It is thought that the basic cell death program is constitutively expressed, and many cell death genes, including apaf-1, appear to be widely expressed. Ark mRNA is broadly distributed in preblastoderm embryos before the major onset of zygotic transcription, apparently due to maternal contribution. However, by stage 7, the highest levels of Ark mRNA were detected in the ventral neurogenic region, and around several invagination furrows. Interestingly, these are regions in which prominent apoptosis occurs during subsequent development. Later, the highest levels of Ark mRNA were found in the ectoderm and mesoderm of the procephalic region. The expression of Ark in the developing head overlaps significantly with that of the proapoptotic gene hid. Again, abundant apoptosis is subsequently observed in this region. The mRNA distribution of Ark is closely mirrored by the pattern of ß-gal expression from the P element insertion l(2)k11502, which carries a lacZ reporter gene. The l(2)k11502 P element transposon is inserted near the transcriptional start site of Ark, 81 bp upstream of the presumptive TATA box. Although a basal level of lacZ expression could be detected in essentially all cells, high levels of expression are again seen in the procephalic region. At later stages, both lacZ reporter expression and Ark mRNA are detected in a segmentally repeated pattern. Finally, ß-gal immunoreactivity is seen in macrophages starting from late stage 11 to the end of embryonic development. These observations indicate that Ark is abundantly expressed in many but not all dying cells and that its expression is transcriptionally regulated during development (Zhou, 2001).
Apaf-1-related-killer, a putative caspase activator, should be expressed in all cells that have the ability to undergo apoptosis. The distribution of Ark mRNA during Drosophila embryogenesis was determined by in situ hybridization. Ark is maternally expressed in the preblastoderm stage, and its expression continues throughout the embryo as it develops. In the larval stage, Ark is also expressed ubiquitously in eye imaginal discs and wing discs, where cell deaths are known to occur through tissue remodeling. A single 7 kb band was detected in Drosophila RNA by Northern hybridization. Although the expression of dapaf-1S, the alternative-splicing variant of Ark, was not detectable in larval tissues or the S2 cell line, a few dapaf-1S transcripts could be detected in developing embryos. These observations suggest that Dapaf-1L is the predominantly expressed isoform in Drosophila (Kanuka, 1999b).
Drosophila IAP1 (DIAP1/Thread) inhibits cell death to facilitate normal embryonic development. Using RNA interference it has been shown that down-regulation of DIAP1 is sufficient to induce cell death in Drosophila S2 cells. Although this cell death process is accompanied by elevated caspase activity, this activation is not essential for cell death. DIAP1 depletion-induced cell death is strongly suppressed by a reduction in the Drosophila caspase DRONC or Dark. RNA interference studies in Drosophila embryos also have demonstrated that the action of Dark is epistatic to that of DIAP1 in this cell death pathway. The cell death caused by down-regulation of DIAP1 is accelerated by overexpression of DRONC and Dark, and a caspase-inactive mutant form of DRONC can functionally substitute the wild-type DRONC in accelerating cell death. These results suggest the existence of a novel mechanism for cell death signaling in Drosophila that is mediated by DRONC and Dark (Igaki, 2002).
The observation that the pan-caspase inhibitor zD-dcb can not suppress the DIAP1 depletion-induced cell death suggests that DRONC may be able to induce cell death independent of its caspase activity. The observation that the caspase-inactive form of DRONC can functionally substitute the wild-type DRONC in accelerating DIAP1 depletion-induced cell death also supports the idea that the cell death can be mediated through non-caspase mechanisms. DRONC might have a protease-independent cell-killing activity that is activated by Dark. It is possible that DRONC is required simply as a bridging or scaffolding protein to bring other proteins together to transmit the cell death signaling. Although the possibility cannot be excluded that zD-dcb can not completely inhibit the caspase activity of DRONC, it is apparent that the mode of cell death caused by the down-regulation of DIAP1 is distinct from Reaper-induced cell death. The effects were assessed of dsRNAs synthesized from reaper, hid, grim, drob-1, and buffy/dborg-2 cDNAs on the diap1 dsRNA-induced cell death; none of them suppresses the cell death. Further in vivo analysis should help elucidate the role of the caspase-independent cell death pathway regulated by DIAP1 (Igaki, 2002).
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. 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 (Lee, 2001).
Morphological studies of developing vertebrate embryos have resulted in the definition of three types of physiological cell death. The first type, widely known as apoptosis, is found in isolated dying cells that exhibit condensation of the nucleus and cytoplasm, followed by fragmentation and phagocytosis by cells that degrade their contents. The second type, known as autophagy, is observed when groups of associated cells or entire tissues are destroyed. These dying cells contain autophagic vacuoles in the cytoplasm that function in the degeneration of cell components. Autophagic cells destroy their own contents, while apoptotic cells depend on phagocytes to accomplish terminal degradation. The third type, known as non-lysosomal cell death, is least common, and is characterized by swelling of cavities with membrane borders followed by degeneration without lysosomal activity. While autophagy fulfills the definition of programmed cell death, occurs during development of diverse organisms, and has been implicated in tumorigenesis, little is known about the molecular genetic mechanisms underlying this type of programmed cell death. The morphological characteristics that distinguish apoptosis and autophagy suggest that these cell deaths are regulated by independent mechanisms. Comparison of biochemical changes during lymphocyte apoptosis and insect intersegmental muscle autophagy also indicate that these physiological cell deaths occur by distinct mechanisms. However, recent studies of steroid-triggered cell death of Drosophila larval salivary glands suggest that these cells utilize genes that are part of the conserved apoptosis pathway, even though these cells exhibit characteristics of autophagy. Specifically, the caspase Dronc and the homolog of ced4/Apaf-1 (Ark), two components of the core apoptotic machinery, increase in transcription immediately prior to salivary gland cell death. Thus, characterization of the mechanisms governing the regulation of autophagy will identify how these cell deaths differ from those that occur by apoptosis (Lee, 2001 and references therein).
Larval salivary glands of Drosophila undergo rapid programmed cell death in response to ecdysone. This cell destruction can be detected using markers that are typically associated with apoptosis including nuclear staining by Acridine Orange, TUNEL to detect DNA fragmentation, and exposure of phosphatidylserine on the outer leaflet of the plasma membrane. The changes in vacuolar structure that immediately precede the synchronous destruction of larval salivary gland cells are clearly more similar to autophagy than heterophagy (apoptosis). Large vacuoles increase in number in prepupal salivary glands, and rearrangement of the cytoskeleton and an increase in acid phosphatase activity are associated with these structures. Dynamic changes in salivary gland structure may reflect important biochemical changes during programmed cell death. Large Eosin-positive vacuoles appear to fragment, a distinct class of Eosin-negative vacuoles are formed that are closely associated with the plasma membrane, and vacuoles containing organelles are observed in the cytoplasm immediately preceding destruction of salivary glands. An increase in transcription of the caspase Dronc occurs at this stage, and inhibition of caspase activity blocks DNA fragmentation and partially prevents changes in vacuoles and plasma membranes, suggesting that these morphological changes may be attributed in part to the activity of enzymes typically associated with apoptosis (Lee, 2001 and references therein).
While morphological analyses of apoptosis and autophagy suggest different mechanisms for these forms of cell death, some genes that function in apoptosis also function during autophagy. Steroid-regulated genes impact distinct cellular changes in dying cells. Ecdysone impacts on the transcription of the cell death genes rpr, hid and diap2. This regulation is mediated by the ecdysone receptor, and a group of ecdysone-activated factors that include the BR-C, E74 and E93 genes. The function of the steroid-regulated BR-C, E74 and E93 genes in salivary gland cell death has been examined. E93 mutant salivary glands exhibit persistence of large vacuoles and plasma membranes, while these structures are destroyed in BR-C and E74A mutants. Two possible explanations exist for the differences in BR-C, E74A and E93 mutant salivary gland cell morphology. E93 mutant salivary glands could be arrested at an earlier stage of cell destruction that is similar to that of 12-hour wild-type cells, while BR-C and E74A mutants are arrested at a stage that is similar to 14.5-hour salivary gland cells. This model is supported by previous studies indicating that E93 function is required for proper regulation of BR-C and E74A transcription. Alternatively, E93 could function to regulate autophagy that results in destruction of vacuoles and plasma membranes, while BR-C and E74A do not function in the regulation of these cellular changes even though these genes are required for salivary gland cell death. The latter interpretation is intriguing when one considers that expression of E93 is sufficient to induce characteristics of apoptosis, and can induce the removal of cells even in the absence of the rpr, hid and grim cell death genes and nuclear apoptotic changes (Lee, 2001).
Several factors indicate that salivary gland autophagy is regulated by genes that also function in apoptosis. (1) Caspases function in salivary gland cell death. Expression of the baculovirus inhibitor of caspases, p35, inhibits destruction of this tissue. Furthermore, p35 expression prevents DNA fragmentation and partially inhibits morphological changes in vacuoles that are associated with autophagy, indicating that caspases are utilized during autophagy. Transcription of the Apaf1 homolog Ark and the caspase, dronc increases immediately preceding salivary gland cell death, and this transcription is blocked in E93 mutants, further supporting that caspases function in salivary gland autophagy. (2) Transcription of the proapoptotic genes, rpr and hid increases immediately prior to salivary gland autophagy, and the transcription of these genes is blocked by mutations in steroid-regulated genes that are involved in this process. Ectopic expression of E93, a critical determinant of salivary gland autophagy, is sufficient to induce cell death with numerous characteristics of apoptosis. In addition, the association of Croquemort (Crq) expression with E93-induced removal of apoptotic cells and autophagy of salivary glands provides yet another link between these morphologically distinct forms of programmed cell death. Combined, these factors indicate that autophagy and apoptosis utilize at least some similar mechanisms (Lee, 2001).
