InteractiveFly: GeneBrief

Head involution defective: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

BIOLOGICAL OVERVIEW

The Wrinkled (W) gene (more often referred to as head involution defective or hid) was described by V. Jollos in a 1936 paper entitled "Mutations observed in Drosophila stocks taken up into the stratosphere." The originally described mutation is dominant. Wings remain small and unexpanded. The phenotype of heterozygotes is not as severe as that of homozygotes. Wings of heterozygotes are often expanded but wrinkled, blistered and the surface finely pebbled and grayish. From the prepupal stage through to the adult, wing bases are abnormally narrow, possibly preventing the flow of body fluids in sufficient quantities to expand the wings (Waddington, 1940, as described in FlyBase). There is a general decrease in apoptosis, or programmed cell death (PCD) throughout the recessive hid mutant embryo. This phenotype is most noticeable in the head region prior to completion of head involution. Striking defects in head morphogenesis occur, in part, from a failure of the dorsal fold to migrate to the anterior in hid mutants (Abbott, 1991). It is possible that these morphogenetic defects result from the decrease of PCD in this region (Grether, 1995). Since the Drosophila literature invariable refers to Wrinkled as hid, this essay will conform to that convention, and not use the more proper name Wrinkled, which is the one recommended by FlyBase.

When hid is expressed from a heat shock promoter, high levels of PCD are observed within 2 hours of heat shock (hs) induction. The hs-hid transgene induces ectopic cell death in wild-type embryos following heat shock. The cell death induced by the hs-hid transgene is lethal to wild-type embryos, and a single heat pulse during embryogenesis kills all flies bearing the hs-hid construct. The ability of hid to kill is not significantly augmented by the presence of an endogenous reaper gene. The embryonic pattern or RPR mRNA is not significantly affected by ectopic expression of hid, even under conditions where large numbers of cells are dying. It is concluded that the induction of PCD by hid occurs independently of rpr (Grether, 1995).

Transformants carrying a single copy of hid expressed from a synthetic glass promoter display a dramatic eye ablation phenotype. Normally, compound eyes consist of about 800 regular units, called ommatidia, each of which consists of several distinct cell types. In hid transformants, only undifferentiated cuticle and a dense band of bristles remain in the places normally occupied by the compound eyes. It appears that these bristles represent the mechanosensory bristles normally found at the corner of each ommatidium. Apparently, these cells are less susceptible to hid-induced death. However, the number of these cells is severely reduced in transformants that are homozygous for the hid transgene, indicating that their survival is sensitive to the dose of hid expression. It is possible that the hid transgene is expressed only weakly in bristle precursors. Alternatively, bristle cells may be better protected against hid-induced PCD. Interestingly, a very similar phenotype is obtained from expression of reaper in the developing retina. The hid-induced eye phenotype is completely suppressed by coexpression of the baculovirus p35 gene (Grether, 1995).

A number of peptide factors including the neurotrophins, insulin-like growth factor 1 (IGF-1), fibroblast growth factor (FGF), and epidermal growth factor (EGF) promote mammalian cell survival by suppressing the intrinsic cell death program. The mechanisms by which survival factors inactivate the intrinsic cell death program are currently the subject of intensive investigation. The growth factors listed above bind to and activate receptor tyrosine kinases (RTKs) at the cell surface; in turn, these factors stimulate the antiapoptotic activity of the proto-oncogene ras (reviewed by Downward, 1998). Ras (Drosophila homolog: Ras oncogene at 85D) controls the activity of a number of effector pathways, two of which result in activation of protein kinases known to mediate its antiapoptotic effect: the mitogen-activated protein kinase p42/44 (MAPK: Drosophila homolog Rolled) of the ERK-type (extracellular signal-related kinase) via Raf and the Akt kinase via Phosphoinositide 3-kinase (PI3-K). Recently, it has been shown that activation of the antiapoptotic PI3-K/Akt-kinase pathway leads to phosphorylation of Bad, a proapoptotic member of the Bcl-2 family (see death executioner Bcl-2 homologue), resulting in its binding to 14-3-3 (Drosophila homolog: Leonardo) as an inactive complex. Activation of the Erk-type MAPK is required to protect PC-12 cells from apoptosis induced by NGF withdrawal. However, a direct mechanistic link between the Raf/MAPK survival pathway and the cell death machinery has not been demonstrated thus far (Bergmann, 1998 and references).

The strong eye ablation phenotype in Drosophila, caused by expressing hid under the control of an eye-specific promoter, was used to perform a genetic screen aimed at identifying components that regulate and mediate Hid activity. Mutations in genes that regulate the EGF receptor (EGFR)/Ras1 (Ras oncogene at 85D) pathway were recovered as strong suppressors of Hid-induced apoptosis. The survival effect of the EGFR/Ras1 pathway is specific for Hid-induced apoptosis, since neither Reaper- nor Grim-induced apoptosis is affected by the EGFR/Ras1 pathway. The Ras1 pathway has been shown to inhibit Hid activity apparently by the direct phosphorylation of Hid by MAPK (Rolled). Alteration of the MAPK phosphorylation sites within the HID sequence blocks the survival signals generated by constitutively activate Ras1 and constitutively active MAPK. It is concluded that the hid gene in Drosophila provides a mechanistic link between the survival activity of Ras1 and the apoptotic machinery. Post-translational modification of Hid is a survival signal regulating Hid activity (Bergmann, 1998).

In addition to a post-translational regulation of Hid, the Ras/MAPK pathway promotes cell survival in Drosophila by downregulating the expression of hid. Conversely, downregulation of the Ras/MAPK pathway induces cell death by upregulating hid expression. Reduction in pointed (pnt) activity has been observed to enhance ectopic Hid induced cell death in the eye. The pointed transcription factor is a target of MAPK function and acts as a positive regulator in the R7 pathway. Like embryos expressing activated Dras1 and activated Draf, pnt2-expressing embryos show decreased hid transcript levels, indicating that the Ras/MAPK pathway, acting through pnt, downregulates hid transcription (Kurada, 1998).

In the embryo, it is likely that Ras protection also acts through non-hid pathways. Even the complete removal of hid, in hid null embryos, results in only a mild decrease in apoptosis (Grether, 1995). This indicates that Ras downregulation of hid cannot account for all of the protection afforded by activated Ras and that Ras must have additional antiapoptotic targets in the embryo. An alternative pathway may be Ras activation of phosphoinositide 3-kinase (PI3-K) and its target Akt/PKB, which have been shown to protect mammalian cells from apoptosis. Drosophila PI3-K isoforms have been cloned and characterized, yet the role of this gene in Drosophila apoptosis is not well understood. Eye-specific expression of a Ras binding PI3-K isoform, Dp110, gives rise to flies with larger eyes (Leevers, 1996). This effect of PI3-K overexpression, however, is not due to inhibiting cell death, but rather due to increased cell size. Nonetheless, recent evidence suggests that the PI3-K target Akt/PKB may produce antiapoptotic activity in the embryo (Staveley, 1998).

We are left with trying to evaluate the relative importance of post-transcriptional modification vs. transcriptional regulation. Perhaps the former response provides a first line of protection against apoptosis, while the transcriptional regulation makes this protection permanent. Transcriptional regulation might also ensure, in instances where the surviving cell generates progeny, that the decisions made in the parent cell are passed on as permanent regulatory changes in the progeny.

Elimination of unfit cells maintains tissue health and prolongs lifespan

Viable yet damaged cells can accumulate during development and aging. Although eliminating those cells may benefit organ function, identification of this less fit cell population remains challenging. Previously, a molecular mechanism, based on 'fitness fingerprints' displayed on cell membranes, was identifed that allows direct fitness comparison among cells in Drosophila. This study reports the physiological consequences of efficient cell selection for the whole organism. The study found that fitness-based cell culling is naturally used to maintain tissue health, delay aging, and extend lifespan in Drosophila. A gene, ahuizotl (azot), was identified that ensures the elimination of less fit cells. Lack of azot increases morphological malformations and susceptibility to random mutations and accelerates tissue degeneration. On the contrary, improving the efficiency of cell selection is beneficial for tissue health and extends lifespan (Merino, 2015).

Individual cells can suffer insults that affect their normal functioning, a situation often aggravated by exposure to external damaging agents. A fraction of damaged cells will critically lose their ability to live, but a different subset of cells may be more difficult to identify and eliminate: viable but suboptimal cells that, if unnoticed, may adversely affect the whole organism (Merino, 2015).

What is the evidence that viable but damaged cells accumulate within tissues? The somatic mutation theory of aging proposes that over time cells suffer insults that affect their fitness, for example, diminishing their proliferation and growth rates, or forming deficient structures and connections. This creates increasingly heterogeneous and dysfunctional cell populations disturbing tissue and organ function. Once organ function falls below a critical threshold, the individual dies. The theory is supported by the experimental finding that clonal mosaicism occurs at unexpectedly high frequency in human tissues as a function of time, not only in adults an embryos (Merino, 2015).

Does the high prevalence of mosaicism in our tissues mean that it is impossible to recognize and eliminate cells with subtle mutations and that suboptimal cells are bound to accumulate within organs? Or, on the contrary, can animal bodies identify and get rid of unfit viable cells (Merino, 2015)?

One indirect mode through which suboptimal cells could be eliminated is proposed by the 'trophic theory,' which suggested that Darwinian-like competition among cells for limiting amounts of surv ead to removal of less fit cells. However, it is apparent from recent work that trophic theories are not sufficient to explain fitness-based cell selection, because there are direct mechanisms that allow cells to exchange 'cell-fitness' information at the local multicellular level (Merino, 2015).

In Drosophila, cells can compare their fitness using different isoforms of the transmembrane protein Flower. The 'fitness fingerprints' are therefore defined as combinations of Flower isoforms present at the cell membrane that reveal optimal or reduced fitness. The isoforms that indicate reduced fitness have been called FlowerLose isoforms, because they are expressed in cells marked to be eliminated by apoptosis called 'Loser cells.' However, the presence of FlowerLose isoforms at the cell membrane of a particular cell does not imply that the cell will be culled, because at least two other parameters are taken into account: (1) the levels of FlowerLose isoforms in neighboring cells: if neighboring cells have similar levels of Lose isoforms, no cell will be killed; (2) the levels of a secreted protein called Sparc, the homolog of the Sparc/Osteonectin protein family, which counteracts the action of the Lose isoforms (Merino, 2015 and references therein).

Remarkably, the levels of Flower isoforms and Sparc can be altered by various insults in several cell types, including: (1) the appearance of slowly proliferating cells due to partial loss of ribosomal proteins, a phenomenon known as cell competition; (2) the interaction between cells with slightly higher levels of d-Myc and normal cells, a process termed supercompetition; (3) mutations in signal transduction pathways like Dpp signaling; or (4) viable neurons forming part of incomplete ommatidia. Intriguingly, the role of Flower isoforms is cell type specific, because certain isoforms acting as Lose marks in epithelial cells are part of the fitness fingerprint of healthy neurons. Therefore, an exciting picture starts to appear, in which varying levels of Sparc and different isoforms of Flower are produced by many cell types, acting as direct molecular determinants of cell fitness. This study aimed to clarify how cells integrate fitness information in order to identify and eliminate suboptimal cells. Subsequently, the physiological consequences were analyzed of efficient cell selection for the whole organism (Merino, 2015).

In order to discover the molecular mechanisms underlying cell selection in Drosophila, this study analyzed genes transcriptionally induced using an assay where WT cells (tub>Gal4) are outcompeted by dMyc-overexpressing supercompetitors (tub>dmyc) due to the increased fitness of these dMyc-overexpressing cells. The expression of CG11165 was strongly induced 24 hr after the peak of flower and sparc expression. In situ hybridization revealed that CG11165 mRNA was specifically detected in Loser cells that were going to be eliminated from wing imaginal discs due to cell competition. The gene, which was named ahuizotl (azot) after a multihanded Aztec creature selectively targeting fishing boats to protect lakes, consists of one exon. azot's single exon encodes for a four EF-hand-containing cytoplasmic protein of the canonical family that is conserved, but uncharacterized, in multicellular animals (Merino, 2015).

To monitor Azot expression, a translational reporter was designed resulting in the expression of Azot::dsRed under the control of the endogenous azot promoter in transgenic flies. Azot expression was not detectable in most wing imaginal discs under physiological conditions in the absence of competition. Mosaic tissue was generated of two clonal populations, which are known to trigger competitive interactions resulting in elimination of otherwise viable cells. Cells with lower fitness were created by confronting WT cells with dMyc-overexpressing cells, by downregulating Dpp signaling, by overexpressing Flower^Lose isoforms, in cells with reduced Wg signaling, by suppressing Jak-Stat signaling or by generating Minute clones. Azot expression was not detectable in nonmosaic tissue of identical genotype, nor in control clones overexpressing UASlacZ. On the contrary, Azot was specifically activated in all tested scenarios of cell competition, specifically in the cells undergoing negative selection. Azot expression was not repressed by the caspase inhibitor protein P35 (Merino, 2015).

Because Flower proteins are conserved in mammals, tests were made to see if they are also able to regulate azot. Mouse Flower isoform 3 (mFlower³) has been shown to act as a 'classical' Lose isoform, driving cell elimination when expressed in scattered groups of cells, a situation where azot was induced in Loser cells but is not inducing cell selection when expressed ubiquitously a scenario where azot was not expressed. This shows that the mouse Flower^Lose isoforms function in Drosophila similarly to their fly homologs (Merino, 2015).

Interestingly, azot is not a general apoptosis-activated gene because its expression is not induced upon eiger, hid, or bax activation, which trigger cell death. Azot was also not expressed during elimination of cells with defects in apicobasal polarity or undergoing epithelial exclusion-mediated apoptosis (dCsk) (Merino, 2015).

azot expression was analyzed during the elimination of peripheral photoreceptors in the pupal retina, a process mediated by Flower-encoded fitness fingerprints. Thirty-six to 38hr after pupal formation (APF), when Flower^Lose-B expression begins in peripheral neurons, no Azot expression was detected in the peripheral edge. At later time points (40 and 44hr APF), Azot expression is visible and restricted to the peripheral edge where photoreceptor neurons are eliminated. This expression was confirmed with another reporter line, azot{KO; gfp}, where gfp was directly inserted at the azot locus using genomic engineering techniques (Merino, 2015).

From these results, it is concluded that Azot expression is activated in several contexts where suboptimal and viable cells are normally recognized and eliminated (Merino, 2015).

