Wrinkled/head involution defective

DEVELOPMENTAL BIOLOGY

Embryonic

Patterns of hid expression are highly dynamic and complex throughout embryogenesis. Significantly, hid is expressed in many regions where cell death occurs. For example, in stage 11 embryos, both acridine orange staining, diagnostic for PCD, and HID mRNA hybridization are observed in the head and gnathal segments, as well as being segmentally repeated throughout the extended germ band. In slightly older embryos undergoing early stages of head involution, a correspondence between the patterns of cell death and HID mRNA expression is observed, particularly in the head. There is evidence of hid expression within macrophages, apparently confined to cell corpses that have been engulfed. Although there is significant overlap between the patterns of hid expression and acridine orange staining, these patterns are not entirely coincident. For example, HID mRNA is found throughout the entire optic lobe primordium, but only some of these cells undergo apoptosis. Not all cells may be equally sensitive to the amount of hid expression. Although there is considerable cell death in the ventral nerve cord during late embryogenesis, little or no hid expression can be detected at this time. Perhaps hid is not required for these deaths; alternatively, hid may be expressed in the ventral nerve cord below the level of detection (Grether, 1995).

Sexually dimorphic development of the gonad is essential for germ cell development and sexual reproduction. The Drosophila embryonic gonad is already sexually dimorphic at the time of initial gonad formation. Male-specific somatic gonadal precursors (msSGPs) contribute only to the testis and express a Drosophila homolog of Sox9 (Sox100B: Loh, 2000), a gene essential for testis formation in humans. The msSGPs are specified in both males and females, but are recruited into only the developing testis. In females, these cells are eliminated via programmed cell death dependent on the sex determination regulatory gene doublesex. This work furthers the hypotheses that a conserved pathway controls gonad sexual dimorphism in diverse species and that sex-specific cell recruitment and programmed cell death are common mechanisms for creating sexual dimorphism (DeFalco, 2003).

To investigate when sexual dimorphism is first manifested in the somatic gonad, expression of SGP markers were examined in embryos whose sex could be unambiguously identified, at a developmental stage (stage 15) soon after gonad coalescence has occurred. Analysis of Eya expression reveals anti-Eya immunoreactivity throughout the female somatic gonad, though Eya expression is somewhat stronger in the posterior. In males, anti-Eya immunoreactivity is also found throughout the somatic gonad. However, the expression at the posterior of the gonad is much more intense than in females, as there appears to be a cluster of Eya-expressing cells at the posterior of the male gonad that is not present in females. In blind experiments, the sex of an embryo could be accurately identified by the Eya expression pattern in the gonad. Thus, sexual dimorphism is already apparent in the somatic gonad soon after initial gonad formation. A sex-specific expression pattern is also observed with Wnt-2 at this stage. As is observed with Eya, Wnt-2 is expressed in the SGPs of the female gonad, but its expression is greatly increased at the posterior of the male gonad. The SGP marker bluetail (see Galloni, 1993) exhibits a similar sex-specific pattern as Eya; however, the SGP marker 68-77 is expressed equally in both sexes (see below). Thus, the somatic gonad is sexually dimorphic by stage 15, but only a subset of SGP markers reveals this sexual dimorphism (DeFalco, 2003).

To investigate how programmed cell death might be controlled in the msSGPs, the genes of the H99 region (head involution defective [hid], reaper [rpr], and grim), which are regulators of apoptosis in Drosophila, were examined. A small deletion (DfH99) removes all three of these genes and blocks most programmed cell death in the Drosophila embryo. In DfH99 mutants, an equivalent cluster of Sox100B-positive cells is observed in both males and females. Again, these posterior cells are also Eya positive. Furthermore, XX embryos mutant for hid alone also contain Sox100B-positive cells in the posterior of the gonad, although the posterior cluster of cells is slightly smaller than in the male. It is concluded that the msSGPs are normally eliminated from females through sex-specific programmed cell death, controlled by hid and possibly also other genes of the H99 region. However, if cell death is blocked in females, these cells can continue to exhibit the normal male behavior of the msSGPs, including proper marker expression and recruitment into the gonad. Therefore, the decision whether or not to undergo apoptosis is likely the crucial event leading to the sexually dimorphic development of these cells at this stage (DeFalco, 2003).

scylla and charybde, homologues of the human apoptotic gene RTP801, are required for head involution in Drosophila

Robotic methods and the whole-genome sequence of Drosophila melanogaster were used to facilitate a large-scale expression screen for spatially restricted transcripts in Drosophila embryos. In this screen, scylla (scyl) and charybde (chrb), which code for dorsal transcripts in early Drosophila embryos and are homologous to the human apoptotic gene RTP801, were identified. In Drosophila, both gene products are transcriptionally regulated targets of Dpp/Zen-mediated signal transduction and appear more generally to be downstream targets of homeobox regulation. Gene disruption studies revealed the functional redundancy of scyl and chrb, as well as their requirement for embryonic head involution. From the perspective of functional genomics, these studies demonstrate that global surveys of gene expression can complement traditional genetic screening methods for the identification of genes essential for development: beginning from their spatio-temporal expression profiles and extending to their downstream placement relative to dpp and zen, these studies reveal roles for the scyl and chrb gene products as links between patterning and cell death (Scuderi, 2006).

Based upon the observations that: (1) simultaneous loss of scyl and chrb function leads to a hid-analogous, cell death defective phenotype and (2) scyl and chrb are homologous to the mammalian apoptotic gene RTP801, it was postulated that the scyl and chrb gene products have pro-apoptotic functions in the embryonic Drosophila head. Two lines of experimentation were employed to test this hypothesis. (1) hid expression was examined in scyl chrb double mutant embryos in situ. The scyl and chrb gene products do not function as transcriptional modulators of hid since hid transcription is unaffected in scyl chrb double mutant embryos. (2) A Caspase-3 activity assay was employed to monitor apoptosis in wild-type and scyl chrb double mutant embryos. Activated Caspase-3 has been used previously to specifically label apoptotic cells in Drosophila. Anti-Caspase-3 staining mirrors cell death patterns previously defined by acridine orange and TUNNEL assays in the Drosophila embryo and pupal retina. In this study, dying cells expressing activated Caspase-3 were evident in the head and the nervous system of 95% of embryos derived from matings of Df(3L)vin4/twi:GFP heterozygotes 0-8 h AEL (n = 278). When GFP screening was used to enrich for similarly staged mutant embryos, it was noted that Caspase-3 activity was greatly diminished in mid-stage scyl chrb double mutants. By 8 AEL, 75% of the mutant-enriched population was caspase-negative, in contrast to the unselected population in which only 8% of the embryos were found to be caspase-negative. No gross differences in Caspase-3 activity were found prior to the onset of germ band retraction and head involution. Since cleaved Caspase-3 is a key executioner (and hence marker) of apoptosis, these data support the hypothesis that Scylla and Charybde have pro-apoptotic roles in Drosophila head involution. More generally, Scylla and Charybde likely function as essential death activators in Drosophila since Caspase-3 activation in scyl chrb double mutants is disrupted in the nervous system as well as in the head. The scylla and charybde gene products are not, however, sufficient for cell death since (1) immunostains reveal wild-type patterns of Caspase-3 activation in embryos derived from dl mutant mothers and in which expression of scylla and charybde is greatly expanded and (2) neither scyl nor chrb (alone or in combination) can mimic hid-induced apoptosis in cultured Cos or Hela cells (Scuderi, 2006).

Several lines of evidence indicate that Scylla and Charybde function in the Hid-mediated cell death pathway. (1) A previous phenotypic analysis of scyl chrb mutants revealed their essential roles in regulating cell death in the developing Drosophila eye. Loss-of-function studies have similarly revealed a requirement for Hid in modulating cell death events in early and late stages of Drosophila eye development. (2) In this study, which relied upon deficiencies and RNAi methodologies to generate scyl chrb null double mutants, an earlier developmental requirement for the scyl and chrb gene products was documented. scyl chrb double mutants suffer an embryonic lethality that is associated with defects in the morphogenetic process of head involution. Drosophila homozygous for loss-of-function hid alleles similarly suffer an embryonic lethality and exhibit signature defects in head involution. (3) Molecular characterization of the embryonic lethality in scyl chrb double mutants revealed that Caspase-3 activation is disrupted not only in the morphogenetically aberrant head, but in the CNS as well. In Drosophila, Hid induces apoptosis in midline glia cells failing to activate the EGFR signaling cascade. Together, the significant homologies of scyl and chrb to the mammalian RTP801 gene product that functions as an apoptotic factor in mammalian cell culture systems, as well as the scyl chrb embryonic and eye phenotype studies establish redundant roles for scyl and chrb in Hid-mediated cell death in both embryonic and post-embryonic stages of the Drosophila life cycle (Scuderi, 2006).

Each of the three cell death proteins, hid, rpr and grim, has been implicated in apoptotic events defining segmental boundaries and/or neuronal fates in the CNS, albeit in different paradigms. In the CNS, specificity in neuronal apoptosis is achieved via differential expression of the BX-C Hox gene abd-A, which prevents neuronal apoptosis in posterior segments. Viewed from this perspective, the finding that the Zen and BX-C Drosophila Hox gene products regulate transcription of the scyl and chrb pro-apoptotic genes (and thereby potentially sculpt head and segment boundaries during development) is reminiscent of the Deformed Drosophila Hox protein functioning as a transcriptional activator of the rpr cell death gene. Together, these studies strengthen the idea that Hox-gene-dependent induction of cell death is a general phenomenon in Drosophila (Scuderi, 2006).

Intriguingly, the pro- and anti-apoptotic roles of the Zen and BX-C Homeobox transcription factors in Drosophila embryogenesis correspond to their activation and repression effects on scyl and chrb gene expression. In this regard, scyl, chrb and cell death are activated by Zen in dorsal domains of the developing embryo, whereas ventrally scyl, chrb and cell death are repressed by one or more of BX-C gene products. Hence, in addition to the pro-apoptotic role of Zen, there is evidence for an anti-apoptotic role for the BX-C gene product(s) and in flies as in mouse related transcription factors function in context-specific fashion (Scuderi, 2006).

As a final point, both TGF-β and BMP mammalian members of the TGF-β cytokine superfamily have been documented to induce cell death in numerous developmental contexts. Along these same lines, previous reports in Drosophila have suggested a link between Dpp and cell death but have stopped short of designating this link as direct. Based on molecular and genetic evidence, it is suggested that the Drosophila pro-apoptotic scyl and chrb gene products serve as direct links between Dpp/Zen-mediated patterning and differentiation, in this case, cell death. Thus, in Drosophila as in vertebrates, cytokines of the TGF-β superfamily control both cell death and cell proliferation within the contexts of their cellular environments (Scuderi, 2006).

Given the importance of cell death regulation in development and disease, it is likely that there are several mechanisms by which cell death can be regulated, and, in like fashion, several nodes where independent regulatory pathways may in specific contexts converge. With respect to members of the RTP801 family of apoptotic factors, evidence points to at least two triggers of regulation: cell death can be a pathologic response to stresses such as hypoxia (as is the case for mammalian RTP801) or cell death can be a developmental response to a spatially and temporally restricted cell signaling pathway, such as the Dpp/TGF-β cytokine-mediated signaling pathway (as is the case for Drosophila Scylla and Charybde). Within the context of pathway convergence nodes, it is particularly notable that several reports document cross-talk between the HIF-1 and TGF-β pathways in regulating gene expression and cell death, and thus it is possible that the RTP801/Scylla/Charybde death effectors represent a point of convergence between these two death activating pathways. Consistent with this model is the demonstration that scyl and chrb are hypoxia-inducible in Drosophila (Reiling, 2004). Viewed from this perspective, the genetically defined roles of Scylla and Charybde as pro-apoptotic effectors establish a clear basis for future genetic and biochemical characterization of the mechanism by which activation of cell death programs might occur via Dpp/TGF-β-mediated signaling (Scuderi, 2006).

Pupal

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).

Ultraviolet (UV) light is absorbed by cellular proteins and DNA, promoting skin damage, aging and cancer. The UV response by cells of the Drosophila retina have been explored. The retina enters a period of heightened UV sensitivity in the young developing pupa, a stage closely associated with its period of normal developmental programmed cell death. Injury to irradiated cells include morphology changes and apoptotic cell death; these defects can be completely accounted for by DNA damage. Cell death, but not morphological changes, is blocked by the caspase inhibitor P35. Utilizing genetic and microarray data, evidence is provided for the central role of Hid expression and for Diap1 protein stability in controlling the UV response. In contrast, Reaper has no effect on UV sensitivity. Surprisingly, Dmp53 is required to protect cells from UV-mediated cell death, an effect attributed to its role in DNA repair. These in vivo results demonstrate that the cellular effects of DNA damage depend on the developmental status of the tissue (Jassim, 2003).

