Genetic regulation of programmed cell death in Drosophila

Programmed cell death plays an important role in maintaining homeostasis during animal development,and has been conserved in animals as different as nematoes and humans. Recent studies of Drosophila have provided valuadle information toward our understanding of genetic regulation of death.Different signals trigger the novel death regulators rpr,hid,and grim,that utilize the evolutionarily conserved iap and ark genes to modulate caspase function.Subsequent removal of dying cells also appears to be accomplished by conserved mechanisms.The similarity between Drosophila and human in cell death signaling pathways illustrate the promise of fruit flies as a model system to elucidatek the mechanisms underlying regulation of programmed cell death.     

EAST and Chromator control the destruction and remodeling of muscles during Drosophila metamorphosis

Metamorphosis involves the destruction of larval, the formation of adult and the transformation of larval into adult tissues. In this study, we demonstrate the role of the Drosophila nuclear proteins EAST and Chromator in tissue destruction and remodeling. To better understand the function of east, we performed a yeast two-hybrid screen and identified the euchromatin associated protein Chromator as a candidate interactor. To analyze the functional significance of our two-hybrid data, we generated a set of novel pupal lethal Chro alleles by P-element excision. The pupal lethal Chro mutants resemble lethal east alleles as homozygous mutants develop into pharates with normal looking body parts, but fail to eclose. The eclosion defect of the Chro alleles is rescued in an east heterozygous background, indicating antagonistic genetic interactions between the two genes. Live cell imaging was applied to study muscle development during metamorphosis. Consistent with the eclosion defects, mutant pharates of both genes show loss and abnormal differentiation of adult eclosion muscles. The two genes have opposite effects on the destruction of larval muscles in metamorphosis. While Chro mutants show incomplete histolysis, muscles degenerate prematurely in east mutants. Moreover east mutants affect the remodeling of abdominal larval muscles into adult eclosion muscles. During this process, loss of east interferes with the spatial coordination of thinning of the larval muscles. Overexpression of EAST-GFP can prevent the disintegration of polytene chromosomes during programmed cell death. We propose that Chro activates and east inhibits processes and genes involved in tissue destruction and remodeling.     

Role of programmed cell death in patterning the Drosophila antennal arista

Programmed cell death is a critical process for the patterning and sculpting of organs during development. The Drosophila arista, a feather-like structure at the tip of the antenna, is composed of a central core and several lateral branches. A homozygous viable mutation in the thread gene, which encodes an inhibitor of apoptosis protein, produces a branchless arista. We have found that mutations in the proapoptotic gene hid lead to numerous extra branches, suggesting that the level of cell death determines the number of branches in the arista. Consistent with this idea, we have found that thread mutants show excessive cell death restricted to the antennal imaginal disc during the middle third instar larval stage. These findings point to a narrow window of development in which regulation of programmed cell death is essential to the proper formation of the arista.     

The Drosophila caspase inhibitor DIAP1 is essential for cell survival and is negatively regulated by HID.

Drosophila Reaper (RPR), Head Involution Defective (HID), and GRIM induce caspase-dependent cell death and physically interact with the cell death inhibitor DIAP1. Here we show that HID blocks DIAP1's ability to inhibit caspase activity and provide evidence suggesting that RPR and GRIM can act similarly. Based on these results, we propose that RPR, HID, and GRIM promote apoptosis by disrupting productive IAP-caspase interactions and that DIAP1 is required to block apoptosis-inducing caspase activity. Supporting this hypothesis, we show that elimination of DIAP1 function results in global early embryonic cell death and a large increase in DIAP1-inhibitable caspase activity and that DIAP1 is still required for cell survival when expression of rpr, hid, and grim is eliminated.     

Identification of a Drosophila melanogaster ICE/CED-3-related protease, drICE.

