To understand the role apoptosis plays in nervous system development and to gain insight into the mechanisms by which steroid hormones regulate neuronal apoptosis, the death of a set of peptidergic neurons was investigated in the CNS of Drosophila. Typically, apoptosis in Drosophila is induced by the expression of the genes reaper, grim, or head involution defective (hid). Genetic evidence is provided that the death of these neurons requires reaper and grim gene function. Consistent with this genetic analysis, these doomed neurons accumulate reaper and grim transcripts prior to the onset of apoptosis. These neurons also accumulate low levels of hid, although the genetic analysis suggests that hid may not play a major role in the induction of apoptosis in these neurons. The death of these neurons is dependent on the fall in the titer of the steroid hormone 20-hydroxyecdysone that occurs at the end of metamorphosis: the accumulation of both reaper and grim transcripts is inhibited by this steroid hormone. These observations support the notion that 20E controls apoptosis by regulating the expression of genes that induce apoptosis (Draizen, 1999).
Hormones and trophic factors provide cues that control neuronal death during development. These developmental cues in some way regulate activation of apoptosis, the mechanism by which most, if not all, developmentally programmed cell deaths occur. In Drosophila, apoptosis can be induced by the expression of the genes reaper, grim, or head involution defective. Prior to the death of a set of identifiable doomed neurons, these neurons accumulate transcripts of the reaper and grim genes, but do not accumulate transcripts of the head involution defective gene. Death of these doomed neurons can be suppressed by two manipulations: either by increasing the levels of the steroid hormone 20-hydroxyecdysone (see Ecdysone receptor) or by decapitation. The impact that these two manipulations have on reaper expression has been investigated. Steroid treatment prevents the accumulation of reaper transcripts, whereas decapitation results in the accumulation of lower levels of reaper transcripts that are not sufficient to activate apoptosis. These data demonstrate that in vivo, reaper, and grim transcripts accumulate coordinately in a set of identified doomed neurons prior to the onset of apoptosis. These observations raise the possibility that products of the reaper and grim genes act in concert in postembryonic neurons to induce apoptosis. That reaper transcript accumulation is regulated by the steroid hormone titer and by the presence of the head is evidence that developmental factors control programmed cell death by regulating the expression of genes that induce apoptosis (Robinow, 1997).
The Drosophila compound eye is formed by selective recruitment of undifferentiated cells into clusters called ommatidia during late larval and early pupal development. Ommatidia at the edge of the eye often lack the full complement of photoreceptors and support cells, and undergo apoptosis during mid-pupation. This cell death is triggered by the secreted glycoprotein Wingless, which activates its own expression in peripheral ommatidia via a positive feedback loop. Wingless signaling elevates the expression of the pro-apoptotic factors head involution defective, grim and reaper, which are required for ommatidial elimination. It is estimated that approximately 6%-8% of the total photoreceptor pool in each eye is removed by this mechanism. In addition, the retinal apoptosis previously reported in apc1 mutants occurs at the same time as the peripheral ommatidial cell death and also depends on head involution defective, grim and reaper. The implications of these findings for eye development and function in Drosophila and other organisms is considered (Lin, 2004).
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).
In Drosophila, the induction of apoptosis requires three closely linked genes, reaper, head involution defective, and grim. The products of these genes induce apoptosis by activating a caspase pathway. Two very similar Drosophila caspases, DCP-1 and drICE, have been previously identified. DCP-1 has a substrate specificity that is remarkably similar to that of human caspase 3 and Caenorhabditis elegans CED-3, suggesting that DCP-1 is a death effector caspase. drICE and DCP-1 have similar yet different enzymatic specificities. Although expression of either in cultured cells induces apoptosis, neither protein is able to induce DNA fragmentation in Drosophila SL2 cells. Ectopic expression of a truncated form of dcp-1 (DeltaN-dcp-1) in the developing Drosophila retina under an eye-specific promoter results in a small and rough eye phenotype, whereas expression of the full-length dcp-1 (fl-dcp-1) has little effect. However, expression of either full-length drICE (fl-drICE) or truncated drICE (DeltaN-drICE) in the retina shows no obvious eye phenotype. Although active DCP-1 protein cleaves full-length DCP-1 and full-length drICE in vitro, GMR-DeltaN-dcp-1 does not enhance the eye phenotype of GMR-fl-dcp-1 or GMR-fl-drICE flies. Significantly, GMR-rpr and GMR-grim, but not GMR-hid, dramatically enhance the eye phenotype of GMR-fl-dcp-1 flies. These results indicate that Reaper and Grim, but not HID, can activate DCP-1 in vivo (Song, 2000).
