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.     

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.     

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.     

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.     

Effector caspase Dcp-1 and IAP protein Bruce regulate starvation-induced autophagy during Drosophila melanogaster oogenesis

A complex relationship exists between autophagy and apoptosis, but the regulatory mechanisms underlying their interactions are largely unknown. We conducted a systematic study of Drosophila melanogaster cell death–related genes to determine their requirement in the regulation of starvation-induced autophagy. We discovered that six cell death genes—death caspase-1 (Dcp-1), hid, Bruce, Buffy, debcl, and p53—as well as Ras–Raf–mitogen activated protein kinase signaling pathway components had a role in autophagy regulation in D. melanogaster cultured cells. During D. melanogaster oogenesis, we found that autophagy is induced at two nutrient status checkpoints: germarium and mid-oogenesis. At these two stages, the effector caspase Dcp-1 and the inhibitor of apoptosis protein Bruce function to regulate both autophagy and starvation-induced cell death. Mutations in Atg1 and Atg7 resulted in reduced DNA fragmentation in degenerating midstage egg chambers but did not appear to affect nuclear condensation, which indicates that autophagy contributes in part to cell death in the ovary. Our study provides new insights into the molecular mechanisms that coordinately regulate autophagic and apoptotic events in vivo.     

An executioner caspase regulates autophagy

The relationships between autophagy and cell death are complex and still not well understood. To advance our understanding of the molecular connections between autophagy and apoptosis, we performed an RNAi-based screen of Drosophila melanogaster apoptosis-related genes for their ability to enhance or suppress starvation-induced autophagy. We discovered that six apoptosis-related genes, Dcp-1, hid, bruce, buffy, debcl and p53 as well as Ras/Raf/MAPK signaling pathway components play a role in autophagy regulation in Drosophila cultured cells. Our study also provides the first in vivo evidence that the effector caspase Dcp-1 and IAP protein Bruce regulate both autophagy and starvation-induced cell death at two nutrient status checkpoints, germarium and mid-oogenesis, in the Drosophila ovary. Analysis of degenerating mid-stage egg chambers in DmAtg1 and DmAtg7 mutants reveal a reduction in TUNEL staining though DNA condensation appears unaffected. Based on these and previous findings, we propose here a putative molecular pathway that might regulate the sensitivity threshold of apoptotic and autophagic responses. We also discuss multiple interpretations of the Atg mutant egg chamber TUNEL phenotype that are consistent with a possible role for autophagy in either suppressing or enhancing the efficiency of cell degradation and/or promoting cell clearance associated with the death process.     

Autophagy as a trigger for cell death: autophagic degradation of inhibitor of apoptosis dBruce controls DNA fragmentation during late oogenesis in Drosophila

Autophagy has been reported to contribute to cell death, but the underlying mechanisms remain largely unknown and controversial. We have: been studying oogenesis in Drosophila melanogaster as a model system to understand the interplay between autophagy and cell death. Using a novel autophagy reporter we found that autophagy occurs during developmental cell death of nurse cells in late oogenesis. Genetic inhibition: of autophagy-related genes atg1, atg13 and vps34 results in late-stage egg chambers containing persisting nurse cell nuclei without fragmented DNA and attenuation of caspase-3 cleavage. We found that Drosophila inhibitor of apoptosis dBruce is degraded by autophagy and this degradation promotes DNA fragmentation and subsequent nurse cell death. These studies demonstrate that autophagic degradation of an inhibitor: of apoptosis is a novel mechanism of triggering cell death.     

Different modes of programmed cell death during oogenesis of the silkmoth Bombyx mori

It is increasingly recognized that programmed cell death includes not only apoptosis and autophagy, but also other types of nonapoptotic cell death, such as paraptosis, which are all characterized by distinct morphological features. Our findings indicate that all three types of programmed cell death occur in the ovarian nurse cell cluster during late vitellogenesis (formation of the egg yolk) of Bombyx mori (Lepidoptera), whereas middle vitellogenesis is exclusively characterized by the presence of a nonapoptotic type of cell death, known as paraptosis. During middle vitellogenesis, nurse cells exhibit clearly cytoplasmic vacuolization, as revealed by ultrastructural examination performed through conventional light and transmission electron microscopy, while no signs of apoptotic or autophagic features are detectable. Moreover, nurse cells of developmental stages 7, 8 and 9 contain autophagic compartments, as well as apoptotic characteristics, such as condensed chromatin, fragmented DNA and activated caspases, as revealed by in vitro assays. We propose that paraptosis precedes both apoptosis and autophagy during vitellogenesis, since its initial activation is detectable during middle vitellogenesis, whereas no apoptotic nor autophagic features are observed. In contrast, at the late stages of Bombyx mori oogenesis, paraptosis, autophagy and apoptosis operate synergistically, resulting in a more efficient elimination of the degenerated nurse cells.

