Cell Cycle Regulation In Oogenesis

卵子发生中的细胞周期调控

• 批准号: 7968603
• 负责人: MARY A LILLY
• 金额: $ 76.32万
• 依托单位: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
• 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/7968603
• 关键词: Actins; Animal Model; Animals; Architecture; CDKN1C gene; Cell Cycle; Cell Cycle Progression; Cell Cycle Regulation; Cell Nucleus; Cells; Cellular biology; Chromosome Segregation; Chromosomes; Congenital Abnormality; Cyclin E; Cyclin-Dependent Kinase Inhibitor; Cyclin-Dependent Kinases; Cyclins; Cyst; DNA Damage; DNA Double Strand Break; DNA biosynthesis; DNA replication origin; Data; Defect; Development; Drosophila genus; Drosophila melanogaster; Event; Female; Female sterility; Future; Gametogenesis; Geminin; Genes; Genome Stability; Genomics; Germ Cells; Goals; Growth; Hormonal; Human; Individual; Laboratories; Licensing; Licensing Factor; Maintenance; Medicine; Meiosis; Meiotic Recombination; Mitosis; Mitotic; Modeling; Mutation; Nuclear; Nuclear Pore; Nuclear Pore Complex Proteins; Nurses; Oocytes; Oogenesis; Ovarian; Ovarian Cysts; Pathway interactions; Phase; Phenotype; Placenta; Plants; Polyploidy; Pre-Replication Complex; Prophase; Proteins; Regulation; Replication Licensing; Reporting; Reproductive Biology; Role; Series; Signal Pathway; Spontaneous abortion; Testing; Variant; Withdrawal; Work; anaphase-promoting complex; cyclin-dependent kinase inhibitor 1B; egg; human CDK2 protein; interest; mutant; programs; protein function; research study; trophoblast; Actins; Animal Model; Animals; Architecture; CDKN1C gene; Cell Cycle; Cell Cycle Progression; Cell Cycle Regulation; Cell Nucleus; Cells; Cellular biology; Chromosome Segregation; Chromosomes; Congenital Abnormality; Cyclin E; Cyclin-Dependent Kinase Inhibitor; Cyclin-Dependent Kinases; Cyclins; Cyst; DNA Damage; DNA Double Strand Break; DNA biosynthesis; DNA replication origin; Data; Defect; Development; Drosophila genus; Drosophila melanogaster; Event; Female; Female sterility; Future; Gametogenesis; Geminin; Genes; Genome Stability; Genomics; Germ Cells; Goals; Growth; Hormonal; Human; Individual; Laboratories; Licensing; Licensing Factor; Maintenance; Medicine; Meiosis; Meiotic Recombination; Mitosis; Mitotic; Modeling; Mutation; Nuclear; Nuclear Pore; Nuclear Pore Complex Proteins; Nurses; Oocytes; Oogenesis; Ovarian; Ovarian Cysts; Pathway interactions; Phase; Phenotype; Placenta; Plants; Polyploidy; Pre-Replication Complex; Prophase; Proteins; Regulation; Replication Licensing; Reporting; Reproductive Biology; Role; Series; Signal Pathway; Spontaneous abortion; Testing; Variant; Withdrawal; Work; anaphase-promoting complex; cyclin-dependent kinase inhibitor 1B; egg; human CDK2 protein; interest; mutant; programs; protein function; research study; trophoblast

We use Drosophila oogenesis as a model to explore the developmental regulation of the cell cycle. In Drosophila, the oocyte develops within the context of a 16-cell germline cyst. Individual cells within the cyst are referred to as cystocytes and are connected by actin-rich ring canals. While all 16 cystocytes enter premeiotic S phase, only a single cell remains in the meiotic cycle and becomes the oocyte. The other 15 cells enter the endocycle and develop as highly polyploid nurse cells. Currently, we are working to understand how cells within ovarian cyst enter and maintain either the meiotic cycle or the endocycle. In addition, we are examining how this cell cycle choice drives the development of the mature egg. The proper execution of premeiotic S phase is essential to both the maintenance of genomic integrity and accurate chromosome segregation during the meiotic divisions. However, the regulation of premeiotic S phase remains poorly defined in metazoa. We identify the p21(Cip1)/p27(Kip1)/p57(Kip2)-like cyclin-dependent kinase inhibitor (CKI) Dacapo (Dap) as a key regulator of premeiotic S phase and genomic stability during Drosophila oogenesis. In dap/ females, ovarian cysts enter the meiotic cycle with high levels of Cyclin E/cyclin-dependent kinase (Cdk)2 activity and accumulate DNA damage during the premeiotic S phase. High Cyclin E/Cdk2 activity inhibits the accumulation of the replication-licensing factor Doubleparked/Cdt1 (Dup/Cdt1). Accordingly, we find that dap/ ovarian cysts have low levels of Dup/Cdt1. Moreover, mutations in dup/cdt1 dominantly enhance the dap/ DNA damage phenotype. Importantly, the DNA damage observed in dap/ ovarian cysts is independent of the DNA double-strands breaks that initiate meiotic recombination. Together, our data suggest that the CKI Dap promotes the licensing of DNA replication origins for the premeiotic S phase by restricting Cdk activity in the early meiotic cycle. We are currently working to define additionally regulators of the premeiotic S phase in Drosophila. The endocycle is a commonly observed variant cell cycle in which cells undergo repeated rounds of DNA replication with no intervening mitosis. Cells that are highly metabolically active, such as the giant trophoblast of the mammalian placenta and the Drosophila ovarian nurse cells, often grow via endoreplication. How the cell cycle machinery is modified to transform a mitotic cycle into endocycle has long been a question of interest. In both plants and animals, the transition from the mitotic cycle to the endocycle requires Fzr/Cdh1, a positive regulator of the Anaphase-Promoting Complex/Cyclosome (APC/C). However, because many of its targets are transcriptionally downregulated upon entry into the endocycle, it remained unclear whether the APC/C functioned beyond the mitotic/endocycle boundary. We have shown that APC/CFzr/Cdh1 activity is required to promote the G/S oscillation of the Drosophila endocycle. We find that compromising APC/C activity, after cells have entered the endocycle, inhibits DNA replication and results in the accumulation of multiple APC/C targets including the mitotic Cyclins and Geminin. Notably our data suggest that the activity of APC/CFzr/Cdh1 during the endocycle is not continuous but cyclic, as demonstrated by the APC/C-dependent oscillation of the pre-replication complex component ORC1. Taken together our data suggest a model in which the cyclic activity of APC/CFzr/Cdh1 during the Drosophila endocycle is driven by the periodic inhibition of Fzr/Cdh1 by Cyclin E/Cdk2. Thus, we propose that as is observed in mitotic cycles, during endocycles APC/CFzr/Cdh1 functions to reduce the levels of the mitotic Cyclins and Geminin in order to facilitate the relicensing of DNA replication origins and cell cycle progression. These studies will allow new models on the minimum cell cycle inputs necessary to construct a G/S oscillator to be formulated and tested. The pathways that control entry into the meiotic cycle and early meiotic progression are poorly understood in metazoans. We previously identified a gene, missing oocyte (mio) that regulates nuclear architecture and meiotic progression in early ovarian cysts. In mio mutants, the oocyte enters the meiotic cycle and progresses to pachytene. However, this meiotic state is not maintained and ultimately the oocyte withdrawals from meiosis, enters the endocycle and becomes polyploid. mio mutants display some of the earliest meiotic defects reported in Drosophila. Moreover, the Mio protein accumulates in the oocyte nucleus in early prophase of meiosis I. Therefore, mio provides an excellent entry point to explore how the unique cell biology of the early ovarian cyst and the establishment of the meiotic program, influence the downstream events of oocyte differentiation and meiotic progression. To better understand the role of mio in oogenesis, we have initiated a series of experiments to identify additionally proteins that function in the Mio pathway. From these studies we have found that Mio physically and genetically interacts with the nucleoporin Nup44A. Moreover, mutations in Nup44A disrupt meiotic progression and female sterility. These observations provide the framework for future studies on how nuclear pore components influence meiotic progression and the maintenance of the oocyte identity.

