Wrinkled/head involution defective
In addition to a post-translational regulation of Hid, the Ras/MAPK pathway
promotes cell survival in Drosophila by downregulating the expression of hid.
Conversely, downregulation of the Ras/MAPK pathway induces cell death by upregulating hid
expression. hid transcript levels are downregulated in dominantly active Dras1- (Dras1Q13) expressing embryos when assayed 3 hr after heat shock. In wild-type embryos, total HID mRNA levels do not change dramatically between stage 11, when Ras expression was ectopically induced, and stage 14, when HID mRNA levels were assayed. This eliminates the concern that developmental arrest might account for the observed difference in HID mRNA levels. It was observed that hid levels return to normal in Dras1Q13 embryos by 5 hr after heat shock. Cell death also resumes in these embryos several hours later. This indicates that a transient increase in Ras activity leads to a transient suppression of hid expression, accompanied by a transient protection from naturally occurring cell death. HID mRNA levels were also assayed through an alternative procedure: whole mount in situ analysis. These results confirm that hid transcript levels decline in dominantly active Dras1- (Dras1Q13) expressing embryos. This is particularly apparent in the midline glia, which strongly express hid. The survival of midline glia is known to depend on the activity of the Epidermal growth factor receptor pathway. To confirm that Ras regulation of hid utilizes the Raf/MAPK pathway, the effect of a constitutively active form of Draf (phlF22) on hid expression has been investigated. In situ analyses were performed on embryos expressing activated Draf under the control of the heat shock promoter. Heat-induced expression of phlF22 results in downregulation of hid transcript levels, suggesting that Ras functions through the Raf/MAPK pathway to downregulate hid expression (Kurada, 1998).
Reduction in pointed (pnt) activity has been observed to enhance ectopic Hid induced cell death in the eye. The pointed transcription factor is a target of MAPK function and acts as a positive regulator in the R7 pathway. The pnt gene encodes two related proteins, pnt1 and pnt2. pnt2 operates downstream of the MAPK rolled in the Ras pathway. Therefore, the consequences of ectopic expression of pnt2 were examined. Embryos were generated that carry UAS-Pnt2 and a midline glia-specific Gal4 driver (52A-Gal4), resulting in the expression of pnt2 in the midline glia cells. Such embryos were tested for hid levels by whole-mount in situ analysis. Like embryos expressing activated Dras1 and activated Draf, pnt2-expressing embryos show decreased hid transcript levels, indicating that the Ras/MAPK pathway, acting through pnt, downregulates hid transcription (Kurada, 1998).
Since upregulation of the Ras/MAPK pathway promotes cell survival and downregulates hid expression, it was predicted that increased hid expression is the cause of the increased apoptosis observed when Ras activity is decreased. Ubiquitous expression of the negative regulator yan is able to induce massive embryonic apoptosis. In these same embryos HID mRNA levels are increased within 2 hr of yanAct induction and continue to rise for many more hours. Thus, downregulation of Ras activity in the embryo results in increased hid transcription and apoptosis, and this transcription is regulated either directly or indirectly by yan. These results imply that Ras activation of MAPK and inactivation of yan is an important cell survival pathway in embryos (Kurada, 1998).
Blocking Epidermal growth factor receptor activity in the developing eye also enhances apoptosis. If hid is a target of Egfr/Ras/MAPK activity in this tissue, then hid levels should increase when Egfr activity is blocked. Expression of a dominant negative Egfr in the developing eye results in a band of increased hid transcription in the eye disc. This band lies several rows posterior to the furrow and corresponds well with the first developmental defects seen in these eye discs. In sum, these data implicate the downregulation of hid transcription as an important component of Egfr antiapoptotic activity. The post-transcriptional modification of Hid appears to be equally important (Kurada, 1998).
The steroid hormone ecdysone signals the stage-specific programmed cell death of the larval salivary glands during Drosophila metamorphosis. This response is
preceded by an ecdysone-triggered switch in gene expression in which the diap2 death inhibitor is repressed and the reaper (rpr) and head involution defective (hid) death activators are induced. rpr is induced directly by the ecdysone-receptor complex through an essential response element in the rpr
promoter. The Broad-Complex (BR-C) is required for both rpr and hid transcription, while E74A is required for maximal levels of hid induction. diap2 induction is
dependent on FTZ-F1, while E75A and E75B are each sufficient to repress diap2. This study identifies transcriptional regulators of programmed cell
death in Drosophila and provides a direct link between a steroid signal and a programmed cell death response (Jiang, 2000).
Although initial studies had indicated that rpr and hid are coordinately induced in the salivary glands approximately 12 hr after puparium formation, more recent work has shown that rpr is induced approximately 1.5 hr earlier than hid, suggesting that these death activators are regulated by distinct mechanisms. The timing of rpr induction is synchronous with the prepupal ecdysone pulse, suggesting that it may be induced as a primary response to the hormone, while the delay in hid induction suggests that it may be a secondary response to ecdysone. These two modes of regulation can be distinguished by their different sensitivity to the inhibition of protein synthesis. Salivary glands were dissected from 10 hr wild-type prepupae and cultured in insect medium supplemented with 20-hydroxyecdysone, either in the presence or absence of cycloheximide. Total RNA was extracted after 0, 2, or 4 hr of culture and analyzed by Northern blot hybridization. Both rpr and hid are induced within 2 hr of hormone treatment, consistent with the proposal that these genes are induced by ecdysone in late prepupal salivary glands. In the presence of the protein synthesis inhibitor cycloheximide, rpr transcription is both delayed and reduced, while hid expression is completely eliminated. These observations indicate that rpr is induced directly by the hormone-receptor complex, although maximal levels of rpr transcription also require the synthesis of ecdysone-induced proteins. In contrast, hid is induced solely as a secondary response to ecdysone. These observations are consistent with the timing of rpr and hid induction in staged salivary glands and provide a framework for defining the molecular mechanisms by which ecdysone regulates rpr and hid transcription, triggering salivary gland cell death (Jiang, 2000).
The BR-C is defined by three genetic functions: broad (br), reduced bristles on palpus (rbp), and l(1)2Bc. Earlier studies have shown that the rbp function of the BR-C is required for salivary gland cell death during metamorphosis. This result has been confirmed by finding that larval salivary glands are not destroyed by 22 hr after puparium formation in pupae that carry the rbp5 null allele. The high penetrance of this mutant phenotype suggests that rpr and hid may not be properly expressed in rbp5 mutant salivary glands (Jiang, 2000).
To test this hypothesis, salivary glands were dissected from staged rbp5 mutants, and rpr and hid expression was examined by Northern blot hybridization. Both rpr and hid transcription is significantly reduced in rbp5 mutant salivary glands, indicating that the failure of salivary gland cell death in this mutant can be attributed to its inability to express these death activators. Both betaFTZ-F1 and the ecdysone-inducible E93 early gene are expressed in rbp5 mutant salivary glands, indicating that the block in rpr and hid transcription is not simply due to developmental arrest of the mutant animals. BR-C is expressed in midprepupal salivary glands and thus would be present in the late prepupal glands used for the cycloheximide experiment described above. This explains why the reduced level of rpr transcription observed in the absence of protein synthesis is not as severe as the rbp5 mutant phenotype (Jiang, 2000).
Both molecular and genetic studies have indicated that the BR-C and E74 function together in common developmental pathways during the onset of metamorphosis. It was therefore asked whether, like the BR-C, E74 might contribute to the ecdysone-triggered destruction of larval salivary glands. In support of this proposal, salivary gland cell death is significantly delayed in E74P[neo] animals. This mutation is a null allele that inactivates the E74A promoter. While salivary glands in control animals are completely destroyed by 16 hr after puparium formation, approximately 20% of E74P[neo]Df(3L)st-81k19 animals have salivary glands at 24 hr after puparium formation. This partially penetrant cell death defect suggests that rpr and hid expression may be reduced in E74A mutant salivary glands. To test this hypothesis, salivary glands were dissected from staged E74P[neo]/Df(3L)st-81k19 mutants, and rpr and hid expression was examined by Northern blot hybridization. Although rpr transcription is unaffected by the E74P[neo] mutation, the levels of hid transcription are significantly reduced. This observation indicates that E74A is required for the maximal induction of hid but not rpr (Jiang, 2000).
The observation that rpr transcription is induced directly by ecdysone in cultured larval salivary glands indicates that one or more EcR/USP binding sites should be present in the rpr promoter. As a first step toward identifying these regulatory elements, the sequences required for ecdysone-inducible rpr transcription in larval salivary glands were mapped. 9.6 kb of the rpr promoter is sufficient to recapitulate certain aspects of the complex pattern of rpr expression during embryogenesis. Four P element constructs were made that carry either 9.5, 6.1, 3.9, or 1.2 kb of DNA upstream from the rpr transcription start site and 125 bp downstream from the transcription start site, with the rpr 5' untranslated region fused to a lacZ reporter gene. These constructs were introduced into the Drosophila germline by P element-mediated transformation, and the patterns of lacZ transcription in staged salivary glands were compared with those of the endogenous rpr gene by Northern blot hybridization. An increased level of rpr promoter activity is seen upon deletion of sequences between -9.5 and -6.1 kb relative to the start site of rpr transcription. The overall level of lacZ transcription is then reduced as more rpr regulatory sequences are deleted. However, 1.3 kb of the rpr promoter is sufficient to direct lacZ induction in synchrony with that of the endogenous rpr gene, indicating that this region contains the sequences required for proper temporal regulation (Jiang, 2000).
DNA fragments from the 1.3 kb rpr promoter region were generated by PCR and tested for their ability bind EcR: a 274 bp fragment extending from -195 bp to +80 bp relative to the rpr transcription start site binds EcR. Sequence analysis has shown a single imperfect palindromic EcR/USP binding site within this fragment. This rpr EcRE matches 10 out of 13 positions with the consensus EcR/USP binding site. The rpr element is not as strong of a binding site as a canonical hsp27 element. This observation is consistent with the deviations from the consensus at positions +2 and +3 in the rpr EcRE. These and other results strongly suggest that the ecdysone-receptor complex directly regulates rpr transcription through at least one binding site in the rpr promoter (Jiang, 2000).
Therefore ecdysone-regulated transcription factors encoded by betaFTZ-F1, BR-C, E74, and E75 function together to direct a burst of the diap2 death inhibitor followed by induction of the rpr and hid death activators. It is proposed that cooperation between rpr and hid allows these genes to overcome the inhibitory effect of diap2, by precisely coordinating when the salivary glands are destroyed. Evidence that the ecdysone-receptor complex directly induces rpr transcription through an essential response element in the promoter, providing a direct link between the steroid signal and a programmed cell death response. The diap2 death inhibitor is expressed briefly in the salivary glands of late prepupae, foreshadowing the imminent destruction of this tissue. This transient expression is directed by at least three ecdysone-regulated transcription factors: betaFTZ-F1, E75A, and E75B. diap2 induction is dependent on the betaFTZ-F1 orphan nuclear receptor. This is consistent with the timing of betaFTZ-F1 expression, which immediately precedes that of diap2, as well as the known role of betaFTZ-F1 as an activator of gene expression in late prepupae (Jiang, 2000).
Steroid hormones trigger dynamic tissue changes during animal development by activating cell proliferation, cell differentiation, and cell death. Steroid regulation of changes have been characterized in midgut structure during the onset of Drosophila metamorphosis. Following an increase in the steroid 20-hydroxyecdysone (ecdysone) at the end of larval
development, future adult midgut epithelium is formed, and the larval midgut is rapidly destroyed. Mutations in the
steroid-regulated genes BR-C and E93 differentially impact larval midgut cell death but do not affect the formation of adult
midgut epithelia. In contrast, mutations in the ecdysone-regulated E74A and E74B genes do not appear to perturb midgut
development during metamorphosis. Larval midgut cells possess vacuoles that contain cellular organelles, indicating that
these cells die by autophagy. While mutations in the BR-C, E74, and E93 genes do not impact DNA degradation during this
cell death, mutations in BR-C inhibit destruction of larval midgut structures, including the proventriculus and gastric caeca,
and E93 mutants exhibit decreased formation of autophagic vacuoles. Dying midguts express the rpr, hid, ark, dronc, and
crq cell death genes, suggesting that the core cell death machinery is involved in larval midgut cell death. The transcription of rpr, hid, and crq are altered in BR-C mutants, and E93 mutants possess altered transcription of the caspase dronc, providing a mechanism for the disruption of midgut cell death in these mutant animals. These studies indicate that ecdysone triggers a two-step hierarchy composed of steroid-induced regulatory genes and apoptosis genes that, in turn, regulate the autophagic death of midgut cells during development (Lee, 2002).
