Kimberly McCall

Induction of apoptosis by Drosophila reaper, hid and grim through inhibition of IAP function

Induction of apoptosis in Drosophila requires the activity of three closely linked genes, reaper, hid and grim. Here we show that the proteins encoded by reaper, hid and grim activate cell death by inhibiting the anti‐apoptotic activity of the Drosophila IAP1 (diap1) protein. In a genetic modifier screen, both loss‐of‐function and gain‐of‐function alleles in the endogenous diap1 gene were obtained, and the mutant proteins were functionally and biochemically characterized. Gain‐of‐function mutations in diap1 strongly suppressed reaper‐, hid‐ and grim‐induced apoptosis. Sequence analysis of these alleles revealed that they were caused by single amino acid changes in the baculovirus IAP repeat domains of diap1, a domain implicated in binding REAPER, HID and GRIM. Significantly, the corresponding mutant DIAP1 proteins displayed greatly reduced binding of REAPER, HID and GRIM, indicating that REAPER, HID and GRIM kill by forming a complex with DIAP1. These data provide strong in vivo evidence for a previously published model of cell death regulation in Drosophila.

Probes of chromatin accessibility in the Drosophila bithorax complex respond differently to Polycomb-mediated repression.

The Polycomb group (PcG) of genes are required for maintenance of the repressed state of the homeotic genes in Drosophila. There are similarities between the PcG repression and mating‐type silencing in yeast or heterochromatic position effect in Drosophila, which suggest that PcG repression may involve a highly compacted chromatin structure. To test for such a structure, heterologous DNA‐ binding proteins were used as probes for DNA accessibility in Drosophila embryos. Binding sites for the yeast transcriptional activator GAL4 and for bacteriophage T7 RNA polymerase were inserted into the bithorax (bx) regulatory region of the endogenous Ultrabithorax (Ubx) gene, which is regulated by the PcG. Ubiquitously expressed GAL4 protein directs transcription through its binding sites only in the posterior segments where the bx region is active. The block to GAL4 activation in the more anterior segments is dependent on Polycomb (Pc) function. In contrast, T7 RNA polymerase can transcribe from its target promoter in all segments of the embryo. Thus, Pc‐mediated repression blocks activated polymerase II transcription, but does not simply exclude all proteins.

Stage-specific regulation of caspase activity in drosophila oogenesis

In Drosophila oogenesis, the programmed cell death of germline cells occurs predominantly at three distinct stages. These cell deaths are subject to distinct regulatory controls, as cell death during early and midoogenesis is stress-induced, whereas the cell death of nurse cells in late oogenesis is developmentally regulated. In this report, we show that the effector caspase Drice is activated during cell death in both mid- and late oogenesis, but that the level and localization of activity differ depending on the stage. Active Drice formed localized aggregates during nurse cell death in late oogenesis; however, active Drice was found more ubiquitously and at a higher level during germline cell death in midoogenesis. Because Drice activity was limited in late oogenesis, we examined whether another effector caspase, Dcp-1, could drive the unique morphological events that occur normally in late oogenesis. We found that premature activation of the effector caspase, Dcp-1, resulted in a disappearance of filamentous actin, rather than the formation of actin bundles, suggesting that Dcp-1 activity must also be restrained in late oogenesis. Overexpression of the caspase inhibitor DIAP1 suppressed cell death induced by Dcp-1 but had no effect on cell death during late oogenesis. This limited caspase activation in dying nurse cells may prevent destruction of the nurse cell cytoskeleton and the connected oocyte.

Cracking open cell death in the Drosophila ovary

The Drosophila melanogaster ovary is a powerful yet simple system with only a few cell types. Cell death in the ovary can be induced in response to multiple developmental and environmental signals. These cell deaths occur at distinct stages of oogenesis and involve unique mechanisms utilizing apoptotic, autophagic and perhaps necrotic processes. In this review, we summarize recent progress characterizing cell death mechanisms in the fly ovary.