我们使用果蝇卵子形成作为模型来探索细胞周期的发育调控。在果蝇中，卵母细胞在16细胞种系囊肿的背景下发展。囊肿中的单个细胞称为膀胱细胞，并通过富含肌动蛋白的环管连接。虽然所有16个膀胱细胞都进入了前虫的相位，但只有一个单元保留在减数分裂周期中，并成为卵母细胞。其他15个细胞进入内猫菌，并作为高度多倍体护士细胞发展。目前，我们正在努力了解卵巢囊肿中的细胞如何进入和维持减数分裂周期或内猫。此外，我们正在研究这种细胞周期选择如何推动成熟卵的发展。 在减数分裂划分期间，适当的前阶段的适当执行对于维持基因组完整性和准确的染色体隔离至关重要。然而，在后生动物中，对前阶段的调节仍然很差。我们确定p21（CIP1）/p27（KIP1）/p57（KIP2）类似细胞周期蛋白依赖性激酶抑制剂（CKI）DACAPO（DAP）作为果蝇期间果蝇期间的前质量稳定性和基因组稳定性的关键调节剂。在DAP/雌性中，卵巢囊肿以高水平的细胞周期蛋白E/ Cyclin依赖性激酶（CDK）2活性进入减数分裂循环，并在预元阶段累积DNA损伤。 高细胞周期蛋白E/CDK2活性抑制了复制许可因子双重标记/CDT1（DUP/CDT1）的积累。因此，我们发现DAP/卵巢囊肿的DUP/ CDT1水平较低。此外，DUP/ CDT1中的突变主要增强了DAP/ DNA损伤表型。重要的是，在DAP/卵巢囊肿中观察到的DNA损伤与启动减数分裂重组的DNA双链破裂无关。总之，我们的数据表明，CKI DAP通过限制早期减数分裂周期中的CDK活性来促进Premeiotic S阶段的DNA复制起源的许可。 我们目前正在努力定义果蝇前阶段的调节剂。 内吞是一个通常观察到的变异细胞周期，其中细胞经历了重复的DNA复制，而无丝分裂。 具有高度代谢活性的细胞，例如哺乳动物胎盘的巨大滋养细胞和果蝇卵巢护士细胞，通常会通过内膜填充而生长。长期以来，如何将细胞周期机械修改以将有丝分裂周期转化为内猫活动一直是一个有趣的问题。 在动植物中，从有丝分裂循环到内吞周期的过渡需要FZR/CDH1，这是促成后期促进酶复合体/循环体（APC/C）的阳性调节剂。 但是，由于其许多目标在进入内鼠时被转录下调，因此尚不清楚APC/C是否在有丝分裂/内循环边界之外运行。 我们已经表明，需要APC/CFZR/CDH1活性来促进果蝇内吞噬的G/S振荡。我们发现损害APC/C活性，在细胞进入内吞后，抑制DNA的复制，并导致多种APC/C靶标积累，包括有丝分裂细胞周期蛋白和Geminin。 值得注意的是，我们的数据表明，在内吞过程中APC/CFZR/CDH1的活性不是连续的，而是循环的，如APC/C依赖性振荡的复制复合物成分ORC1的振荡所证明。综上所述，我们的数据表明了一个模型，其中果蝇内环过程中APC/CFZR/CDH1的环状活性是由Cyclin E/CDK2对FZR/CDH1的周期性抑制驱动的。因此，我们提出，如在有丝分裂周期中所观察到的，在内幼体APC/CFZR/CDH1期间起作用以降低有丝分裂细胞周期蛋白和双子素的水平，以促进DNA复制起源和细胞循环进展的重新化。这些研究将允许对构建和测试构建G/S振荡器的最小细胞周期输入的新模型。 在后生动物中，控制进入减数分裂周期和早期减数分裂进展的途径知之甚少。 我们以前鉴定了一个基因，缺失卵母细胞（MIO），该基因调节早期卵巢囊肿的核结构和减数分裂进程。 在mio突变体中，卵母细胞进入减数分裂周期并发展为pachytene。 但是，这种减数分裂状态无法保持，最终从减数分裂中提取卵母细胞，进入内吞并变成多倍体。 Mio突变体显示果蝇中报告的一些最早的减数分裂缺陷。 此外，MIO蛋白在减数分裂的早期预言中积聚在卵母细胞核中。因此，MIO提供了一个极好的入口点，以探索早期卵巢囊肿的独特细胞生物学如何影响减数分裂程序的建立，影响卵巢分化和减数分裂进展的下游事件。 为了更好地了解MIO在卵子发生中的作用，我们启动了一系列实验，以鉴定在MIO途径中起作用的蛋白质。 从这些研究中，我们发现MIO在物理和遗传上与核孔NUP44A相互作用。 此外，NUP44A中的突变破坏了减数分裂进展和女性不育。 这些观察结果为将来的研究提供了关于核孔成分如何影响减数分裂进程和维持卵母细胞身份的框架。