The proapoptotic proteins encoded by rpr, hid, and grim all require caspase activity to kill cells. Whether coexpression of caspases and these proapoptotic genes could lead to significantly enhanced cell killing was investigated. For this purpose, flies carrying GMR-rpr, GMR-hid, and GMR-grim were crossed to GMR-fl-dcp-1 and GMR-fl-drICE flies. Two different GMR-fl-dcp-1 transgenic fly lines were crossed to GMR-rpr46, GMR-hid1M, and GMR-grim flies, with identical results. Likewise, two GMR-fl-drICE transgenic fly lines were crossed to GMR-rpr46, GMR-hid1M, and GMR-grim flies, again with identical results. Flies carrying one copy of GMR-fl-dcp-1 or GMR-fl-drICE have almost normal eye morphology. Flies transgenic for GMR-hid1M, GMR-grim, or GMR-rpr46 have a mild but easily detectable eye phenotype. The coexpression of hid and full-length drICE produces no obvious enhancement of the eye phenotype, but rather an additive effect of the two transgenes. Also, the expression of hid together with full-length dcp-1 enhanced the eye phenotype only weakly, comparable to what is seen for coexpression of many other proapoptotic gene combinations. In stark contrast, expression of either rpr or grim together with GMR-fl-dcp-1 yields a dramatically enhanced eye phenotype that cannot be simply explained by additive effects. rpr produces a stronger effect than grim. The expression of rpr also enhances the eye phenotype of GMR-fl-drICE flies, whereas grim is not very effective. This finding is consistent with the observation that drICE is activated in rpr-transfected S2 cells. Among the different cell types of the Drosophila retina, the pigment cells appear to be particularly sensitive to DCP-1. Both the truncated and the full-length DCP-1 cause pigment cell death. Judging by the complete loss of eye color, all pigment cells are eliminated in flies that coexpress DCP-1 with either rpr and grim. In order to further investigate the specificity of this interaction, GMR-fl-dcp-1 flies were also crossed to a transgenic line with strong hid expression: GMR-hid 10 flies. Again, the eye phenotype observed for this combination is not significantly enhanced. Overall, rpr and grim were found to interact with dcp-1 much more strongly than hid and interact more effectively with dcp-1 than with drICE. Taken together, these observations suggest that dcp-1 is rate limiting for cell killing by rpr and grim, but not hid. Therefore, it is proposed that rpr and grim function upstream of dcp-1 in vivo (Song, 2000).
These results indicate that Reaper and Grim, but not Hid, can lead to DCP-1 activation. Several other observations also indicate that rpr and grim have cell killing properties that are distinct from those of hid. For example, the Ras/MAPK pathway inhibits hid-induced cell death but has no effect on rpr- or grim-induced death (Bergmann, 1998). In addition, mutations in the diap1 gene of Drosophila have been isolated that enhance rpr- and grim-induced cell killing but suppress hid-induced cell killing (J. Agapite, K. McCall, and H. Steller, unpublished data cited in Song, 2000). The easiest interpretation of all these observations is that rpr and grim kill cells by activating the same (set of) caspases and that hid activates a distinct caspase. Since it has been recently shown that rpr, hid, and grim induce cell death by inhibiting the antiapoptotic activity of diap1, diap1 must control at least two distinct caspase pathways. According to this model, Reaper and Grim and HID would interact selectively with specific DIAP1-(pro)caspase complexes. The binding of Reaper, Grim, or HID to the relevant DIAP1-(pro)caspase complex is thought to result in caspase activation. This model is consistent with a variety of findings from both invertebrate and vertebrate systems. However, the possibility that rpr and grim may also activate DCP-1 through a DIAP1-independent pathway cannot be ruled out. Although several Drosophila caspases have been described, these results indicate that additional caspases, in particular ones activated by HID, remain to be identified (Song, 2000).
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, 2001a).
Analyses of R/Grim and G/Reaper chimeras have indicated that the closely related RHG motifs of Reaper and Grim are not functionally interchangeable. Instead, the four amino acid substitutions between their RHG motifs help determine the unique cell killing abilities of Reaper and Grim. For example, unlike Grim, R/Grim resembles Reaper and is unable to induce cell death in the CNS midline. In contrast, one P[UAS-g/reaper] strain induces significant midline cell death, implying that the presence of the Grim RHG motif can confer Grim-like cell killing abilities on Reaper. It is important to note, however, that the identity of the RHG motif does not completely transform the cell killing properties of the chimeras, indicating that other regions of Reaper and Grim proteins are also crucial for their distinct actions. In this regard, like Grim, both R/Grim and G/Reaper are able to act synergistically with Reaper to induce CNS midline cell death (Wing, 2001a).