To understand Azot function in cell elimination, azot knockout (KO) flies were generated by deleting the entire azot gene. Next, Azot function was analyzed using dmyc-induced competition. In the absence of Azot function, loser cells were no longer eliminated, showing a dramatic 100-fold increase in the number of surviving clones. Loser cells occupied more than 20% of the tissue 72hr after clone induction (ACI). Moreover, using azot{KO; gfp} homozygous flies (that express GFP under the azot promoter but lack Azot protein), it was found that loser cells survived and showed accumulation of GFP. From these results, it is concluded that azot is expressed by loser cells and is essential for their elimination (Merino, 2015).

In addition, clone removal was delayed in an azot heterozygous background (50-fold increase, 15%), compared to control flies with normal levels of Azot. Cell elimination capacity was fully restored by crossing two copies of Azot::dsRed into the azot^-/- background demonstrating the functionality of the fusion protein. Silencing azot with two different RNAis was similarly able to halt selection during dmyc-induced competition. Next, in order to determine the role of Azot's EF hands, a mutated isoform of Azot (Pm4Q12) was generated and overexpressed, that carryed, in each EF hand, a point mutation known to abolish Ca²⁺ binding. Although overexpression of wild-type azot in negatively selected cells did not rescue the elimination, overexpression of the mutant AzotPm4Q12 reduced cell selection, functioning as a dominant-negative mutant. This shows that Ca²⁺ binding is important for Azot function. Finally, staining for apoptotic cells corroborated that the lack of Azot prevents cell elimination, because cell death was reduced 8-fold in mosaic epithelia containing loser cells (Merino, 2015).

The role of azot in elimination of peripheral photoreceptor neurons in the pupal retina was examined using homozygous azot KO flies. Pupal retinas undergoing photoreceptor culling (44hr APF) of azot^+/+ and azot^-/- flies were stained for the cell death marker and the proapoptotic factor. Consistent with the expression pattern of Azot, the number of Hid and TUNEL-positive cells was dramatically decreased in azot^-/- retinas compared to azot^+/+ retinas (Merino, 2015).

Those results show that Azot is required to induce cell death and Hid expression during neuronal culling. Therefore, tests were performed to see that was also the case in the wing epithelia during dmyc-induced competition. Hid was found to be expressed in loser cells and the expression was found to be strongly reduced in the absence of Azot function (Merino, 2015).

Finally, forced overexpression of Flower^Lose isoforms from Drosophila were unable to mediate WT cell elimination when Azot function was impaired by mutation or silenced by RNAi (Merino, 2015).

These results suggested that azot function is dose sensitive, because heterozygous azot mutant flies display delayed elimination of loser cells when compared with azot WT flies. Therefore advantage was taken of the functional reporter Azot::dsRed to test whether cell elimination could be enhanced by increasing the number of genomic copies of azot. Tissues with three functional copies of azot were more efficient eliminating loser cells during dmyc-induced competition and most of the clones were culled 48hr ACI. From these results, it is concluded that azot expression is required for the elimination of Loser cells and unwanted neurons (Merino, 2015).

Next, it was asked what could be the consequences of decreased cell selection at the tissue and organismal level. To this end, advantage was taken of the viability of homozygous azot KO flies. An increase of several developmental aberrations was observed. Focus was placed on the wings, where cell competition is best studied and, because aberrations, including melanotic areas, blisters, and wing margin nicks, were quantified. Wing defects of azot mutant flies could be rescued by introducing two copies of azot::dsRed, showing that the phenotypes are specifically caused by loss of Azot function (Merino, 2015).

Next, it was reasoned that mild tissue stress should increase the need for fitness-based cell selection after damage. First, in order to generate multicellular tissues scattered with suboptimal cells, larvae were exposed to UV light and Azot expression was monitored in wing discs of UV-irradiated WT larvae that were stained for cleaved caspase-3, 24hr after treatment. Under such conditions, Azot was found to be expressed in cleaved caspase-3-positive cells. All Azot-positive cells showed caspase activation and 17% of cleaved caspase-positive cells expressed Azot. This suggested that Azot-expressing cells are culled from the tissue. To confirm this, later time points (3 days after irradiation) were examined; the increase in Azot-positive cells was no longer detectable. The elimination of azot-expressing cells after UV irradiation required azot function, because cells revealed by reporter azot{KO; gfp}, that express GFP instead of Azot, persisted in wing imaginal discs from azot-null larvae. Tests were performeed to see if lack of azot leads to a faster accumulation of tissue defects during organ development upon external damage. azot^-/- pupae 0 stage were irradiated, and the number of morphological defects in adult wings was compared to those in nonirradiated azot KO flies. It was found that aberrations increased more than 2-fold when compared to nonirradiated azot^-/- flies (Merino, 2015).

In order to functionally discriminate whether azot belongs to genes regulating apoptosis in general or is dedicated to fitness-based cell selection, whether azot silencing prevents Eiger/TNF-induced cell death was exanubed. Inhibiting apoptosis (UASp35) or eiger (UASRNAieiger) rescued eye ablation, whereas azot silencing and overexpression of AzotPm4Q12 did not. Furthermore, azot silencing did not impair apoptosis during genitalia rotation or cell death of epithelial precursors in the retina. These results highlight the consequences of nonfunctional cell-quality control within developing tissues (Merino, 2015).

The next part of the analysis demonstrated that the azot promoter computes relative Flower^Lose and Sparc Levels. Epistasis analyses were performed to understand at which level azot is transcriptionally regulated. For this purpose, the assay where WT cells are outcompeted by dMyc-overexpressing supercompetitors was used. It was previously observed that azot induction is triggered upstream of caspase-3 activation and accumulates in outcompeted cells unable to die. Then, upstream events of cell selection were genetically modified. Silencing fwe^Lose transcripts by RNAi or overexpressing Sparc both blocked the induction of Azot::dsRed in WT loser cells. In contrast, when outcompeted WT cells were additionally 'weakened' by Sparc downregulation using RNAi, Azot is detected in almost all loser cells compared to its more limited induction in the presence of endogenous Sparc. Inhibiting JNK signaling with UASpuc did not suppress Azot expression (Merino, 2015).

The activation of Azot upon irradiation was examined. Strikingly, it was found that all Azot expression after irradiation was eliminated when Flower Lose was silenced and also when relative differences of Flower Lose where diminished by overexpressing high levels of Lose isoforms ubiquitously. On the contrary, Azot was not suppressed after irradiation by expressing the prosurvival factor Bcl-2 or a p53 dominant negative. These results show that Azot expression during competition and upon irradiation requires differences in Flower Lose relative levels (Merino, 2015).

Finally, the regulation of Azot expression in neurons was analyzed. Silencing fwe transcripts by RNAi blocked the induction of Azot::dsRed in peripheral photoreceptors. Because Wingless signaling induces Flower^Lose-B expression in peripheral photoreceptors, tests were performed to see if overexpression of Daxin, a negative regulator of the pathway, affected Azot levels. Axin overespression completely inhibited Azot expression. Similarly, overexpression of the cell competition inhibitor Sparc also fully blocked Azot endogenous expression in the retina. Finally, ectopic overexpression of Flower^Lose-B in scattered cells of the retina was sufficient to trigger ectopic Azot activation. These results show that photoreceptor cells also can monitor the levels of Sparc and the relative levels of Flower^Lose-B before triggering Azot expression (Merino, 2015).

These results suggest that the azot promoter integrates fitness information from neighboring cells, acting as a relative 'cell-fitness checkpoint.'

To test if fitness-based cell selection is a mechanism active not only during development, but also during adult stages, WT adult flies were exposed to UV light and monitor Azot and Flower expression were monitored in adult tissues. UV irradiation of adult flies triggered cytoplasmic Azot expression in several adult tissues including the gut and the adult brain. Likewise, UV irradiation of adult flies triggered Flower Lose expression in the gut and in the brain. Irradiation-induced Azot expression was unaffected by Bcl-2 but was eliminated when Flower Lose was silenced or when relative differences of Flower Lose where diminished in the gut. This suggests that the process of cell selection is active throughout the life history of the animal. Further confirming this conclusion, Azot function was essential for survival after irradiation, because more than 99% of azot mutant adults died 6 days after irradiation, whereas only 62.4% of WT flies died after the same treatment. The percentage of survival correlated with the dose of azot because adults with three functional copies of azot had higher median survival and maximum lifespan than WT flies, or null mutant flies rescued with two functional azot transgenes (Merino, 2015).

The next part of the study addressed the role of cell selection during aging. Lack of cell selection could affect the whole organism by two nonexclusive mechanisms. First, the failure to detect precancerous cells, which could lead to cancer formation and death of the individual. Second, the time-dependent accumulation of unfit but viable cells could lead to accelerated tissue and organ decay. We therefore tested both hypotheses (Merino, 2015).

It has been previously shown that cells with reduced levels for cell polarity genes like scrib or dlg are eliminated but can give rise to tumors when surviving. Therefore this study checked if azot functions as a tumor suppressing mechanism in those cells. Elimination of dlg and scrib mutant cells was not affected by RNAi against azot or when Azot function was impaired by mutation, in agreement with the absence of azot induction in these mutant cells. However, azot RNAi or the same azot mutant background efficiently rescued the elimination of clones with reduced Wg signaling (Merino, 2015).

Moreover, the high number of suboptimal cells produced by UV treatment did not lead to tumoral growth in azot-null background. Thus, tumor suppression mechanisms are not impaired in azot mutant backgrounds, and tumors are not more likely to arise in azot-null mutants (Merino, 2015).

Also tests were performed to see whether the absence of azot accelerates tissue fitness decay in adult tissues. Focused was placed on the adult brain, where neurodegenerative vacuoles develop over time and can be used as a marker of aging. The number was compared of vacuoles appearing in the brain of flies lacking azot (azot^-/-), WT flies (azot^+/+), flies with one extra genomic copy of the gene (azot^+/+; azot⁺), and mutant flies rescued with two genomic copies of azot (azot^-/-;azot^+/+). For all the genotypes analyzed, a progressive increase was observed in the number and size of vacuoles in the brain over time. Interestingly, azot^-/- brains showed higher number of vacuoles compared to control flies (azot^+/+ and azot^-/-;azot^+/+) and a higher rate of vacuole accumulation developing over time. In the case of flies with three genomic copies of the gene (azot^+/+; azot⁺), vacuole number tended to be the lowest (Merino, 2015).

The cumulative expression of azot was analyzed during aging of the adult brain. Positive cells were detected as revealed by reporter azot{KO; gfp}, in homozygosis, that express GFP instead of Azot. A time-dependent accumulation of azot-positive cells was observed (Merino, 2015).

From this, it is concluded that azot is required to prevent tissue degeneration in the adult brain and lack of azot showed signs of accelerated aging. This suggested that azot could affect the longevity of adult flies. Flies lacking azot (azot^-/-) had a shortened lifespan with a median survival of 7.8 days, which represented a 52% decrease when compared to WT flies (azot^+/+), and a maximum lifespan of 18 days, 25% less than WT flies (azot^+/+). This effect on lifespan was azot dependent because it was completely rescued by introducing two functional copies of azot. On the contrary, flies with three functional copies of the gene (azot^+/+; azot⁺) showed an increase in median survival and maximum lifespan of 54% and 17%, respectively (Merino, 2015).

In conclusion, azot is necessary and sufficient to slow down aging, and active selection of viable cells is critical for a long lifespan in multicellular animals (Merino, 2015).

The next part of the study demonstrates that death of unfit cells is sufficient and required for multicellular fitness maintenance. The results cited above show the genetic mechanism through which cell selection mediates elimination of suboptimal but viable cells. However, using flip-out clones and MARCM, this study found that Azot overexpression was not sufficient to induce cell death in wing imaginal discs. Because Hid is downstream of Azot, it was wondered whether expressing Hid under the control of the azot regulatory regions could substitute for Azot function (Merino, 2015).

In order to test this hypothesis, the whole endogenous azot protein-coding sequence was replaced by the cDNA of the proapoptotic gene hid (azot{KO; hid}) flies. In a second strategy, the whole endogenous azot protein-coding sequence was replaced by the cDNA of transcription factor Gal4, so that the azot promoter can activate any UAS driven transgene (azot{KO; Gal4} flies. The number of morphological aberrations was compared in the adult wings of six genotypes: first, homozygous azot{KO; Gal4} flies that lacked Azot; second, azot{KO; hid} homozygous flies that express Hid with the azot pattern in complete absence of Azot; third, azot^+/+ WT flies as a control; and finally three genotypes where the azot{KO; Gal4} flies were crossed with UAShid, UASsickle, another proapoptotic gene, or UASp35, an apoptosis inhibitor. In the case of UASsickle flies, a second azot mutation was introduced to eliminate azot function. Interestingly, the number of morphological aberrations was brought back to WT levels in all the situations where the azot promoter was driving proapoptotic genes (azot{KO; hid}, azot{KO; Gal4} × UAShid, azot{KO; Gal4} × UASsickle with or without irradiation. On the contrary, expressing p35 with the azot promoter was sufficient to produce morphological aberrations despite the presence of one functional copy of azot. Likewise, p35-expressing flies (azot{KO; Gal4}/azot⁺; UASp35) did not survive UV treatments, whereas a percentage of the flies expressing hid (26%) or sickle (28%) in azot-positive cells were able to survive (Merino, 2015).

From this, it is concluded that specifically killing those cells selected by the azot promoter is sufficient and required to prevent morphological malformations and provide resistance to UV irradiation (Merino, 2015).

The next part of the study demonstrated that death of unfit cells extends lifespan It was asked whether the shortened longevity observed in azot^-/- flies could be also rescued by killing azot-expressing cells with hid in the absence of Azot protein. It was found that azot{KO; hid} homozygous flies had dramatically improved lifespan with a median survival of 27 days at 29°C, which represented a 125% increase when compared to azot^-/- flies, and a maximum lifespan of 34 days, 41% more than mutant flies (Merino, 2015).

Similar results were obtained at 25°C. It was found that flies lacking azot (azot^-/-) had a shortened lifespan with a median survival of 25days, which represented a 24% decrease when compared to WT flies (azot^+/+), and a maximum lifespan of 40 days, 31% less than WT flies (azot^+/+). On the contrary, flies with three functional copies of the gene (azot^+/+; azot⁺) or flies where azot is replaced by hid (azot{KO; hid} homozygous flies) showed an increase in median survival of 54% and 63% and maximum lifespan of 12% and 24%, respectively (Merino, 2015).