Transcription of rpr, hid, ark, dronc, and crq increases in
wild-type animals following the late larval pulse of ecdysone
that triggers larval midgut cell death. Since
mutations in the BR-C and E93 genes prevent proper
destruction of larval midguts, Northern blots
were prepared from midguts of these mutants at stages
preceding and during cell death. BR-C 2Bc2 mutants have
altered transcription of rpr, hid, and crq, but do not impact
the transcription of ark and dronc. In contrast, E93
mutants possess altered transcription of dronc, but do not
change the transcript levels of the other cell death genes
known to be expressed in dying midguts. Although
midguts die by autophagy, they transcribe core apoptosis
regulators during this cell death, and mutants that prevent
autophagy alter transcription of apoptosis genes (Lee, 2002).
The distributed association of future adult cells within
the epithelium of larval midguts is another
important difference between ecdysone-regulated
midgut and salivary gland programmed cell death. The
close association of larval and adult midgut cells may be
one of the reasons why larval midgut exhibits a less
synchronized cell death than salivary glands. Both salivary
glands and midguts require the function of the E93 and
BR-C genes. However, mutations in these genes appear to
result in different effects in salivary glands and midguts;
BR-C appears to play a more important role in midguts. While both salivary glands and midguts express the cell
death genes rpr, hid, ark, dronc, and crq, the impact of
mutations in BR-C and E93 are very different in the midgut
than in salivary glands. BR-C affects transcription of rpr,
hid, and crq, but E93 mutants only affect dronc transcription
in midguts. In contrast, mutations in E93
prevent proper transcription of all of these cell death genes
in dying salivary glands. Clearly, many
more genes may be involved in the complicated autophagic
cell death of midguts. While several
similarities and differences have been identified between salivary gland and
midgut death, future analyses are needed to clarify the
mechanism by which the steroid ecdysone triggers midgut
programmed cell death (Lee, 2002).
Much of what is known about apoptosis in human cells stems from pioneering genetic studies in the nematode C. elegans. However, one important way in which the regulation of mammalian cell death appears to differ from that of its nematode counterpart is in the employment of TNF and TNF receptor superfamilies. No members of these families are present in C. elegans, yet TNF factors play prominent roles in mammalian development and disease. The cloning and characterization of Eiger, a unique TNF homolog in Drosophila, is described. Like a subset of mammalian TNF proteins, Eiger is a potent inducer of apoptosis. Unlike its mammalian counterparts, however, the apoptotic effect of Eiger does not require the activity of the caspase-8 homolog DREDD, but it completely depends on its ability to activate the JNK pathway. Eiger-induced cell death requires the caspase-9 homolog DRONC and the Apaf-1 homolog DARK. These results suggest that primordial members of the TNF superfamily can induce cell death indirectly by triggering JNK signaling, which, in turn, causes activation of the apoptosome. A direct mode of action via the apical FADD/caspase-8 pathway may have been coopted by some TNF signaling systems only at subsequent stages of evolution (Moreno, 2002).
The mechanism by which JNK signaling triggers cell death in response to TNF is poorly understood in mammals and is unknown in Drosophila. It was therefore of interest to identify the apoptotic machinery responsible for Eiger-induced cell death. Having excluded the caspase-8-like FADD/DREDD branch, focus was placed on the involvement of caspase-9, which represents another major pathway that leads to apoptosis. The key event for caspase-9 activation is its association with the protein cofactor Apaf-1 to form an active complex referred to as the apoptosome. Since many cell intrinsic insults can trigger this pathway, it has been termed the 'intrinsic death pathway'. Expression of a dominant-negative form of the Drosophila caspase-9 homolog DRONC, comprising only the CARD domain, fully blocks Eiger-induced apoptosis in a dose-dependent manner. Moreover, genetic removal of DARK, the homolog of Apaf-1, suppresses Eiger-dependent phenotypes. These results indicate that the presumptive Drosophila apoptosome is essential for the ability of Eiger to induce cell death. In agreement with this conclusion, overexpression of Thread, the Drosophila inhibitor of apoptosis protein 1 (DIAP1/Thread) blocks Eiger function. Thread/DIAP1 has been shown to bind DRONC and target it for degradation. Most instances of programmed cell death that have been analyzed in Drosophila are triggered by, and require, the genes reaper, hid, or grim, which encode small proteins that bind to and inactivate IAPs, such as Thread/DIAP1. The removal of one copy of a chromsosomal segment that includes the genes hid, grim, and reaper rescues eye ablation, and Eiger induces a strong transcriptional activation of hid and a weak activation of reaper. These results suggest, therefore, that Eiger/JNK signaling triggers DRONC by inactivating the IAPs via a transcriptional upregulation of hid (Moreno, 2002).
An important issue in Metazoan development is to understand the mechanisms that lead to stereotyped patterns of programmed cell death. In particular, cells programmed to die may arise from asymmetric cell divisions. The mechanisms underlying such binary cell death decisions are unknown. A Drosophila sensory organ lineage is described that generates a single multidentritic neuron in the embryo. This lineage involves two asymmetric divisions. Following each division, one of the two daughter cells expresses the pro-apoptotic genes reaper and grim and subsequently dies. The protein Numb appears to be specifically inherited by the daughter cell that does not die. Numb is necessary and sufficient to prevent apoptosis in this lineage. Conversely, activated Notch is sufficient to trigger death in this lineage. These results show that binary cell death decision can be regulated by the unequal segregation of Numb at mitosis. This study also indicates that regulation of programmed cell death modulates the final pattern of sensory organs in a segment-specific manner (Orgogozo, 2002).
The vmd1a neuron is located within a cluster of five multidendritic (md) neurons in the ventral region of abdominal segments A1-A7. The vmd1a neuron can be distinguished from the other ventral md neurons (vmd1-4) using the B6-2-25 enhancer-trap marker. The origin of this vmd1a neuron is not known. vmd1-4 neurons are generated by the four vp1-4 external sensory (es) organ primary precursor (pI) cells. Each vp1-4 pI cell follows a lineage called the md-es lineage. This lineage is composed of four successive asymmetric cell divisions that generate five distinct cells, the four cells of the es organ at the position where the pI cell has formed and one md neuron that will then migrate to the ventral md cluster. In the md-es lineage, the membrane-associated protein Numb is segregated into one of the two daughter cells at each cell division. Numb establishes a difference in cell fate by antagonizing Notch in the Numb-receiving cell. Because no es organ is found in the vicinity of the vmd1a neuron, this neuron is probably not generated by a md-es lineage (Orgogozo, 2002).
rpr and grim, but not hid, are expressed specifically in the pIIa and pIIIb cells of the vmd1a lineage. By contrast, these genes are not expressed in cells of the vp1-4 lineages. In embryos in which a pIIb cell divides at the vp1 position in at least one abdominal segment, most segments contain a vmd1a pIIa-pIIb pair with one cell expressing rpr or grim. This cell is the pIIa cell fated to die. In some other segments, neither of these two cells accumulates rpr (25%) or grim (8%). Since the development of segments is not perfectly synchronous, it is assumed that this represents a situation preceding the onset of rpr and grim expression in the pIIa cell. In the remaining segments, a single Cut-positive cell is detected indicating that the pIIa cell has died. In those segments, expression of rpr and grim is never detected in the remaining pIIb cell (Orgogozo, 2002).
During the pIIb division, Numb was shown to segregate into the dorsal pIIb daughter cell. This cell is not fated to die and differentiates as a vmd1a neuron. By contrast, it could not be directly determined which one of the two pI daughter cells inherits Numb. Indeed, since the orientation of the vmd1a pI cell division is random, the pIIa and pIIb cells could not be identified from their relative positions. Nevertheless the vmd1a pIIa and pIIIb cells appear to generate ectopic shaft/socket and neuron/sheath cell pairs when cell death is prevented. In the md-es lineage, these cell pairs are the progeny of the cells that do not inherit Numb. This suggests that both the vmd1a pIIIb cell and the pIIa cell do not inherit Numb. Thus, Numb appears to segregate in the cells that do not die in the vmd1a lineage (Orgogozo, 2002).
The role of Numb was tested in regulating rpr and grim expression as well as cell death in the vmd1a lineage. In numb mutant embryos in which a secondary precursor cell divides at the vp1 position in at least one abdominal segment, it was observed that the two Cut-positive vmd1a pI daughter cells accumulate rpr or grim transcripts (54% of the segments for rpr, 52% for grim). In other segments a single Cut-positive pI daughter cell was found accumulating rpr or grim. In these segments one pI daughter cell has already died and the other one is undergoing apoptosis. These two phenotypes are not seen in wild-type embryos. Thus, in the absence of numb, both pI daughter cells undergo programmed cell death. Consistently, no Cut-positive cell is observed at the vmd1a position in numb mutant embryos in most segments. It is concluded that numb is required to inhibit the expression of rpr and grim and to prevent cell death in the pIIb cell (Orgogozo, 2002).
To test whether numb is sufficient to prevent cell death, the progeny of the vmd1a pI cell was analyzed in arm-Gal4 UAS-numb embryos that express high levels of Numb. In wild-type embryos in which a vp1 pIIIb cell is dividing in at least one segment, one or two Cut-positive cells are observed at the vmd1a position. In contrast, four Cut-positive cells are observed in 50% of the segments in arm-Gal4 UAS-numb embryos at the same stage. In 8 out of the 9 segments with four cells, two cells accumulating high levels of Pros and two cells accumulating low levels of Pros are seen, suggesting that these cells are two vmd1a neurons and two pIIIb cells. These data indicate that the pIIa cell death is inhibited and that the pIIa cell is transformed into a pIIb-like cell (Orgogozo, 2002).
Numb is known to function by antagonizing Notch activity. This therefore suggests that Notch promotes cell death in the vmd1a lineage and that Numb blocks this activity of Notch. Unfortunately, the strong effect of Notch loss-of-function alleles on the selection of the vmd1a pI cell means that it was not possible to test directly whether Notch is required for cell death in the vmd1a lineage. Therefore the conditional Notchts1 allele was used. However, when Notchts1 embryos are shifted to a restrictive temperature (31°C) soon after the specification of the vmd1a pI cell (i.e., at 13-14.5 hours after egg laying at 19°C), no significant reduction was seen in the number of rpr- or grim-expressing pIIa cells. A stronger Notchts1/Notch55e11 combination causes the appearance of additional vmd1a pI cells even at the permissive temperature (19°C). It is therefore not possible to determine whether an increase in the number of rpr- or grim-negative cells results from a lack of Notch-dependent apoptosis or from an excess of vmd1a pI cells due to reduced Notch signaling during lateral inhibition (Orgogozo, 2002).
Therefore a test was performed to see whether an activated form of Notch, Nintra, can promote the death of the pIIb cell when expressed around the time of the vmd1a pI cell division. In 6% of the segments from embryos in which at least one segment shows a dividing vp1 pIIb cell, rpr or grim transcripts accumulate in both vmd1a pI daughter cells. In other segments, a single Cut-positive cell remains at the vmd1a position and accumulates rpr or grim. These expression patterns are not seen in heat-shocked control embryos. Importantly, these observations are similar to those made in numb mutant embryos. Thus, both loss of numb activity and ectopic Notch signaling lead to transcriptional activation of pro-apoptotic genes in the pIIb cell. Finally, a similar effect of Nintra on rpr and grim expression is seen in the vmd1a pIIb daughter cells when Nintra expression was induced at a later stage, i.e., when the vmd1a pIIb cell is dividing. Together, these results indicate that Notch signaling is sufficient to promote cell death in the vmd1a lineage (Orgogozo, 2002).