The location and type of cell appears to be an important determinant for the type of programmed cell death that occurs in the context of animal development. Autophagy occurs when groups of cells or entire tissues die, while apoptosis occurs in isolated dying cells. These studies are consistent with these criteria; salivary gland destruction occurs by autophagy and requires E93 function, while ectopic induction of cell death by expression of E93 during embryogenesis has the characteristics of apoptosis. It is hypothesized that this is due to similarities between autophagy and apoptosis. Alternatively, autophagy and apoptosis may be mechanistically distinct, and the ability to induce ectopic cell death by expression of E93 is simply due to activating a death program in different cell types. This explanation is supported by data demonstrating that p35 inhibits salivary gland cell death, but that p35 is not capable of inhibiting E93-induced cell death in embryos. However, several possibilities exist to explain the disparity of these data. (1) Ectopic expression of E93 during embryogenesis may lead to higher than normal levels of this protein. In side-by-side comparisons with the proapoptotic genes rpr and hid, expression of E93 results in greater cell death and lethality. Thus, the strong killing potential of E93 may be sufficient to overcome inhibition of cell death by p35. (2) Other cell death genes are not inhibited by expression of p35, including cell death that is induced by ectopic expression of the caspase Dronc. (3) Inhibition of vacuolar changes by expression of p35 during salivary gland cell death is incomplete, even though DNA fragmentation is inhibited in this tissue. Thus, caspases may play a role in salivary gland cell death, and both p35 experiments and the transcription of dronc during salivary gland autophagy support this conclusion. However, it is possible that other proteolytic mechanisms act in concert with caspases in the bulk degradation of salivary gland cells (Lee, 2001).
It is concluded that Autophagy and apoptosis are morphologically distinct, suggesting that the mechanisms underlying the regulation of these forms of programmed cell death are different. Nearly all of the large polytenized larval cells die during Drosophila metamorphosis. The synchrony and volume of these cell deaths suggests that engulfment of each dying cell may be limited by the number of available phagocytes. One obvious distinction between autophagy and apoptosis is the location of the lysosomal machinery that degrades the dying cell. Autophagic cells destroy their own contents, while apoptotic cells depend on phagocytes to accomplish terminal degradation. This distinction may account for much of the differences in the morphological appearance of these two forms of dying cells, but does not exclude the possibility that a single autophagic cell utilizes the mechanisms that exist in distinct apoptotic and phagocytic cells. The specific expression of Crq during autophagy supports this possibility, but genetic studies of crq function are needed to test this hypothesis. Future studies of autophagy, and its relationship to apoptosis, will illustrate the similarities and differences between these forms of programmed cell death (Lee, 2001).
The insertion of P element transposon l(2)k11502 appears to disrupt the expression of Ark since embryos homozygous for the insertion display no head-specific in situ signals above the basal levels derived from maternally contributed mRNA. In order to examine whether zygotic Ark function is required for normal cell death, embryos heterozygous or homozygous for the l(2)k11502 insertion were labeled with TUNEL to visualize apoptotic cells. In wild-type and heterozygous embryos, a large number of TUNEL-positive cells were consistently observed in the head region of stage 12 embryos. In contrast, significantly fewer TUNEL-positive cells were present in the head region of embryos homozygous for l(2)k11502. This phenotype is highly penetrant, but some variation among homozygous mutant embryos is seen, possibly due to differences in the amount or perdurance of maternal Ark. Therefore, although many cells can still die in homozygous mutant embryos, zygotic Ark product is required for the normal pattern of apoptosis (Zhou, 1999).
It is unlikely that elimination of zygotic Ark function in l(2)k11502 homozygous embryos represents the true null phenotype, since Ark mRNA is maternally contributed. To inactivate both zygotic and maternal Ark mRNA, the RNA interference assay (RNAi) was used. Double-stranded Ark RNA corresponding to either the first 723 amino acids, or amino acids 134-332, encompassing the most closely conserved region between APAF-1 and CED-4, was made and injected into syncytial wild-type embryos. The patterns of cell death in these embryos were compared to uninjected and lacZ RNAi control embryos by TUNEL and acridine orange staining. When injected into the posterior of the embryo, both Ark dsRNAs significantly reduce the number of apoptotic cells in the injected part of the embryo. Injecting the anterior of the embryo yields similar results, with the reduction of cell death restricted to the injected half of the embryo. Since RNAi produced a more severe reduction of apoptosis than elimination of zygotic Ark function, it appears that maternal Ark product contribute to the induction of cell death during normal embryonic development (Zhou, 1999).
At least some of the 'undead' cells in the l(2)k11502 mutant embryos appear to develop into extra neurons. When stained with the anti-Elav antibody, which recognizes all differentiated neurons, brains of l(2)k11502 homozygous mutant embryos contain more Elav-positive cells than wild type. This phenotype is very similar to that seen in null mutants of the proapoptotic gene head involution defective (hid). In addition, like loss of hid function, many Ark mutant embryos show defects in head involution. These findings suggest that Ark, like hid, is required to eliminate cells for proper morphogenesis of the Drosophila embryo (Zhou, 1999).
The ectopic expression of reaper, hid, grim, and several Drosophila caspases can lead to ectopic apoptosis. For example, when these genes are expressed in the developing retina under the control of an eye-specific promoter, GMR, cell death is induced in a dosage-dependent manner, and this produces various degrees of eye ablation. This provides a highly sensitized background to investigate genetic interactions with other components of the cell death pathway. For example, eliminating one copy of the antiapoptotic gene diap1 suppresses the eye ablation phenotypes of GMR-reaper and GMR-hid, and mutations in various components of the Ras-signaling pathway dominantly modify the GMR-hid phenotype. Potential interactions of Ark with several other proapoptotic genes were investigated by crossing the l(2)k11502 P insertion strain with transgenic fly strains carrying GMR-reaper, GMR-hid, GMR-grim, and GMR-dcp-1. Heterozygosity for Ark results in only a very mild suppression of the eye phenotypes of GMR-reaper/hid/grim. In contrast, l(2)k11502 robustly suppresses the eye phenotype of GMR-dcp-1. This indicates that endogenous Ark is rate limiting for cell killing by DCP-1, but not for apoptosis induced by reaper, hid, and grim under these circumstances. Similar effects were seen upon expression of a truncated form of dcp-1 lacking the prodomain (GMR-dcp-1-N). This suggests that Ark functions at a step either downstream of, or in parallel to, the removal of the dcp-1 prodomain (Zhou, 1999).
Genetic evidence suggests that reaper and grim induce apoptosis by activating dcp-1. For example, when both reaper and dcp-1 are expressed in the eye, they synergize to induce cell death. Expression of full-length dcp-1 results in a weak phenotype, presumably due to poor activation of the zymogen in these conditions. In contrast, coexpression of reaper and grim with dcp-1 leads to an eye ablation phenotype far more severe than the expression of any of these genes alone. Whether this synergy depends on Ark was investigated. Since the eye phenotype of GMR-dcp-1 is suppressed by heterozygosity for Ark, a significant modification of the GMR-reaper GMR-dcp-1 eye phenotype was expected. However, this was not observed. Upon introduction of l(2)k11502 into a GMR-reaper GMR-dcp-1 background, only a very mild change in eye morphology could be detected. It is concluded that the activation of dcp-1 by reaper is independent of Ark function (Zhou, 1999).
Strong genetic interactions were observed between Ark and the Drosophila caspase dcp-1, but only weak interactions with reaper, grim, and hid. In particular, the dosage of endogenous Ark is rate limiting for ectopic cell killing in the fly retina induced by dcp-1 but has little effect on killing by reaper, hid, and grim. However, reaper and grim interact genetically with dcp-1. Furthermore, the synergy between reaper and dcp-1 for the induction of cell death in the retina does not depend on the dosage of Ark. The lack of significant genetic interactions between Ark and reaper, hid, and grim indicates that these molecules act at some distance from one another in the cell death pathway, since genetic interactions are typically strongest among proximal components of a pathway. Therefore, it is proposed that Ark promotes caspase activation through a pathway that is distinct from the one used by reaper, hid, and grim. According to this model, the activation of caspases during apoptosis in Drosophila, and presumably also in mammals, is controlled by the simultaneous action of two separate pathways. In one case, Reaper, Hid, and Grim can activate caspases by inhibiting the antiapoptotic activity of diap1, one of the Drosophila IAPs. In the other case, Ark appears to promote caspase activation by a mechanism that is similar to APAF-1. If one activating pathway is strongly induced, for example by overexpression of Reaper, the activity of components in the other pathway may become less important. This would explain why a reduction of Ark dosage has little effect on reaper, grim, and hid-induced eye ablation. In addition, it is considered likely that the activation of dcp-1 by Ark involves one or more upstream caspases. A number of caspase-like sequences have been identified in Drosophila, but the precise order in which they may act within a caspase cascade remains to be determined. Therefore, it is possible that reaper, grim, hid, and Ark initially activate distinct upstream caspases. It is expected that the activity of Ark may also be controlled in a manner similar to that of APAF-1 and CED-4. Specifically, Ark may be regulated by CED-9/BCL-2-like proteins. The recent identification of BCL-2-like sequences in Drosophila should allow a critical test of this hypothesis. Significantly, Ark is also regulated at the transcriptional level. These results demonstrate that zygotic Ark expression is not at all uniform and becomes restricted to specific regions of the embryo. Expression of Ark in the cephalic region is similar to the expression of hid and correlates overall well with regions in which major morphogenetic cell death occurs subsequently. Furthermore, Ark transcription is induced by both X-ray and UV irradiation in a manner similar to what has been previously reported for reaper. Therefore, it appears that at least some proapoptotic stimuli induce cell death by simultaneously turning on two different pathways for caspase activation (Zhou, 1999).