Finally, the effects of dietary restriction on longevity of those flies was tested. It was found that dietary restriction could extend both the median survival and the maximum lifespan of all genotypes. Interestingly, dietary restricted flies with three copies of the gene azot showed a further increase in maximum lifespan of 35%. This shows that dietary restriction and elimination of unfit cells can be combined to maximize lifespan (Merino, 2015).

In conclusion, eliminating unfit cells is sufficient to increase longevity, showing that cell selection is critical for a long lifespan in Drosophila (Merino, 2015).

This study has shown that active elimination of unfit cells is required to maintain tissue health during development and adulthood. The gene (azot), whose expression is confined to suboptimal or misspecified but morphologically normal and viable cells. When tissues become scattered with suboptimal cells, lack of azot increases morphological malformations and susceptibility to random mutations and accelerates age-dependent tissue degeneration. On the contrary, experimental stimulation of azot function is beneficial for tissue health and extends lifespan. Therefore, elimination of less fit cells fulfils the criteria for a hallmark of aging (Merino, 2015).

Although cancer and aging can both be considered consequences of cellular damage, no evidence was found for fitness-based cell selection having a role as a tumor suppressor in Drosophila. The results rather support that accumulation of unfit cells affect organ integrity and that, once organ function falls below a critical threshold, the individual dies (Merino, 2015).

Azot expression in a wide range of 'less fit' cells, such as WT cells challenged by the presence of 'supercompetitors,' slow proliferating cells confronted with normal proliferating cells, cells with mutations in several signaling pathways (i.e., Wingless, JAK/STAT, Dpp), or photoreceptor neurons forming incomplete ommatidia. In order to be expressed specifically in 'less fit' cells, the transcriptional regulation of azot integrates fitness information from at least three levels: (1) the cell's own levels of Flower^Lose isoforms, (2) the levels of Sparc, and (3) the levels of Lose isoforms in neighboring cells. Therefore, Azot ON/OFF regulation acts as a cell-fitness checkpoint deciding which viable cells are eliminated. It is proposed that by implementing a cell-fitness checkpoint, multicellular communities became more robust and less sensitive to several mutations that create viable but potentially harmful cells. Moreover, azot is not involved in other types of apoptosis, suggesting a dedicated function, and - given the evolutionary conservation of Azot - pointing to the existence of central cell selection pathways in multicellular animals (Merino, 2015).

REGULATION

Transcriptional Regulation

In addition to a post-translational regulation of Hid, the Ras/MAPK pathway promotes cell survival in Drosophila by downregulating the expression of hid. Conversely, downregulation of the Ras/MAPK pathway induces cell death by upregulating hid expression. hid transcript levels are downregulated in dominantly active Dras1- (Dras1^Q13) expressing embryos when assayed 3 hr after heat shock. In wild-type embryos, total HID mRNA levels do not change dramatically between stage 11, when Ras expression was ectopically induced, and stage 14, when HID mRNA levels were assayed. This eliminates the concern that developmental arrest might account for the observed difference in HID mRNA levels. It was observed that hid levels return to normal in Dras1^Q13 embryos by 5 hr after heat shock. Cell death also resumes in these embryos several hours later. This indicates that a transient increase in Ras activity leads to a transient suppression of hid expression, accompanied by a transient protection from naturally occurring cell death. HID mRNA levels were also assayed through an alternative procedure: whole mount in situ analysis. These results confirm that hid transcript levels decline in dominantly active Dras1- (Dras1^Q13) expressing embryos. This is particularly apparent in the midline glia, which strongly express hid. The survival of midline glia is known to depend on the activity of the Epidermal growth factor receptor pathway. To confirm that Ras regulation of hid utilizes the Raf/MAPK pathway, the effect of a constitutively active form of Draf (phlF22) on hid expression has been investigated. In situ analyses were performed on embryos expressing activated Draf under the control of the heat shock promoter. Heat-induced expression of phlF22 results in downregulation of hid transcript levels, suggesting that Ras functions through the Raf/MAPK pathway to downregulate hid expression (Kurada, 1998).

Reduction in pointed (pnt) activity has been observed to enhance ectopic Hid induced cell death in the eye. The pointed transcription factor is a target of MAPK function and acts as a positive regulator in the R7 pathway. The pnt gene encodes two related proteins, pnt1 and pnt2. pnt2 operates downstream of the MAPK rolled in the Ras pathway. Therefore, the consequences of ectopic expression of pnt2 were examined. Embryos were generated that carry UAS-Pnt2 and a midline glia-specific Gal4 driver (52A-Gal4), resulting in the expression of pnt2 in the midline glia cells. Such embryos were tested for hid levels by whole-mount in situ analysis. Like embryos expressing activated Dras1 and activated Draf, pnt2-expressing embryos show decreased hid transcript levels, indicating that the Ras/MAPK pathway, acting through pnt, downregulates hid transcription (Kurada, 1998).

Since upregulation of the Ras/MAPK pathway promotes cell survival and downregulates hid expression, it was predicted that increased hid expression is the cause of the increased apoptosis observed when Ras activity is decreased. Ubiquitous expression of the negative regulator yan is able to induce massive embryonic apoptosis. In these same embryos HID mRNA levels are increased within 2 hr of yan^Act induction and continue to rise for many more hours. Thus, downregulation of Ras activity in the embryo results in increased hid transcription and apoptosis, and this transcription is regulated either directly or indirectly by yan. These results imply that Ras activation of MAPK and inactivation of yan is an important cell survival pathway in embryos (Kurada, 1998).

Blocking Epidermal growth factor receptor activity in the developing eye also enhances apoptosis. If hid is a target of Egfr/Ras/MAPK activity in this tissue, then hid levels should increase when Egfr activity is blocked. Expression of a dominant negative Egfr in the developing eye results in a band of increased hid transcription in the eye disc. This band lies several rows posterior to the furrow and corresponds well with the first developmental defects seen in these eye discs. In sum, these data implicate the downregulation of hid transcription as an important component of Egfr antiapoptotic activity. The post-transcriptional modification of Hid appears to be equally important (Kurada, 1998).

The steroid hormone ecdysone signals the stage-specific programmed cell death of the larval salivary glands during Drosophila metamorphosis. This response is preceded by an ecdysone-triggered switch in gene expression in which the diap2 death inhibitor is repressed and the reaper (rpr) and head involution defective (hid) death activators are induced. rpr is induced directly by the ecdysone-receptor complex through an essential response element in the rpr promoter. The Broad-Complex (BR-C) is required for both rpr and hid transcription, while E74A is required for maximal levels of hid induction. diap2 induction is dependent on FTZ-F1, while E75A and E75B are each sufficient to repress diap2. This study identifies transcriptional regulators of programmed cell death in Drosophila and provides a direct link between a steroid signal and a programmed cell death response (Jiang, 2000).

Although initial studies had indicated that rpr and hid are coordinately induced in the salivary glands approximately 12 hr after puparium formation, more recent work has shown that rpr is induced approximately 1.5 hr earlier than hid, suggesting that these death activators are regulated by distinct mechanisms. The timing of rpr induction is synchronous with the prepupal ecdysone pulse, suggesting that it may be induced as a primary response to the hormone, while the delay in hid induction suggests that it may be a secondary response to ecdysone. These two modes of regulation can be distinguished by their different sensitivity to the inhibition of protein synthesis. Salivary glands were dissected from 10 hr wild-type prepupae and cultured in insect medium supplemented with 20-hydroxyecdysone, either in the presence or absence of cycloheximide. Total RNA was extracted after 0, 2, or 4 hr of culture and analyzed by Northern blot hybridization. Both rpr and hid are induced within 2 hr of hormone treatment, consistent with the proposal that these genes are induced by ecdysone in late prepupal salivary glands. In the presence of the protein synthesis inhibitor cycloheximide, rpr transcription is both delayed and reduced, while hid expression is completely eliminated. These observations indicate that rpr is induced directly by the hormone-receptor complex, although maximal levels of rpr transcription also require the synthesis of ecdysone-induced proteins. In contrast, hid is induced solely as a secondary response to ecdysone. These observations are consistent with the timing of rpr and hid induction in staged salivary glands and provide a framework for defining the molecular mechanisms by which ecdysone regulates rpr and hid transcription, triggering salivary gland cell death (Jiang, 2000).

The BR-C is defined by three genetic functions: broad (br), reduced bristles on palpus (rbp), and l(1)2Bc. Earlier studies have shown that the rbp function of the BR-C is required for salivary gland cell death during metamorphosis. This result has been confirmed by finding that larval salivary glands are not destroyed by 22 hr after puparium formation in pupae that carry the rbp⁵ null allele. The high penetrance of this mutant phenotype suggests that rpr and hid may not be properly expressed in rbp⁵ mutant salivary glands (Jiang, 2000).

To test this hypothesis, salivary glands were dissected from staged rbp⁵ mutants, and rpr and hid expression was examined by Northern blot hybridization. Both rpr and hid transcription is significantly reduced in rbp⁵ mutant salivary glands, indicating that the failure of salivary gland cell death in this mutant can be attributed to its inability to express these death activators. Both betaFTZ-F1 and the ecdysone-inducible E93 early gene are expressed in rbp⁵ mutant salivary glands, indicating that the block in rpr and hid transcription is not simply due to developmental arrest of the mutant animals. BR-C is expressed in midprepupal salivary glands and thus would be present in the late prepupal glands used for the cycloheximide experiment described above. This explains why the reduced level of rpr transcription observed in the absence of protein synthesis is not as severe as the rbp⁵ mutant phenotype (Jiang, 2000).

Both molecular and genetic studies have indicated that the BR-C and E74 function together in common developmental pathways during the onset of metamorphosis. It was therefore asked whether, like the BR-C, E74 might contribute to the ecdysone-triggered destruction of larval salivary glands. In support of this proposal, salivary gland cell death is significantly delayed in E74^P[neo] animals. This mutation is a null allele that inactivates the E74A promoter. While salivary glands in control animals are completely destroyed by 16 hr after puparium formation, approximately 20% of E74^P[neo]Df(3L)st-81k19 animals have salivary glands at 24 hr after puparium formation. This partially penetrant cell death defect suggests that rpr and hid expression may be reduced in E74A mutant salivary glands. To test this hypothesis, salivary glands were dissected from staged E74^P[neo]/Df(3L)st-81k19 mutants, and rpr and hid expression was examined by Northern blot hybridization. Although rpr transcription is unaffected by the E74^P[neo] mutation, the levels of hid transcription are significantly reduced. This observation indicates that E74A is required for the maximal induction of hid but not rpr (Jiang, 2000).

The observation that rpr transcription is induced directly by ecdysone in cultured larval salivary glands indicates that one or more EcR/USP binding sites should be present in the rpr promoter. As a first step toward identifying these regulatory elements, the sequences required for ecdysone-inducible rpr transcription in larval salivary glands were mapped. 9.6 kb of the rpr promoter is sufficient to recapitulate certain aspects of the complex pattern of rpr expression during embryogenesis. Four P element constructs were made that carry either 9.5, 6.1, 3.9, or 1.2 kb of DNA upstream from the rpr transcription start site and 125 bp downstream from the transcription start site, with the rpr 5' untranslated region fused to a lacZ reporter gene. These constructs were introduced into the Drosophila germline by P element-mediated transformation, and the patterns of lacZ transcription in staged salivary glands were compared with those of the endogenous rpr gene by Northern blot hybridization. An increased level of rpr promoter activity is seen upon deletion of sequences between -9.5 and -6.1 kb relative to the start site of rpr transcription. The overall level of lacZ transcription is then reduced as more rpr regulatory sequences are deleted. However, 1.3 kb of the rpr promoter is sufficient to direct lacZ induction in synchrony with that of the endogenous rpr gene, indicating that this region contains the sequences required for proper temporal regulation (Jiang, 2000).

DNA fragments from the 1.3 kb rpr promoter region were generated by PCR and tested for their ability bind EcR: a 274 bp fragment extending from -195 bp to +80 bp relative to the rpr transcription start site binds EcR. Sequence analysis has shown a single imperfect palindromic EcR/USP binding site within this fragment. This rpr EcRE matches 10 out of 13 positions with the consensus EcR/USP binding site. The rpr element is not as strong of a binding site as a canonical hsp27 element. This observation is consistent with the deviations from the consensus at positions +2 and +3 in the rpr EcRE. These and other results strongly suggest that the ecdysone-receptor complex directly regulates rpr transcription through at least one binding site in the rpr promoter (Jiang, 2000).

Therefore ecdysone-regulated transcription factors encoded by betaFTZ-F1, BR-C, E74, and E75 function together to direct a burst of the diap2 death inhibitor followed by induction of the rpr and hid death activators. It is proposed that cooperation between rpr and hid allows these genes to overcome the inhibitory effect of diap2, by precisely coordinating when the salivary glands are destroyed. Evidence that the ecdysone-receptor complex directly induces rpr transcription through an essential response element in the promoter, providing a direct link between the steroid signal and a programmed cell death response. The diap2 death inhibitor is expressed briefly in the salivary glands of late prepupae, foreshadowing the imminent destruction of this tissue. This transient expression is directed by at least three ecdysone-regulated transcription factors: betaFTZ-F1, E75A, and E75B. diap2 induction is dependent on the betaFTZ-F1 orphan nuclear receptor. This is consistent with the timing of betaFTZ-F1 expression, which immediately precedes that of diap2, as well as the known role of betaFTZ-F1 as an activator of gene expression in late prepupae (Jiang, 2000).

Steroid hormones trigger dynamic tissue changes during animal development by activating cell proliferation, cell differentiation, and cell death. Steroid regulation of changes have been characterized in midgut structure during the onset of Drosophila metamorphosis. Following an increase in the steroid 20-hydroxyecdysone (ecdysone) at the end of larval development, future adult midgut epithelium is formed, and the larval midgut is rapidly destroyed. Mutations in the steroid-regulated genes BR-C and E93 differentially impact larval midgut cell death but do not affect the formation of adult midgut epithelia. In contrast, mutations in the ecdysone-regulated E74A and E74B genes do not appear to perturb midgut development during metamorphosis. Larval midgut cells possess vacuoles that contain cellular organelles, indicating that these cells die by autophagy. While mutations in the BR-C, E74, and E93 genes do not impact DNA degradation during this cell death, mutations in BR-C inhibit destruction of larval midgut structures, including the proventriculus and gastric caeca, and E93 mutants exhibit decreased formation of autophagic vacuoles. Dying midguts express the rpr, hid, ark, dronc, and crq cell death genes, suggesting that the core cell death machinery is involved in larval midgut cell death. The transcription of rpr, hid, and crq are altered in BR-C mutants, and E93 mutants possess altered transcription of the caspase dronc, providing a mechanism for the disruption of midgut cell death in these mutant animals. These studies indicate that ecdysone triggers a two-step hierarchy composed of steroid-induced regulatory genes and apoptosis genes that, in turn, regulate the autophagic death of midgut cells during development (Lee, 2002).