The major inhibitor of caspase activity in Drosophila is Diap1. Stability of Diap1 is the central point of cell death regulation in the developing retina and this is also true during UV irradiation in the retina. Genetic and microarray results further suggest that the retina requires Hid as a primary regulator of Diap1 stability during UV irradiation. Hid may represent the primary regulator of Diap1 during UV (versus ionizing) irradiation response by the fly. Alternatively, the retina utilizes Hid as its major RHG factor during its development, and this preference may simply extend to its response to UV; other tissues may exploit different Diap1 regulators that reflect their use during development (Jassim, 2003).

Together, these results identify two points of regulation during a retinal cell's response to UV irradiation. The early step involves pyrimidine dimers, and requires proper repair from factors that include XPG and p53. The second step involves activation of caspases and requires regulation of Diap1 stability; interommatidial cells utilize Hid at this step, and the remaining cells employ a different (unknown) regulator. One challenge will be to connect these two points of regulation. Multiple signaling pathways are suggested by the microarray data. These include EGFR/Ras1 signaling (a central regulator of Hid), JNK pathway signaling and TGFß pathway signaling. The role of these factors is not known, but understanding them may help to connect early and late events (Jassim, 2003).

Programmed cell death and context dependent activation of the EGF pathway regulate gliogenesis in the Drosophila olfactory system

In the Drosophila antenna, sensory lineages selected by the basic helix-loop-helix transcription factor Atonal are gliogenic while those specified by the related protein Amos are not. What are the mechanisms that cause the two lineages to act differentially? Ectopic expression of the Baculovirus inhibitor of apoptosis protein (p35) rescues glial cells from the Amos-derived lineages, suggesting that precursors are removed by programmed cell death. In the wildtype, glial precursors express the extracellular-signal regulated kinase (phosphoERK) transiently, and antagonism of Epidermal growth factor pathway signaling compromises their development. It is suggested that all sensory lineages on the antenna are competent to produce glia but only those specified by Atonal respond to EGF signaling and survive. These results underscore the importance of developmental context of cell lineages in their responses to non-autonomous signaling in the choice between survival and death (Sen, 2004).

Several lines of investigation have ascertained that the first cells to divide in the sensory lineages are the secondary progenitors: PIIa, PIIb and PIIc. The numbers of sensory cells undergoing division at different times in the developing antenna were estimated by staining mitotic nuclei with antibodies against phosphorylated histone. A peak of cell division was observed between 16 and 24 h after puparium formation (APF). It has been considered that only in those sensory lineages specified by Ato, PIIb produces a glial cell and a tertiary progenitor, PIIIb, which in turn divides to form the sheath cell and a neuron. In Amos dependent lineages, PIIb is believed to directly give rise to a neuron and a sheath cell. The difference between the two lineages could be entirely dependent on the nature of the proneural genes activated; Amos, for example, could direct a non-gliogenic lineage. Alternatively, the two proneural genes could specify similar division patterns but the glial cell precursor in Amos-lineages could be removed by PCD, resulting in non-gliogenic lineages (Sen, 2004).

To test the latter possibility, cell death profiles were examined in developing pupal antennae using the terminal transferase assay (TUNEL) and attempts were made to correlate the timing of PCD with cell division profiles discussed above. The appearance of TUNEL-positive cells peaked between 22 and 24 h APF consistent with the occurrence of PCD immediately after division of secondary progenitors (Sen, 2004).

TUNEL reactions were performed on 22-24 h APF antennae from lz-Gal4; UAS-lacZnls and ato-Gal4; UAS-lacZnls animals. Double labeling with antibodies against ß-galactosidase marked sensory cells arising from the Lz and Ato lineages. Lz::lacZ overlaps the regions of the antennal disc where amos expression occurs and labels all the basiconic and trichoid sensilla in the mature (36 h APF) antenna. Hence for the purpose of this study, all cells in which lz-Gal4 expresses will be considered to belong to the Amos-dependent lineages. ato-Gal4 drives reporter activity in proneural domains of the disc and subsequently in all cells of the coeloconic sense organs (Sen, 2004).

Most of the apoptotic nuclei observed during olfactory sense organ development co-localized with Lz::LacZ suggesting that death occurred mainly within the 'Amos-dependent' sensory clusters. Only very few TUNEL-positive cells were detected in regions where ato-lacZ expressed and these did not co-localize with the reporter expression. If PCD is the mechanism used to remove glial precursors from Amos lineages, then their rescue would be expected to result in additional peripheral glia in the antenna (Sen, 2004).

The GAL4/UAS system was used to target ectopic expression of baculovirus inhibitor of apoptosis protein (p35) to different cell types within the developing antennal disc. distalless⁹⁸¹-Gal4 (henceforth called dll-Gal4), which drives expression in all cells of the antennal disc, resulted in the formation of >300 glial cells as compared to ~100 in the wildtype. Other sensory cells--neurons, sheath, socket and shaft cells--within sense organs were unaffected. Ectopic expression of p35 specifically in Ato lineages (ato::p35) did not alter glial number. This means that the `additional' glial cells rescued in dll::p35 must arise from lineages other than Ato. Mis-expression of p35 in Amos-dependent lineages using lz-Gal4, on the other hand, resulted in a significant increase in glial number. While other explanations are possible, it is believed that the somewhat lower number of glia obtained in lz::p35 as compared to dll::p35 could be accounted for by the strength of the P(Gal4) driver (Sen, 2004) (Sen, 2004).

In order to identify the cell within the Amos lineage that is fated to die, the cellular events during development of sense organs were re-examined. At approximately 12-14 h APF, most sensory cells are associated in clusters of secondary progenitors. Two cells in each cluster -- PIIb and PIIc -- express the homeodomain protein Prospero (Pros). pros-Gal4;UAS-GFP recapitulates Pros expression at this stage and marks PIIb and PIIc and their progeny in all olfactory lineages. In the wildtype, a Repo-positive cell was associated with only a few of the total sensory clusters, these were all located within the coeloconic domain of the antenna. Targeted expression of p35 using pros-Gal4 increased glial number indicating that cells which are the progeny of either PIIb or PIIc could be rescued from apoptosis. In the pros-Gal UAS-2XEGFP/UAS-p35 genotype, a glial cell was associated with most clusters at 18 h APF rather than in Ato lineages alone (Sen, 2004).

In order to directly visualize the cell undergoing apoptosis, 22-24 h APF antenna from the neu^A101 strain were stained with antibodies against ß-galactosidase to mark the sensory cells and with TUNEL. Sensory clusters located in basiconic and trichoid domains of the pupal antenna each had a single associated TUNEL positive cell. Since TUNEL reactivity data does not reflect the initiation of the death program, developing antennae were also stained at different time points with an antibody that recognized the activated caspase -- Drice. At 20 h APF, a single Drice-positive cell was found within each sensory cluster within the basiconic and trichoid domains of the pupal antenna. This cell also expressed low levels of Pros suggesting that it could arise from either PIIb or PIIc. This means that the PIIb/c in Amos lineages, like that in Ato, divides to give rise to a PIIIb and its sibling. The sibling in the former lineage was not previously detected because it expresses only low levels of Pros and soon dies. Since this cell is capable of expressing the glial-identity gene repo when rescued from death, it is denoted as a glial precursor (Sen, 2004).

How is apoptosis of a specific cell within the lineage regulated? In Drosophila three genes [reaper (rpr), grim and head involution defective (hid)] which all map under the Df(3L)H99 are necessary for the initiation of the death program. Heterozygotes of Df(3L)H99 show a small but significant increase in glial number over that of normal controls. hid-lacZ was used to follow expression during antennal development; reporter activity occurs at low levels ubiquitously including in glial cells. Levels of reporter expression indicate somewhat higher hid transcription in glia rescued by p35 mis-expression. The presence of Hid in the 'normal' glial precursors suggests a mechanism dependent on possible trophic factors to keep cells alive. In several other systems signaling, mainly through the EGFR pathway, results in an antagonism of Hid action and transcription. The sustained levels of hid transcription in the rescued glia, is not unexpected since inhibitors of apoptosis act by antagonizing a downstream event of caspase activation, rather than on Hid itself (Sen, 2004).

Myc regulates organ size by inducing cell competition executed via induction of the proapoptotic gene hid

Experiments in both vertebrates and invertebrates have illustrated the competitive nature of growth and have led to the idea that competition is a mechanism for regulating organ and tissue size. Competitive interactions between cells were assessed in a developing organ and their effect on its final size were examined. Local expression of the Drosophila growth regulator dMyc, a homolog of the c-myc proto-oncogene, induces cell competition and leads to the death of nearby wild-type cells in developing wings. Cell competition is executed via induction of the proapoptotic gene hid and both competition and hid function are required for the wing to reach an appropriate size when dMyc is expressed. Moreover, evidence is provided that reproducible wing size during normal development requires apoptosis. Modulating dmyc levels to create cell competition and hid-dependent cell death may be a mechanism used during normal development to control organ size (de la Cova, 2004).

This work leads to three major conclusions. (1) Expression of the c-myc protooncogene homolog dMyc in small populations of wing disc cells induces cell competition, leading to the elimination of nearby cells via induction of the proapoptotic gene hid. (2) The competition induced by dMyc and the elimination of cells that results is required for control of proper wing size. (3) Studies reveal that apoptosis is required for the fidelity of size during normal wing development, suggesting that the modulation of hid expression by competitive interactions between cells may be used as an endogenous mechanism of size control (de la Cova, 2004).

These experiments demonstrate that expression of dMyc in some cells of a developing organ leads to elimination of nonexpressing cells through apoptosis. The growth disadvantage induced by dMyc-expressing cells fulfills the classic definition of cell competition: viable but slower-growing cells in an organ are eliminated by an encroaching faster-growing cell population, proximity to the fast-growing cell population dictates the severity of the disadvantage in the slow-growing cells, cells are protected from cell competition by developmental compartment boundaries, and appropriate organ size is reached at the end of development. Relative differences in dMyc levels lead to competitive situations between cells -- dmyc mutant cells are outcompeted by neighboring nonmutant cells; wild-type cells, with a normal complement of endogenous dmyc, are also subject to competition if surrounded by cells expressing a dMyc transgene. However, wild-type cells appear to be subject to competition only if they lie within about eight cell diameters of dMyc-expressing cells, and they must reside in the same developmental compartment. Thus, proximity, compartmental provenance, and the relative levels of dmyc are particularly important aspects of the competitive effects of dMyc (de la Cova, 2004).

During the process of cell competition induced by dMyc, the proapoptotic gene hid is induced in the growth-disadvantaged cells. Since a reduction of hid function protects cells from competition-induced death, it is believed that hid upregulation is a consequence of the sensing of competitive stress. An intriguing question that remains is how cells are able to sense competition. One possibility is that cells compete for sufficient levels of a survival factor that normally blocks hid expression. Dpp signaling promotes cell survival in the wing disc but appears to be unaffected in discs expressing dMyc. Alternatively, some cells in competition may be deprived of adequate nutrients, although in these experiments, cells at a growth disadvantage retain a normal nucleolar size, arguing that their biosynthetic rates are not abnormally low. However, the results suggest that dMyc provokes competition and hid expression via a short-range signal, since close proximity is required for the perception of competitive effects. Perhaps the most intriguing feature of this signal is that it is not perceived by nearby cells across a compartment boundary, although dMyc induces competition between cells within the posterior compartment as well as within the anterior. One possibility is that cells expressing dMyc acquire adhesive properties that transmit a competitive signal to neighboring cells, which is not compatible with the adhesive barrier that maintains the compartment boundary (de la Cova, 2004).

