Germline cell death is inhibited by P-element insertions disrupting the dcp-1/pita nested gene pair in Drosophila

Germline cell death in Drosophila oogenesis is controlled by distinct signals. The death of nurse cells in late oogenesis is developmentally regulated, whereas the death of egg chambers during mid-oogenesis is induced by environmental stress or developmental abnormalities. P-element insertions in the caspase gene dcp-1 disrupt both dcp-1 and the outlying gene, pita, leading to lethality and defective nurse cell death in late oogenesis. By isolating single mutations in the two genes, we have found that the loss of both genes contributes to this ovary phenotype. Mutants of pita, which encodes a C2H2 zinc-finger protein, are homozygous lethal and show dumpless egg chambers and premature nurse cell death in germline clones. Early nurse cell death is not observed in the dcp-1/pita double mutants, suggesting that dcp-1+ activity is required for the mid-oogenesis cell death seen in pita mutants. dcp-1 mutants are viable and nurse cell death in late oogenesis occurs normally. However, starvation-induced germline cell death during mid-oogenesis is blocked, leading to a reduction and inappropriate nuclear localization of the active caspase Drice. These findings suggest that the combinatorial loss of pita and dcp-1 leads to the increased survival of abnormal egg chambers in mutants bearing the P-element alleles and that dcp-1 is essential for cell death during mid-oogenesis.     

The Drosophila caspases Strica and Dronc function redundantly in programmed cell death during oogenesis

Programmed cell death (PCD) in the Drosophila ovary occurs either during mid-oogenesis, resulting in degeneration of the entire egg chamber or during late oogenesis, to facilitate the development of the oocyte. PCD during oogenesis is regulated by mechanisms different from those that control cell death in other Drosophila tissues. We have analyzed the role of caspases in PCD of the female germline by examining caspase mutants and overexpressing caspase inhibitors. Imprecise P-element excision was used to generate mutants of the initiator caspase strica. While null mutants of strica or another initiator caspase, dronc, display no ovary phenotype, we find that strica exhibits redundancy with dronc, during both mid- and late oogenesis. Ovaries of double mutants contain defective mid-stage egg chambers similar to those reported previously in dcp-1 mutants, and mature egg chambers with persisting nurse cell nuclei. In addition, the effector caspases drice and dcp-1 also display redundant functions during late oogenesis, resulting in persisting nurse cell nuclei. These findings indicate that caspases are required for nurse cell death during mid-oogenesis, and participate in developmental nurse cell death during late oogenesis. This reveals a novel pathway of cell death in the ovary that utilizes strica, dronc, dcp-1 and drice, and importantly illustrates strong redundancy among the caspases.     

Mechanisms of programmed cell death during oogenesis in <Emphasis Type="Italic">Drosophila virilis</Emphasis>

We describe the features of programmed cell death occurring in the egg chambers of Drosophila virilis during mid-oogenesis and late oogenesis. During mid-oogenesis, the spontaneously degenerating egg chambers exhibit typical characteristics of apoptotic cell death. As revealed by propidium iodide, rhodamine-conjugated phalloidin staining, and the TUNEL assay, respectively, the nurse cells contain condensed chromatin, altered actin cytoskeleton, and fragmented DNA. In vitro caspase activity assays and immunostaining procedures demonstrate that the atretic egg chambers possess high levels of caspase activity. Features of autophagic cell death are also observed during D. virilis mid-oogenesis, as shown by monodansylcadaverine staining, together with an ultrastructural examination by transmission electron microscopy. During the late stages of oogenesis in D. virilis, once again, the two mechanisms, viz., nurse cell cluster apoptosis and autophagy, operate together, manifesting features of cell death similar to those detailed above. Moreover, an altered form of cytochrome c seems to be released from the mitochondria in the nurse cells proximal to the oocyte. We propose that apoptosis and autophagy function synergistically during oogenesis in D. virilis in order to achieve a more efficient elimination of the degenerated nurse cells and abnormal egg chambers. The present study was co-financed within Op. Education by the European Social Fund and by National Resources via a grant (HRAKLEITOS 70/3/7164) to Professor L.H. Margaritis.     

