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

VRK-1 在秀丽隐杆线虫胚胎减数分裂中的作用

• 批准号: 7967206
• 负责人: Andy Golden
• 金额: $ 35.89万
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7967206
• 关键词: Accounting; Affect; Alleles; Antibodies; Behavior; Caenorhabditis elegans; Cell Cycle; Chimeric Proteins; Chromatin; Chromosomes; Code; DNA; Defect; Development; Double-Stranded RNA; Drosophila genus; Embryo; Embryonic Development; FUS-1 Protein; Failure; Family; Fertilization; Genes; Genetic; Genetic Recombination; Genetic screening method; Genome; Germ Cells; Germ Lines; Goals; Heterozygote; Histones; Human; Image; Infertility; Lead; Life; Meiosis; Missense Mutation; Mitogen-Activated Protein Kinases; Mitotic; Molecular; Morphogenesis; Morphology; Mothers; Mutation; Nuclear Envelope; Oocytes; Oogenesis; Organism; Orthologous Gene; Pathway interactions; Pattern; Phenotype; Phosphotransferases; Preparation; Process; Protamine Kinase; Proteins; RNA Interference; Role; Screening procedure; Spontaneous abortion; Staging; Staining method; Stains; Sterility; Time; Transgenes; Vaccinia; histone modification; immunocytochemistry; interest; mutant; oocyte maturation; Accounting; Affect; Alleles; Antibodies; Behavior; Caenorhabditis elegans; Cell Cycle; Chimeric Proteins; Chromatin; Chromosomes; Code; DNA; Defect; Development; Double-Stranded RNA; Drosophila genus; Embryo; Embryonic Development; FUS-1 Protein; Failure; Family; Fertilization; Genes; Genetic; Genetic Recombination; Genetic screening method; Genome; Germ Cells; Germ Lines; Goals; Heterozygote; Histones; Human; Image; Infertility; Lead; Life; Meiosis; Missense Mutation; Mitogen-Activated Protein Kinases; Mitotic; Molecular; Morphogenesis; Morphology; Mothers; Mutation; Nuclear Envelope; Oocytes; Oogenesis; Organism; Orthologous Gene; Pathway interactions; Pattern; Phenotype; Phosphotransferases; Preparation; Process; Protamine Kinase; Proteins; RNA Interference; Role; Screening procedure; Spontaneous abortion; Staging; Staining method; Stains; Sterility; Time; Transgenes; Vaccinia; histone modification; immunocytochemistry; interest; mutant; oocyte maturation

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.

VRK-1是秀丽隐杆线虫中与疫苗相关的激酶，在整个发育过程中具有多个功能。 在胚胎发生过程中，核包膜形成以及在幼虫发育过程中形成核管形成和外阴形态发生所必需。 果蝇直系同源物NHK-1是一种组蛋白激酶，突变等位基因会影响卵母细胞中的染色体形态。 我们对影响秀丽隐杆线虫减数分裂分裂期间染色体形态和行为的蛋白质有持续的兴趣。 我们一直在使用RNAi和我们最近确定的错义突变来表征VRK-1基因耗尽的胚胎的表型。 该突变是胚胎致死的，必须将应变保持为杂合子。 我们已经观察到该突变的母亲和受到RNAi的野生型母亲的高度渗透性胚胎致死性。 这些胚胎在减数分裂分裂以及未能解除染色质的情况下显示出缺陷。 结果，这些胚胎将其作为多细胞胚胎的DNA很少。 该缺陷不是发育中的卵母细胞中核包膜缺陷的结果，而是确实影响了染色质与卵母细胞核包膜的关联。 我们正在使用表达GFP标记组蛋白的活胚的实时成像，以遵循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包膜的关联受到干扰。 我们还使用免疫细胞化学来确定目前是否会扰动任何特定的组蛋白修饰或染色质相关因素，以说明观察到的减数分裂缺陷。 现在，这个新的突变等位基因使我们能够通过询问其他基因中的突变是否可以增强或抑制VRK-1突变体（例如MAP激酶）来执行更多定向的基因检测。 我们通过在种系中表达GFP :: VRK-1融合蛋白来挽救VRK-1突变体。 我们正在计划一个遗传抑制器筛选，以识别在VRK-1途径中起作用的其他因素。 在另一项研究以探讨染色体如何为减数分裂裂片准备的研究中，我们正在研究秀丽隐杆线虫Myt1直系同源物的功能。 MYT1属于激酶的WEE1家族，被认为可以在细胞周期中调节CDK1。 RNAi对MYT1直系同源物WEE-1.3进行研究导致不育。 注射dsRNA的母亲迅速变得不育；卵母细胞染色体不再在减数分裂的炎性I。这些染色体具有许多有丝分裂的标志。它们用多种有丝分裂标记抗体染色。 卵母细胞的成熟似乎也很早熟。 我们建议WEE-1.3通常功能以使母体CDK-1在卵子发生过程中保持不活跃，并且在受精后，CDK-1被激活以允许胚胎的减数分裂和有丝分裂分裂。 在没有WEE-1.3的情况下，CDK-1的活跃性就变得活跃，并在未完全区分的未成熟卵母细胞中驱动卵母细胞成熟和染色体成熟。 这些卵母细胞可能没有受精，因为它们没有合成他们进一步开发所需的所有适当的卵母细胞/胚胎产品。 我们最近构建并表达了针对Wee-1.3 :: GFP融合蛋白编码的转基因编码。 这些转基因在种系和核包膜中表达，将有助于识别扰动这种表达模式的突变体。 我们还开始使用RNAi筛选，以筛选与WEE-1.3共同消耗的基因，不再引起无菌表型，以识别该途径的其他组件。