To examine genetic interactions between Nedd2-like caspase (Dronc) and other apoptotic pathway genes, two UAS-dronc transgenic lines (#23 and #80) were chosen that result in relatively low lethality when crossed to GMR-GAL4 and a recombinant second chromosome was generated for each of these transgenes with GMR-GAL4. When GMR-GAL4 UAS-dronc#80 was crossed to wild type w1118 flies at 25°C, adult flies that exhibited slightly rough and mottled eyes were observed. A similar phenotype has been observed in previous studies and has been shown to be due to ablation of the pigment and photoreceptor cells. Similar results were observed for GMR-GAL4, UAS-dronc#23. This phenotype became more severe when expression of dronc via GMR-GAL4 was increased by raising the temperature to 29°C. Because this eye phenotype can be modified by increasing the expression of dronc, it provided a dosage-sensitive system for examining genetic interactions between dronc and other genes of the apoptosis pathway. To test this further, whether co-expression of the baculovirus caspase inhibitor P35 from the GMR enhancer was able to suppress the eye phenotype of GMR-dronc at 29°C was examined. Co-expression of GMR-p35 dramatically improves the eye ablation phenotype of GMR-dronc. Thus, in this system, Dronc is sensitive to P35 in the Drosophila eye (Quinn, 2000).
Whether the GMR-dronc eye phenotype is sensitive to halving the dosage of the various Drosophila apoptosis-regulatory genes was tested. To assess whether the GMR-dronc eye phenotype is sensitive to the dosage of the H99 genes (reaper, hid, and grim), GMR-dronc flies were crossed to a deficiency removing the H99 genes, Df(3L)H99, at 29°C. The H99 deficiency dominantly suppressed the GMR-dronc eye phenotype. Thus, the cell death-inducing activity of dronc is sensitive to the dosage of the H99 genes. Furthermore, halving the dosage of dronc using a deficiency modifies the ablated eye phenotype of GMR-hid and GMR-rpr, suggesting that dronc is downstream of hid and rpr. To determine whether there was a genetic interaction with dronc and dark, whether decreasing the dosage of dark modified the eye phenotype of GMR-dronc at 29°C was examined. Three different P-element alleles of dark (darkCD4, darkCD8, and darkl(2)k11502) show suppression of the GMR-dronc eye phenotype, indicating that Dark plays a role in promoting Dronc-induced cell death in the eye. Halving the dosage of diap1 using deficiencies or the specific allele thread5 dominantly enhances the GMR-dronc eye phenotype at 25°C . In addition, these diap1 mutations dominantly enhance the lethality associated with GMR-dronc, resulting in at least 10-fold lower numbers of GMR-dronc/+; Df(diap1)/+ adult flies than expected. In contrast, a deficiency removing diap2 showed no effect on the GMR-dronc phenotype, and no lethal effects were observed. Thus diap1, but not a deficiency removing diap2, shows a dosage-sensitive interaction with dronc. By contrast, ectopic expression of diap1 or diap2 from the GMR promoter shows suppression of the GMR-dronc ablated eye phenotype, although GMR-diap2 results in much weaker suppression than GMR-diap1. Thus, both Diap1 and Diap2 are capable of directly or indirectly blocking Dronc-mediated cell death (Quinn, 2000).
Mutations that remove DRONC are not available. Therefore, to examine a possible role for DRONC as a cell death effector a form of DRONC, DRONCC318S, was generated in which the active site cysteine was altered to serine. Expression of similar forms of other caspases results in a suppression of caspase activity and caspase-dependent cell death. This may occur as a result of interaction of DRONCC318S with the Drosophila homolog of the caspase-activating protein Apaf-1, thus preventing the Drosophila Apaf-1 from binding to wild type DRONC and promoting its activation in a manner similar to that described for mammalian Apaf-1 and caspase-9. Transgenic Drosophila were generated in which DRONCC318S was expressed under the control of a promoter, known as GMR, that drives transgene expression specifically in the developing fly eye. The eyes of these flies, known as GMR-DRONCC318S flies, appear similar to those of wild type flies. To assay the ability of DRONCC318S to block cell death, GMR-DRONCC318S flies were crossed to flies overexpressing rpr (GMR-rpr), hid (GMR-hid), or grim (GMR-grim) under the control of the same promoter. GMR-driven expression of rpr, hid, or grim results in a small eye phenotype due to activation of caspase-dependent cell death. However, flies coexpressing GMR-DRONCC318S and one of the cell death activators showed a dramatic suppression of the small eye phenotype, indicating that cell death had been suppressed. The possibility cannot be ruled out that this suppression is a result of DRONCC318S forming nonproductive interactions with the Drosophila Apaf-1 that block its ability to activate other long prodomain caspases such as DCP-2/DREDD. However, these possibilities notwithstanding, these results suggest that DRONC activity is important for bringing about rpr-, hid-, and grim-dependent cell death (Hawkins, 2000).
In mammals and Drosophila, apoptotic caspases are under positive control via the CED-4/Apaf-1/Dark adaptors and negative control via IAPs (inhibitor of apoptosis proteins). However, the in vivo genetic relationship between these opposing regulators is not known. In this study, it has been demonstrated that a dark mutation reverses catastrophic defects seen in Diap1 mutants and rescues cells specified for Diap1-regulated cell death in development and in response to genotoxic stress. dark function is required for hyperactivation of caspases that occurs in the absence of Diap1. Since the action of dark is epistatic to that of Diap1, these findings demonstrate that caspase-dependent cell death requires concurrent positive input through Apaf-1-like proteins together with disruption of IAP-caspase complexes (Rodriguez, 2002).
Mutations in dark cause a variety of developmental defects, including an enlarged larval CNS. In the absence of dark function, cells specified to die actually survive and also differentiate. The embryonic CNS midline glia is a well-studied cell lineage in the fly embryo and, though only this particular lineage was studied, it is likely that supernumerary cell types in other lineages also persist in dark mutants. Several important conclusions derive from these observations. (1) The persistence of extra cells excludes trivial explanations for reduced TUNEL labeling in dark mutants (e.g. cell death in the absence of TUNEL labeling or redirected cell fates that prevent the specification of PCD) and reinforces models that favor a fundamental role for dark in embryonic PCD. (2) As shown in C.elegans and in studies of Reaper interval Drosophila mutants, rescue from PCD can uncover a cryptic differentiation program. (3) Most important, the same midline glial cells that fail to die in dark mutants also require the induced action of reaper, grim and hid. There is a requirement for dark function in normal PCD when reaper, grim and/or hid are expressed at physiological levels. It is noteworthy that Diap1 and at least two of the cell death initiators (i.e. reaper and hid) are expressed in pools of progenitor cells, which give rise to the embryonic midline glia and also play an important role in specifying the death of these cells. Thus, although midline glia presumably receive death signals from reaper and hid leading to the disruption of Diap1-caspase interactions, these cells still fail to die and are even capable of differentiating normally in the absence of dark function. This inference supports conclusions from epistasis studies indicating that a disruption of caspase-Diap1 interactions alone is insufficient for apoptosis, and suggests that dark functions as a co-effector of cell death signaling along with reaper, grim and/or hid. It is worth noting that the mammalian ortholog of dark, Apaf-1, functions downstream from the point at which mitochondrial factors including cytochrome c are released from mitochondria. If the same holds true for the fly protein, it follows that embryonic midline glia (and perhaps other cell types) can survive and differentiate beyond this mitochondrial 'point of no return' (Rodriguez, 2002).
Additional evidence that PCD requires coordinated dark-dependent caspase activation in conjuction with the release of IAP-mediated caspase inhibition comes from tests of dark mutants under conditions of stress that elicit apoptotic responses by the embryonic cell death initiators. Like the Reaper interval mutants, dark mutants also exhibit profound failures in cell death in response to ionizing radiation. Interestingly, the dark mutation itself does not interfere with the radiation induction of reaper mRNA. Therefore the possibility that resistance to damage-induced apoptosis evident in irradiated dark mutants is caused by a disruption of upstream elements in the signaling pathway can be excluded. Instead, the results raise the possibility that dark, like reaper, may function as an important effector of Drosophila p53-mediated apoptosis. In addition, these findings reinforce an apoptotic requirement for dark (even when reaper is transcriptionally induced) and provide evidence for models where cell death signals do not converge on Diap1 alone. In future studies, it will be interesting to determine the range of damage signals that can engage dark activity, since this locus might also function in a broader range of damage signals beyond those provoked by radiation (Rodriguez, 2002).
In flies, Diap1 is thought to function as a rate-limiting brake on apoptotic cell death. If, however, Diap1 were the most proximal rate-limiting regulator of apoptosis, then the presence or absence of dark function should have no influence on Diap1-dependent effects. If, however, dark and Diap1 exert interdependent functions, then the opposite outcome is predicted. The results from these studies provide consistent and compelling evidence favoring the latter scenario since, wherever tested, the influence of Diap1 upon apoptotic signaling is heavily dependent upon intact dark function. In the darkCD4 mutant, for instance, the Diap15 mutation fails to enhance grim- and hid-induced cell killing in the eye. These experiments examined Diap1 in the heterozygous condition, but the same outcome is also true when Diap1 is tested in the homozygous state. Early in embryonic development, Diap1 homozygotes exhibit catastrophic phenotypes including morphological arrest soon after gastrulation, extensive TUNEL-positive nuclei and hyperactivation of caspases. Each of these defects is profoundly dependent upon dark function since each is dramatically reversed by the dark mutation. In darkCD4; Diap15 double mutant embryos, no evidence is found for widespread apoptosis and, in fact, the large majority of these double mutants proceed through stages of embryogenesis well beyond the point at which single Diap1 mutants arrest. Thus, loss of dark not only suppresses apoptotic deaths that would otherwise occur, but definitively reverses a profound morphogenetic arrest that ensues in the absence of Diap1. Consequently, homozygosity at dark not only prevents the onset of apoptotic markers (i.e. DNA fragmentation and/or caspase activation), but actually preserves cells that are otherwise fated to die when normal checks upon caspases are removed. Rescue from inappropriate apoptosis in this instance is consistent with what was observed in the midline glia but, rather than preserving cells fated for programmed death, rescue occurs even when signaling from IAP antagonists in the Reaper region is bypassed. A similar result, with similar implications, was obtained in the ovary, where ~90% of heteroallelic Diap16/8 mutants show abnormal degeneration of early staged egg chambers and associated sterility. Again, loss of dark function not only suppresses the pathological effect, but actually reverses these degenerative defects to the extent that half of the darkCD4; Diap16/8 double mutant females produce normal egg chambers and are fertile. Thus, in both the ovary and the embryo, loss of dark rescues functional cells from apoptosis caused by misregulated Diap1 (Rodriguez, 2002).