These studies reveal that cell competition is not invariably induced whenever rapidly growing cells populate regions of a developing organ. Both the PI3K Dp110 and cyclin D/Cdk4 potently promote growth when overexpressed, yet they do not induce competition in any of these assays. These observations also demonstrate that balanced growth -- growth that simultaneously drives cell division and cellular growth -- is not required to induce cell competition. dMyc expression increases clonal mass solely by increasing cell size. Thus, this trait of cell competition may be related to a size-measuring mechanism that recognizes total mass rather than cell number. However, Dp110 also promotes growth primarily by increasing cell size, indicating that qualitative differences exist in the cellular response to expression of dMyc and Dp110. Although both growth regulators increase protein synthesis, Orian (2003) suggests that dMyc probably does so by increasing components of the protein synthetic machinery (initiation factors and ribosomal proteins, etc.) whereas PI3K signaling is thought to function by increasing the utilization of existing machinery. Regardless of the mechanism, these experiments argue against the notion that apposed populations of fast- and slow-growing cells always result in cell competition (de la Cova, 2004).

Three lines of evidence have been provided that indicate that cell competition leading to cell death is required for control of wing size. (1) Growth induced by local expression of either Dp110 or cyclin D + Cdk4 does not induce competition and causes wing overgrowth. (2) When dMyc is expressed in all cells of the wing disc, the wing overgrows, whereas the introduction of clones lacking dMyc leads to cell competition and to wings approaching normal size. (3) Genetic reduction of hid prevents the cell death associated with competition and leads to overgrowth of the compartment in which the dMyc-expressing cells reside (de la Cova, 2004).

An important conclusion of this work is that apoptosis is critical for appropriate wing development. These experiments demonstrate that apoptosis has two roles in regulating wing size. One role is uncovered when the disc is challenged by local changes in dMyc levels, conditions in which cells are exceptionally sensitive to hid gene dosage: the full hid complement is necessary for the disc to respond properly to competition and eliminate cells. However, a second role of apoptosis is revealed when it is abolished: this role regulates uniformity of disc size, and its loss is manifested as a widening of the range of disc sizes within a given population. This second role of apoptosis indicates that organ overgrowth is distinct from loss of organ size control. Wing overgrowth -- observed when cell competition is not executed during local growth perturbations -- occurs such that, although larger than normal, wing size still falls within a uniform range. In contrast, loss of size control is the absence of a discrete and reproducible size population and results from a failure to induce apoptosis during the process of growth. Based on these observations, it is proposed that hid-regulated apoptosis contributes to a disc-intrinsic mechanism that limits variation in size by allowing elimination of cells. This mechanism may serve as negative feedback to the positive aspects of growth during development. Loss of feedback control could allow stochastic variation in size, as has been observed. Although it has been proposed that overall organ mass rather than cell number is sensed by the intrinsic size mechanism, these experiments imply that size control is implemented at least in part by reduction of cell number via apoptosis (de la Cova, 2004).

Is cell competition also part of the intrinsic size control program? If cell competition has a role in normal development, growth rate variations should be observed within developing organs. Indeed, both spatial and temporal differences in cell proliferation rates exist in the wing disc, and cell size also varies across the disc, suggesting differences in cellular growth rates. dmyc is regulated both by Wingless and Dpp, which direct the majority of disc patterning. Minor alterations in their signaling could plausibly cause subtle competitive effects by influencing levels of dmyc expression, which in turn would modulate hid expression and allow for the correction of patterning mistakes that occur during development. In this sense, cell competition, on a small scale, might be a surveillance or 'quality control' mechanism to guarantee that organs reach a body-proportional, reproducible size with the appropriate complement of cell fates (de la Cova, 2004).

Cell competition is likely a common mechanism used in organs under many conditions, including those that are adverse. Competitive mechanisms are known to be important to reestablish homeostasis in lymphoid tissue after an immune response. During tumorigenesis, cancer cells may compete with normal tissue and ultimately overtake the organ, leading to overgrowth of the tumor. In addition, cell competition could prove important therapeutically for many diseases. For example, when liver cells are transplanted into a diseased host liver, cell competition would be critical for the replacement of viable but damaged liver cells with the regenerating donor cells. Although of the three growth regulators tested only dMyc induced cell competition, other growth-promoting genes that induce cell competition probably exist. The identification of these genes holds promise for a further elucidation of the role of cell competition in organ development (de la Cova, 2004).

Apoptotic cells can induce compensatory cell proliferation through the JNK and the Wingless signaling pathways: DIAP1 antagonists reaper and hid can activate the JNK pathway which in turn is required for inducing wg and cell proliferation

In many metazoans, damaged and potentially dangerous cells are rapidly eliminated by apoptosis. In Drosophila, this is often compensated for by extraproliferation of neighboring cells, which allows the organism to tolerate considerable cell death without compromising development and body size. Despite its importance, the mechanistic basis of such compensatory proliferation remains poorly understood. Apoptotic cells are shown to express the secretory factors Wingless and Decapentaplegic. When cells undergoing apoptosis were kept alive with the caspase inhibitor p35, excessive nonautonomous cell proliferation is observed. Significantly, Wg signaling is necessary and, at least in some cells, also sufficient for mitogenesis under these conditions. Finally, evidence is provided that the DIAP1 antagonists reaper and hid can activate the JNK pathway and that this pathway is required for inducing wg and cell proliferation. These findings support a model where apoptotic cells activate signaling cascades for compensatory proliferation (Ryoo, 2004).

To investigate how the inhibition of diap1 may lead to mitogen expression, attention was focused on Dronc and the Jun N-terminal Kinase (JNK) pathway. Dronc has been implicated in compensatory proliferation, and its activity can be inhibited by the expression of dronc^DN. In addition, the JNK signaling pathway was considered as a candidate, since its activity is known to correlate with many forms of stress-provoked apoptosis, including disruption of morphogens, cell competition, and rpr expression. In Drosophila, the JNK pathway can be effectively blocked by the expression of puckered (puc), which encodes a phosphatase that negatively regulates JNK (Ryoo, 2004).

To induce patches of undead cells, wing imaginal discs were generated with mosaic clones expressing hid and p35. 48 hr after induction, these imaginal discs contained hid-expressing clones that autonomously induced wg. Using this experimental setup, it was asked whether additional expression of either dronc^DN or puc would block wg induction in undead cells. When dronc^DN was coexpressed, a subset of the hid-expressing population was still able to induce wg. In contrast, when puc was coexpressed, wg induction by hid was almost completely blocked. These results provide evidence that the JNK pathway is required for wg induction under these conditions but fail to uncover a similar requirement for Dronc (Ryoo, 2004).

To independently investigate the role of puc and dronc^DN in compensatory proliferation, the size of wing discs harboring undead cells was measured and they were compared with those of the sibling controls. Under the experimental conditions, wing discs harboring hid- and p35-expressing clones were on average 53% larger than their sibling controls. Coexpression of puc within these undead clones significantly limited growth, resulting in only a small increase in wing disc size that was not statistically significant. In contrast, coexpression of dronc^DN did not limit growth. Wing size measurements also correlated with the degree of wg induction. The larger size of discs harboring hid- and p35-expressing cells is not due simply to extra cell survival: (1) these undead cells are derived from the normal lineage; (2) the size of wing discs expressing hid, p35, and puc serves as a control. In this case, although a large number of undead cells were generated, no significant increase in disc size was observed, in stark contrast to the discs expressing hid and p35 only. It is concluded that the JNK pathway is required for the nonautonomous growth promoting activity of the undead cells (Ryoo, 2004).

To confirm a role of puc in imaginal disc growth, rpr and p35 werecoexpressed in wild-type and puc^−/+ imaginal discs. Like hid, rpr is a DIAP1 antagonist, but with a weaker cell killing activity when overexpressed in imaginal disc cells. In a puc^+/+ background, a small amount of ectopic wg expression was observed, indicative of rpr's weaker DIAP1 inhibiting activity. In contrast, ectopic wg expression was strongly enhanced in puc^−/+ discs. Because the puc allele used, puc^E69, also acts as a lacZ reporter, JNK pathway induction could be monitored simultaneously. wg induction in undead cells correlates very well with puc-lacZ expression, with a stronger induction at the center of the wing pouch. These results further support the role of JNK in the induction of wg (Ryoo, 2004).

Next to be tested was whether the reduction of puc had an effect on apoptosis-induced cell proliferation. Whereas puc^−/+ discs expressing only p35 had BrdU incorporation similar to wild-type discs, coexpression of rpr and p35 in puc^−/+ led to a significant increase in BrdU incorporation. Also, the size of these discs were on average 41% larger than those coexpressing rpr and p35 in a puc^+/+ background. Taken together, these results show that diap1 inhibition leads to JNK activation and that JNK activity promotes wg induction and cell proliferation (Ryoo, 2004).

To directly test if JNK signaling can activate wg and dpp expression, hep^CA, a constitutively active form of hemipterous (hep), the Drosophila JNK kinase was conditionally expressed. Expression of hep^CA causes induction of wg-lacZ within 22 hr and to a lesser extent also dpp-lacZ. These ß-gal-expressing cells shifted basally and were apoptotic as assayed by anti-active caspase-3 antibody labeling. Hid protein levels were also elevated in these cells. Significantly, since p35 was not use to block apoptosis in this experiment, this demonstrates that wg and dpp can be induced not only in undead cells, but also in 'real' apoptotic cells (Ryoo, 2004).

This study provides evidence that the central apoptotic regulators can control the activity of mitogenic pathways. In particular, inhibition of DIAP1, either via expression of Reaper and Hid or by mutational inactivation, leads to the induction of the putative mitogens wg and dpp. When apoptosis was initiated through DIAP1 inhibition but cells were kept alive by blocking caspases, the resulting 'undead cells' exhibited strong mitogenic activity and stimulated tissue overgrowth. Inhibiting wg signaling with a conditional TCF^DN blocked cell proliferation in imaginal discs, indicating that wg has an essential mitogenic function. Finally, evidence was provided that the JNK pathway mediates mitogen expression and imaginal disc overgrowth in response to rpr and hid. Based on these results, it is proposed that apoptotic cells actively signal to induce compensatory proliferation. DIAP1 inhibits both caspases as well as dTRAF1. According to this model, when DIAP1 is inhibited in response to cellular injury, the JNK pathway is activated and wg/dpp are induced in apoptotic cells. Secretion of these factors stimulates growth of proliferation-competent neighboring cells and leads to compensatory proliferation (Ryoo, 2004).

This study provides clear genetic evidence that diap1 is involved in compensatory proliferation. Overall, similar results were obtained with hypomorphic diap1 alleles (diap1^22-8s, diap1^33-1s), a null allele (diap1^th5), and inactivation of diap1 by expression of Reaper and Hid. However, whereas expression of p35 effectively blocked apoptosis of diap1^22-8s/22-8s cells and in response to Reaper/Hid, it only partially suppressed the death of diap1^th5/th5 cells. Consequently, the generation of undead cells was less efficient with the diap1^th5 mutation. Moreover, these results suggest that the JNK pathway transduces the signal to activate mitogen expression and cell proliferation. Since IAPs have been shown to ubiquitylate TRAFs in both mammals and Drosophila and since no evidence was found for Dronc in growth promotion, it is attractive to speculate that JNK is regulated through direct DIAP1/TRAF1 interaction (Ryoo, 2004).

An important unresolved question is why compensatory proliferation is seen only in response to cellular injury, but not during normal developmental apoptosis. In particular, inactivation of DIAP1 by Reaper, Hid, and Grim is restricted not only to injury-provoked apoptosis, but also underlies most developmental cell deaths. One possible explanation is that activation of the JNK pathway is key to mitogenic signaling of apoptotic cells. Consistent with this idea, the JNK pathway is activated in response to tissue stress and injury, but not during developmental apoptosis. Furthermore, this study shows that JNK signaling can induce the expression of wg/dpp and nonautonomous cell proliferation. Therefore, it is possible that robust JNK activation and compensatory proliferation require the combined input of stress and apoptotic signals (Ryoo, 2004).

Compensatory proliferation in Drosophila imaginal discs requires Dronc-dependent p53 activity

The p53 transcription factor directs a transcriptional program that determines whether a cell lives or dies after DNA damage. Animal survival after extensive cellular damage often requires that lost tissue be replaced through compensatory growth or regeneration. In Drosophila, damaged imaginal disc cells can induce the proliferation of neighboring viable cells, but how this is controlled is not clear. This paper provides evidence that Drosophila p53 has a previously unidentified role in coordinating the compensatory growth response to tissue damage. The sole p53 ortholog in Drosophila, is required for each component of the response to cellular damage, including two separate cell-cycle arrests, changes in patterning gene expression, cell proliferation, and growth. These processes are regulated by p53 in a manner that is independent of DNA-damage sensing but that requires the initiator caspase Dronc. These results indicate that once induced, p53 amplifies and sustains the response through a positive feedback loop with Dronc and the apoptosis-inducing factors Hid and Reaper. How cell death and cell proliferation are coordinated during development and after stress is a fundamental question that is critical for an understanding of growth regulation. These data suggest that p53 may carry out an ancestral function that promotes animal survival through the coordination of responses leading to compensatory growth after tissue damage (Wells, 2006; full text of article).