Mutations in the midway gene disrupt a Drosophila acyl coenzyme A: diacylglycerol acyltransferase

During Drosophila oogenesis, defective or unwanted egg chambers are eliminated during mid-oogenesis by programmed cell death. In addition, final cytoplasm transport from nurse cells to the oocyte depends upon apoptosis of the nurse cells. To study the regulation of germline apoptosis, we analyzed the midway mutant, in which egg chambers undergo premature nurse cell death and degeneration. The midway gene encodes a protein similar to mammalian acyl coenzyme A: diacylglycerol acyltransferase (DGAT), which converts diacylglycerol (DAG) into triacylglycerol (TAG). midway mutant egg chambers contain severely reduced levels of neutral lipids in the germline. Expression of midway in insect cells results in high levels of DGAT activity in vitro. These results show that midway encodes a functional DGAT and that changes in acylglycerol lipid metabolism disrupt normal egg chamber development in Drosophila.     

Stage-specific regulation of programmed cell death during oogenesis of the medfly Ceratitis capitata (Diptera, Tephritidae)

In the present study, we describe novel features of programmed cell death in developing egg chambers occurring during mid- and late-oogenesis of the medfly Ceratitis capitata. During mid-oogenesis, the spontaneously degenerated egg chambers exhibit typical characteristics of apoptotic cell death. Their nurse cells contain fragmented DNA and fragmented actin, as revealed by TUNEL assay and immunolabelling, respectively. In vitro caspase activity assays and immunostaining procedures demonstrated that the atretic egg chambers acquired high levels of caspase activity. Distinct features of autophagic cell death were also observed during C. capitata mid-oogenesis, as revealed by the monodansylcadaverine staining approach and ultrastructural examination performed by transmission electron microscopy. Additionally, atretic egg chambers exhibit an upregulation of lysosomal proteases, as demonstrated by a procathepsin L immunolabelling procedure. At the late stages of C. capitata oogenesis, apoptosis and autophagy coexist, manifesting cell death features that are similar to the ones mentioned above, being also chaperoned by the involvement of an altered cytochrome c conformational display. We propose that apoptosis and autophagy operate synergistically during C. capitata oogenesis for a more efficient elimination of the degenerated nurse cells and abnormal egg chambers.     

Apoptosis and Autophagy Function Cooperatively for the Efficacious Execution of Programmed Nurse Cell Death During Drosophila virilis Oogenesis

Programmed cell death consists of two major types, apoptotic and autophagic, both of which are mainly defined by morphological criteria. Our findings indicate that both types of programmed cell death occur in the ovarian nurse cells during middle and late oogenesis of Drosophila virilis. During mid-oogenesis, the spontaneously degenerated egg chambers exhibit typical characteristics of apoptotic cell death. Their nurse cells contain condensed chromatin and fragmented DNA, whereas active caspase assays and immunostaining procedures demonstrate the presence of highly activated caspases. Distinct features of autophagic cell death are also observed during D. virilis mid-oogenesis, as shown by monodansylcadaverine staining and ultrastructural examination performed by transmission electron microscopy. Additionally, atretic egg chambers exhibit an accumulation of lysosomal proteases. At the late stages of D. virilis oogenesis, apoptosis and autophagy coexist, manifesting cell death features that are similar to the ones described above, being also escorted by the involvement of an altered cytochrome c conformational display. We propose that apoptosis and autophagy operate synergistically during D. virilis oogenesis for a more efficient elimination of the degenerated nurse cells.

Addendum to:

Mechanisms of Programmed Cell Death During Oogenesis in Drosophila virilis

A.D. Velentzas, I.P. Nezis, D.J. Stravopodis, I.S. Papassideri and L.H. Margaritis

Cell Tissue Res 2006; doi: 10.1007/s00441-006-0298-x     

Apoptosis and autophagy function cooperatively for the efficacious execution of programmed nurse cell death during Drosophila virilis oogenesis

Programmed cell death consists of two major types, apoptotic and autophagic, both of which are mainly defined by morphological criteria. Our findings indicate that both types of programmed cell death occur in the ovarian nurse cells during middle- and late-oogenesis of Drosophila virilis. During mid-oogenesis, the spontaneously degenerated egg chambers exhibit typical characteristics of apoptotic cell death. Their nurse cells contain condensed chromatin and fragmented DNA, whereas active caspase assays and immunostaining procedures demonstrate the presence of highly activated caspases. Distinct features of autophagic cell death are also observed during D. virilis mid-oogenesis, as shown by monodansylcadaverine staining and ultrastructural examination performed by transmission electron microscopy. Additionally, atretic egg chambers exhibit an accumulation of lysosomal proteases. At the late stages of D. virilis oogenesis, apoptosis and autophagy coexist, manifesting cell death features that are similar to the ones described above, being also escorted by the involvement of an altered cytochrome c conformational display. We propose that apoptosis and autophagy operate synergistically during D. virilis oogenesis for a more efficient elimination of the degenerated nurse cells.     

