RNA blot analysis of dronc detected a 2.2-kb transcript, consistent with the size of cDNA, at all embryonic stages. Between first and third instar larva stages dronc transcript was barely detectable, however, from late third instar larva stage, dronc mRNA is remarkably up-regulated. dronc expression was examined by in situ hybridization to Drosophila embryos and larval tissues by using a digoxigenin-labeled antisense mRNA probe. Dronc is expressed highly in stage 1-4 syncitial embryos, representing maternally derived mRNA. In stage 8 cellularized embyros, dronc mRNA is ubiquitously expressed, but as development proceeds expression levels generally are reduced. Expression of the Drosophila caspase dredd has been shown to be up-regulated in embryonic cells undergoing programmed cell death. However, unlike dredd, dronc expression is not up-regulated in apoptotic cells in embryos (Dorstyn, 1999).
dronc expression was further examined in second and third instar larval tissues. High levels of dronc expression are observed in midgut and salivary glands from late third instar larvae, but not from second instar larvae. Massive apoptosis of midgut tissues occurs at the onset of pupariation although small numbers of apoptotic cells can be detected in the gastric caeca in late second instar larvae, whereas apoptosis of the salivary glands begins 13.5 hr after pupariation. Thus, high expression of dronc precedes apoptosis in these tissues. Low levels of dronc expression are observed throughout third instar larval eye discs and brain lobes, which contain apoptotic cells at this stage. However, up-regulation of dronc expression is not observed in eye disc or brain lobe cells that should be undergoing apoptosis. The cells strongly staining with dronc in the eye disc are blood cells, which often associate with imaginal discs. High expression of dronc in a subset of blood cells is consistent with programmed cell death also occurring in these cells. dronc expression is checked in ovaries. During oogenesis in adult flies, nurse cells undergo apoptosis in stage 12 oocytes, which is required for the deposition of nurse cell cytoplasm into the oocytes. Strong dronc expression was observed in egg chambers after stage 10, but also in earlier stages, indicating that dronc expression precedes apoptosis during oogenesis (Dorstyn, 1999).
Regulated cell death and survival play important roles in neural development. Extracellular signals are presumed to regulate seven apparent caspases to determine the final structure of the nervous system. In the eye, the EGF receptor, Notch, and intact primary pigment and cone cells have been implicated in survival or death signals. An antibody (CM1) raised against a peptide from human caspase 3 was used to investigate how extracellular signals control spatial patterning of cell death. The antibody crossreacts specifically with dying Drosophila cells and labels the activated effector caspase Drice. The initiator caspase Dronc and the proapoptotic gene head involution defective are important for activation in vivo. Dronc may play roles in dying cells in addition to activating downstream effector caspases. Epistasis experiments ordered the EGF receptor, Notch, and primary pigment and cone cells into a single pathway that affects caspase activity in pupal retina through hid and Inhibitor of Apoptosis Proteins. None of these extracellular signals appear to act by initiating caspase activation independently of hid. Taken together, these findings indicate that in eye development spatial regulation of cell death and survival is integrated through a single intracellular pathway (Yu, 2002).
A particularly useful feature of the CM1 antiserum is the detection of cells that would otherwise be marked for death but which are protected by baculovirus p35 expression. The morphological protection provided by p35 may permit better investigation of the location and autonomy of death and survival signals. On Western blots the CM1 antibody detects activated Drice but not the Drice zymogen; Drice is the Drosophila sequence most similar to the immunizing peptide. Definitive evidence that CM1-stained cells are apoptotic comes from the dependence of embryonic CM1-staining on the 75C1,2 chromosome region, and from the p35-sensitivity of larval and pupal CM1-stained cells. The morphology of all CM1-stained cells is altered by p35 expression. In the presence of p35, CM1-stained cells become indistinguishable from normal cells by morphological criteria, and are not distinguishable except by CM1 staining. Since baculovirus p35 blocks cell death by inhibiting caspase activity, p35-dependent morphology of CM1-stained cells shows that such morphology depends on caspase activity in the cells, which are therefore apoptotic. These results show that only apoptotic cells are labelled by the CM1 antiserum. Apoptotic cells that are unlabelled by CM1 might also exist, although none have been noticed (Yu, 2002).
The results indicate that effector caspases such as Drice can be processed in the presence of baculovirus p35. The initiator caspase Dronc is responsible for effector caspase processing in p35 expressing cells. p35-insensitive caspases are also thought to initiate cell death in other insect cells. In Drosophila eye development, Dronc always functions redundantly with other, p35-sensitive initiator caspases. Such redundancy explains why Dronc-DN had no effect in earlier studies of eye development. Dronc has been implicated in embryonic cell death by RNA interference studies (Yu, 2002).
If feedback of effector caspases on their own activation is essential for cell death, it would be expected that Dronc alone is insufficient for CM1 labelling in p35-expressing cells. By contrast, CM1 labelling persists in most cells, indicating that feedback of effector caspases is dispensable for the activation of at least one effector caspase. However, the results do not exclude the possibility that feedback makes a quantitative contribution to the pace of death or is required for a subset of effector caspases. Results are different for neuronal photoreceptor cells. R8 photoreceptor survival is rescued in the absence of EGFR by p35 expression, but such rescued R8 cells are not labelled strongly by CM1. It is possible that amplification of the apoptotic cascade might be more important for apoptosis of R8 precursors than for unspecified cells. It is also possible that R8 cell apoptosis involves different effector caspases or is initiated independently of Dronc (Yu, 2002).
These findings suggest that Dronc may cleave other substrates in dying cells in addition to activating p35-sensitive effector caspases. Mild eye roughening, seen when p35 is expressed in the eye, is found to depend on Dronc activity. It seems unlikely that the rough eye could be due to some downstream effector caspases escaping the p35 inhibition, because DIAP1 overexpression blocks cell death less effectively than p35 but does not cause eye roughening. It is speculated that Dronc might have cellular targets other than downstream caspases and that cleavage of such targets affects eye morphology. However, these data provide no evidence for Dronc activity, except in cells that would normally die. The simplest model is that Dronc might have another role in cell death in addition to activating effector caspases. The data do not support any effect of p35 other than its inhibition of caspases, since the eye roughening caused by p35 is suppressed by co-expression of DIAP1 or Dronc-DN (Yu, 2002).
Mutations of hid reduce cell death in pupal eye development. hid is absolutely required for caspase activation in both eye disc and pupal retina. Cell death is reduced even in hid/+ heterozygotes, consistent with dominant effects of hid in modifier screens. hid is required for caspase processing, which is redundantly mediated by Dronc and other initiator caspases. Therefore, in principle, Hid is a candidate for regulating initiator caspases, however, in embryos, hid functions to sequester DIAPs. In pupal retinas, DIAP overexpression mimics hid mutation, consistent with sequestration of DIAPs by Hid. In the eye imaginal disc, however, proapoptotic Hid function is not overcome by DIAP1 overexpression, since targeted DIAP1 expression neither reduces cell death in normal eye discs nor protects against cell death when EGFR function is removed in egfr mutant clones. It seems unlikely that endogenous Hid levels are too high for DIAP1 to be effective, because hid is haploinsufficient for eye disc cell death. Instead, these findings raise the possibility of a proapoptotic activity of Hid that is not blocked by DIAP1. This could involve inhibiting a pathway parallel to DIAP1 that also inhibits caspase activation, or promoting activation of caspase zymogens in other ways, for which there is precedent in vertebrates (Yu, 2002).
Two other proapoptotic genes, rpr and grim, induce eye cell death on ectopic expression. Whether rpr or grim are required for cell death in normal eye development is uncertain because point mutants are not available. The absolute requirement for hid may indicate that rpr and grim are not active during normal eye development. Since hid has been shown to be required for eye death in response to ectopic rpr, however, it is also possible that rpr and grim have activities that depend on hid function (Yu, 2002).
Experiments using the egfrts1a allele have confirmed that Egfr is required for survival of pupal retinal cells, as suggested by misexpression experiments. Egfr is also required for survival of eye imaginal disc cells. Consistent with the model that Egfr prevents cell death by inactivating hid, hid is absolutely required for caspase activation in egfr mutant clones. Similar results have been obtained using TUNEL experiments to assess Egfr-DN-induced cell death (Yu, 2002).
Survival in pupal retina is regulated by two further extracellular signals that are not involved in eye imaginal discs. In principle, such signals might act to modulate Egfr signaling, to regulate Hid or DIAP activity in parallel to Egfr, or to activate initiator caspases. Notch (N) is required for caspase activation in the pupal retina. Epistasis experiments show that N is not required for pupal cell death in the absence of Egfr function, and therefore that the normal function of N is to inhibit the Egfr survival signaling pathway in pupae. Such results place N upstream of Egfr and indicate that N acts ultimately through hid and the anti-apoptotic DIAP proteins that prevent caspase activation, rather than through N-mediated caspase activation. Survival in pupal retinas also depends on signals from primary pigment cells and/or cone cells. Such signals must antagonize proapoptotic N activity, since N is epistatic to the primary pigment cell/cone cell signal. The data now imply a pathway in which primary pigment cells and/or cone cells promote survival by inhibiting activation of N, thus preventing N antagonism of Egfr activity in the interommatidial cells (Yu, 2002).
The essential role of Egfr now seems to be downstream of N, whereas the cone cell/primary pigment cell signal must act upstream. Downstream Egfr function raises anew the question of identity of the primary pigment cell/cone cell signal. Primary pigment cells or cone cells do not seem essential for Egfr activation, because N is still required for apoptosis after ablation of these cells. Pupal photoreceptor cells express the Egfr ligand SPI and its processing/presenting factor Rhomboid, and are one possible source of Egfr activation. One model suggests that primary pigment cells and/or cone cells are the source of an unidentified signal or mechanism that prevents N activation (in particular interommatidial cells) so that Egfr survival signaling can continue (Yu, 2002).
According to one view, survival signals are the critical extracellular regulators of developmental cell death. By contrast, results from C. elegans and mammals indicate that cell death depends on activation of initiator caspases to trigger the apoptotic cascade. Homologs of the activatory components exist in Drosophila. Studies of eye development place three extracellular signals in a pathway acting through Egfr and hid to regulate survival, in part through IAPs. The only evidence consistent with positive regulation of apoptosis is that in eye imaginal discs, hid appears to promote cell death through an unidentified mechanism independent of DIAPs, and, in this case, the role of EGF receptor signaling is still to promote survival by inhibiting Hid (Yu, 2002).
These findings do not rule out other pathways that activate initiator caspases during eye development, or that such activation might be required for cell death. Since hid is essential for cell death, however, pathways that activate initiator caspases independently of hid cannot be sufficient for any of the cell death that normally occurs during eye development. Because loss of Egfr survival signaling is sufficient for cell death, and Egfr survival signaling is only important to inhibit Hid, these data imply that release of hid is sufficient as well as necessary for normally occurring cell death. The data do not rule out any parallel Egfr-dependent signal to suppress caspase activation independently of hid, but such a pathway cannot be sufficient for cell death in the absence of hid. These findings suggest that positive activators of caspase processing may not be the direct targets of extracellular regulation. However, it will be important to investigate survival and death signals in other organs, including cell deaths that occur independently of reaper, grim and Hid in ovarian nurse cells and during autophagy, the mechanisms of which have yet to be determined (Yu, 2002).