The repair of tissue after cellular damage can be critical to the survival of the animal. Previous studies demonstrated that undead cells stimulate the proliferation of neighboring cells, providing a model for how damaged and dying cells contribute to the replacement of lost tissue. With this model, it was found that the wing imaginal disc responds to this damage as a whole by deploying a multi-step process that ends with compensatory growth. p53 functions in a dronc-dependent manner at each step of the tissue-replacement process. Furthermore, p53 and the initiator caspase dronc may be generally required for tissue recovery in imaginal discs, because it was found that blastema formation was significantly impaired during regeneration induced in either p53 or dronc mutant leg discs (Wells, 2006).

The data suggest that p53 is induced and becomes functional in undead cells by a mechanism that does not require DNA-damage sensing or activation of the stress kinase AMPK. Rather, Dronc, an initiator caspase homologous to caspase-9, is necessary and sufficient to induce all aspects of the growth regulation by p53. It is not known how Dronc activity results in p53 expression and activity in these cells, but many caspase substrates are not directly involved in apoptosis. As an example, one of the first caspase substrates identified was the cytokine IL-1β, which regulates many aspects of the inflammatory response. Induction of p53 mRNA in undead cells is prevented in dronc mutant discs, and thus it is possible that a regulator of p53 is cleaved by Dronc, leading to its expression and ultimately to its ability to regulate the compensatory growth response in the imaginal discs. Regardless of the molecular mechanism, the data argue for direct communication between Dronc and p53 in response to tissue damage (Wells, 2006).

Collectively, these experiments imply that p53 serves as a master coordinator of tissue repair in imaginal discs, regulating both cell-autonomous and non-cell-autonomous cell-cycle arrests, the expression of the pattern-regulating genes wg and dpp, and compensatory cell proliferation and growth. Based on these results, it is suggested that cellular damage activates Dronc, which in a nonapoptotic role causes the induction of p53 mRNA and leads to p53 activity. It is proposed that p53 then acts as an overall damage monitor, in a role that includes its conserved functions in apoptosis (here, induction of hid and rpr expression) and growth arrest (by repression of stg/cdc25), but also allows for induction of signals that promote compensatory growth of the disc. The results suggest that p53 monitors tissue damage through a feed-forward loop with Dronc and the pro-apoptotic genes hid and rpr, which both amplifies and sustains the growth-regulating signal (Wells, 2006).

An intriguing puzzle left unanswered by these results is why the growth response to undead cells occurs only several days after they are generated: both HhGal4 and EnGal4 drive expression of Hid or Rpr from early embryonic stages, yet even with careful observation no growth phenotype was detected until the middle part of the third instar. Caspases are active in cells expressing Hid or Rpr + P35 at early time points, indicating that these cells are not immune to the apoptotic response early in development. The genes involved in the apoptotic response are subject to many levels of control, including that by micro-RNAs (miRNAs). Hid protein expression, for example, is suppressed by Bantam, a miRNA highly expressed early in imaginal disc development, but declining as development progresses. It is likely that rpr is also regulated by miRNA gene silencing. Hence, the delay of the growth response in discs with undead cells may reflect a requirement for threshold levels of these factors to fully activate the feedback loop. At the very least it emphasizes that the regulation of growth and cell death during wing disc development is complex and has multiple inputs, many of which are poorly understood (Wells, 2006).

Activity thresholds appear to play an important role in the processes induced by undead cells. Dronc, for instance, is haploinsufficient for its effect in compensatory proliferation. It is possible that the apoptotic functions of Dronc require a relatively low activity level, but that high Dronc activity allows activation of the p53-dependent tissue-damage response. Regulation of Dronc by critical activity thresholds could provide the animal some regenerative capacity and increase its chances for survival when conditions are appropriate for tissue repair (Wells, 2006).

As expected given its role in coordinating many cellular behaviors, p53 modulates the activity or expression of myriad effectors. Regulatory effectors of Drosophila p53 are only beginning to be identified, and these data add stg/cdc25 to the list. One of the first detectable disc responses to undead cells is G2 arrest, mediated by loss of stg mRNA. Cdc25 is also regulated by vertebrate p53 but is inhibited post-transcriptionally by p53-dependent 14-3-3 activity (Levine, 2006). Experiments with irradiated p53 mutant animals have not revealed a cell-cycle arrest role. However, recent work indicates that dp53 also regulates a G1 checkpoint under conditions of metabolic stress; thus, like vertebrate p53, Drosophila p53 can activate both a G1 and a G2 checkpoint in response to tissue stress. Other effectors and targets involved in the compensatory proliferation process remain unknown, although expression profiling experiments from irradiated p53 mutants identified several potential targets, several of which do not have obvious roles in cell death or DNA repair (Wells, 2006).

How does Drosophila p53 control the signaling that leads to compensatory proliferation? The events observed — G2 arrests in two different cell populations, ectopic expression of wg, and compensatory growth — are all regulated by p53. It is possible that p53 directly and coordinately controls each of these processes by regulating the expression of specific effectors. However, because the response is both cell autonomous and non-cell autonomous, the idea is favored that these processes are interdependent, but sequentially activated. It is envisioned that as a result of Dronc activation in undead cells, p53 induces loss of stg, leading to G2 arrest, and hid and rpr expression, initiating the feedback loop. It is postulate that cells then synthesize factors that stimulate their survival and proliferation. The non-cell-autonomous arrest in the anterior compartment may be a secondary effect of undead cells in the posterior. High levels of TUNEL activity was observed in the anterior cells of these discs, which could feasibly activate p53 in those cells. However, no p53 mRNA was detected in anterior cells. One possibility is that the DNA fragmentation resulting from dying anterior cells could activate ATM and Chk2 in those cells. Consistent with this, although loss of either of these kinases did not affect undead cell induction of Wg expression or compensatory growth, the cell-cycle arrest in anterior cells was reduced in a fraction of atm and chk2 mutants (Wells, 2006).

What is the growth-stimulating signal induced by undead cells? While its identity is still unclear, both Wg and Dpp have been implicated in this role. This makes sense, because Wg and Dpp are the major pattern organizers of all imaginal discs and are also involved in regulating their growth, and furthermore they are known to be induced in disc regeneration. However, although wg and dpp are ectopically expressed in undead cells, it was found that targets of both are sharply downregulated, specifically in the undead cells. These data also show that undead cells are able to proliferate and contribute to the compensatory growth. Thus, although the nonautonomous stimulation of growth (anterior cells near the A/P boundary) could be due to increased Dpp signaling, it is suspected that the autonomous growth stimulation is due to other, unidentified factors (Wells, 2006).

This study identified a growth-regulatory role for p53 that seems counter to its role as a tumor suppressor in vertebrates. However, it is speculated that the ability of p53 to sense and respond to tissue damage and promote compensatory proliferation and regeneration in Drosophila reflects an ancestral function, aspects of which have been appropriated for developmental processes and distributed among p53, p63, and p73 during vertebrate evolution. Although p63 and p73 initially were proposed to have evolved as duplications of p53, reanalysis of the phylogenetic relationship between the three family members has suggested that p63 may be the ancestral gene. p63 and p73 are structurally similar to p53 but contain an additional SAM domain. p53 is the sole member of the family encoded in the Drosophila genome, and although dp53 does not contain a SAM domain, based on the sequence of the DNA binding domain, the most highly conserved region of p53, it is more related to vertebrate p63 than to p53. After irradiation, cell-cycle arrest is not p53 dependent in either Drosophila or the nematode C. elegans, and therefore it has been proposed that the ancestral p53 function is apoptosis, rather than a “repair, then death” response when damage cannot be repaired. The experiments argue that as in vertebrates, p53 plays a role in cell-cycle arrest after tissue damage. The additional functions of p53 in promoting cell proliferation may have been conserved in p63, which regulates progenitor cell renewal in the epidermis. Other processes that require cell renewal may also be regulated by p53. For example, p53 mutants are reported to have fertility defects, so it is tempting to speculate that stem cell renewal in the gonad requires this previously unappreciated role of Drosophila p53 (Wells, 2006).

Multiple apoptotic caspase cascades are required in nonapoptotic roles for Drosophila spermatid individualization

Spermatozoa are generated and mature within a germline syncytium. Differentiation of haploid syncytial spermatids into single motile sperm requires the encapsulation of each spermatid by an independent plasma membrane and the elimination of most sperm cytoplasm, a process known as individualization. Apoptosis is mediated by caspase family proteases. Many apoptotic cell deaths in Drosophila utilize the REAPER/HID/GRIM family proapoptotic proteins. These proteins promote cell death, at least in part, by disrupting interactions between the caspase inhibitor DIAP1 and the apical caspase DRONC, which is continually activated in many viable cells through interactions with ARK, the Drosophila homolog of the mammalian death-activating adaptor APAF-1. This leads to unrestrained activity of DRONC and other DIAP1-inhibitable caspases activated by DRONC. This study demonstrates that ARK- and HID-dependent activation of DRONC occurs at sites of spermatid individualization and that all three proteins are required for this process. dFADD, the Drosophila homolog of mammalian FADD, an adaptor that mediates recruitment of apical caspases to ligand-bound death receptors, and its target caspase DREDD are also required. A third apoptotic caspase, DRICE, is activated throughout the length of individualizing spermatids in a process that requires the product of the driceless locus, which also participates in individualization. These results demonstrate that multiple caspases and caspase regulators, likely acting at distinct points in time and space, are required for spermatid individualization, a nonapoptotic process (Huh, 2004; full text of article).

Effects of Mutation or Deletion

The head involution defective locus is located within the chromosomal region 75B8-C1,2. During the morphogenetic reorganization of the embryonic head region, hid+ function is necessary for the movement of the dorsal fold across the procephalon and clypeolabrum, a process that forms the frontal sac. The absence of the frontal sac in the hid mutant embryos affects the formation of the dorsal bridge and disrupts the development of the larval cephalopharyngeal skeleton. In addition to its embryonic role, this same hid function is also required during pupal development for the 360 degrees rotation about the anterior-posterior body axis of the male terminalia, and for a late step of wing blade morphogenesis. Although the abnormal wing phenotype caused by the Wrinkled (W) mutation is quite different from the one resulting from the loss-of-function hid mutations, the characterization of EMS-induced W revertants reveals that W is actually an antimorphic allele of hid (Abbott, 1991).

Deletions of chromosomal region 75C1,2 block virtually all programmed cell death (PCD) in the Drosophila embryo. A second gene, in addition to reaper, has now been identified in this region. head involution defective (hid) plays a similar role in PCD. hid mutant embryos have decreased levels of cell death and contain extra cells in the head. hid mutant embryos have extra cells in the head region, in particular, extra larval photoreceptor cells. There are also extra cells in the abdominal segments. Expression of the hid gene is sufficient to induce PCD in cell death defective mutants. The hid gene appears to encode a novel 410-amino-acid protein, and its mRNA is expressed in regions of the embryo where cell death occurs. Ectopic expression of hid in the Drosophila retina results in eye ablation. This phenotype can be suppressed completely by expression of the anti-apoptotic p35 protein from baculovirus, indicating that p35 may act genetically downstream from hid (Grether, 1995).

Expression of the cell death regulatory protein Reaper (Rpr) in the developing Drosophila eye results in a smaller than normal eye owing to excess cell death. Mutations in thread (th) are dominant enhancers of Rpr-induced cell death. thread encodes a protein homologous to baculovirus inhibitors of apoptosis (IAPs), called Drosophila IAP1 (DIAP1). Overexpression of DIAP1 (or a related protein, DIAP2) in the eye suppresses normally occurring cell death as well as death due to overexpression of rpr or head involution defective. IAP death-preventing activity localizes to the N-terminal baculovirus IAP repeats, a motif found in both viral and cellular proteins associated with death prevention (Hay, 1995).

A new activator of apoptosis, grim, maps between two previously identified cell death genes in this region: reaper and head involution defective. Expression of Grim RNA coincides with the onset of programmed cell death at all stages of embryonic development, whereas ectopic induction of grim triggers extensive apoptosis in both transgenic animals and in cell culture. Cell killing by Grim was blocked by coexpression of p35, a viral product that inactivates ICE-like proteases, and does not require the function of either rpr or hid. The predicted Grim protein shares an amino-terminal motif in common with RPR. However, Grim is sufficient to elicit apoptosis in at least one context, where Rpr is not. The grim gene product might thus function in a parallel circuit of cell death signaling that ultimately activates a common set of downstream apoptotic effectors (Chen, 1996).