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 (Reaper, Hid, Grim) 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, 2001a).
As with Reaper or Grim, P[GMR-gal4]-targeted expression of R/Grim or G/Reaper is very effective at inducing cell death. However, the actions of the chimeras are distinct from those of Reaper or Grim. In particular, the cell death phenotypes resulting from R/Grim or G/Reaper expression are completely blocked by Diap1 and partially blocked by Diap2. In contrast, both Diap1 and Diap2 completely block the effects of Reaper expression, but do not affect cell death induced by Grim. Thus, as a result of the presence of the Reaper RHG motif, R/Grim exhibits an increased sensitivity to repression by the Diaps compared with Grim. Similarly, the presence of the Grim RHG motif in G/Reaper results in decreased sensitivity to repression by Diaps compared with Reaper. These results indicated that the sequence differences in the RHG motifs of Reaper and Grim may strongly influence functional interactions with the Diaps (Wing, 2001a).
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, 2001a).
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, 2001a 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, 2001a).
The Drosophila reaper, head involution defective, 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. The Gal4/UAS targeted gene expression system has been 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 reaper or hid to induce higher levels of midline cell death than observed for any of the genes individually. Finally the function was analyzed of a truncated Reaper-C protein that 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, 2001b).
An overt alteration in cytochrome c anticipates programmed cell death (PCD) in Drosophila tissues, occurring at a time that considerably precedes other known indicators of apoptosis. The altered configuration is manifested by display of an otherwise hidden epitope and occurs without release of the protein into the cytosol. Conditional expression of the Drosophila death activators, reaper or grim, provoke apoptogenic cytochrome c display and, surprisingly, caspase activity is necessary and sufficient to induce this alteration. In cell-free studies, cytosolic caspase activation is triggered by mitochondria from apoptotic cells but identical preparations from healthy cells are inactive. These observations provide compelling validation of an early role for altered cytochrome c in PCD and suggest propagation of apoptotic physiology through reciprocal, feed-forward amplification involving cytochrome c and caspases (Varkey, 1999).
Previous studies on Drosophila SL2 cells have shown that conditional expression of rpr or grim triggers apoptosis in cultured cells and in transgenic animals. Transiently transfected SL2 cells were induced for rpr or grim and, at various time intervals after induction, the preparations were examined for cytochrome c immunoreactivity with mAb 2G8. Apoptotic cultures exhibit profound staining with the antibody. To test the possibility that cytochrome c might be released into the cytosol during apoptosis, healthy SL2 cells and apoptotic rpr- or grim-expressing cells were fractionated, and assayed for cytochrome c in the mitochondrial and cytosolic fractions. Surprisingly, these cells showed no difference in cytochrome c distribution and no evidence was found for the transit of cytochrome c to the cytosol as a correlate to apoptosis. Biochemical data indicating retention of cytochrome c in mitochondria during apoptosis is consistent with cytological studies. These observations indicate that appreciable efflux of cytochrome c from mitochondria does not occur during apoptosis in Drosophila cells (Varkey, 1999).
Mitochondria isolated from apoptotic cells trigger caspase activation in vitro. Caspase activation was measured in L2 cell cytosol that had been coincubated with mitochondria isolated from parental L2 cells or from pre-apoptotic cells (induced either for rpr or grim). Caspase activation was detected, as measured by signature cleavage of a bovine substrate, PARP. Cleavage of PARP in this assay is indistinguishable from the signature activity reported in many mammalian systems and is readily detected in the cytosol of pre-apoptotic cells but not in cytosol from parental L2. These observations emphasize the importance of one or more mitochondrial factors in the activation of caspase function triggered by rpr or grim (Varkey, 1999).
The Drosophila death activators, rpr and grim, activate one or more caspases to elicit apoptosis. To study the temporal relation of cytochrome c display with respect to caspase activity, SL2 cells were cotransfected with rpr and p35 plasmids. Six hours after induction, cells induced for rpr alone show pronounced labeling with mAb 2G8 whereas cells expressing rpr together with p35 are prevented from apoptosis and do not bind the mAb. These observations suggest that apoptogenic cytochrome c display requires caspase activity, a presumption that is further substantiated when rpr-expressing cells are treated with the peptide caspase inhibitors zDEVD-fmk and zVAD-fmk. As seen for p35-blocked cells, these inhibitors similarly prevent mAb 2G8 labeling and subsequent apoptosis. Parallel results are observed in grim-expressing cells (Varkey, 1999).