Eggs over easy: cell death in the Drosophila ovary

Programmed cell death is the most common fate of female germ cells in Drosophila and many animals. In Drosophila, oocytes form in individual egg chambers that are supported by germline nurse cells and surrounded by somatic follicle cells. As oogenesis proceeds, 15 nurse cells die for every oocyte that is produced. In addition to this developmentally regulated cell death, groups of germ cells or entire egg chambers may be induced to undergo apoptosis in response to starvation or other insults. Recent findings suggest that these different types of cell death involve distinct genetic pathways. This review focuses on progress towards elucidating the molecular mechanisms acting during programmed cell death in Drosophila oogenesis.     

Identification and time of synthesis of chorion proteins in Drosophila melanogaster.

The chorion of Drosophila melanogaster consists of proteins secreted by the follicular epithelium during late oogenesis. Petri, Wyman and Kafatos (1976) have described six major protein components of the Drosophila chorion and reported the synthesis of these proteins in vitro by mass-isolated egg chambers. We have used two-dimensional gel electrophoresis to identify approximately twenty components in highly purified chorion preparations. The synthesis patterns of these proteins in vivo were determined by isolating egg chambers of different developmental stages from flies injected with 14C amino acids. Chorion proteins constitute a large fraction of the protein synthesized by ovarian egg chambers in stages 12--14. The sizes and times of synthesis of the chorion proteins correlate closely with the production of poly(A)-containing RNAs by the follicle cells (Spradling and Mahowald, 1979).     

Ecdysone response genes govern egg chamber development during mid-oogenesis in Drosophila.

The steroid hormone ecdysone regulates larval development and metamorphosis in Drosophila melanogaster through a complex genetic hierarchy that begins with a small set of early response genes. Here, we present data indicating that the ecdysone response hierarchy also mediates egg chamber maturation during mid-oogenesis. E75, E74 and BR-C are expressed in a stage-specific manner while EcR expression is ubiquitous throughout oogenesis. Decreasing or increasing the ovarian ecdysone titer using a temperature-sensitive mutation or exogenous ecdysone results in corresponding changes in early gene expression. The stage 10 follicle cell expression of E75 in wild-type, K10 and EGF receptor (Egfr) mutant egg chambers reveals regulation of E75 by both the Egfr and ecdysone signaling pathways. Genetic analysis indicates a germline requirement for ecdysone-responsive gene expression. Germline clones of E75 mutations arrest and degenerate during mid-oogenesis and EcR germline clones exhibit a similar phenotype, demonstrating a functional requirement for ecdysone responsiveness during the vitellogenic phase of oogenesis. Finally, the expression of Drosophila Adrenodoxin Reductase increases during mid-oogenesis and clonal analysis confirms that this steroidogenic enzyme is required in the germline for egg chamber development. Together these data suggest that the temporal expression profile of E75, E74 and BR-C may be a functional reflection of ecdysone levels and that ecdysone provides temporal signals regulating the progression of oogenesis and proper specification of dorsal follicle cell fates.     

Drosophila quail, a villin-related protein, bundles actin filaments in apoptotic nurse cells

Drosophila Quail protein is required for the completion of fast cytoplasm transport from nurse cells to the oocyte, an event critical for the production of viable oocytes. The abundant network of cytoplasmic filamentous actin, established at the onset of fast transport, is absent in quail mutant egg chambers. Previously, we showed that Quail is a germline-specific protein with sequence homology to villin, a vertebrate actin-regulating protein. In this study, we combined biochemical experiments with observations in egg chambers to define more precisely the function of this protein in the regulation of actin-bundle assembly in nurse cells. We report that recombinant Quail can bind and bundle filamentous actin in vitro in a manner similar to villin at a physiological calcium concentration. In contrast to villin, Quail is unable to sever or cap filamentous actin, or to promote nucleation of new actin filaments at a high calcium concentration. Instead, Quail bundles the filaments regardless of the calcium concentration. In vivo, the assembly of nurse-cell actin bundles is accompanied by extensive perforation of the nurse-cell nuclear envelopes, and both of these phenomena are manifestations of nurse-cell apoptosis. To investigate whether free calcium levels are affected during apoptosis, we loaded egg chambers with the calcium indicator Indo-1. Our observations indicate a rise in free calcium in the nurse-cell cytoplasm coincident with the permeabilization of the nuclear envelopes. We also show that human villin expressed in the Drosophila germline could sense elevated cytoplasmic calcium; in nurse cells with reduced levels of Quail protein, villin interfered with actin-bundle stability. We conclude that Quail efficiently assembles actin filaments into bundles in nurse cells and maintains their stability under fluctuating free calcium levels. We also propose a developmental model for the fast phase of cytoplasm transport incorporating findings presented in this study.     

