The role of VRK-1 during the meiotic divisions of C. elegans embryos

VRK-1是秀丽隐杆线虫中一种与牛痘相关的激酶，在整个发育过程中具有多种功能。它是胚胎发育过程中核膜形成和幼虫发育过程中种系增殖和外阴形态形成所必需的。果蝇同源基因NHK-1是一种组蛋白激酶，其突变等位基因影响卵母细胞的染色体形态。我们对秀丽隐杆线虫减数分裂过程中影响染色体形态和行为的蛋白质有持续的兴趣。我们一直在用RNAi和我们最近发现的一个错义突变来表征vrk-1基因缺失的胚胎的表型。这种突变是胚胎致死性的，必须保持该菌株为杂合子。我们观察到具有这种突变的纯合子母株和受RNAi影响的野生型母株的高渗透性胚胎致死率。这些胚胎在减数分裂中表现出缺陷以及去致密染色质的失败。结果，这些胚胎停滞为多细胞胚胎，只有很少的DNA。这种缺陷不是发育中的卵母细胞核膜缺陷的结果，但似乎确实影响了染色质与卵母细胞核膜的关联。我们正在使用表达gfp标记组蛋白的活胚胎的实时成像来跟踪vrk -1缺失的卵母细胞和胚胎中卵母细胞成熟过程中减数分裂染色体的行为。减数分裂染色体与核膜的结合受到干扰。我们还使用免疫细胞化学来确定是否有任何特定的组蛋白修饰或染色质相关因子在这个时候受到干扰，以解释观察到的减数分裂缺陷。

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.