Mary A. Lilly

New insights into cell cycle control from the Drosophila endocycle

During metazoan development, the organization of the cell cycle is often modified in response to developmental signals. The endocycle provides a dramatic example of this phenomenon. In the endocycle, also referred to as the endoreplicative cycle, cells undergo successive rounds of DNA replication without an intervening mitosis. Often the endocycle is used to expand the genome of a group of specialized cells that are highly biosynthetically active. In these circumstances, large polyploid cells are produced in organisms that are primarily comprised of diploid cells. However, many organisms achieve growth by increasing cell size, rather than cell number. This strategy is more generally exploited in insects and plants. For instance, in the insect Drosophila melanogaster, the majority of the larval tissues, as well as many adult tissues, enter the endocycle and become polyploid. Therefore, Drosophila has been a rich source for studies on endocycle regulation. Recent work from Drosophila is beginning to reveal how developmental signals promote the transition from the mitotic cycle to the endocycle, as well as what drives endocycle progression. In addition, studies on the endocycle have provided insight into the regulatory principles underlying the once per cell cycle replication of the genome, as well as the relationship between S phase and mitosis.

APC/CFzr/Cdh1 promotes cell cycle progression during the Drosophila endocycle

The endocycle is a commonly observed variant cell cycle in which cells undergo repeated rounds of DNA replication with no intervening mitosis. How the cell cycle machinery is modified to transform a mitotic cycle into endocycle has long been a matter 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, because many of its targets are transcriptionally downregulated upon entry into the endocycle, it remains unclear whether the APC/C functions beyond the mitotic/endocycle boundary. Here, we report that APC/CFzr/Cdh1 activity is required to promote the G/S oscillation of the Drosophila endocycle. We demonstrate that compromising APC/C activity, after cells have entered the endocycle, inhibits DNA replication and results in the accumulation of multiple APC/C targets, including the mitotic cyclins and Geminin. Notably, our data suggest that the activity of APC/CFzr/Cdh1 during the endocycle is not continuous but is cyclic, as demonstrated by the APC/C-dependent oscillation of the pre-replication complex component Orc1. Taken together, our data suggest a model in which the cyclic activity of APC/CFzr/Cdh1 during the Drosophila endocycle is driven by the periodic inhibition of Fzr/Cdh1 by Cyclin E/Cdk2. We propose that, as is observed in mitotic cycles, during endocycles, APC/CFzr/Cdh1 functions to reduce the levels of the mitotic cyclins and Geminin in order to facilitate the relicensing of DNA replication origins and cell cycle progression.

twin, a CCR4 homolog, regulates cyclin poly(A) tail length to permit Drosophila oogenesis

Cyclins regulate progression through the cell cycle. Control of cyclin levels is essential in Drosophila oogenesis for the four synchronous divisions that generate the 16 cell germ line cyst and for ensuring that one cell in each cyst, the oocyte, is arrested in meiosis, while the remaining fifteen cells become polyploid nurse cells. Changes in cyclin levels could be achieved by regulating transcription, translation or protein stability. The proteasome limits cyclin protein levels in the Drosophila ovary, but the mechanisms regulating RNA turnover or translation remain largely unclear. Here, we report the identification of twin, a homolog of the yeast CCR4 deadenylase. We show that twin is important for the number and synchrony of cyst divisions and oocyte fate. Consistent with the deadenylase activity of CCR4 in yeast, our data suggest that Twin controls germ line cyst development by regulating poly(A) tail lengths of several targets including Cyclin A (CycA) RNA. twin mutants exhibit very low expression of Bag-of-marbles (Bam), a regulator of cyst division, indicating that Twin/Ccr4 activity is necessary for wild-type Bam expression. Lowering the levels of CycA or increasing the levels of Bam suppresses the defects we observe in twin ovaries, implicating CycA and Bam as downstream effectors of Twin. We propose that Twin/Ccr4 functions during early oogenesis to coordinate cyst division, oocyte fate specification and egg chamber maturation.