Reversal of Diap1-dependent defects by dark is also evident when caspase activity is directly assayed in early embryos. In contrast to the 'hyperactivated' caspase levels detected in Diap1 single mutant lysates, Diap15; darkCD4 lysates show levels of caspase activity that are suppressed >90% relative to Diap1 mutants. The unusually high caspase activity detected in Diap1 mutant embryos is thought to reflect the action of these proteolytic enzymes unimpeded by native inhibitors. However, studies here demonstrate that removing a negative regulator alone is not sufficient to achieve caspase hyperactivation, since dark is clearly required for this unrestrained activity. It is worth mentioning that the basal DEVDase activity in double mutants is still somewhat elevated compared with control embryos. This might indicate dark-independent caspase activity due to caspase auto-activation in the absence of Diap1 or, since it is possible that the darkCD4 allele used here is not a complete null, it could reflect hypomorphic dark function. Alternatively, since a role for Diap1 in cytokinesis has not been ruled out, it is possible that this residual caspase activity ensues because of secondary developmental defects leading to cell death. Nevertheless, these enzymatic data extend the analyses to the biochemical level in a manner fully consistent with phenotypic studies (Rodriguez, 2002).
Interestingly, since Apaf-1/Dark adaptor proteins act upon procaspases while IAPs preferentially inhibit processed caspases, these results further suggest that pre-existing levels of processed caspases in most cells are probably not high enough to achieve an apoptotic threshold. Instead, a positive cell death stimulus from dark is required for the unusually high levels of caspase activation seen in Diap1-/- embryos. It is also evident that Dark-dependent 'basal' levels of DEVDase are detected in these assays several hours before the onset of embryonic PCD, and therefore authentic effector caspase (e.g. DrICE, Dcp-1) activity occurs even in the absence of overt apoptotic signals. These data indicate that constitutive levels of active effector caspases, not derived from autoproteolysis but instead promoted by Apaf-1-like adaptor proteins, may exist in many and perhaps all viable cells (Rodriguez, 2002).
Taken together, a strict Diap-1-caspase 'liberation' model does not explain sufficiently the evidence described. The action of dark is epistatic to that of Diap1, demonstrating an order of gene action whereby Dark functions either downstream or parallel to Diap1. Therefore, simple derepression of caspases via an IAP inhibitory bridge does not account adequately for epistasis between Diap1 and dark. Put another way, these results support the notion that apoptotic cell death in vivo results from the simultaneous activation of caspases by dark and the derepression of caspases by reaper, grim and/or hid. Accordingly, the findings are inconsistent with models that presume that Diap1 is the sole effector of reaper, grim and hid and that cells are 'pre-loaded' with sufficient levels of IAP-inhibited processed caspases to achieve cell killing. Instead, a 'gas and brake' model is favored whereby positive input from Apaf-1/Dark adaptors, together with removal of IAP inhibition, drives caspase activation to levels that exceed a threshold necessary for apoptosis (Rodriguez, 2002).
Much of what is known about apoptosis in human cells stems from pioneering genetic studies in the nematode C. elegans. However, one important way in which the regulation of mammalian cell death appears to differ from that of its nematode counterpart is in the employment of TNF and TNF receptor superfamilies. No members of these families are present in C. elegans, yet TNF factors play prominent roles in mammalian development and disease. The cloning and characterization of Eiger, a unique TNF homolog in Drosophila, is described. Like a subset of mammalian TNF proteins, Eiger is a potent inducer of apoptosis. Unlike its mammalian counterparts, however, the apoptotic effect of Eiger does not require the activity of the caspase-8 homolog DREDD, but it completely depends on its ability to activate the JNK pathway. Eiger-induced cell death requires the caspase-9 homolog DRONC and the Apaf-1 homolog DARK. These results suggest that primordial members of the TNF superfamily can induce cell death indirectly by triggering JNK signaling, which, in turn, causes activation of the apoptosome. A direct mode of action via the apical FADD/caspase-8 pathway may have been coopted by some TNF signaling systems only at subsequent stages of evolution (Moreno, 2002).
The mechanism by which JNK signaling triggers cell death in response to TNF is poorly understood in mammals and is unknown in Drosophila. It was therefore of interest to identify the apoptotic machinery responsible for Eiger-induced cell death. Having excluded the caspase-8-like FADD/DREDD branch, focus was placed on the involvement of caspase-9, which represents another major pathway that leads to apoptosis. The key event for caspase-9 activation is its association with the protein cofactor Apaf-1 to form an active complex referred to as the apoptosome. Since many cell intrinsic insults can trigger this pathway, it has been termed the 'intrinsic death pathway'. Expression of a dominant-negative form of the Drosophila caspase-9 homolog DRONC, comprising only the CARD domain, fully blocks Eiger-induced apoptosis in a dose-dependent manner. Moreover, genetic removal of DARK, the homolog of Apaf-1, suppresses Eiger-dependent phenotypes. These results indicate that the presumptive Drosophila apoptosome is essential for the ability of Eiger to induce cell death. In agreement with this conclusion, overexpression of Thread, the Drosophila inhibitor of apoptosis protein 1 (DIAP1) blocks Eiger function. Thread/DIAP1 has been shown to bind DRONC and target it for degradation. Most instances of programmed cell death that have been analyzed in Drosophila are triggered by, and require, the genes reaper, hid, or grim, which encode small proteins that bind to and inactivate IAPs, such as Thread/DIAP1. The removal of one copy of a chromsosomal segment that includes the genes hid, grim, and reaper rescues eye ablation, and Eiger induces a strong transcriptional activation of hid and a weak activation of reaper. These results suggest, therefore, that Eiger/JNK signaling triggers DRONC by inactivating the IAPs via a transcriptional upregulation of hid (Moreno, 2002).
Border cell migration in the Drosophila ovary is a relatively simple and genetically tractable model for studying the conversion of epithelial cells to migratory cells. Like many cell migrations, border cell migration is inhibited by a dominant-negative form of the GTPase Rac. To identify new genes that function in Rac-dependent cell motility, a screen was performed for genes that when overexpressed suppressed the migration defect caused by dominant-negative Rac. Overexpression of the Drosophila inhibitor of apoptosis 1 (DIAP1), which is encoded by the thread (th) gene, suppresses the migration defect. Moreover, loss-of-function mutations in th causes migration defects but, surprisingly, did not cause apoptosis. Mutations affecting the Dark protein, an activator of the upstream caspase Dronc, also rescues RacN17 migration defects. These results indicate an apoptosis-independent role for DIAP1-mediated Dronc inhibition in Rac-mediated cell motility (Geisbrecht, 2004).
The observation that two different dark mutant alleles cause mild border cell migration defects suggests that Dronc, which is thought to be constitutively active at a low level in most cells, contributes to normal migration. In fact, caspases have been shown to function in cell proliferation and differentiation in a variety of cell types, in addition to their better known role in promoting apoptosis. In some cases, caspase activity is required for terminal differentiation events that resemble incomplete apoptosis. For example, terminal differentiation of Drosophila sperm requires removal of much of the cytoplasm and requires caspase activity. Similarly, differentiation of mammalian lens cells and erythrocytes requires caspase activity. Other differentiation events, such as those of macrophages and skeletal muscle, do not overtly resemble apoptosis and yet require caspase activity. There must be some mechanism in such cells, and in border cells, to restrict the caspase activity to selected substrates so that apoptosis does not occur (Geisbrecht, 2004).
Cytochrome C has two apparently separable cellular functions: respiration and caspase activation during apoptosis. While a role of the mitochondria and cytochrome C in the assembly of the apoptosome and caspase activation has been established for mammalian cells, the existence of a comparable function for cytochrome C in invertebrates remains controversial. Drosophila possesses two cytochrome c genes, Cytochrome c proximal and Cytochrome c distal. cyt-c-d is required for caspase activation in an apoptosis-like process during spermatid differentiation, whereas cyt-c-p is required for respiration in the soma. However, both cytochrome C proteins can function interchangeably in respiration and caspase activation, and the difference in their genetic requirements can be attributed to differential expression in the soma and testes. Furthermore, orthologues of the apoptosome components, Ark (Apaf-1) and Dronc (caspase-9), are also required for the proper removal of bulk cytoplasm during spermatogenesis. Finally, several mutants that block caspase activation during spermatogenesis were isolated in a genetic screen, including mutants with defects in spermatid mitochondrial organization. These observations establish a role for the mitochondria in caspase activation during spermatogenesis (Arama, 2006).
In order to identify genes required for caspase activation during spermatid differentiation in Drosophila, attempts were made to identify mutants that lacked activated caspase-3 staining, as detected using CM1 antibody, which detects the active form of the effector casepase drICE. For this purpose, an existing collection was screened of more than 1000 male-sterile mutant lines defective in spermatid individualization that were previously identified among a collection of about 6000 viable mutants. Dissected testes from each line were stained with CM1: 33 lines were identified that were CM1-negative. However, the vast majority of male-sterile lines remained CM1-positive, even though many displayed severe defects in spermatid individualization. Therefore, caspase activation at the onset of spermatid individualization appears to be independent of other aspects of sperm differentiation, such as the assembly of the individualization complex or its movement. One of the mutants, line Z2-1091, failed to complement the sterility of bln1, a P-element insertion in cyt-c-d, and was CM1-negative as a homozygote, in trans to a small deletion removing the cyt-c-d locus [Df(2L)Exel6039], or in trans to the cyt-c-dbln1 allele. In contrast, Z2-1091 complemented the lethality of K13905, a P-element insertion in cyt-c-p, and K13905 complemented the sterility of Z2-1091. Genomic sequence analyses of the transcription units of both cyt-c-d and cyt-c-p in Z2-1091 flies revealed a point mutation of TGG to TGA at codon 62 in cyt-c-d, causing a change of Trp62 into a stop codon that results in a truncation of almost half of the protein. Henceforth this allele will be referred to as cyt-c-dZ2-1091. Given the molecular nature of cyt-c-dZ2-1091, it is very unlikely that this allele affects the function of genes adjacent to cyt-c-d (Arama, 2006).
