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Mol Cell Biol. Oct 2006; 26(19): 7258–7268.
PMCID: PMC1592896

DRONC Coordinates Cell Death and Compensatory Proliferation

Abstract

Accidental cell death often leads to compensatory proliferation. In Drosophila imaginal discs, for example, γ-irradiation induces extensive cell death, which is rapidly compensated by elevated proliferation. Excessive compensatory proliferation can be artificially induced by “undead cells” that are kept alive by inhibition of effector caspases in the presence of apoptotic stimuli. This suggests that compensatory proliferation is induced by dying cells as part of the apoptosis program. Here, we provide genetic evidence that the Drosophila initiator caspase DRONC governs both apoptosis execution and subsequent compensatory proliferation. We examined mutants of five Drosophila caspases and identified the initiator caspase DRONC and the effector caspase DRICE as crucial executioners of apoptosis. Artificial compensatory proliferation induced by coexpression of Reaper and p35 was completely suppressed in dronc mutants. Moreover, compensatory proliferation after γ-irradiation was enhanced in drice mutants, in which DRONC is activated but the cells remain alive. These results show that the apoptotic pathway bifurcates at DRONC and that DRONC coordinates the execution of cell death and compensatory proliferation.

Developing animal tissues have striking regenerative capacity. If a developing tissue is accidentally injured and a large number of cells undergo cell death, the final shape and size of the tissue are often invariant, owing to additional cell proliferation that compensates for the cell loss. The developing Drosophila larval imaginal disc is a tissue with extremely high regenerative capacity. Even when more than half of the cells are lost, the remaining cells undergo extra cell divisions and compensate for the loss (22). This “compensatory proliferation” is, at least in part, a local event that does not depend on the size control mechanism that governs the whole disc. For example, when apoptosis is locally induced by ectopic toxin expression, elevated cell proliferation is observed only around the site of apoptosis (40), suggesting that cells can perceive apoptosis occurring in the vicinity and undergo extra cell divisions until the original cell number is restored. Currently, little is known about how compensatory proliferation is regulated.

One attractive hypothesis is that dying cells actively induce proliferation of the neighboring cells by secreting mitogens before they disappear. This hypothesis assumes that the non-cell-autonomous induction of compensatory proliferation is a process that is under the control of the intracellular signaling pathway of apoptosis. According to this model, if cells could be stimulated to undergo apoptosis but artificially kept alive, the intracellular apoptosis signaling pathway would be activated and the “undead cells” produce mitogens indefinitely as long as the apoptotic stimulus persists. As a consequence, the neighboring cells would be misguided into conducting unrestrained compensatory proliferation. Several recent studies investigated this hypothesis using the Drosophila wing disc (25, 45, 51). The trick they used is to stimulate cells to undergo apoptosis and simultaneously block cell death by overexpressing p35, a potent inhibitor of effector caspases. Effector caspases are proteases that lie at the final step in the apoptosis signaling cascade. Thus, in such “undead cells,” the signaling cascade up to effector caspases is activated, but the cells fail to undergo apoptosis. Indeed, they found that excessive non-cell-autonomous proliferation is induced by p35-expressing cells if they are stimulated to undergo apoptosis (25, 45, 51). “Undead cells” ectopically express Wingless (WG) and Decapentaplegic (DPP), two major morphogens that shape the wing disc, of which DPP was found to be required for the abnormal overgrowth in a subsequent study (46).

What remains unanswered is which molecule of the apoptosis signaling pathway triggers compensatory proliferation. Two previous studies addressed this issue, with different conclusions (25, 51). Apoptosis in Drosophila is typically induced by transcriptional activation of the proapoptotic genes named reaper, hid, and grim (RHG) (1, 36, 37). Before apoptotic induction, caspases are constantly inhibited by the ubiquitin ligase DIAP1. The RHG proteins trigger apoptosis by blocking the function of DIAP1, which allows autocatalytic activation of the initiator caspase DRONC (Nedd2-like caspase-FlyBase). Interestingly, DRONC is insensitive to p35 inhibition (21, 39), which suggests that DRONC is activated in p35-expressing “undead cells.” Huh et al. suggested that the activation of DRONC is required for compensatory proliferation (25). They found that unrestrained compensatory proliferation induced by “undead” cells is suppressed by dominant-negative DRONC. On the other hand, Ryoo et al. suggested that DIAP1 downregulation activates JNK signaling and mitogen production independently of DRONC (51). They showed that unrestrained compensatory proliferation is accompanied by activation of JNK signaling and ectopic WG expression. The ectopic WG expression is suppressed by overexpression of the JNK-inhibiting phosphatase Puckered, but not by dominant-negative DRONC. The sources of these discrepancies are not clear. The two studies used different dominant-negative forms of DRONC, different GAL4 drivers, and different UAS reporters, making it difficult to directly compare the results.