The neuropeptide eclosion hormone (EH) is a key regulator of insect ecdysis. The role of the two EH-producing neurons in Drosophila was determined by using an EH cell-specific enhancer to activate cell death genes reaper and head involution defective in order to ablate the EH cells. In the EH cell knockout flies, larval and adult ecdyses are disrupted, yet a third of the knockouts emerge as adults, demonstrating that EH has a significant but nonessential role in ecdysis. The EH cell knockouts have discrete behavioral deficits, including slow, uncoordinated eclosion and an insensitivity to ecdysis-triggering hormone. The knockouts lack the lights-on eclosion response despite having a normal circadian eclosion rhythm. This study represents a novel approach to the dissection of neuropeptide regulation of a complex behavioral program (McNabb, 1997).

In Drosophila, the chromosomal region 75C1-2 contains at least three genes (reaper, head involution defective, and grim) that have important functions in the activation of programmed cell death. To better understand how cells are killed by these genes, a well defined set of embryonic central nervous system midline cells have been used that normally exhibit a specific pattern of glial cell death. Most of the developing midline glia die and are quickly phagocytosed by migrating macrophages, whereas none of the ventral unpaired median neurons die during embryogenesis. Both rpr and hid are expressed in dying midline cells; the normal pattern of midline cell death requires the function of multiple genes in the 75C1-2 interval. The P[UAS]/P[Gal4] system was used to target expression of rpr and hid to midline cells. Targeted expression of rpr or hid alone is not sufficient to induce ectopic midline cell death. However, expression of both rpr and hid together rapidly induces ectopic midline cell death, resulting in axon scaffold defects characteristic of mutants with abnormal midline cell development. Midline-targeted expression of the baculovirus p35 protein, a caspase inhibitor, blocks both normal and ectopic rpr- and hid-induced cell death. Taken together, these results suggest that rpr and hid are expressed together and cooperate to induce programmed cell death during development of the central nervous system midline (Zhou, 1997).

The Drosophila larva modulates its pattern of locomotion when exposed to light. Modulation of locomotion can be measured as a reduction in the distance traveled and by a sharp change of direction when the light is turned on. When the light is turned off this change of direction, albeit significantly smaller than when the light is turned on, is still significantly larger than in the absence of light transition. Mutations that disrupt adult phototransduction disrupt a subset of these responses. In larvae carrying these mutations the magnitude of change of direction when the light is turned on is reduced to levels indistinguishable from that recorded when the light is turned off, but it is still significantly higher than in the absence of any light transition. Similar results are obtained when these responses are measured in strains where the larval photoreceptor neurons have been ablated by mutations in the glass (gl) gene or by the targeted expression of the cell death gene head involution defective (hid). A mutation in the homeobox gene sine oculis (so) that ablates the larval visual system, or the targeted expression of the reaper (rpr) cell death gene, abolishes all responses to light detected as a change of direction. The existence of an extraocular light perception that does not use the same phototransduction cascade as the adult photoreceptors is proposed. The results indicate that this novel visual function depends on the blue-absorbing rhodopsin Rh1 and is specified by the so gene (Busto, 1999).

Three genes---reaper, grim, and hid---are crucial to the regulation of programmed cell death in Drosophila. Mutations involving all three genes virtually abolish apoptosis during development, and homozygous hid mutants die as embryos with extensive defects in apoptosis. Although Hid is central to apoptosis in Drosophila, it has no mammalian homolog identified to date. Evidence is presented that expression of Drosophila Hid in mammalian cells induces apoptosis. This activity is subject to regulation by inhibitors of mammalian cell death. The N terminus of Hid, which is a region of homology with Reaper and Grim, is essential for Hid's function in mammalian cells. Hid is localized to the mitochondria via a hydrophobic region at its C terminus and functionally interacts with BclXL. This study shows that the function of Hid as a death inducer in Drosophila is conserved in mammalian cells and argues for the existence of a mammalian homologue of this critical regulator of apoptosis (Haining, 1999).

Some Bcl2 family members have potent antiapoptotic effects. The antiapoptotic members include BclXL and the adenoviral protein E1B19k. Although homologs of this family exist in C. elegans and mammals, no Drosophila counterpart has yet been identified. It was therefore of interest to ascertain whether the apoptosis pathway triggered by Hid in mammalian cells is susceptible to Bcl2-family inhibition. BclXL shows a pronounced effect on reducing Hid-induced apoptosis (35% to 11%), whereas E1B19k shows a more modest effect (to 23%). These results demonstrate that Bcl2-type antiapoptotic genes can inhibit Hid-induced apoptosis in mammalian cells (Haining, 1999).

Given the functional interaction between Hid and BclXL, a protein that can target the mitochondria, it was of interest to determine the cellular localization of Hid. A monoclonal antibody was raised to full-length Hid protein and used to label transfected cells immunohistochemically. Hid immunostaining is predominantly punctate and perinuclear. To better identify the subcellular distribution of Hid, transfected cells were colabeled with a fluorescent dye that accumulates inside mitochondria. The pattern of mitochondrial staining is very similar to that of Hid. Merged images of Hid- and mitochondrially stained cells show a striking concordance in the distribution of these two stains. This result demonstrates that Drosophila Hid localizes to mitochondria when expressed in mammalian cells. Further magnified views of dually stained cells shows that, although the pattern of staining is very similar, it is not overlapping; rather, the Hid-staining appears on the outside of the mitochondrion whereas the mitochondrial dye labels the inner portion. Despite the lack of Hid-induced apoptosis in 293 cells, it is noteworthy that Hid's distribution in these cells is also mitochondrial (Haining, 1999).

Because the mitochondrial localization of Hid had not been previously demonstrated in insect cells, Hid was expressed by transient transfection in the insect cell line SF9. This cell line was found to be susceptible to apoptosis from Hid overexpression. Cells colabeled with mitochondrial dye and Hid antibody again showed a predominantly mitochondrial pattern of Hid staining (Haining, 1999).

To assess the effect of apoptosis inhibition on the pattern of Hid staining, immunohistochemistry was performed on HeLa cells cotransfected with Hid and BclXL. The mitochondrial localization of Hid is disrupted in these cells, and Hid fluorescence is found in a diffuse pattern, suggestive of cytoplasmic distribution. This effect is not seen in cells cotransfected with p35, DIAP1, or XIAP or in those treated with the inhibitor of apoptosis BOC-D-fmk (Haining, 1999).

To investigate which portions of the Hid molecule are required for its proapoptotic activity and subcellular localization, two Hid mutant proteins encoded by alleles A206 and A329 were studied. These mutations in the hid gene locus were induced in flies by chemical mutagenesis, and they cause a mild reduction in Hid function in Drosophila. Each mutation is the result of a single nucleotide change that causes a premature stop codon at amino acid position 261 and position 304 in alleles A206 and A329, respectively. Both of these prematurely truncated proteins induce apoptosis in HeLa cells at levels comparable to those caused by wild-type Hid. This may be because of the high levels of Hid expression achieved in HeLa cells. A reduction of Hid function that may be sufficient to reduce its proapoptotic activity in Drosophila cells may not be noticeable in HeLa cells because of the large amounts of Hid protein expressed. Immunohistochemistry of cells transfected with each of these mutants, however, shows a marked alteration of cellular localization. Whereas levels of expression are comparable, the mitochondrial targeting of wild-type Hid is completely lost, and the mutant Hid-transfected cells shows a diffuse cytoplasmic pattern of staining. Although Hid appears to have neither a signal sequence nor a mitochondrial localization signal, close scrutiny of the C terminus reveals a stretch of hydrophobic residues (amino acid position 393-409). Deletion of these residues is sufficient to abolish mitochondrial localization. However, this mutation does not impair apoptosis induction. These results suggest that when expressed at high levels in HeLa cells, Hid does not require mitochondrial localization to effect cell death. However, the fact that mutations that delete the C terminus of Hid were identified as loss-of-function in Drosophila suggests that this domain, and possibly mitochondrial localization, is important for Hid's proapoptotic function (Haining, 1999).

Sequence analysis of Hid, Reaper, and Grim reveals similarities among the three proteins restricted to their N-terminal 14 amino acids. Deleting residues 2-14 of Hid abolishes its ability to initiate apoptosis in mammalian cells. Immunostaining of mutant-transfected cells shows levels of expression comparable to cells transfected with wild-type Hid. The deletion does not impair the mutant's ability to localize to the mitochondria. Because the deleted region is that required for DIAP1 binding, one interpretation of this result is that binding to IAPs (presumably endogenous mammalian IAPs) in these experiments is essential for Hid's ability to induce cell death in HeLa cells (Haining, 1999).

During development, signaling pathways coordinate cell fates and regulate the choice between cell survival or programmed cell death. The well-conserved Wingless/Wnt pathway is required for many developmental decisions in all animals. One transducer of the Wingless/Wnt signal is Armadillo/ß-catenin. Drosophila Armadillo not only transduces Wingless signal, but also acts in cell-cell adhesion via its role in the epithelial adherens junction. While many components of both the Wingless/Wnt signaling pathway and adherens junctions are known, both processes are complex, suggesting that unknown components influence signaling and junctions. A genetic modifier screen was carried out to identify some of these components by screening for mutations that can suppress the armadillo mutant phenotype. Twelve regions of the genome were identified that have this property. From these regions and from additional candidate genes tested, four genes were identified that suppress arm: dTCF, puckered, head involution defective (hid), and presenilin. The interaction with hid, a known regulator of programmed cell death, was further investigated. The data suggest that Wg signaling modulates Hid activity and that Hid regulates programmed cell death in a dose-sensitive fashion (Cox, 2000).

It has been known for more than a decade that PCD plays an important role in the segment polarity phenotype resulting from inactivation of either the Hedgehog or Wg pathways. Detailed analysis of this process has been carried out, quantitating cell death in wg, arm, gooseberry, and naked. The elevation in cell death affects particular cells. Since the first reports of cell death in segment polarity mutants, the machinery that drives PCD in embryos has begun to be identified. Homozygosity for the small chromosomal Deficiency, Df(3L)H99, blocks essentially all PCD. Within this interval, three genes play roles in PCD: grim, reaper, and hid. Ectopic expression of any of these can trigger PCD, but loss-of-function mutations are only available for hid (Cox, 2000 and references therein).

Given the role of PCD in the segment polarity phenotype, it is perhaps not surprising that elimination of PCD would alter it. Several aspects of the effect of PCD reduction were unexpected, however. First, and most striking, the phenotypes of arm and wg mutants were very sensitive to relatively small changes in the dose of hid and the other cell-death promoters. For example, while heterozygosity for hid has no known effects on normal development, it strongly suppresses arm. Further reductions in the levels of hid or the other cell-death regulators have no additional effect on arm, suggesting that reducing the Hid dose by half eliminates the relevant ectopic PCD that occurs in an arm mutant. The wg phenotype is also suppressed in a highly dose-sensitive fashion, but in a different dosage range. A 50% reduction of hid causes slight but detectable effects; a 50% reduction in all three death promoters causes greater suppression, while homozygosity for the deletion removing all three genes results in the strongest wg suppression (Cox, 2000).

Recent observations regarding the role of Hid in PCD in the eye may explain this. Signaling through the ras/mitogen-activated protein kinase (MAPK) pathway promotes cell survival by antagonizing Hid. It has been suggested that Hid serves as a rheostat, with its levels determining the probability of PCD. It has been further suggested that Hid activity has to exceed a threshold to trigger PCD; the accumulation of hid mRNA in cells that are not programmed to die is consistent with this. Current observations further support this model. Wg signaling may normally antagonize Hid, potentially by regulating its expression. In embryos where Wg signaling is attenuated, elevated Hid activity may trigger PCD when it rises above a critical threshold. A threshold model could explain why the segment polarity phenotype is so sensitive to the dose of Hid and its partners (Cox, 2000 and references therein).