Effector caspases, such as drICE, can display DEVD cleaving activity. Therefore, it was asked whether wild-type adult testes also contain DEVDase activity, and whether this activity is affected in cyt-c-d mutant testes. Lysates of wild-type testes indeed display detectable levels of DEVDase activity, which were significantly reduced upon treatment with the potent DEVDase inhibitor Z-VAD.fmk. Importantly, this activity was highly reduced in cyt-c-dZ2-1091 mutant testes. These results provide independent evidence for effector caspase activity in wild-type sperm, and they support a role of cytochrome C-d in caspase activation in this system (Arama, 2006).
In mammals, mitochondria are important for the regulation of apoptosis, and it has been shown that they can release several proapoptotic proteins into the cytosol in response to apoptotic stimuli. The best-studied case is the release of cytochrome C, an essential component of the respiratory chain. Cytosolic cytochrome C can bind to and activate Apaf-1, which in turn leads to the activation of caspase-9. However, no comparable role of mitochondrial factors for caspase activation has yet been established in invertebrates. The elimination of cytoplasm during terminal differentiation of spermatids in Drosophila involves an apoptosis-like process that requires caspase activity; a P-element insertion (bln1) in one of the two Drosophila cytochrome c genes, cyt-c-d, has been shown to be associated with male-sterility and loss of effector caspase activation during spermatid individualization. This study demonstrates that the defects in caspase activation and spermatid individualization of bln1 mutant males can be rescued by transgenic expression of the ORF of cyt-c-d. Furthermore, from screening more than a thousand male-sterile lines with defects in sperm individualization for defects in active-caspase (CM1) staining, a nonsense point mutation was identified in cyt-c-d, that recapitulates all the phenotypes observed for bln1. Taken together, these results unequivocally demonstrate that cyt-c-d is necessary for effector caspase activation and sperm terminal differentiation in Drosophila (Arama, 2006).
Two decades ago, the mouse cytochrome c gene was used as a probe for screening a Drosophila genomic library and a fragment was isolated that carried two distinct cytochrome c genes. Northern blot analyses indicated high levels of cyt-c-p expression, while cyt-c-d was reported to be expressed at much lower levels in all stages of development. However, neither the exon/intron organization nor the boundaries of the 5' and 3' UTRs of these genes were determined at the time. As a result, the original Northern analyses were performed with a probe corresponding to the untranscribed genomic region between the two cytochrome c genes that was not suitable to properly assess the size and distribution of cytochrome c transcripts. Unfortunately, this has caused considerable confusion in the field from the start, as even the original report noted that the size of the observed cyt-c-d transcript differed more than two-fold from the predicted size. More recently, relying on the incorrect assumption that cyt-c-d is ubiquitously expressed in the fly, it has been suggested that a loss-of-function mutation in cyt-c-d should lead to severe developmental defects and lethality rather than merely male sterility. However, using a specific cyt-c-d 3' UTR probe reveals a transcript of the predicted size that is absent in cyt-c-dbln1 mutants. Furthermore, the RT–PCR and immunofluorescence analyses presented in this study indicate that cyt-c-d is mainly expressed in the male germ line and is completely absent during embryonic and larval development, while cyt-c-p is expressed in the soma during all stages of development. In light of these findings, it is not surprising that loss-of-function mutations in cyt-c-d cause male sterility, whereas cyt-c-p mutations lead to embryonic lethality. RT–PCR results suggest that cyt-c-p is also expressed in the testis, although to a much lower extent than cyt-c-d. This expression is attributed primarily to the somatic cells of the testis, since no cytochrome C protein is detected in cyt-c-dbln1 elongating spermatids, while cyt-c-p RNA is expressed in cyt-c-dbln1 mutant flies. However, the very low cyt-c-d expression detected in the soma of adult females leaves room for the possibility that cyt-c-d might function in caspase activation in some somatic cells as well (Arama, 2006).
In mammalian cells, release of cytochrome C into the cytosol in response to proapoptotic stimuli can be readily demonstrated. However, previous attempts to detect a similar phenomenon in Drosophila have been unsuccessful. In contrast, apoptotic stimuli can lead to increased cytochrome C immuno-reactivity. A possible limitation is that all these studies were conducted using mammalian antibodies with questionable specificity and sensitivity, and only in a small number of cell types and paradigms. Using an antibody that was raised against Drosophila cytochrome C-d, an increase in a 'grainy signal' was detected upon the onset of individualization, with the highest staining observed in the vicinity of the individualization comple (IC). Since it is highly unlikely that additional cytochrome C-d is being transcribed and imported to the mitochondria at this late stage, the explanation is favored that a conformational change or an exposure of a hidden epitope causes the increase in the intensity of the signal. The activation of Dronc, the Drosophila caspase-9 orthologue, also occurs in association with the IC and depends on the presence of the Drosophila Apaf-1 orthologue, Ark. Moreover, the proapoptotic Hid protein is localized in a similar fashion. What are these structures then, which accumulate apoptotic factors in the vicinity of the IC? One plausible suggestion from the literature is that these structures correspond to 'mitochondrial whorls', which result from the extrusion of material from the minor mitochondrial derivative and constitute the leading component of the IC. These 'whorls' can be labeled using a testes-specific mitochondrial-expressed GFP line. Using this GFP marker, it was found that cytochrome C-d is indeed closely associated with mitochondrial whorls. Therefore, it is possible that an active apoptosome forms in the vicinity of the IC in response to dramatic changes in the mitochondrial architecture that occur at this stage of spermatid differentiation. Similarly, studying the response of Drosophila flight muscle cells to oxygen stress, have recently reported that the cristae within individual mitochondria become locally rearranged in a pattern that they termed a 'swirl'. This process was associated with widespread apoptotic cell death in the flight muscle, which was correlated with a conformational change of cytochrome C manifested by the display of an otherwise hidden epitope. Collectively, these observations suggest that apoptosome-like complexes composed of cytochrome C-d, Ark, and Dronc might be associated with unique mitochondrial swirl-like structures. Consistent with this idea, it was found that the long isoform of Ark that contains the WD40 repeats, the target for cytochrome C binding to mammalian Apaf-1, is the major form detectably expressed in testes (Arama, 2006).
The fact that cytochrome C-d immunoreactivity increases in the vicinity of the IC suggests that the extensive mitochondrial organizations preceding individualization may be partially required for caspase activation. Consistent with this idea, several mutants, such as plnZ2-0516, which display defects in Nebenkern differentiation and caspase activation. However, not all mitochondrial differentiation events are required for caspase activation. For example, CM1 staining is seen in fuzzy onions, a mutant defective in the mitochondrial fusion event that generates the Nebenkern. In contrast, analysis of the pln mutant indicates that proper elongation of the Nebenkern is essential for caspase activation. Therefore, characterization of other mitochondrial mutants may shed light on the connection between mitochondrial organization and caspase activation during sperm differentiation (Arama, 2006).
What are the mechanisms by which cytochrome C-d activates caspases during late spermatogenesis? In vertebrate cells, following its release into the cytosol, cytochrome C binds to the WD40 domain of the adaptor molecule Apaf-1, which in turn multimerizes and recruits the initiator caspase, caspase-9 via interaction of their CARD domains. This complex, known as the apoptosome, further cleaves and activates effector caspases like caspase-3. Although this model has become the prevailing dogma in the field, the phenotype of mice mutant for a Cyt c with drastically reduced apoptogenic function ('KA allele') suggests that the mechanisms for caspase activation may be more complex than what was previously thought. In particular, this study suggests that cytochrome C-independent mechanisms for the activation of Apaf-1 and caspase-9 exist, as well as cytochrome C-dependent but Apaf-1-independent mechanisms for apoptosis. These analyses of ark (Apaf-1) and dronc (caspase-9) loss-of-function mutants demonstrate that both genes are required for spermatid individualization, and that their phenotypes, in particular their failure to properly remove the spermatid cytoplasm into the WB, resemble cyt-c-d mutant spermatids and expression of the caspase inhibitor p35 in the testes. However, some caspase-3-like activity could still be detected in these mutant testes. This may suggest that either the ark and dronc alleles are not null, or that cytochrome C-d also functions in an apoptosome-independent pathway to promote caspase-3 activation. Therefore, the regulation of caspase activation and apoptosis may be more similar between insects and mammals than has been previously appreciated. Further genetic analysis of this pathway in Drosophila may provide general insights into diverse mechanisms of apoptosis activation (Arama, 2006).
Previous observations raised the possibility that the two distinct cytochrome c genes may have evolved to serve distinct functions in respiration and caspase regulation. In order to address this hypothesis, it was asked whether expression of one protein might rescue mutations in the other cytochrome c gene. Surprisingly, it was found that transgenic expression of the cyt-c-p ORF in germ cells rescues caspase activation, spermatid individualization, and sterility of cyt-c-d-/- flies. Therefore, the ability to activate caspases is not restricted to the cytochrome C-d protein, and it is possible that cytochrome C-p functions in apoptosis in at least some somatic cells (Arama, 2006).