The idea that DRONC, the most crucial component of the apoptosis signaling pathway, also regulates compensatory proliferation is particularly interesting. However, the prior studies are at odds with each other over the role of DRONC in compensatory proliferation. Unfortunately, the demonstration by Huh et al. is not conclusive because it is conceivable that the dominant-negative DRONC, which is a catalytically inactive form that can still bind to DIAP1, blocks compensatory proliferation not by inhibiting DRONC but by antagonizing DIAP1. Ideally, the requirement of DRONC and other caspases in compensatory proliferation would be unambiguously determined by testing whether the massive overgrowth induced by coexpression of p35 and RHG can be completely suppressed in null mutants of caspases. In the present study, we address whether caspases are required for compensatory proliferation by mutant analysis. There are seven caspases in the Drosophila genome: three initiator caspases (Dredd, DRONC, and STRICA [Dream-FlyBase]) and four effector caspases (DCP-1, DRICE [Ice-FlyBase], DECAY, and DAMM) (7, 11, 12, 13, 14, 19, 52). Biochemical and genetic studies have shown that DRONC is a major initiator caspase required for the execution of apoptosis (8, 10, 11, 20, 39, 48, 55, 60). Although another initiator caspase DREDD has been reported to be specifically expressed in apoptotic cells (7), null mutants of DREDD have no defect in apoptosis but show defective innate immunity (34). Biochemical studies have suggested that DRICE is a major effector death caspase (14, 15), but its role in vivo has been elusive owing to the lack of drice mutants. Here we report on the isolation of null mutations in dronc, drice, and decay and show that execution of apoptosis predominantly depends on DRONC and DRICE. Using these mutants, we show that the initiator caspase DRONC is also a crucial regulator of compensatory proliferation.

MATERIALS AND METHODS

Fly stocks and genetics.

The following fly stocks were used: EY08574 (2); GE284897 and GE2731 (from EP collection established by GenExel, Inc., Korea), GMR-Dmp53 (43), GMR-reaper (57), GMR-hid (17), GMR-grim (6), dreddB118 (34), hs-FLP (18), ey-FLP (41), dcp-1prev1 (31), yw; ey-GAL4 UAS-FLP/CyO y+; GMR-hid l(3)CL3L FRT2A/TM6 y+ (53), w; P[ovoD1-18]3L FRT2A/st betaTub85DD ss e/TM3 Sb (9), UAS-reaper (17), UAS-p35 (62), and hh-GAL4 (kindly provided by Tetsuya Tabata).

Deletion alleles of dronc, drice, and decay were obtained by remobilization of EY08574, GE284897, and GE2731, respectively. For each caspase locus, up to 500 candidate excision events were screened by genomic PCR that amplifies the whole locus. Four deletion lines—droncΔA8, droncΔB3, droncΔD8, and droncΔE10—were obtained at the dronc locus. A precise excision line, droncΔA1 was used as a wild-type control in compensatory proliferation after γ-irradiation. Two deletion lines, driceΔ1E4 and driceΔ2C8 were obtained at the drice locus. Both of them delete the entire open reading frame and are null alleles. We could not obtain a precise excision line. driceΔ1A1 is an imprecise excision that retains the drice open reading frame. driceΔ1A1 animals are viable and fertile, showing no defect in apoptosis. It was also used as wild-type control in compensatory proliferation after γ-irradiation. Two deletion lines, decayΔH10 and decayΔK2, were obtained at the decay locus. The regions deleted in decayΔH10 and decayΔK2 include the transcription start site and part of the catalytic subunit; thus, we believe both of them are null alleles. A precise excision line decayΔS3 was also obtained.

For eye-specific mosaic analysis of dronc, yw ey-FLP; droncΔA8 FRT2A/TM6B was crossed to GMR-X/CyO; GMR-hid l(3)CL3L FRT2A/TM6B (see Fig. Fig.3).3). Maternal/zygotic dronc embryos were obtained by the FLP-DFS system (see Fig. Fig.2B2B).

