The unit on cell cycle regulation uses 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 program of the Drosophila ovarian cyst is coordinated with the developmental events of oogenesis. The Drosophila 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. The cyst is produced through a series of four synchronous mitotic divisions during which cytokinesis is incomplete. 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 the laboratory is 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. mio oocytes eventually abandon the meiotic cycle, enter the endocycle and develop as nurse cells. We have molecularly characterized the mio gene. mio is predicted to encode a protein of 867 amino acids that contains four WD40 repeats and is conserved from yeast to humans. We have determined that the mio protein is present at high levels in the oocyte nucleus soon after it enters 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 Drosophila 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. Germline cysts from mio, egl double mutants have a significantly stronger phenotype. The 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. However, exactly when mio is required to maintain the meiotic cycle has not yet been determined. Further studies of mio may help elucidate the poorly characterized pathways that control meiotic progression and the maintenance of oocyte identity. Dacapo is a vital gene that encodes a p21CIP/p27KIP1/p57KIP2 like cyclin dependent kinase inhibitor (cki) 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 (de Nooij et al., 2000). 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 resetting 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. We have entered into collaboration with the laboratory of Jennifer Lippincott-Schwartz to use live imaging of Green Fluorescent Protein (GFP) tagged proteins to study the structure and function of the Drosophila fusome. The fusome is a membranous organelle comprised of microtubules and an array of membrane-associated cytoskeletal proteins that grows along the remnants of the mitotic spindle after each cyst division. In the absence of the fusome, which physically connects all of the cells in the cyst, mitotic synchrony between cystocytes is lost, the number of cystocyte divisions is reduced and oocyte differentiation does not occur. However, the molecular mechanisms by which the fusome influences the cystocyte cell cycle and oocyte differentiation are poorly understood. This work has lead to three important conclusions: 1) The fusomal membranes are derived from the endoplasmic reticulum (ER); 2) The fusome is continuous with the cytoplasmic ER; and, 3) All the cells within a single ovarian cyst share a common ER. We predict that the interconnectivity of the cyst ER, through the fusome, may be important to the synchronization of the mitotic cyst divisions as well as other signaling events within the cyst. Mutations in the twin gene cause an array of phenotypes that suggest the cell cycle program of the ovarian cyst has been altered. First, twin egg chambers frequently contain either too few or two many cells indicating the cyst has undergone an inappropriate number of mitotic divisions. Second, approximately 5% of twin 16-cell cysts contain two oocytes. Finally, the nurse cells in twin mutants frequently have abnormal chromatin structure. In collaboration with the laboratory of Ruth Lehmann we have determined that twin is the putative ortholog of the S. cerivisiae CCR4 gene. In yeast, CCR4 is the catalytic subunit of the major cytoplasmic mRNA deadenylsase. Deadenylation influences both mRNA function and stability. In addition CCR4 has been implicated in transcriptional initiation and elongation. The catalytic domain that contains 3prime-5prime poly(A)RNA and ssDNA exonuclease activity is highly conserved between CCR4 and twin. We are currently examining if the twin phenotype is dominantly modified by mutations in known cell cycle genes as well as components of the transcriptional machinery that genetically interact with CCR4 in yeast.
Showing the most recent 10 out of 11 publications