Although cyt-c-d is almost exclusively expressed in the male germ cells, ectopic expression of this protein in the soma can rescue the respiration defect and lethality of cyt-c-p-/- mutant flies, demonstrating that cytochrome C-d can function in energy metabolism. This raises the question whether the lack of caspase activation could be due to reduced ATP-levels. Although this is a formal possibility, this explanation is considered very unlikely since mutant spermatids complete many other energy-intensive cellular processes. These include the extensive transformation from round spermatids to 1.8 mm long elongated spermatids, a process that involves extensive remodeling and movement of actin filaments, generation of the axonemal tail, mitochondrial reorganization, plasma/axonemal membranes reorganization, and nuclear condensation and elongation. Since all of these processes can occur in the absence of cytochrome C-d, there is no overt shortage of ATP in cyt-c-d mutants. It is therefore considered very unlikely that ATP has become limiting in these mutant cells. Since earlier stage spermatids express cytochrome C-p, sufficient ATP seems to persist to late developmental stages. In mammalian cells, cellular ATP concentration is sufficiently high (around 2 mM) to keep cultured cell alive for several days upon ATP synthase inhibition. Furthermore, cells in which cytochrome c expression is decreased by RNAi still undergo apoptosis in response to various stimuli. Likewise, it appears that cytochrome C is not essential for the function of mature murine sperm, since mice deficient for the testis specific form of cytochrome C, Cyt cT, are fertile. Taken together, all these observations argue strongly against the possibility that ATP levels in cyt-c-d-/- mutant spermatids would be insufficient for caspase activation (Arama, 2006).
In conclusion, the results presented in this study definitively demonstrate that cytochrome C-d is essential for caspase activation and spermatid individualization. Both cytochrome C proteins of Drosophila are, at least to some extent, functionally interchangeable. The results also indicate that cytochrome C can promote caspase activation in the absence of a functional apoptosome. Given the powerful genetic techniques available, late spermatogenesis of Drosophila promises to be a powerful system to identify novel pathways for mitochondrial regulation of caspase activation (Arama, 2006).
The Apaf-1 protein is essential for cytochrome c-mediated caspase-9 activation in the intrinsic mammalian pathway of apoptosis. Although Apaf-1 is the only known mammalian homologue of the Caenorhabditis elegans CED-4 protein, the deficiency of apaf-1 in cells or in mice results in a limited cell survival phenotype, suggesting that alternative mechanisms of caspase activation and apoptosis exist in mammals. In Drosophila melanogaster, the only Apaf-1/CED-4 homologue, ARK, is required for the activation of the caspase-9/CED-3-like caspase DRONC. Using specific mutants that are deficient for ark function, it has been demonstrated that ARK is essential for most programmed cell death (PCD) during Drosophila development, as well as for radiation-induced apoptosis. ark mutant embryos have extra cells, and tissues such as brain lobes and wing discs are enlarged. These tissues from ark mutant larvae lack detectable PCD. During metamorphosis, larval salivary gland removal is severely delayed in ark mutants. However, PCD occurs normally in the larval midgut, suggesting that ARK-independent cell death pathways also exist in Drosophila (Mills, 2006).
ark alleles were obtained in a screen conducted using mitotic recombination for mutations that appear in an increased relative representation of mutant over wild-type (WT) tissue. In these mutants, the mutant clones were larger than the corresponding WT twin spots. The screen of the right arm of chromosome 2 identified mutations in the hippo locus. Four alleles of ark were also obtained from the same screen; these alleles were all lethal at the pupal stage of development as homozygotes or in trans to each other. Sequencing revealed point mutations or deletions in the coding sequence of the ark gene in each of the mutant chromosomes. ark1 had a G to A mutation, resulting in the truncation of the protein after residue 206; ark2 had a C to T mutation, causing protein truncation after residue 660, and ark3 had a deletion after residue 592, generating a frameshift mutation, whereas ark4 possessed a T to G mutation, causing protein truncation after residue 1,357. The mutation in ark1 is predicted to affect both of the reported alternately spliced transcripts of the ark gene. Because all ark mutants were lethal at a similar stage, only ark1 and ark2 were analyzed in these studies (Mills, 2006).
Similar to Apaf-1, ARK consists of a CARD, a nucleotide-binding NB-ARC domain, and multiple WD40 repeats. ark1 mutation truncates the protein in the NB-ARC (CED-4 domain), whereas ark2 leads to a protein lacking most of the WD40 repeats. Both mutants are lethal and have very similar phenotypes, suggesting that they are strong loss-of-function alleles. The phenotypes also indicate that both the NB-ARC and the WD40 domains are essential for ARK function. Unlike the published hypomorphs, all homozygous ark1 and ark2 animals die as pupae. Despite the similar overall phenotypes for ark1 and ark2 alleles, development of ark1 mutants to pupation was significantly delayed when compared with WT or ark2 alleles, suggesting that ark1 may be a stronger allele than ark2. Consequently, the survival of ark1-null animals to early pupae stage was lower than that of the heterozygotes. Although larvae and pupae from both ark mutants appear grossly normal externally, some larval tissues derived from late third instar animals show hyperplasia. For example the larval central nervous system (CNS) was enlarged in both ark mutants. This was particularly evident in the ventral ganglion that appeared to be elongated and contained longer nerve fibers. In ~40% of ark1 and most of the ark2 animals, the wing discs were enlarged. In a small number of both mutants, the eye discs were also enlarged (Mills, 2006).
dronc mutant embryos contain extra cells, and the removal of maternal dronc abolishes most cell death during embryogenesis. dronc-deficient embryos also show an enlargement of the CNS, which is presumably caused by reduced PCD. By staining embryos with anti-embryonic lethal abnormal visual protein (ELAV) antibody to visualize neurons in the CNS and peripheral nervous system, extra neurons were found in chordotonal cell clusters in ark mutant embryos. There were up to three extra cells per cluster in most ark mutant embryos analyzed. Staining of embryos with BP102 antibody, which recognizes CNS axons, showed gross abnormalities in many mutant animals, with ark2 animals often showing more dramatic features. Stronger staining of CNS axons was consistently observed in ark mutant embryos compared with WT animals, which could result from more densely packed axons. In many mutant animals, the ventral nerve cord appeared to be improperly compacted and the spacing between longitudinal axonal tracts was enlarged. This could be attributable to additional cells in the mutants caused by reduced PCD (Mills, 2006).
Since ark mutants essentially phenocopy the loss-of-dronc function, the data argue that these proteins act in a common pathway. Previous experiments using RNA interference have shown that ARK is required for DRONC activation. These results suggest that the primary function of ARK is to facilitate DRONC activation. The observation that metamorphic midgut cell death occurs normally, whereas salivary gland PCD is significantly delayed, suggests that the midgut may provide a model system for studying novel caspase activation and cell death pathways that are independent of the evolutionarily conserved canonical pathway (Mills, 2006).
In Drosophila, the APAF-1 homolog ARK is required for the activation of the initiator caspase DRONC, which in turn cleaves the effector caspases DRICE and DCP-1. While the function of ARK is important in stress-induced apoptosis in Drosophila S2 cells, since its removal completely suppresses cell death, the decision to undergo apoptosis appears to be regulated at the level of caspase activation, which is controlled by the IAP proteins, particularly DIAP1. This study further dissects the apoptotic pathways induced in Drosophila S2 cells in response to stressors and in response to knock-down of DIAP1. The induction of apoptosis is dependent in each case on expression of ARK and DRONC and surviving cells continue to proliferate. A difference was noted in the effects of silencing the executioner caspases DCP-1 and DRICE; knock-down of either or both of these have dramatic effects to sustain cell survival following depletion of DIAP1, but have only minor effects following cellular stress. These results suggest that the executioner caspases are essential for death following DIAP1 knock-down, indicating that the initiator caspase DRONC may lack executioner functions. The apparent absence of mitochondrial outer membrane permeabilization (MOMP) in Drosophila apoptosis may permit the cell to thrive when caspase activation is disrupted (Kiessling, 2006).
This study further dissected the apoptotic pathways induced in Drosophila S2 cells in response to stressors and in response to knock-down of DIAP1. The induction of apoptosis is dependent on expression of the APAF-1 homolog ARK, and the initiator caspase, DRONC. Knock-down of either ARK or DRONC led not only to short term cell survival, as is also observed in mammalian cells lacking APAF-1 or caspase-9, but also to long term survival, seen as cellular accumulation as the cells continued to proliferate. This is in striking contrast to observations in mammalian cells lacking APAF-1 or caspase-9, where cells ultimately succumb to “caspase-independent cell death” and do not proliferate. This difference is most easily explained by the difference in mitochondrial involvement: in mammals, MOMP is associated with the release of potentially toxic factors, such as AIF, endoG, Omi, and others, and with an eventual loss of mitochondrial function, any of which can contribute to death, even when downstream caspase activation is blocked or defective. The apparent absence of MOMP in Drosophila apoptosis may permit the cell to thrive when caspase activation is disrupted (Kiessling, 2006).
Studies in ARK mutants clearly demonstrate that ARK is required for cell death in vivo, since these mutants display developmental defects, including an enlarged nervous system, and resist death induced by transgenic expression of Grim. Furthermore, genetic studies revealed an epistatic relationship between ARK and DIAP1 by demonstrating that loss of ARK reverses catastrophic defects seen in DIAP1 mutants and rescues developing tissues that would otherwise die from DAIP1 inactivation. The function of ARK is required for hyperactivation of caspases which occurs in the absence of DIAP-1. One might argue that the current findings are therefore merely confirmatory. However, it should be noted that profound developmental defects are observed in mice lacking APAF-1, caspase-9, or caspase-3, which are nevertheless dispensable for stress or oncogene-induced cell death in MEFs and lymphocytes from these mice in vitro and cells of the interdigital web in vitro or in vivo. In fact, there is currently no evidence that a cell capable of proliferation can do so following MOMP, and alternative explanations of developmental defects in these knockout mice (other than survival and proliferation following MOMP) have been offered (Kiessling, 2006).
A rapid loss of the DIAP1 is observed in the S2 cells, when treated with various stressors. The full length DIAP1 protein disappears rapidly and a smaller, 27 kDa fragment accumulates over time. Interestingly, the broad spectrum caspase inhibitor zVAD-fmk does not suppress the degradation of DIAP1, but the 27 kDa cleavage product could not be detected when caspase activation was inhibited. The differences between DIAP1 degradation with or without caspase activity could be explained by the notion that the degradation of DIAP1 after treatment with apoptosis-inducing stimuli is mediated by a combination of cleavage by caspases and proteasomal degradation. Thus, the continued degradation of DIAP1 in the presence of activated caspases produces the 27 kDa fragment. It has been recently reported that caspase-dependent cleavage of DIAP1 is required for DIAP1 loss in an early stage of apoptosis and that cleavage of DIAP1 is required for degradation. Similarly, it was observed that if caspases are inhibited following apoptosis induction, DIAP1 levels remain unaltered for a number of hours, however, the inhibition of caspases does not block DIAP1 degradation at longer times (Kiessling, 2006).
