Cell Cycle Regulation In Oogenesis

卵子发生中的细胞周期调控

• 批准号: 8149283
• 负责人: MARY A LILLY
• 金额: $ 127.55万
• 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/8149283
• 关键词: Cell Cycle Regulation; Oogenesis

We use Drosophila oogenesis as a model to explore the developmental regulation of the cell cycle. As is observed in mammals and Xenopus, the Drosophila oocyte initiates meiosis within a germline cyst. Drosophila ovarian cysts are produced through a series of four synchronous mitotic divisions during which cytokinesis is incomplete. Soon after the completion of the mitotic divisions, all 16 cells enter premeiotic S phase. However, only the single oocyte remains in meiosis and goes on to produce a functional gamete. The other 15 cells lose their meiotic features, enter the endocycle, and develop as polyploid nurse cells. Throughout much of oogenesis, the oocyte remains faithfully arrested in prophase of meiosis I. Late in oogenesis, the single oocyte undergoes meiotic maturation and proceeds to the first meiotic metaphase. In contrast, the nurse cells transfer their contents to the growing oocyte and undergo apoptosis. To understand the regulatory inputs that control early meiotic progression, we are working to determine how the oocyte initiates and then maintains the meiotic cycle within the challenging environment of the ovarian cyst. Our studies focus on questions that are relevant to the development of all animal oocytes. What strategies does the oocyte use to protect itself from inappropriate DNA replication? How does the oocyte inhibit mitotic activity prior to meiotic maturation and the full growth and development of the egg? Finally, how does cell cycle status within the ovarian cyst influence the differentiation of the oocyte? To answer these questions my laboratory has undertaken studies to determine the basic cell cycle program of the Drosophila ovarian cyst 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 Seh1 a component of the nuclear pore complex (NPC). The NPC mediates transport of macromolecules between the nucleus and the cytoplasm. Recent evidence indicates that structural nucleoporins, the building blocks of the NPC, have a variety of unanticipated cellular functions. We have defined an unexpected tissue specific requirement for the structural nucleoporin Seh1 during Drosophila oogenesis. Seh1 is a component of the Nup107-160 complex, the major structural subcomplex of the NPC. We find that Seh1 associates with the product of the mio gene. Like mio, the nucleoporin seh1 has a critical germline function during oogenesis. In both mio and seh1 ovaries a fraction of oocytes fail to maintain the meiotic cycle and develop as pseudo-nurse cells. Moreover, we find that the accumulation of the Mio protein is greatly diminished in the seh1 mutant background. Surprisingly, our characterization of a seh1 null allele indicates that while required in the female germline, seh1 is dispensable for the development of somatic tissues. Our work represents the first examination of seh1 function within the context of a multicellular organism. Our studies demonstrate that Mio is a novel interacting partner of the conserved nucleoporin Seh1 and add to the growing body of evidence that structural nucleoporins can have novel tissue-specific roles. Additionally, our observations provide the framework for future studies on how nuclear pore components influence meiotic progression and the maintenance of the oocyte identity. 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 metazoans. We have determined that the p21(Cip1)/p27(Kip1)/p57(Kip2)-like cyclin-dependent kinase inhibitor (CKI) Dacapo (Dap) is a key regulator of premeiotic S phase and genomic stability during Drosophila oogenesis. In dap mutant 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 in the absence of Dap, ovarian cysts have low levels of Dup/Cdt1. Moreover, mutations in dup/cdt1 dominantly enhance the DNA damage phenotype of dap mutants. 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 as well as other early meiotic events in oogenesis.

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。 正如在哺乳动物和爪蟾中观察到的那样，果蝇卵母细胞在种系囊肿内引发减数分裂。果蝇卵巢囊肿是通过一系列四个同步有丝分裂分裂产生的，在此过程中，细胞因子不完整。 有丝分裂分裂完成后不久，所有16个细胞都进入前阶段。但是，只有单个卵母细胞保留在减数分裂中，并继续产生功能性配子。 其他15个细胞失去了其减数分裂特征，进入内吞以多倍体护士细胞发展。在整个卵子发生的大部分时间里，卵母细胞仍然忠实地以减数分裂的预言。 相比之下，护士细胞将其含量转移到生长的卵母细胞中并经历凋亡。 为了了解控制早期减数分裂进程的调节输入，我们正在努力确定卵母细胞如何启动，然后在卵巢囊肿的挑战性环境中保持减数分裂周期。 我们的研究集中于与所有动物卵母细胞发展有关的问题。 卵母细胞用于保护自身免受不适当的DNA复制而采用哪些策略？ 卵母细胞在减数分裂成熟之前如何抑制有丝分裂活性以及卵的全部生长和发育？ 最后，卵巢囊肿内的细胞周期状态如何影响卵母细胞的分化？ 为了回答这些问题，我的实验室已经进行了研究以确定果蝇卵巢囊肿的基本细胞周期计划 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SEH1相互作用是核孔复合物（NPC）的成分。 NPC介导细胞核和细胞质之间的大分子的转运。 最近的证据表明，结构性核孔蛋白（NPC的构件）具有多种意外的细胞功能。 我们已经定义了果蝇卵子发生过程中对结构核孔蛋白SEH1的意外组织特异性需求。 SEH1是NUP107-160复合物的组成部分，NPC的主要结构子复杂。 我们发现SEH1与MIO基因的产物相关联。 像MIO一样，核孔SEH1在卵子发生过程中具有关键的生殖功能。在MIO和SEH1卵巢中，卵母细胞的一部分都无法维持减数分裂周期，并以伪核细胞的形式发展。此外，我们发现在SEH1突变体背景下，MIO蛋白的积累大大减少。令人惊讶的是，我们对SEH1无效等位基因的表征表明，尽管在女性种系中需要SEH1，但对于体细胞组织的发展是可分配的。 我们的工作代表了在多细胞生物的背景下对SEH1功能的首次检查。 我们的研究表明，MIO是保守的核孔SEH1的一种新型相互作用伴侣，并增加了越来越多的证据表明结构性核孔可以具有新型组织特异性作用。 此外，我们的观察结果为将来的研究提供了有关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。 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。但是，在后生动物中，对前阶段的调节仍然很差。我们已经确定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）是果蝇期间的prepeoiotic S相和基因组稳定性的关键调节剂。在DAP突变雌性中，卵巢囊肿以高水平的细胞周期蛋白E/细胞周期蛋白依赖性激酶（CDK）2活性进入减数分裂循环，并在预局部阶段累积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，我们发现在没有DAP的情况下，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突变体的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前虫阶段以及其他早期减数分裂事件的调节剂。