We use Drosophila oogenesis as a model to explore the developmental regulation of the cell cycle. The long-term goal of the laboratory is to understand how the cell cycle events of meiosis are coordinated with the developmental events of gametogenesis. 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 influences the nurse cell/oocyte fate decision. In order to identify the pathways that direct entry into and maintenance of the meiotic cycle in the single pro-oocyte we screened for mutants in which all 16 cells enter the endocycle and develop as nurse cells. From this screen we identified a new gene, missing oocyte (mio) that is required for the maintenance of the meiotic cycle. In mio mutants the oocyte enters the meiotic cycle, forms mature synaptonemal complexes and progresses to pachytene. However this meiotic state is not maintained. Ultimately, mio oocytes abandon the meiotic cycle, enter the endocycle and develop as nurse cells. We molecularly characterized the mio gene. From this analysis we determined that mio is predicted to encode a protein of 867 amino acids that is highly conserved from yeast to humans. In higher eukaryotes, all Mio family members share a similar domain structure. The amino termini contain a series of four to six well-conserved WD40 repeats. WD40 repeats often provide a surface for protein-protein interactions The WD40 repeats found in mio family members are most similar to those present in the chromatin binding protein CAF1p48/RbAp48, which is a component of numerous chromatin-remodeling complexes. Specifically, CAF1p48/RbAp48 is found in complexes that modify chromatin through the acetylation and deacetylation of histones. In addition to the WD40 repeats, Mio family members contain a highly conserved 50 amino acid domain near their C termini that share structural similarities to two well-characterized zinc binding domains, the RING finger and the PHD finger. RING finger domains are present in a subclass of E3 ubiquitin ligases while PHD fingers have been implicated in chromatin binding. While the ?Mio domain? does share structural similarities to these zinc-binding domains, it does not fit the exact consensus of either a canonical RING finger or a canonical PHD finger. Therefore, the biochemical function of this highly conserved domain remains to be empirically determined. Mio accumulates to high levels in the oocyte nucleus during early prophase of meiosis I. Double labeling with anti-Mio antibodies and an antibody against the synaptonemal complex protein C(3)G, indicate that Mio specifically localizes to the nucleus of the oocyte soon after the completion of premeiotic S phase. This makes Mio one of the earliest nuclear markers for the oocyte in that is not a known component of the synaptonemal complex. Genetic interaction studies indicate that Mio functions early in meiosis, prior to the onset of pachytene. In egalitarian (egl) mutants all 16-cyst cells enter the meiotic cycle and progress to pachytene as assayed by the presence of mature synaptonemal complexes. However, this meiotic state can?t be maintained and eventually all the cells exit the meiotic and enter the endocycle. Intriguingly, germline cysts from mio, egl double mutants have a significantly stronger phenotype than either single mutant. Ovarian cysts from mio, egl double mutants never form mature synaptonemal complexes and do not progress past zygotene. These data indicate that mio influences the oocyte cell cycle in early prophase of meiosis I, prior to the formation of the mature synaptonemal complex. The mio ovarian phenotype is suppressed by inhibiting the formation of the double-stranded breaks (DSB) that initiate meiotic recombination during meiosis. In mio single mutants the oocyte frequently enters the endocycle and becomes polyploid. However, when placed in a genetic background in which DSB formation is inhibited, the majority of mio egg chambers retain an oocyte and develop to late stages of oogenesis. The simplest interpretation of the data is that mio is required to repair the DSBs that initiate meiotic recombination and that the inability to repair DSBs significantly contributes to the mio phenotype. While clearly important, the inability to repair DSBs during meiosis is unlikely to be sole cause of the mio phenotype. Considering that Mio localizes to the oocyte nucleus early in meiosis and functions before the construction of the mature SC, we speculate that Mio acts upstream of the enzymology of DSB repair. Thus, the inability of mio mutants to repair DSB may be due to a general alteration in meiotic chromosome structure or alternatively subtle alterations in the meiotic program. Further studies of mio may help elucidate the poorly characterized pathways that control meiotic progression and the maintenance of oocyte identity. Animal oocytes undergo a highly conserved developmental arrest in prophase of meiosis I. Often this marks a period of rapid growth for the oocyte and is necessary to coordinate meiotic progression with the developmental events of oogenesis. In Drosophila the oocyte develops within a 16-cell germline cyst. Throughout much of oogenesis the oocyte remains in prophase of meiosis I. In contrast, its 15 mitotic sisters enter the endocycle and become polyploid in preparation for their role as nurse cells. How germline cysts establish and maintain these two independent cell cycles is unknown. We have shown that the p21CIP/p27Kip1/p57Kip2 like cyclin-dependent kinase inhibitor (CKI) Dacapo maintains the prophase I meiotic arrest of the Drosophila oocyte. dacapo is a vital gene that that specifically inhibits the activity of CycE/Cdk2 complexes. CycE/Cdk2 activity is required for S phase in Drosophila. Throughout much of the growth phase of Drosophila oogenesis the levels of the cki Dacapo oscillate in the 15-polyploid nurse cells but remain persistently high in the single oocyte. We have shown that both modes of Dacapo regulation are functionally important. In the oocyte the prophase I arrest is lost, or not properly established, in germline cysts that lack Dacapo. This is the first demonstration of a cip/kip family member functioning in a normal meiotic cycle. In addition, our data indicate that Dacapo is part of the biochemical oscillator that drives the nurse cell endocycle. Specifically, we find that in polyploid nurse cells the oscillations of Dacapo facilitate the relicencing of DNA replication origins during endoreplication by inhibiting CycE/Cdk2 activity at the end of each endocycle S phase. Our data are consistent with recently proposed models that suggest the periodic expression of members of the cip/kip family of Cdk inhibitors direct entry into the Gap phase during endoreplicative cycles. We propose that it is through the differential regulation of the cki Dacapo that two dramatically different cell cycles, the meiotic cycle and the endocycle, are independently maintained within the common cytoplasm of the ovarian cyst. Currently, we are performing genetic screens to identify additional genes that regulate CycE/Cdk2 during oogenesis.
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