A Mutation in the Drosophila Profilin Homolog Gene Affects Gametogenesis and Bristle Development in Both Sexes

We have isolated a Drosophila melanogaster mutant, allelic to the profilin gene reported as chickadee . We named the allele chickadee^bin , in which the oogenesis and the spermatogenesis are disrupted, and the bristles are malformed. In the mutant nurse cells, cytoplasmic actin filaments fail to polymerize, and nuclei are displaced. The flow of cytoplasm from nurse cells to the oocyte is abortive. These ovarian phenotypes are principally the same as those reported in chickadee^{wc57 and WF57} (2). In addition, the egg chamber of chickadee^bin contains a reduced number of cystocytes that are binucleafed. In some egg chambers, the oocyte fails to differentiate. All cystocytes in such an egg chamber are morphologically similar to nurse cells with polyploid nuclei. Mutant male flies have defective testes in which the spermatocyst is deficient or reduced in number. Mutant adults have shortened and forked bristles. We discuss the function of profilin in the gametogenesis and bristle development.     

Lethal(2)giant larvae is required in the follicle cells for formation of the initial AP asymmetry and the oocyte polarity during Drosophila oogenesis

The intricately regulated differentiation of the somatic follicle cell lineages into distinct subpopulations with specific functions plays an essential role in Drosophila egg development. At early oogenesis, induction of the stalk cells generates the first anteroposterior (AP) asymmetry in the egg chamber by inducing the posterior localization of the oocyte. Later, the properly specified posterior follicle cells signal to polarize the oocyte along the AP and dorsoventral (DV) axes at mid-oogenesis. Here, we show that lethal(2)giant larvae (lgl), a Drosophila tumor suppressor gene, is required in the follicle cells for the differentiation of both stalk cells and posterior follicle cells. Loss-of-function mutations in lgl cause oocyte mispositioning in the younger one of the fused chambers, due to lack of the stalk. Removal of lgl function from the posterior follicle cells using the FLP/FRT system results in loss of the oocyte polarity that is elicited by the failure of those posterior cells to differentiate normally. Thus, we provide the first demonstration that lgl is implicated in the formation of the initial AP asymmetry and the patterning of the AP and DV axes in the oocyte by acting in the specification of a subset of somatic follicle cells.     

The Drosophilaovarian tumorGene Is Required for the Organization of Actin Filaments during Multiple Stages in Oogenesis

Theovarian tumorgene is required during both early and late stages of oogenesis. Mutations produce a range of phenotypes, including agametic ovarioles, tumorous egg chambers, and late stage oogenic arrest. We demonstrate that each of these phenotypes is associated with specific aberrations in actin distribution. In the earliest case,ovarian tumormutations cause actin filaments to accumulate ectopically in the fusome. This correlates with abnormal fusome morphology and arrested germ cell development in the germaria. Similarly,ovarian tumorfunction is required for the localization of actin that is essential for the maturation of ring canals. This defect gives rise to tumorous egg chambers in which germ cell numbers and morphology are profoundly aberrant. We also confirm thatovarian tumoris required for the formation of the nurse cell cytoplasmic actin array that is essential for the nonspecific transport of cytoplasmic contents to the oocyte during late oogenesis. Our data suggest that at this stageovarian tumorcontrols the site where actin filaments initiate. Taken together, these studies suggest that the diverseovarian tumormutant phenotypes derive from the mislocalization of actin filaments, indicating a role for this gene in organizing the female germline cytoskeleton, and that the misregulation of actin can have profound effects on germ cell division and differentiation.     