Another surprise was the qualitative difference in the effect of cell death reduction on wg and arm mutants. While the resulting cell number is likely increased in both double-mutant genotypes in the arm; hid double mutant, the reduction in PCD restored an almost wild-type-length cuticle, while in the wg;hid double mutant, the increase in cell number is not reflected in an increase in cuticle length. The reason for this remains a mystery. One possible explanation for this discrepancy is the difference in the degree to which Wg signal is compromised in the two situations and the embryonic stage at which this disruption occurs. In the wg null, Wg signaling is totally eliminated from the beginning of development. In contrast, perdurance of maternal Arm substantially rescues early defects in Wg signaling in arm zygotic nulls. arm mutants remain more normal in morphology than wg mutants through the onset of germband retraction and retain remnant denticle diversity. Thus when one eliminates PCD in an arm mutant a more normal pattern is restored. The difference in amount and timing of Wg signaling in the two backgrounds may also explain why arm mutants are affected by smaller alterations in Hid level. The remaining Wg signaling in an arm zygotic mutant may promote cell survival to some extent, meaning that a smaller reduction in Hid activity prevents ectopic PCD (Cox, 2000).

It is also surprising that reduction in cell death alleviates arm's dorsal closure defect. It has been suspected that this defect is due solely to Arm's role as a catenin. However, recent data suggest that dorsal closure is promoted by Wg signaling. It is now suspected that defects in Wg signaling and catenin function combine to block dorsal closure in arm mutants. Restoring either rescues the arm dorsal closure defect. However, blocking PCD alone should not restore Wg signaling or catenin function. Perhaps the excess cell death in the head region or in the amnioserosa of an arm mutant contributes to its dorsal closure defect (Cox, 2000).

Mutations that remove DRONC are not available. Therefore, to examine a possible role for DRONC as a cell death effector a form of DRONC, DRONC^C318S, was generated in which the active site cysteine was altered to serine. Expression of similar forms of other caspases results in a suppression of caspase activity and caspase-dependent cell death. This may occur as a result of interaction of DRONC^C318S with the Drosophila homolog of the caspase-activating protein Apaf-1, thus preventing the Drosophila Apaf-1 from binding to wild type DRONC and promoting its activation in a manner similar to that described for mammalian Apaf-1 and caspase-9. Transgenic Drosophila were generated in which DRONC^C318S was expressed under the control of a promoter, known as GMR, that drives transgene expression specifically in the developing fly eye. The eyes of these flies, known as GMR-DRONC^C318S flies, appear similar to those of wild type flies. To assay the ability of DRONC^C318S to block cell death, GMR-DRONC^C318S flies were crossed to flies overexpressing rpr (GMR-rpr), hid (GMR-hid), or grim (GMR-grim) under the control of the same promoter. GMR-driven expression of rpr, hid, or grim results in a small eye phenotype due to activation of caspase-dependent cell death. However, flies coexpressing GMR-DRONC^C318S and one of the cell death activators showed a dramatic suppression of the small eye phenotype, indicating that cell death had been suppressed. The possibility cannot be ruled out that this suppression is a result of DRONC^C318S forming nonproductive interactions with the Drosophila Apaf-1 that block its ability to activate other long prodomain caspases such as DCP-2/DREDD. However, these possibilities notwithstanding, these results suggest that DRONC activity is important for bringing about rpr-, hid-, and grim-dependent cell death (Hawkins, 2000).

Apoptosis plays a major role in vertebrate and invertebrate development. The adult Drosophila thoracic microchaete is a mechanosensory organ whose development has been extensively studied as a model of how cell division and cell determination intermingle. This sensory organ arises from a cell lineage that produces a glial cell and four other cells that form the organ. In this study, using an in vivo approach as well as fixed material, it has been shown that the glial cell undergoes nucleus fragmentation shortly after birth. Fragmentation was blocked after overexpression of the caspase inhibitor p35 or removal of the pro-apoptotic genes reaper, hid and grim, showing that the glial cell undergoes apoptosis. Moreover, it seems that fragments are eliminated from the epithelium by mobile macrophages. Forcing survival of the glial cells induces precocious axonal outgrowth but does not affect final axonal patterning and connectivity. However, under these conditions, glial cells do not fragment but leave the epithelium by a mechanism that is reminiscent of cell competition. Finally, evidence is presented showing that glial cells are committed to apoptosis independently of gcm and prospero expression. It is suggested that apoptosis is triggered by a cell autonomous mechanism (Fichelson, 2003).

Genetic and microarray analyses have been used to determine how ionizing radiation (IR) induces p53-dependent transcription and apoptosis in Drosophila melanogaster. IR induces MNK/Chk2-dependent phosphorylation of p53 without changing p53 protein levels, indicating that p53 activity can be regulated without an Mdm2-like activity. In a genome-wide analysis of IR-induced transcription in wild-type and mutant embryos, all IR-induced increases in transcript levels required both p53 and the Drosophila Chk2 homolog MNK. Proapoptotic targets of p53 include hid, reaper, sickle, and the tumor necrosis factor family member EIGER. Overexpression of Eiger is sufficient to induce apoptosis, but mutations in Eiger do not block IR-induced apoptosis. Animals heterozygous for deletions that span the reaper, sickle, and hid genes exhibited reduced IR-dependent apoptosis, indicating that this gene complex is haploinsufficient for induction of apoptosis. Among the genes in this region, hid plays a central, dosage-sensitive role in IR-induced apoptosis. p53 and MNK/Chk2 also regulate DNA repair genes, including two components of the nonhomologous end-joining repair pathway, Ku70 and Ku80. These results indicate that MNK/Chk2-dependent modification of Drosophila p53 activates a global transcriptional response to DNA damage that induces error-prone DNA repair as well as intrinsic and extrinsic apoptosis pathways (Brodsky, 2004).

Role of programmed cell death in patterning the Drosophila antennal arista

Programmed cell death is a critical process for the patterning and sculpting of organs during development. The Drosophila arista, a feather-like structure at the tip of the antenna, is composed of a central core and several lateral branches. A homozygous viable mutation in the thread gene, which encodes an inhibitor of apoptosis protein, produces a branchless arista. Mutations in the proapoptotic gene hid led to numerous extra branches, suggesting that the level of cell death determines the number of branches in the arista. Consistent with this idea, it was found that thread mutants show excessive cell death restricted to the antennal imaginal disc during the middle third instar larval stage. These findings point to a narrow window of development in which regulation of programmed cell death is essential to the proper formation of the arista (Cullen, 2004).

Analysis of the th¹ mutant has revealed a decrease in cell number by pupal stages, suggesting that excessive apoptosis could have occurred earlier in development. Indeed, TUNEL analysis revealed that th¹ mutants show a dramatic increase in apoptosis compared to wild-type at a specific developmental timepoint, the middle third larval instar. Interestingly, caspase activity was found to be more extensive than TUNEL labeling, suggesting that caspases are activated in many antennal cells, but only a fraction succumb to apoptosis. This may indicate that there are other protectors acting downstream of caspase activation when inhibition by Thread fails. Alternatively, because this antibody detects processed effector caspases, the th¹ mutant may not be able to inhibit caspase processing but may be able to inhibit enough caspase activity to prevent apoptosis. The increase in caspase activity that was observed is limited both spatially and temporally, such that by the late third larval instar, th¹ discs show wild-type levels of immunolabeling. The ectopic caspase activity is also limited to the antennal portion of the eye-antennal disc, suggesting that thread activity or caspase inhibition is regulated differently in the eye and the antenna (Cullen, 2004).

One of the best-characterized activators of apoptosis in Drosophila is head involution defective or hid. Hid is thought to promote apoptosis by binding to Th, displacing it from caspases and triggering its auto-ubiquitination. hid mutants have been shown to have excessive cell numbers in the embryonic CNS and the adult eye. Here, hid mutants have numerous ectopic lateral branches in the posterior antennal arista. Mitotic clones of Df(3L)H99 dp not appear to have more branches than hid mutants alone, suggesting that hid is the primary regulator of cell death in the arista, as it is in the eye. Consistent with this idea, reaper mutants show only a mild aristal phenotype. Attempts were made to alter the amount of cell death in the arista by expression of reaper, hid, grim, or dcp-1, but high levels of expression tended to result in lethality and lower levels of expression did not produce phenotypes (Cullen, 2004).

hid mutants or H99 mosaics did not show any ectopic laterals on the anterior side of the arista, indicating that the anterior laterals could be regulated by a distinct apoptotic activator, or may be formed through an apoptosis-independent mechanism. However, because th¹ mutants lack anterior laterals, and dark; th¹ double mutants show ectopic anterior laterals, it is likely that an apoptotic mechanism is indeed involved. Rescue experiments indicate that a higher level of thread expression is required for anterior lateral formation, suggesting that there may be a potent apoptotic activator that can overcome low levels of Th present in the cells that give rise to the anterior laterals. Alternatively, different cohorts of caspases may be activated in the anterior and posterior cells, and the caspases in the anterior cells could require a higher level of Th for inhibition (Cullen, 2004).

The results with hid and th mutants suggest that an inhibition of cell death is required for lateral formation. This could be a direct effect, with a particular dying cell influencing the fate of a neighboring cell. Alternatively, the role for cell death could be more indirect, simply affecting the total number of cells, which in turn could determine whether a lateral will form or not. Indeed, th¹ mutants have reduced cell numbers in the pupal aristae compared to wild-type, consistent with the observation of considerable apoptosis in the mid-third instar larval stage. Further support for the cell number hypothesis comes from observations of non-autonomy in H99 mitotic clones in the arista. While it was possible to detect ectopic branches in the H99 mosaics, these branches were not always marked with yellow, suggesting that they arose from heterozygous (or homozygous) yellow⁺ cells. Thus, the H99 clones may increase the total cell number in the developing aristae, but the specific cells that give rise to branches could be either homozygous or heterozygous for H99. How cell number influences lateral formation is unclear. It could involve lateral inhibition or lateral specification, where signaling cells induce adjacent cells to produce branches, and branch-producing cells block that fate in their neighbors. There may be a minimal number of cells required for basic support and extension of the arista; th¹ mutants may have only this minimal number of cells, with no extra cells available for branch production. Alternatively, the th¹ cells could be unable to produce lateral extensions due to cellular damage from insufficient caspase inhibition (Cullen, 2004).

The ectopic laterals observed in hid mutant aristae are intermediate in length and thickness compared to the long and short wild-type laterals. In addition, the normal longer branches in hid mutants are often shorter and thinner than wild-type. Since the laterals are thought to be formed as actin-rich projections of single cells, it is unclear how perturbing apoptosis could influence the length of the lateral. One possibility is that an increased cell number could lead to crowding or an overall decrease in cell size. The cell size could then influence the amount of cellular material available for the lateral projection. Alternatively, the 'undead' cells that survive abnormally may have ill-defined cell fates or lack sufficient cytoskeletal proteins to generate long lateral branches. Several caspase targets are regulators of the actin cytoskeleton, so increases in caspase activity might perturb the cytoskeleton, even though the caspase activity is not high enough to cause apoptosis. Similarly, the split laterals seen in the dark; th¹ mutants could arise from cellular abnormalities (Cullen, 2004).

th¹ antennal imaginal discs show increased apoptosis at a specific developmental timepoint, suggesting that regulation of th is critical in these cells. This developmental stage is characterized by rapid cell divisions and the establishment of cell fates. Key regulators of distal antennal fates are the transcription factors Distalless (Dll) and Homothorax (Hth). Coexpression of Dll and hth is sufficient to induce aristal transformations in leg, wing, head, and genital disc derivatives, accompanied by misexpression of spalt, a gene normally expressed in antennal but not leg discs. spalt and several other genes have been identified as targets of Dll and/or hth, however, most of these genes appear to be expressed in the proximal antenna, largely excluded from the presumptive arista. One exception is spineless, which is expressed in the aristal primordia during larval stages. spineless mutants show antennal to leg transformations, suggesting that its normal function is to repress leg and promote antennal fates. It remains unclear how such patterning genes could produce cell fates that are specifically susceptible to loss of Th. Perhaps these genes could directly regulate th levels transcriptionally or post-transcriptionally, and the th¹ mutant may have a mutation in a corresponding region (Cullen, 2004).

The molecular nature of the th¹ mutation is currently unknown. The th¹ mutation behaves like a loss-of-function allele, displaying the aristal phenotype in trans to a deficiency and being rescued by a duplication for the chromosomal region. The coding sequence of the th¹ allele is reported to lack any obvious mutations, although the appropriate background strain is unknown. Further investigation will be required to determine if any observed amino acid changes affect the protein function. There are three reported transcripts of th initiating from distinct promoters, but the tissue-specificity of these transcripts has not been reported in detail. Perhaps, the th¹ mutation could disrupt one of the transcript variants that is primarily expressed in the presumptive arista, lowering the Th protein levels below a certain level necessary for maintaining caspase inhibition. The spontaneous nature of the th¹ mutation suggests that it could be caused by the insertion of a transposable element, which could potentially disrupt specific transcripts. Future molecular analysis of the th¹ mutation will contribute to the understanding of the role of cell death in patterning the antennal arista (Cullen, 2004).