These data demonstrate that caspase activity is required for apoptogenic cytochrome c display. To determine if caspase function is sufficient to trigger this change, apoptosis was induced in SL2 cells by conditional expression of an activated version of the Drosophila caspase, dcp-1. If deleted for its prodomain, this caspase provokes considerable apoptosis in mammalian cells and SL2 cells. When labeled with mAb 2G8, cells transfected and induced for dcp-1 expression exhibit profound punctate cytochrome c staining with features indistinguishable from those associated with expression of the death activators (Varkey, 1999).
Two potential explanations reconcile the in vivo observations reported here on apoptogenic cytochrome c with reports from mammalian cell-free systems that cytochrome c can trigger caspase activation. One possibility is that the order and/or nature of cytochrome c apoptotic function is not conserved between mammals and insects and thus, relative to caspase action, cytochrome c is upstream in the former case and downstream in the latter case. This scenario, however, seems unlikely given the widespread conservation of apoptotic components, the fact that display of fly cytochrome c in the animal significantly precedes all signs of programmed cell death, and reports from mammalian systems that upstream caspases can trigger cytochrome c release. Therefore, a more likely interpretation of the results reported here is that cytochrome c propagates apoptotic physiology by functioning together with caspases in a feed-forward amplification loop. In this scenario, altered cytochrome c and caspase activity exert positive and reciprocal feedback on one another, similar to observations recently reported for caspase 8. Thus, agents that restrain caspase action (p35) are also predicted to suppress pro-apoptotic display of cytochrome c, which behaves as an amplifier of caspase function. This interpretation is also consistent with recent studies on Fas signaling in type II cells, where molecular ordering studies found that activation of an initiator caspase (caspase 8/Flice) occurs upstream of changes associated with cytochrome c (Varkey, 1999).
A novel grim-reaper gene, termed sickle, has been identified that resides adjacent to reaper. The sickle gene, like reaper and grim, encodes a small protein which contains an RHG motif and a Trp-block. In wild-type embryos, sickle expression is detected in cells of the developing central nervous system. Unlike reaper, hid, and grim, the sickle gene is not removed by Df(3L)H99, and strong ectopic sickle expression is detected in the nervous system of this cell death mutant. sickle very effectively induced cell death in cultured Spodoptera Sf-9 cells, and this death is antagonized by the caspase inhibitors p35 or DIAP1. Strikingly, unlike the other grim-reaper genes, targeted sickle expression does not induce cell death in the Drosophila eye. However, sickle strongly enhanced the eye cell death induced by expression of either an reaper/grim chimera or reaper (Wing, 2002).
To test sickle's ability to induce cell death, transient transfection assays were performed using cultured Spodoptera Sf-9 cells. Survival of the transfected cells was monitored using a LacZ reporter construct. Compared to the empty vector, transfection with the sickle expression construct results in a dramatic increase in cell death levels, as evidenced by an 18-fold reduction in LacZ expression. Transfection of a reaper expression construct also results in significant cell loss, although not to the same extent as is observed with sickle. The cell death induced by either sickle or reaper is suppressed ~3- to 6-fold by coexpression of the genes encoding the caspase inhibitory proteins p35 or DIAP1. These data imply that like other Grim-Reaper proteins, Sickle acts upstream of caspases and induces apoptosis via a mechanism involving inhibition of IAP function. The cell death-inducing capabilities of sickle were also investigated in Drosophila. Surprisingly, P[GMR-gal4]-targeted sickle expression using P[UAS-sickle] strains is ineffective at inducing ectopic cell death in the adult eye. Thus, P[GMR-gal4]/P[UAS-sickle] animals survive to adulthood, and nearly all exhibit normal eye morphology. (A few of these flies did have slightly roughened eyes, suggesting weak cell killing effects of sickle expression.) This result is in stark contrast to the lethality and complete loss of eye tissue seen for P[GMR-gal4]-targeted expression of reaper, hid, or grim. The use of several additional P[gal4] strains also failed to yield any evidence for sickle-induced ectopic cell death. While the basis for the distinct effects of sickle expression in cultured cells and Drosophila tissue is not yet clear, cell-specific effects of ectopic grim-reaper expression have been previously noted (Wing, 2002).