Transcription of rpr, hid, ark, dronc, and crq increases in wild-type animals following the late larval pulse of ecdysone that triggers larval midgut cell death. Since mutations in the BR-C and E93 genes prevent proper destruction of larval midguts, Northern blots were prepared from midguts of these mutants at stages preceding and during cell death. BR-C 2Bc² mutants have altered transcription of rpr, hid, and crq, but do not impact the transcription of ark and dronc. In contrast, E93 mutants possess altered transcription of dronc, but do not change the transcript levels of the other cell death genes known to be expressed in dying midguts. Although midguts die by autophagy, they transcribe core apoptosis regulators during this cell death, and mutants that prevent autophagy alter transcription of apoptosis genes (Lee, 2002).

The distributed association of future adult cells within the epithelium of larval midguts is another important difference between ecdysone-regulated midgut and salivary gland programmed cell death. The close association of larval and adult midgut cells may be one of the reasons why larval midgut exhibits a less synchronized cell death than salivary glands. Both salivary glands and midguts require the function of the E93 and BR-C genes. However, mutations in these genes appear to result in different effects in salivary glands and midguts; BR-C appears to play a more important role in midguts. While both salivary glands and midguts express the cell death genes rpr, hid, ark, dronc, and crq, the impact of mutations in BR-C and E93 are very different in the midgut than in salivary glands. BR-C affects transcription of rpr, hid, and crq, but E93 mutants only affect dronc transcription in midguts. In contrast, mutations in E93 prevent proper transcription of all of these cell death genes in dying salivary glands. Clearly, many more genes may be involved in the complicated autophagic cell death of midguts. While several similarities and differences have been identified between salivary gland and midgut death, future analyses are needed to clarify the mechanism by which the steroid ecdysone triggers midgut programmed cell death (Lee, 2002).

Much of what is known about apoptosis in human cells stems from pioneering genetic studies in the nematode C. elegans. However, one important way in which the regulation of mammalian cell death appears to differ from that of its nematode counterpart is in the employment of TNF and TNF receptor superfamilies. No members of these families are present in C. elegans, yet TNF factors play prominent roles in mammalian development and disease. The cloning and characterization of Eiger, a unique TNF homolog in Drosophila, is described. Like a subset of mammalian TNF proteins, Eiger is a potent inducer of apoptosis. Unlike its mammalian counterparts, however, the apoptotic effect of Eiger does not require the activity of the caspase-8 homolog DREDD, but it completely depends on its ability to activate the JNK pathway. Eiger-induced cell death requires the caspase-9 homolog DRONC and the Apaf-1 homolog DARK. These results suggest that primordial members of the TNF superfamily can induce cell death indirectly by triggering JNK signaling, which, in turn, causes activation of the apoptosome. A direct mode of action via the apical FADD/caspase-8 pathway may have been coopted by some TNF signaling systems only at subsequent stages of evolution (Moreno, 2002).

The mechanism by which JNK signaling triggers cell death in response to TNF is poorly understood in mammals and is unknown in Drosophila. It was therefore of interest to identify the apoptotic machinery responsible for Eiger-induced cell death. Having excluded the caspase-8-like FADD/DREDD branch, focus was placed on the involvement of caspase-9, which represents another major pathway that leads to apoptosis. The key event for caspase-9 activation is its association with the protein cofactor Apaf-1 to form an active complex referred to as the apoptosome. Since many cell intrinsic insults can trigger this pathway, it has been termed the 'intrinsic death pathway'. Expression of a dominant-negative form of the Drosophila caspase-9 homolog DRONC, comprising only the CARD domain, fully blocks Eiger-induced apoptosis in a dose-dependent manner. Moreover, genetic removal of DARK, the homolog of Apaf-1, suppresses Eiger-dependent phenotypes. These results indicate that the presumptive Drosophila apoptosome is essential for the ability of Eiger to induce cell death. In agreement with this conclusion, overexpression of Thread, the Drosophila inhibitor of apoptosis protein 1 (DIAP1/Thread) blocks Eiger function. Thread/DIAP1 has been shown to bind DRONC and target it for degradation. Most instances of programmed cell death that have been analyzed in Drosophila are triggered by, and require, the genes reaper, hid, or grim, which encode small proteins that bind to and inactivate IAPs, such as Thread/DIAP1. The removal of one copy of a chromsosomal segment that includes the genes hid, grim, and reaper rescues eye ablation, and Eiger induces a strong transcriptional activation of hid and a weak activation of reaper. These results suggest, therefore, that Eiger/JNK signaling triggers DRONC by inactivating the IAPs via a transcriptional upregulation of hid (Moreno, 2002).

An important issue in Metazoan development is to understand the mechanisms that lead to stereotyped patterns of programmed cell death. In particular, cells programmed to die may arise from asymmetric cell divisions. The mechanisms underlying such binary cell death decisions are unknown. A Drosophila sensory organ lineage is described that generates a single multidentritic neuron in the embryo. This lineage involves two asymmetric divisions. Following each division, one of the two daughter cells expresses the pro-apoptotic genes reaper and grim and subsequently dies. The protein Numb appears to be specifically inherited by the daughter cell that does not die. Numb is necessary and sufficient to prevent apoptosis in this lineage. Conversely, activated Notch is sufficient to trigger death in this lineage. These results show that binary cell death decision can be regulated by the unequal segregation of Numb at mitosis. This study also indicates that regulation of programmed cell death modulates the final pattern of sensory organs in a segment-specific manner (Orgogozo, 2002).

The vmd1a neuron is located within a cluster of five multidendritic (md) neurons in the ventral region of abdominal segments A1-A7. The vmd1a neuron can be distinguished from the other ventral md neurons (vmd1-4) using the B6-2-25 enhancer-trap marker. The origin of this vmd1a neuron is not known. vmd1-4 neurons are generated by the four vp1-4 external sensory (es) organ primary precursor (pI) cells. Each vp1-4 pI cell follows a lineage called the md-es lineage. This lineage is composed of four successive asymmetric cell divisions that generate five distinct cells, the four cells of the es organ at the position where the pI cell has formed and one md neuron that will then migrate to the ventral md cluster. In the md-es lineage, the membrane-associated protein Numb is segregated into one of the two daughter cells at each cell division. Numb establishes a difference in cell fate by antagonizing Notch in the Numb-receiving cell. Because no es organ is found in the vicinity of the vmd1a neuron, this neuron is probably not generated by a md-es lineage (Orgogozo, 2002).

rpr and grim, but not hid, are expressed specifically in the pIIa and pIIIb cells of the vmd1a lineage. By contrast, these genes are not expressed in cells of the vp1-4 lineages. In embryos in which a pIIb cell divides at the vp1 position in at least one abdominal segment, most segments contain a vmd1a pIIa-pIIb pair with one cell expressing rpr or grim. This cell is the pIIa cell fated to die. In some other segments, neither of these two cells accumulates rpr (25%) or grim (8%). Since the development of segments is not perfectly synchronous, it is assumed that this represents a situation preceding the onset of rpr and grim expression in the pIIa cell. In the remaining segments, a single Cut-positive cell is detected indicating that the pIIa cell has died. In those segments, expression of rpr and grim is never detected in the remaining pIIb cell (Orgogozo, 2002).

During the pIIb division, Numb was shown to segregate into the dorsal pIIb daughter cell. This cell is not fated to die and differentiates as a vmd1a neuron. By contrast, it could not be directly determined which one of the two pI daughter cells inherits Numb. Indeed, since the orientation of the vmd1a pI cell division is random, the pIIa and pIIb cells could not be identified from their relative positions. Nevertheless the vmd1a pIIa and pIIIb cells appear to generate ectopic shaft/socket and neuron/sheath cell pairs when cell death is prevented. In the md-es lineage, these cell pairs are the progeny of the cells that do not inherit Numb. This suggests that both the vmd1a pIIIb cell and the pIIa cell do not inherit Numb. Thus, Numb appears to segregate in the cells that do not die in the vmd1a lineage (Orgogozo, 2002).

The role of Numb was tested in regulating rpr and grim expression as well as cell death in the vmd1a lineage. In numb mutant embryos in which a secondary precursor cell divides at the vp1 position in at least one abdominal segment, it was observed that the two Cut-positive vmd1a pI daughter cells accumulate rpr or grim transcripts (54% of the segments for rpr, 52% for grim). In other segments a single Cut-positive pI daughter cell was found accumulating rpr or grim. In these segments one pI daughter cell has already died and the other one is undergoing apoptosis. These two phenotypes are not seen in wild-type embryos. Thus, in the absence of numb, both pI daughter cells undergo programmed cell death. Consistently, no Cut-positive cell is observed at the vmd1a position in numb mutant embryos in most segments. It is concluded that numb is required to inhibit the expression of rpr and grim and to prevent cell death in the pIIb cell (Orgogozo, 2002).

To test whether numb is sufficient to prevent cell death, the progeny of the vmd1a pI cell was analyzed in arm-Gal4 UAS-numb embryos that express high levels of Numb. In wild-type embryos in which a vp1 pIIIb cell is dividing in at least one segment, one or two Cut-positive cells are observed at the vmd1a position. In contrast, four Cut-positive cells are observed in 50% of the segments in arm-Gal4 UAS-numb embryos at the same stage. In 8 out of the 9 segments with four cells, two cells accumulating high levels of Pros and two cells accumulating low levels of Pros are seen, suggesting that these cells are two vmd1a neurons and two pIIIb cells. These data indicate that the pIIa cell death is inhibited and that the pIIa cell is transformed into a pIIb-like cell (Orgogozo, 2002).

Numb is known to function by antagonizing Notch activity. This therefore suggests that Notch promotes cell death in the vmd1a lineage and that Numb blocks this activity of Notch. Unfortunately, the strong effect of Notch loss-of-function alleles on the selection of the vmd1a pI cell means that it was not possible to test directly whether Notch is required for cell death in the vmd1a lineage. Therefore the conditional Notch^ts1 allele was used. However, when Notch^ts1 embryos are shifted to a restrictive temperature (31°C) soon after the specification of the vmd1a pI cell (i.e., at 13-14.5 hours after egg laying at 19°C), no significant reduction was seen in the number of rpr- or grim-expressing pIIa cells. A stronger Notch^ts1/Notch^55e11 combination causes the appearance of additional vmd1a pI cells even at the permissive temperature (19°C). It is therefore not possible to determine whether an increase in the number of rpr- or grim-negative cells results from a lack of Notch-dependent apoptosis or from an excess of vmd1a pI cells due to reduced Notch signaling during lateral inhibition (Orgogozo, 2002).

Therefore a test was performed to see whether an activated form of Notch, Nintra, can promote the death of the pIIb cell when expressed around the time of the vmd1a pI cell division. In 6% of the segments from embryos in which at least one segment shows a dividing vp1 pIIb cell, rpr or grim transcripts accumulate in both vmd1a pI daughter cells. In other segments, a single Cut-positive cell remains at the vmd1a position and accumulates rpr or grim. These expression patterns are not seen in heat-shocked control embryos. Importantly, these observations are similar to those made in numb mutant embryos. Thus, both loss of numb activity and ectopic Notch signaling lead to transcriptional activation of pro-apoptotic genes in the pIIb cell. Finally, a similar effect of Nintra on rpr and grim expression is seen in the vmd1a pIIb daughter cells when Nintra expression was induced at a later stage, i.e., when the vmd1a pIIb cell is dividing. Together, these results indicate that Notch signaling is sufficient to promote cell death in the vmd1a lineage (Orgogozo, 2002).

In summary, the lineage generating the vmd1a neuron has been described. This lineage is composed of two asymmetric divisions following which one daughter cell undergoes apoptosis. These two binary cell death decisions are regulated by the unequal segregation of Numb at mitosis. Therefore, the data provide the first experimental evidence that alternative cell death decision can be regulated by the unequal segregation of a cell fate determinant. The conserved role of Numb and Notch in neuronal specification in flies and vertebrates suggests that Numb-mediated inhibition of Notch may play a similar role in regulating cell death decisions in vertebrates (Orgogozo, 2002).

Molecular mechanism of Reaper-Grim-Hid-mediated suppression of DIAP1-dependent Dronc ubiquitination

The inhibitor of apoptosis protein DIAP1 inhibits Dronc-dependent cell death by ubiquitinating Dronc. The pro-death proteins Reaper, Hid and Grim (RHG) promote apoptosis by antagonizing DIAP1 function. This study reports the structural basis of Dronc recognition by DIAP1 as well as a novel mechanism by which the RHG proteins remove DIAP1-mediated downregulation of Dronc. Biochemical and structural analyses revealed that the second BIR (BIR2) domain of DIAP1 recognizes a 12-residue sequence in Dronc. This recognition is essential for DIAP1 binding to Dronc, and for targeting Dronc for ubiquitination. Notably, the Dronc-binding surface on BIR2 coincides with that required for binding to the N termini of the RHG proteins, which competitively eliminate DIAP1-mediated ubiquitination of Dronc. These observations reveal the molecular mechanisms of how DIAP1 recognizes Dronc, and more importantly, how the RHG proteins remove DIAP1-mediated ubiquitination of Dronc (Chai, 2003).

E2F1 and E2F2 have opposite effects on radiation-induced p53-independent apoptosis in Drosophila

The ability of ionizing radiation (IR) to induce apoptosis independent of p53 is crucial for successful therapy of cancers bearing p53 mutations. p53-independent apoptosis, however, remains poorly understood relative to p53-dependent apoptosis. IR induces both p53-dependent and p53-independent apoptoses in Drosophila, making studies of both modes of cell death possible in a genetically tractable model. Previous studies have found that Drosophila E2F proteins are generally pro-death or neutral with regard to p53-dependent apoptosis. This study reports that dE2F1 promotes IR-induced p53-independent apoptosis in larval imaginal discs. Using transcriptional reporters, evidence is provided that, when p53 is mutated, dE2F1 becomes necessary for the transcriptional induction of the pro-apoptotic gene hid after irradiation. In contrast, the second E2F homolog, dE2F2, as well as the net E2F activity, which can be depleted by mutating the common cofactor, dDp, is inhibitory for p53-independent apoptosis. It is concluded that p53-dependent and p53-independent apoptoses show differential reliance on E2F activity in Drosophila (Wichmann, 2010).