Hid and cell death in the eye disc

To examine genetic interactions between Nedd2-like caspase (Dronc) and other apoptotic pathway genes, two UAS-dronc transgenic lines (#23 and #80) were chosen that result in relatively low lethality when crossed to GMR-GAL4 and a recombinant second chromosome was generated for each of these transgenes with GMR-GAL4. When GMR-GAL4 UAS-dronc#80 was crossed to wild type w¹¹¹⁸ flies at 25°C, adult flies that exhibited slightly rough and mottled eyes were observed. A similar phenotype has been observed in previous studies and has been shown to be due to ablation of the pigment and photoreceptor cells. Similar results were observed for GMR-GAL4, UAS-dronc#23. This phenotype became more severe when expression of dronc via GMR-GAL4 was increased by raising the temperature to 29°C. Because this eye phenotype can be modified by increasing the expression of dronc, it provided a dosage-sensitive system for examining genetic interactions between dronc and other genes of the apoptosis pathway. To test this further, whether co-expression of the baculovirus caspase inhibitor P35 from the GMR enhancer was able to suppress the eye phenotype of GMR-dronc at 29°C was examined. Co-expression of GMR-p35 dramatically improves the eye ablation phenotype of GMR-dronc. Thus, in this system, Dronc is sensitive to P35 in the Drosophila eye (Quinn, 2000).

Whether the GMR-dronc eye phenotype is sensitive to halving the dosage of the various Drosophila apoptosis-regulatory genes was tested. To assess whether the GMR-dronc eye phenotype is sensitive to the dosage of the H99 genes (reaper, hid, and grim), GMR-dronc flies were crossed to a deficiency removing the H99 genes, Df(3L)H99, at 29°C. The H99 deficiency dominantly suppressed the GMR-dronc eye phenotype. Thus, the cell death-inducing activity of dronc is sensitive to the dosage of the H99 genes. Furthermore, halving the dosage of dronc using a deficiency modifies the ablated eye phenotype of GMR-hid and GMR-rpr, suggesting that dronc is downstream of hid and rpr. To determine whether there was a genetic interaction with dronc and dark, whether decreasing the dosage of dark modified the eye phenotype of GMR-dronc at 29°C was examined. Three different P-element alleles of dark (dark^CD4, dark^CD8, and dark^l(2)k11502) show suppression of the GMR-dronc eye phenotype, indicating that Dark plays a role in promoting Dronc-induced cell death in the eye. Halving the dosage of diap1 using deficiencies or the specific allele thread⁵ dominantly enhances the GMR-dronc eye phenotype at 25°C . In addition, these diap1 mutations dominantly enhance the lethality associated with GMR-dronc, resulting in at least 10-fold lower numbers of GMR-dronc/+; Df(diap1)/+ adult flies than expected. In contrast, a deficiency removing diap2 showed no effect on the GMR-dronc phenotype, and no lethal effects were observed. Thus diap1, but not a deficiency removing diap2, shows a dosage-sensitive interaction with dronc. By contrast, ectopic expression of diap1 or diap2 from the GMR promoter shows suppression of the GMR-dronc ablated eye phenotype, although GMR-diap2 results in much weaker suppression than GMR-diap1. Thus, both Diap1 and Diap2 are capable of directly or indirectly blocking Dronc-mediated cell death (Quinn, 2000).

Regulated cell death and survival play important roles in neural development. Extracellular signals are presumed to regulate seven apparent caspases to determine the final structure of the nervous system. In the eye, the EGF receptor, Notch, and intact primary pigment and cone cells have been implicated in both survival and death signals. An antibody (CM1) raised against a peptide from human caspase 3 was used to investigate how extracellular signals control spatial patterning of cell death. The antibody crossreacts specifically with dying Drosophila cells and labels the activated effector caspase Drice. The initiator caspase Dronc and the proapoptotic gene head involution defective are important for activation in vivo. Dronc may play roles in dying cells in addition to activating downstream effector caspases. Epistasis experiments ordered the EGF receptor, Notch, and primary pigment and cone cells into a single pathway that affects caspase activity in pupal retina through hid and Inhibitor of Apoptosis Proteins (IAPs). None of these extracellular signals appear to act by initiating caspase activation independently of hid. Taken together, these findings indicate that in eye development spatial regulation of cell death and survival is integrated through a single intracellular pathway (Yu, 2002).

A particularly useful feature of the CM1 antiserum is the detection of cells that would otherwise be marked for death but which are protected by baculovirus p35 expression. The morphological protection provided by p35 may permit better investigation of the location and autonomy of death and survival signals. On Western blots the CM1 antibody detects activated Drice but not the Drice zymogen; Drice is the Drosophila sequence most similar to the immunizing peptide. Definitive evidence that CM1-stained cells are apoptotic comes from the dependence of embryonic CM1-staining on the 75C1,2 chromosome region, and from the p35-sensitivity of larval and pupal CM1-stained cells. The morphology of all CM1-stained cells is altered by p35 expression. In the presence of p35, CM1-stained cells become indistinguishable from normal cells by morphological criteria, and are not distinguishable except by CM1 staining. Since baculovirus p35 blocks cell death by inhibiting caspase activity, p35-dependent morphology of CM1-stained cells shows that such morphology depends on caspase activity in the cells, which are therefore apoptotic. These results show that only apoptotic cells are labelled by the CM1 antiserum. Apoptotic cells that are unlabelled by CM1 might also exist, although none have been noticed (Yu, 2002).

The results indicate that effector caspases such as Drice can be processed in the presence of baculovirus p35. The initiator caspase Dronc is responsible for effector caspase processing in p35 expressing cells. p35-insensitive caspases are also thought to initiate cell death in other insect cells. In Drosophila eye development, Dronc always functions redundantly with other, p35-sensitive initiator caspases. Such redundancy explains why Dronc-DN had no effect in earlier studies of eye development. Dronc has been implicated in embryonic cell death by RNA interference studies (Yu, 2002).

If feedback of effector caspases on their own activation is essential for cell death, it would be expected that Dronc alone is insufficient for CM1 labelling in p35-expressing cells. By contrast, CM1 labelling persists in most cells, indicating that feedback of effector caspases is dispensable for the activation of at least one effector caspase. However, the results do not exclude the possibility that feedback makes a quantitative contribution to the pace of death or is required for a subset of effector caspases. Results are different for neuronal photoreceptor cells. R8 photoreceptor survival is rescued in the absence of EGFR by p35 expression, but such rescued R8 cells are not labelled strongly by CM1. It is possible that amplification of the apoptotic cascade might be more important for apoptosis of R8 precursors than for unspecified cells. It is also possible that R8 cell apoptosis involves different effector caspases or is initiated independently of Dronc (Yu, 2002).

These findings suggest that Dronc may cleave other substrates in dying cells in addition to activating p35-sensitive effector caspases. Mild eye roughening, seen when p35 is expressed in the eye, is found to depend on Dronc activity. It seems unlikely that the rough eye could be due to some downstream effector caspases escaping the p35 inhibition, because DIAP1 overexpression blocks cell death less effectively than p35 but does not cause eye roughening. It is speculated that Dronc might have cellular targets other than downstream caspases and that cleavage of such targets affects eye morphology. However, these data provide no evidence for Dronc activity, except in cells that would normally die. The simplest model is that Dronc might have another role in cell death in addition to activating effector caspases. The data do not support any effect of p35 other than its inhibition of caspases, since the eye roughening caused by p35 is suppressed by co-expression of DIAP1 or Dronc-DN (Yu, 2002).

Mutations of hid reduce cell death in pupal eye development. hid is absolutely required for caspase activation in both eye disc and pupal retina. Cell death is reduced even in hid/+ heterozygotes, consistent with dominant effects of hid in modifier screens. hid is required for caspase processing, which is redundantly mediated by Dronc and other initiator caspases. Therefore, in principle, Hid is a candidate for regulating initiator caspases, however, in embryos, hid functions to sequester DIAPs. In pupal retinas, DIAP overexpression mimics hid mutation, consistent with sequestration of DIAPs by Hid. In the eye imaginal disc, however, proapoptotic Hid function is not overcome by DIAP1 overexpression, since targeted DIAP1 expression neither reduces cell death in normal eye discs nor protects against cell death when EGFR function is removed in egfr mutant clones. It seems unlikely that endogenous Hid levels are too high for DIAP1 to be effective, because hid is haploinsufficient for eye disc cell death. Instead, these findings raise the possibility of a proapoptotic activity of Hid that is not blocked by DIAP1. This could involve inhibiting a pathway parallel to DIAP1 that also inhibits caspase activation, or promoting activation of caspase zymogens in other ways, for which there is precedent in vertebrates (Yu, 2002).

Two other proapoptotic genes, rpr and grim, induce eye cell death on ectopic expression. Whether rpr or grim are required for cell death in normal eye development is uncertain because point mutants are not available. The absolute requirement for hid may indicate that rpr and grim are not active during normal eye development. Since hid has been shown to be required for eye death in response to ectopic rpr, however, it is also possible that rpr and grim have activities that depend on hid function (Yu, 2002).

Experiments using the egfr^ts1a allele have confirmed that Egfr is required for survival of pupal retinal cells, as suggested by misexpression experiments. Egfr is also required for survival of eye imaginal disc cells. Consistent with the model that Egfr prevents cell death by inactivating hid, hid is absolutely required for caspase activation in egfr mutant clones. Similar results have been obtained using TUNEL experiments to assess Egfr-DN-induced cell death (Yu, 2002).

Survival in pupal retina is regulated by two further extracellular signals that are not involved in eye imaginal discs. In principle, such signals might act to modulate Egfr signaling, to regulate Hid or DIAP activity in parallel to Egfr, or to activate initiator caspases. Notch (N) is required for caspase activation in the pupal retina. Epistasis experiments show that N is not required for pupal cell death in the absence of Egfr function, and therefore that the normal function of N is to inhibit the Egfr survival signaling pathway in pupae. Such results place N upstream of Egfr and indicate that N acts ultimately through hid and the anti-apoptotic DIAP proteins that prevent caspase activation, rather than through N-mediated caspase activation. Survival in pupal retinas also depends on signals from primary pigment cells and/or cone cells. Such signals must antagonize proapoptotic N activity, since N is epistatic to the primary pigment cell/cone cell signal. The data now imply a pathway in which primary pigment cells and/or cone cells promote survival by inhibiting activation of N, thus preventing N antagonism of Egfr activity in the interommatidial cells (Yu, 2002).

The essential role of Egfr now seems to be downstream of N, whereas the cone cell/primary pigment cell signal must act upstream. Downstream Egfr function raises anew the question of identity of the primary pigment cell/cone cell signal. Primary pigment cells or cone cells do not seem essential for Egfr activation, because N is still required for apoptosis after ablation of these cells. Pupal photoreceptor cells express the Egfr ligand SPI and its processing/presenting factor Rhomboid, and are one possible source of Egfr activation. One model suggests that primary pigment cells and/or cone cells are the source of an unidentified signal or mechanism that prevents N activation (in particular interommatidial cells) so that Egfr survival signaling can continue (Yu, 2002).

According to one view, survival signals are the critical extracellular regulators of developmental cell death. By contrast, results from C. elegans and mammals indicate that cell death depends on activation of initiator caspases to trigger the apoptotic cascade. Homologs of the activatory components exist in Drosophila. Studies of eye development place three extracellular signals in a pathway acting through Egfr and hid to regulate survival, in part through IAPs. The only evidence consistent with positive regulation of apoptosis is that in eye imaginal discs, hid appears to promote cell death through an unidentified mechanism independent of DIAPs, and, in this case, the role of EGF receptor signaling is still to promote survival by inhibiting Hid (Yu, 2002).