Because of the synergistic activities of reaper, hid, and grim in embryonic CNS midline, attempts were made to determine if sickle might enhance the actions of other grim-reaper genes. Since P[GMR-gal4]-targeted sickle expression failed to induce ectopic cell death, this issue was addressed by coexpression of either sickle and an reaper/grim chimera or reaper in the adult eye. P[GMR-gal4]-targeted expression of reaper/grim results in viable adults that exhibit a temperature-sensitive loss of eye tissue and pigmentation. In contrast, at either 25°C or 21°C P[GMR-gal4]-targeted coexpression of sickle and reaper/grim results in complete lethality. At 18°C, where the reaper/grim effects are reduced, a few flies coexpressing sickle and reaper/grim did emerge, and these exhibited a much greater loss of eye tissue than flies expressing either sickle or reaper/grim alone. Thus, sickle exhibits strong synergistic actions with reaper/grim. As expected, the effects of reaper/grim, as well as reaper/grim and sickle, are repressed by coexpression of p35; these animals are viable when raised at 25°C and exhibit essentially normal eye size and pigmentation. To demonstrate that sickle-dependent synergism is not restricted to the reaper/grim chimera, whether P[GMR-gal4]-targeted sickle expression would enhance cell death induced by P[GMR-reaper] was also examined. Flies bearing a single copy of P[GMR-reaper] exhibit a moderate loss of eye tissue. In contrast, flies bearing one copy each of P[GMR-reaper], P[GMR-gal4], and P[UAS-sickle], exhibit much more severe eye cell death, with greatly reduced eye size and pigmentation. This ectopic cell death is repressed by expression of p35. Synergistic eye cell death effects are also observed for coexpression of sickle and a g/reaper chimera, as well as sickle and grim. These results indicate that sickle can potentiate the cell killing effects of grim-reaper genes, and they provide the first examples of synergistic action for grim-reaper genes outside of the embryonic CNS midline (Wing, 2002).
In summary, these data demonstrate that sickle is a novel member of the grim-reaper family of cell death activators and suggest that functional interactions may be a general mechanism underlying the actions of Grim-Reaper proteins. The sequence of the Sickle protein strongly suggests that it has unique RHG motif-dependent and RHG motif-independent functions. Overall, the identification of sickle reveals further complexity in the regulation of cell death activation in Drosophila and provides additional evidence that these linked genes at 75C be considered a genetic complex (Wing, 2002).
Although programmed cell death (PCD) plays a crucial role throughout Drosophila CNS development, its pattern and incidence remain largely uninvestigated. This study provides a detailed analysis of the occurrence of PCD in the embryonic ventral nerve cord (VNC). The spatio-temporal pattern of PCD was traced and the appearance of, and total cell numbers in, thoracic and abdominal neuromeres of wild-type and PCD-deficient H99 mutant embryos were compared. Furthermore, the clonal origin and fate of superfluous cells in H99 mutants was examined by DiI labeling almost all neuroblasts, with special attention to segment-specific differences within the individually identified neuroblast lineages. These data reveal that although PCD-deficient mutants appear morphologically well-structured, there is significant hyperplasia in the VNC. The majority of neuroblast lineages comprise superfluous cells, and a specific set of these lineages shows segment-specific characteristics. The superfluous cells can be specified as neurons with extended wild-type-like or abnormal axonal projections, but not as glia. The lineage data also provide indications towards the identities of neuroblasts that normally die in the late embryo and of those that become postembryonic and resume proliferation in the larva. Using cell-specific markers it was possible to precisely identify some of the progeny cells, including the GW neuron, the U motoneurons and one of the RP motoneurons, all of which undergo segment-specific cell death. The data obtained in this analysis form the basis for further investigations into the mechanisms involved in the regulation of PCD and its role in segmental patterning in the embryonic CNS (Rogulja-Ortmann. 2007).
In this analysis of PCD distribution it was found that, macroscopically, the CNS of wt and PCD-deficient (H99) embryos do not show large differences. These observations indicate that the supernumerary cells do not disturb developmental events in the CNS of H99 embryos, such as cell migration and axonal pathfinding. The glial cells mostly find their appropriate positions accurately. The DiI-labeled NB lineages were, in the majority of cases, easily identifiable based on their shape, position and axonal pattern, despite the supernumerary cells. The FasII pattern showed that the axonal projections form and extend along their usual paths. In fact, the supernumerary cells themselves are capable of differentiating i.e. expressing marker genes and extending axons, as shown by clones of several NBs and by cell marker expression analysis in H99 (e.g. NB7-3) (Rogulja-Ortmann. 2007).