In summary, the lineage generating the vmd1a neuron has been described. This lineage is composed of two asymmetric divisions following which one daughter cell undergoes apoptosis. These two binary cell death decisions are regulated by the unequal segregation of Numb at mitosis. Therefore, the data provide the first experimental evidence that alternative cell death decision can be regulated by the unequal segregation of a cell fate determinant. The conserved role of Numb and Notch in neuronal specification in flies and vertebrates suggests that Numb-mediated inhibition of Notch may play a similar role in regulating cell death decisions in vertebrates (Orgogozo, 2002).
To discover whether expression of apoptosis activators reaper, grim and hid triggers the accumulation of Death related ced-3/Nedd2-like protein (DREDD) mRNA, the three apoptosis activators were ectopically expressed in mesoderm, and the expression of DREDD mRNA examined. Expression of the apoptosis activators triggers excessive apoptosis in mesoderm. During stage 13 and beyond, DREDD mRNA is not widely expressed in the developing musculature in wild-type flies. However, when misexpression of each of the death activators is directed to these tissues, prominent levels of ectopic DREDD mRNA are detected. Expression of grim in the ectoderm also results in DREDD mRNA accumulation. DREDD mRNA accumulation has also been examined in embryos homozygous for crumbs (crb). In crb mutants, reaper is ectopically expressed in the disorganized epidermis. As anticipated, ectopic accumulation of DREDD mRNA is found scattered throughout the ectoderm in crb embryos, coincident with widespread patterns of rpr expression. Perhaps the most compelling evidence for a direct role for Dredd in apoptosis comes from an examination of accumulation of DREDD mRNA in embryos carrying a homozygous deletion of the entire reaper region (mutated for rpr, hid, and grim). No apoptosis occurs in these deletion mutants. The selective accumulation of DREDD mRNA fails to occur in these mutants. This is the first report of a molecular activity that is completely blocked by the absence of H99-associated signaling (Chen, 1998).
Inhibitor of apoptosis (IAP) proteins suppress apoptosis and inhibit caspases. Several IAPs also function as ubiquitin-protein ligases. Regulators of IAP auto-ubiquitination, and thus IAP levels, have yet to be identified. Head involution defective (Hid), Reaper (Rpr) and Grim downregulate
Drosophila melanogaster IAP1 (DIAP) protein levels. Hid stimulates DIAP1
polyubiquitination and degradation. In contrast to Hid, Rpr and Grim can
downregulate DIAP1 through mechanisms that do not require DIAP1 function as a
ubiquitin-protein ligase. Observations with Grim suggest that one mechanism by which these proteins produce a relative decrease in DIAP1 levels is to promote a general suppression of protein translation. These observations define two mechanisms through which DIAP1 ubiquitination controls cell death: first, increased ubiquitination promotes degradation directly; second, a decrease in global protein synthesis results in a differential loss of short-lived proteins such as DIAP1. Because loss of DIAP1 is sufficient to promote caspase activation, these mechanisms should promote apoptosis (Yoo, 2002).
Inhibitors of apoptosis (IAPs) inhibit caspases, thereby preventing proteolysis
of apoptotic substrates. IAPs occlude the active sites of caspases to which they
are bound and can function as ubiquitin ligases. IAPs are also reported to
ubiquitinate themselves and caspases. Several proteins induce apoptosis, at
least in part, by binding and inhibiting IAPs. Among these are the Drosophila
melanogaster proteins Reaper (Rpr), Grim, and HID, and the mammalian proteins
Smac/Diablo and Omi/HtrA2, all of which share a conserved amino-terminal
IAP-binding motif. Rpr not only inhibits IAP function, but
also greatly decreases IAP abundance. This decrease in IAP levels results from a combination of increased IAP degradation and a previously unrecognized ability of Rpr to repress total protein translation. Rpr-stimulated IAP degradation required both IAP ubiquitin ligase activity and an unblocked Rpr N terminus. In contrast, Rpr lacking a free N terminus still inhibits protein translation. Since the abundance of short-lived proteins are severely affected after translational inhibition, the coordinated dampening of protein synthesis and the ubiquitin-mediated destruction of IAPs can effectively reduce IAP levels to lower the threshold for apoptosis (Holley, 2002).
Trophic mechanisms in which neighboring cells mutually control their survival by secreting extracellular factors play an important role in determining cell number. However, how trophic signaling suppresses cell death is still
poorly understood. The survival of a subset of midline glia cells in Drosophila depends upon direct suppression of the proapoptotic protein Hid via the Egf receptor/RAS/MAPK pathway. The TGF alpha-like ligand Spitz is activated in the neurons, and glial cells compete for limited amounts of secreted Spitz to survive. In midline glia that fail to activate the Egfr pathway, Hid induces apoptosis by
blocking a caspase inhibitor, Diap1. Therefore, a direct pathway linking a specific extracellular survival factor with a caspase-based death program has been established (Bergmann, 2002).
The reduction in midline glia (MG) cell number due to apoptosis, as well as the requirement of the RAS/MAPK pathway for MG survival, has been documented using various MG-specific enhancer trap lines and reporter fusion constructs. The MG are visualized using a reporter fusion construct for the slit gene (sli-lacZ) in which a 1 kb fragment of the slit promoter confers expression specifically to the MG. Using a ß-gal antibody to monitor the developmental profile of the MG, about ten cells per segment expressing sli-lacZ are detectable at midembryogenesis (stage 13). By the end of embryogenesis at stage 17, the number of sli-lacZ-positive cells is reduced to approximately three per segment. Since the sli-lacZ expression is specific for the MG, sli-lacZ-expressing cells are referred to as MG (Bergmann, 2002).
Prominent activation of MAPK has been identified in the MG cells, but its functional role has not been determined. The fate of the MG in mapk-deficient embryos has been analyzed. Compared to wild-type embryos, the initial generation of the MG appears to be normal. However, by stage 17 (the end of embryogenesis), none of the MG in mapk-deficient embryos survive. This finding suggests that MAPK is required for MG survival in wild-type embryos (Bergmann, 2002).
The genetic requirement of mapk for MG survival and of hid for MG apoptosis prompted the assumption that MAPK promotes survival of the MG by inhibition of HID activity. According to this model, the MG would be unprotected from HID-induced apoptosis in mapk-deficient embryos, and die. Consistent with this idea, HID protein is detectable in the MG of late stage wild-type embryos. To test this further, embryos that were mutant for both mapk and hid were examined. In early stage mapk;hid double mutant embryos, the initial generation of the MG appears to be normal. However, in contrast to mapk mutants alone, the MG is rescued in mapk;hid double mutant embryos although the survival function of MAPK is missing in these embryos. Dissection revealed that the MG are located directly at the cuticle of the embryos. Because segmental fusions occur in these embryos, some of the MG cluster in groups of up to 20 cells. In individual segments, five to six MG are visible. This number is larger compared to wild-type (three MG per segment), and is remarkably similar to the number of surviving MG in hid mutant embryos alone, indicating that MAPK promotes MG survival largely through inhibition of HID (Bergmann, 2002).
The mutant analysis revealed that MAPK is required to suppress the activity of HID for MG survival. If hid is mutant in mapk-deficient embryos (i.e., in mapk; hid double mutants), MAPK is no longer needed for the survival of the MG. Thus, MAPK-mediated survival of the MG functions through inhibition of HID (Bergmann, 2002).
In hid mutant embryos there is a 2-fold increase of the MG compared to wild-type. Approximately six MG per segment survive in hid embryos compared to the 2.8 MG per segment in wild-type, indicating that hid is genetically required for MG cell death. MAPK activity is required for MG survival. Does the level of MAPK activity determine the final number of surviving MG cells? Mutational activation of MAPK, using a dominant allele of MAPK termed Sevenmaker or mapkSem, promotes survival of extra MG. About 6.0 MG per segment survive in stage 17 mapkSem embryos, providing additional evidence that MAPK is required for MG survival. Remarkably, the number of surviving MG in mapkSem embryos is very similar to the number of surviving MG in hid mutant embryos. In both cases, approximately six MG survive per segment. Therefore, it was determined whether the six surviving MG in mapkSem embryos correspond to the same MG that survive in hid mutant embryos by double mutant analysis. Stage 17 mapkSem; hid double mutant embryos contain on average 6.6 MG, or slightly more than the single mutants alone. This result strongly suggests that hid expression and MAPK activation occur in largely the same set of MG, that is, in a group of about six MG. If MAPK activation and hid expression would occur in different MG independently of each other, then the mapkSem;hid double mutant would be expected to be the composite of the individual mutants and a total of about ten to twelve MG would survive in the double mutant, similar to what has been observed in H99 mutant embryos. It is inferred from the double mutant analysis that the survival of approximately six MG is regulated by MAPK-dependent inhibition of HID. As long as MAPK is activated, these MG survive (as seen in the activated mapkSem mutant). However, MG in this group that does not maintain activated MAPK are eliminated by HID-induced apoptosis. Thus, the coordinated expression of HID and activation of MAPK regulate the final MG cell number (Bergmann, 2002).
MAPK suppresses hid activity in two ways: via downregulation of its transcription and via phosphorylation of HID protein. However, hid mRNA and protein are readily detectable in the surviving MG of wild-type embryos. Therefore, transcriptional downregulation of hid does not account for MG survival. This prompted a test to see whether inhibitory phosphorylation of HID by MAPK might be critical for MG survival. For this purpose, advantage was taken of an observation that overexpression of HID in the MG using the MG-specific sli-GAL4 driver and UAS-hid transgenes is not sufficient to induce MG apoptosis. Even two copies of the UAS-hid transgenes are not able to ablate the MG. This is contrary to findings in other tissues in which expression of hid induces cell death very well. However, since MAPK is activated in the MG and required for MG survival, it was hypothesized that even overexpressed HID might be inactivated via MAPK phosphorylation (Bergmann, 2002).
To examine this further, UAS-hid transgenes were generated that alter the five phosphoacceptor residues of the MAPK phosphorylation sites to nonphosphorylatable Ala residues (UAS-hidAla5). The UAS-hidAla5 transgenes driven by sli-GAL4 induce apoptosis in the MG very efficiently. One copy of a UAS-hidAla5 transgene is sufficient for the ablation of the MG. Occasionally, some embryos are recovered in which the ablation of the MG is incomplete. However, nerve cord preparations reveal that in these embryos, only a small fraction of the MG survives compared to wild-type. Some segments completely lack MG cells, while others just contain one remaining MG. The MG is required for separation of the commissural axon tracts of the CNS. Consistently, expression of the UAS-hidAla5 transgenes and consequently ablation of the MG causes a fused commissure phenotype. In summary, this analysis demonstrates that MG survival requires suppression of HID activity by MAPK. The MAPK phosphorylation sites in HID are critical for this response, providing an important mechanism for the regulation of MG number (Bergmann, 2002).
Activation of MAPK usually requires activation of RAS, which in turn is activated by receptor tyrosine kinase (RTK) signaling: this demonstrates that MG survival depends on RAS, which is consistent with the model. Within the embryonic CNS, the Drosophila homolog of the Epidermal growth factor receptor (Egfr) is specifically expressed and required for MG differentiation. The requirement of Egfr signaling for MG survival was examined (Bergmann, 2002).
Due to severe developmental defects in egfr mutants, only a few MG start forming at stage 11, and none of them survive. Thus, it is difficult to directly study the requirement of the Egfr for MG survival. To overcome this problem, a dominant-negative mutant of the Egfr (UAS-EgfrDN) was expressed in the MG using the sli-GAL4 driver in otherwise wild-type embryos. In this way, Egfr activity is specifically diminished in the MG after their generation. As expected, the MG form normally in these embryos. However, most of the MG die during subsequent developmental stages and only a few survive to the end of embryogenesis, indicating a direct requirement of the Egfr for MG survival. To determine whether the MG death in this experimental condition is due to failure to inhibit HID, EgfrDN was expressed in the MG of hid mutants. In this genetic background, on average 6.1 MG cells survive, demonstrating that MG survival requires functional Egfr signaling to suppress HID activity (Bergmann, 2002).
The spi gene is required for MG survival and encodes a candidate trophic factor for MG survival. To prove that spi function is required to suppress hid activity, the fate of the MG in spi;hid double mutant embryos was determined. The MG survive in spi embryos if hid is removed as well, and it is concluded that the survival function of spi is mediated through suppression of hid-induced apoptosis (Bergmann, 2002).