Drosophila IAP1 (DIAP1) 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).
Self-digestion of cytoplasmic components is the hallmark of autophagic programmed cell death. This auto-degradation appears to be distinct from what occurs in apoptotic cells that are engulfed and digested by phagocytes. Although much is known about apoptosis, far less is known about the mechanisms that regulate autophagic cell death. Autophagic cell death is regulated by steroid activation of caspases in Drosophila salivary glands. Salivary glands exhibit some morphological changes that are similar to apoptotic cells, including fragmentation of the cytoplasm, but do not appear to use phagocytes in their degradation. Changes in the levels and localization of filamentous Actin, alpha-Tubulin, alpha-Spectrin and nuclear Lamins precede salivary gland destruction, and coincide with increased levels of active Caspase 3 and a cleaved form of nuclear Lamin. Mutations in the steroid-regulated genes ßFTZ-F1, E93, BR-C and E74A that prevent salivary gland cell death possess altered levels and localization of filamentous Actin, alpha-Tubulin, alpha-Spectrin, nuclear Lamins and active Caspase 3. Inhibition of caspases, by expression of either the caspase inhibitor p35 or a dominant-negative form of the initiator caspase Dronc, is sufficient to inhibit salivary gland cell death, and prevent changes in nuclear Lamins and alpha-Tubulin, but not to prevent the reorganization of filamentous Actin. These studies suggest that aspects of the cytoskeleton may be required for changes in dying salivary glands. Furthermore, caspases are not only used during apoptosis, but also function in the regulation of autophagic cell death (Martin, 2004).
Studies of dying salivary glands indicate that autophagic cell death has
both similarities to and differences from apoptosis. Several markers of
apoptosis, including nuclear staining by Acridine Orange and DNA
fragmentation, appear 2 hours following the rise in steroid hormone that
triggers salivary gland cell death. The cytoplasm and membranes of salivary gland cells bleb and fragment as these cells degrade, and these are characteristics that are common to apoptotic cells. These common morphologies raise the questions of what distinguishes apoptosis from autophagic cell death, and what common and different regulatory mechanisms mediate these forms of cell killing (Martin, 2004).
The mechanisms of cell degradation and removal appear to provide the
clearest distinction between apoptosis and autophagic cell death. In salivary
glands, dynamic changes in vacuole structure immediately precede their demise,
and such changes have not been reported in apoptotic cells. Within one hour of salivary gland DNA degradation, large vacuoles appear to break into smaller vacuoles, and this occurs within 2 hours of complete tissue destruction. As
these large vacuoles fragment, smaller but distinct vacuoles accumulate near
the plasma membrane, and autophagic vacuoles containing components of the
cytoplasm, including mitochondria, are formed. Salivary gland cells then begin
to fragment, and nuclei and components of the cytoplasm then disperse within
the haemocoel.
Although a role for phagocytes in autophagic cell death cannot be excluded,
salivary gland cells proceed to late stages of degradation without the
assistance of phagocytes. It has been suggested that phagocytes may play a
secondary role in the removal of cellular debris toward the end of autophagic
cell death, and the results are consistent with this possible
conclusion. The presence of autophagic vacuoles in degrading salivary glands
further indicates that autophagic cells use their own lysosomes for
degradation, whereas apoptotic cells depend on phagocytes for the bulk
degradation of long-lived cellular proteins (Martin, 2004).
Dying cells exhibit dynamic changes in cell shape.
The changes in cell organization that occur during salivary gland cell death
are likely to be controlled by the modification of structural protein
organization. Indeed, dynamic changes in the abundance and localization of
filamentous Actin, alpha-Tubulin, alpha-Spectrin and nuclear Lamins
precede the death of salivary glands. At least two possible explanations exist for how such changes
in protein expression are regulated in dying cells. One possibility is that
proteases cleave structural proteins, and that this results in the changes in cell shape. Alternatively, changes in cell shape could be regulated by the assembly of cytoskeletal proteins, such as filamentous Actin, through signaling that is mediated by small GTPases (Martin, 2004).
Proteolysis and changes in the assembly of the cytoskeleton both appear to
be involved in the regulation of changes that occur during autophagic cell
death of salivary glands. Although caspases play an important role in the cell
death of salivary glands, several lines of evidence suggest that some changes
in the structure of the cytoskeleton may occur in a caspase-independent
manner. First, whereas changes in filamentous Actin localization occur in
synchrony with changes in proteins such as nuclear Lamins that are cleaved by
caspases, changes in Actin protein levels are delayed by 4 hours. Second, mutations in steroid-signaling genes, such as ßFTZ-F1, that prevent
expression of active caspase-3 and cleavage of nuclear Lamins do not prevent
changes in filamentous Actin localization. Third, although
inhibition of caspases by expression of either p35 or a dominant-negative form
of Dronc is sufficient to prevent changes in nuclear Lamins and
alpha-Tubulin, these inhibitors are not sufficient to block changes in
filamentous Actin. These data are further supported by the observation that numerous small GTPases increase their expression immediately prior to salivary gland cell death. Although previous studies have suggested that changes in the Actin cytoskeleton are required for autophagic cell death, the
failure to distinguish between cytoskeleton proteolysis and rearrangement has
made it difficult to interpret the potential significance of maintenance of
the cytoskeleton during cell death (Martin, 2004).
Several possibilities exist to explain why the cytoskeleton is maintained
during cell death. The cytoskeleton could be used to restrict the subcellular
location and activity of pro-apoptotic regulators of the Bcl-2 family, and
activation of apoptosis. This mechanism seems unlikely during salivary gland cell
death because the Actin cytoskeleton is maintained after caspase-dependent
cleavage of substrates, including nuclear Lamins. Alternatively, the Actin
cytoskeleton could be maintained as a substrate to localize proteins,
membranes and vacuoles within the cell. Intracellular trafficking plays an
important role in autophagy, as evidenced by the observation that membrane-bound cytoplasmic components
(autophagic vacuoles) are transported to the lysosome for degradation. Since
autophagic vacuoles are observed at stages after caspase activation and cleavage of
substrates such as nuclear Lamins, it is possible that the Actin cytoskeleton is maintained to enable transport of vacuoles to lysosomes (Martin, 2004).
Studies of salivary glands indicate that caspases play an important role in
their autophagic cell death. The caspase-encoding genes dronc and
drice show an increase in their transcription following the rise in
steroid that triggers salivary gland autophagic cell death. This
increase in caspase transcription corresponds to the increase in active
caspase protein levels and in the cleavage of substrates such as nuclear
Lamins in dying salivary glands. Mutations in the
steroid-regulated ßFTZ-F1, E93 and BR-C genes, which
prevent salivary gland cell death, exhibit little or no active Caspase-3/Drice
expression, and have altered alpha-Tubulin, alpha-Spectrin and nuclear
Lamin expression in salivary glands. Although E74A
mutants prevent salivary gland cell death, they have elevated Caspase-3/Drice
levels and degraded nuclear Lamins. Although these data are consistent with the partially degraded morphology of E74A mutant salivary glands, it
remains unclear what factor(s) E74A may regulate that are required
for normal cell death. However, the data indicate that ßFTZ-F1,
E93 and BR-C play a crucial role in determining caspase levels
in dying salivary gland cells, and this is supported by the impact of these
genes on the transcription of dronc.
Significantly, inhibition of caspases by expression of either p35 or
dominant-negative Dronc is sufficient to prevent DNA fragmentation, changes in
nuclear Lamins and alpha-Tubulin, and death of salivary glands (Martin, 2004).
The morphologies of dying cells indicate that apoptosis and autophagy are
distinct. However, the difference between these cells becomes less apparent
when one considers characteristics that were previously considered to be
specific to apoptosis. Clearly, markers such as DNA fragmentation, expression
and function of caspases, and cleavage of caspase substrates can exist in
cells that possess the morphology of autophagic cell death. As expression of
dominant-negative Dronc is sufficient to block caspase-dependent changes in
salivary glands, these studies also indicate that the mechanism for caspase
activation during autophagic cell death is similar to apoptosis during
Drosophila development; the initiator caspase Dronc regulates the
activation of the executioner caspase Drice and cleavage of cell substrates. It is
surprising how little is known about the activity of caspases in developing
animals; these proteases have been a subject of substantial investigation.
Recent studies indicate that caspases do not only function during autophagic
and apoptotic cell death, but that they are also used to degrade proteins
during the differentiation of sperm in Drosophila. Studies
of salivary glands indicate that the distinction between apoptosis and
autophagic cell death may be more subtle than their morphology suggests, and
raise the question of what makes these cells look so different. Restriction of
caspase activity within compartments of the dying cell may provide one
possible explanation, but it is also possible that other mechanisms of
proteolysis occur during autophagic cell death. This possibility is supported
by the large increase in transcription of non-caspase proteases just before
cell death of salivary glands, and by the fact that, unlike apoptotic cells that require phagocyte lysosomes, salivary gland cells appear to degrade themselves through autophagy. Future studies should provide important insights into the
similarities and differences in the mechanisms that regulate apoptosis and
autophagic programmed cell death (Martin, 2004).
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 work reported here demonstrates a new function for DIAP1 in promoting cell migration. The strongest evidence for this is that border cells lacking DIAP1 fail to migrate. This finding is surprising since there has been no previous indication that IAP proteins contribute to cell motility. However, in most cells, it would be difficult or impossible to uncover a requirement for DIAP1 in cell migration because loss of the protein typically results in cell death (Geisbrecht, 2004).
The effect of DIAP1 on cell migration appears to be through the small GTPase Rac and its effects on the actin cytoskeleton based on genetic, biochemical, and cell culture experiments. First, overexpression of DIAP1 suppresses RacN17 migration defects specifically and does not rescue border cell migration defects that are due to other causes. Moreover, overexpression of either actin5C or profilin, both of which would be expected to increase the amount of polymerization competent G-actin in the cell, also rescue RacN17 border cell migration defects. The association of DIAP1 protein with Rac and profilin in S2 cells together with the finding that overexpression of DIAP1 can enhance activated Rac's effects on the actin cytoskeleton in cultured cells further support the conclusion that DIAP1 affects cell migration via Rac and the actin cytoskeleton. Additional support is provided by the finding that overexpression of Rac in border cells results in increased accumulation of DIAP1 protein and F-actin in vivo (Geisbrecht, 2004).
The effect of DIAP1 on border cell migration is clearly independent of its role in preventing apoptosis. The lack of apoptosis in th mutant follicle cell clones is striking since at other stages of Drosophila development, cells fail to survive in the absence of DIAP1. However, IAP proteins are thought to play a less critical role in survival of certain mammalian cells as well, where the current view is that the balance between proapoptotic and antiapoptotic BCL-2 family proteins is the deciding factor between life and death. The results presented here suggest that DIAP1 is not required for survival of every cell and tissue of the fly either. It may be that in both flies and mammals, different cell types have distinct requirements for particular classes of survival molecules (Geisbrecht, 2004).
