Cell Cycle Regulation in Oogenesis

Mary Lilly, PhD, Head, Unit on Cell Cycle Regulation
Eva Decotto, PhD, Visiting Fellow
Karine Narbonne, PhD, Visiting Fellow
Stefania Senger, PhD, Visiting Fellow
Guillermo Gonzalez, BS, Technician

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 the ovarian cyst enter and maintain either the meiotic cycle or the endocycle. In addition, we are examining how this cell cycle choice drives development of the mature egg.

Regulation of developmentally programmed endocycles

Narbonne, Senger; in collaboration with Asano, Deák, Richardson, Aladjem, Riesgo-Escovar

The endocycle is a commonly observed variant cell cycle in which cells undergo repeated rounds of DNA replication with no intervening mitosis. Cells that are highly metabolically active, such as the giant trophoblast of the mammalian placenta and Drosophila ovarian nurse cells, often grow via endoreplication. How the cell cycle machinery is modified to transform a mitotic cycle into endocycle has long been a question of interest. In both plants and animals, the transition from the mitotic cycle to the endocycle requires Fzr/Cdh1, a positive regulator of the anaphase-promoting complex/cyclosome (APC/C). However, with many of APC/C’s targets transcriptionally downregulated upon entry into the endocycle, it remained unclear whether the APC/C functioned beyond the mitotic/endocycle boundary. We have now shown that APC/C^Fzr/Cdh1 activity is required to promote G/S oscillation of the Drosophila endocycle. Compromised APC/C activity following cell entry into the endocycle inhibits DNA replication and results in the accumulation of several APC/C targets, including the mitotic cyclins and geminin. Notably, our data suggest that the activity of APC/C^Fzr/Cdh1 during the endocycle is not continuous but rather cyclic, as demonstrated by the APC/C-dependent oscillation of the prereplication complex component ORC1. Taken together, our data suggest a model in which the cyclic activity of APC/C^Fzr/Cdh1 during the Drosophila endocycle is driven by the periodic inhibition of Fzr/Cdh1 by cyclin E/Cdk2. Thus, we propose that, as observed in mitotic cycles, APC/C^Fzr/Cdh1 functions during endocycles to reduce levels of the mitotic cyclins and geminin in order to facilitate the relicensing of DNA replication origins and cell cycle progression. Our work has provided important new insights into the basic cell requirements of the endoreplicative cycle.

In Drosophila, the oscillations of cyclin E/Cdk2 activity drive the endocycle. How the periodicity of cyclin E/Cdk2 activity is achieved during endocycles is poorly understood. We have determined that the p21^cip1/p27^kip1/p57^kip2-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 their 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 premeiotic S phase. 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. Our work represents the first example of a CKI acting to promote replication licensing in a metazoan.

Hong A, Narbonne-Reveau K, Riesgo-Escovar J, Fu H, Aladjem MI, Lilly MA. The cyclin-dependent kinase inhibitor Dacapo promotes replication licensing during Drosophila endocycles. EMBO J 2007;26:2071-2082.
Narbonne-Reveau K, Senger S, Pal M, Herr A, Asano M, Richardson HE, Deak P, Lilly MA. APC/C^Fzr/Cdh1 promotes cell cycle progression during the Drosophila endocycle. Development 2008;135:1451-1461.

Meiotic progression and oocyte differentiation in early ovarian cysts

Senger, Decotto

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, it does not maintain the meiotic state and ultimately withdraws from meiosis, enters the endocycle, and becomes polyploid. The mio gene is predicted to encode a protein of 867 amino acids that is highly conserved from yeast to humans. Conservation of the Mio protein can be observed in two main blocks. First, the N-terminal block contains four to six WD-40 repeats, which often function as protein-protein interaction domains. Second, the C-terminal block contains a putative U box, which is structurally similar to the RING finger domain and has been implicated in ubiquitylation. The Mio protein accumulates in the oocyte nucleus in early prophase of meiosis I. Moreover, mio mutants display some of the earliest meiotic defects reported in Drosophila. Therefore, mio provides an excellent entry point for exploring the unique cell biology of the early ovarian cyst and how the establishment of the meiotic program influences the downstream events of oocyte differentiation and meiotic progression.

We are using both biochemical and genetic strategies to identify additional genes that act in the mio pathway. To identify proteins that physically interact with Mio, we used tandem affinity purification (TAP) and mass spectrometry. From these studies, we determined that Mio strongly associates with the nucleoporin Nup44A. In support of the biological relevance of the Mio/Nup44A interaction, nup44A mutations act as strong dominant suppressors of the mio 16 nurse cell phenotype. Moreover, we determined that mutations in Nup44A specifically disrupt meiotic progression and female sterility. Our work provides the framework for future studies on how nuclear pore components influence meiotic progression and maintenance of oocyte identity.

Mavrakis M, Rikhy R, Lilly MA, Lippincott-Schwartz J. Fluorescence imaging techniques for studying Drosophila development. Curr Protoc Cell Biol 2008;4:Unit 4.18.

Collaborators

Mirit Aladjem, PhD, Laboratory of Molecular Pharmacology, NCI, Bethesda, MD
Maki Asano, MD, PhD, Duke University Medical Center, Durham, NC
Péter Deák, PhD, Institute of Biochemistry, Biological Research Center, Szeged, Hungary
Helena Richardson, PhD, Peter MacCallum Cancer Center, Melbourne, Australia
Juan Riesgo-Escovar, PhD, Neurobiology Institute, Campus UNAM-Juriquilla, Querétaro, Mexico

For further information, contact mary_lilly@nih.gov or visit http://cbmp.nichd.nih.gov/sgd/index.html