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The last step of cell death is cell clearance, a process critical for tissue homeostasis. For efficient cell clearance to occur, phagocytes and dead cells need to reciprocally signal to each other. One important phenomenon that is under-investigated, however, is that phagocytes not only engulf corpses but contribute to cell death progression. The aims of this study were to determine how the phagocytic receptor Draper non-autonomously induces cell death, using the Drosophila ovary as a model system. We found that Draper, expressed in epithelial follicle cells, requires its intracellular signaling domain to kill the adjacent nurse cell population. Kinases Src42A, Shark and JNK (Bsk) were required for Draper-induced nurse cell death. Signs of nurse cell death occurred prior to apparent engulfment and required the caspase Dcp-1, indicating that it uses a similar apoptotic pathway to starvation-induced cell death. These findings indicate that active signaling by Draper is required to kill nurse cells via the caspase Dcp-1, providing novel insights into mechanisms of phagoptosis driven by non-professional phagocytes. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 C[sub 2]H[sub 2] 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[sup +] 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. [ABSTRACT FROM AUTHOR]

The death and clearance of nurse cells is a consequential milestone in Drosophila melanogaster oogenesis. In preparation for oviposition, the germline-derived nurse cells bequeath to the developing oocyte all their cytoplasmic contents and undergo programmed cell death. The death of the nurse cells is controlled non-autonomously and is precipitated by epithelial follicle cells of somatic origin acquiring a squamous morphology and acidifying the nurse cells externally. Alternatively, stressors such as starvation can induce the death of nurse cells earlier in mid-oogenesis, manifesting apoptosis signatures, followed by their engulfment by epithelial follicle cells. To identify and contrast the molecular pathways underlying these morphologically and genetically distinct cell death paradigms, both mediated by follicle cells, we compared their genome-wide transcriptional, translational, and secretion profiles before and after differentiating to acquire a phagocytic capability, as well as during well-fed and nutrient-deprived conditions. By coupling the GAL4-UAS system to Translating Ribosome Affinity Purification (TRAP-seq) and proximity labeling (HRP-KDEL) followed by Liquid Chromatography tandem mass-spectrometry, we performed high-throughput screens to identify pathways selectively activated or repressed by follicle cells to employ nurse cell-clearance routines. We also integrated two publicly available single-cell RNAseq atlases of the Drosophila ovary to define the transcriptomic profiles of follicle cells. In this report, we describe the genes and major pathways identified in the screens and the striking consequences to Drosophila melanogaster oogenesis caused by RNAi perturbation of prioritized candidates. To our knowledge, our study is the first of its kind to comprehensively characterize two distinct apoptotic and non-apoptotic cell death paradigms in the same multi-cellular system. Beyond molecular differences in cell death, our investigation may also provide insights into how key systemic trade-offs are made between survival and reproduction when faced with physiological stress. Author summary: Cell death occurs throughout development and homeostasis to removed damaged or obsolete cells. Typically dying cells are engulfed by neighboring phagocytic cells. In some cases, the phagocytic cells directly promote the death of neighboring cells, in a process called "phagoptosis." The ovary of the vinegar fly, Drosophila melanogaster, provides a valuable model to investigate the differences between cell death mechanisms. The oocyte is normally supported by nurse cells during its development, and at the end of oocyte development the nurse cells undergo phagoptosis by surrounding follicle cells. Nurse cells can also undergo a form of cell death called apoptosis earlier in development in response to starvation, and apoptotic nurse cells are phagocytosed by follicle cells. To identify and contrast the molecular pathways underlying these morphologically and genetically distinct cell death paradigms, we compared their genome-wide transcriptional, translational, and secretion profiles. In this report, we describe the genes and major pathways identified in the screens and the striking consequences to Drosophila melanogaster oogenesis caused by genetic disruption of these genes. To our knowledge, this study is the first of its kind to comprehensively characterize two distinct apoptotic and non-apoptotic cell death paradigms in the same tissue. [ABSTRACT FROM AUTHOR]