Although the ability of DIAP1 to rescue RacN17 border cell migration defects is independent of its function in preventing cell death, and independent of its inhibition of effector caspases, the effects result from inhibition of the initiator caspase Dronc. The finding that inhibition of Dronc can rescue RacN17 border cell migration defects indicates that Dronc activity has a negative effect on migration. Since Dronc is a protease, the most parsimonious hypothesis would be that Dronc cleaves one or more proteins required for Rac-mediated cell motility. Previous studies have shown that Rac can be cleaved and inactivated by caspase 3 in lymphocytes. Therefore one possibility is that Rac itself is a Dronc substrate in border cells. A number of cytoskeleton-associated proteins are cleaved by caspases, including actin. Since reduced profilin levels was observed in th mutant follicle cells, it is also possible that profilin is a Dronc substrate, though no increased accumulation of actin or profilin was detected in cells overexpressing DIAP1. Further study will be required to pinpoint the physiologically relevant Dronc substrate in border cells (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).
DIAP1 is a member of an evolutionarily conserved family of proteins that contain BIR domains. BIR domain-containing proteins are found in organisms from yeast to man and seem to have arisen in evolution prior to the apoptotic machinery. For example, in yeast, BIR1p is a protein required for proper chromosome segregation and cytokinesis. Yet the yeast genome does not encode an obvious caspase and yeast are not known to undergo apoptosis. In C. elegans, there is a BIR domain protein that does not suppress apoptosis when overexpressed. Reduction in the expression of this protein by RNA interference leads to defective cytokinesis and a phenotype that is very similar to loss of the worm formin protein. This is interesting since formin homology proteins can bind Rac, stimulate actin polymerization in concert with profilin, and promote cell migration. However it is not known if the C. elegans BIR domain protein interacts with formin or profilin, or whether it functions downstream of Rac. Taken together, these observations suggest that a primitive function of BIR domain proteins may have been regulation of cell division and the cytoskeleton (Geisbrecht, 2004).
It is well known that growth factors promote both survival and proliferation, as well as migration of specific cells. For example, Steel factor acting through the c-kit receptor tyrosine kinase regulates survival and proliferation of primordial germ cells and melanocytes in the mouse embryo. In addition, Steel factor and c-kit may contribute to guiding the embryonic migrations of these two cell populations. Conversely, overexpression of a factor that functions in repulsive guidance of Drosophila primordial germ cells causes excessive germ cell death. This intimate relationship between guidance and survival may exist to ensure that only those cells that migrate to the appropriate location survive and proliferate (Geisbrecht, 2004).
Rho family GTPases have also been demonstrated to affect both migration and survival. By activating gene expression through the JNK pathway, Rac1 protects COS 7 cells from apoptosis induced by ultraviolet light. Rac is also required for survival of cerebellar granule neurons. Another pathway required for both cell survival and Rac-mediated cell migration is the phosphatidylinositol 3-kinase pathway (PI3K). Activation of PI3K, and its downstream effector Akt, is capable of promoting neuronal survival in the absence of growth factors. Akt is also essential for Rac-mediated motility in mammalian fibroblasts. Akt is activated by Rac, and phosphorylated Akt colocalizes with Rac at the leading edge of fibroblasts. Therefore, there are several biochemical pathways that control, and possibly coordinate, cell survival and cell motility.
The mammalian formin homology protein FRL functions as a survival signal, in addition to its role in Rac-mediated regulation of the cytoskeleton. Overexpression of a truncated form of FRL, containing only the N-terminal Rac binding site, results in inhibition of cell growth and apoptosis in the macrophage cell line P388D1. These lines of evidence support the view that regulation of the cytoskeleton and cell survival are intertwined. The present study demonstrates that the inhibitor of apoptosis proteins, well known for their role in cell survival, can also promote cell migration, thus demonstrating a new and unexpected molecular link between survival and migration (Geisbrecht, 2004).
The p53 transcription factor directs a transcriptional program that determines whether a cell lives or dies after DNA damage. Animal survival after extensive cellular damage often requires that lost tissue be replaced through compensatory growth or regeneration. In Drosophila, damaged imaginal disc cells can induce the proliferation of neighboring viable cells, but how this is controlled is not clear. This paper provides evidence that Drosophila p53 has a previously unidentified role in coordinating the compensatory growth response to tissue damage.
The sole p53 ortholog in Drosophila, is required for each component of the response to cellular damage, including two separate cell-cycle arrests, changes in patterning gene expression, cell proliferation, and growth. These processes are regulated by p53 in a manner that is independent of DNA-damage sensing but that requires the initiator caspase Dronc. These results indicate that once induced, p53 amplifies and sustains the response through a positive feedback loop with Dronc and the apoptosis-inducing factors Hid and Reaper. How cell death and cell proliferation are coordinated during development and after stress is a fundamental question that is critical for an understanding of growth regulation. These data suggest that p53 may carry out an ancestral function that promotes animal survival through the coordination of responses leading to compensatory growth after tissue damage (Wells, 2006; full text of article).
The repair of tissue after cellular damage can be critical to the survival of the animal. Previous studies demonstrated that undead cells stimulate the proliferation of neighboring cells, providing a model for how damaged and dying cells contribute to the replacement of lost tissue. With this model, it was found that the wing imaginal disc responds to this damage as a whole by deploying a multi-step process that ends with compensatory growth. p53 functions in a dronc-dependent manner at each step of the tissue-replacement process. Furthermore, p53 and the initiator caspase dronc may be generally required for tissue recovery in imaginal discs, because it was found that blastema formation was significantly impaired during regeneration induced in either p53 or dronc mutant leg discs (Wells, 2006).
The data suggest that p53 is induced and becomes functional in undead cells by a mechanism that does not require DNA-damage sensing or activation of the stress kinase AMPK. Rather, Dronc, an initiator caspase homologous to caspase-9, is necessary and sufficient to induce all aspects of the growth regulation by p53. It is not known how Dronc activity results in p53 expression and activity in these cells, but many caspase substrates are not directly involved in apoptosis. As an example, one of the first caspase substrates identified was the cytokine IL-1β, which regulates many aspects of the inflammatory response. Induction of p53 mRNA in undead cells is prevented in dronc mutant discs, and thus it is possible that a regulator of p53 is cleaved by Dronc, leading to its expression and ultimately to its ability to regulate the compensatory growth response in the imaginal discs. Regardless of the molecular mechanism, the data argue for direct communication between Dronc and p53 in response to tissue damage (Wells, 2006).
Collectively, these experiments imply that p53 serves as a master coordinator of tissue repair in imaginal discs, regulating both cell-autonomous and non-cell-autonomous cell-cycle arrests, the expression of the pattern-regulating genes wg and dpp, and compensatory cell proliferation and growth. Based on these results, it is suggested that cellular damage activates Dronc, which in a nonapoptotic role causes the induction of p53 mRNA and leads to p53 activity. It is proposed that p53 then acts as an overall damage monitor, in a role that includes its conserved functions in apoptosis (here, induction of hid and rpr expression) and growth arrest (by repression of stg/cdc25), but also allows for induction of signals that promote compensatory growth of the disc. The results suggest that p53 monitors tissue damage through a feed-forward loop with Dronc and the pro-apoptotic genes hid and rpr, which both amplifies and sustains the growth-regulating signal (Wells, 2006).
An intriguing puzzle left unanswered by these results is why the growth response to undead cells occurs only several days after they are generated: both HhGal4 and EnGal4 drive expression of Hid or Rpr from early embryonic stages, yet even with careful observation no growth phenotype was detected until the middle part of the third instar. Caspases are active in cells expressing Hid or Rpr + P35 at early time points, indicating that these cells are not immune to the apoptotic response early in development. The genes involved in the apoptotic response are subject to many levels of control, including that by micro-RNAs (miRNAs). Hid protein expression, for example, is suppressed by Bantam, a miRNA highly expressed early in imaginal disc development, but declining as development progresses. It is likely that rpr is also regulated by miRNA gene silencing. Hence, the delay of the growth response in discs with undead cells may reflect a requirement for threshold levels of these factors to fully activate the feedback loop. At the very least it emphasizes that the regulation of growth and cell death during wing disc development is complex and has multiple inputs, many of which are poorly understood (Wells, 2006).
Activity thresholds appear to play an important role in the processes induced by undead cells. Dronc, for instance, is haploinsufficient for its effect in compensatory proliferation. It is possible that the apoptotic functions of Dronc require a relatively low activity level, but that high Dronc activity allows activation of the p53-dependent tissue-damage response. Regulation of Dronc by critical activity thresholds could provide the animal some regenerative capacity and increase its chances for survival when conditions are appropriate for tissue repair (Wells, 2006).
As expected given its role in coordinating many cellular behaviors, p53 modulates the activity or expression of myriad effectors. Regulatory effectors of Drosophila p53 are only beginning to be identified, and these data add stg/cdc25 to the list. One of the first detectable disc responses to undead cells is G2 arrest, mediated by loss of stg mRNA. Cdc25 is also regulated by vertebrate p53 but is inhibited post-transcriptionally by p53-dependent 14-3-3 activity (Levine, 2006). Experiments with irradiated p53 mutant animals have not revealed a cell-cycle arrest role. However, recent work indicates that dp53 also regulates a G1 checkpoint under conditions of metabolic stress; thus, like vertebrate p53, Drosophila p53 can activate both a G1 and a G2 checkpoint in response to tissue stress. Other effectors and targets involved in the compensatory proliferation process remain unknown, although expression profiling experiments from irradiated p53 mutants identified several potential targets, several of which do not have obvious roles in cell death or DNA repair (Wells, 2006).
How does Drosophila p53 control the signaling that leads to compensatory proliferation? The events observed — G2 arrests in two different cell populations, ectopic expression of wg, and compensatory growth — are all regulated by p53. It is possible that p53 directly and coordinately controls each of these processes by regulating the expression of specific effectors. However, because the response is both cell autonomous and non-cell autonomous, the idea is favored that these processes are interdependent, but sequentially activated. It is envisioned that as a result of Dronc activation in undead cells, p53 induces loss of stg, leading to G2 arrest, and hid and rpr expression, initiating the feedback loop. It is postulate that cells then synthesize factors that stimulate their survival and proliferation. The non-cell-autonomous arrest in the anterior compartment may be a secondary effect of undead cells in the posterior. High levels of TUNEL activity was observed in the anterior cells of these discs, which could feasibly activate p53 in those cells. However, no p53 mRNA was detected in anterior cells. One possibility is that the DNA fragmentation resulting from dying anterior cells could activate ATM and Chk2 in those cells. Consistent with this, although loss of either of these kinases did not affect undead cell induction of Wg expression or compensatory growth, the cell-cycle arrest in anterior cells was reduced in a fraction of atm and chk2 mutants (Wells, 2006).
What is the growth-stimulating signal induced by undead cells? While its identity is still unclear, both Wg and Dpp have been implicated in this role. This makes sense, because Wg and Dpp are the major pattern organizers of all imaginal discs and are also involved in regulating their growth, and furthermore they are known to be induced in disc regeneration. However, although wg and dpp are ectopically expressed in undead cells, it was found that targets of both are sharply downregulated, specifically in the undead cells. These data also show that undead cells are able to proliferate and contribute to the compensatory growth. Thus, although the nonautonomous stimulation of growth (anterior cells near the A/P boundary) could be due to increased Dpp signaling, it is suspected that the autonomous growth stimulation is due to other, unidentified factors (Wells, 2006).
