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 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, Morris, 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 also be able to activate the mitotic kinase later in oogenesis when they resume the meiotic cycle. Although some of the most highly conserved events of gametogenesis, the 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 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 inhibits the expression of the mitotic cyclins after meiotic entry. Our data indicate that Bruno accomplishes this in part by binding to bruno response elements (BREs) present in the cyclin A 3´ UTR and by inhibiting cyclin translation. In Drosophila, Cyclin A is the primary positive regulatory subunit of Cdk1. Clams and fish use a similar strategy for maintaining prophase I arrest and the required low levels of Cdk1 activity, as do 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 of 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. 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 germline development and gametogenesis. In the future, studies of the regulation of Twin, along with the identification of additional Twin targets, will help further our understanding of how translational controls help coordinate cell cycle regulation with gamete formation during oogenesis.
Morris JZ, Hong A, Lilly MA, Lehmann R. twin, a CCR4 homolog, regulates cyclin poly(A) tail length to permit Drosophila oogenesis. Development 2005;132:1165-1174.
Sugimura I, Lilly MA. Bruno restricts the accumulation of the mitotic cyclins during the prophase I meiotic arrest of the Drosophila oocyte. Dev Cell (in press).
Coordinating meiotic progression with oocyte differentiation
Senger, Sokolowski
A long-term goal of the laboratory has been to identify factors that are concentrated or activated in the oocyte and that promote meiotic progression and/or the 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 all 16 cells enter the endocycle and develop as nurse cells. From the 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 it does not maintain the meiotic state. Ultimately, mio oocytes abandon the meiotic cycle, enter the endocycle, and develop as nurse cells.
We have found that mio homologues from yeast to humans share a common 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. In addition to the WD40 repeats, Mio family members contain a highly conserved 50–amino acid domain near their C termini that shares 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. Although the “Mio domain” bears structural similarities to the 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.
Intriguingly, the mio ovarian phenotype is suppressed by inhibiting the formation of the double-stranded breaks (DSB) that initiate meiotic recombination during meiosis. In miosingle 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 mioegg chambers retain an oocyte and develop to late stages of oogenesis. The simplest interpretation of the data is that miois required to repair the DSBs that initiate meiotic recombination and that the inability to repair DSBs significantly contributes to the mio phenotype. To obtain further insight into the pathway(s) in which Mio functions, we have undertaken a screen to identify dosage-sensitive modifiers of the mio phenotype. Our preliminary data indicate that mio is dominantly suppressed by mutations in the Rad51 homologue spnA. Rad51 is required for the repair of DSB in both yeast and mammals. Furthermore, we have identified at least one additional region of the genome that encodes a dosage-sensitive suppressor of mio. We have begun to characterize Mio biochemically and have determined that it is present in a stable multiprotein complex of approximately 650 kDa. Currently, we are working to identify additional proteins of the Mio complex.
Anderson LK, Lai A, Royer S, Page S, McKim K, Lilly MA, Hawley RS. Juxtaposition of C(2)M and the transverse filament protein C(3)G within the central region of Drosophila synaptonemal complex. Proc Natl Acad Sci USA 2005;102:4482-4487.
Iida T, Lilly MA. missing oocyte encodes a highly conserved nuclear protein required for the maintenance of the meiotic cycle and oocyte identity in Drosophila. Development2004;131:1029-1039.
A dual function for p27/Dacapo in oogenesis
Hong, Narbone; in collaboration with Aladjem
Although physically connected via intercellular bridges, the nurse cells and the oocyte of Drosophilavarian cysts maintain markedly different cell cycles throughout much of oogenesis. As mentioned above, the oocyte faithfully executes the meiotic cycle in order to produce the genetic material for the egg. In contrast, the biosynthetically active nurse cells enter the endocycle and become highly polyploid. During oogenesis, these apparently incompatible cell cycles must coexist for several days within the shared cytoplasm of the cyst. As a model for the developmental regulation of the cell cycle, we are working to understand how DNA replication is inhibited in the meiotic oocyte while simultaneously promoted in the adjacent nurse cells.
We have found that the p21CIP/p27Kip1/p57Kip2-like Cyclin-dependent kinase inhibitor (CKI) Dacapo maintains the prophase I meiotic arrest of the Drosophilaoocyte. dacapo is a vital gene that specifically inhibits the activity of Cyclin E/Cdk2 complexes; however, Cyclin E/Cdk2 activity is required for DNA replication in Drosophila. Throughout much of the growth phase of Drosophilaoogenesis, the levels of Dacapo oscillate in the 15-polyploid nurse cells but remain persistently high in the single oocyte. In dacapo mutants, the prophase I arrest of the oocyte is lost, and the oocyte enters the endocycle and becomes polyploid. Furthermore, our data indicate that inappropriate entry into the endocycle inhibits oocyte differentiation and promotes the nurse cell developmental pathway.
Driven by the oscillations of the single Cyclin/Cdk combination Cyclin E/Cdk2, the Drosophila endocycle appears to be the simplest of cell cycles. Yet, how the oscillations of Cyclin E/Cdk2 activity are achieved during the endocycle has proven difficult to delineate. We find that Dacapo is part of the biochemical oscillator that drives the nurse cell endocycle. Specifically, through the cyclic inhibition of Cyclin E/Cdk2 kinase activity, Dacapo oscillations promote entry into the Gap phase as well as the formation of pre-replication complexes (pre–RCs). In dacapo mutants, endocycling cells exhibit reduced levels of Double-Parked (Dup/Cdt1), a pre–RC component that is required to load the MCM2-7 complex onto chromatin. Consistent with low levels of Dup/Cdt1, dacapo nurse cells contain dramatically lower levels of chromatin-bound MCM2-7 complex than wild-type nurse cells, although the total available nucleoplasmic pool of MCM2-7 is equivalent to that found in wild-type cells. In support of the hypothesis that dacapo nurse cells assemble fewer pre–RCs during the Gap phase, mutant nurse cells exhibit lower rates of BrdU incorporation than wild-type nurse cells. Moreover, dacapo nurse cells demonstrate increased levels of DNA damage, as measured by gammaH2Av staining, consistent with the inability to complete genomic replication. Our data suggest a model in which Dacapo 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 proposed for the CKI Sic1 in promoting replication origin licensing in late G1 in S. cerevisiae. In yeast, hypomorphic alleles of cdt1 are synthetically lethal with a sic1 deletion. Similarly, dacapo and dup/Cdt1 genetically interact in Drosophila, revealing a general role for dacapoin promoting the assembly of pre–RCs during the endocycle.
Calvi B, Lilly MA. Fluorescent BrdU labeling and nuclear flow sorting of the Drosophila ovary. Methods Mol Biol 2004;247:203-213.
Hong A, Lee-Kong S, Iida T, Sugimura I, Lilly MA. The p27cip/kip ortholog dacapo maintains the Drosophila oocyte in prophase of meiosis I. Development 2003;130:1235-1242.
Lilly MA, Duronio RJ. New insights into cell cycle control from the Drosophila endocycle. Oncogene 2005;24:2765-2775.
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
Jason Z. Morris, PhD, Fordham University, New Rochelle, NY
For further information, contact mary_lilly@nih.gov.