Embryonic morphogenesis in C. elegans is the process by which an ovoid mass of cells reshapes itself to take on the final elongated larval shape. Embryonic cells are quite dynamic during this process, undergoing both positional and morphological changes as they differentiate and form mature tissues. Many genes required for this dynamic behavior are also required earlier in development, complicating the identification of specific morphogenetic requirements using either non-conditional alleles or RNAi. Conditional alleles can bypass early genetic requirements and thus provide an excellent tool for dissecting later stage genetic pathways. We have isolated a collection of ~1,000 temperature-sensitive embryonic lethal (TS-Emb) mutants as a valuable resource for the identification of genes that influence embryonic morphogenesis. We now seek to identify all of the morphogenesis defective mutants within this collection, and to positionally clone as many of them as possible. In the longer term, we propose to build a systems-level model of embryonic morphogenesis.
To date, we have identified 68 mutants with penetrant embryonic morphogenesis defects. Most of these mutants arrest without extensive elongation, while others arrest after significant elongation (≥2-fold stage). We are initially classifying these terminal phenotypes using DIC microscopy. We also are scoring heterozygous embryonic lethality at the restrictive temperature and segregation frequency after outcross to ensure we are working with single gene, loss of function alleles. So far, nearly all alleles observed have behaved as expected for recessive, loss of function mutations.
We are using a high throughput whole genome sequencing-based approach to positionally clone mutants identified in this screen. This approach has been previously described1,2 and applied to TS-Emb mutants affecting various stages of development3,4, and both provides genome wide SNP mapping data and uncovers all homozygous SNPs within the mapping region. Genetic complementation tests against known loss of function alleles are then used to determine gene identity. So far, we have cloned 10 alleles representing 7 genes: glp-1 (3 alleles), let-19 (2 alleles), klp-18, chaf-1, zwl-1, rib-1, hlh-1. We anticipate that some of these genes will have direct roles in morphogenesis, while others may represent partial loss of function mutations in genes with earlier more general requirements in metabolism or cell division. Ultimately, we aim to clone 200+ mutants using this approach. These alleles will provide a resource that augments other efforts to build a detailed model of embryonic morphogenesis in C. elegans.