Addendum to: Mpakou VE, Nezis IP, Stravopodis DJ, Margaritis LH, Papassideri IS. Programmed cell death of the ovarian nurse cells during oogenesis of the silkmoth Bombyx mori. Dev Growth Differ 2006; 48:419–28.     

<Emphasis Type="Italic">Drosophila</Emphasis> Chk2 and p53 proteins induce stage-specific cell death independently during oogenesis

In Drosophila, the checkpoint protein-2 kinase (DmChk2) and its downstream effector protein, Dmp53, are required for DNA damage-mediated cell cycle arrest, DNA repair and apoptosis. In this study we focus on understanding the function of these two apoptosis inducing factors during ovarian development. We found that expression of Dmp53, but not DmChk2, led to loss of ovarian stem cells. We demonstrate that expression of DmChk2, but not Dmp53, induced mid-oogenesis cell death. DmChk2 induced cell death was not suppressed by Dmp53 mutant, revealing for the first time that in Drosophila, over-expression of DmChk2 can induce cell death which is independent of Dmp53. We found that over-expression of caspase inhibitors such as DIAP1, p35 and p49 did not suppress DmChk2- and Dmp53-induced cell death. Thus, our study reveals stage-specific effects of Dmp53 and DmChk2 in oogenesis. Moreover, our results demonstrate that although DmChk2 and Dmp53 affect different stages of ovarian development, loss of ovarian stem cells by p53 expression and mid-oogenesis cell death induced by DmChk2 do not require caspase activity.     

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.     

Apoptosis of nurse cells at the late stages of oogenesis of Drosophila melanogaster

 In Drosophila a remarkable feature of oogenesis is the regression of the nurse cells after dumping their cytoplasmic contents into the oocyte. We have studied the nature of this process at the late stages of egg chamber development. In egg chambers DAPI staining shows highly condensed chromatin from stage 12 and TUNEL labelling shows DNA fragmentation up to stage 14. Gel electrophoresis of the end-labelled DNA, extracted from isolated egg chambers at the same stages of development, shows a ladder typical of apoptotic nuclei. This provides evidence that, during Drosophila oogenesis, the nurse cells undergo apoptosis. Apoptotic nuclei have also been detected in dumping-defective egg chambers, indicating that the cytoplasmic depletion of nurse cells is concurrent with but apparently not the cause of the process. Received: 12 December 1997 / Accepted: 6 January 1998     

Autophagic degradation of dBruce controls DNA fragmentation in nurse cells during late Drosophila melanogaster oogenesis

Autophagy is an evolutionarily conserved pathway responsible for degradation of cytoplasmic material via the lysosome. Although autophagy has been reported to contribute to cell death, the underlying mechanisms remain largely unknown. In this study, we show that autophagy controls DNA fragmentation during late oogenesis in Drosophila melanogaster. Inhibition of autophagy by genetically removing the function of the autophagy genes atg1, atg13, and vps34 resulted in late stage egg chambers that contained persisting nurse cell nuclei without fragmented DNA and attenuation of caspase-3 cleavage. The Drosophila inhibitor of apoptosis (IAP) dBruce was found to colocalize with the autophagic marker GFP-Atg8a and accumulated in autophagy mutants. Nurse cells lacking Atg1 or Vps34 in addition to dBruce contained persisting nurse cell nuclei with fragmented DNA. This indicates that autophagic degradation of dBruce controls DNA fragmentation in nurse cells. Our results reveal autophagic degradation of an IAP as a novel mechanism of triggering cell death and thereby provide a mechanistic link between autophagy and cell death.     

Control of non-apoptotic nurse cell death by engulfment genes in Drosophila

Programmed cell death occurs as a normal part of oocyte development in Drosophila. For each egg that is formed, 15 germline-derived nurse cells transfer their cytoplasmic contents into the oocyte and die. Disruption of apoptosis or autophagy only partially inhibits the death of the nurse cells, indicating that other mechanisms significantly contribute to nurse cell death. Recently, we demonstrated that the surrounding stretch follicle cells non-autonomously promote nurse cell death during late oogenesis and that phagocytosis genes including draper, ced-12, and the JNK pathway are crucial for this process. When phagocytosis genes are inhibited in the follicle cells, events specifically associated with death of the nurse cells are impaired. Death of the nurse cells is not completely blocked in draper mutants, suggesting that other engulfment receptors are involved. Indeed, we found that the integrin subunit, αPS3, is enriched on stretch follicle cells during late oogenesis and is required for elimination of the nurse cells. Moreover, double mutant analysis revealed that integrins act in parallel to draper. Death of nurse cells in the Drosophila ovary is a unique example of programmed cell death that is both non-apoptotic and non-cell autonomously controlled.     