This study identified a growth-regulatory role for p53 that seems counter to its role as a tumor suppressor in vertebrates. However, it is speculated that the ability of p53 to sense and respond to tissue damage and promote compensatory proliferation and regeneration in Drosophila reflects an ancestral function, aspects of which have been appropriated for developmental processes and distributed among p53, p63, and p73 during vertebrate evolution. Although p63 and p73 initially were proposed to have evolved as duplications of p53, reanalysis of the phylogenetic relationship between the three family members has suggested that p63 may be the ancestral gene. p63 and p73 are structurally similar to p53 but contain an additional SAM domain. p53 is the sole member of the family encoded in the Drosophila genome, and although dp53 does not contain a SAM domain, based on the sequence of the DNA binding domain, the most highly conserved region of p53, it is more related to vertebrate p63 than to p53. After irradiation, cell-cycle arrest is not p53 dependent in either Drosophila or the nematode C. elegans, and therefore it has been proposed that the ancestral p53 function is apoptosis, rather than a “repair, then death” response when damage cannot be repaired. The experiments argue that as in vertebrates, p53 plays a role in cell-cycle arrest after tissue damage. The additional functions of p53 in promoting cell proliferation may have been conserved in p63, which regulates progenitor cell renewal in the epidermis. Other processes that require cell renewal may also be regulated by p53. For example, p53 mutants are reported to have fertility defects, so it is tempting to speculate that stem cell renewal in the gonad requires this previously unappreciated role of Drosophila p53 (Wells, 2006).
Ubiquitin-proteasome system (UPS) is a multistep protein degradation machinery implicated in many diseases. In the nervous system, UPS regulates remodeling and degradation of neuronal processes and is linked to Wallerian axonal degeneration, though the ubiquitin ligases that confer substrate specificity remain unknown. Having shown previously that class IV dendritic arborization (C4da) sensory neurons in Drosophila undergo UPS-mediated dendritic pruning during metamorphosis, an E2/E3 ubiquitinating enzyme mutant screen was conducted, revealing that mutation in ubcD1, an E2 ubiquitin-conjugating enzyme encoding Effete, resulted in retention of C4da neuron dendrites during metamorphosis. Further, UPS activation likely leads to UbcD1-mediated degradation of DIAP1, a caspase-antagonizing E3 ligase. This allows for local activation of the Dronc caspase, thereby preserving C4da neurons while severing their dendrites. Thus, in addition to uncovering E2/E3 ubiquitinating enzymes for dendrite pruning, this study provides a mechanistic link between UPS and the apoptotic machinery in regulating neuronal process remodeling (Kuo, 2006).
The ubiquitin-proteasome system (UPS), evolutionarily conserved for the regulation of protein turnover, targets proteins for degradation via a complex, temporally regulated process that results in proteasome-mediated destruction of polyubiquitinated proteins. There are two distinct steps involved: first, the covalent conjugation of ubiquitin polypeptide to the protein substrates, and second, the destruction of tagged proteins in the proteasome complex. The transfer of ubiquitin to a target molecule slated for degradation involves at least three enzymatic modifications: ubiquitin is first activated by the ubiquitin-activating enzyme E1; ubiquitin is then transferred to a carrier protein, a ubiquitin-conjugating enzyme E2, and finally, ubiquitin is transferred to a protein substrate bound by a ubiquitin ligase E3. There are minor variations to this enzymatic cascade, but overall, these highly specific protein-protein interactions ensure ubiquitin targeting specificity and regulate many aspects of housekeeping protein turnover and cellular maintenance. However, with the multiple regulatory layers, different parts of this complex machinery can break down. Mutations in the UPS pathway causing accumulation of nondegraded proteins have been implicated in a variety of human diseases (Kuo, 2006).
In the nervous system, aberrations in the UPS pathway have been implicated in disorders such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, and other neurodegenerative diseases. One of the common pathological features of neurodegenerative diseases, besides neuronal loss, is local axon degeneration. For example, in the case of Wallerian degeneration in vertebrates, distal parts of a severed axon remain viable and conduct action potentials in vivo for some time before a rapid dismantling of cytoskeletal proteins and axon degeneration, and the initiation of this rapid axon degeneration involves the UPS pathway. It is thought that UPS activation can lead to microtubule depolymerization and subsequent neurofilament degradation, possibly acting in conjunction with the Ca2+-dependent protease calpain. Moreover, inhibiting UPS activity in neurons prior to severing their axons can dramatically retard degradation of the severed axons. These results suggest that a cell-intrinsic UPS pathway regulates axon stability and that pharmaceutical inactivation of the UPS may prevent axonal degeneration in disease states (Kuo, 2006 and references therein).
In Drosophila, the remodeling of neuronal processes during normal development closely resembles the pathological phenotypes in Wallerian degeneration. In the mushroom body γ neurons, extensive pruning of larval axons occurs during metamorphosis in a process regulated by glia engulfment and neuron-intrinsic UPS activity. Similarly, in the fly peripheral nervous system, the class IV dendritic arborization (C4da) neurons undergo complete pruning of their extensive larval dendrites during metamorphosis, in a process that is also regulated by UPS activity (Kuo, 2005). In both of these examples, severing of neuronal processes is preceded by microtubule depolymerization and followed by cytoplasmic blebbing and degeneration, all phenotypes resembling Wallerian degeneration. Therefore, these fly neurons represent excellent systems in which to understand the roles of the UPS in regulating neuronal axon/dendrite integrity, given the rather limited knowledge of how the UPS participates in the degradation of neuronal processes. It is not known which specific E2 ubiquitin-conjugating enzyme(s) and E3 ubiquitin ligase(s) are involved in UPS-mediated remodeling/degradation of neuronal processes, or their specific downstream target(s) (Kuo, 2006).
It has been shown that mutations in the fly ubiquitin activation enzyme (uba1) and the proteasome complex (mov34) can prevent efficient pruning of C4da neuron larval dendrites during metamorphosis (Kuo, 2005). To further investigate the role of UPS in C4da neuron dendrite remodeling, a candidate gene screen was conducted to identify the E2 ubiquitin-conjugating enzyme and the E3 ubiquitin ligase required for this process. Analysis of genetic mutants showed that UPS activation in C4da neurons likely results in UbcD1 (an E2 ubiquitin-conjugating enzyme) mediated degradation of Drosophila inhibitor of apoptosis protein 1 (DIAP1), an E3 ligase that antagonizes caspase activity. Degradation of DIAP1 leads to activation of caspase Dronc, which results in local caspase activation and cleavage of proximal dendrites in C4da neurons during metamorphosis. In addition to the identification of a set of E2/E3 ubiquitinating enzymes for C4da neuron dendrite remodeling—with the surprising finding that the UPS mediates degradation of the potent protease inhibitor DIAP1—this study also establishes a mechanistic link between the UPS and caspase pathways in regulating C4da neuron dendrite pruning (Kuo, 2006).
To identify the E2 ubiquitin-conjugating enzyme and E3 ubiquitin ligase mediating dendrite pruning of C4da neurons during metamorphosis, a candidate gene approach was taken to systematically test the roles of known E2/E3 ubiquitinating enzymes in Drosophila. A set of putative E2/E3 ubiquitinating enzyme mutations was assembled, and live imaging was used to visualize C4da neurons carrying the E2/E3 mutation via the pickpocket(ppk)-EGFP marker, which specifically labels C4da neurons during Drosophila development. Those mutants with an early lethal phase were characterized by generating mosaic analysis with a repressible cell marker (MARCM) mutant neuronal clones. Since wild-type (wt) C4da neurons during metamorphosis do not retain any larval dendrites following head eversion, as imaged 18–20 hr after puparium formation (APF), mutations that caused larval dendrite retention in C4da neurons at this stage were sought. The candidate genes tested mostly showed no defects in dendrite pruning or neuronal cell death. However, one candidate, ubcD1, showed a modest level of larval dendrite retention at 18 hr APF (Kuo, 2006).
Live imaging of wt C4da neuron MARCM clones at the start of pupariation (white pupae, WP) showed primary and secondary dendritic branching patterns typical of C4da neurons. Consistent with previous reports (Kuo, 2005; Williams, 2005), wt C4da neurons sever their larval dendrites during early metamorphosis and by 18 hr APF are devoid of any dendrites. The ubcD1 mutant C4da MARCM clones showed similar dendritic morphology to the wt clones at the onset of metamorphosis. However, at 18 hr APF, the mutant clones consistently retained intact, nonsevered larval dendrites. Thus, the UbcD1 E2 ubiquitin-conjugating enzyme is required for proper UPS-mediated dendrite pruning in C4da neurons during metamorphosis (Kuo, 2006).
UbcD1, encoded by the gene effete, regulates UPS-mediated degradation of the antiapoptotic protein DIAP1 (Treier, 1992; Wang, 1999; Ryoo, 2002). In protecting cells from apoptosis, the DIAP1 E3 ubiquitin ligase antagonizes Dronc caspase activity by regulating ubiquination and degradation of the Dronc protein. Following apoptotic stimuli, UbcD1 mediates self-ubiquination and degradation of DIAP1, allowing for subsequent Dronc caspase activation. The biochemical and genetic interactions between these molecules are well established. The baculovirus p35, which is commonly used to inhibit caspase activity in Drosophila, and does not block C4da neuron dendrite pruning (Kuo, 2005). This may seem to make the involvement of caspases in this process unlikely; however, p35 has only limited activity against the caspase Dronc. To study the effects of dronc mutation on C4da neuron dendrite pruning, two null alleles of Dronc, dronc51 and dronc11, were used. MARCM analysis of dronc mutant clones revealed that the dendrites of mutant C4da neurons appeared normal at larval stages. However, unlike wt clones, without Dronc these neurons failed to properly prune their larval dendrites during metamorphosis, and most showed relatively intact primary and secondary larval dendritic arbors at 18 hr APF. These results show that severing of primary larval dendrites from C4da neurons during early metamorphosis requires the Dronc caspase (Kuo, 2006).
During apoptosis, Dronc activation requires the degradation of the antiapoptotic/anticaspase protein DIAP1, which is downstream of UbcD1. The requirement of UbcD1 for C4da neuron larval dendrite pruning during metamorphosis, together with the finding that Dronc caspase activity is also essential, raised the question of whether UPS-mediated DIAP1 degradation is a key step that allows for the severing of larval dendrites. Because loss of DIAP1 function causes C4da neuron cell death prior to the onset of metamorphosis, this question was approached using a gain-of-function allele of diap1, diap16-3s, which has a single amino acid mutation that makes DIAP1 an inefficient substrate for UPS-mediated degradation. ppk-EGFP was crossed into the gain-of-function mutant background and live imaging was used to follow C4da neuron dendrite pruning during metamorphosis. The diap16-3s mutation did not significantly affect the ability of C4da neurons to elaborate larval dendrites. However, unlike wt C4da neurons that completely pruned their larval dendrites by 18 hr APF, C4da neurons in the diap16-3s gain-of-function mutants failed to efficiently sever larval dendrites at 18 hr APF. These results suggest that the degradation of DIAP1 during early metamorphosis is required for proper C4da neuron larval dendrite pruning. Quantitatively, mutations in the UPS pathway that modulate Dronc activity (diap16-3s and ubcD1) resulted in less severe dendrite pruning defects than dronc mutants, both in terms of total number of large dendrites attached to soma and in the length of the longest attached dendrite at 18 hr APF (Kuo, 2006).