Cysteine proteases of the ICE/CED-3 family (caspases) are required for the execution of programmed cell death (PCD) in a wide range of multicellular organisms. Caspases are implicated in the execution of apoptosis in Drosophila melanogaster by the observation that expression of baculovirus p35, a caspase inhibitor, blocks cell death in vivo in Drosophila. We report here the identification and characterization of drICE, a D. melanogaster caspase. We show that overexpression of drICE sensitizes Drosophila cells to apoptotic stimuli and that expression of an N-terminally truncated form of drICE rapidly induces apoptosis in Drosophila cells. Induction of apoptosis by rpr overexpression or by cycloheximide or etoposide treatment of Drosophila cells results in proteolytic processing of drICE. We further show that drICE is a cysteine protease that cleaves baculovirus p35 and Drosophila lamin DmO in vitro and that drICE is expressed at all the stages of Drosophila development at which PCD can be induced. Taken together, these results strongly argue that drICE is an apoptotic caspase that acts downstream of rpr. drICE is therefore the first unequivocal link between the molecular machinery of Drosophila cell death and the conserved machinery of Caenorhabditis elegans and vertebrates. Identification of drICE should facilitate the elucidation of upstream regulators and downstream targets of caspases by genetic screening.     

Programmed cell death mechanisms of identifiable peptidergic neurons in Drosophila melanogaster

The molecular basis of programmed cell death (PCD) of neurons during early metamorphic development of the central nervous system (CNS) in Drosophila melanogaster are largely unknown, in part owing to the lack of appropriate model systems. Here, we provide evidence showing that a group of neurons (vCrz) that express neuropeptide Corazonin (Crz) gene in the ventral nerve cord of the larval CNS undergo programmed death within 6 hours of the onset of metamorphosis. The death was prevented by targeted expression of caspase inhibitor p35, suggesting that these larval neurons are eliminated via a caspase-dependent pathway. Genetic and transgenic disruptions of ecdysone signal transduction involving ecdysone receptor-B (EcR-B) isoforms suppressed vCrz death, whereas transgenic re-introduction of either EcR-B1 or EcR-B2 isoform into the EcR-B-null mutant resumed normal death. Expression of reaper in vCrz neurons and suppression of vCrz-cell death in a reaper-null mutant suggest that reaper functions are required for the death, while no apparent role was found for hid or grim as a death promoter. Our data further suggest that diap1 does not play a role as a central regulator of the PCD of vCrz neurons. Significant delay of vCrz-cell death was observed in mutants that lack dronc or dark functions, indicating that formation of an apoptosome is necessary, but not sufficient, for timely execution of the death. These results suggest that activated ecdysone signaling determines precise developmental timing of the neuronal degeneration during early metamorphosis, and that subsequent reaper-mediated caspase activation occurs through a novel DIAP1-independent pathway.     

The CARD-carrying caspase Dronc is essential for most, but not all, developmental cell death in Drosophila

The initiator caspase Dronc is the only Drosophila caspase that contains a caspase activation and recruitment domain (CARD). Although Dronc has been implicated as an important effector of apoptosis, the genetic function of dronc in normal development is unclear because dronc mutants have not been available. In an EMS mutagenesis screen, we isolated four point mutations in dronc that recessively suppress the eye ablation phenotype caused by eye-specific overexpression of hid. Homozygous mutant dronc animals die during pupal stages; however, at a low frequency we obtained homozygous adult escapers. These escapers have additional cells in the eye and wings that are less transparent and slightly curved down. We determined that this is due to lack of apoptosis. Our analyses of dronc mutant embryos suggest that dronc is essential for most apoptotic cell death during Drosophila development, but they also imply the existence of a dronc-independent cell death pathway. We also constructed double mutant flies for dronc and the apoptosis inhibitor diap1. dronc mutants can rescue the ovarian degeneration phenotype caused by diap1 mutations, confirming that dronc acts genetically downstream of diap1.     

Drosophila sickle is a novel grim-reaper cell death activator

The Drosophila genes reaper, head involution defective (hid), and grim all reside at 75C on chromosome three and encode related proteins that have crucial functions in programmed cell death (reviewed in ). In this report, we describe a novel grim-reaper gene, termed sickle, that resides adjacent to reaper. The sickle gene, like reaper and grim, encodes a small protein which contains an RHG motif and a Trp-block. In wild-type embryos, sickle expression was detected in cells of the developing central nervous system. Unlike reaper, hid, and grim, the sickle gene is not removed by Df(3L)H99, and strong ectopic sickle expression was detected in the nervous system of this cell death mutant. sickle very effectively induced cell death in cultured Spodoptera Sf-9 cells, and this death was antagonized by the caspase inhibitors p35 or DIAP1. Strikingly, unlike the other grim-reaper genes, targeted sickle expression did not induce cell death in the Drosophila eye. However, sickle strongly enhanced the eye cell death induced by expression of either an r/grim chimera or reaper.     