The question arises as to which cells process mSPI and provide a source of sSPI for MG survival. Since spi is ubiquitously expressed, it is difficult to determine histochemically where sSPI, the active ligand, is generated. Therefore, a genetic approach was used; whether the loss of MG in spi mutant embryos can be rescued by expression of the membrane bound inactive precursor (UAS-mSPI) either in the MG (using the sim-GAL4 driver) or in neuronal axons (using the elav-GAL4 driver) was examined. It was reasoned that the MG would be rescued in spi mutant embryos only if mSPI is presented in the location where it is normally processed for MG survival in wild-type embryos. Presentation of mSPI by the MG itself does not result in rescue of the MG in spi mutant embryos, ruling out an autocrine mechanism. In contrast, expression of mSPI in neuronal axons appears to be sufficient for MG survival in spi embryos. This argues in favor of a paracrine mechanism. In control experiments, sSPI was examined using these two Gal4 drivers in wild-type embryos. With both GAL4 drivers an increase in the number of MG cells is detected, indicating that they are expressed at the right time and that the MG does not fail to secrete Spi once it has been processed (Bergmann, 2002).
A key regulator of Spi activation is rhomboid, a gene encoding a cell surface, seven-pass transmembrane protein that appears to function as a serine protease directly cleaving mSPI. rhomboid has been implicated in suppression of MG apoptosis. Ectopic expression of Rho in neurons (elav-Gal4/UAS-Rhomboid) promotes an excess of MG, suggesting that neurons have the capacity to process endogenous mSPI. Another essential protein for Spi processing is Star. Star mutants display an MG phenotype similar to spi. STAR regulates intracellular trafficking of mSPI. Expression of Star from the neurons but not from the MG rescues the Star phenotype in the MG. Thus, this analysis clearly demonstrates that the sSPI signal for MG survival is generated and secreted by neurons (Bergmann, 2002).
The surviving MG in late stage embryos are in close contact to commissural axons. In embryos lacking the commissureless (comm) gene, the commissural axons are absent. In comm embryos the MG die prematurely, and some survivors become misplaced laterally along the longitudinal axon tracts. The location of the MG along the longitudinal axons in comm mutant embryos as well as their close contact to commissural axons in wild-type embryos has prompted the suggestion that axon contact is required for MG survival. Axon contact appears to permit the MG to respond to trophic signaling, which is necessary for its survival. Consistent with this notion trophic signaling provided by sSPI/Egfr is present only in MG associated with longitudinal axons in comm;hid double mutants. Thus, it was asked whether axon contact-mediated Egfr signaling in the MG is required to activate MAPK, which in turn suppresses the cell death-inducing ability of HID (Bergmann, 2002).
To address this question, the fate of the MG was examined in comm mutant embryos which are at the same time mutant for hid (comm;hid double mutants) or carry the dominant active mapk allele, mapkSem (mapkSem;comm double mutants). Strikingly, a substantial number of the MG survive even in the absence of axonal contact if hid function is removed or if MAPK is activated. This strongly suggests that axon contact is necessary to suppress HID via MAPK. Only MG in proximity to neurons undergo Egfr signaling. The additional MG that survive along the midline in comm;hid mutants do not express spry, that is, do not receive an Egfr signal, and survive only because hid is absent in this experimental condition (Bergmann, 2002).
It is noted that active MAPK is capable of rescuing a total of six MG based on analysis of mapkSem embryos. Presumably, this MAPK activation in mapkSem embryos is inherited from the differentiation period of the MG. However, only three MG survive by stage 17 in wild-type embryos. It is proposed that of the group of six MG that require MAPK for survival, only the three surviving cells make adequate axon contacts necessary to receive sufficient quantities of the survival factor sSPI. According to this model, the remaining three MG die because they lose the competition for axon contact and do not receive levels of sSPI that are high enough to inactivate HID via phosphorylation by MAPK. If additional sSPI is provided in the midline, additional MG can be rescued. The limited availability of axon-derived sSPI would serve to match the number of MG to the length of commissural axons requiring ensheathment. Thus, the regulation of MG number and survival represents a genetically defined example of the classical trophic theory of cell survival (Bergmann, 2002).
The regulation of MG apoptosis in Drosophila bears striking overall similarity to the regulation of glial cell death in the rat optic nerve. There is an early dependence of the oligodendroglia in the rat optic nerve on growth factors for differentiation followed by a dependence on axon contact for survival. However, it is not clear how the oligodendroglia in the rat optic nerve survive upon axon contact. Since mammalian homologs for many of the components in the apoptotic pathway both upstream and downstream of Drosophila HID are known, it will be interesting to analyze whether similar molecules regulate apoptosis and cell number in the mammalian nervous system. Therefore, molecular genetic studies in Drosophila promise considerable insight for advancing an understanding of the basic control mechanisms involved in the regulation of apoptosis in the context of a developing organism in vivo (Bergmann, 2002).
E2F transcription factors are generally believed to be positive regulators of apoptosis. This study shows that dE2F1 and dDP are important for the normal pattern of DNA damage-induced apoptosis in Drosophila wing discs. Unexpectedly, the role played by E2F varies depending on the position of the cells within the disc. In irradiated wild-type discs, intervein cells show a high level of DNA damage-induced apoptosis, while cells within the D/V boundary are protected. In irradiated discs lacking E2F regulation, intervein cells are largely protected, but apoptotic cells are found at the D/V boundary. The protective effect of E2F at the D/V boundary is due to a spatially restricted role in the repression of hid. These loss-of-function experiments demonstrate that E2F cannot be classified simply as a pro- or anti-apoptotic factor. Instead, the overall role of E2F in the damage response varies greatly and depends on the cellular context (Moon, 2005).
One of the major difficulties in studying the biological functions of E2F is that E2F complexes affect the expression of a large number of genes and can act in a variety of different ways. It is difficult to assess the overall role of E2F regulation in a given process by studying an individual E2F gene, or a single E2F target. The rate-limiting targets for E2F function most likely vary from context to context, and they may not always be the usual suspects. In the D/V boundary of the developing wing disc, in which E2F/DP complexes protect from DNA damage-induced apoptosis, E2F/DP proteins are needed specifically to limit the expression of hid. Remarkably, the loss of E2F/DP leads to an upregulation of hid in this one part of the disc. This change occurs prior to irradiation, and it alters the cellular sensitivity to DNA damage. No apoptosis was found in unirradiated dDP mutant wing discs, implying that the change in hid expression in the dDP mutant wing disc is not, by itself, sufficient to induce apoptosis. The elevated hid expression is clearly important, because reducing the gene dosage of hid almost completely eliminated DNA damage-induced apoptosis in dDP mutant discs, but not in wild-type discs (Moon, 2005).
What is the connection between dE2F1 and hid? Since dE2F1 binds to sequences upstream of the hid transcription start site, the transcription of hid is most likely reduced by the direct action of E2F complexes. Previous studies in mammalian cells have shown that E2F1 can directly repress transcription of some E2F1-specific targets, although the mechanisms underlying these effects are not well understood. The dE2F1 binding site upstream of hid has two interesting features that may be significant. (1) Unlike most dE2F-regulated promoters that have been examined to date, this binding site is bound specifically by dE2F1, but not by dE2F2. This specificity fits with the genetic evidence that de2f1, rather than de2f2, is important for protection from DNA damage-induced apoptosis, and it may explain why hid is not generally repressed by dE2F2 complexes. (2) Another curious feature is that the dE2F1 binding site upstream of hid is surprisingly distal from the transcription start site. In most E2F-induced promoters, E2F binding sites are typically within 500 bp of the transcriptional start site. The position of the E2F1 binding site in the hid promoter, at −1.4 kb, may be part of the reason why hid differs from other dE2F1 targets and is not activated in a cell cycle-dependent manner. It is noted that the pattern of hid expression that sensitizes cells to apoptosis in dDP mutants occurs in the absence of E2F regulation; therefore, dE2F1 does not directly contribute to the pattern itself, but it presumably serves to interfere with another transcription factor (Factor X) that is specifically active within this region. As the hid promoter is largely uncharacterized, the possibility that dE2F1 may act through additional sites or that it may also repress hid expression via an indirect mechanism cannot be excluded. In order to test this model, it will be necessary to identify the factors that control hid expression in vivo (Moon, 2005).
A simple model is presented for the context-specific effects of dE2F1. In the intervein region, dE2F1 increases the expression of proapoptotic genes. In doing so, dE2F1 helps set a level of sensitivity for DNA damage-induced apoptosis, and this threshold is reduced when dE2F1 or dDP are removed. At the D/V boundary, dE2F1/dDP complexes are also needed, most likely in conjunction with RBF, to limit the expression of hid. When E2F regulation is removed, the increase in hid expression outweighs the changes in expression of other E2F targets, making cells more sensitive to apoptosis (Moon, 2005).
If E2F's contribution to the DNA damage response varies in mammalian cells as much as it does in Drosophila, then this would have implications for the use of general E2F inhibitors that are currently under development. These results suggest that a global inhibitor of E2F activity, or even a specific inhibitor of activator E2Fs, may have the unintended consequence of making some normal cell types very sensitive to DNA damage-induced apoptosis (Moon, 2005).
Growth of tissues and organs during animal development involves careful coordination of the rates of cell proliferation and cell death. Cell proliferation depends on signals to stimulate cell growth and cell division. In addition, cells compete for intercellular survival signals which are required to prevent them from undergoing apoptosis in response to growth stimuli. How these cellular processes are coordinated with pattern formation during animal development is a challenging question in developmental biology. The bantam gene of Drosophila has been found to encode a 21 nucleotide microRNA (miRNA) that promotes tissue growth. bantam expression is temporally and spatially regulated in response to patterning cues. bantam microRNA simultaneously stimulates cell proliferation and prevents apoptosis. The pro-apoptotic gene hid has been identified as a target for regulation by bantam miRNA, providing an explanation for bantam's anti-apoptotic activity (Brennecke 2003).
The three proapoptotic genes hid, reaper, and grim downregulate levels of the IAP proteins in Drosophila, thereby preventing caspase activation. Unlike reaper and grim, whose activity appears to be regulated primarily at the transcriptional level, hid mRNA is also detected in cells that do not undergo apoptosis. Evidence has been presented for transcriptional regulation of hid and for posttranslational regulation of Hid activity by the MAPK signaling pathway. By showing that bantam blocks the activity of Hid(Ala5), which is insensitive to MAPK regulation, an indirect effect of bantam mediated by regulation of the MAPK pathway is excluded. The hid 3'UTR confers bantam-mediated regulation on a heterologous reporter. These findings provide evidence that hid is subject to translational regulation in vivo by the bantam miRNA (Brennecke 2003).
hid is known to play an important role in regulating apoptosis in eye development. Removing one copy of the endogenous bantam gene enhances the severity of the Hid-induced apoptosis phenotype in the eye, whereas the severity of the reaper-induced apoptosis phenotype is affected much less strongly. Similarly, overexpression of bantam suppresses both the GMR-hid and GMR-reaper phenotypes, but has a stronger effect on hid. The severity of the GMR-reaper phenotype is sensitive to the levels of hid activity. By overexpressing bantam, Hid levels are reduced, providing an explanation for the observed suppression of the GMR-reaper phenotype. Similarly, by removing one copy of bantam an increase in endogenous hid activity in the eye would be expected. By altering the level of Hid, bantam can indirectly alter the threshold for reaper-induced apoptosis. This provides an explanation for the slight increase in severity of the GMR-reaper phenotype observed. There are no bantam target sites in the reaper gene, suggesting that bantam's effect on the GMR-reaper phenotype must be indirect. Finally, no increase in apoptosis was observed in bantam mutant clones in the wing disc. Endogenous hid has not been implicated in developmental control of cell death in the wing (Brennecke 2003).