The UbcD1-DIAP1-Dronc pathway in apoptosis is well established. Thus, it may be necessary for C4da neurons to restrict the action of this pathway to specific cellular locations in order to prune unwanted dendrites without triggering apoptosis. To address this possibility, the subcellular distribution of DIAP1 and Dronc proteins was examined in ppk-EGFP C4da neurons. During the transition from third instar larvae to white pupae at the onset of metamorphosis, as well as 2 hr APF, there was a consistent induction of nuclear DIAP1 in GFP-labeled C4da neurons. During the same period a concurrent decrease was detected in Dronc staining in the soma of C4da neurons, unlike those from the neighboring cells at 2 hr APF. These results are consistent with previous observations that C4da neurons survive through this stage of metamorphosis. However, the level of antibody staining made it difficult to monitor the distribution of DIAP1 and Dronc within the dendritic structures of the C4da neurons. Because overexpression of Dronc caused C4da neuron to undergo apoptosis prior to metamorphosis, it was not possible to use GFP-tagged Dronc to examine its distribution in these neurons during pupariation. It was therefore necessary to search for alternative means to visualize activated Dronc or its downstream caspases (Kuo, 2006).
An antibody generated against activated mammalian caspase 3 has been shown to be effective in recognizing activated caspases in Drosophila. Whereas this antibody reportedly recognizes the Drosophila effector caspase Drice, it may also cross react with other activated Drosophila caspases such as Dronc during tissue staining, because of similarities in the sequences of these caspases in the region corresponding to the peptide used to generate this antibody. Therefore this antibody was used to determine whether activated caspase is localized to the dendrites of C4da neurons during the initial severing event. At 4 hr APF, just prior to dendrite severing, antibody staining for activated caspase was consistently observed within the proximal larval dendrites of C4da neurons, especially within dendritic swellings. In the diap16-3s gain-of-function mutant that inhibits Dronc activity, as well as in ubcD1 and dronc mutant MARCM clones, C4da neurons did not show dendritic swellings or activated caspase staining in dendrites during early metamorphosis. Consistent with previous observation that C4da neurons do not remodel their axons during concurrent dendrite pruning, no activated caspase staining was seen within the axons of C4da neurons during dendrite severing. Since overexpression of p35 in these neurons did not block dendrite pruning (Kuo, 2005), it is believed this antibody staining likely recognizes activated Dronc directly or recognizes a p35-resistant caspase that is activated by Dronc. These results show that, concurrent with the nuclear upregulation of DIAP1 in C4da neurons that prevents apoptosis, there is a local activation of caspases in the dendrites, likely as a result of UPS-mediated degradation of DIAP1. The spatially restricted activation of caspases then allows the severing of proximal larval dendrites from the soma (Kuo, 2006).
This study has shown that the UPS regulates pruning of larval dendrites from C4da neurons in a cell-intrinsic manner. To better understand the molecular pathways regulating UPS-mediated pruning, a candidate E2/E3 ubiquitinating enzyme screen was conducted. In this screen an E2 ubiquitin-conjugating enzyme mutation in was uncovered ubcD1, causing dendrite pruning defects. Taken together with the extensive biochemical characterization of interactions between UbcD1, DIAP1, and Dronc, this study suggests that in C4da neurons, UPS activation leads to UbcD1-mediated degradation of E3 ubiquitin ligase DIAP1, thereby allowing Dronc caspase activation and the subsequent cleavage of larval dendrites. This work not only identifies a set of E2/E3 ubiquitinating enzymes regulating neuronal process remodeling, it also links the UPS to a hitherto unappreciated mechanism for local caspase activation in dendrites during Drosophila metamorphosis (Kuo, 2006).
The mechanistic link between the UPS and caspase activity in regulating C4da neuron dendrite pruning is unexpected. Although the UPS is known to regulate remodeling and degradation of neuronal processes, it is generally believed that this process is accomplished by degradation of cellular proteins (such as microtubules and neurofilaments) that are required to keep dendrites and axons intact. However, it was found that the UPS in C4da neurons is in fact causing the degradation of an E3 ligase, DIAP1, thereby allowing for subsequent dendrite pruning. In this case, UPS-mediated degradation of a protein does not in and of itself lead to a structural compromise in dendrites, but rather it leads to the activation of another protease that executes dendrite pruning. This two-step activation cascade, which involves both the UPS and the apoptotic machinery, may provide an additional level of control and flexibility that would not be possible if UPS alone regulated the pruning program. After all, these C4da neurons, which are specified during fly embryogenesis, maintain a highly elaborate dendritic field to receive sensory inputs throughout larval development, which lasts for several days. The maintenance of these dendrites over time requires a network of finely tuned cell-intrinsic and -extrinsic pathways. Just as important, the dendritic pruning program enables dramatic neuronal remodeling in response to profound environmental changes during metamorphosis. It is conceivable that C4da neurons evolved this dual control mechanism to prevent any accidental triggering of dendrite pruning prior to metamorphosis. Initiation of C4da neuron dendrite pruning requires cell-intrinsic ecdysone signaling, and ecdysone receptors have been shown to regulate Dronc expression. It will be of interest to determine how this UPS/caspase dendritic pruning pathway is related to the ecdysone signaling cascade (Kuo, 2006).
During metamorphosis, C4da neurons upregulate DIAP1 expression in the nucleus, which is consistent with this class of neurons surviving early stages of the metamorphosis (only one of the three C4da neurons per hemisegment, the ventral neuron, is lost at a later stage of pupariation). Remarkably, there are activated caspases within the dendrites prior to severing, and a gain-of-function diap1 mutation can block dendrite pruning, strongly implicating a local dendritic program that can activate caspases without causing apoptosis of the neuron. Although mutations in both the Dronc caspase and the UPS pathway that modulate Dronc activity (UbcD1 and DIAP1) result in retention of larval dendrites, their dendrite pruning defects differed somewhat quantitatively. Compared to dronc mutants, diap1 gain-of-function and especially ubcD1 mutants showed less retention of larval dendrites during metamorphosis. This is not surprising for diap1 gain-of-function, as it is an effective Dronc inhibitor but unlikely to be 100% efficient. UbcD1, as an E2 ubiquitin-conjugating enzyme, has wider substrate specificity than E3 ligases. Previous study showed that UbcD1 is involved in mushroom body neuroblast proliferation, so it may be involved in other UPS-mediated pathways during dendrite pruning. It is also conceivable that in the absence of UbcD1 another E2 may trigger a low level of DIAP1 degradation, allowing residual Dronc activation which results in a milder dendrite pruning phenotype in ubcD1 mutants. It is currently unclear whether UbcD1 is also required during DIAP1-mediated degradation of Dronc. However, pruning defects in the ubcD1 mutants suggest that it may not be absolutely required, since undegraded DIAP1 continues to inhibit Dronc, presumably via interaction with another E2 protein (Kuo, 2006).
How is the specificity of dendrite pruning achieved? Several possible mechanisms are proposed: first, C4da neurons do not change their axonal projections during dendrite pruning, so there could be dendrite-specific trafficking of components of the UPS, such as UbcD1, and/or the caspase Dronc. Of the known proteins that are preferentially trafficked to dendrites, these molecules have not been implicated but warrant further investigation. Second, it is also possible that activated Dronc, or another p35-resistant protease activated by Dronc, could cleave a dendrite-specific substrate. Examples are now emerging from other cellular systems, such as in sperm formation and border cell migration, in which caspases can participate in cleavage of proteins not resulting in apoptosis. Third, the dendritic pruning program takes place during drastic environmental changes that include concurrent degradation and regrowth of the overlying epidermis, activation of extracellular matrix metalloproteases, and blood phagocytes. These environmental cues likely complement the neuronal intrinsic pruning programs, but their exact relationships are not known. Experiments addressing these and other possible mechanisms should provide a greater insight into how the large-scale remodeling of C4da neuron dendrites is achieved (Kuo, 2006).
In vertebrates, the UPS pathway has been implicated in Wallerian degeneration of severed axons. In the fly, mushroom body γ neurons undergo extensive remodeling of their processes during metamorphosis. The initial stages of axon pruning in these mushroom body neurons closely resemble Wallerian degeneration, and the UPS again plays a critical role. To date, the specific ubiquitin-conjugating enzymes and ligases that mediate target protein degradation have not been identified in these systems. It will be interesting to see whether the UbcD1-DIAP1-Dronc pathway implicated in C4da neuron dendrite pruning also participates in remodeling/degradation of neuronal processes in other systems. It seems likely that more than one pathway would be employed in remodeling different neurons; a previous study excluded UbcD1 as a possible ubiquitin-conjugating enzyme regulating mushroom body γ neuron remodeling, and normal remodeling of mushroom body neuron processes in is seen dronc mutant MARCM clones during metamorphosis (Kuo, 2006).
A multilayered regulatory machinery for remodeling neurons, as uncovered in this study for C4da neurons, offers versatility and flexibility. It is conceivable that another ubiquitin ligase/caspase pair may function in an analogous UPS pathway during mushroom body neuron remodeling, potentially affording differential regulation of neuronal remodeling. Although pharmacological inhibition of mammalian caspases showed no effect on Wallerian degeneration, it would be important to assess the in vivo effectiveness of the inhibitors against a comprehensive panel of caspases. Moreover, a dual control mechanism, similar to what is proposed for C4da neuron remodeling, may coordinately regulate UPS and another protease that executes axon degradation. Conceivably, instead of having the target of the UPS directly involved in maintaining dendrite/axon stability, the executor of neuronal process degradation may involve a different protease: in the case of C4da neurons it is the caspase Dronc, and in Wallerian degeneration the relevant protease might be the Ca2+-responsive calpain. Future experiments along these lines of thinking may accelerate the identification of specific ubiquitinating enzymes involved in other areas of developmental neuronal remodeling and in diseases where the UPS pathway has been implicated. As target-specific E3 ligases are excellent candidates for pharmaceutical intervention, this approach may also help to find effective treatments for developmental and neurodegenerative diseases that involve degeneration of neuronal processes (Kuo, 2006).
Spermatozoa are generated and mature within a germline syncytium. Differentiation of haploid syncytial spermatids into single motile sperm requires the encapsulation of each spermatid by an independent plasma membrane and the elimination of most sperm cytoplasm, a process known as individualization. Apoptosis is mediated by caspase family proteases. Many apoptotic cell deaths in Drosophila utilize the REAPER/HID/GRIM family proapoptotic proteins. These proteins promote cell death, at least in part, by disrupting interactions between the caspase inhibitor DIAP1 and the apical caspase DRONC, which is continually activated in many viable cells through interactions with ARK, the Drosophila homolog of the mammalian death-activating adaptor APAF-1. This leads to unrestrained activity of DRONC and other DIAP1-inhibitable caspases activated by DRONC. This study demonstrates that ARK- and HID-dependent activation of DRONC occurs at sites of spermatid individualization and that all three proteins are required for this process. dFADD, the Drosophila homolog of mammalian FADD, an adaptor that mediates recruitment of apical caspases to ligand-bound death receptors, and its target caspase DREDD are also required. A third apoptotic caspase, DRICE, is activated throughout the length of individualizing spermatids in a process that requires the product of the driceless locus, which also participates in individualization. These results demonstrate that multiple caspases and caspase regulators, likely acting at distinct points in time and space, are required for spermatid individualization, a nonapoptotic process (Huh, 2004; full text of article).
