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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

基本信息

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
项目类别：
财政年份：
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

项目摘要

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 是秀丽隐杆线虫中的一种牛痘相关激酶，在整个发育过程中具有多种功能。它是胚胎发生过程中核膜形成以及幼虫发育过程中种系增殖和外阴形态发生所必需的。果蝇直系同源物 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。我们对影响线虫减数分裂期间染色体形态和行为的蛋白质一直感兴趣。我们一直在使用 RNAi 和我们最近发现的错义突变来表征缺乏 vrk-1 基因的胚胎的表型。这种突变是胚胎致死的，并且该菌株必须保持为杂合子。我们观察到该突变纯合子母体和接受 RNAi 的野生型母体具有高度渗透性胚胎致死率。这些胚胎在减数分裂中表现出缺陷，并且无法解凝结染色质。结果，这些胚胎作为多细胞胚胎而停滞，DNA 非常少。这种缺陷不是发育中卵母细胞核膜缺陷的结果，但似乎确实影响染色质与卵母细胞核膜的结合。我们使用表达 GFP 标记组蛋白的活胚胎的实时成像来跟踪 VRK-1 耗尽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关联受到干扰。我们还使用免疫细胞化学来确定此时是否有任何特定的组蛋白修饰或染色质相关因子受到干扰，以解释观察到的减数分裂缺陷。现在，这种新的突变等位基因使我们能够通过询问其他基因的突变是否可以增强或抑制 vrk-1 突变体（例如 MAP 激酶）来进行更有针对性的基因测试。我们通过在种系中表达 gfp::vrk-1 融合蛋白来拯救 vrk-1 突变体。我们正在计划进行基因抑制筛选，以确定在 VRK-1 通路中发挥作用的其他因素。在另一项探索染色体如何为减数分裂做好准备的单独研究中，我们正在检查线虫 Myt1 直系同源物的功能。 Myt1 属于 Wee1 激酶家族，被认为在细胞周期中下调 Cdk1。使用 Myt1 直向同源物 wee-1.3 进行的 RNAi 研究导致不育。注射 dsRNA 的母亲很快就会变得不孕；卵母细胞染色体不再暂停在减数分裂 I 的终变过程中。这些染色体具有许多有丝分裂的特征；它们用许多有丝分裂标记抗体染色。卵母细胞成熟似乎也早熟。我们认为，WEE-1.3 通常的作用是在卵子发生过程中保持母体 CDK-1 不活跃，并且在受精时，CDK-1 被激活以允许胚胎的减数分裂和有丝分裂。在缺乏 WEE-1.3 的情况下，CDK-1 变得早熟活跃，并驱动未完全分化的未成熟卵母细胞的卵母细胞成熟和染色体成熟。这些卵母细胞未能受精，可能是因为它们没有合成进一步发育所需的所有适当的卵母细胞/胚胎产物。我们最近构建并表达了编码 WEE-1.3::GFP 融合蛋白的转基因。这些转基因在种系和核膜中表达，可用于鉴定扰乱这种表达模式的突变体。我们还开始进行 RNAi 筛选，通过筛选与 wee-1.3 共同耗尽时不再导致不育表型的基因来鉴定该途径的其他成分。