MicroRNAs are small noncoding RNAs that control gene function posttranscriptionally through mRNA degradation or translational inhibition. Much has been learned about the processing and mechanism of action of microRNAs, but little is known about their biological function. Injection of 2′O-methyl antisense oligoribonucleotides (2'OM-ORNs) into early Drosophila embryos leads to specific and efficient depletion of microRNAs and thus permits systematic loss-of-function analysis in vivo. Twenty-five of the forty-six embryonically expressed microRNAs show readily discernible defects; pleiotropy is moderate and family members display similar yet distinct phenotypes. Processes under microRNA regulation include cellularization and patterning in the blastoderm, morphogenesis, and cell survival. The largest microRNA family in Drosophila (miR-2/6/11/13/308) is required for suppressing embryonic apoptosis; this is achieved by differential posttranscriptional repression of the proapoptotic factors hid, grim, reaper, and sickle. These findings demonstrate that microRNAs act as specific and essential regulators in a wide range of developmental processes (Leaman, 2005).
miR-2/13 and miR-6 depletion results in catastrophic apoptosis: Embryos injected with miR-2/13 and miR-6 antisense 2′OM-ORNs fail to differentiate normal internal and external structures. At the end of embryogenesis, the embryos fall apart on touch, and no cuticle is recovered. To determine the onset of these problems, blastoderm embryos were examined, and it was found that cellularization and early pattern formation along the anteroposterior axis occur normally for both miRNAs, indicating that early fating and morphogenesis are intact. Interestingly, in miR-6, but not miR-2/13 depleted embryos, pole cell formation at the posterior end is disrupted (Leaman, 2005).
One possible cause of the catastrophic defects observed in miR-2/13 and miR-6 depleted embryos is excessive and widespread apoptosis. In both miR-2/13 and miR-6 antisense injected embryos, the number of apoptotic cells is greatly increased compared to wild-type by stage 13. Notably, the overall morphology of miR-6 depleted embryos is much more affected than that of miR-2/13 depleted embryos. miR-6 depleted embryos are generally smaller in size and have fewer and abnormally large (para-) segments, suggesting greater excess or earlier onset of apoptosis (Leaman, 2005).
To determine the specificity of the effects of miR-6 and miR-2/13 antisense injections, genomic rescue experiments were carried out. Embryos ubiquitously overexpressing mir-6 or mir-2 (Actin-Gal4;UAS-mir6-3/2b-2) show normal cell-death patterns. When injected with miR-6 or miR-2/13 antisense, they show significant rescue of miR-6 antisense by mir-6, with respect to both cell death and morphology, and of miR-2/13 antisense by mir-2. Interestingly, crossrescue of miR-6 antisense by mir-2 overexpression and of miR-2/13 antisense by mir-6 is weak (Leaman, 2005).
The miRNA sequence family miR-6 and miR-2/13 belong to has two additional members, miR-11 and miR-308. Depletion of miR-11 results in a moderate and of miR-308 in a mild increase in apoptosis in midembryogenesis. Thus, for all members of the miR-2 family, antisense-induced depletion results in excess embryonic cell death, but with marked differences in phenotypic strength. This differential could be due to differences in expression level or to sequence divergence and thus differential interaction with target mRNAs (Leaman, 2005).
The miR-2 family regulates cell survival by translational repression of proapoptotic factors: In Drosophila, three pathways are known to control caspase activity. The main control is thought to come from the proapoptotic factors Hid, Grim, and Reaper (Rpr), which are transcriptionally activated in response to a range of natural and toxic conditions; they promote caspase activation through inhibition of the caspase inhibitor Diap1. The three factors appear to act independently, with each being sufficient to drive apoptosis. When miR-2/13 and miR-6 antisense 2′OM-ORNs are injected into embryos deficient for the hid, grim, and rpr genes (H99 deficiency), they are unable to trigger apoptosis, indicating that these miRNAs act through hid, grim, and/or rpr (Leaman, 2005).
To determine whether the regulation of the three proapoptotic factors occurs at the transcriptional or at the posttranscriptional level, their RNA expression was examined in miR-2/13 and miR-6 depleted embryos using in situ hybridization and quantitative PCR. No significant increase was found in the expression level or broadening of the pattern compared to control embryos for any of three transcripts, either at embryonic stage 13 or 1 hr earlier at embryonic stage 12. By contrast, the protein expression of Hid is dramatically increased in miR-6 depleted embryos and modestly in miR-2/13 depleted embryos. These results strongly argue against a transcriptional and in favor of a posttranscriptional regulation of the proapoptotic factors by miR-2/13 and miR-6 (Leaman, 2005).
To test this directly, two existing translation control assays were adapted to the embryonic paradigm. In the first assay, full-length 3′UTRs are fused to a ubiquitously transcribed sensor (tub-GFP); transgenic embryos are injected with sense or antisense 2′OM-ORNs, and GFP fluorescence is measured. The 3′UTRs of hid, grim, rpr, and sickle (skl, a structurally related but less potent proapoptotic factor display marked differences in sensor expression, with rpr showing no expression, hid and skl low uniform expression, and grim strong and spatially modulated expression, indicating that these proapoptotic factors experience quite different levels of translation control. To gauge the efficacy of the assay, hid GFP sensor embryos were injected with bantam antisense 2′OM-ORNs, and mild but statistically significant derepression of GFP expression was found as compared to control, consistent with the weak cell-death phenotype of bantam depleted embryos. Antisense injection of miR-2 family members reveals strong derepression of the hid GFP sensor by miR-6 antisense, but not by miR-2/13, 11, or 308 antisense. Conversely, the grim GFP sensor shows significant derepression as a result of miR-2/13, 11, and 308, but not miR-6 depletion. Finally, the skl GFP sensor shows significant derepression for all four family members (Leaman, 2005).
To assess effects on rpr, a second, more sensitive assay was developed that employs transient expression of a dual-luciferase vector in injected embryos. For initial comparison with the GFP assay, a hid luciferase sensor was tested against the entire miR-2 family and the same profile was found. The rpr luciferase sensor shows strong derepression in miR-6 and 2/13, moderate derepression in miR-11, and no significant effect in miR-308 depleted embryos. Thus, the 3′UTRs of all four proapoptotic factors are subject to translational repression by the miR-2 family, but each miRNA displays a distinct interaction profile. The interaction preferences correlate well with the observed differences in phenotype: miR-6 has the most severe death phenotype and is the only family member to regulate hid, the factor with the broadest expression and the strongest proapoptotic effect. mir-2/13 and miR-11 have the same overall profile, but they differ in the strength of their interaction with rpr and show a corresponding differential in phenotypic strength. Finally, miR-308, which has the mildest death phenotype, interacts only with the weakly proapoptotic skl and with grim (Leaman, 2005).
The differences in target interaction profile between the miR-2 family members are pronounced and do not merely reproduce differences in the strength or onset of miRNA expression. This suggests that differential pairing outside the 5′ core sequence shared by all members has an important role in target selection. Computational predictions indicate that miR-2 family binding sites are present in the 3′UTRs of all four proapoptotic factors: rpr and grim have one, hid and skl two predicted sites. All six miRNA target sites lie in sequence blocks that are conserved between the six sequenced Drosophilid species, spanning an evolutionary distance of 40 Myr. Interestingly, for all sites, absolute conservation extends well beyond the bases complementary to the 5′ core of the miRNA and includes adjacent stretches suitable for pairing with the 3′ end. All but one of the sites show Watson-Crick pairing with miRNA positions 2-7 and variable pairing at the 3′ end. One of the hid sites (hid468) has a mismatch in the core but shows strong pairing with miR-6 at the 3′ end. The rules for 3′ pairing between miRNAs and their targets are not yet well understood, but it is clear that the miR-2 family members differ considerably in their ability to form 3′ matches with the six target sites. Further experimentation will be required to better understand how the observed differences in regulatory effect relate to differences in sequence pairing (Leaman, 2005).
Although mitochondrial proteins play well-defined roles in caspase activation in mammalian cells, the role of mitochondrial factors in caspase activation in Drosophila is unclear. Using cell-free extracts, it is demonstrated that mitochondrial factors play no apparent role in Drosophila caspase activation. Cytosolic extract from apoptotic S2 cells, in which caspases were inhibited, induced caspase activation in cytosolic extract from normal S2 cells. Mitochondrial extract does not activate caspases, nor does it influence caspase activation by cytosolic extract. Silencing of Hid, Reaper, or Grim reduces caspase activation by apoptotic cell extract. Furthermore, a peptide representing the amino terminus of Hid is sufficient to activate caspases in cytosolic extract, and this activity is not enhanced by addition of mitochondria or mitochondrial lysate. The Hid peptide also induces apoptosis when introduced into S2 cells. These results suggest that caspase activation in Drosophila is regulated solely by cytoplasmic factors and does not involve any mitochondrial factors (Means, 2006).
Exposure of phosphatidylserine is a conserved feature of apoptotic cells and is thought to act as a signal for engulfment of the cell corpse. A putative receptor for phosphatidylserine (PSR) was previously identified in mammalian systems. This receptor is proposed to function in engulfment of apoptotic cells, although gene ablation of PSR has resulted in a variety of phenotypes. The role of the predicted Drosophila homolog of PSR (phosphatidylserine receptor;
dPSR) in apoptotic cell engulfment was examined and no obvious role for dPSR in apoptotic cell engulfment by phagocytes was found in the embryo. In addition, dPSR is localized to the nucleus, inconsistent with a role in apoptotic cell recognition. However, it was surprisind to find that overexpression of dPSR protects from apoptosis, while loss of dPSR enhances apoptosis in the developing eye. The increased apoptosis is mediated by the head involution defective (Wrinkled) gene product. In addition, the data suggest that dPSR acts through the c-Jun-NH2 terminal kinase pathway to alter the sensitivity to cell death (Krieser, 2007).
Evidence against a role for PSR in engulfment also comes
from two other knockout models and from data on the localization of the
protein. One of the reported mouse knockouts showed no difference in
engulfment of apoptotic cells by macrophages in the mutant, although
PSR-/- macrophages were generally inhibited in their release of
pro- and anti-inflammatory cytokines. In addition, fibroblast lines established from
PSR-/- embryos showed no defect in apoptotic cell engulfment or in
their response to apoptotic cells. Zebrafish lacking PSR accumulated dead cells, but were not definitively shown to have defects in apoptotic cell engulfment. Finally,
localization data from the current work and from a number of labs strongly supports a
nuclear localization for the protein. This is not consistent with a role for PSR as a surface receptor for the recognition for apoptotic cells, although PS could
theoretically modulate the activity of this protein within the cell (Krieser, 2007).
The observations support a role for dPSR in cell survival. In zebrafish,
reduction of PSR resulted in an increase in the number of apoptotic cells
present during development. In particular, the brains of these fish were shrunken and
had an increase in apoptotic cells. In two of the mouse knockout models an
increase in apoptotic cells was detected. However, all three knockouts resulted in perinatal lethality, with defects in differentiation in a variety of tissues. It is
speculated that defects in engulfment detected in some of the gene ablation models could reflect a role for PSR in the proper differentiation of macrophages. Increased apoptosis seen in the current studies and by others might also be due to defects in proper differentiation in the absence of PSR (Krieser, 2007).
What insight can be gained from these studies into the function of dPSR in
differentiation and cell survival? Increased dPSR results
in a cell survival phenotype that is suppressed by activation of the JNK
pathway, while loss of dPSR results in apoptosis, activated by the
cell death regulator Hid, a known target of JNK activation in apoptosis.
Taken together, these data suggest that dPSR may normally act to suppress JNK
activation of Hid-induced apoptosis (Krieser, 2007).
JNK activation is important for many processes in cells, including cell
death, proliferation and differentiation. A role for JNK in apoptosis was
found in many mammalian cell types. Data from mouse knockouts of JNK also suggest a role for JNK in proliferation and differentiation. In
addition, JNK activation in dying cells is required for proliferative signals
originating from apoptotic cells in Drosophila.
Interestingly, defects in proliferation and differentiation of many tissues
were observed in mice that lack PSR. Taken together with observations of increased cell
death in dPSR mutant flies, these observations suggest that some of
the phenotypes seen in mouse and fish models of PSR gene ablation might be due
to inappropriate activation of the JNK pathway (Krieser, 2007).
Based on genetic assays, it is proposed that one function of dPSR is to
suppress Hid activation. Flies that lack dPSR show increased
apoptosis in the developing pupal eye, which is suppressed in the absence of
hid, while overexpression of dPSR results in ectopic cell survival.
Hid function is required for the death of the interommatidial cells in the
pupal retina. The results also showed that expression of dPSR can inhibit
death induced by the expression of Hid- or Grim in the eye, and that loss of
dPSR enhances Rpr-, Hid- or Grim-induced death in the eye. Interestingly, loss of one copy of hid can also suppress cell death induced by Rpr or Hid expression in the eye. Therefore alterations of dPSR levels in the eye may be altering Hid activity to modify the Grim- and Rprinduced eye phenotypes (Krieser, 2007).