It has been shown that a large number of CNS cells undergo PCD during embryonic development. The distribution of activated Caspase-3-positive cells in wt embryos suggests that the death of some cells is under tight spatial and temporal control, as revealed by their regular, segmentally repeated occurrence. Other dying cells were rather randomly distributed, suggesting a certain amount of developmental plasticity. The overall counts of Caspase-3-positive cells give an estimate of the numbers of dying cells at a given time. They indicate that PCD becomes evident in the CNS at stage 11 and is most abundant in the late embryo (from stage 14). It is however difficult to estimate the total number of apoptotic cells throughout CNS development by anti-Caspase-3 labeling, because the cell corpses are removed fairly quickly. Therefore the total number of cells were counted per thoracic and abdominal hemineuromere in the late embryo. Comparison between stage 16 and stage 17 wt embryos indicates that 25-30 % of all cells are removed in both tagmata after stage 16, which in turn suggests that the total percentage of removed cells must be high, since PCD occurs at high levels already from stage 14 on. In comparison to the developing nervous system of C. elegans, where PCD removes about 10% of cells, and of mammals, where this number can be as high as 50-90%, PCD in the fly CNS appears to show an intermediate prevalence. This lends support to the hypothesis of an increasing contribution of PCD in shaping more advanced nervous systems during evolution (Rogulja-Ortmann. 2007).
Comparisons between wt and H99 reveal, as expected, a greater number of cells in both tagmata of H99 embryos (151% increase in the thorax and 162% in the abdomen at stage 17). These additional cells in H99 may reflect the total number of cells normally undergoing cell death until stage 17. However, there is a large variability in the total number of cells, especially within the H99 strain. In wt embryos, it seems to be more pronounced in the thorax and at stage 17, which might be a consequence of variable amounts of PCD occurring until this stage. The even higher variability within the H99 strain (both in thorax and abdomen) is likely to reflect variable numbers of additional cell divisions. The great majority of abdominal NBs are normally removed by PCD after they have generated their embryonic progeny, whereas in the thoracic neuromeres most of the NBs enter quiescence at the end of embryogenesis and continue dividing as postembryonic NBs in larval stages. Thus, there are few mitoses occurring in the wt CNS from stage 16 onwards. BrdU labeling experiments revealed a high number of BrdU-positive cells in some H99 embryos injected at early stage 17. It is assumed that these are progeny of mitotic NBs and/or GMCs that survive and continue dividing, generating cells that do not exist in wt. Clones obtained by DiI labeling in H99 confirm this conclusion. The finding that surviving cells divide already in the embryo complement results that showed that, in reaper mutants, NBs in the abdominal neuromeres survive and generate progeny in larval stages (Rogulja-Ortmann. 2007).
Among the DiI-labeled clones in H99 embryos, very few NB lineages were obtained which did not differ from their wt counterparts. The majority contained, as expected, supernumerary cells. In some cases axons projected by these cells could be identified, showing that they are specified as neurons. In fact, in three cases (NB4-2, NB5-3 and NB7-3), these additional cells were found to be specified as motoneurons. As additional axons within a fascicle were generally difficult to identify, it is possible that these are not the only lineages which make additional motoneurons in H99. Whether these cells are normally born and apoptose, or originate from additional divisions of surviving NBs or GMCs, cannot be determined from these experiments, but similar observations have been made for both cases. It is interesting that none of these cells, regardless of their origin, are specified as glia. No additional glia were observed in the NB clones in H99 embryos, and equal numbers of Repo-expressing glial cells were found in wt and H99. It is concluded that PCD occurs almost exclusively in neurons and/or undifferentiated cells, and that lateral glia are not produced in excess numbers in the embryo. Furthermore, because it is likely that NBs, which normally die, stay in a late temporal window in H99, one could speculate that NBs in this window normally do not give rise to glia. These results are not in agreement with the notion that LG are overproduced, and their numbers adjusted through axon contact. Occasional apoptotic LG have been observed and it is possible that the current method of counting does not allow a resolution fine enough to account for an occasional additional Repo-positive cell in H99 embryos. However, if LG were consistently overproduced, a higher number of glia in would be expected H99 embryos. It is assumed that LG cell death may reflect a small variability in the number of cells needed, and not a general mechanism for adjusting glial cell numbers (Rogulja-Ortmann. 2007).
Generally, no difference was found between Repo-expressing glia numbers in wt and H99. However, a small difference does become apparent when one separates the total cell counts into those in the CNS and those in the periphery: 25.67±0.45 cells/hs and 28.42±0.64 cells/hs for wt and H99, respectively, were counted in the CNS, whereas 8.50±0.28 cells/hs and 6.35±0.82 cells/hs for wt and H99, respectively, were found in the periphery. The reasons for this difference might be the greater width of the CNS in H99 embryos, and that the cues required for proper migration of the peripheral glia are disturbed by additional cells. Alternatively, the difference might be due to differentiation defects in these cells (Rogulja-Ortmann. 2007).