The p27cip/kip ortholog dacapo maintains the Drosophila oocyte in prophase of meiosis I.

Animal oocytes undergo a highly conserved developmental arrest in prophase of meiosis I. Often this marks a period of rapid growth for the oocyte and is necessary to coordinate meiotic progression with the developmental events of oogenesis. In Drosophila, the oocyte develops within a 16-cell germline cyst. Throughout much of oogenesis, the oocyte remains in prophase of meiosis I. By contrast, its 15 mitotic sisters enter the endocycle and become polyploid in preparation for their role as nurse cells. How germline cysts establish and maintain these two independent cell cycles is unknown. We demonstrate a role for the p21CIP/p27Kip1/p57Kip2-like cyclin-dependent kinase inhibitor (cki) dacapo in the maintenance of the meiotic cycle in Drosophila oocytes. Our data indicate that it is through the differential regulation of the cki Dacapo that two modes of cell-cycle regulation are independently maintained within the common cytoplasm of ovarian cysts.

The cyclin-dependent kinase inhibitor Dacapo promotes replication licensing during Drosophila endocycles

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. Here, we demonstrate that the p21cip1/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 minichromosome maintenance (MCM2–7) complex. Moreover, 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. Finally, genetic interaction studies suggest that dap functions to promote replication licensing in a subset of Drosophila mitotic cycles.

DRP1-dependent mitochondrial fission initiates follicle cell differentiation during Drosophila oogenesis

Reduced Drp1-mediated mitochondrial fission decreases cell cycle exit and prevents Notch-dependent follicle cell differentiation during oogenesis.

Fluorescence Imaging Techniques for Studying Drosophila Embryo Development

This unit describes fluorescence‐based techniques for noninvasive imaging of development in living Drosophila embryos, discussing considerations for fluorescent imaging within living embryos and providing protocols for generation of flies expressing fluorescently tagged proteins and for preparation of embryos for fluorescent imaging. The unit details time‐lapse confocal imaging of live embryos and discusses optimizing image acquisition and performing three‐dimensional imaging. Finally, the unit provides a variety of specific methods for optical highlighting of specific subsets of fluorescently tagged proteins and organelles in the embryo, including fluorescence recovery after photobleaching (FRAP), fluorescence loss in photobleaching (FLIP), and photoactivation techniques, permitting analysis of specific movements of fluorescently tagged proteins within cells. These protocols, together with the relative ease of generating transgenic animals and the ability to express tagged proteins in specific tissues or at specific developmental times, provide powerful means for examining in vivo behavior of any tagged protein in embryos in myriad mutant backgrounds. Curr. Protoc. Cell Biol. 39:4.18.1‐4.18.43.

The nucleoporin Seh1 forms a complex with Mio and serves an essential tissue-specific function in Drosophila oogenesis

The nuclear pore complex (NPC) mediates the transport of macromolecules between the nucleus and cytoplasm. Recent evidence indicates that structural nucleoporins, the building blocks of the NPC, have a variety of unanticipated cellular functions. Here, we report an unexpected tissue-specific requirement for the structural nucleoporin Seh1 during Drosophila oogenesis. Seh1 is a component of the Nup107-160 complex, the major structural subcomplex of the NPC. We demonstrate that Seh1 associates with the product of the missing oocyte (mio) gene. In Drosophila, mio regulates nuclear architecture and meiotic progression in early ovarian cysts. Like mio, seh1 has a crucial germline function during oogenesis. In both mio and seh1 mutant ovaries, a fraction of oocytes fail to maintain the meiotic cycle and develop as pseudo-nurse cells. Moreover, the accumulation of Mio protein is greatly diminished in the seh1 mutant background. Surprisingly, our characterization of a seh1 null allele indicates that, although required in the female germline, seh1 is dispensable for the development of somatic tissues. Our work represents the first examination of seh1 function within the context of a multicellular organism. In summary, our studies demonstrate that Mio is a novel interacting partner of the conserved nucleoporin Seh1 and add to the growing body of evidence that structural nucleoporins can have novel tissue-specific roles.

missing oocyte encodes a highly conserved nuclear protein required for the maintenance of the meiotic cycle and oocyte identity in Drosophila.