This study has taken advantage of the relative simplicity of Drosophila E2F and p53 families to study the role of E2Fs in p53-independent apoptosis. The results indicate that Drosophila E2F homologs play opposing roles in regulating p53-independent apoptosis in response to IR. dE2F1, a homolog of the mammalian 'activator' E2Fs, is required for Chk2-/p53-independent apoptosis, while dE2F2, a homolog of the mammalian 'repressor' E2Fs, limits p53-independent apoptosis. The net E2F activity in the cell, reduced by mutations in dDP, is inhibitory towards p53-independent apoptosis (Wichmann, 2010).

One surprising finding from these studies is that 2 kb of hid promoter confers IR-induced transcriptional activation in a p53-dependent manner. This is surprising because in embryos, transcriptional activation of hid by IR in a p53-dependent manner requires the IRER (irradiation responsive enhancer region) that lies next to rpr, ~ 200 kb away from hid, and is regulated epigenetically by histone modification (Zhang, 2008). Yet, as shown previously, 2 kb of hid promoter is enough to allow IR-induced GFP expression in eye and wing imaginal discs (Tanaka-Matakatsu, 2009). This study shows that this induction is p53-dependent. Clearly, regulation of hid by IR is different between embryos and larval discs (Wichmann, 2010).

Mammalian p53 consensus is a tandem repeat of 10 nucleotides with the sequence RRRCWWGYYY where R = G/A, W = A/T and Y = T/C and invariant C and G are shown in bold. Drosophila p53 binds to a DNA damage response element at the rpr locus that differs from the mammalian consensus at one position shown in lower case; tGACATGTTT GAACAAGTCg. Manual examination of 2 kb of hid promoter fragment that responds to p53 status shows a potential binding sequence at −2006 from the start of hid transcription that deviates from the mammalian consensus at two positions, and another at −1667 that deviates at three positions. These are ttGCATGCTC GctCATGTTC and GtGCAAGagT GtGCTTGaat respectively. Since the consensus for Drosophila p53 has not been determined, it is possible that either or both of these are responsible for the effect of p53 on hid-driven GFP reporter (Wichmann, 2010).

The 2 kb hid enhancer includes E2F consensus sequences. Rb has been shown to repress the expression of hid-driven GFP reporter when E2F binding sequences are intact but not when these are mutated (Tanaka-Matakatsu, 2009). That is, E2F binding sites allow for repression of hid by Rb although which E2F mediates this repression is not known. In any case, the finding that net E2F activity is inhibitory towards apoptosis would be consistent with the published result that Rb inhibits hid expression via E2F consensus sites. It is not known if dE2F1 plays a permissive role (e.g. by allowing elevated basal expression of pro-apoptotic genes) or an instructive role (e.g. by allowing for induction of pro-apoptotic genes by IR), or both. The results with hid-driven GFP reporter are consistent with an instructive role but do not rule out a permissive role (Wichmann, 2010).

In the absence of p53, dE2F1 is needed for the transcriptional induction of hid> GFP reporter by IR. This can explain two published results: that hid is necessary for IR-induced p53-independent apoptosis (McNamee, 2009), and that hid is transcriptionally induced in p53 mutants after a time delay. Human E2F1 can bind to the promoter of a hid ortholog, Smac/DIABLO, and can, when ectopically expressed, activate the transcription of the latter in vivo. The role of p53 status in this process or the significance of this mode of regulation was not investigated. It is speculated that the role of E2F1 in IR-induced, p53-independent transcriptional activation of Smac/DIABLO genes may be conserved in mammals (Wichmann, 2010).

Previous work has shown that dE2F1 and dE2F2 exhibit antagonistic functions, with dE2F1 activating and dE2F2 repressing the transcription of a reporter containing canonical E2F sites from the PCNA promoter. dE2F1 and dE2F2 occupy the PCNA promoter and the ratio of the two E2F proteins influenced the degree of transcriptional activation or repression. In wild type, PCNA expression is tightly coupled to the pattern of S phase, in eye imaginal discs for example. In de2f2 mutants, PCNA is no longer down-regulated outside pattern of the S phase. The loss of all E2F activities, either in de2f1, de2f2 double mutants or in dDP single mutants, results in de-repression of PCNA such that a low but significant level is expressed throughout the cell cycle. Thus the net result of opposing E2F activities is the cyclical expression of PCNA in concert with DNA replication (Wichmann, 2010).

The paradigm of E2F-dependent regulation of PCNA aids in understanding the role of E2F proteins in p53-independent apoptosis. dE2F1 and dE2F2 might similarly influence p53-independent apoptosis by regulating pro-apoptotic gene(s) such as hid. According to this model, dE2F2 (with dDp) provides a net repressive activity that inhibits IR-induced apoptosis. This activity must be operative only in the absence of p53;dE2F2 mutations that have no effect on apoptosis when p53 is present. In p53 mutants, dE2F1 counteracts dE2F2 after irradiation and thus promotes apoptosis. Disabling transcriptional activation by dE2F1, which is what the allelic combination de2f1^i2/de2f1⁷¹⁷² is predicted to cause, would result in the failure to overcome dE2F2/Dp. Removal of dE2F2 with null alleles, would result in increased gene expression and more apoptosis. Reducing the ability of dDP to interact with dE2Fs, which is what the allelic combination dDP^a1/dDP^a2 is predicted to cause, would reduce dE2F1 and dE2F2 activities simultaneously. Since this results in more apoptosis, the net E2F activity is inhibitory on apoptosis when p53 is absent (Wichmann, 2010).

This study and published studies in wing imaginal discs reveal significant differences in the effect of E2F/DP mutations on p53-dependent apoptosis (typically assayed at 4–6 h post irradiation) and p53-independent apoptosis (18–24 h after irradiation in p53 mutants). The clearest difference is that dE2F2 null mutations have little or no effect on p53-dependent apoptosis, but increase the level of p53-independent apoptosis. dDP loss-of-function mutations decrease p53-dependent apoptosis throughout eye imaginal discs and in most cells of the wing pouch, whereas they increase the level of p53-independent apoptosis. In contrast, loss-of-function mutations in dE2F1 reduced both p53-dependent apoptosis in most cells of the wing imaginal disc and p53-independent apoptosis in the wing imaginal disc. These differences raise the question ‘how does p53 status alter the role of dE2F2 and dDP in IR-induced apoptosis?’ In the presence of p53, dE2F2 has little effect and dDP is stimulatory. In the absence of p53, dE2F2 and dDP play inhibitory roles. Perhaps the occupancy of transcriptional factors at the target loci such as the hid promoter is sensitive to p53 status (Wichmann, 2010).

In the eye imaginal disc, mutations in ago, a ubiquitin ligase, result in elevated apoptosis. This mode of cell death occurs via elevated E2F1 activity, increased expression of hid and rpr and is independent of apoptosis. Thus, the role of dE2F1 in promoting p53-independent apoptosis is conserved in another tissue of the larvae (Wichmann, 2010).

Previous studies found that the role of Drosophila E2F transcription factors in apoptosis is context-dependent and is influenced by, for example, whether the cells are in the wing pouch or at the dorsal/ventral margin of the wing disc and whether apoptosis is induced by radiation or by the loss of a tumor suppressor homolog, Rb. The current study addresses the role of E2F family members in IR-induced p53-independent apoptosis. The most significant finding in this study is that reducing E2F activity, as in the case of dDP mutants, allows p53-null cells to die following IR exposure. This is in clear contrast to the finding that a similar reduction of E2F activity prevents p53 wild type cells from dying following IR exposure. Several E2F antagonists are being considered in cancer therapy. The current results from Drosophila studies would caution that p53 status must be considered when using such therapies in conjunction with radiation treatment. If the findings in Drosophila apply to human cancers, an E2F antagonist would help kill p53-deficient cancer cells following radiation treatment, but would help p53-wild type cancer cells survive. In addition, an E2F antagonist may be particularly suitable for combination therapy with radiation to eradicate p53-deficient tumors because it may sensitize p53-deficient cancer cells to radiation while protecting p53-wild type somatic cells from the cell-killing effects of IR (Wichmann, 2010).

S phase-coupled E2f1 destruction ensures homeostasis in proliferating tissues

Precise control of cell cycle regulators is critical for normal development and tissue homeostasis. E2F transcription factors are activated during G1 to drive the G1-S transition and are then inhibited during S phase by a variety of mechanisms. The single Drosophila activator E2F (E2f1) was genetically manipulate to explore the developmental requirement for S phase-coupled E2F down-regulation. Expression of an E2f1 mutant that is not destroyed during S phase drives cell cycle progression and causes apoptosis. Interestingly, this apoptosis is not exclusively the result of inappropriate cell cycle progression, because a stable E2f1 mutant that cannot function as a transcription factor or drive cell cycle progression also triggers apoptosis. This observation suggests that the inappropriate presence of E2f1 protein during S phase can trigger apoptosis by mechanisms that are independent of E2F acting directly at target genes. The ability of S phase-stabilized E2f1 to trigger apoptosis requires an interaction between E2f1 and the Drosophila pRb homolog, Rbf1, and involves induction of the pro-apoptotic gene, hid. Simultaneously blocking E2f1 destruction during S phase and inhibiting the induction of apoptosis results in tissue overgrowth and lethality. It is proposed that inappropriate accumulation of E2f1 protein during S phase triggers the elimination of potentially hyperplastic cells via apoptosis in order to ensure normal development of rapidly proliferating tissues (Davidson, 2012).

Thus stabilizing the single Drosophila activator E2f1 in S phase results in apoptosis is necessary to prevent hypertrophy of wing imaginal discs. It is concluded from these data that hyper-accumulation of E2f1 during S phase represents a form of proliferative stress during development that is sensed by the apoptotic machinery and results in the elimination of cells with excess E2f1 activity to maintain homeostasis during tissue growth (Davidson, 2012).

What might be the function of a Drosophila cell's ability to detect abnormal accumulation of E2f1 protein during S phase and subsequently trigger apoptosis? One possibility is that accumulation of E2f1 during S phase resembles instances of abnormally high E2f1 activity that might occur sporadically during rapid growth of a population of precursor cells such as those in the wing imaginal disc. These events could be caused by stochastic or even genetic changes that affect either E2f1 gene transcription or the ability of the CRL4^Cdt2/PCNA pathway to destroy E2f1 after replication factor genes are activated in late G1. The cell's ability to detect E2f1 accumulation in S phase clears these potentially hyperplastic cells from developing tissues via apoptosis, consequently contributing to the balance between cell proliferation and cell death that is necessary for normal tissue growth (Davidson, 2012).

Growing Drosophila imaginal discs possess another mechanism of homeostasis in which a process of compensatory proliferation is activated in order to achieve normal tissue development when as many as 50% of cells are killed by external stimuli like radiation-induced DNA damage. Indeed, in spite of high levels of apoptosis (15% of the cells), 50% of en-Gal4>E2f1^Stable progeny survive until adulthood with about 2/3 of these surviving flies containing wings with somewhat mild morphological defects. Blocking apoptosis with baculovirus p35 when E2f1^Stable is expressed shifts the cell proliferation/apoptosis balance too strongly in favor of cell proliferation, resulting in massive hypertrophy and 100% lethality (Davidson, 2012).

p35 is a broad caspase inhibitor that blocks effector caspase activity at a step downstream of their proteolytic activation. Therefore, cells expressing p35 can initiate apoptosis, but lack the capacity to complete it and are referred to as 'undead cells.' These undead cells produce signals that stimulate unaffected neighboring cells to proliferate. Thus, the dramatic hypertrophy seen in E2f1^Stable/p35 wing discs might be the result of two synergizing growth signals: hyper-active E2f1 and compensatory proliferation from undead cells. The current experiments cannot precisely discern the relative contribution of these two inputs, but E2f1 activity appears to make a larger contribution because E2f1^{Stable/DBD Mut} expression does not cause dramatic overgrowth (Davidson, 2012).

What might explain the 32% of en-Gal4>E2f1^Stable discs that displayed a reduced posterior compartment rather than an overgrown one? The DNA damage observed in eye discs experiments provides a possible answer. Perhaps early in development the 'arrest' class of wing discs sustained enough genomic damage to prevent proliferation, resulting in too small a pool of cells that could respond to the hyper-active E2f1 and undead cell signals to support disc overgrowth. Thus, the wide range of phenotypes that were observed in E2f1^Stable/p35 wing discs may result from multiple influences that act stochastically within the population (Davidson, 2012).

Because endogenous E2f1 is quantitatively destroyed only in S phase, the relative amount of hyper-accumulation of E2f1^Stable is greater during S phase than during any other stage of the cell cycle. Therefore, one possibility is that E2f1^Stable-induced phenotypes result from the stability of E2f1 protein in S phase, and not from general over-expression throughout the cell cycle. Failure to detect E2f1^Stable induced apoptosis in G1-arrested embryonic cells is consistent with this possibility. However, another difference between these embryonic cells and wing discs cells is that the former are cell cycle arrested and the latter are continuingly proliferating during larval development. Thus, another possibility is that S phase-destruction of E2f1 modulates the levels of E2f1 in proliferating cells, and cells that fail to destroy E2f1 during S phase have an increased chance of activating apoptosis at any point in the cell cycle. In either model, S phase E2f1 destruction is not essential for proliferation per se. In marked contrast, E2f1^Stable expression blocks endocycle progression, suggesting that knocking in E2f1^Stable to the endogenous locus would be lethal during development, perhaps even dominant lethal (Davidson, 2012).

E2f1^Stable induces apoptosis at least in part through expression of the pro-apoptotic gene hid. Surprisingly, these events still occur after expression of an E2f1^Stable variant that cannot bind DNA and therefore lacks the ability to stimulate transcription of replication factor genes or cell cycle progression. Instead, E2f1^Stable requires the ability to bind Rbf1 to induce hid gene expression and apoptosis. Genetic disruption of Rbf1 is well known to result in increased hid expression. It is therefore proposed that the inappropriate accumulation of E2f1 in S phase disrupts some aspect of Rbf1 function leading to hid expression and apoptosis (Davidson, 2012).