Because specific mutations in dronc are currently not available, the
technique of RNAi was used to ablate dronc gene function during
embryogenesis. RNAi, a technique developed in Caenorhabditis elegans, has recently been successfully used in
Drosophila and mammalian cells to specifically ablate gene
function. Dronc double-stranded
mRNA was injected into precellularized embryos, and samples were
aged until stage 13. Embryos were analyzed by Dronc antibody staining
to assess the efficiency of the Dronc protein ablation and by TUNEL
assays to reveal apoptotic cells. In addition, embryos were also
stained with neural differentiation marker monoclonal antibody 22C10 to reveal whether ablation of dronc affects neural development. At stage 13, uninjected embryos show Dronc expression throughout the embryo and a
large number of TUNEL-positive cells. In
contrast, in stage 13 dronc RNAi embryos, Dronc protein is
undetectable, and very few TUNEL-positive cells are observed. Buffer-injected control embryos show no decrease in cell death, but rather more cells are
TUNEL-positive. At least 400 dronc RNAi-injected embryos were examined, and the results are consistent for all embryos. Although dronc RNAi-injected embryos fail to hatch, examination of embryonic structures using Nomarski optics shows no apparent gross structural defects. Furthermore, staining with neural
marker 22C10 shows that neural differentiation is normal. These results show that dronc is essential for induction of cell death during
embryogenesis. Because Dronc shares very limited (<25%) nucleotide
sequence homology with all Drosophila caspases, dronc RNAi is unlikely to affect the function of other caspases (Quinn, 2000).
Does DRONC acts in an Rpr-dependent or an Rpr-independent death pathway? To investigate this question more carefully, whether Rpr-induced cell death is sensitive to dronc gene dosage was examined. Because no single gene mutations in dronc are currently available, mutant flies were used with a larger chromosomal deletion that includes the dronc locus [Df(3L)AC1]. Df(3L)AC1 was crossed to GMR-rpr flies and it was found that flies carrying Df(3L)AC1 show a significant suppression of the Rpr eye phenotype. Furthermore, Df(3L)AC1 also suppresses Hid-mediated cell killing in the eye. To investigate further whether this observed suppression is due specifically to loss of dronc, whether the expression of dominant-negative DRONC mutants (pro-DRONC CdeltaA and DRONC-CARD) also suppresses the Rpr eye phenotype was assessed. Pro-DRONC CdeltaA strongly suppresses Rpr cell killing, and, surprisingly, the pro-domain of DRONC on its own (DRONC-CARD) completely rescues the Rpr eye phenotype. These results, in which DRONC function is ablated either by the Df(3L)AC1 deletion or by the action of dominant-negative DRONC, are consistent with the notion that DRONC is a rate-limiting caspase in the Rpr and Hid death pathway (Meier, 2000).
Studies have indicated that Rob-1 (aka Debcl) can induce apoptosis by a caspase independent mechanism. Although Rob-1 strongly stimulates caspase activation when overexpressed in S2 cells, its ability to kill cells is barely antagonized by the caspase-related apoptosis inhibitors p35 and DIAP2, or by an active-site mutant of a Drosophila apical caspase, DRONC (Nedd2-like caspase). Furthermore, in transgenic flies, rough-eye phenotype caused by overexpression of Rob-1 is not completely suppressed by coexpression of p35. Similarly, previous studies on the proapoptotic Bax, Bak, and Mtd suggest that they all induce apoptosis in the presence of broad caspase inhibitors. Moreover, both Bax and Bak can induce mitochondrial dysfunction and also kill yeast, which lack endogenous caspases. Both Rpr- and Hid-induced cell death are blocked by coexpression of baculovirus p35. In contrast, overexpression of p35 shows no effect on Rob-1-induced apoptosis. Higher levels of p35 expression exhibit only a slight protective effect against Rob-1-induced cell killing. Similarly, DIAP2, which inhibits both Rpr- and Hid-induced apoptosis, or an active-site mutant of the CARD (caspase recruitment domain)-containing Drosophila caspase, DRONC, shows little effect on Rob-1-induced apoptosis, suggesting that the Rob-1-stimulated cell-death pathway is downstream or independent of DIAP2 and DRONC. Thus, overexpression of Rob-1 induces apoptosis, probably through a caspase-independent pathway partly distinct from that used by other known Drosophila killer proteins (Rpr, Hid, and Grim). Rob-1 overexpressed in human embryonic kidney 293T cells also exhibits a proapoptotic activity that is only slightly inhibited by p35. Thus, Rob-1, like mammalian Bax, Bak, and Mtd, may activate cell death by inducing both caspase-dependent and -independent pathways. The latter pathways probably can be antagonized by an as-yet-unidentified antiapoptotic member of the Drosophila Bcl-2 family. In the nematode C. elegans, in which two Bcl-2/CED-9 family members, CED-9 and EGL-1, have been identified, all cell deaths occur in a caspase-dependent manner. The presence of a Bax-like protein, Rob-1, in Drosophila might indicate the acquisition of a caspase-independent cell death pathway through evolution (Igaki, 2000).
Among the seven caspases encoded in the fly genome, only dronc contains a caspase recruitment domain. To assess the function of this gene in development, a null mutation in dronc was produced. Animals lacking zygotic dronc are defective for programmed cell death (PCD) and arrest as early pupae. These mutants present a range of defects, including extensive hyperplasia of hematopoietic tissues, supernumerary neuronal cells, and head involution failure. dronc genetically interacts with the Ced4/Apaf1 counterpart, Dark, and adult structures lacking dronc are disrupted for fine patterning. Furthermore, in diverse models of metabolic injury, dronc− cells are completely insensitive to induction of cell killing. These findings establish dronc as an essential regulator of cell number in development and illustrate broad requirements for this apical caspase in adaptive responses during stress-induced apoptosis (Chew, 2004).
In many metazoans, damaged and potentially dangerous cells are rapidly eliminated by apoptosis. In Drosophila, this is often compensated for by extraproliferation of neighboring cells, which allows the organism to tolerate considerable cell death without compromising development and body size. Despite its importance, the mechanistic basis of such compensatory proliferation remains poorly understood. Apoptotic cells are shown to express the secretory factors Wingless and Decapentaplegic. When cells undergoing apoptosis were kept alive with the caspase inhibitor p35, excessive nonautonomous cell proliferation is observed. Significantly, Wg signaling is necessary and, at least in some cells, also sufficient for mitogenesis under these conditions. Finally, evidence is provided that the DIAP1 antagonists reaper and hid can activate the JNK pathway and that this pathway is required for inducing wg and cell proliferation. These findings support a model where apoptotic cells activate signaling cascades for compensatory proliferation (Ryoo, 2004).
To investigate how the inhibition of diap1 may lead to mitogen expression, attention was focused on Dronc and the Jun N-terminal Kinase (JNK) pathway. Dronc has been implicated in compensatory proliferation, and its activity can be inhibited by the expression of droncDN. In addition, the JNK signaling pathway was considered as a candidate, since its activity is known to correlate with many forms of stress-provoked apoptosis, including disruption of morphogens, cell competition, and rpr expression. In Drosophila, the JNK pathway can be effectively blocked by the expression of puckered (puc), which encodes a phosphatase that negatively regulates JNK (Ryoo, 2004).
To induce patches of undead cells, wing imaginal discs were generated with mosaic clones expressing hid and p35. 48 hr after induction, these imaginal discs contained hid-expressing clones that autonomously induced wg. Using this experimental setup, it was asked whether additional expression of either droncDN or puc would block wg induction in undead cells. When droncDN was coexpressed, a subset of the hid-expressing population was still able to induce wg. In contrast, when puc was coexpressed, wg induction by hid was almost completely blocked. These results provide evidence that the JNK pathway is required for wg induction under these conditions but fail to uncover a similar requirement for Dronc (Ryoo, 2004).
To independently investigate the role of puc and droncDN in compensatory proliferation, the size of wing discs harboring undead cells was measured and they were compared with those of the sibling controls. Under the experimental conditions, wing discs harboring hid- and p35-expressing clones were on average 53% larger than their sibling controls. Coexpression of puc within these undead clones significantly limited growth, resulting in only a small increase in wing disc size that was not statistically significant. In contrast, coexpression of droncDN did not limit growth. Wing size measurements also correlated with the degree of wg induction. The larger size of discs harboring hid- and p35-expressing cells is not due simply to extra cell survival: (1) these undead cells are derived from the normal lineage; (2) the size of wing discs expressing hid, p35, and puc serves as a control. In this case, although a large number of undead cells were generated, no significant increase in disc size was observed, in stark contrast to the discs expressing hid and p35 only. It is concluded that the JNK pathway is required for the nonautonomous growth promoting activity of the undead cells (Ryoo, 2004).
To confirm a role of puc in imaginal disc growth, rpr and p35 werecoexpressed in wild-type and puc−/+ imaginal discs. Like hid, rpr is a DIAP1 antagonist, but with a weaker cell killing activity when overexpressed in imaginal disc cells. In a puc+/+ background, a small amount of ectopic wg expression was observed, indicative of rpr's weaker DIAP1 inhibiting activity. In contrast, ectopic wg expression was strongly enhanced in puc−/+ discs. Because the puc allele used, pucE69, also acts as a lacZ reporter, JNK pathway induction could be monitored simultaneously. wg induction in undead cells correlates very well with puc-lacZ expression, with a stronger induction at the center of the wing pouch. These results further support the role of JNK in the induction of wg (Ryoo, 2004).
Next to be tested was whether the reduction of puc had an effect on apoptosis-induced cell proliferation. Whereas puc−/+ discs expressing only p35 had BrdU incorporation similar to wild-type discs, coexpression of rpr and p35 in puc−/+ led to a significant increase in BrdU incorporation. Also, the size of these discs were on average 41% larger than those coexpressing rpr and p35 in a puc+/+ background. Taken together, these results show that diap1 inhibition leads to JNK activation and that JNK activity promotes wg induction and cell proliferation (Ryoo, 2004).
To directly test if JNK signaling can activate wg and dpp expression, hepCA, a constitutively active form of hemipterous (hep), the Drosophila JNK kinase was conditionally expressed. Expression of hepCA causes induction of wg-lacZ within 22 hr and to a lesser extent also dpp-lacZ. These ß-gal-expressing cells shifted basally and were apoptotic as assayed by anti-active caspase-3 antibody labeling. Hid protein levels were also elevated in these cells. Significantly, since p35 was not use to block apoptosis in this experiment, this demonstrates that wg and dpp can be induced not only in undead cells, but also in 'real' apoptotic cells (Ryoo, 2004).
