Mary Lilly, PhD, Head, Unit on Cell Cycle Regulation
Karine Narbonne, PhD, Visiting Fellow
Stefania Senger, PhD, Visiting Fellow
Isamu Sugimura, PhD, Visiting Fellow
Lily Nguyen, BS, Postbaccalaureate Fellow

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.

Translational control of mitotic Cyclin expression in meiotic and premeiotic ovarian cysts

Sugimura; in collaboration with Lehmann

Arrest of the oocyte cell cycle in prophase of meiosis I (prophase I) is a universally conserved feature of animal oogenesis. During prophase I arrest, animal oocytes must perform two seemingly contradictory tasks. First, they must accumulate and store large quantities of mRNAs and proteins that are required to drive the two future meiotic divisions as well as the mitotic divisions of the early embryo. Second, they must remain fully arrested in order to maintain the integrity of the genome and avoid producing an aneuploid gamete. An important factor in maintaining prophase I arrest is the inhibition of the mitotic kinase Cdk1. The precocious activation of Cdk1 might force the oocyte back into the mitotic cycle or alternatively result in premature entry into the first meiotic metaphase. Thus, oocytes must inhibit Cdk1 activity at the onset of meiosis but be able to activate the mitotic kinase later in oogenesis when they resume the meiotic cycle. While it is one of the most highly conserved events of gametogenesis, the precise developmental and cell cycle events that initiate and maintain prophase I arrest are not fully understood.

We have determined that the translational inhibitor Bruno, which is encoded by the arrest gene, maintains mitotic quiescence during 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. After meiotic entry, however, the levels of 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 to Bruno Response Elements (BREs) present in the cyclin A 3′ UTR and inhibiting its translation. In Drosophila, Cyclin A is the primary positive regulatory subunit of Cdk1. A similar strategy for maintaining prophase I arrest, and the required low levels of Cdk1 activity, is employed in clams and fish as well as in many amphibians, whose translation of Cyclin B is inhibited until meiotic maturation. Bruno has previously been implicated in the translational inhibition of gurken and oskar, two genes involved in the differentiation of the egg and embryo. The dual function of Bruno in regulating the translation of genes that influence both the meiotic program and oocyte differentiation suggests 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.

In collaboration with the laboratory of Ruth Lehmann, we have characterized twin, the homologue of the yeast cytoplasmic deadenylase CCR4. The gene twin regulates the number and synchrony of the premeiotic ovarian cyst divisions as well as the differentiation of the oocyte. Consistent with its biochemical function in yeast, Drosophila Twin regulates the poly(A) tail length of several targets, including Cyclin A and Cyclin E. The translational regulation of gene expression is a central regulatory mechanism controlling germ line development and gametogenesis. Future studies on the regulation of Twin, as well as the identification of additional Twin targets, will further our understanding of how translational controls help coordinate cell cycle regulation with gamete formation during oogenesis.

Coordinating meiotic progression and oocyte differentiation

Senger, Nguyen

A long-term goal of the laboratory has been to identify factors that are concentrated or activated in the oocyte and promote meiotic progression and/or establishment of the oocyte identity. To identify the pathways that direct entry into and maintenance of the meiotic cycle, we screened for mutants in which ovarian cysts develop with 16 nurse cells and no oocyte. From this screen, we identified a new gene, missing oocyte (mio), which is required for the maintenance of the meiotic cycle. In mio mutants, the oocyte enters the meiotic cycle and forms mature synaptonemal complexes but the meiotic state is not maintained. Ultimately, mio oocytes abandon the meiotic cycle, enter the endocycle, and develop as nurse cells. Intriguingly, inhibiting the formation of the double-stranded breaks that initiate meiotic recombination strongly suppresses the mio 16-nurse cell phenotype. The production of the double-stranded breaks during meiosis requires mei-W68 and mei-P22. We find that mio, mei-W68 double mutants show a four-fold reduction in the production of cysts with 16-nurse cells while mio, mei-P22 double mutants show a three-fold reduction. These data suggest that mio interacts with pathways that influence DNA metabolism. The mei-41 gene encodes an ATM/ATR homologue that is proposed to be a component of a pathway that delays meiotic progression in response to unrepaired double-stranded breaks. However, mio is not suppressed by null mutations in mei-41. The data indicate that the presence of unrepaired double-stranded breaks contributes to the mio mutant phenotype independent of the meiotic checkpoint. In summary, we find that mio is required to maintain the meiotic cycle and oocyte fate in the presence of the double-stranded breaks that initiate meiotic recombination.

To gain a molecular understanding of how mio influences early meiotic progression and oocyte differentiation, we cloned the mio gene, which is predicted to encode a protein of 867 amino acids that is highly conserved from yeast to humans. In the yeast S. cerevisiae, the putative mio orthologue YBL104C is not a vital gene; however, YBL104C knockouts are slow-growing. The conservation of the Mio protein is present in two main blocks. The N-terminal block contains four to six WD-40 repeats that often function as protein-protein interaction domains. The C-terminus of the protein contains a putative U box that is structurally similar to the RING finger domain and has been implicated in ubiquitin-dependent protein degradation. We have begun to characterize Mio biochemically and have determined that Mio is present in a stable multiprotein complex of approximately 550 kDa. We are currently working to identify additional proteins present in the Mio complex.

p27/Dacapo and the licensing of DNA replication origins in Drosophila

Narbonne; in collaboration with Aladjem, Riesgo-Escovar

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 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 exhibit reduced levels of the licensing factor Double-Parked/Cdt1 (Dup/Cdt1) as well as decreased levels of chromatin-bound MCM2-7 complex. In addition, mutations in dup/cdt1 dominantly enhance the dap phenotype in several polyploid cell types. Consistent with reduced ability to complete genomic replication, dap mutants accumulate increased levels of DNA damage during the endocycle S phase. Finally, genetic interaction studies indicate that dap functions to promote replication licensing in a subset of Drosophila mitotic cycles. 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. Intriguingly, a similar role has been defined for 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.

Collaborators

Mirit Aladjem, PhD, Laboratory of Molecular Pharmacology, NCI, Bethesda, MD
Ruth Lehmann, PhD, Howard Hughes Medical Institute, Skirball Institute of Biomolecular Medicine, New York University School of Medicine, New York, NY
Juan Riesgo-Escovar, PhD, Neurobiology Institute, Campus UNAM-Juriquilla, Universidad Nacional Antonoma de Mexico, Queretaro, Mexico

For further information, contact mary_lilly@nih.gov.