While a requirement for ARK and DRONC was observed under all of the pro-apoptotic conditions in S2 cells, a difference was noted in the effects of knock-down of the executioner caspases DCP-1 and DRICE. Knock-down of either or both of these has dramatic effects to sustain cell survival following knock-down of DIAP1, but has only minor effects following cellular stress (Kiessling, 2006).
Several possible explanations were envisioned for this difference. One possibility is that knock-down of DIAP1 leads to caspase activation uniquely through permitting DRONC function to activate DCP-1 and DRICE, while stressors somehow engage other caspase activation pathways (and other caspases). This, however, is inconsistent with the observation that stress-induced apoptosis is clearly dependent on ARK and DRONC. Alternatively, it may be that stress-induced death also involves inhibition of other IAPs, such as DIAP-2 and dBRUCE, which may have a wider spectrum of effects to engage additional caspases not affected by DIAP1 alone. Previously, however, it was noted that knock-down of DIAP-2 does not trigger apoptosis, but greatly enhances susceptibility to death induced by stressors such as were used in this study. This argues that DIAP-2 function, at least, continues following such stress (such that its knock-down has an effect) and thus it is less likely to be an important explanation for the current effects (Kiessling, 2006).
The final possibility is perhaps the most interesting. The knock-down of DIAP1 leads to death, presumably through permitting low levels of ongoing (and otherwise repressed) caspase activation to function and any subsequent effect may depend on amplification, as active executioner caspases cleave and activate others. Therefore, knock-down of even one caspase in the cell may dampen this amplification so that cells survive. In contrast, the induction of apoptosis by stress may involve not only blockade of DIAP1 function (through the N-termini of Reaper, Hid, Grim and Sickle) but also another signal that amplifies caspase activation upstream of ARK. Such an upstream effect has been suggested by studies of the so-called 'GH3' region in these proteins, that appears to be required for death in Drosophila cells and can function to promote the mitochondrial pathway in vertebrate systems. Reaper, Hid, Grim, and probably Sickle are necessary for stress-induced apoptosis in Drosophila, and therefore their effects are likely to depend on ARK and ARK-DRONC interactions. Nevertheless, this line of reasoning suggests that they function not only to de-repress caspases (though blocking DIAP1), but also to do something else to bypass full dependence on DCP-1 and DRICE, perhaps by amplifying caspase activation at the level of the ARK-DRONC interaction. While speculative, this possibility is intriguing, and suggests that the induction of apoptosis in Drosophila may prove to be more complex than the simple models indicate (Kiessling, 2006).
Post-eclosion elimination of the Drosophila wing epithelium was examined in vivo where collective 'suicide waves' promote sudden, coordinated death of epithelial sheets without a final engulfment step. Like apoptosis in earlier developmental stages, this unique communal form of cell death is controlled through the apoptosome proteins, Dronc and Dark, together with the IAP antagonists, Reaper, Grim, and Hid. Genetic lesions in these pathways caused intervein epithelial cells to persist, prompting a characteristic late-onset blemishing phenotype throughout the wing blade. This phenotype wase leveraged in mosaic animals to discover relevant genes. homeodomain interacting protein kinase (HIPK) was shown to be required for collective death of the wing epithelium. Extra cells also persisted in other tissues, establishing a more generalized requirement for HIPK in the regulation of cell death and cell numbers (Link, 2007).
Elimination of cells by programmed cell death (PCD) is a universal feature of development and aging. In both vertebrates and invertebrates, dying cells often progress through a stereotyped set of transformations referred to as apoptosis. In this form of PCD the nucleus condenses, and the collapsing cell corpse fragments into 'apoptotic bodies' that are engulfed by specialized phagocytes or neighboring cells. Apoptosis requires autonomous genetic functions within the dying cell, and extrinsic cues that elicit apoptosis have been investigated in numerous experimental models. Other forms of death are also thought to contribute during development and differ from apoptosis with respect to cellular morphology, mechanism, or mode of activation. These may include necrosis, characterized by swelling of the plasma membrane, or autophagic cell death, which is linked to extensive vacuolization in the cytoplasm. These forms of cell death can be caspase dependent or independent and may or may not be under deliberate genetic control (Link, 2007).
Two conserved protein families comprise central elements of the apoptotic machinery. Orthologous proteins represented by Ced4 in the nematode, Apaf1 in mammals, and Drosophila Ark (Dark) function as activating adaptors for CARD-containing apical caspases. During apoptosis, Ced4/Apaf1/Dark adaptors associate with pro-caspase partners (Ced3, Caspase 9, and Dronc) in a multimeric complex referred to as the 'apoptosome'. This complex is regulated by Bcl2 proteins, but apparently through a diverse group of mechanisms (Link, 2007).
Components of the Drosophila apoptosome have been genetically examined. dark and dronc are recessive, lethal genes. Both exert global functions during PCD and in stress-induced apoptosis. However, their roles in apoptosis are not absolute because rare cell deaths occurred in embryos lacking maternal and zygotic product of either gene. Elimination of dronc in the wing causes a unique, age-dependent phenotype associated with late-onset blemishing throughout the wing blade (Chew, 2004). This study shows that this progressive phenotype is characteristic for wing epithelia that lack apoptogenic functions and is caused by defects in a communal form of PCD where epithelial cells are collectively and rapidly eliminated. These findings were leveraged to discover additional genes required for PCD, and a limited set of loci, many of which were previously unknown to function in cell death, were recovered. This study establish that homeodomain interacting protein kinase (HIPK) is essential for coordinated death in the wing epithelium and, consistent with PCD functions in earlier developmental stages, regulates proper cell number in diverse tissue types (Link, 2007).
Wings mosaic for dronc- tissue exhibit normal morphology at eclosion but develop progressive, melanized blemishes with age (Chew, 2004). Similar methods were applied to determine whether lesions in other apoptogenic genes present a similar phenotype. After eclosion, wings mosaic for Df(H99), a deletion removing the apoptotic activators reaper (rpr), grim, and hid, were morphologically normal at eclosion, but over 3-7 d, melanized blemishes appeared at random throughout the wing (Link, 2007).
Likewise, homozygous driceDelta1 adult 'escapers' deficient for the effector caspase Drice also presented normal wings at eclosion but developed blemishes with age. Wings mosaic for dark82, a null allele of dark, were indistinguishable from wild-type (WT) at eclosion, but within 4 d developed wing blemishes. These late-onset blemishes became markedly more severe as animals aged. Similar yet less severe wing blemishes occurred in adults homozygous for darkCD4, a hypomorphic allele of dark. Together, these observations establish that late-onset progressive blemishing in mosaic wings is a characteristic phenotype shared among mutants in canonical PCD pathways (Link, 2007).
In the wing of newly eclosed adults, PCD removes the epithelium that forms the dorsal and ventral cuticles. To determine whether the cause of the blemish phenotype might trace to defective death in the wing epithelium, this tissue was examined in dark mutants. For these studies, wings of darkCD4 adults were prepared for light and electron microscopy. Histological analyses at the light level showed that on the first day of eclosion, the dorsal and ventral cuticles of WT animals became tightly merged with no intervening tissue evident between these layers. However, even 14 d after eclosion, cells and cell remnants remained situated between the dorsal and ventral cuticles in dark mutants. This 'undead' tissue was most easily visualized in lateral sections through melanized blemishes. Further examination of the persisting epithelium at the EM level showed evidence of intact cells soon after eclosion and ectopic cellular material 24 h after eclosion (Link, 2007).
To directly examine the death of wing epithelial cells in vivo, a transgenic nuclear DsRed reporter was used that driven by vestigial-Gal4 (vg:DsRed), allowing visualization of the fate of these cells soon after eclosion. Observations with this pan-epithelial marker in the wing confirmed earlier studies. Within 1 h of eclosion, intact epithelial cells are clearly present and regularly patterned throughout the wing. 1-2 h later (2-3 h after eclosion), the entire intervein epithelium disappears, manifested here by the abrupt loss of DsRed throughout the wing blade. Live, real-time imaging of the wing in newly eclosed adults revealed unexpected features associated with elimination of the intervein epithelium. Epithelial cells, labeled by nuclear fluorescence, were arranged in a regular, predictable pattern throughout the wing. Then, consistent with nuclear breakdown, fluorescence became redistributed throughout the cell followed by indications of blebbing and the appearance of fragmenting cells. Occasionally, weak fluorescence enclosed in cell corpses condensed to bright punctate bodies. This series of apoptogenic changes spread extremely rapidly throughout the epithelium, appearing here as a collective wave initiating from the peripheral edge and moving across the wing blade (Link, 2007).
Within just 4 min, virtually all nuclei (~450 cells) within a space of ~114 mm2 converted from viable to apoptotic morphology. The process involved tight coordination at the group level because the likelihood of a single cell apoptosing was clearly linked to similar behaviors by nearest neighboring cells over short time frames. Also, the direction and size of the cell death wave may not be fixed in every region of the wing, but centrally located cell groups were generally eliminated earlier (Link, 2007).
Unlike conventional examples of PCD in development, no indication was found that overt engulfment of apoptotic corpses occurred at the site of death. Instead, DsRed-labeled cell remnants were passively swept en masse toward the nearest wing vein where, apparently under hydrostatic pressure, cell debris streamed proximally toward the body through the wing or along the wing vein. Together, these observations describe a communal form of PCD that rapidly eliminates the wing epithelium through coordinated group behavior (Link, 2007).