JNK activation has been shown to increase hid activity.
However, Hid activity is also modulated by activation of the Ras/Erk pathway. Ras
activation results in the survival of ectopic interommatidial cells, through
the downregulation of Hid activity. Ectopic Ras activation also results in genital
rotation defects, similar to those seen with dPSR overexpression. This suggests that PSR overexpression might activate the Ras/Erk pathway. Based on the current data, it is not clear whether dPSR might activate Ras and thus suppress JNK activity, whether dPSR could suppress JNK and thus activate Ras, or whether dPSR might act independently in an opposing manner on the JNK and Ras pathways (Krieser, 2007).
By examining the function of dPSR in the Drosophila system, new insight has been provided into the controversy regarding this protein.
Although no evidence was found that this protein plays a role in engulfment,
it is important in cell survival. This is consistent with
phenotypes seen in gene ablation models in other organisms. Furthermore, dPSR affects the JNK pathway, and this may provide a clue as to its diverse functions in mammals (Krieser, 2007).
Drosophila embryos are highly sensitive to gamma-ray-induced apoptosis at early but not later, more differentiated stages during development. Two proapoptotic genes, reaper and hid, are upregulated rapidly following irradiation. However, in post-stage-12 embryos, in which most cells have begun differentiation, neither proapoptotic gene can be induced by high doses of irradiation. The sensitive-to-resistant transition is due to epigenetic blocking of the irradiation-responsive enhancer region (IRER), which is located upstream of reaper but is also required for the induction of hid in response to irradiation. This IRER, but not the transcribed regions of reaper/hid, becomes enriched for trimethylated H3K27/H3K9 and forms a heterochromatin-like structure during the sensitive-to-resistant transition. The functions of histone-modifying enzymes Hdac1(Rpd3) and Su(var)3-9 and PcG proteins Su(z)12 and Polycomb are required for this process. Thus, direct epigenetic regulation of two proapoptotic genes controls cellular sensitivity to cytotoxic stimuli (Zhang, 2008).
Irradiation responsiveness appears to be a highly conserved feature of reaper-like IAP antagonists. A recently identified functional ortholog of reaper in mosquito genomes, michelob_x (mx), is also responsive to irradiation. These results highlighted that stress responsiveness is an essential aspect of functional regulation of upstream proapoptotic genes such as reaper/hid. It is also worth mentioning that several mammalian BH3 domain-only proteins, the upstream proapoptotic regulators of the Bcl-2/Ced-9 pathway, are also regulated at the transcriptional level (Zhang, 2008).
This study shows that the irradiation responsiveness of reaper and hid is subject to epigenetic regulation during development. The epigenetic regulation of the IRER is fundamentally different from the silencing of homeotic genes in that the change of DNA accessibility is limited to the enhancer region while the promoter of the proapoptotic genes remains open. Thus, it seems more appropriate to refer this as the 'blocking' of the enhancer region instead of the 'silencing' of the gene. This region, containing the putative P53RE and other essential enhancer elements, is required for mediating irradiation responsiveness. ChIP analysis indicates that histones in this enhancer region are quickly trimethylated at both H3K9 and H3K27 at the sensitive-to-resistant transition period, accompanied by a significant decrease in DNA accessibility. DNA accessibility in the putative P53RE locus (18,368k), when measured by the DNase I sensitivity assay, did not show significant decrease until sometime after the transition period. It is possible that other enhancer elements, in the core of IRER_left, are also required for radiation responsiveness. An alternative explanation is that the strong and rapid trimethylation of H3K27 and association of PRC1 at 18,366,000-18, 368,000 are sufficient to disrupt DmP53 binding and/or interaction with the Pol II complex even though the region remains relatively sensitive to DNase I. Eventually, the whole IRER is closed with the exception of an open island around 18,387,000 (Zhang, 2008).
The finding that epigenetic regulation of the enhancer region of proapoptotic genes controls sensitivity to irradiation-induced cell death may have implications in clinical applications involving ionizing irradiation. It suggests that applying drugs that modulate epigenetic silencing may help increase the efficacy of radiation therapy. It also remains to be seen whether the hypersensitivity of some tumors to irradiation is due to the dedifferentiation and reversal of epigenetic blocking in cancer cells. In contrast, loss of proper stress response to cellular damage is implicated in tumorigenesis. The fact that the formation of heterochromatin in the sensitizing enhancer region of proapoptotic genes is sufficient to convey resistance to stress-induced cell death suggests it could contribute to tumorigenesis. In addition, it could also be the underlying mechanism of tumor cells' evading irradiation-induced cell death. This is a likely scenario given that it has been well documented that oncogenes such as Rb and PML-RAR fusion protein cause the formation of heterochromatin through recruiting of a human ortholog of Su(v)3-9. In this regard, the reaper locus, especially the IRER, provides an excellent genetic model system for understanding the cis- and trans-acting mechanisms controlling the formation of heterochromatin associated with cellular differentiation and tumorigenesis (Zhang, 2008).
The developmental consequence of epigenetic regulation of the IRER is the tuning down (off) of the responsiveness of the proapoptotic genes, thus decreasing cellular sensitivity to stresses such as DNA damage. Epigenetic blocking of the IRER corresponds to the end of major mitotic waves when most cells begin to differentiate. Similar transitions were noticed in mammalian systems. For instance, proliferating neural precursor cells are extremely sensitive to irradiation-induced cell death while differentiating/differentiated neurons become resistant to γ-ray irradiation, even though the same level of DNA damage was inflicted by the irradiation. These findings here suggest that such a dramatic transition of radiation sensitivity could be achieved by epigenetic blocking of sensitizing enhancers (Zhang, 2008).
Later in Drosophila development, around the time of pupae formation, the organism becomes sensitive to irradiation again, with LD50 values similar to what was observed for the 4–7 hr AEL embryos. Interestingly, it has also been found that during this period, the highly proliferative imaginal discs are sensitive to irradiation-induced apoptosis, which is mediated by the induction of reaper and hid through P53 and Chk2. However, it remains to be studied whether the reemergence of sensitive tissue is due to reversal of the epigenetic blocking in the IRER or the proliferation of undifferentiated stem cells that have an unblocked IRER (Zhang, 2008).
The blocking of the IRER differs fundamentally from the silencing of homeotic genes in several aspects. (1) The change of DNA accessibility and histone modification is largely limited to the enhancer region. The promoter regions of reaper (and hid) remain open, allowing the gene to be responsive to other stimuli. Indeed, there are a few cells in the central nervous system that could be detected as expressing reaper long after the sensitive-to-resistant transition. Even more cells in the late-stage embryo can be found having hid expression. Yet, the irradiation responsiveness of the two genes is completely suppressed in most if not all cells, transforming the tissues into a radiation-resistant state (Zhang, 2008).
(2) The histone modification of the IRER has a mixture of features associated with pericentromeric heterochromatin formation and canonic PcG-mediated silencing. Both H3K9 and H3K27 are trimethylated in the IRER. Both HP1, the signature binding protein of the pericentromeric heterochromatin, and PRC1 are bound to the IRER. As demonstrated by genetic analysis, the functions of both Su(var)3-9 and Su(z)12/Pc are required for the silencing. Preliminary attempts to verify specific binding of PRC2 proteins to this region were unsuccessful. The fact that none of the mutants tested could completely block the transition seems to suggest that there is a redundancy of the two pathways in modifying/blocking the IRER. It is also possible that the genes tested are not the key regulators of IRER blocking but only have participatory roles in the process (Zhang, 2008).
(3) Within the IRER, there is a small region around 18,386,000 to 18,188,000 that remains relatively open until the end of embryogenesis. Interestingly, this open region is flanked by two putative noncoding RNA transcripts represented by EST sequences. If they are indeed transcribed in the embryo as suggested by the mRNA source of the cDNA library, then the 'open island' within the closed IRER will likely be their shared enhancer/promoter region. Sequences of both cDNAs revealed that there is no intron or reputable open reading frame in either sequence. Despite repeated efforts, their expression was not confirmed via ISH or northern analysis. Overexpression of either cDNA using an expression construct also failed to show any effect on reaper/hid-induced cell death in S2 cells. Yet, sections of the two noncoding RNAs are strongly conserved in divergent Drosophila genomes. The potential role of these two noncoding RNAs in mediating reaper/hid expression and/or blocking of the IRER remains to be studied (Zhang, 2008).
Drosophila Reaper (Rrp), Head involution defective (Hid), and Grim induce caspase-dependent cell death and physically interact with the cell death inhibitor
DIAP1. Hid blocks Diap1's ability to inhibit caspase activity and evidence is provided suggesting that Rpr and Grim can act similarly. Based on
these results, it is proposed that Rpr, Hid, and Grim promote apoptosis by disrupting productive IAP-caspase interactions and that Diap1 is required to block
apoptosis-inducing caspase activity. Supporting this hypothesis, it is shown that elimination of Diap1 function results in global early embryonic cell death and a large
increase in Diap1-inhibitable caspase activity and that Diap1 is still required for cell survival when expression of rpr, hid, and grim is eliminated (Wang, 1999).
Induction of apoptosis in Drosophila requires the activity of three closely linked genes: reaper, hid and grim. The proteins encoded by reaper, hid
and grim activate cell death by inhibiting the anti-apoptotic activity of the Drosophila IAP1 (Diap1, also known as Thread) protein. In a genetic modifier screen, both loss-of-function and
gain-of-function alleles in the endogenous diap1 gene were obtained, and the mutant proteins were functionally and biochemically characterized. Gain-of-function
mutations in diap1 strongly suppress reaper-, hid- and grim-induced apoptosis. Sequence analysis of these diap1 alleles reveals that they are caused by single amino
acid changes in the baculovirus IAP repeat domains of Diap1, a domain implicated in binding Reaper, Hid and Grim. Significantly, the corresponding mutant
Diap1 proteins display greatly reduced binding of Reaper, Hid and Grim, indicating that Reaper, Hid and Grim kill by forming a complex with Diap1. Collectively, these data provide strong support for the idea that Reaper, Hid and Grim kill by
inhibiting DIAP1's ability to antagonize caspase function (Goyal, 2000).
It is thought that the previously
proposed function of IAPs upstream of reaper, hid and grim is simply an artifact of unphysiologically high levels of protein expression in heterologous systems.
When IAP expression constructs are introduced into cultured cells under the control of strong promoters and at high copy numbers, the levels of proteins expressed
far exceed those of the endogenous cellular IAP proteins. Under these unphysiological conditions, cellular IAPs can display properties that do not reflect their normal
mechanism of action. In particular, the current results demonstrate that mutant proteins that completely lack anti-apoptotic
activity in vivo can still inhibit cell death in vitro as long as they can bind to Reaper, Hid and Grim. Conversely, gain-of-function diap1 alleles
that display reduced binding to Reaper, Hid and Grim have strongly increased anti-apoptotic function in vivo, but show reduced protection in
heterologous cell transfection assays. These results clearly reveal the limitations of overexpression studies in cultured cells for determining the normal
mechanism of action of these proteins in the cell death pathway (Goyal, 2000).
Dronc (Nedd2-like caspase) was isolated through its interaction with the effector caspase drICE. Ectopic
expression of Dronc induces cell death in Schizosaccharomyces pombe, mammalian fibroblasts and the developing Drosophila eye.
The caspase inhibitor p35 fails to rescue Dronc-induced cell death in vivo and is not cleaved by Dronc in vitro, making Dronc
the first identified p35-resistant caspase. The Dronc pro-domain interacts with Drosphila inhibitor of apoptosis protein 1 (Diap1: known as Thread),
and co-expression of DIAP1 in the developing Drosophila eye completely reverts the eye ablation phenotype induced by pro-Dronc
expression. In contrast, Diap1 fails to rescue eye ablation induced by Dronc lacking the pro-domain, indicating that interaction of Diap1 with the pro-domain of
Dronc is required for suppression of Dronc-mediated cell death. Heterozygosity at the Diap1 locus enhances the pro-Dronc eye phenotype, consistent with
a role for endogenous Diap1 in suppression of Dronc activation. Both heterozygosity at the Dronc locus and expression of dominant-negative Dronc mutants
suppress the eye phenotype caused by Reaper (Rpr) and Head involution defective (Hid), consistent with the idea that Dronc functions in the Rpr and Hid
pathway (Meier, 2000).