In Drosophila, a single oocyte develops within a 16-cell germline cyst. Although all 16 cells initiate meiosis and undergo premeiotic S phase, only the oocyte retains its meiotic chromosome configuration and remains in the meiotic cycle. The other 15 cells in the cyst enter the endocycle and develop as polyploid nurse cells. A longstanding goal in the field has been to identify factors that are concentrated or activated in the oocyte, that promote meiotic progression and/or the establishment of the oocyte identity. We present the characterization of the missing oocyte gene, an excellent candidate for a gene directly involved in the differentiation of the oocyte nucleus. The missing oocyte gene encodes a highly conserved protein that preferentially accumulates in pro-oocyte nuclei in early prophase of meiosis I. In missing oocyte mutants, the oocyte enters the endocycle and develops as a polyploid nurse cell. Genetic interaction studies indicate that missing oocyte influences meiotic progression prior to pachytene and may interact with pathways that control DNA metabolism. Our data strongly suggest that the product of the missing oocyte gene acts in the oocyte nucleus to facilitate the execution of the unique cell cycle and developmental programs that produce the mature haploid gamete.

TORC1 regulators Iml1/GATOR1 and GATOR2 control meiotic entry and oocyte development in Drosophila

Significance The target of rapamycin complex 1 (TORC1) promotes cell growth and anabolic metabolism. In yeast, entry into meiosis is contingent on the down-regulation of TORC1 activity by the increased minichromosome loss 1/GTPase-activating proteins toward Rags 1 (Iml1/GATOR1) complex in response to amino acid starvation. Here we define the developmental requirements for the TORC1 regulators Iml1/GATOR1 and GATOR2 during Drosophila oogenesis. We demonstrate that, as is observed in yeast, the Iml1/GATOR1 complex down-regulates TORC1 activity to facilitate the mitotic/meiotic transition in Drosophila ovarian cysts. Later in oogenesis, components of the GATOR2 complex oppose the activity of GATOR1 to enable a rise in TORC1 activity that drives oocyte development and growth. Thus, a conserved nutrient stress pathway has been incorporated into a developmental program that regulates meiotic progression in Drosophila. In single-cell eukaryotes the pathways that monitor nutrient availability are central to initiating the meiotic program and gametogenesis. In Saccharomyces cerevisiae an essential step in the transition to the meiotic cycle is the down-regulation of the nutrient-sensitive target of rapamycin complex 1 (TORC1) by the increased minichromosome loss 1/ GTPase-activating proteins toward Rags 1 (Iml1/GATOR1) complex in response to amino acid starvation. How metabolic inputs influence early meiotic progression and gametogenesis remains poorly understood in metazoans. Here we define opposing functions for the TORC1 regulatory complexes Iml1/GATOR1 and GATOR2 during Drosophila oogenesis. We demonstrate that, as is observed in yeast, the Iml1/GATOR1 complex inhibits TORC1 activity to slow cellular metabolism and drive the mitotic/meiotic transition in developing ovarian cysts. In iml1 germline depletions, ovarian cysts undergo an extra mitotic division before meiotic entry. The TORC1 inhibitor rapamycin can suppress this extra mitotic division. Thus, high TORC1 activity delays the mitotic/meiotic transition. Conversely, mutations in Tor, which encodes the catalytic subunit of the TORC1 complex, result in premature meiotic entry. Later in oogenesis, the GATOR2 components Mio and Seh1 are required to oppose Iml1/GATOR1 activity to prevent the constitutive inhibition of TORC1 and a block to oocyte growth and development. To our knowledge, these studies represent the first examination of the regulatory relationship between the Iml1/GATOR1 and GATOR2 complexes within the context of a multicellular organism. Our data imply that the central role of the Iml1/GATOR1 complex in the regulation of TORC1 activity in the early meiotic cycle has been conserved from single cell to multicellular organisms.