FIG. 2.
Requirement of caspases in developmental apoptosis. (A to F) Embryos were stained by using TUNEL. (A) Wild type; (B to F) maternal/zygotic caspase mutants. Genotypes: B, droncΔA8/droncΔA8; C, dreddB118/dreddB118; D, driceΔ2C8/ ...
FIG. 3.
Requirement of caspases in RHG-induced apoptosis. (A to L) Eyes of adult flies. GMR-hid induces dramatic reduction in eye size (B). These phenotypes are suppressed in dronc (C) and drice (E) backgrounds but not in dredd (D), dcp-1 (F), and decay (G) backgrounds. ...

Immunohistochemistry.

Immunohistochemistry of imaginal discs was done as described previously (49). The following antibodies were used: rabbit anti-phospho-histone H3 (1:1,000; Upstate Technologies), mouse anti-WG 4D4 (1:20; Hybridoma Bank), rat anti-ELAV 7E8A10 (1:20; Hybridoma Bank), rabbit anti-Bar (1:200) (24), rabbit anti-p35 (1:100) (54), and rabbit anti-DIAP1 (1:1,000) (30). Fluorescein isothiocyanate-, Alexa 488-, Cy3-, and Cy5-conjugated secondary antibodies were obtained from Jackson Immunoresearch and Molecular Probes and used at 1:100 to 101:500. Pictures were taken using a confocal microscope. Acridine orange staining of imaginal discs was done as described previously (27). Pictures were taken on an epifluorescence microscope. TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling) staining of embryos was done as described previously (28). Cy3-conjugated streptavidin was used for detection. Pictures were taken on a confocal microscope. Either a Zeiss LSM5 Pa or Zeiss LSM510 META microscope was used for confocal microscopy.

γ-irradiation.

Wandering third-instar larvae were irradiated with 20 Gy or 40 Gy of γ rays at 1 Gy/min using a 137Cs source. They were subsequently allowed to recover at 25°C.

RESULTS

Phenotypic analysis of caspase mutants.

Previous studies have shown that compensatory proliferation is induced by the apoptosis signaling pathway. In order to understand the underlying molecular mechanisms, we need to know which component of the apoptosis signaling pathway triggers compensatory proliferation. We chose to examine the requirement of caspases for compensatory proliferation because these proteases may be able to activate a variety of substrates through their cleavage. There are seven caspases in the Drosophila genome. At the time we started the present study, mutants were available only for dredd and dcp-1 (31, 34). We created null mutants of three other caspases, decay, drice, and dronc by imprecise excision of P-element insertions and analyzed their phenotypes. Figure Figure11 summarizes the extent of deletions in the mutant alleles.

FIG. 1.
Generation of caspase mutants. The genomic structures of caspases are schematized. A P-element was remobilized to create deletions in each caspase gene. The translated and untranslated regions are depicted as empty and filled rectangles, respectively. ...

First, we examined apoptosis in initiator caspase mutants. Four deletions were recovered at the dronc locus. All alleles except for droncΔE10 showed indistinguishable phenotypes. Homozygous dronc animals died as early pupae. A very low fraction (<0.1%) of dronc homozygous animals emerged as adult flies. Their external morphology was apparently normal, except that their wings were opaque due to the failure of the wing cells to undergo apoptosis. Maternal/zygotic dronc mutant animals were embryonic lethal, with almost complete absence of apoptosis (Fig. (Fig.2B).2B). Developmental apoptosis was also abrogated in the mutant larval imaginal discs (Fig. (Fig.2K).2K). Such developmental apoptosis was normal in dredd mutants (Fig. 2C and L). These results indicate that DRONC is a major initiator caspase required for apoptosis, although the other putative initiator caspase, STRICA, which was excluded from our current analysis, may also have minor contribution to developmental apoptosis (33). One dronc allele, droncΔE10, behaved differently from the other alleles. Homozygous droncΔE10 animals showed late larval lethality with a prolonged larval stage and occasional formation of melanotic tumors, phenotypes similar to those of dronc51 (8). Both droncΔE10 and dronc51 remove the putative promoter region of the adjacent gene CG6685 along with the first exon of dronc. Their phenotypes likely result from the combined effect of inactivating both dronc and CG6685.