The finding that Diap1 directly binds to and inhibits cell death caused by ectopic expression of Dronc, as well as by Rpr, Grim and Hid, underscores the key
role played by Diap1 in the regulation of apoptosis in D. melanogaster and raises the possibility that Rpr, Hid or Grim may exert some, or all, of their
pro-apoptotic action through displacement of Diap1 from the pro-domain of Dronc, thereby allowing activation of the caspase and consequent cell death. This idea is strongly supported by the successful isolation of Diap1 mutants that display greatly reduced binding for Rpr, Hid and Grim and significantly suppress Rpr, Hid and Grim cell killing. According to this model, IAPs function as 'guardians' of the apoptotic machinery: they act to suppress the chance of
spontaneous activation of the intrinsic cell death machinery by neutralizing pro-apoptotic caspases, thereby establishing a buffered threshold that must be either exceeded
or neutralized in order to initiate the destruction of a cell (Meier, 2000).
The proapoptotic genes reaper (rpr), grim, and head involution defective (hid) are required for virtually all embryonic apoptosis. The proteins encoded by these genes share a short region of homology at their amino termini. The Drosophila IAP homolog Thread/Diap1 (Th/Diap1) negatively regulates apoptosis during development. It has been proposed that Rpr, Grim, and Hid induce apoptosis by binding and inactivating TH/Diap1. The region of homology between the three proapoptotic proteins has been proposed to bind to the conserved BIR2 domain of TH/Diap1. An analysis of loss-of-function and gain-of-function alleles of th indicates that additional domains of Th/Diap1 are necessary to allow th to inhibit death induced by Rpr, Grim, and Hid. In addition, analysis of loss-of-function mutations demonstrates that th is necessary to block apoptosis very early in embryonic development. This may reflect a requirement to block maternally provided Rpr and Hid, or it may indicate another function of the Th/Diap1 protein (Lisi, 2000).
Several mechanisms of action have been suggested for the antiapoptotic properties of the IAP family of proteins. Among these are the binding of the Drosophila IAPs to the proapoptotic proteins Rpr, Grim, and Hid. This interaction has been demonstrated in overexpression systems, and has been proposed to involve the homologous amino-terminal 14 amino acid sequences of the apoptosis initiators with the second BIR domain of the IAPs. The data presented here suggest that this is an oversimplification. Another mechanism that has been proposed for IAP antiapoptotic activity is the direct binding and inhibition of caspases. Th/Diap1 binds to the Drosophila caspases drICE and DCP-1 and functions to inhibit their ability to induce apoptosis. Here again, this binding activity appears to rest within BIR2 (Lisi, 2000).
These physical interactions support a simple model of IAP action. In this model, IAPs act within viable cells to inhibit caspase function. The action of Rpr, Hid, and Grim interferes with the ability of IAPs to inhibit caspases, thus inducing apoptosis. On the basis of the model, the LOF mutations identified in this study would be predicted to interfere with the ability of the Th/Diap1 protein to inhibit caspase function. This is likely to be true for th109.07, which lacks most of the protein, as well as for th5 and th4, which affect conserved residues in BIR2. BIR2 is sufficient to inhibit apoptosis induced by the active form of the Drosophila caspase drICE. The th9 mutation in BIR1 suggests that this BIR is also important for the full function in caspase inhibition. Alternatively, this change in BIR1 might have long-range effects on BIR2 structure or on protein stability (Lisi, 2000).
It is interesting to note that th7, which acts as a very strong LOF mutation and seems to show some dominant-negative properties, has only the BIR1 attached to the spacer and ring domains. Thus, despite the extensive homologies between the two BIR domains of the protein, a single BIR is not sufficient for Th/Diap1 function, at least in the presence of an attached ring domain. BIR2 of Th/Diap1 and Op-IAP, as well as the single BIR of survivin, are able to inhibit apoptosis (Lisi, 2000).
Again, on the basis of the model above, the GOF mutations identified would be predicted to bind to caspases, but not to the inducers. The thSL mutation maps to a weakly conserved residue in BIR1 and does not result in increased th protein levels. This suggests that BIR1 is important for Rpr and Grim binding, but not for Hid binding, as Hid activity is unaffected in this mutation. Even in the context of overexpression in the eyes of transgenic flies, this mutant IAP retains some specificity for Rpr and Grim killing. This implies that the simple model of BIR2 binding to the conserved NH2-terminal sequences of Rpr, Grim, and Hid is not accurate, and that other residues in the protein are differentially important for Rpr and Grim, as opposed to Hid binding (Lisi, 2000).
The importance of regions outside of BIR2 for Diap1 activity is supported by the analysis of the GOF1 class of mutations, th6B and th81.03. Both of these mutations suppress Hid killing and would be predicted to inhibit Hid binding. These mutations change conserved cysteines in the ring domain to tyrosines. This suggests that the ring is important for Hid/Diap1 interaction. However, the region of Hid binding to Diap1 and Op-IAP has been mapped to BIR2, while the ring does not show any ability to bind to Hid. In addition, mutations in the ring, including those in conserved cysteines, have little effect on the ability of Op-IAP to protect against Hid killing. These data, together with the finding that both GOF1 mutations are cysteine-to-tyrosine changes, suggest that these mutations might have a novel ability to interfere with binding of Hid to BIR2. In addition, the observation that the GOF1 mutations slightly enhance Rpr and Grim killing suggests that these mutants are less potent inhibitors of caspases. This might result from weaker binding to caspases or from proteins that are slightly less stable. This second attribute would be predicted to enhance killing by any inducer that binds the IAP, but not to have an effect on Hid, which is unable to bind (Lisi, 2000 and references therein).
In conclusion, the data support a model where Rpr, Grim, and Hid interact with Th/Diap1 to induce apoptosis. Mutations that affect killing by Rpr and Grim or by Hid can be isolated, indicating that these inducers interact with Th/Diap1 in different ways. The GOF mutations that have been identified also provide useful tools to examine the roles of IAPs, rpr, grim, and hid during Drosophila development. The other Drosophila IAP homolog, DIAP2, has been shown to selectively inhibit Rpr- and Hid-induced but not Grim-induced death (Lisi, 2000).
In LOF th alleles, a developmental arrest occurs at the blastoderm stage and, subsequently, a synchronous apoptosis of all the nuclei. Earlier reports that homozygous th embryos show no ectopic apoptosis probably reflects the very early stage at which this apoptosis occurs. At this time, a direct requirement for th to block apoptosis cannot be distinguised from a requirement for th in another developmental process. This developmental defect could then result in secondary apoptosis. The latter possibility is reasonable, as many failures in development result in ectopic apoptosis. A BIR containing protein from Caenorhabditis elegans is required for cytokinesis in embryos. However, it is also possible that developmental arrest occurs as a result of the initiation of apoptosis, which is manifest only as DNA damage several hours later (Lisi, 2000).
Does this early requirement for th reflect a need to inhibit apoptosis induced by rpr, grim, and hid? Double mutants of th and Df(3L)H99, the deletion that removes rpr, grim, and hid, show a phenotype similar to th alone. This indicates that Th/Diap1 is not required to suppress zygotic Rpr, Grim, and Hid activity. However, hid and rpr mRNA can be seen in a subset of cells in the blastoderm embryo, as judged by in situ analysis. This may indicate that these gene products are supplied maternally. Th/Diap1 may be required to suppress maternally supplied Rpr, Grim, or Hid. Allelic differences in the stage at which apoptosis begins in the th mutants parallel the general ability of the alleles to inhibit apoptosis induced by Rpr, Hid, and Grim. The strong LOF alleles arrest at the blastoderm stage; the GOF1 alleles arrest much later, and the GOF2 allele is completely viable (Lisi, 2000).
Activation of Ras inhibits apoptosis during Drosophila development. Genetic evidence indicates that Ras antiapoptotic
activity in the developing eye is regulated by the Drosophila EGF receptor and operates through the Raf/MAPK
pathway. Decreased activity of this pathway enhances (and increased activity suppresses) apoptosis induced by
ectopic expression of the cell death regulators reaper (rpr) and head involution defective (hid). In addition, ectopic
activation of the Ras/MAPK pathway in the developing embryo and in the developing eye suppresses naturally
occurring apoptosis and regulates the transcription of the proapoptotic gene hid. Null alleles of hid recapitulate the
antiapoptotic activities of Ras/MAPK, providing genetic evidence that downregulation of hid is an important
mechanism by which Ras promotes survival (Kurada, 1998).
Extracellular growth factors are required for the survival of most animal cells. They often signal through the activation
of the Ras pathway. However, the molecular mechanisms by which Ras signaling inhibits the intrinsic cell death
machinery are not well understood. Evidence is presented that in Drosophila, activation of the Ras pathway
specifically inhibits the proapoptotic activity of the gene hid. By using transgenic animals
and cultured cells, it has been shown that MAPK phosphorylation sites in Hid are critical for this response. These findings
define a novel mechanism by which growth factor signaling directly inactivates a critical component of the intrinsic cell
death machinery. These studies provide further insight into the function of ras as an oncogene (Bergmenn, 1998).
Reaper (Rpr), Hid, and Grim activate apoptosis in cells programmed to die during Drosophila development. Transient overexpression of Rpr in the lepidopteran SF-21 cell line induces apoptosis. Members
of the inhibitor of apoptosis (IAP) family of antiapoptotic proteins can inhibit Rpr-induced apoptosis and physically interact
with Rpr through the BIR (baculovirus IAP repeat) motifs of IAP family members. Transient overexpression of HID and GRIM also induces apoptosis in the
SF-21 cell line. Baculovirus and Drosophila IAPs block HID- and GRIM-induced apoptosis and also physically interact
with them through the BIR motifs of IAP family members. The region of sequence similarity shared by Rpr, Hid, and Grim (the
N-terminal 14 amino acids of each protein) is required for the induction of apoptosis by Hid and its binding to IAPs. When
stably overexpressed by fusion to an unrelated, nonapoptotic polypeptide, the N-terminal 37 amino acids of Hid and Grim are sufficient to induce apoptosis and confer IAP binding activity. However, Grim is more complex than HID since the
C-terminal 124 amino acids of Grim retain apoptosis-inducing and IAP binding activity, suggesting the presence of two
independent apoptotic motifs within Grim. Coexpression of IAPs with Hid stabilizes Hid levels and results in the
accumulation of Hid in punctate perinuclear locations that coincide with IAP localization. The physical interaction of IAPs
with Rpr, Hid, and Grim provides a common molecular mechanism for IAP inhibition of these Drosophila proapoptotic
proteins (Vucic, 1998).
Drosophila genes reaper, grim, and head-involution-defective (hid) induce apoptosis in several cellular contexts.
N-terminal sequences of these proteins are highly conserved and are similar to N-terminal inactivation domains of
voltage-gated potassium (K+) channels. Synthetic Reaper and Grim N terminus peptides induce fast inactivation of
Shaker-type K+ channels when applied to the cytoplasmic side of the channel. This inactivation is qualitatively similar to the
inactivation produced by other K+ channel inactivation particles. Mutations that reduce the apoptotic activity of Reaper
also reduced the synthetic peptide's ability to induce channel inactivation, indicating that K+ channel inactivation
correlates with apoptotic activity. Coexpression of Reaper mRNA or direct injection of full length Reaper protein causes
near irreversible block of the K+ channels. These results suggest that Reaper and Grim may participate in initiating
apoptosis by stably blocking K+ channels (Avdonin, 1998).
The Drosophila reaper, head involution defective (hid), and grim genes play key roles in regulating the
activation of programmed cell death. Two useful systems for studying the functions of these genes are
the embryonic CNS midline and adult eye. In this study the Gal4/UAS targeted gene
expression system was used to demonstrate that unlike reaper or hid, expression of grim alone is sufficient to
induce ectopic CNS midline cell death. In both the midline and eye, grim-induced
cell death is not blocked by the Drosophila anti-apoptosis protein Diap2, which does block both reaper-
and hid-induced cell death. grim can also function synergistically with either reaper or hid to induce higher
levels of midline cell death than those observed for any of the genes individually. Analysis was made of the
function of a truncated Reaper-C protein, which lacks the NH2-terminal 14 amino acids that are
conserved between Reaper, Hid, and Grim. Ectopic expression of Reaper-C reveals cell killing
activities distinct from full length Reaper, and indicates that the conserved NH2-terminal domain acts
in part to modulate Reaper activity (Wing, 1998).