The vg:DsRed reporter was used to track the fate of mosaic wing epithelia where mutant clones were induced. In sharp contrast to WT wings, abnormally persisting cells could be readily detected as patches of DsRed in the nuclei of epithelial cells in mosaic tissues. For example, wings mosaic for dronc- clones retained extensive patches of persisting DsRed-labeled cells. Here, cells and nuclei were readily detected 4 d after eclosion, and even at 11 d post-eclosion, extensive evidence of cell debris was seen (not depicted). Wings mosaic for the H99 deletion gave identical results. Likewise, adults mutated for dark exhibited persisting cells throughout the wing blade. Consistent with this, rare driceDelta1 escapers also showed evidence of persisting cells after eclosion. These observations link failures in PCD to progressive melanized wing blemishes, raising the possibility that other apoptogenic mutants might also produce this phenotype (Link, 2007).
Unlike previously described wing defects, which are congenital and evident at eclosion, the age-dependent phenotype described in in this study is characteristic of mutations in genes that function in canonical PCD pathways. Moreover, when the dosage of dronc was reduced by half in darkCD4 adults or if WT Dmp53 was removed from these same animals, melanized blemishes became far more severe. These genetic interactions are highly specific because wing defects were never observed in Dmp53- homozygotes or in dronc51 heterozygotes. Numerous other mutants showed no such effects in combination with a dark hypomorph. It was reasoned that, if genetically eliminated, additional regulators and effectors in PCD pathways should phenocopy wings mosaic for dark- or dronc- tissue. A collection of preexisting transposon mutants was screened to capture insertions that exhibit normal wings at eclosion but develop melanized blemishes with age. This strategy exploits the FLP/FRT system together with wing-specific drivers to interrogate animals bearing wing genotypes mosaic for clones of P element-derived lethal mutations. Progeny with mosaic wings were examined for late-onset wing blemishes at 1, 7, and 14 d post-eclosion. Over 1,000 lethal insertions were screened, representing 356 2nd chromosome mutations and 707 3rd chromosome mutations (Link, 2007).
The majority of insertions (87%) produced no visible defects as wing mosaics. 13% of insertions tested produced abnormalities, and these were scored for the phenotypic categories. Congenital defects including notched, blistered, or wrinkled wings occurred alone or occasionally as compound phenotypes. The candidate strains that developed wing blemishing were further subdivided based on phenotypic severity. Insertions in class A developed pronounced blemishes within a week of eclosion, whereas those in class B developed relatively light-colored patches between 1 and 2 wk after eclosion. Mutant lines exhibiting class A phenotypes were rare (~2%). All members of this class lacked blemishes at eclosion and displayed progressive blemishing occasionally associated with fragile and sometimes broken wings. A new allele of dark (l(2)SH0173) was recovered in this class, providing reassuring validation for the screening strategy. Some members among these classes exhibited congenital notches or blisters, but congenital blemishes present at eclosion were not found (Link, 2007).
Inverse PCR was applied to map or confirm insertion sites of many class A and B strains. In addition to darkl(3)SH0173, several mutations associated with genes previously implicated in PCD were isolated. For example, l(2)SH2275 contains an insertion 2 kb upstream of mir-14, a microRNA capable of modulating Rpr-induced cell death. Likewise, l(3)S048915 maps to the first intron of DIAP1 and may represent a hypermorphic allele at this locus. l(3)S055409 maps near misshapen, a gene implicated in cell killing triggered by Rpr or Eiger, the fly counterpart of TNF. Several insertions map in or near transcriptional or translational regulators that might alter the expression of cell death genes. For example, grunge (l(3)S146907), an Atrophin-like protein, functions as a transcriptional repressor, while belle (l(3)S097074) belongs to the DEAD-box family of proteins often implicated in translational regulation and RNA processing. A portion of the class A and B hits were also directly examined for defective PCD by applying the vg:DsRed reporter in mosaic wings. Of the 29 strains tested, 14 showed obvious evidence for persisting cells in the wing epithelium (Link, 2007).
Mutants identified that exhibit both blemishing and persisting cells are likely candidates for PCD genes. One strain, l(3)S134313, produced severe late-onset blemishing and a persisting cell phenotype. After mapping this insertion to the first intron of the HIPK, null alleles at this locus were produced (Link, 2007).
Two FRT-containing P element insertions flanking the coding region of HIPK were used to generate a novel deletion. PCR verified recombination between P elements, and 8 deletion strains were recovered. These validated alleles eliminate exons 4-12, removing over 92% of coding sequence in the predicted HIPK open reading frame. Deletions at the HIPK locus were uniformly lethal before the 3rd instar stage. However, zygotic HIPK is not essential to complete embryogenesis because ~70% of HIPK homozygotes hatch to 1st instar larvae. HIPKD1 was recombined on the FRT79 chromosome to generate adult wings mosaic for this allele, and like the original insertion, these animals also developed robust progressive blemishes and a persisting cell phenotype. Both phenotypes were more severe than the original P insertion, suggesting that the l(3)S134313 allele is hypomorphic for HIPK. These findings link loss of HIPK function to the query phenotypes, establishing that the action of HIPK is essential for post-eclosion PCD in the wing epithelium (Link, 2007).
Using general stains (acridine orange) or TUNEL methods, embryonic PCD was not overtly disturbed in HIPK mutants. To investigate the possibility of more subtle or specific phenotypes, the nervous system was examined using antibodies that label specific populations of neurons affected by the H99 deletion. Using anti-Kruppel antibody, it was confirmed that stage 14-15 WT embryos contained 9-12 Kruppel-positive cells in the Bolwig's Organ. However, a portion of animals lacking maternal HIPK contained as many as 15 cells per organ at a penetrance comparable to H99 animals, which are completely cell death defective. Neurons expressing dHb9, a homeodomain protein marking a subset of cells that persist in cell death-defective H99 embryos, was examined (Rogulja-Ortmann, 2007). In germline clones, distinct classes of dHb9 staining patterns emerged. A subset of animals exhibited extreme patterning defects. Other animals displayed a striking increase in dHb9-positive cell numbers when compared with WT embryos of the parental strain. These data establish that HIPK fundamentally regulates cell numbers in the nervous system, and because the same subpopulation of cells are affected by the H99 mutation, they implicate HIPK as a more general regulator of PCD (Link, 2007).
The pupal eye undergoes reorganization involving cell death of interommatidial cells after pupation. To determine if HIPK regulates cell death in the retina, whole eye clones were generated and the anti-Dlg (discs large) antibody was used to outline cell borders in dissected pupal eyes after pupation. Extra interommatidial cells were frequently retained in whole eye HIPK- clones. This phenotype is overtly similar to animals lacking the apical caspase Dronc and consistent with an essential role in retinal PCD (Link, 2007).
Elimination of the wing epithelium in newly eclosed adults is predictable, easily visualized, and experimentally tractable. The major histomorphologic events involve cell death, delamination, and clearance of corpses and cell remnants. Recent studies established that post-eclosion PCD is under hormonal control and involves the cAMP/PKA pathway (Kimura, 2004). While dying cells in the adult wing present apoptotic features (e.g., sensitivity to p35 and TUNEL positive), elimination of the epithelium is distinct from classical apoptosis in several important respects. (1) Unlike most in vivo models, overt engulfment of cell corpses does not occur at the site of death. Instead, dead or dying cells and their remnants are washed into the thoracic cavity via streaming of material along and through wing veins. (2) Extensive vacuolization is seen in ultrastructural analyses, which could indicate elevated autophagic activity. (3) Widespread and near synchronous death that occurs in this context defines an abrupt group behavior. The process affects dramatic change at the tissue level, causing wholesale loss of intervein cells and coordinated elimination of the entire layer of epithelium. Rather than die independently, these cells die communally, as if responding to coordinated signals propagated throughout the entire epithelium, perhaps involving intercellular gap junctions. This group behavior contrasts with canonical in vivo models where a single cell, surrounded by viable neighbors, sporadically initiates apoptosis (Link, 2007).
It has been proposed that an epithelial-to-mesenchymal transition (EMT) accounts for the removal of epithelial cells after eclosion (Kiger, 2007). Although the results do not exclude EMT associated changes in the newly eclosed wing epithelium, compelling lines of evidence establish that post-eclosion loss of the wing epithelium occurs by PCD in situ -- before cells are removed from the wing. First, before elimination, wing epithelial cells label prominently with TUNEL. Second, every mutation in canonical PCD genes so far tested failed to effectively eliminate the wing epithelium, and at least two of these were recovered in the screen described in this paper. Third, elimination of the wing epithelium was reversed by induction of p35, a broad-spectrum caspase inhibitor. Fourth, using time-lapse microscopy, condensing or pycnotic nuclei, followed by the rapid removal of all cell debris in time frames was detected that was not consistent with active migration. Instead, removal of cell remnants occurred by a passive streaming process, involving perhaps hydrostatic flow of the hemolymph (Link, 2007).
This study sampled over one fifth of all lethal genes and nearly 10% of all genes in the fly genome for the progressive blemish phenotype, a reliable indicator of PCD failure in the wing epithelium. Nearly half of the mutants that produced melanized wing blemishing also displayed a cell death-defective phenotype when examined with the vg:DsRed reporter. The precise link between these defects is unclear, but a likely explanation suggests that as the surrounding cuticle fuses, persisting cells, now deprived for nutrients and oxygen, become necrotic and may initiate melanization. Mutants could arrest at upstream steps, involving the specification or execution of PCD, or they might affect proper clearance of cell corpses from the epithelium. New alleles were recovered of dark (l(2)SH0173) and a likely hypermorph of thread (l(3)S048915), which provides reassuring validation of this prediction (Link, 2007).
By leveraging this distinct phenotype, novel cell death genes were captured, including the Drosophila orthologue of HIPK. Though first identified as an NK homeodomain binding partner, this gene is an essential regulator of PCD and cell numbers in diverse tissue contexts. Of the four mammalian HIPK genes, HIPK2, the predicted ortholog of Drosophila HIPK, has been placed in the p53 stress-response apoptotic pathway (D'Orazi, 2002; Hofmann, 2002; Di Stefano, 2004; Di Stefano, 2005), but whether the Drosophila counterpart similarly impacts this network is not yet known (Link, 2007).
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date revised: 15 May 2007
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