We next examined effector caspase mutants. decay mutants were completely viable and fertile without any obvious morphological abnormalities. Developmental apoptosis was normal in decay mutants (Fig. 2F and J). drice mutants were mostly pupal lethal, and their lethal phase was later than that of dronc mutants (data not shown). Many of the drice mutant animals developed into pharate adults, with occasional adult escapers emerging at a frequency of <10%. In maternal/zygotic drice mutant embryos and mutant imaginal discs, apoptosis was greatly reduced but was not completely abolished (Fig. 2D and H). Another effector caspase, dcp-1 is a closely related homolog of drice and is not essential for viability (31, 52). Biochemical studies have shown that both drice and dcp-1 are substrates of dronc. Although dcp-1 mutants are completely viable, we found that developmental apoptosis in the larval eye disc was significantly reduced (Fig. (Fig.2I).2I). Therefore, we speculated that the milder phenotype of drice would be due to its functional redundancy with dcp-1. Indeed, developmental apoptosis in the eye disc was completely suppressed in drice/dcp-1 double mutants (Fig. (Fig.2M),2M), and the lethal phase of drice/dcp-1 double mutants was now indistinguishable from dronc mutants (data not shown), suggesting that drice and dcp-1 cooperatively act downstream of dronc, with drice playing a major role.

In Drosophila, transcriptional upregulation of RHG proteins is an essential step in the execution of all apoptosis. Accordingly, apoptosis requires specific transcription factors that regulate the expression of RHG genes. For example, Dmp53, the Drosophila homolog of the tumor suppressor p53, binds to the enhancer of reaper and activates its transcription during stress-induced apoptosis (5, 43, 44). Ectopic overexpression of RHG proteins or Dmp53 in the developing eye imaginal disc using the GMR promoter induces extensive apoptosis and causes dramatic reduction in adult eye size (6, 17, 43, 57; Fig. 3B, H, K, and M). To test the requirement of caspases in RHG-induced apoptosis, we overexpressed RHG proteins in each caspase mutant background. The eye size reduction caused by GMR-reaper, GMR-hid, and GMR-grim was all suppressed in dronc and drice backgrounds (Fig. 3C, E, I, and J; also see Fig. S1 in the supplemental material). In contrast, we could not observe any phenotypic suppression in other caspase mutants (Fig. 3D, F, and G). Mutations in dronc and drice also suppressed apoptosis induced by overexpression of RHG proteins (Fig. 3H to J). Thus, apoptosis triggered by the RHG proteins requires the activity of caspases DRONC and DRICE. Although the dronc mutation also suppressed apoptosis induced by overexpression of Dmp53, it did not suppress the eye size reduction (Fig. 3K, L, M, and N). Indeed, the eye discs of GMR-Dmp53 dronc−/− larvae showed a severe defect in neuronal differentiation (Fig. 3O and P). Targets of Dmp53 aside from RHG proteins likely affect neuronal differentiation, even when apoptosis is completely blocked.

DRONC is required for excessive compensatory proliferation.

When RHG proteins and the baculoviral caspase inhibitor p35 are locally overexpressed, enormous tissue overgrowth is observed in and around the area of ectopic expression. It has been proposed that apoptosis signaling is constitutively activated in the “undead cells” overexpressing RHG proteins and p35, which induces unrestrained compensatory proliferation (25, 51). We chose to use this phenotype as an indicator for the level of compensatory proliferation. We overexpressed Reaper and p35 in the posterior compartment of the wing disc using hh-GAL4. The ectopic expression driven by hh-GAL4 is much higher than en-GAL4, another posterior-specific GAL4 driver, and the resultant overgrowth phenotype was indeed severer than the previously reported phenotypes that were induced by en-GAL4 (25, 51). As previously reported, WG was ectopically expressed in the posterior compartment (Fig. (Fig.4C).4C). Interestingly, overexpression of p35 alone often induced local overgrowths (Fig. (Fig.4B).4B). During larval development, sporadic apoptosis occurs in the wing disc (40). We assume that suppression of this naturally occurring apoptosis led to the observed local overgrowths.