Reaper, Hid, and Grim are three Drosophila cell death activators that each contain a conserved NH2 -terminal Reaper-Hid-Grim (RHG)
motif. The importance of the RHG motifs in Reaper and Grim have been examined for their different abilities to activate cell death during
development. Analysis of chimeric R/Grim and G/Reaper proteins indicates that the Reaper and Grim RHG motifs are functionally distinct
and help to determine specific cell death activation properties. A truncated GrimC protein lacking the RHG motif retains an ability to induce
cell death, and unlike Grim, R/Grim, or G/Reaper, its actions are not efficiently blocked by the cell death inhibitors Diap1, Diap2, p35, or a
dominant/negative Dronc caspase. Finally, a second region of sequence similarity was identified in Reaper, Hid, and Grim, that may be
important for shared RHG motif-independent activities (Wing, 2001).
While the Grim-Reaper proteins do not contain defined structural domains, they
each share sequence similarity in the 14 amino acids at
their NH2-termini. This RHG motif is
most similar between Reaper and Grim (71.4% identity),
and least similar between Hid and Grim (21.4% identity).
The RHG motif plays a key role in interactions between
Grim-Reaper proteins and members of the Inhibitor-of-Apoptosis-Protein (IAP) family, including Drosophila
Diap1 and Diap2. Like other IAPs, Diap1 and Diap2 both
contain related baculovirus IAP repeat (BIR) motifs, as well
as a Really Interesting New Gene (RING) finger. Diap1 is an essential cell death
regulator and diap1 mutants exhibit early embryonic lethality due to massive ectopic cell death. The functions of Diap2
in regulating cell death are less clear; however, it does share
a number of functional properties with Diap1. Diap1 can
directly bind caspases and repress their proteolytic activities. Significantly, caspase inhibition by Diap1 is antagonized by Hid, suggesting
a double-repression model where the Grim-Reaper proteins
promote cell death by binding to Diaps, suppressing
their ability to inhibit caspases. Recent studies have indicated
that the vertebrate Diablo/SMAC protein also promotes cell
death activation by binding to IAPs and suppressing their
death inhibitory activities. Thus, IAP suppression may be an evolutionarily
conserved cell death regulatory mechanism. In this regard,
while Grim-Reaper orthologs have not been identified, the
expression of each protein can induce vertebrate cells to die, implying that they may suppress vertebrate IAPs (Wing, 2001).
Diap1, like Diap2, exhibits distinct abilities to repress cell death
induced by Reaper, Hid, or Grim. In the CNS midline,
Diap1 more effectively blocks Grim-induced cell death
than cell death induced by Reaper and Hid. In contrast,
when examined in the adult eye, Diap1 is most effective
at blocking Reaper-induced cell death, moderately effective
against Hid, and ineffective against Grim. Similar results
were obtained using the thsl
gain-of-function diap1 mutant allele, which represses Reaper-induced
eye cell death more effectively than death induced by Hid or
Grim. Importantly, these data indicate that Diap1 has
distinct, tissue-specific effects on cell death induced by
Grim-Reaper proteins, and that these effects differ from
those of Diap2. The basis for these functional distinctions
are not yet clear. One possibility is that the associations
between each Diap and Grim-Reaper protein may differ
in strength, or be influenced by specific ancillary factors.
Differences have been noted between Diap1 and Diap2 in
their ability to bind and repress the actions of certain
caspases, and Reaper, Hid, and Grim
can act through different downstream caspases. Taken together, these findings suggest potentially
complex functional interactions between Grim-Reaper
proteins, Diaps, and caspases. It is likely that distinct activities of individual Grim-Reaper and Diap proteins provide
enhanced capabilities for regulating cell death processes in
different developmental and physiological contexts (Wing, 2001).
Do Reaper, Hid, and Grim share RHG-independent functions? Both truncated ReaperC and GrimC proteins induce cell death in developing tissues, indicating that regions outside the RHG motif also have death-inducing activities. Surprisingly, it was found that cell death induced by GrimC or ReaperC is only partially repressed by p35, suggesting a distinct mode of action compared with native Reaper or Grim. Similar to Reaper, Hid and Grim, GrimC does apparently act through the p35-insensitive caspase, Dronc, as GrimC-induced death is partially suppressed by a dominant/negative DroncC318S protein. However, the persistence of some eye cell death in the presence of DroncC318S indicates that GrimC and ReaperC also act through alternate pathways. Perhaps GrimC acts through pro-apoptotic Drosophila Bcl-2 orthologs that may induce cell death which is not blocked by p35. Another interesting possibilty is that GrimC might act via a Drosophila ortholog of Scythe, a Xenopus cell death regulator that binds Reaper, Hid, and Grim independently of the RHG motif (Wing, 2001 and references therein).
A second region of sequence similarity, the 30 amino acid Trp-block, has been identified that is present once in Reaper and Grim, and four times in Hid. The Trp-blocks may be important for the cell death activation capabilities of GrimC and ReaperC, as well as for potentially shared RHG motif-independent activities of native Grim-Reaper proteins. This
additional sequence similarity also suggests a modular organization of the Grim-Reaper proteins, where distinct functions may be afforded by the RHG motif and Trp-block. Taken together, the sequence similarities of the Grim-Reaper proteins, as well as the organization and chromosomal location of the corresponding genes, imply that the grim-reaper genes arose from duplication of a common ancestor and have diverged to assume overlapping yet
distinct cell death activation functions. It will be of interest
to determine the representation of grim-reaper orthologs in
other species, information that could provide important insights into the evolution of cell death control mechanisms. This is of particular relevance given that inhibition of IAP activity is likely to constitute a conserved mechanism to regulate cell death activation (Wing, 2001).
The inhibitor of apoptosis protein DIAP1 suppresses apoptosis in Drosophila, with the second BIR domain (BIR2) playing an important role. Three proteins, Hid, Grim, and Reaper, promote apoptosis, in part by binding to DIAP1 through their conserved N-terminal sequences. The crystal structures of DIAP1-BIR2 by itself and in complex with the N-terminal peptides from Hid and Grim reveal that these peptides bind a surface groove on DIAP1, with the first four amino acids mimicking the binding of the Smac tetrapeptide to XIAP. The next 3 residues also contribute to binding through hydrophobic interactions. Interestingly, peptide binding induces the formation of an additional alpha helix in DIAP1. This study reveals the structural conservation and diversity necessary for the binding of IAPs by the Drosophila Hid/Grim/Reaper and the mammalian Smac proteins (Wu, 2001).
Many members of the inhibitor of apoptosis (IAP) family of proteins suppress programmed cell death, at least in part, by physically interacting with and inhibiting the catalytic activity of caspases. An important functional unit in all death-inhibiting IAP proteins is the so-called baculoviral IAP repeat (BIR), which contains approximately 80 amino acids folded around a zinc atom. The Drosophila genome contains four genes that encode proteins with BIR domains. The overexpression of two of these, DIAP1 and DIAP2, inhibit both normal developmental cell death and apoptosis induced by expression of proapoptotic genes. In addition, DIAP1 is required for cell survival in the embryo and in a number of adult tissues. These observations, in conjunction with others showing that DIAP1 binds and inactivates several Drosophila caspases and that loss of DIAP1 results in an increase in caspase activity in vivo, argue that DIAP1's function as a caspase inhibitor is required for cell survival. DIAP1 contains two N-terminal BIR repeats and a C-terminal RING domain. DIAP1 fragments containing the BIR2 domain are sufficient to prevent cell death in a number of contexts. Interestingly, fragments consisting of the BIR2 and surrounding linker sequences also bind multiple proapoptotic proteins, including the apical caspase DRONC, and Hid, Grim, and Reaper (Wu, 2001 and references therein).
One mechanism by which Hid, Grim, and Reaper promote cell death is by binding to DIAP1, thereby inhibiting its function as a caspase inhibitor. Although Hid, Grim, and Reaper perform a similar function in promoting cell death, they only share homology in the N-terminal 14 residues of their primary sequences. These N-terminal sequences are sufficient to mediate interactions with DIAP1 and with several mammalian IAPs. In the case of Hid in insects, and Hid and Reaper in mammalian cells, these N-terminal sequences are essential for proapoptotic function (Wu, 2001 and references therein).
In mammalian cells, caspase inhibition by IAPs is negatively regulated by a mitochondrial protein Smac/DIABLO, which is released from the mitochondrial intermembrane space into the cytosol upon apoptotic stimuli. Smac/DIABLO physically interacts with multiple IAPs and relieves their inhibitory effect on both initiator and effector caspases. Thus, Smac/DIABLO represents the mammalian functional homolog of the Drosophila Hid, Grim, and Reaper proteins. Recent structural studies reveal that the N-terminal tetrapeptide of Smac/DIABLO binds a surface groove on XIAP-BIR3, thus competitively removing the inhibition of caspase-9 by XIAP. Smac/DIABLO shares sequence homology with Hid, Grim, and Reaper only in the N-terminal 4 residues, prompting the hypothesis that Hid, Grim, and Reaper interact with DIAP1 using similar tetrapeptides and binding to a similar surface groove on DIAP1 (Wu, 2001 and references therein).
There is currently no structural information on DIAP1 or Hid, Grim, or Reaper. To investigate the structural mechanisms of DIAP1 recognition by the Drosophila Hid, Grim, and Reaper proteins, the DIAP1-BIR2 domain was crystalized by itself and in complex with the N-terminal peptides from both Hid and Grim (these structures were determined at 2.7, 2.7, and 1.9 Angstrom resolution, respectively). By analogy to the Smac-XIAP interactions, the first four amino acids of Hid and Grim bind an evolutionarily conserved surface groove on DIAP1-BIR2. The next 3 conserved residues of Hid and Grim also contribute to the interactions with DIAP1 through extensive van der Waals contacts. Interestingly, peptide binding to DIAP1-BIR2 appears to induce the formation of an additional alpha helix, which appears to stabilize peptide binding. In conjunction with biochemical analysis, this structural study reveals a molecular basis for the conservation and diversity necessary for the recognition of IAPs by the Drosophila Hid/Grim/Reaper and the mammalian Smac proteins. These results have important ramifications for the design of IAP inhibitors toward therapeutic applications (Wu, 2001).
It has been suggested that the Drosophila Hid protein interacts with the baculovirus Op-IAP protein in a manner similar to that of human Smac binding to XIAP, based largely on amino acid sequence homology. The interaction between Hid and Op-IAP has been precisely mapped; the biochemical interactions between the amino terminus of Hid and BIR2 of Op-IAP are highly similar to those found between the processed amino terminus of Smac and BIR3 of XIAP. Also similar to Smac, the amino terminus of Hid must be processed to bind Op-IAP. In addition, the data also suggest that a second interaction between Hid and Op-IAP exists that does not involve the amino terminus of Hid. The evolutionary conservation of this mechanism of binding underscores its importance in apoptotic regulation. Nevertheless, interaction with Hid is not sufficient for Op-IAP to inhibit apoptosis induced by Hid overexpression or by treatment with actinomycin D, indicating that additional sequence elements are required for the anti-apoptotic function of Op-IAP (Wright, 2002).
Members of the IAP family block activation of the intrinsic cell death machinery by binding to and neutralizing the activity of pro-apoptotic caspases. In Drosophila melanogaster, the pro-apoptotic proteins Reaper Rpr, Grim and Hid all induce cell death by antagonizing the anti-apoptotic activity of Drosophila IAP1 (DIAP1), thereby liberating caspases. In vivo, the RING finger of DIAP1 is essential for the regulation of apoptosis induced by Rpr, Hid and Dronc. Furthermore, the RING finger of DIAP1 promotes the ubiquitination of both itself and of Dronc. Disruption of the DIAP1 RING finger does not inhibit its binding to Rpr, Hid or Dronc, but completely abrogates ubiquitination of Dronc. These data suggest that IAPs suppress apoptosis by binding to and targeting caspases for ubiquitination (Wilson, 2002).
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