Follicular atresia during Dacus oleae oogenesis

Programmed cell death, constitutes a common fundamental incident that occurs during oogenesis in a variety of different animals. It plays a significant role in the maturation process of the female gamete and also in the removal of abnormal and superfluous cells at certain checkpoints of development. In the present study, we demonstrate the existence of follicular atresia during mid-oogenesis in the olive fruit fly Dacus oleae (Tephritidae). The number of atretic follicles increases following the age of the fly, suggesting for the presence of an age-susceptible process. The atretic follicles contain nurse cells that exhibit chromatin condensation, DNA fragmentation and actin cytoskeleton alterations, as revealed by propidium iodide staining, TUNEL labeling and phalloidin-FITC staining. Conventional light and electron microscopy disclose that the nurse cell remnants are phagocytosed by the adjacent follicle cells. The follicular epithelium also eliminates the oocyte through phagocytosis, resulting to an egg chamber with no compartmentalized organization. The data presented herein are very similar compared to previous reported results in other Diptera species, strongly suggesting the occurrence of a phylogenetically conserved mechanism of follicular atresia. All these observations also support the notion that mid-oogenesis in D. oleae may be the critical regulation point at which superfluous and defective egg chambers are selectively eliminated before they reach maturity.     

chickadee encodes a profilin required for intercellular cytoplasm transport during Drosophila oogenesis.

The entire cytoplasmic contents of 15 highly polyploid nurse cells are transported rapidly to the oocyte near the end of Drosophila oogenesis. chickadee is one of a small group of genes whose mutant phenotype includes a disruption of this nurse cell cytoplasm transport. We have cloned the chickadee gene and found that cDNA clones encode a protein 40% identical to yeast and Acanthamoeba profilin. The nurse cells from chickadee egg chambers that lack ovary-specific profilin fail to synthesize cytoplasmic actin networks correctly. In addition, the nurse cell nuclei in chickadee egg chambers become displaced and often partially stretched through the channels leading into the oocyte, blocking the flow of cytoplasm. We suggest that the newly synthesized cytoplasmic actin networks are responsible for maintaining nuclear position in the nurse cells.     

The KASH domain protein MSP-300 plays an essential role in nuclear anchoring during Drosophila oogenesis

During late stages of Drosophila oogenesis, the cytoplasm of nurse cells in the egg chamber is rapidly transferred ("dumped") to oocytes, while the nurse cell nuclei are anchored by a mechanism that involves the actin cytoskeleton. The factors that mediate this interaction between nuclei and actin cytoskeleton are unknown. MSP-300 is the likely Drosophila ortholog of the mammalian Syne-1 and -2 and C. elegans ANC-1 proteins, contained both actin-binding and nuclear envelope localization domains. By using an antibody against C-terminus of MSP-300, we find that MSP-300 is distributed throughout the cytoplasm and accumulates at the nuclear envelope of nurse cells and the oocyte. A GFP fusion protein containing the C-terminal region of MSP-300 is also sufficient to localize protein on the nuclear envelope in oocytes. To eliminate the maternal gene activity during oogenesis, we generated homozygous germ-line clones of a loss-of-function mutation in msp-300 in otherwise heterozygous mothers. In the mutant egg chambers that develop from such clones, cytoplasmic dumping of nurse cells is severely disturbed. The nuclei of nurse cells and the oocyte are mislocalized and the usually well-organized actin structures are severely disrupted. These results indicate that maternal MSP-300 plays an important role in actin-dependent nuclear anchorage during cytoplasmic transport.     

Femcoat, a novel eggshell protein in Drosophila: functional analysis by double stranded RNA interference.

In Drosophila oogenesis, follicle cells derived from somatic tissue surround the oocyte and play key roles in generating properly polarized oocytes. During the later steps of oogenesis, follicle cells are involved in secretion of proteins that make the eggshell, an essential protective layer for the oocyte. Although studies on the signaling processes to make polarized oocytes have been progressed very far, studies on the mechanisms for eggshell formation is not clear yet. To elucidate the underlying mechanism in eggshell formation, we used a differential display screen to isolate genes that are specifically expressed during the later stages of oogenesis, and isolated a novel gene, Femcoat. Femcoat encodes a putative chorion membrane protein that contains many highly charged residues and has a putative signal peptide. Femcoat is expressed specifically in the follicle cells with a punctate staining pattern typical of secreted proteins, and becomes cross-linked heavily at the final steps of oogenesis. To identify the developmental role of Femcoat in eggshell formation, we performed an inducible double stranded RNA mediated interference (dsRNAi) method to specifically reduce Femcoat expression during oogenesis in adult flies. Electron microscopy analysis of egg chambers from these flies showed defects in chorion formation. These pieces of evidence demonstrated that Femcoat is necessary for eggshell formation, especially during chorion synthesis. Our results demonstrate that inducible dsRNAi analysis can be effective in determining the developmental function of novel genes.