FIG. 4.
DRONC is required for artificially induced compensatory proliferation. (A to E) Single confocal sections of wing discs stained with anti-WG, anti-p35, and anti-DIAP1. Anti-p35 was used to visualize the posterior compartment of the wing disc (B to E). ...

p35 is a broad-specificity caspase inhibitor, but it does not inhibit DRONC (39). Thus, we assume that the following events take place in the “undead” cells expressing Reaper and p35. First, Reaper downregulates DIAP1, which leads to activation of DRONC. Subsequently, DRONC transforms DRICE into an active form by internal cleavage, but the cleaved DRICE is quickly inactivated by p35. Therefore, there are four candidate events that lead to the production of a compensatory proliferation signal: expression of Reaper, downregulation of DIAP1, activation of DRONC, and cleavage of DRICE. To test these possibilities, we overexpressed Reaper and p35 in dronc and drice backgrounds. The overgrowth of the posterior compartment and the ectopic WG expression were not suppressed in a drice background (Fig. (Fig.4E),4E), indicating that neither the caspase activity of DRICE nor the cleaved DRICE protein is required for compensatory proliferation. In contrast, they were completely suppressed in a dronc background, clearly demonstrating that compensatory proliferation requires DRONC (Fig. (Fig.4D4D).

In agreement with our results, Huh et al. showed that the abnormal overgrowth and ectopic WG expression induced by “undead cells” is suppressed by a dominant-negative form of DRONC (25). However, under a different experimental regime, cells overexpressing HID, p35, and dominant-negative DRONC were still able to induce ectopic WG expression (51). These contrasting results likely arose from the use of dominant-negative forms of DRONC. Because dominant-negative proteins act by competing with normal proteins, inactivation of endogenous protein can be incomplete (39).

Overexpression of RHG proteins has been shown to induce reduction of DIAP1 protein (23, 29, 50, 58, 59, 61). Indeed, DIAP1 protein was reduced in “undead cells” of the posterior compartment of the wing disc, which overexpressed Reaper and p35 (Fig. (Fig.4C).4C). The reduction of DIAP1 protein in “undead cells” was unaffected in a dronc mutant background, despite the fact that the tissue overgrowth was completely suppressed (Fig. (Fig.4E).4E). Therefore, DIAP1 downregulation alone is not sufficient to induce compensatory proliferation. In contrast, overexpression of DRONC induces hyperproliferation without reducing the DIAP1 protein levels (25; see Fig. S2 in the supplemental material). Taken together, these observations suggest that activation of DRONC is sufficient to induce compensatory proliferation and that the major role of DIAP1 downregulation in compensatory proliferation is to activate DRONC.

Endogenous compensatory proliferation requires DRONC.

Apoptotic cells are quickly removed by macrophages, usually within a few hours. Accordingly, compensatory proliferation should also occur immediately after apoptosis, since the mitogens that induce compensatory proliferation are unlikely to be produced after clearance of the apoptotic cells. The overgrowth of the wing disc caused by RHG proteins and p35 is a result of their overexpression throughout the larval stages, which amounts to more than 100 h. In the “undead cells” that are artificially kept alive for such a long period of time, many intracellular changes are likely to take place. Therefore, we cannot rule out the possibility that the overgrowth phenotype is a secondary consequence of apoptosis inhibition that occurs independently of the immediate compensatory proliferation.

To test the role of DRONC in naturally occurring compensatory proliferation, we chose to examine the response of the wing imaginal disc upon damage caused by γ-irradiation (4, 26). High-dose γ-irradiation triggers immediate cell cycle arrest of the wing disc cells within 1 h (4) (Fig. (Fig.6).6). During the cell cycle arrest, the DNA repair machinery attempts to repair double-strand breaks in the genomic DNA inflicted by the γ rays. If the damage is so extensive that it cannot be repaired, cells choose to undergo apoptosis. The cell loss is quickly restored by compensatory proliferation, which manifests as elevated cell proliferation during the recovery period (26).

FIG. 6.
Compensatory proliferation is accelerated in drice mutants. Third-instar larvae were irradiated with 40 Gy of γ-irradiation and allowed to recover at 25°C. At the indicated times after irradiation, wing discs were dissected and stained ...

