VRK-1, a vaccinia-related kinase in C. elegans, has multiple functions throughout development. It is required for nuclear envelope formation during embryogenesis and for germ line proliferation and vulval morphogenesis during larval development. The Drosophila ortholog, NHK-1, is a histone kinase, mutant alleles of which affect chromosome morphology in oocytes. We have an ongoing interest in proteins that influence chromosome morphology and behavior during the meiotic divisions in C. elegans. We have been characterizing the phenotypes of embryos depleted of the vrk-1 gene, using RNAi and a missense mutation that we recently identified. This mutation is embryonic lethal and the strain must be maintained as a heterozygote. We have observed highly penetrant embryonic lethality from mothers homozygous for this mutation and from wildtype mothers subjected to RNAi. These embryos display defects in the meiotic divisions as well as a failure to decondense chromatin. As a result, these embryos arrest as multicellular embryos with very little DNA. This defect is not a result of nuclear envelope defects in the developing oocytes but does appear to affect the association of chromatin with the oocyte nuclear envelope. We are using real time imaging of live embryos expressing GFP-tagged histones to follow the behavior of the meiotic chromosomes during oocyte maturation in VRK-1-depleted oocytes and embryos. The association of meiotic chromosomes with the nuclear envelope is perturbed. We are also using immunocytochemistry to determine whether any specific histone modifications or chromatin-associated factors are perturbed at this time to account for the observed meiotic defects. This new mutant allele now allows us to perform more directed genetic tests by asking whether mutations in other genes can enhance or suppress the vrk-1 mutant, such as MAP kinase. We have rescued the vrk-1 mutant by expressing a gfp::vrk-1 fusion protein in the germline. We are planning a genetic suppressor screen to identify other factors that function in the VRK-1 pathway. In a separate study to explore how chromosomes prepare for the meiotic divisions, we are examining the function of the C. elegans Myt1 ortholog. Myt1 belongs to the Wee1 family of kinases and is thought to down regulate Cdk1 during the cell cycle. RNAi studies with the Myt1 ortholog, wee-1.3, result in sterility. Mothers injected with dsRNA quickly become infertile;the oocyte chromosomes are no longer paused in diakinesis of meiosis I. These chromosomes have many hallmarks of being mitotic;they stain with a number of mitotic marker antibodies. Oocyte maturation also appears to be precocious. We propose that WEE-1.3 normally functions to keep maternal CDK-1 inactive during oogenesis, and that upon fertilization, CDK-1 becomes activated to allow for the meiotic and mitotic divisions of the embryo. In the absence of WEE-1.3, CDK-1 becomes precociously active and drives oocyte maturation and chromosome maturation in immature oocytes that are not fully differentiated. These oocytes fail to be fertilized presumably because they have not synthesized all the proper oocyte/embryo products they need for further development. We have recently constructed and expressed transgenes coding for WEE-1.3::GFP fusion proteins. These transgenes are expressed in the germline and nuclear envelope and will be useful for identifying mutants that perturb this expression pattern. We have also begun an RNAi screens to identify other components of this pathway by screening for genes that when co-depleted with wee-1.3 no longer cause a sterile phenotype.
|Arur, Swathi; Ohmachi, Mitsue; Nayak, Sudhir et al. (2009) Multiple ERK substrates execute single biological processes in Caenorhabditis elegans germ-line development. Proc Natl Acad Sci U S A 106:4776-81|