In addition to NB clones with too many cells and wild-type-like axon projections in H99, some lineages were obtained whose clones exhibited atypical projection patterns. These projections were found to belong both to motoneurons (e.g. in NB4-2) and interneurons (e.g. NB5-3, NB7-2 and NB-7-4). NB4-2 normally produces two motoneurons (RP2 and 4-2Mar) and 8-14 interneurons. In two out of three NB4-2 clones in H99 two additional motoneurons that project anteriorly were found, similar to RP2. One of the two clones was found in the thorax and had a normal cell number (16), whereas the other was abdominal and had too many cells (25). Thus, the two additional motoneurons are likely to be the progeny of divisions occurring in the wt, and not of an additional NB or GMC mitosis. The fact that the third NB4-2 clone (found in the abdomen and comprising 17 cells) did not show the same motoneuronal projections could be due to these cells not being differentiated at the time of fixation (clones of different ages were occasionally observed in the same embryo), or they may not have differentiated at all. It would be interesting to determine the target(s) of these additional motoneurons and thereby perhaps gain insight into physiological reasons for their death. However, such an experiment has to await tools that allow us to specifically label the NB4-2 lineage, or these motoneurons, in the H99 mutant background (Rogulja-Ortmann. 2007).
The other three lineages (NB5-3, NB7-2 and NB7-4) all have atypical interneuronal projections. The cells which these atypical axons belong to may represent evolutionary remnants that are not needed in the Drosophila CNS. Alternatively, they might have a function earlier in development and be removed when this function is fulfilled. Such a role has been shown for the dMP2 and MP1 neurons, which are born in all segments and pioneer the longitudinal axon tracts. At the end of embryogenesis these neurons undergo PCD in all segments except A6 to A8, where their axons innervate the hindgut. It is known that some cells of the NB5-3 lineage express the transcription factor Lbe, and that H99 mutants show about three additional Lbe-positive neurons per hemisegment, which mostly likely belong to NB5-3. The DiI-labeling results complement this finding in that four or more additional neurons were also found in H99 clones. The supernumerary Lbe-positive neurons in H99 could possibly be the ones producing the atypical axonal projections (Rogulja-Ortmann. 2007).
In the wt embryo, only eight NB lineages show obvious tagma-specific differences in cell number and composition. Tagma-specific differences among serially homologous CNS lineages have been shown to be controlled by homeotic genes. Therefore, these lineages provide useful models for studying homeotic gene function on segment-specific PCD. In H99 embryos, further lineages were observed that were differently affected in the thorax and abdomen. How these tagma-specific differences arise in a PCD-deficient background is an interesting question. For example, NB4-3 shows a wild-type cell number in the thorax (8 and 12-13), but has too many cells in the abdomen (15, 15 and 22). There are a couple of plausible scenarios to explain this observation. (1) The development of the NB4-3 lineage, including the involvement of PCD, could actually differ in the thorax and abdomen of wt embryos, with the final cell number being similar by chance. The DiI-labeled clones allow determination of the final cell number, but do not reveal how this number is achieved. The difference would become obvious in an H99 mutant background, at least regarding the involvement of PCD. (2) This possibility does not exclude the first one, the thoracic NB4-3 could become a postembryonic NB (pNB) and the abdominal NB4-3 might undergo PCD after generating the embryonic lineage. In H99, the abdominal NB would be capable of undergoing a variable number of additional divisions to generate a variable number of progeny. This would easily explain larger discrepancies in cell number between individual clones in H99 (e.g. the abdominal NB4-3 clone with 22 cells), and is in agreement with occasional observations of H99 embryos with a very high CNS cell number per segment, and with the two observed classes of H99 embryos with high and low numbers of BrdU-positive cells (Rogulja-Ortmann. 2007).
NB6-2 is another lineage whose clones differ in the two tagmata of H99 embryos. In this case, the abdominal clones showed no difference to their wt counterparts, whereas the thoracic clones did (18 and 19 cells). Although no difference in cell number between thoracic and abdominal clones was reported for this lineage, a rather large count range (8-16 cells) was given, which would allow for a thorax-specific PCD of two to three postmitotic progeny. Alternatively, the thoracic NB6-2 might undergo cell death upon generating its progeny, which would make it the first identified apoptotic NB in the thorax. When PCD is prevented, this NB may undergo a few additional rounds of division. The data obtained in these experiments do not counter this notion, but the number of clones obtained in the thorax was not sufficient to draw a definite conclusion. As the abdominal NB6-2 lineage in H99 did not differ from the one in wt, its NB may be one of the few abdominal postembryonic NBs (Rogulja-Ortmann. 2007).