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.     

Illuminating the role of caspases during Drosophila oogenesis

Cell death is a prominent feature of animal germline development. In Drosophila, the death of 15 nurse cells is linked to the development of each oocyte. In addition, females respond to poor environmental conditions by inducing egg chamber death prior to yolk uptake by the oocyte. To study these two forms of cell death, we analyzed caspase activity in the germline by expressing a transgene encoding a caspase cleavage site flanked by cyan fluorescent protein and yellow fluorescent protein. When expressed in ovaries undergoing starvation-induced apoptosis, this construct was an accurate reporter of caspase activity. However, dying nurse cells at the end of normal oogenesis showed no evidence of cytoplasmic caspase activity. Furthermore, although expression of the caspase inhibitors p35 or Drosophila inhibitor of apoptosis protein 1 blocked starvation-induced death, it did not affect normal nurse cell death or overall oogenesis in well-fed females. Our data suggest that caspases play no role in developmentally programmed nurse cell death.     

Proteasome inhibition induces developmentally deregulated programs of apoptotic and autophagic cell death during Drosophila melanogaster oogenesis

Ubiquitin/proteasome‐mediated degradation of eukaryotic proteins is critically implicated in a number of signalling pathways and cellular processes. To specifically impair proteasome activities, in vitro developing Drosophila melanogaster egg chambers were exposed to the MG132 or epoxomicin proteasome inhibitors, while a GAL4/UAS binary genetic system was employed to generate double transgenic flies overexpressing β2 and β6 conditional mutant proteasome subunits in a cell type‐specific manner. MG132 and epoxomicin administration resulted in severe deregulation of in vitro developing egg chambers, which was tightly associated with precocious induction of nurse cell‐specific apoptotic and autophagic death programmes, featured by actin cytoskeleton disorganization, nuclear chromatin condensation, DRICE caspase activation and autophagosome accumulation. In vivo targeted overexpression of β2 and β6 conditional mutants, specifically in the nurse cell compartment, led to a notable up‐regulation of sporadic apoptosis potency during early and mid‐oogenesis ‘checkpoints’, thus reasonably justifying the observed reduction in eclosion efficiency. Furthermore, in response to the intracellular abundance of β2 and β6 conditional mutant forms, specifically in numerous tissues of third instar larval stage, the developmental course was arrested, and lethal phenotypes were obtained at this particular embryonic period, with the double transgenic heterozygote embryos being unable to further proceed to complete maturation to adult flies. Our data demonstrate that physiological proteasome function is required to ensure normal oogenesis and embryogenesis in D. melanogaster, since targeted and cell type‐dependent proteasome inactivation initiates developmentally deregulated apoptotic and autophagic mechanisms.     

Reversible response of protein localization and microtubule organization to nutrient stress during Drosophila early oogenesis

The maturation of animal oocytes is highly sensitive to nutrient availability. During Drosophila oogenesis, a prominent metabolic checkpoint occurs at the onset of yolk uptake (vitellogenesis): under nutrient stress, egg chambers degenerate by apoptosis. To investigate additional responses to nutrient deprivation, we studied the intercellular transport of cytoplasmic components between nurse cells and the oocyte during previtellogenic stages. Using GFP protein-traps, we showed that Ypsilon Schachtel (Yps), a putative RNA binding protein, moved into the oocyte by both microtubule (MT)-dependent and -independent mechanisms, and was retained in the oocyte in a MT-dependent manner. These data suggest that oocyte enrichment is accomplished by a combination of MT-dependent polarized transport and MT-independent flow coupled with MT-dependent trapping within the oocyte. Under nutrient stress, Yps and other components of the oskar ribonucleoprotein complex accumulated in large processing bodies in nurse cells, accompanied by MT reorganization. This response was detected as early as 2 h after starvation, suggesting that young egg chambers rapidly respond to nutrient stress. Moreover, both Yps aggregation and MT reorganization were reversed with re-feeding of females or the addition of exogenous insulin to cultured egg chambers. Our results suggest that egg chambers rapidly mount a stress response by altering intercellular transport upon starvation. This response implies a mechanism for preserving young egg chambers so that egg production can rapidly resume when nutrient availability improves.