The clearance of dead cells is a fundamental process in the maintenance of tissue homeostasis. Genetic studies in Drosophila melanogaster, Caenorhabditis elegans, and mammals have identified two evolutionarily conserved signaling pathways that act redundantly to regulate this engulfment process: the ced-1/-6/-7 and ced-2/-5/-12 pathways. Of these engulfment genes, only the ced-7/ABCA1 ortholog remains to be identified in D. melanogaster. Homology searches have revealed a family of putative ced-7/ABCA1 homologs encoding ATP-binding cassette (ABC) transporters in D. melanogaster. To determine which of these genes functions similarly to ced-7/ABCA1, we analyzed mutants for engulfment phenotypes in oogenesis, during which nurse cells (NCs) in each egg chamber undergo programmed cell death (PCD) and are removed by neighboring phagocytic follicle cells (FCs). Our genetic analyses indicate that one of the ABC transporter genes, which we have named Eato (Engulfment ABC Transporter in the ovary), is required for NC clearance in the ovary and acts in the same pathways as drpr, the ced-1 ortholog, and in parallel to Ced-12 in the FCs. Additionally, we show that Eato acts in the FCs to promote accumulation of the transmembrane receptor Drpr, and promote membrane extensions around the NCs for their clearance. Since ABCA class transporters, such as CED-7 and ABCA1, are known to be involved in lipid trafficking, we propose that Eato acts to transport membrane material to the growing phagocytic cup for cell corpse clearance. Our work presented here identifies Eato as the ced-7/ABCA1 ortholog in D. melanogaster, and demonstrates a role for Eato in Drpr accumulation and phagocytic membrane extensions during NC clearance in the ovary. [ABSTRACT FROM AUTHOR]

Abstract: Organized boundaries between different cell fates are critical in patterning and organogenesis. In some tissues, long-range signals position a boundary, and local Notch signaling maintains it. How Notch activity is restricted to boundary regions is not well understood. During Drosophila oogenesis, the long-range signals EGF and Dpp regulate expression of bunched (bun), which encodes a homolog of mammalian transcription factors TSC-22 and GILZ. Here, we show that bun establishes a boundary for Notch signaling in the follicle cell epithelium. Notch signaling is active in anterior follicle cells and is required for concurrent follicle cell reorganizations including centripetal migration and operculum formation. bun is required in posterior columnar follicle cells to repress the centripetal migration fate, including gene expression, cell shape changes and accumulation of cytoskeletal components. bun mutant clones adjacent to the centripetally migrating follicle cells showed ectopic Notch responses. bun is necessary, but not sufficient, to down-regulate Serrate protein levels throughout the follicular epithelium. These data indicate that Notch signaling is necessary, but not sufficient, for centripetal migration and that bun regulates the level of Notch stimulation to position the boundary between centripetally migrating and stationary columnar follicle cells. [Copyright &y& Elsevier]

Billions of cells die in our bodies on a daily basis and are engulfed by phagocytes. Engulfment, or phagocytosis, can be broken down into five basic steps: attraction of the phagocyte, recognition of the dying cell, internalization, phagosome maturation, and acidification. In this study, we focus on the last two steps, which can collectively be considered corpse processing, in which the engulfed material is degraded. We use the Drosophila ovarian follicle cells as a model for engulfment of apoptotic cells by epithelial cells. We show that engulfed material is processed using the canonical corpse processing pathway involving the small GTPases Rab5 and Rab7. The phagocytic receptor Draper is present on the phagocytic cup and on nascent, phosphatidylinositol 3-phosphate (PI(3)P)- and Rab7-positive phagosomes, whereas integrins are maintained on the cell surface during engulfment. Due to the difference in subcellular localization, we investigated the role of Draper, integrins, and downstream signaling components in corpse processing. We found that some proteins were required for internalization only, while others had defects in corpse processing as well. This suggests that several of the core engulfment proteins are required for distinct steps of engulfment. We also performed double mutant analysis and found that combined loss of draper and αPS3 still resulted in a small number of engulfed vesicles. Therefore, we investigated another known engulfment receptor, Crq. We found that loss of all three receptors did not inhibit engulfment any further, suggesting that Crq does not play a role in engulfment by the follicle cells. A more complete understanding of how the engulfment and corpse processing machinery interact may enable better understanding and treatment of diseases associated with defects in engulfment by epithelial cells. [ABSTRACT FROM AUTHOR]