This study provides evidence that the central apoptotic regulators can control the activity of mitogenic pathways. In particular, inhibition of DIAP1, either via expression of Reaper and Hid or by mutational inactivation, leads to the induction of the putative mitogens wg and dpp. When apoptosis was initiated through DIAP1 inhibition but cells were kept alive by blocking caspases, the resulting 'undead cells' exhibited strong mitogenic activity and stimulated tissue overgrowth. Inhibiting wg signaling with a conditional TCFDN blocked cell proliferation in imaginal discs, indicating that wg has an essential mitogenic function. Finally, evidence was provided that the JNK pathway mediates mitogen expression and imaginal disc overgrowth in response to rpr and hid. Based on these results, it is proposed that apoptotic cells actively signal to induce compensatory proliferation. DIAP1 inhibits both caspases as well as dTRAF1. According to this model, when DIAP1 is inhibited in response to cellular injury, the JNK pathway is activated and wg/dpp are induced in apoptotic cells. Secretion of these factors stimulates growth of proliferation-competent neighboring cells and leads to compensatory proliferation (Ryoo, 2004).
This study provides clear genetic evidence that diap1 is involved in compensatory proliferation. Overall, similar results were obtained with hypomorphic diap1 alleles (diap122-8s, diap133-1s), a null allele (diap1th5), and inactivation of diap1 by expression of Reaper and Hid. However, whereas expression of p35 effectively blocked apoptosis of diap122-8s/22-8s cells and in response to Reaper/Hid, it only partially suppressed the death of diap1th5/th5 cells. Consequently, the generation of undead cells was less efficient with the diap1th5 mutation. Moreover, these results suggest that the JNK pathway transduces the signal to activate mitogen expression and cell proliferation. Since IAPs have been shown to ubiquitylate TRAFs in both mammals and Drosophila and since no evidence was found for Dronc in growth promotion, it is attractive to speculate that JNK is regulated through direct DIAP1/TRAF1 interaction (Ryoo, 2004).
An important unresolved question is why compensatory proliferation is seen only in response to cellular injury, but not during normal developmental apoptosis. In particular, inactivation of DIAP1 by Reaper, Hid, and Grim is restricted not only to injury-provoked apoptosis, but also underlies most developmental cell deaths. One possible explanation is that activation of the JNK pathway is key to mitogenic signaling of apoptotic cells. Consistent with this idea, the JNK pathway is activated in response to tissue stress and injury, but not during developmental apoptosis. Furthermore, this study shows that JNK signaling can induce the expression of wg/dpp and nonautonomous cell proliferation. Therefore, it is possible that robust JNK activation and compensatory proliferation require the combined input of stress and apoptotic signals (Ryoo, 2004).
Proteases of the caspase family play key roles in the execution of apoptosis. In Drosophila there are seven caspases, but their roles in cell death have not been studied in detail due to a lack of availability of specific mutants. This study describes the generation of a specific mutant of the Drosophila gene encoding Dronc, the only caspase recruitment domain (CARD) containing apical caspase in the fly. dronc mutants are pupal lethal and these studies show that Dronc is required for many forms of developmental cell deaths and apoptosis induced by DNA damage. Furthermore, Dronc is required for the autophagic death of larval salivary glands during metamorphosis, but not for histolysis of larval midguts. These results indicate that Dronc is involved in specific developmental cell death pathways and that in some tissues, effector caspase activation and cell death can occur independently of Dronc (Daish, 2004).
A Drosophila P element line, KG02994, was obtained with an insertion 113 bp upstream of the 5' UTR of the dronc gene. Analysis of KG02994 shows that this line is a hypomorphic allele of dronc. The KG02994 insertion was used to generate P element excision mutants in the attempt to delete dronc. Three potential dronc mutant lines were identified as containing a deletion. One line, named droncZ, was selected for further studies of DRONC function. The breakpoints were confirmed by sequencing as being 1926 bp upstream of the dronc transcription start site and within the intron of dronc, 890 bp downstream from the ATG. Inspection of the Drosophila genome sequence indicates that a gene of unknown function, CG6685, is within this deleted region. This confounds any conclusions that could be drawn from the observed larval lethal phenotype of the droncZ deletion line regarding Dronc function (Daish, 2004).
The dronc deletion mutants (droncZ) arrest at the late larval stage with melanotic tumors and developmental defects in larval tissues. CG66854 animals fail to form a normal puparium following gut clearance and arrest as partially contracted larvae with melanotic tumors. All droncd5 animals pupate but arrest prior to the pharate adult stage (Daish, 2004).
It was necessary to assess, in a droncZ background, the ability of the respective complementation transgenes to emulate the endogenous expression profiles and restore viability when combined in the same animal. The CG66854/droncd5; droncZ/droncZ animals survived to adult with an eclosion rate of 32.67% of expected. The remaining noneclosed animals develop to an advanced pharate adult stage. These results demonstrate that the discreet phenotypes observed for droncd5 and CG66854 are attributed primarily to the gene product of the nonfunctional gene. Since a detailed expression profile of the CG6685 transcript in development is not known, it is not possible to demonstrate definitively that its correct expression has been restored in droncd5 animals. However, it is believed that the phenotype and lethality observed for droncd5 animals can be primarily attributed to the absence of Dronc and not disruption to the CG6685 expression profile for the following reasons: (1) each of the transgenes is associated with discreet developmental defect profiles and survival boundaries, (2) combining the two complementation transgenes in the same animal significantly rescues droncZ lethality along with the individual lethalities of droncd5 and CG66854; (3) Dronc is present in CG66854 animals; (4) droncd5 animals have CG6685 transcriptional activity, and (5) CG6685 transcript is barely detectable in larval midguts and absent from salivary glands prior to their destruction (Daish, 2004).
To assess the role of dronc in development, the survival and developmental progression of these mutant and complementation lines were analyzed. No significant lethality was found during embryonic stages, as measured by hatching frequency, in any of the mutant or complementation lines. The survival rate of KG02994 homozygotes to the 3L stage was decreased (70% of expected, and eclosion was delayed compared to their sibling heterozygotes. While droncZ and CG66854 animals had a reduced survival frequency to 3L, this was not observed for droncd5 animals. This suggests that the increased larval lethality associated with droncZ and CG66854 is due to the loss of CG6685 gene function and not dronc. Additionally, reduced developmental delay in droncd5 and KG02994 animals than in droncZ and CG66854 animals was observed, with the majority reaching the late 3L stage 1.5 days after wild-type (wt) animals (Daish, 2004).
dronc is transcriptionally upregulated in salivary glands following the late prepupal ecdysone pulse triggering gland histolysis. Larval salivary gland cell death is delayed in KG02994 animals. To further asses the role of Dronc in salivary gland PCD, droncd5 and wt animals were staged to 20 and 30 hr relative to puparium formation (RPF) and sections analyzed by light microscopy. droncd5 animals contained persistent salivary glands with an overall appearance similar to prehistolysed controls including an intact lumen surrounded by nonrounded cells. Adult structures form in droncd5 animals as in the control, indicating continuing pupal development. Progression of autophagy judged by vacuolar dynamics prior to histolysis (13 hr RPF) shows wt salivary gland cells containing variable sized eosin-positive vacuoles while droncd5 cells primarily contain larger vacuoles typical of earlier developmental stages. Ultrastructural analysis of droncd5 salivary glands at 14 hr RPF showed a lack of membrane bound autophagic bodies observed in wt salivary glands, suggesting a lack of characteristic autophagy in the droncd5 salivary glands (Daish, 2004).
No TUNEL-positive nuclei were observed in droncd5 salivary glands at the time when all wt gland cells were TUNEL positive. Hoechst staining of persistent glands showed that nuclear integrity is maintained in droncd5 salivary glands 30 hr RPF. If a dronc-dependent mechanism contributes to salivary gland histolysis, droncd5 glands should be deficient in caspase activity. Consistent with this, caspase activity is reduced in droncd5 salivary glands at 14 hr RPF relative to wt. An increase in active DRICE-like immunostaining was seen in wt salivary glands at 14 hr RPF. However, active DRICE-like immunostaining in droncd5 salivary glands at 14 hr RPF was significantly lower, even though the levels of total DRICE protein in droncd5 and wt were comparable. These results indicate that effector caspase activation in salivary glands is Dronc dependent (Daish, 2004).
To assess whether the genetic regulatory hierarchy upstream of dronc is intact in droncd5 animals, cell death gene expression was analyzed in droncd5 salivary glands. A key ecdysone-regulated transcription factor E93 (which is required for maximal dronc transcription) is upregulated in droncd5 salivary glands at a time similar to wt. hid transcript was also present, indicating that the ecdysone-regulated PCD hierarchy is intact in droncd5 salivary glands (Daish, 2004).
dronc is upregulated following the late 3L ecdysone pulse, which triggers midgut histolysis. However, in contrast to salivary glands, droncd5 midgut histolysis is initiated normally. The fact that DNA fragmentation and caspase activity precedes the time of maximal dronc transcription suggests that aspects of apoptosis are initiated in the midgut in the absence of this apical caspase (Daish, 2004).
Dronc has been shown to function in the Rpr, Hid, and Grim cell death pathway. Since droncd5 is a null mutant and Dronc acts downstream of Rpr, Hid, and Grim, suppression of the Rpr-, Hid-, and Grim-induced phenotypes was expected in a genetic interaction cross with droncd5. As expected, the Rpr-, Hid-, and Grim-induced eye phenotypes are suppressed in the droncd5 heterozygous background, thus confirming that Dronc is required in Rpr-, Grim-, and Hid-mediated death pathways (Daish, 2004).
To investigate the role of Dronc in DNA damage-induced PCD, droncd5 and wt 3L animals were irradiated with 8 Gy γ and the effect on apoptosis and caspase activity was analyzed. Following irradiation, wt larvae showed significant increases in eye disc cell death as observed by AO staining. No similar increase in AO staining was observed in droncd5 eye discs following irradiation. Whole-animal lysates from the same experiment showed an increase in caspase activity. Consistent with the lack of AO staining following irradiation, no significant increase in VDVAD or VEID cleavage was observed for droncd5. Although DEVD showed a marginal increase in activity, it was less than half the control increase (Daish, 2004).
It is concluded that although caspase function is been implicated in Drosophila cell death pathways, most of the previous studies have relied on overexpression of wild-type or dominant-negative transgenes. These studies, while informative, do not provide conclusive evidence for caspase function in developmental PCD. This paper describes the analyses of a dronc mutant alleles in Drosophila. The mutant is a specific point mutation in the dronc gene replacing the deleted allele. The upstream gene CG6685 is essential for development, and deletion of dronc may have effects on the regulation of CG6685 transcription by removal of essential promoter elements. Therefore, the dronc mutant described in this study is an effective way of generating a specific gene disruption if employing a P element excision strategy (Daish, 2004).
While some of the phenotypes in dronc mutants are consistent with previous observations, a number of unexpected findings were uncovered in this study. These include: (1) Dronc plays an essential role in development; (2) despite the fact that Dronc is the only CARD-containing caspase in Drosophila, there are Dronc-independent caspase activation and cell death pathways, (3) Dronc is dispensible for midgut histolysis even though it is highly upregulated by ecdysone in this tissue prior to its histolysis, and (4) evidence that Dronc may be required for some aspects of autophagy is provided. As Dronc has also been recently implicated in some nonapoptotic events, the availability of specific dronc mutants now makes it possible to further explore alternative roles of this apical caspase in diverse developmental events (Daish, 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 simple models indicate (Kiessling, 2006).
