胞）也严重影响斑点动物的事实，因此我们的研究预计不仅对ENS领域而且对更广泛的发育生物学领域产生非常显著和持久的影响。