It has been shown that γ-irradiation induces reaper mRNA expression (42), and RHG deletion mutants are resistant to irradiation-induced apoptosis (56). Thus, one would expect that γ-irradiation brings about apoptosis by upregulating RHG proteins, which promote degradation of DIAP1 and subsequent activation DRONC and DRICE. To test whether γ-ray-induced apoptosis depends on DRONC and DRICE, we irradiated wild-type and caspase mutant third-instar larvae with 40 Gy of γ-irradiation. Between 4 and 7 h after irradiation, extensive apoptosis is induced in wild-type wing discs (Fig. 5A,D). γ-ray-induced apoptosis was completely suppressed in dronc and drice mutants (Fig. 5B, C, E, and F), whereas apoptosis was normally induced in dredd, dcp-1, and decay mutants (Fig. 5G to I). These results establish that DRONC and DRICE are necessary for the induction of apoptosis induced by DNA damage.

FIG. 5.
DRONC and DRICE are required for γ-ray-induced apoptosis. (A to I) Wing imaginal disc stained with acridine orange before and after γ irradiation. Wandering third-instar larvae were irradiated with 40 Gy of γ rays and allowed to ...

If compensatory proliferation is indeed triggered by DRONC, we can predict that, in drice mutants, cell proliferation after γ- irradiation will be enhanced compared to irradiated wild-type animals. Irradiated drice cells do not undergo apoptosis, but induction of RHG proteins, downregulation of DIAP1, and activation of DRONC should normally occur, since these processes lie upstream of DRICE in the apoptosis signaling cascade. Thus, in the “undead cells,” activated DRONC would induce continuous production of mitogens, which enhances compensatory proliferation. To test this possibility, we compared the time course of cell proliferation after irradiation between the wild-type and caspase mutant animals. We irradiated wandering third-instar larvae with 40 Gy of γ-irradiation and allowed them to recover at room temperature. Both in the wild-type and in the caspase mutants, cell cycle arrest occurred normally when examined at 1 h after irradiation. At this dose, mitotic cells appeared around 8 h after irradiation in the wild-type wing disc (data not shown). In drice mutants, mitotic cells were already visible at 6 h after irradiation (Fig. (Fig.6),6), suggesting that the resumption of mitosis was accelerated by the accumulated mitogens produced by the “undead cells.” In contrast, dronc mutants were indistinguishable from the wild type (Fig. (Fig.6).6). To test whether the accelerated resumption of mitosis depends on DRONC, we examined dronc/drice double mutants. The acceleration in recovery was suppressed in dronc/drice double mutants (Fig. (Fig.6),6), indicating that DRONC is responsible for the accelerated resumption of mitosis that occurs in drice mutants. These results demonstrate that DRONC is required for endogenous compensatory proliferation that is induced by γ-irradiation.

DISCUSSION

In Drosophila imaginal discs, accidental cell loss due to damage-induced apoptosis is rapidly recovered by a process known as compensatory proliferation (40). Previous studies have shown that apoptotic cells actively induce proliferation of the neighboring cells, which suggests that induction of compensatory proliferation is part of the apoptosis signaling pathway (25, 45, 51). There are seven caspases in the Drosophila genome. We created mutations in three of them, dronc, drice and decay and showed that dronc and drice play major roles in apoptosis. Using these mutants, we tested the requirement of DRONC for compensatory proliferation. We found that the ability of “undead cells” to induce excessive compensatory proliferation depends on DRONC. We then showed that DRONC is required for immediate compensatory proliferation that occurs after extensive apoptosis induced by high-dose γ-irradiation. Our results provide unambiguous genetic evidence that the apoptosis signaling pathway bifurcates at DRONC and that DRONC is required for both DRICE activation and compensatory proliferation (Fig. (Fig.77).

FIG. 7.
Schematic diagram showing the proposed signaling cascade that regulates apoptosis and compensatory proliferation. First, apoptotic stimuli given to the cell activates RHG proteins. RHG proteins activate DRONC through inhibition of DIAP1 function. The ...

Downstream events of DRONC activation.

Activation of the apoptosis signaling pathway leads to transcriptional upregulation of secreted factors WG and DPP, of which DPP is believed to induce proliferation of the neighboring cells (46). Since DRONC is a protease, it is unlikely that it activates the transcription directly. A candidate event responsible for the transcription of dpp and wg is activation of JNK signaling. Ryoo et al. found that JNK signaling is activated in “undead cells” and that it is required for the ectopic WG expression and the disc size enlargement (51). Indeed, our preliminary data also show that the massive tissue overgrowth induced by “undead cells” can be suppressed by blocking JNK signaling (our unpublished observation).