The data do not discern either the function of Rbf1 that is disrupted by E2f1^Stable or the mechanism of hid induction. While the mechanism connecting Rbf1/E2f1 function and hid may be indirect, some studies suggest that Rbf1 and/or E2f1 could regulate hid directly. It has been demonstrated that Drosophila wing disc cells undergo apoptosis in response to ionizing radiation independently of p53 and that this response requires E2f1 and is triggered by hid expression. In eye discs, loss of Rbf1 function in the MF results in apoptosis that requires E2f1 transactivation function and is accompanied by hid expression. However, whether these effects represent a direct induction of hid by E2f1 is not clear. E2f1 binding at the hid locus has been observed, but the binding site is located ~1.4 kb upstream of the of the start of hid transcription, which is more distal than in well characterized E2F-regulated promoters. When located this far upstream the hid E2f1 binding site fails to activate gene expression in S2 cell reporter assays. hid is also a target of p53, and so any DNA damage resulting from stabilizing E2f1 during S phase, as was observed in eye discs, may also contribute to the activation of hid expression via p53-mediated DNA damage response pathways (Davidson, 2012).

Another possibility is that E2f1, in combination with Rbf1, plays primarily a repressive role at the hid locus. In this model, the result that E2f1^Stable or E2f1^{Stable/DBD Mut} both induce apoptosis would be explained by disruption of Rbf1/E2f1 repressive complexes at the hid locus causing de-repression of hid expression. This model has interesting caveats: what protects the Rbf1/E2f1 complex at the hid locus from being disrupted by Cyclin E/Cdk2, which is active during S phase and inactivates Rbf1-mediated repression of E2f1, or by CRL4^Cdt2-mediate destruction of E2f1? Recent data indicate that the dREAM/MMB complex is required for the stability of E2F/Rbf1 repressive complexes in S phase, and acts to protect these complexes from CDK-mediated phosphorylation at non-cell cycle-regulated genes. While there is yet no evidence that dREAM/MMB regulates hid , this work provides precedent for gene specific Rbf1 regulation during S phase (Davidson, 2012).

Finally, while hid might be a critical player in the response to E2f1^Stable, there are likely other mechanisms responsible for sensing and modulating the apoptotic response to E2f1 levels. For instance, it has been demonstrated that a micro-RNA, mir-11, which is located within the last intron of the Drosophila E2f1 gene, acts to dampen expression of pro-apoptotic E2f1 target genes following DNA damage. In this way, the normal controls of E2f1 gene expression modulate apoptosis. In addition, transgenic constructs lack the normal E2f1 3' UTR, which serves as a site for suppression of E2f1 expression by pumilio translational repressor complexes. Thus, several modes of E2f1 regulation have been bypassed via transgenic expression of E2f1^Stable (Davidson, 2012).

The finding that stabilized Drosophila E2f1 can induce apoptosis independently of transcription and cell cycle progression parallels previous observations made in mammalian cells, albeit with important differences. In mammalian cells, E2F1 can induce apoptosis independently of transcription and cell cycle progression, but apoptosis required E2F1 DNA binding activity, unlike in the current experiments. These studies suggested that DNA binding by E2F1 prevented pro-apoptotic promoters from binding repressor E2F family members (Davidson, 2012).

This comparison of results highlights the way similar phenotypic outcomes in different species can arise from different mechanisms. While mammalian activator E2Fs are also inhibited during S phase, they are not subject to CRL4^Cdt2-mediated, S phase-coupled destruction like Drosophila E2f1. Instead, mammalian activator E2Fs are inhibited by direct Cyclin A/Cdk2 phosphorylation, targeted for destruction by SCF^Skp2, and functionally antagonized by E2F7 and E2F8. The regulation provided by E2F7 and E2F8 is of particular note, as it is essential for mouse development. These atypical E2Fs homo and hetero-dimerize and act redundantly to repress E2F1 target genes independently of pRb family proteins, thus blocking E2F1 from inducing apoptosis. Moreover, the E2F7 and E2F8 genes are E2F1 targets, consequently creating a negative feedback loop that limits E2F1 activity after the G1/S transition. A similar negative feedback loop among factors that regulate G1/S transcription exists in yeast. The analogous Drosophila negative feedback loop involves E2f1 inducing its own destruction by stimulating Cyclin E transcription, which triggers S phase. Therefore, the evolution of eukaryotes has resulted in the use of different molecular mechanism to achieve negative feedback regulation of G1/S-regulated transcription, and in the case of activator E2Fs this regulation is essential for normal development (Davidson, 2012).

JAK/STAT autocontrol of ligand-producing cell number through apoptosis

During development, specific cells are eliminated by apoptosis to ensure that the correct number of cells is integrated in a given tissue or structure. How the apoptosis machinery is activated selectively in vivo in the context of a developing tissue is still poorly understood. In the Drosophila ovary, specialised follicle cells [polar cells (PCs)] are produced in excess during early oogenesis and reduced by apoptosis to exactly two cells per follicle extremity. PCs act as an organising centre during follicle maturation as they are the only source of the JAK/STAT pathway ligand Unpaired (Upd), the morphogen activity of which instructs distinct follicle cell fates. This study shows that reduction of Upd levels leads to prolonged survival of supernumerary PCs, downregulation of the pro-apoptotic factor Hid, upregulation of the anti-apoptotic factor Diap1 and inhibition of caspase activity. Upd-mediated activation of the JAK/STAT pathway occurs in PCs themselves, as well as in adjacent terminal follicle and interfollicular stalk cells, and inhibition of JAK/STAT signalling in any one of these cell populations protects PCs from apoptosis. Thus, a Stat-dependent unidentified relay signal is necessary for inducing supernumerary PC death. Finally, blocking apoptosis of PCs leads to specification of excess adjacent border cells via excessive Upd signalling. These results therefore show that Upd and JAK/STAT signalling induce apoptosis of supernumerary PCs to control the size of the PC organising centre and thereby produce appropriate levels of Upd. This is the first example linking this highly conserved signalling pathway with developmental apoptosis in Drosophila (Borensztejn, 2013).

A role for STAT in cell death and survival has been clearly documented in mammals, and depending on which of the seven mammalian Stat genes is considered and on the cellular context, both pro- and anti-apoptotic functions have been characterised. In the Drosophila developing wing, phosphorylated Stat92E has been shown to be necessary for protection against stress-induced apoptosis, but not for wing developmental apoptosis. This study provides evidence that Upd and the JAK/STAT pathway control developmental apoptosis during Drosophila oogenesis (Borensztejn, 2013).

This study demonstrated that the JAK/STAT pathway ligand, Upd, and all components of the JAK/STAT transduction cascade (the receptor Dome, JAK/Hop and Stat92E) are involved in promoting apoptosis of supernumerary PCs produced during early oogenesis. It is argued that The JAK/STAT pathway is essential for this event for several reasons. Indeed, in the strongest mutant context tested, follicle poles containing large TFC and PC clones homozygous for Stat92E amorphic alleles, almost all of these (95%) maintained more than two PCs through oogenesis. Also, RNAi-mediated reduction of upd, dome and hop blocked PC number reduction and deregulated several apoptosis markers, inhibiting Hid accumulation, Diap1 downregulation and caspase activation in supernumerary PCs. Altogether, these data, along with what has already been shown for JAK/STAT signalling in this system, fit the following model. Upd is secreted from PCs and diffuses in the local environment. Signal transduction via Dome/Hop/Stat92E occurs in nearby TFCs, interfollicular stalks and PCs themselves, leading to specific target gene transcription in these cells, as revealed by a number of pathway reporters. An as-yet-unidentified Stat92E-dependent pro-apoptotic relay signal (X) is produced in TFCs, interfollicular stalks and possibly PCs, which promotes supernumerary PC elimination via specific expression of hid in these cells, consequent downregulation of Diap1 and finally caspase activation. An additional cell-autonomous role for JAK/STAT signal transduction in supernumerary PC apoptosis of these cells is also consistent with, though not demonstrated by, the results (Borensztejn, 2013).

Relay signalling allows for spatial and temporal positioning of multiple signals in a tissue and thus exquisite control of differentiation and morphogenetic programmes. In the Drosophila developing eye, the role of Upd and the JAK/STAT pathway in instructing planar polarity has been shown to require an as-yet-uncharacterised secondary signal. In the ovary, the fact that JAK/STAT-mediated PC apoptosis depends on a relay signal may provide a mechanism by which PC apoptosis and earlier JAK/STAT-dependent stalk-cell specification can be separated temporally (Borensztejn, 2013).

Although neither the identity, nor the nature, of the relay signal are known, it is possible to propose that the signal is not likely to be contact-dependent, and could be diffusible at only a short range. Indeed, Stat92E homozygous mutant TFC clones in contact with PCs, as well as those positioned up to three cell diameters away from PCs, are both associated with prolonged survival of supernumerary PCs, whereas clones further than three cell diameters away from PCs are not. In addition, fully efficient apoptosis of supernumerary PCs may require participation of all surrounding TFCs, stalk cells and possibly PCs, for production of a threshold level of relay signal. In support of this, large stat mutant TFC clones are more frequently associated with prolonged survival of supernumerary PCs, and the effects of removing JAK/STAT signal transduction in several cell populations at the same time are additive. Interestingly, the characterisation of two other Drosophila models of developmental apoptosis, interommatidial cells of the eye and glial cells at the midline of the embryonic central nervous system, also indicates that the level and relative position of signals (EGFR and Notch pathways) is determinant in selection of specific cells to be eliminated by apoptosis (Borensztejn, 2013).

The results indicate that only the supernumerary PCs respond to the JAK/STAT-mediated pro-apoptotic relay signal, whereas two PCs per pole are always protected. Indeed, this study found that overexpression of Upd did not lead to apoptosis of the mature PC pairs and delayed rather than accelerated elimination of supernumerary PCs. Recently, it was reported that selection of the two surviving PCs requires high Notch activation in one of the two cells and an as-yet-unknown Notch-independent mechanism for the second cell. Intriguingly, expression of both Notch and Stat reporters is dynamic in PC clusters and PC survival and death fates are associated with respective activation of the Notch and JAK/STAT pathways. However, this study found that RNAi-mediated downregulation of upd did not affect either expression of Notch or that of two Notch activity reporters. Therefore, JAK/STAT does not promote supernumerary PC apoptosis by downregulating Notch activity in these cells. Identification of the relay signal and/or of Stat target genes should help further elucidate the mechanism underlying the induction of apoptosis in selected PCs (Borensztejn, 2013).

Interfollicular stalk formation during early oogenesis has been shown to depend on activation of the JAK/STAT pathway. The presence of more than two PCs during these stages may be important to produce the appropriate level of Upd ligand to induce specification of the correct number of stalk cells. Later, at stages 7-8 of oogenesis, correct specification of anterior follicle cell fates (border, stretch and centripetal cells) depends on a decreasing gradient of Upd signal emanating from two PCs positioned centrally in this field of cells. Attaining the correct number of PCs per follicle pole has been shown to be relevant to this process and border cells (BC) specification seems to be particularly sensitive to the number of PCs present. Previously work has shown apoptosis of supernumerary PCs is physiological necessary for PC organiser function, as blocking caspase activity in PCs such that more than two PCs are present from stage 7 leads to defects in PC/BC migration and stretch cell morphogenesis. This study now shows that the excess PCs produced by blocking apoptosis lead to increased levels of secreted Upd and induce specification of excess BCs compared with the control, and these exhibit inefficient migration. These results indicate that reduction of PC number to two is necessary to limit the amount of Upd signal such that the correct numbers of BCs are specified for efficient migration to occur. Taken together with the role shown for Upd and JAK/STAT signalling in promoting PC apoptosis, it is possible to propose a model whereby Upd itself controls the size of the Upd-producing organising centre composed of PCs by inducing apoptosis of supernumerary PCs. Interestingly, in the polarising region in the vertebrate limb bud, which secretes the morphogen Sonic Hedgehog (Shh), Shh-induced apoptosis counteracts Fgf4-stimulated proliferation to maintain the size of the polarising region and thus stabilise levels of Shh. It is likely that signal autocontrol via apoptosis of signal-producing cells will prove to be a more widespread mechanism as knowledge of apoptosis control during development advances (Borensztejn, 2013).

dLin52 is crucial for dE2F and dRBF mediated transcriptional regulation of pro-apoptotic gene hid

Drosophila lin52 (dlin52) is a member of Myb transcription regulator complex and it shows a dynamic pattern of expression in all Drosophila tissues. Myb complex functions to activate or repress transcription in a site-specific manner; however, the detailed mechanism is yet to be clearly understood. Members of the Drosophila melanogaster Myb-MuvB/dREAM complex have been known to regulate expression of a wide range of genes including those involved in regulating apoptosis. E2F and its corepressor RBF also belong to this complex and together they regulate expression of genes involved in cell cycle progression, apoptosis, differentiation, and development. This study examined whether the depletion of dlin52 in developing photoreceptor neurons results in enhanced apoptosis and disorganisation of the ommatidia. Strikingly, it was found that dLin52 is essential for transcriptional repression of the pro-apoptotic gene, hid; decrease in dlin52 levels led to dramatic induction of hid and apoptosis in eye-antennal discs. Reduction of Rpd3 (HDAC1), another member of the dREAM complex, also led to marginal upregulation of Hid. In addition, it was demonstrated that an optimum level of dLin52 is needed for dE2F1/2 activity on the hid promoter. dlin52 cooperates with dRBF and dE2F1/2 for recruitment of repressor complex on the hid promoter. Preliminary data indicates that Rpd3/HDAC1 also contributes to hid repression. Based on the findings, it is concluded that dLin52 functions as a co-factor and modulates activity of members of dMyb/dREAM complex at hid promoter, thus, regulating apoptosis by repressing this pro-apoptotic gene in the developing Drosophila eye (Bhaskar, 2014).