These findings do not rule out other pathways that activate initiator caspases during eye development, or that such activation might be required for cell death. Since hid is essential for cell death, however, pathways that activate initiator caspases independently of hid cannot be sufficient for any of the cell death that normally occurs during eye development. Because loss of Egfr survival signaling is sufficient for cell death, and Egfr survival signaling is only important to inhibit Hid, these data imply that release of hid is sufficient as well as necessary for normally occurring cell death. The data do not rule out any parallel Egfr-dependent signal to suppress caspase activation independently of hid, but such a pathway cannot be sufficient for cell death in the absence of hid. These findings suggest that positive activators of caspase processing may not be the direct targets of extracellular regulation. However, it will be important to investigate survival and death signals in other organs, including cell deaths that occur independently of reaper, grim and Hid in ovarian nurse cells and during autophagy, the mechanisms of which have yet to be determined (Yu, 2002).

So far, relatively few mechanisms have been shown to be capable of regulating both cell proliferation and cell death in a coordinated manner. In a screen for Drosophila mutations that result in tissue overgrowth, salvador (sav), a gene that promotes both cell cycle exit and cell death was identified. Elevated Cyclin E and DIAP1 levels are found in mutant cells, resulting in delayed cell cycle exit and impaired apoptosis. Salvador contains two WW domains and binds to the Warts protein kinase. The human ortholog of salvador (hWW45) is mutated in several cancer cell lines. Thus, salvador restricts cell numbers in vivo by functioning as a dual regulator of cell proliferation and apoptosis (Tapon, 2002).

In wild-type eyes, excessive interommatidial cells are eliminated by a wave of apoptosis that is evident in 38 hr pupal retinas. Even in sav mutant clones, cell proliferation, as assessed by BrdU incorporation, has ceased within 24 hr APF. When mosaic retinas were examined 38 hr APF, cell death is mostly confined to the wild-type portions of the retina. Thus, the apoptotic cell deaths that are part of normal retinal development appear to require sav function (Tapon, 2002).

Apoptosis in the pupal retina requires hid function, since hid mutants display additional interommatidial cells. Hid is thought to induce caspase activation by binding to the DIAP1 protein and preventing it from inhibiting caspase function. Overexpression of hid using the eye-specific GMR promoter generates a small eye. The induction of cell death by hid is severely impaired in sav mutant clones. As a consequence, eyes derived from GMR-hid-expressing discs that contain sav mutant clones are larger than those derived from wild-type discs that express GMR-hid. Since sav function is required for hid-induced cell death, sav is likely to function either downstream of hid or in a parallel pathway (Tapon, 2002).

Several studies have shown that Hid and Rpr activate caspases by another mechanism in which they induce the autoubiquitination of DIAP1 and target it for degradation by the proteasome. DIAP1 levels are markedly elevated in sav clones in the larval eye disc and remain elevated in the interommatidial cells in mutant clones in the pupal eye disc. Thus, increased levels of DIAP1 in sav cells may be able to overcome the effect of many proapoptotic signals (Tapon, 2002).

To examine DIAP1 RNA levels, in situ hybridization was used to examine 20 wild-type discs and 20 mutant discs. The presence of sav (GFP^-) clones in the mutant discs was confirmed by examining the discs by fluorescence microscopy prior to hybridization. There is a modest level of DIAP1 RNA expression posterior to the furrow in both populations of discs and no evidence of increased DIAP1 RNA in the discs containing sav clones. Thus, at least at this level of detection, the increased DIAP1 expression in sav cells does not appear to result from increased transcription (Tapon, 2002).

In wild-type eye discs, DIAP1 protein is expressed at higher levels posterior to the morphogenetic furrow. DIAP1 protein levels are downregulated by GMR-rpr or, to a lesser extent, by GMR-hid expression. In sav mutant clones expressing GMR-rpr, DIAP1 protein levels remain elevated. Similar results are observed with GMR-hid. Thus, neither GMR-rpr nor GMR-hid appears capable of downregulating the elevated levels of DIAP1 sufficiently in sav clones to activate caspases (Tapon, 2002).

Expression of hid or reaper (rpr) in the eye imaginal disc results in activation of the effector caspase Drice. An antibody that recognizes the cleaved (activated) form of Drice was used to stain eye discs expressing GMR-hid or GMR-rpr. In wild-type cells, Drice is activated by GMR-hid or GMR-rpr. However, in clones of sav tissue, Drice activation by either GMR-hid or GMR-rpr is almost completely blocked. These experiments indicate that sav blocks activation of Drice by both rpr and hid (Tapon, 2002).

A mutant form of Hid (Hid-Ala5) is resistant to inactivation by MAP kinase phosphorylation. GMR-hid-Ala5 is a more potent inducer of cell death than is GMR-hid, as assessed by the extent of Drice activation in the eye disc. Cell death induced by GMR-hid-Ala5 is only partially blocked in sav clones, indicating that the increased potency of Hid-Ala-5 may be able to overcome increased DIAP1 levels (Tapon, 2002).

Elevated DIAP1 levels are likely to underlie the absence of the developmentally regulated apoptosis in sav clones in the pupal retina as well as the resistance to hid-induced and rpr-induced apoptosis in the larval imaginal disc. The elevated DIAP1 levels appear to result from alterations in posttranscriptional regulation of DIAP1 expression. Recent work has shown that both Rpr and Hid can downregulate DIAP1 levels either by promoting the autoubiquitination of DIAP1 or by causing a generalized inhibition of translation that especially impacts proteins with a short half-life such as DIAP1. Either of these mechanisms is likely to be less efficient in cells that already have elevated levels of DIAP1 (Tapon, 2002).

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).

Programmed cell death (PCD) is utilized in a wide variety of tissues to refine structure in developing tissues and organs. However, little is understood about the mechanisms that, within a developing epithelium, combine signals to selectively remove some cells while sparing essential neighbors. One popular system for studying this question is the developing Drosophila pupal retina, where excess interommatidial support cells are removed to refine the patterned ommatidial array. Data is presented indicating that PCD occurs earlier within the pupal retina than previously demonstrated. As with later PCD, this death is dependent on Notch activity. Surprisingly, altering Drosophila Epidermal Growth Factor Receptor or Ras pathway activity has no effect on this death. Instead, a role for Wingless signaling is indicated in provoking this cell death. Together, these signals regulate an intermediate step in the selective removal of unneeded interommatidial cells that is necessary for a precise retinal pattern (Cordero, 2004).

In the course of examining hid mutant retinae, it was noticed that blocking cell death in the earliest pupal stages -- prior to known stages of cell death -- led to a clear increase in the number of interommatidial cells. With this in mind, pupal retinas was examined at earlier developmental stages from 14 to 24 h APF by using an antibody to the junction protein Armadillo; apoptotic cell death was also directly assessed with TUNEL staining. Prior to approximately 20 h APF, the retina is composed of a loosely patterned array of ommatidia consisting of photoreceptor neurons and cone cells; primary pigment cells (1°s) first emerge and enwrap the cone cells at 20 h APF, and secondary and tertiary pigment cells (2°/3°s) begin organizing at about this stage as well. Approximately one third of the interommatidial cells observed at 24 h APF (25°C) are selected to die by PCD during the following 10-12 h (Cordero, 2004).

Prior to 18 h APF, no significant amount of death was observed. At 18 h APF, a sharp band of death was observed in the anterior portion of the retina; some of this death is within the retina, and some is just outside the retinal field. Between 20 and 24 h APF, additional death is observed towards the middle of the retina in addition to the anterior death band. Levels of apoptosis are highest in anterior regions of the eye, but the center of the eye, for example, also contains significant levels of death. At 24 h APF, this early wave of death rapidly declines; the remaining interommatidial cells have reorganized end-to-end by this stage. At 26 h APF, the known, previously described burst of death commences. The increasing amount of TUNEL staining correlates with a decrease in the number of interommatidial cells. These results indicated that the pupal retina undergoes two separate surges of cell death that occur between 18-24 and 26-36 h APF; for convenience, these events are referred to as 'early-stage' and 'late-stage' cell death events in the pupal eye, respectively. During the early-stage death 1.8 cells are removed per ommatidia. The early-stage has not been described previously, and it was examined whether the pathways known to regulate late-stage death also regulate its predecessor (Cordero, 2004).

The baculovirus protein P35 interferes with apoptosis by binding to and inhibiting caspase activity; it is effective in inhibiting cell death including late-stage death in the Drosophila eye. Targeted over-expression of P35 with the eye-specific promoter GMR led to a near complete block of early-stage death: only a line of anterior cell death remained in GMR-p35 retinas. This result indicates that the early-stage cell death occurred by caspase-dependent apoptosis. In addition, it confirmed the assessment, based on TUNEL staining, that some of the anterior-most apoptotic cell death occurs in a region of future head cuticle just anterior to the retina (and is therefore outside of the expression domain of the GMR promoter (Cordero, 2004).

The head involution defective hid gene is a central regulator of cell death in Drosophila including late-stage cell death pathway in the retina. Hid induces PCD through activation of caspases. Retinas lacking functional hid activity looses all evidence of early-stage PCD. The number of cells within the GMR-p35 and hid^-/- retinas at 20 and 21 h APF, respectively was in fact higher than the number of cells in a 18 h APF control retina. In these mutant genotypes the ommatidia are disorganized when compared with the control retinas due to the excess of cells. It was often found that hid^-/- retinas are attached to what seems to be the antennal disc, suggesting that this early-stage death may include events required for separation of the eye-antennal discs. Together these results suggest that, similar to late-stage death, early-stage death is regulated by a caspase-mediated apoptosis pathway (Cordero, 2004).

The Egfr/Ras-1 pathway has been implicated in multiple stages of fly eye development including cell proliferation, survival and differentiation. Loss of function mutations in the Egfr leads to excessive cell death of the interommatidial cells. Activation of Egfr leads to activation of dRas1, which promotes cell survival by repressing the activity and expression of hid (Cordero, 2004).

Activated Egfr and dRas1^V12 was expressed under the control of an inducible, heat shock promoter. As expected, late-stage cell death (26 h APF) is almost completely blocked by each transgene. Surprisingly, no alteration was seen in either the pattern of death or the cell number in 21 h APF retinas, suggesting that early-stage death is insensitive to the Egfr/dRas1 pathway. Consistent with these results, no effect on cell death was seen upon over-expression of the Egfr antagonist Argos. These findings are especially surprising because of the results indicating that hid is required for early-stage death: unlike larval or late-stage death, hid activity appears to be regulated by a Egfr-independent mechanism during early-stage cell death (Cordero, 2004).

Mis-specified cells die by an active gene-directed process, and inhibition of this death results in cell fate transformation in Drosophila

Incorrectly specified or mis-specified cells often undergo cell death or are transformed to adopt a different cell fate during development. The underlying cause for this distinction is largely unknown. In many developmental mutants in Drosophila, large numbers of mis-specified cells die synchronously, providing a convenient model for analysis of this phenomenon. The maternal mutant bicoid is a particularly useful model with which to address this issue because its mutant phenotype is a combination of both transformation of tissue (acron to telson) and cell death in the presumptive head and thorax regions. A subset of these mis-specified cells die through an active gene-directed process involving transcriptional upregulation of the cell death inducer hid. Upregulation of hid also occurs in oskar mutants and other segmentation mutants. In hid bicoid double mutants, mis-specified cells in the presumptive head and thorax survive and continue to develop, but they are transformed to adopt a different cell fate. Evidence is provided that the terminal torso signaling pathway protects the mis-specified telson tissue in bicoid mutants from hid-induced cell death, whereas mis-specified cells in the head and thorax die, presumably because equivalent survival signals are lacking. These data support a model whereby mis-specification can be tolerated if a survival pathway is provided, resulting in cellular transformation (Werz, 2005).

Although this study largely focus on the maternal effect mutants bicoid and oskar, it is likely that the principles uncovered are of broader significance. Segmentation mutants acting downstream of bicoid and oskar, including mutants of gap genes (Krüppel, knirps), pair-rule genes (odd, fushi-tarazu) and segment polarity genes (wg, hedgehog, engrailed) induce expression of hid. These mutants are characterized by loss of larval tissue. As in the case of bicoid and oskar, hid expression is upregulated during stage 9 of embryogenesis in the regions of the mutant embryos that are later deleted in the larvae. In addition, hid mutants rescue the cuticle phenotype of armadillo mutants. Finally, hid expression accompanied by TUNEL-positive cell death was found in dorsal and Toll^10b mutants, which cause dorsalizing and ventralizing phenotypes, respectively, along the dorsoventral axis of Drosophila embryos. Thus, these data support the notion that upregulation of hid appears to be a common trigger for a caspase-dependent cell death program in mis-specified cells of patterning mutants (Werz, 2005).

Furthermore, mutations affecting imaginal disc development result in loss of the adult appendage due to inappropriate cell death. It is currently being determined whether these mutants also require <