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.
Showing the most recent 10 out of 11 publications