Then, how is JNK signaling activated? It has been previously shown that a molecular pathway exists whereby downregulation of DIAP1 induces JNK activation through DTRAF1 (29, 47). However, downregulation of DIAP1 in “undead cells” is not sufficient to induce ectopic WG expression and massive overgrowth if DRONC activity is blocked (25) (Fig. (Fig.4C).4C). Therefore, we propose that activation of JNK signaling during compensatory proliferation is a downstream event of DRONC activation. Ryoo et al. showed that JNK signaling is required for the ectopic WG expression induced by overexpression of HID and p35 (51). On the other hand, Huh et al. showed that overexpression of DRONC is sufficient to induce ectopic WG expression (25). The simplest interpretation of these observations is that DRONC and JNK act in a single linear pathway to induce WG expression during compensatory proliferation. Indeed, coexpression of DRONC and p35 in the wing disc induces JNK activation and abnormal overgrowth (see Fig. S2 in the supplemental material). In mammals, Mst1, an upstream kinase of the JNK pathway, is activated through cleavage by caspase (16, 32). A similar upstream kinase may exist in Drosophila, which is specifically cleaved by DRONC and activates JNK signaling. Recently, it was reported that activation of JNK signaling accompanies compensatory proliferation of the liver (35). Thus, the mechanism uncovered in Drosophila may also apply to compensatory proliferation in mammals.

Compensatory proliferation during Drosophila development.

During normal development, a large number of cells undergo developmentally programmed apoptosis, whereby specific cells are invariably removed according to the developmental program of the organism. We have shown that compensatory proliferation is induced by “unprogrammed” apoptosis, such as accidental stress-induced apoptosis and RHG-induced artificial apoptosis of cells that normally do not undergo developmentally programmed cell death. On the other hand, there is little evidence that compensatory proliferation is induced by developmentally programmed apoptosis. In fact, there are obvious cases in which no proliferation is induced by apoptosis. For example, although a large number of cells that are produced in excess undergo developmentally programmed apoptosis in the pupal retina of Drosophila, there is no accompanying cell proliferation (3). This may be because there are no surrounding cells that respond to the mitogens released from the dying cell. It is also possible that developmentally programmed apoptosis occurs only in a context that does not induce compensatory proliferation.

Interestingly, however, our result that inhibition of apoptosis by overexpression of p35 in an otherwise normal genetic background induces ectopic WG expression and overgrowth (Fig. (Fig.4A)4A) indicates that there are apoptotic cells that induce compensatory proliferation even during normal development. The sites of the ectopic overgrowth were not consistent, which suggests that the inhibited apoptosis was not developmentally programmed apoptosis but accidental stress-induced apoptosis. This notion is further supported by the fact that sensitizing cells to stress-induced apoptosis simply by half-dose reduction of the puckered gene induces massive tissue overgrowth upon inhibition of apoptosis by p35 (38).

In nature, animals are constantly exposed to environmental stressors such as UV radiation, dynamic temperature change, and low nutritional conditions. Our finding suggests that, even under laboratory conditions, stress-induced apoptosis occurs during normal development to induce compensatory proliferation. Since there are more environmental stressors in nature, induction of compensatory proliferation during normal development is probably a frequent event and is not limited to extremely stressful conditions. Although the signaling pathway downstream of DRONC in compensatory proliferation is not yet clear, it likely involves secreted molecules, such as WG and DPP, which are also used for developmental patterning and proliferation. Thus, the developing tissue must interpret two signals: spatiotemporal information within the tissue for morphogenesis and the signal from apoptotic cells. The Drosophila imaginal disc is likely equipped with a mechanism that integrates these two signals, by which a tissue with the normal pattern is restored after injury.

Supplementary Material

[Supplemental material]

Acknowledgments

We thank the Bloomington Drosophila Stock Center; the Developmental Studies Hybridoma Bank; GenExel, Inc.; Kimberly McCall; Tetsuya Tabata; and Takaomi Saido for reagents. We thank Hoshio Eguchi for assistance with γ-irradiation. We thank members of the Miura and Hiromi laboratories for helpful discussions.

This study was supported by grants from the Japanese Ministry of Education, Science, Sports, Culture, and Technology (to M.M. and Y.H.); the CREST program of the Japan Science and Technology Agency (to Y.H.); and the Cell Science Research Foundation (to M.M.).

Footnotes

Supplemental material for this article may be found at http://mcb.asm.org/.

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