Systems Analysis of Conditional C. elegans Mutants with Late Embryonic Defects

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 DESCRIPTION (provided by applicant): We propose to combine powerful new technologies to investigate at a systems level an almost entirely neglected area of developmental biology research-late embryogenesis in the nematode Caenorhabditis elegans-and to greatly expand the availability of conditional mutations in essential genes for the investigation of biological processes in this model animal. We will use Illumina-based whole genome sequencing to rapidly identify the causal mutations in a collection of 250 temperature-sensitive, embryonic-lethal C. elegans mutants that progress normally through the early stages of embryogenesis but arrest late in embryogenesis. About 30 of these mutants arrest with severe defects in morphogenesis - the elongation of an oval mass of largely post-mitotic cells into a long thin worm. The remainder arrest later in development after extensive morphogenesis. While embryonic morphogenesis in C. elegans has been a subject of investigation for many years, our understanding of it remains incomplete. Our mutant collection promises important new insights into the genetic networks that regulate and mediate this complex process. In addition to identifying the causal genes, we will systematically identify the developmental defects in late embryos at cellular resolution, which has been almost entirely neglected due to technical difficulties. To characterize the developmental defects at high resolution in both classes of mutants, and assemble our data into a systems view of late embryogenesis, we will (i) apply recently developed automated embryonic cell lineage analysis as well as new methods for quantitative image analysis to identify abnormalities throughout embryogenesis, (ii) use CRISPR/Cas9 technology to generate transgenic strains bearing translational fusions of GFP to the genes we identify so as to determine when and where in embryogenesis they are expressed, and to guide our phenotype analysis, and (iii) assemble the phenotype and expression data into a multi-scale view of late embryonic development from genes to organismal morphology. We also will take advantage of the conditional mutations we have isolated to determine when in development the affected genes perform their essential embryonic functions, and to identify additional essential functions throughout the nematode life cycle. Genome-wide RNA interference screens have identified about 2500 essential genes in C. elegans, most of which are conserved in other animals. Temperature-sensitive mutations provide a uniquely powerful tool for investigating the requirements for essential genes, as many if not most of them have multiple functions throughout the life of the animal. Moreover, C. elegans is unique as an animal model in which one can feasibly isolate large numbers of relatively rare conditional mutations in essential genes, and yet conditional mutations have been identified in only about one hundred of the 2500 essential C. elegans genes. In addition to substantially advancing our understanding of morphogenesis and late embryogenesis, we also will greatly expand the availability of these powerful genetic tools to investigators throughout th world who use C. elegans as an animal model.