Regulated progression through the cell cycle is essential for ordered cell proliferation. Changes in the balance between cell cycle-driving proto-oncogene-dependent pathways and inhibiting signals from tumour suppressors are a common cause for cancer. One of the best characterised tumour suppressors is the retinoblastoma protein pRB, the first cloned tumour suppressor. This gene was first described as a susceptibility gene for retinoblastoma, an eye tumour in children; it is now known to be mutated in many cancers. Currently, more than 120 proteins have been reported to be associated with pRb, and a wide assortment of chromatin-modifying and binding complexes have been implicated in pRB mediated repression. The association of these complexes with pRb has broadened its range of functions. In humans, one of the complexes known as the DREAM complex is required to arrest expression of genes essential for cell cycle progression. A similar complex is also found in C.elegans (synMuvB complementation group or DRM complex) as well as in Drosophila (dREAM complex). The function of this complex seems to be conserved across taxa and has definitive control over normal cell development. In the past few years, the function and composition of this complex has been unravelled. The role of the dREAM complex has now been extended to development, differentiation, and apoptosis. Its composition has also been reported to vary according to the function and site of the action (Bhaskar, 2014).

The smallest member of dREAM/MMB complex, dLin52 is found in Drosophila. The dynamic pattern of expression of dLin52 in various tissues has been reported and a strong conservation was found of this protein across taxa (Bhaskar, 2012). Hence, this research was extended to explore the functional characterisation of dlin52. GMR-GAL4 and UAS-dlin52-RNAi were used to deplete dLin52 in the developing eye and examine its role in the developing photoreceptors (Bhaskar, 2014).

A recent study, Lewis (2012), has shown that dLin52 is needed for viability, adult eye development and embryogenesis via its maternal effect. The mutant phenotypes could be rescued by heterozygous deletion of mip120 or loss of function allele of mip130. It was concluded that Lin-52 and Myb proteins counteract against the repressive activities of the other members of the MMB/dREAM complex at specific genomic loci in a developmentally controlled manner (Bhaskar, 2014).

The findings of the current study showed that down regulation of dlin52 leads to rough eye phenotype, which is primarily caused by enhanced apoptosis. this observation is in accordance with a study of mammalian cells where depletion of LIN52 sensitised gastrointestinal stromal cells to imatinib induced apoptosis, suggesting a similar mechanism for regulation of apoptosis by LIN52 in higher organisms (Boichuk, 2013). A connection between E2Fs, regulation of hid, and apoptosis has also been found in Drosophila. While dE2F1 activates, dE2F2 has been found to represses hid in wing discs. However, it has been reported that the depletion of dE2F1 in S2 cells leads to activation of hid, showing for the first time the ability of dE2F1 to repress or negatively regulate transcription (Bhaskar, 2014).

The present study established that apoptosis due to dlin52 down regulation is cell autonomous. The components of Drosophila cell death regulatory pathway are conserved in higher organisms. This includes inhibitor of apoptosis proteins (IAPs) which bind to caspases and pro-apoptotic proteins. This study observed that over-expression of DIAPI rescued dlin52-RNAi phenotype. Further, only loss of function alleles of hid suppressed dlin52 rough eye phenotype but not rpr or grim alleles. Similarly, depletion of dLin52 also led to increase in hid transcript levels but not that of rpr or grim. The pattern of hid activation caused by dlin52-RNAi is in agreement with the earlier studies which reported the loss of dRBF and dE2F1; similar to those of previous studies, the cells immediately posterior to the MF were more sensitised to dLin52 downregulation in the eye-antennal discs. Therefore, these findings, supported by the previous observations with loss of RBF and E2F1, suggest that the reduction of dLin52 results in apoptosis is mediated via upregulation of Hid. Additionally, dlin52 also showed robust genetic interaction with Rbf, E2f, and E2f2alleles. It is evident that dLin52 works synergistically with dRBF and dE2F1/2 to mediate transcriptional repression of hid. Previous studies revealed that loss of dRBF and dE2F1/2 activates hid and increases apoptosis, while this tudy identified a third important component, dLin52, vital for hid regulation (Bhaskar, 2014).

Although Lin52 itself is not DNA binding, in humans, Lin52 has been shown to be essential for the formation of transcription repressor complex. Thiss tudy demonstrated that ablated dLin52 levels diminish both dE2F1 and dE2F2 recruitment on the hid promoter. As mutated dE2F binding site on the hid promoter fails to enhance dlin52-RNAi phenotype, it indicates that dE2F1 mediates repression of hid, aided by dLin52 and dRBF1. It was observed that recruitment of dE2F2 was also affected with loss of dLin52; it is likely that both E2F1 and E2F2 function to repress hid, and the stoichiometry of both E2F1 and E2F2 are important for their regulatory function. Taken together these data suggest dLin52 as the essential factor for dRBF, dE2F1/2 mediated repression of hid expression. It was also found that transcriptional regulation of hid is mediated via the dE2F binding site present in the 5' UTR of hid. Previous studies showed that dE2F1 not only binds to a unique site in promoter region of hid but also regulates its transcription in presence of dRBF. Therefore, it is concluded that optimum level of dLin52 is needed for recruitment of dE2F1/2 to the hid promoter; reducing dLin52 dramatically compromises with the dE2F1/2 binding to the cis-acting sequences on the hid promoter (Bhaskar, 2014).

Histone deacetylase1 (HDAC1) has been the most thoroughly studied HDAC at the biochemical and functional levels. HDACs are known to promote heterochromatin silencing by deacetylating H3. They target hundreds of genes in the genome and play a major role as a direct transcriptional repressor. HDAC1 controls segmentation genes through interaction with the Groucho corepressor and has also been linked to silencing by Polycomb repressors. HDAC3 and HDAC1 mutants together can suppress position-effect variegation (PEV), indicating the ability of both the HDACs in mediating heterochromatin silencing while working together. Recently, Zhu, found Drosophila HDAC3 regulating the wing imaginal disc size through suppression of apoptosis, while HDAC3 mutants shows ectopic Hid levels in wing discs. Mammalian HDAC1 mutant also shows increased apoptosis. Rpd3, the Drosophila homologue of HDAC1/2, has been purified along with dMyb/MMB/dREAM complex. Function of both HDAC3 and Rpd3 appears to overlap in regulating silencing and chromatin modification. In this study, it was observed that down-regulation of Rpd3 alone in eye-antennal discs led to a rough eye phenotype and induced marginal increase of Hid; however, ectopic expression was not limited to a few rows of cells posterior to the MF but traversed the differentiating photoreceptors, implying that Rpd3 mediated hid regulation is not limiting to cells specifically regulated by dLin52, dRbF and dE2F1/2 but expands to a much broader area in the developing photoreceptors. Similarly, lowering of both dlin52 and Rpd3 at the same time led to substantial increase in Hid levels with enhanced disorganisation of the ommatidia. Although Rpd3 in Drosophila lacks LXCXE motif, it might be recruited at the repressed sites by other members of the complex. Hence, it may be concluded that both dLin52 and Rpd3 (HDAC1) have a major role to play in negative regulation of hid expression. However, this is only a preliminary report suggesting that dLin52 and Rpd3 can work in parallel to regulate hid expression. Further experiments like biochemical purification of dREAM complex in the presence and absence of dLin52 would help in elaborating function of Rpd3 in regulating apoptosis (Bhaskar, 2014).

This study is in agreement with the earlier findings that Lin52 plays an important role in forming dREAM complex. Human Lin52 phosphorylation is needed for assembling of the dREAM complex. Earlier, it was reported that the Serine-28 residue in the human Lin52 is conserved in dLin52. This study proposes that limiting dLin52 might actually be equivalent to un-phosphorylated dLin52 which is non-functional, resulting in assembling defects of the dREAM complex. Therefore, it is hypothesised that dLin52 is a vital survival signal, needed for suppressing hid transcription and apoptosis and conclude that dLin52 is a crucial cofactor essential for assembling members of dREAM/MMB complex (dRBF, dE2F1/2). In addition, this study has also presented preliminary data indicating that Rpd3 functions together with dLin52, dRBF, and dE2F1/2 for mediating transcriptional repression of hid (Bhaskar, 2014).

A proposed model shows that dLin52 and Rpd3 (HDAC1) together with dRBF and dE2Fs are part of a repressor complex, repressing hid activation. When dLin52 becomes limiting, E2F1/2 cannot be recruited to the consensus E2F1/2 binding site on the hid promoter, resulting in the inability of the repressor complex to assemble. This leads to derepression and activation of hid, followed by increased apoptosis (Bhaskar, 2014).

Chemical inhibitors that block HDAC activity are of considerable interest in cancer research because of their ability to induce tumour cell killing by activating cell death pathway leading to apoptosis. Hence, it is proposed that Lin52 may also be selectively inhibited in inducing apoptosis in tumour cells (Bhaskar, 2014).

This study established the important role of dLin52 in repressing apoptosis. This leads to the belief that dLin52 is needed for maintenance of proper development, differentiation, normal physiology, and homeostasis in Drosophila (Bhaskar, 2014).

This study has found down regulation of dlin52 resulting in a rough eye phenotype. Based on these findings, it is suggested that the rough eye phenotype is due to increase in apoptosis. This study established through genetic analysis that downregulation of dlin52 increases hid expression. As dLin52 by itself is not DNA binding, it is predicted that it may be regulating hid along with the members of dREAM complex. Based on the observations, it can be concluded that dRBF, dE2F1, dE2F2, and Rpd3 work together with dLin52 for repressing hid activation, because the loss of function alleles of these genes led to strong enhancement of dlin52-RNAi eye phenotype. ChIP experiment demonstrated that reduced dlin52 levels also affect dE2F1 and dE2F2 binding to its consensus binding site on hid promoter (Bhaskar, 2014).

Furthermore, it can be concluded that dLin52 regulates apoptosis in eye-antennal discs by repressing hid transcription; loss of dLin52 induces hid expression. This suggests that dLin52 is a co-factor, needed for repression of hid along with dE2Fs and dRBF. Additionally, loss of dLin52 also affected binding of dE2F1 and dE2F2 to their consensus sequence, which means like DP, dLin52 can also affect DNA binding capacity of RBF and E2Fs. Taken together these data suggest that dLin52, dRBF, dE2F1, dE2F2, and dRpd3 cooperate to negatively regulate hid transcription and apoptosis. Further studies in understanding the role of Drosophila lin52 in apoptosis will shed light on the role of LIN52 in higher organisms (Bhaskar, 2014).

Yorkie drives supercompetition by non-autonomous induction of autophagy via bantam microRNA in Drosophila

Mutations in the tumor-suppressor Hippo pathway lead to activation of the transcriptional coactivator Yorkie (Yki), which enhances cell proliferation autonomously and causes cell death non-autonomously. The mechanism by which Yki causes cell death in nearby wild-type cells, a phenomenon called supercompetition, and its role in tumorigenesis remained unknown. This study shows that Yki-induced supercompetition is essential for tumorigenesis and is driven by non-autonomous induction of autophagy. Clones of cells mutant for a Hippo pathway component fat activate Yki and cause autonomous tumorigenesis and non-autonomous cell death in Drosophila eye-antennal discs. This study found that mutations in autophagy-related genes or NF-κB genes in surrounding wild-type cells block both fat^-induced tumorigenesis and supercompetition. Mechanistically, fat mutant cells upregulate Yki-target microRNA bantam, which elevates protein synthesis levels via activation of TOR signaling. This induces elevation of autophagy in neighboring wild-type cells, which leads to downregulation of IκB Cactus and thus causes NF-κB-mediated induction of the cell death gene hid. Crucially, upregulation of bantam is sufficient to make cells to be supercompetitors and downregulation of endogenous bantam is sufficient for cells to become losers of cell competition. These data indicate that cells with elevated Yki-bantam signaling cause tumorigenesis by non-autonomous induction of autophagy that kills neighboring wild-type cells (Nagata, 2022).

The data reveal that the Hippo pathway mutant fat clones cause supercompetition by inducing autophagy-mediated cell death in surrounding wild-type cells via NF-κB-mediated induction of hid. The autophagy induction in wild-type cells depends on Yki-bantam-mediated activation of TOR signaling in neighboring fat mutant cells. This mechanism is similar to what was observed in the elimination of ribosomal protein or Hel25E mutant loser clones when surrounded by wild-type cells. This is particularly interesting in two ways: first, it suggests that different types of cell competition, namely elimination of unfit cells by wild-type cells and elimination of wild-type cells by supercompetitors, are driven by the common mechanism, and second, it indicates that induction of autophagy in loser cells is non-autonomous, as even wild-type cells elevate autophagy when juxtaposed to supercompetitors. Although the mechanism by which autophagy is induced in loser cells nearby winner cells remains unknown, observations in this study in conjunction with the previous data on the elimination of ribosomal protein or Hel25E mutant clones suggest the possibility that relative difference in protein synthesis levels between cells plays a critical role in autophagy induction (Nagata, 2022).

The mechanism by which elevated autophagy induces hid expression via NF-κB still remains to be elucidated. Elevated autophagy results in downregulation of IκB protein Cactus. IκB is known to be degraded by the ubiquitin-proteasome system. On the other hand, elevated autophagy by starvation or rapamycin treatment was shown to cause degradation of IκB and thus activate NF-κB in mouse fibroblast. Together, the data suggest the possibility that IκB is degraded by selective autophagy in losers of cell competition (Nagata, 2022).

The observations of this studsy intriguingly show that non-autonomous cell death in wild-type cells promotes fat-induced tumorigenesis. This supports the idea that cancer cells expand their territories within the tissue by cell competition during malignant progression of tumors. While the mechanism by which wild-type cell death fuels neighboring tumorigenesis is an important open question, it may involve compensatory proliferation triggered by mitogenic factors secreted from dying cells. Intriguingly, it has been reported in Drosophila eye-antennal discs that clones of malignant tumors caused by Ras activation and cell polarity defects induce autophagy in surrounding wild-type cells, which in this case do not cause cell death but provide nutrient such as amino acids to neighboring tumors to promote their growth. Clones of cells overexpressing activated form of Yki were also shown to induce autophagy in neighboring cells, but in this case non-autonomous autophagy does not have a role in promoting tumorigenesis. Thus, non-autonomous autophagy may have multiple roles and mechanisms in regulating tissue homeostasis and tumorigenesis (Nagata, 2022).

Given that the Hippo pathway is conserved throughout evolution and that YAP-mediated cell competition occurs in mammalian systems as well, autophagy-mediated cell death may play an important role in mammalian cell competition. Notably, in a mouse liver cancer model, hyperactivation of YAP in peritumoral hepatocytes triggers regression of primary liver tumors and melanoma-derived liver metastases. Thus, further studies on the mechanism of Hippo-signaling-mediated supercompetition in Drosophila may provide a novel therapeutic strategy against human cancers (Nagata, 2022).