A specific set of NBs undergoes PCD in the late embryo, whereas surviving NBs resume proliferation in the larva as pNBs, after a period of mitotic quiescence. The identities of the individual NBs undergoing PCD versus those surviving as pNBs are still unknown. The sizes of NB lineages obtained in H99 embryos may provide hints for identifying candidate pNBs in the abdomen [12 NBs/hs in A1, four in A2 and three in A3 to A7, and NBs that undergo PCD in the thorax at the end of embryogenesis [seven NBs/hs in T1 to T3. In the abdomen, NB1-1a and NB6-2 are obvious candidates for pNBs, as they remained consistently unchanged in H99 embryos. Two other NBs, NB1-2 and NB3-2, are also potential abdominal pNBs as they mostly did not differ from their wt counterparts, and only occasionally contained one additional cell. On the other hand, clones which showed more than twice the cell number in H99 (NB2-1, NB5-4a and NB7-3) than in wt, strongly suggest that these NBs normally undergo PCD in the abdomen (but perform additional divisions in H99), because, even if one daughter cell of each GMC undergoes PCD, they still cannot account for all cells found in H99 clones (Rogulja-Ortmann. 2007).
Regarding thoracic NBs, it can only be speculated on account of low sample numbers. NBs which seem to become pNBs in the thorax, as they showed no difference between wt and H99 clones, are NB3-2, NB4-3 and NB4-4. Potential candidates for NBs which do not become pNBs, but undergo PCD in the thorax, are expected to consistently have a significant increase in cell number in H99. These are NB5-1 and NB5-5. In addition, lineages for which one clone was obtained in H99 but which also showed many more cells in the thorax than normal are NB2-2t, NB5-4t and NB7-3 (Rogulja-Ortmann. 2007).
In order to investigate the developmental signals and mechanisms involved in the regulation of PCD in the embryonic CNS, some of the apoptotic cells were identified which will be used as single-cell PCD models. These are the dHb9-positive RP neuron from NB3-1, Lbe-positive neurons from NB5-3, the Eg-positive GW neuron from NB7-3 and the Eve-positive U neurons from NB7-1. As not much is known about the dying RP motoneuron or the Lbe-positive neurons, the first goal will be to characterize each of these cells more closely, based on the combination of expressed molecular markers (Rogulja-Ortmann. 2007).
Some of the dying NB7-3 cells are already known to be undifferentiated daughter cells of the second and third GMC, which undergo PCD shortly after birth. Notch has been identified as the signal initiating PCD. The surviving daughters receive the asymmetrically distributed protein Numb, which counteracts the PCD-inducing Notch signal. The same had been shown in a sensory organ lineage of the embryonic peripheral nervous system, where cells produced in two subsequent divisions undergo Notch-dependent PCD. Both the PCD in the NB7-3 lineage and in the sensory organ lineage require the Hid, rpr and grim genes. It will be interesting to see whether the Notch-Numb interaction also plays a role in the segment-specific PCD of the differentiated GW motoneuron, or if another signal is used for the removal of this, and possibly other, differentiated cells (Rogulja-Ortmann. 2007).
The U motoneurons also show a segment-specific cell death pattern (they apoptose in A6 to A8), thus somewhat resembling the MP1 and dMP2 neurons. However, in contrast to MP1 and dMP2, the U neurons survive in the anterior segments and undergo PCD in the posterior ones. Whether homeotic genes play any role in the survival or death of these cells remains to be investigated (Rogulja-Ortmann. 2007).
In summary, this study has presented descriptions of PCD in the developing CNS of the wt Drosophila embryo, and of the CNS of PCD-deficient embryos. The pattern of Caspase-dependent PCD is partly very orderly, suggesting tight spatio-temporal control of cell death, and partly random, which suggests a certain amount of plasticity already in the embryo. The CNS of PCD-deficient embryos is nevertheless well organized, despite the presence of too many cells. These superfluous cells come from both a block in PCD and from additional divisions that surviving NBs go through. It was possible to link the occurence of cell death to identified NB lineages by clonal analysis in PCD-deficient embryos, to uncover segment-specific differences, and to establish single-cell PCD models that will be used in further studies to investigate mechanisms responsible for controlling PCD in the embryonic CNS (Rogulja-Ortmann. 2007).
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date revised: 25 July 2007
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