Development of the enteric nervous system

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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.