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: 2017
• 资助国家: 加拿大
• 起止时间: 2017-01-01 至 2018-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=629009
• 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(ENS)是周围神经系统中最大、最复杂的部分。它遍布整个肠道，并被组织成两个相互连接的神经节网络，这两个神经节主要负责肠道运动。ENS是在胚胎发育过程中形成的，来源于从背侧神经管迁移而来的神经脊细胞(NCC)。前肠最初被定植，然后，肠道(E)NCC从前到后迁移到中肠和后肠。不完全定植导致远端肠道长度不等的神经神经节缺失，导致出生后不久的致死性便秘(无神经节细胞巨结肠)。ENS的形成还需要细胞的增殖、存活和分化等多个过程。在分子水平上，两条信号通路被认为是ENS发生的关键分子：一条由胶质细胞源性神经营养因子(GDNF)控制，另一条由内皮素-3(ET3)控制。然而，基于对人类和模式生物的遗传学研究，很明显，对ENS发展至关重要的补充角色尚未确定。为了识别这些新的基因/位点，我们在转基因小鼠中进行了插入突变筛选。由于在基因组中随机插入转基因DNA，该屏幕允许识别三个品系的小鼠，命名为“TashT”、“Spot”和“Holstein”，它们表现出无神经节细胞的大结肠表型。基因组DNA的深度测序表明，对于每一株系，转基因插入位点都位于一个完全新的基因/位点上(TashT和Spot)，或者是以前没有与ENS形成相关的基因/位点(Holstein)。因此，这一独特的小鼠突变体集合代表着在该领域取得重大突破的极其难得的机会。MAIN目标：这项研究计划的目的是识别和表征我们每个突变系的致病基因。具体目标：现在要求延长NSERC的资金用于SPOT和荷斯坦品系的工作，并根据以下具体目标特别关注SPOT：1)表征导致SPOT巨结肠表型的发育缺陷：我们将确定在SPOT eNCC中哪个细胞过程(迁移、增殖、生存或分化)是放松调控的。我们还将确定候选基因(A830082K12Rik)在关键GDNF和ET3信号通路中的层级位置。此外，通过比较野生型和突变型胚胎的转录组图谱，将通过RNAseq揭示eNCC中受点突变影响的完整基因集。2)确定A830082K12Rik为斑点致病基因：建立A830082K12Rik靶向突变的小鼠模型，并与该斑点进行等位基因检测。3)新的长非编码RNA基因A830082K12Rik的特征：基因表达模式将通过原位杂交进行评估，而亚细胞定位将使用RNA标记系统和荧光标记相结合的方法进行分析。在eNCC中筛选蛋白质相互作用物将使用下拉实验和质谱学相结合的方法进行。结论：这项工作是增加我们对一类令人兴奋的新型调控分子(即lncRNA)的总体理解的极好机会。加之其他NCC衍生结构(如黑素细胞和内耳感觉上皮)在斑点动物中也受到严重影响，因此我们的研究有望不仅对ENS领域，而且对更广泛的发育生物学领域产生非常重大和持久的影响。