Steroid regulation of autophagic programmed cell death during development

Apoptosis and autophagy are morphologically distinct forms of programmed cell death. While autophagy occurs during the development of diverse organisms and has been implicated in tumorigenesis, little is known about the molecular mechanisms that regulate this type of cell death. Here we show that steroid-activated programmed cell death of Drosophila salivary glands occurs by autophagy. Expression of p35 prevents DNA fragmentation and partially inhibits changes in the cytosol and plasma membranes of dying salivary glands, suggesting that caspases are involved in autophagy. The steroid-regulated BR-C, E74A and E93 genes are required for salivary gland cell death. BR-C and E74A mutant salivary glands exhibit vacuole and plasma membrane breakdown, but E93 mutant salivary glands fail to exhibit these changes, indicating that E93 regulates early autophagic events. Expression of E93 in embryos is sufficient to induce cell death with many characteristics of apoptosis, but requires the H99 genetic interval that contains the rpr, hid and grim proapoptotic genes to induce nuclear changes diagnostic of apoptosis. In contrast, E93 expression is sufficient to induce the removal of cells by phagocytes in the absence of the H99 genes. These studies indicate that apoptosis and autophagy utilize some common regulatory mechanisms.     

Overlapping functions of microRNAs in control of apoptosis during Drosophila embryogenesis

Regulation of apoptosis is crucial for tissue homeostasis under normal development and environmental stress. In Drosophila, cell death occurs in different developmental processes including embryogenesis. Here, we report that two members of the miR-2 seed family of microRNAs, miR-6 and miR-11, function together to limit the level of apoptosis during Drosophila embryonic development. Mutants lacking both miR-6 and miR-11 show embryonic lethality and defects in the central nervous system (CNS). We provide evidence that miR-6/11 functions through regulation of the proapoptotic genes, reaper (rpr), head involution defective (hid), grim and sickle (skl). Upregulation of these proapoptotic genes is responsible for the elevated apoptosis and the CNS defects in the mutants. These findings demonstrate that the activity of the proapoptotic genes is kept in check by miR-6/11 to ensure normal development.     

Steroid Hormone Control of Cell Death and Cell Survival: Molecular Insights Using RNAi

The insect steroid hormone ecdysone triggers programmed cell death of obsolete larval tissues during metamorphosis and provides a model system for understanding steroid hormone control of cell death and cell survival. Previous genome-wide expression studies of Drosophila larval salivary glands resulted in the identification of many genes associated with ecdysone-induced cell death and cell survival, but functional verification was lacking. In this study, we test functionally 460 of these genes using RNA interference in ecdysone-treated Drosophila l(2)mbn cells. Cell viability, cell morphology, cell proliferation, and apoptosis assays confirmed the effects of known genes and additionally resulted in the identification of six new pro-death related genes, including sorting nexin-like gene SH3PX1 and Sox box protein Sox14, and 18 new pro-survival genes. Identified genes were further characterized to determine their ecdysone dependency and potential function in cell death regulation. We found that the pro-survival function of five genes (Ras85D, Cp1, CG13784, CG32016, and CG33087), was dependent on ecdysone signaling. The TUNEL assay revealed an additional two genes (Kap-α3 and Smr) with an ecdysone-dependent cell survival function that was associated with reduced cell death. In vitro, Sox14 RNAi reduced the percentage of TUNEL-positive l(2)mbn cells (p<0.05) following ecdysone treatment, and Sox14 overexpression was sufficient to induce apoptosis. In vivo analyses of Sox14-RNAi animals revealed multiple phenotypes characteristic of aberrant or reduced ecdysone signaling, including defects in larval midgut and salivary gland destruction. These studies identify Sox14 as a positive regulator of ecdysone-mediated cell death and provide new insights into the molecular mechanisms underlying the ecdysone signaling network governing cell death and cell survival.