Cell Cycle Regulation In Oogenesis

Child Health And Human Development

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Abstract

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 influences oocyte differentiation. [unreadable] Arrest of the oocyte cell cycle in prophase of meiosis I (prophase I) is a universally conserved feature of animal oogenesis. We have determined that the translational inhibitor Bruno, which is encoded by the arrest gene, maintains mitotic quiescence during the prophase I meiotic arrest of the Drosophila oocyte. In arrest(bruno) mutants, ovarian cysts enter the meiotic cycle and progress to pachytene, as indicated by the formation of mature synaptonemal complexes. However, after meiotic entry the levels of the mitotic Cyclins increase and the germ cells reenter the mitotic cycle and continue to proliferate. Thus, Bruno functions to inhibit the expression of the mitotic Cyclins after meiotic entry. Our data indicate that Bruno accomplishes this task in part by binding Bruno Response Elements (BREs) present in the cyclin A 3UTR and inhibiting its translation. In Drosophila, Cyclin A is the primary positive regulatory subunit of Cdk1. A similar strategy for maintaining the prophase I arrest, and the required low levels of Cdk1 activity, is employed in clams and fish, as well as many amphibians, where the translation of Cyclin B is inhibited until meiotic maturation. Bruno has previously been implicated in the translational inhibition of two genes involved in the differentiation of the egg and embryo, gurken and oskar. The dual function of Bruno in regulating the translation of genes that influence both the meiotic program and oocyte differentiation suggest a model for how cell cycle regulation and gamete differentiation are coordinated during oogenesis. Our findings represent a major step forward in understanding the regulation of the early meiotic cycle in a genetically tractable metazoan and will provide a framework for future studies on the regulation of this highly-conserved cell cycle arrest. To further define the developmental inputs that control meiotic progression during oogenesis, we have initiated genetic screens to identify the pathways that regulate Bruno expression and activity during the prophase I meiotic arrest. [unreadable] [unreadable] The endocycle is a developmentally programmed variant cell cycle in which cells undergo repeated rounds of DNA replication with no intervening mitosis. In Drosophila the endocycle is driven by the oscillations of Cyclin E/Cdk2 activity. How the periodicity of Cyclin E/Cdk2 activity is achieved during endocycles is poorly understood. We have determined that the p21cip/p27kip1/p57kip2-like Cyclin-dependent Kinase Inhibitor (CKI) Dacapo (Dap) promotes replication licensing during Drosophila endocycles by reinforcing low Cdk activity during the endocycle Gap-phase. In dap mutants, cells in the endocycle have reduced levels of the licensing factor Double-Parked/Cdt1 (Dup/Cdt1), as well as decreased levels of chromatin bound MCM2-7 complex. Additionally, mutations in dup/cdt1 dominantly enhance the dap phenotype in several polyploid cell types. Consistent with a reduced ability to complete genomic replication, dap mutants accumulate increased levels of DNA damage during the endocycle S phase. Intriguingly, we find that dap also promotes replication licensing and genomic stability during pre-meiotic S phase. For both the meiotic cycle and the endocycle our data suggest a model in which Dap inhibits Cyclin E/Cdk2 activity during the Gap phase and thus promotes the efficient licensing of DNA replication origins. A similar role has been defined for the CKI SIC1 in promoting replication origin licensing in late G1 in S. cerevisiae. However, our work represents the first report of a CKI acting to promote replication licensing in a metazoan.[unreadable] [unreadable] We have previously established that missing oocyte (mio) is required for the maintenance of the meiotic cycle during Drosophila oogenesis. In mio mutants, the oocyte enters the meiotic cycle and forms mature synaptonemal complexes. However, this meiotic state is not maintained and ultimately the oocyte withdrawals from meiosis, enters the endocycle and becomes polyploid. Intriguingly, inhibiting the formation of the double-stranded breaks that initiate meiotic recombination, strongly suppresses the mio 16-nurse cell phenotype, suggesting that mio interacts with pathways that influence DNA metabolism. Recently, we have initiated a biochemical characterization of Mio. From these studies, we determined that Mio is present in a stable multi-protein complex of approximately 550 kDa. Additionally, we have found that Mio physically and genetically interacts with components of the nuclear pore complex. These studies will provide the framework for future studies on how nuclear pore components influence the events of meiosis.