Differentiated cells assume complex shapes through polarized cell migration and growth. These processes require the restricted organization of the actin cytoskeleton at limited subcellular regions. IKKε
is a member of the IκB kinase family, and its developmental role has not been clear. Drosophila IKKε localizes to the ruffling membrane of cultured cells and is required for F actin turnover at the cell margin. In IKKε mutants, tracheal terminal cells, bristles, and arista laterals, which require accurate F actin assembly for their polarized elongation, all exhibit aberrantly branched morphology. These phenotypes are sensitive to a change in the dosage of Drosophila inhibitor of apoptosis protein 1 (DIAP1) and the caspase DRONC without apparent change in cell viability. In contrast to this, hyperactivation of IKKε destabilizes F actin-based structures. Expression of a dominant-negative form of IKKε increases the amount of DIAP1. The results suggest that at the physiological level, IKKε acts as a negative regulator of F actin assembly and maintains the fidelity of polarized elongation during cell morphogenesis. This IKKε function involves the negative regulation of the nonapoptotic activity of DIAP1 (Oshima, 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).
A genome-wide RNA interference screen was performed to systematically identify regulators of apoptosis induced by DNA damage in Drosophila cells. Forty-seven double- stranded RNAs were identified that target a functionally diverse set of genes, including several with a known function in promoting cell death. Further characterization uncovers 10 genes that influence caspase activation upon the removal of Drosophila inhibitor of apoptosis 1. This set includes the Drosophila initiator caspase Dronc and, surprisingly, several metabolic regulators, a candidate tumor suppressor, Charlatan, and an N-acetyltransferase, ARD1. Importantly, several of these genes show functional conservation in regulating apoptosis in mammalian cells. These data suggest a previously unappreciated fundamental connection between various cellular processes and caspase-dependent cell death (Yi, 2007).
The genes that are specifically involved in caspase-dependent cell death were classified. Substantial induction of caspase activity was observed 8 h after treatment with a topoisomerase II inhibitor, doxorubicin (dox), to induce dose-dependent cell death. Any RNAi suppressing this activity implicates the target gene in early regulation of caspase activation. In addition to dcp-1 RNAi, knockdown of dronc and jra (the Drosophila homolog of c-Jun) significantly suppressed caspase-3/7-like activity in the presence of dox, whereas the negative control, RNAi against calpain A, a calcium-dependent cysteine protease, did not affect this pathway (Yi, 2007).
This analysis was expanded to all of the genes identified in the initial RNAi screen and 20 dsRNAs were discovered that suppressed caspase activation induced by DNA damage. Interestingly, 12 of these genes were found to be epistatic to diap1 (Yi, 2007).
diap1 epistatic analysis was performed to further categorize the genes. DIAP1, the fly orthologue of the mammalian inhibitors of apoptosis proteins, is a direct inhibitor of caspases, and deficiency in DIAP1 leads to rapid caspase activation and apoptosis in vivo. Thus, apoptosis induced by the loss of DIAP1 presents an alternative apoptotic assay independent of DNA damage. Silencing of genes that regulate activation of the core apoptotic machinery may provide protection against apoptosis induced by both DNA damage and the loss of DIAP1. RNAi against dcp-1 partially suppressed cell death induced by the depletion of DIAP1 in Kc cells. Also, dronc RNAi potently protected cells against apoptosis induced by deficiency in DIAP1. Altogether, 32 of the genes confirmed from the primary screen provided significant protection against cell death induced by the silencing of DIAP1 (Yi, 2007).
Interestingly, 12 dsRNAs suppressed caspase-3/7-like activity after dox treatment and protected against cell death induced by diap1 RNAi, suggesting that these genes are required for apoptosis induced by multiple stimuli. To confirm that these genes are necessary for the full activation of caspases, it was determined whether these dsRNAs could suppress spontaneous caspase activity induced by diap1 RNAi. Maximal induction of caspase activity by diap1 RNAi was observed after 24 h, and this effect was completely suppressed by dsRNA against dcp-1. Importantly, ablating 10/12 dsRNAs resulted in the significant suppression of caspase activity compared with diap1 RNAi only (Yi, 2007).
In addition to dronc RNAi, dsRNAs targeting chn and dARD1 provided the strongest suppression of spontaneous caspase activity. Consistent with the observation that RNAi against chn protects against DNA damage-induced cell death, the mammalian orthologue neuron-restrictive silencer factor (NRSF)/RE1-silencing transcription factor (REST) was recently identified as a candidate tumor suppressor in epithelial cells (Westbrook, 2005). Previous work indicates that Chn and NRSF/REST function as a transcriptional repressor of neuronal-specific genes (Chong, 1995; Schoenherr, 1995; Tsuda, 2006), suggesting that cellular differentiation may render cells refractory to caspase activation and apoptosis. Also, several metabolic genes, CG31674, CG14740, and CG12170, were identified that may be involved in the general regulation of caspase activation. It has been demonstrated that NADPH produced by the pentose phosphate pathway regulates the activation of caspase-2 in nutrient-deprived Xenopus laevis oocytes. Together with these results, these observations provide further evidence for an intimate link between the regulation of metabolism and induction of apoptosis (Yi, 2007).
To further explore the significance of these findings, whether silencing the mammalian orthologues of the fly genes identified from the RNAi screen confers protection against dox-induced cell death was investigated in mammalian cells. A set of mammalian orthologues was selected that are believed to be nonredundant. The list includes the orthologues of dMiro, which functions as a Rho-like GTPase; dARD1, which functions as an N-acetyltransferase; CG12170, which functions as a fatty acid synthase; and Chn, which functions as a transcriptional repressor (RHOT1, hARD1, OXSM, and REST, respectively; FlyBase). In addition, Plk3, a mammalian orthologue of Polo, was tested since dsRNA targeting polo potently protected against dox treatment (Yi, 2007).
The ability of siRNAs targeting a gene of interest to protect against DNA damage was tested in HeLa cells. As a positive control, cells were transfected with siRNAs targeting Bax or Bak, two central regulators of mammalian cell death. Indeed, silencing of Bax or Bak resulted in significant protection against dox- induced cell death. It was observed that plk3 RNAi provided partial protection against dox treatment, which is consistent with previous studies implicating Plk3 in stress-induced apoptosis. Interestingly, the knockdown of hARD1 dramatically enhanced cell survival in the presence of dox to levels similar to that of Bak. This protective effect was also evident at the morphological level. In cells transfected with a nontargeting control siRNA, dox treatment resulted in typical apoptotic morphology, including cell rounding and membrane blebbing. In direct contrast, cells transfected with siRNAs against hARD1 maintained a normal and healthy morphology and continued to proliferate in the presence of dox (Yi, 2007).
To examine whether the protection provided by siRNAs targeting hARD1 and plk3 is associated with the suppression of caspase activation, caspase activity was measured in these cells treated with dox. RNAi against plk3 provided partial suppression of caspase activity, again supporting the observed protection phenotype. Interestingly, the depletion of REST resulted in some suppression of caspase activity in the presence of dox even though the protection against cell death was not statistically significant. Consistent with the viability assay, complete suppression of caspase-3/7 activity was observed in cells transfected with hARD1 siRNA. These results indicate that hARD1 is required for caspase-dependent cell death induced by DNA damage. Furthermore, all four siRNAs targeting hARD1 were individually capable of providing robust protection against cell death, strongly suggesting that these siRNAs target hARD1 specifically (Yi, 2007).
Because the silencing of hARD1 dramatically suppressed activation of the downstream caspases, whether activation of the upstream caspases in response to dox treatment is also perturbed was also examined. Remarkably, hARD1 RNAi inhibited the cleavage of caspase-2 and -9 in cells treated with dox, whereas caspase cleavage was readily detected in control cells. Thus, it is proposed that hARD1 regulates the signal transduction pathway apical to the apoptotic machinery in the DNA damage response itself or the activation of upstream caspases (Yi, 2007).
Consistent with the results of the caspase-3/7 assay, silencing of hARD1 completely inhibited the appearance of activated caspase-3 induced by dox. This assay was used for a hARD1 complementation experiment to demonstrate the proapoptotic role of hARD1 in response to DNA damage. A new siRNA pool was used, targeting the 5' untranslated region of hARD1 (5'si); this treatment inhibited caspase-3 cleavage induced by dox treatment. Furthermore, caspase-3 cleavage was observed in reconstituted hARD1 knockdown cells. Because six out of six siRNAs against hARD1 provided strong protection against DNA damage-induced apoptosis and complementation of hARD1-sensitized cells to caspase activation, it is concluded that the functional role of ARD1 for dox-induced apoptosis is evolutionally conserved from Drosophila to mammals (Yi, 2007).
In summary, this study used an unbiased RNAi screening platform in Drosophila cells to identify genes involved in promoting DNA damage-induced apoptosis. Forty-seven dsRNAs were isolated that suppress cell death induced by dox. These genes encode for known apoptotic regulators such as Dronc, the Drosophila orthologue of the known proapoptotic transcriptional factor c-Jun, and an ecdysone-regulated protein, Eip63F-1, thereby validating the primary screen. Furthermore, this study implicates a large class of metabolic genes that were previously not suspected to have a role in modulating caspase activation and apoptosis, such as genes involved in fatty acid biosynthesis (CG11798), amino acid/carbohydrate metabolism (CG31674), citrate metabolism (CG14740), complex carbohydrate metabolism (CG10725), and ribosome biosynthesis (CG6712). These results support the proposal that the cellular metabolic status regulates the threshold for activation of apoptosis and thus plays a critical role in the decision of a cell to live or die (Yi, 2007).
Of particular interest is the identification of ARD1. Evidence is presented that RNAi against ARD1 provides protection against cell death and leads to the suppression of caspase activation induced by DNA damage in fly cells and HeLa cells. Furthermore, deficiency in dARD1 renders fly cells resistant to the spontaneous caspase activity and cell death associated with loss of Diap1. Importantly, substantial evidence is provided that hARD1 is required for caspase activation in the presence of DNA damage in mammalian cells. Cleavage of initiator and executioner caspases are suppressed in hARD1 RNAi cells treated with dox, suggesting that hARD1 functions further upstream of caspase activation, and the complementation of hARD1 knockdown cells restores caspase-3 cleavage. These data indicate that ARD1 is necessary for DNA damage-induced apoptosis in flies and mammals (Yi, 2007).
ARD1 functions in a complex with N-acetyltransferase to catalyze the acetylation of the Nα-terminal residue of newly synthesized polypeptides and has been implicated in the regulation of heterochromatin, DNA repair, and the maintenance of genomic stability in yeast. These studies suggest that ARD1 may be involved in regulating an early step in response to DNA damage. It is anticipated that future studies will focus on determining whether ARD1 functions in similar processes in mammals. The diversity of genes identified in this screen illustrates the complex cellular integration of survival and death signals through multiple pathways (Yi, 2007).
To investigate the possible causes of lethality in droncd5, the morphology of various larval organs was examined and acridine orange (AO) staining was used to investigate cell death. Although larger in some animals, no obvious morphological defect in droncd5 larval brain lobes was apparent. However, there was significant reduction in AO staining in droncd5 brain lobes compared to wt, which showed many AO-positive cells. Larval eye discs also displayed a dramatic decrease in cell death compared to wt. It is therefore concluded that the loss of Dronc results in a decrease in cell death in the larval brain lobes and eye discs (Daish, 2004).
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date revised: 25 October 2009
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