Bcl-2 proteins and autophagy regulate mitochondrial dynamics during programmed cell death in the Drosophila ovary

The Bcl-2 family has been shown to regulate mitochondrial dynamics during cell death in mammals and C. elegans, but evidence for this in Drosophila has been elusive. Here, we investigate the regulation of mitochondrial dynamics during germline cell death in the Drosophila melanogaster ovary. We find that mitochondria undergo a series of events during the progression of cell death, with remodeling, cluster formation and uptake of clusters by somatic follicle cells. These mitochondrial dynamics are dependent on caspases, the Bcl-2 family, the mitochondrial fission and fusion machinery, and the autophagy machinery. Furthermore, Bcl-2 family mutants show a striking defect in cell death in the ovary. These data indicate that a mitochondrial pathway is a major mechanism for activation of cell death in Drosophila oogenesis.

Diversity of cell death pathways: insight from the fly ovary

Multiple types of cell death exist including necrosis, apoptosis, and autophagic cell death. The Drosophila ovary provides a valuable model to study the diversity of cell death modalities, and we review recent progress to elucidate these pathways. At least five distinct types of cell death occur in the ovary, and we focus on two that have been studied extensively. Cell death of mid-stage egg chambers occurs through a novel caspase-dependent pathway that involves autophagy and triggers phagocytosis by surrounding somatic epithelial cells. For every egg, 15 germline nurse cells undergo developmental programmed cell death, which occurs independently of most known cell death genes. These forms of cell death are strikingly similar to cell death observed in the germlines of other organisms.

Detection of apoptosis in Drosophila.

Drosophila has unique genetic and cell biological advantages as a model system for the study of apoptosis. Many cell death genes are evolutionarily conserved between flies and mammals. Cell death can be induced by environmental stimuli and normally occurs during diverse developmental processes in Drosophila. Here, we review several approaches for detecting cell death in Drosophila. We provide detailed protocols for labeling apoptotic cells in the embryo and ovary using terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling and acridine orange. Additionally, we describe methods for ectopically expressing cell death genes in the eye and the use of transgenic flies for the detection of genetic interactions among cell death genes.

Combined inhibition of autophagy and caspases fails to prevent developmental nurse cell death in the Drosophila melanogaster ovary.

During the final stages of Drosophila melanogaster oogenesis fifteen nurse cells, sister cells to the oocyte, degenerate as part of normal development. This process involves at least two cell death mechanisms, caspase-dependent cell death and autophagy, as indicated by apoptosis and autophagy markers. In addition, mutations affecting either caspases or autophagy partially reduce nurse cell removal, leaving behind end-stage egg chambers with persisting nurse cell nuclei. To determine whether apoptosis and autophagy work in parallel to degrade and remove these cells as is the case with salivary glands during pupariation, we generated mutants doubly affecting caspases and autophagy. We found no significant increase in either the number of late stage egg chambers containing persisting nuclei or in the number of persisting nuclei per egg chamber in the double mutants compared to single mutants. These findings suggest that there is another cell death mechanism functioning in the ovary to remove all nurse cell remnants from late stage egg chambers.

Phagocytosis genes nonautonomously promote developmental cell death in the Drosophila ovary.

Significance Programmed cell death is usually considered a cell-autonomous suicide program, synonymous with apoptosis. Here we demonstrate that a specific example of large-scale nonapoptotic developmental programmed cell death in the Drosophila ovary occurs by an alternative cell death program where surrounding epithelial cells nonautonomously promote the death of the germ line. We find that genes normally required for engulfment of dying cells act to promote the death of the germ line. Developmental programmed cell death in the Drosophila ovary is an intriguing example of nonapoptotic, nonautonomous cell death, providing insight on the diversity of cell death mechanisms. Programmed cell death (PCD) is usually considered a cell-autonomous suicide program, synonymous with apoptosis. Recent research has revealed that PCD is complex, with at least a dozen cell death modalities. Here, we demonstrate that the large-scale nonapoptotic developmental PCD in the Drosophila ovary occurs by an alternative cell death program where the surrounding follicle cells nonautonomously promote death of the germ line. The phagocytic machinery of the follicle cells, including Draper, cell death abnormality (Ced)-12, and c-Jun N-terminal kinase (JNK), is essential for the death and removal of germ-line–derived nurse cells during late oogenesis. Cell death events including acidification, nuclear envelope permeabilization, and DNA fragmentation of the nurse cells are impaired when phagocytosis is inhibited. Moreover, elimination of a small subset of follicle cells prevents nurse cell death and cytoplasmic dumping. Developmental PCD in the Drosophila ovary is an intriguing example of nonapoptotic, nonautonomous PCD, providing insight on the diversity of cell death mechanisms.

Drosophila 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.