Tie-mediated signal from apoptotic cells protects stem cells in Drosophila melanogaster

Many types of normal and cancer stem cells are resistant to killing by genotoxins, but the mechanism for this resistance is poorly understood. This study shows that adult stem cells in Drosophila melanogaster germline and midgut are resistant to ionizing radiation (IR) or chemically induced apoptosis; the mechanism for this protection was dissected. Upon IR the receptor tyrosine kinase Tie/Tie-2 is activated, leading to the upregulation of microRNA bantam that represses FOXO-mediated transcription of pro-apoptotic Smac/DIABLO orthologue, Hid in germline stem cells. Knockdown of the IR-induced putative Tie ligand, PDGF- and VEGF-related factor 1 (Pvf1), a functional homologue of human Angiopoietin, in differentiating daughter cells renders germline stem cells sensitive to IR, suggesting that the dying daughters send a survival signal to protect their stem cells for future repopulation of the tissue. If conserved in cancer stem cells, this mechanism may provide therapeutic options for the eradication of cancer (Xing, 2015).

A form of programmed cell death, apoptosis, is characterized as controlled, caspase-induced degradation of cellular compartments to terminate the activity of the cell. Apoptosis plays a vital role in various processes including normal cell turnover, proper development and function of the immune system and embryonic development. Apoptosis is also induced by upstream signals, such as DNA double-strand breaks (DSB), to destruct severely damaged cells. DSB activate ATM checkpoint kinase and Chk2 kinase-dependent p53 phosphorylation and induction of repair genes. However, if DSB are irreparable, p53 activation will result in pro-apoptotic gene expression and cell death. However, aggressive cancers contain cells that show inability to undergo apoptosis in response to stimuli that trigger apoptosis in sensitive cells. This feature is responsible for the resistance to anticancer therapies, as well as the relapse of tumours after treatment, yet the molecular mechanism of this resistance is poorly understood (Xing, 2015).

As the cell type that constantly regenerates and gives rise to differentiated cell types in a tissue, stem cells share high similarities with cancer stem cells, including unlimited regenerative capacity and resistance to genotoxic agents. Adult stem cells in model organisms such as Drosophila melanogaster, have been utilized to study stem cell biology and for conducting drug screens, thanks to their intrinsic niche, which provides authentic in vivo microenvironment. This study shows that Drosophila adult stem cells are resistant to radiation/chemical-induced apoptosis, and the mechanism for this protection was dissected. A previously reported cell survival gene with a human homologue, pineapple eye (pie) , acts in both stem cells and in differentiating cells to repress the transcription factor FOXO. Elevated FOXO levels in pie mutants lead to apoptosis in differentiating cells, but not in stem cells, indicating the presence of an additional anti-apoptotic mechanism(s) in the latter. We show that this mechanism requires Tie, encoding a homologue of human receptor tyrosine kinase Tie-2, and its target, bantam, encoding a microRNA. The downstream effector of FOXO, Tie and ban, is show to be Hid, encoding a Smac/DIABLO orthologue. Knocking down the ligand Pvf1/PDGF/VEGF/Ang in differentiating daughter cells made stem cells more sensitive to radiation-induced apoptosis, suggesting that Pvf1 from the apoptotic differentiating daughter cells protects stem cells (Xing, 2015).

This study shows that an anti-apoptotic gene, pie, is required for stem cell self-renewal but not for resistance to apoptosis, indicating a compensatory anti-apoptotic mechanism in stem cells. The cell cycle marker profile of pie GSCs resembles that of InR deficient GSCs, leading to the finding that pie controls GSC, as well as ISC self-renewal/division through FOXO protein levels. Surprisingly, pie targets FOXO as well in differentiating cells, failing to explain why the loss of pie does not induce apoptosis in stem cells. However, while the upregulation of FOXO leads to the upregulation of its apoptotic target Hid in differentiating cells, in adult stem cells Hid is not upregulated. Hence additional regulatory pathway is in place to repress Hid and thereby apoptosis in stem cells. This study identified Tie-receptor as the key gatekeeper for the process in the GSCs. The signal (Pvf1) from the dying daughter cells activates Tie in GSCs to upregulate bantam microRNA that represses Hid, thereby protecting the stem cells. Bantam is known to repress apoptosis and activate the cell cycle. However, while protected from apoptosis in this manner, the stem cells do not activate the cell cycle but rather stay in protective quiescence through FOXO activity. When the challenge is passed, stem cells repopulate the tissue (Xing, 2015).

The mammalian pie homologue, G2E3 was reported to be an ubiquitin ligase with amino terminal catalytic PHD/RING domains. G2E3 is essential for early embryonic development (Brooks, 2008). Importantly, microarray data show significant enrichment of G2E3 expression levels in human embryonic stem (ES) cell lines. These observations suggest a critical role of G2E3 in embryonic development, potentially in maintaining the pluripotent capacity. Since FOXO is shown to be an important ESC regulator, it will be interesting to test whether defects in G2E3 result in changes in FOXO levels. Furthermore, future studies are required to test whether human ES cells also are protected from apoptosis due to external signals from dying neighbouring cells (Xing, 2015).

The cell cycle defects of pie mutant stem cells, such as abnormal cell cycle marker profile, can be a consequence of elevated FOXO levels, since FOXO is a transcription factor with wide array of target genes, many of which are involved with cell cycle progress, such as the cyclin-dependent kinase inhibitor p21/p27 (Dacapo in Drosophila). This may be critical when bantam function is considered in the stem cells. Bantam is known to function as anti-apoptotic and cell cycle inducing microRNA. While in GSC bantam is critical through its anti-apoptotic function as a Hid repressor, it has no capacity to induce GSC cell cycle after irradiation. In a challenging situation, such as irradiation, an additional protection mechanism for the tissue is to keep the stem cell in a quiescent state during challenge. bantam's pro-cell division activity may be dampened by FOXO's capacity to upregulate p21/Dacapo (Xing, 2015).

The FOXO family is involved in diverse cellular processes such as tumor suppression, stress response and metabolism. The FOXO group of human Forkhead proteins contains four members: FOXO1, FOXO3a, FOXO4, and FOXO6. Studies to elucidate their function in various stem cell types in vivo using knockout mice have shown some potential redundancy of FOXO proteins. Recent publications have demonstrated a requirement for some of the FOXO family members in mouse hematopoietic stem cell proliferation, mouse neural stem cells, leukaemia stem cells and human and mouse ES cells in vitro. However, FOXO is shown to be dispensable in the early embryonic development in mouse. Drosophila genome has only one FOXO, allowing a definitive study of FOXO's function in stem cells. This study now demonstrates that tight regulation of FOXO protein levels is essential for in vivo GSC and ISC self-renewal in Drosophila. While the loss of FOXO function generates supernumerary stem cells, inappropriately high level of FOXO results in stem cell loss. Under challenge, such as exposure to irradiation, stem cells depleted of FOXO fail to stay quiescent and become more sensitive to the damage, leading to the loss of GSC population. These data demonstrate the importance of the balanced FOXO expression level for stem cell fate (Xing, 2015).

Previous studies have shown that multiple adult stem cell types manage to avoid cell death in response to severe DNA damage. This work has studied the mechanisms that stem cells utilize to avoid apoptosis in absence of pie and revealed that apoptosis is protected through a receptor, Tie and its target miRNA bantam that can repress the pro-apoptotic gene Hid. The ligand for Tie is likely secreted from the dying neighbours since Tie is essential in GSC only after irradiation challenge, IR induces Tie's potential ligand Pvf1 expression in cystoblasts and knockdown of Pvf1 in cystoblasts eliminates stem cells' protection against apoptosis. Further studies will reveal whether the same protective pathway is utilized in other stem cells. Community phenomenon have been described previously around dying cells: compensatory proliferation, Phoenix rising, bystander effect and Mahakali. While Bystander effect describes dying cells inducing death in the neighbours, compensatory proliferation, Phoenix rising and Mahakali describe positive effects in cells neighbouring the dying cells. The present work shows that adult stem cell can survive but show no immediate induction of proliferation when neighboured by dying cells. However, since adult stem cells can repopulate the tissue when death signals have passed, it is proposed that in adult stem cells these phenomenon merge. First, the GSCs survive by bantam repressing the apoptotic inducer, Hid, and later repopulate the tissue by activating cell cycle. Recent findings have suggested that p53 might play an important role in re-entry to cell cycle in stem cells51. The results from the current studies shed light on the general understanding of stem cell behaviour in response to surrounding tissue to ensure the normal tissue homeostasis. It is also plausible that cancer stem cells hijack these normal capacities of stem cells (Xing, 2015).

Targets of Activity

To discover whether expression of apoptosis activators reaper, grim and hid triggers the accumulation of Death related ced-3/Nedd2-like protein (DREDD) mRNA, the three apoptosis activators were ectopically expressed in mesoderm, and the expression of DREDD mRNA examined. Expression of the apoptosis activators triggers excessive apoptosis in mesoderm. During stage 13 and beyond, DREDD mRNA is not widely expressed in the developing musculature in wild-type flies. However, when misexpression of each of the death activators is directed to these tissues, prominent levels of ectopic DREDD mRNA are detected. Expression of grim in the ectoderm also results in DREDD mRNA accumulation. DREDD mRNA accumulation has also been examined in embryos homozygous for crumbs (crb). In crb mutants, reaper is ectopically expressed in the disorganized epidermis. As anticipated, ectopic accumulation of DREDD mRNA is found scattered throughout the ectoderm in crb embryos, coincident with widespread patterns of rpr expression. Perhaps the most compelling evidence for a direct role for Dredd in apoptosis comes from an examination of accumulation of DREDD mRNA in embryos carrying a homozygous deletion of the entire reaper region (mutated for rpr, hid, and grim). No apoptosis occurs in these deletion mutants. The selective accumulation of DREDD mRNA fails to occur in these mutants. This is the first report of a molecular activity that is completely blocked by the absence of H99-associated signaling (Chen, 1998).

Inhibitor of apoptosis (IAP) proteins suppress apoptosis and inhibit caspases. Several IAPs also function as ubiquitin-protein ligases. Regulators of IAP auto-ubiquitination, and thus IAP levels, have yet to be identified. Head involution defective (Hid), Reaper (Rpr) and Grim downregulate Drosophila melanogaster IAP1 (DIAP) protein levels. Hid stimulates DIAP1 polyubiquitination and degradation. In contrast to Hid, Rpr and Grim can downregulate DIAP1 through mechanisms that do not require DIAP1 function as a ubiquitin-protein ligase. Observations with Grim suggest that one mechanism by which these proteins produce a relative decrease in DIAP1 levels is to promote a general suppression of protein translation. These observations define two mechanisms through which DIAP1 ubiquitination controls cell death: first, increased ubiquitination promotes degradation directly; second, a decrease in global protein synthesis results in a differential loss of short-lived proteins such as DIAP1. Because loss of DIAP1 is sufficient to promote caspase activation, these mechanisms should promote apoptosis (Yoo, 2002).

Inhibitors of apoptosis (IAPs) inhibit caspases, thereby preventing proteolysis of apoptotic substrates. IAPs occlude the active sites of caspases to which they are bound and can function as ubiquitin ligases. IAPs are also reported to ubiquitinate themselves and caspases. Several proteins induce apoptosis, at least in part, by binding and inhibiting IAPs. Among these are the Drosophila melanogaster proteins Reaper (Rpr), Grim, and HID, and the mammalian proteins Smac/Diablo and Omi/HtrA2, all of which share a conserved amino-terminal IAP-binding motif. Rpr not only inhibits IAP function, but also greatly decreases IAP abundance. This decrease in IAP levels results from a combination of increased IAP degradation and a previously unrecognized ability of Rpr to repress total protein translation. Rpr-stimulated IAP degradation required both IAP ubiquitin ligase activity and an unblocked Rpr N terminus. In contrast, Rpr lacking a free N terminus still inhibits protein translation. Since the abundance of short-lived proteins are severely affected after translational inhibition, the coordinated dampening of protein synthesis and the ubiquitin-mediated destruction of IAPs can effectively reduce IAP levels to lower the threshold for apoptosis (Holley, 2002).

Hid is regulated by the Egf receptor/RAS/MAPK pathway

Trophic mechanisms in which neighboring cells mutually control their survival by secreting extracellular factors play an important role in determining cell number. However, how trophic signaling suppresses cell death is still poorly understood. The survival of a subset of midline glia cells in Drosophila depends upon direct suppression of the proapoptotic protein Hid via the Egf receptor/RAS/MAPK pathway. The TGF alpha-like ligand Spitz is activated in the neurons, and glial cells compete for limited amounts of secreted Spitz to survive. In midline glia that fail to activate the Egfr pathway, Hid induces apoptosis by blocking a caspase inhibitor, Diap1. Therefore, a direct pathway linking a specific extracellular survival factor with a caspase-based death program has been established (Bergmann, 2002).

The reduction in midline glia (MG) cell number due to apoptosis, as well as the requirement of the RAS/MAPK pathway for MG survival, has been documented using various MG-specific enhancer trap lines and reporter fusion constructs. The MG are visualized using a reporter fusion construct for the slit gene (sli-lacZ) in which a 1 kb fragment of the slit promoter confers expression specifically to the MG. Using a ß-gal antibody to monitor the developmental profile of the MG, about ten cells per segment expressing sli-lacZ are detectable at midembryogenesis (stage 13). By the end of embryogenesis at stage 17, the number of sli-lacZ-positive cells is reduced to approximately three per segment. Since the sli-lacZ expression is specific for the MG, sli-lacZ-expressing cells are referred to as MG (Bergmann, 2002).

Prominent activation of MAPK has been identified in the MG cells, but its functional role has not been determined. The fate of the MG in mapk-deficient embryos has been analyzed. Compared to wild-type embryos, the initial generation of the MG appears to be normal. However, by stage 17 (the end of embryogenesis), none of the MG in mapk-deficient embryos survive. This finding suggests that MAPK is required for MG survival in wild-type embryos (Bergmann, 2002).

The genetic requirement of mapk for MG survival and of hid for MG apoptosis prompted the assumption that MAPK promotes survival of the MG by inhibition of HID activity. According to this model, the MG would be unprotected from HID-induced apoptosis in mapk-deficient embryos, and die. Consistent with this idea, HID protein is detectable in the MG of late stage wild-type embryos. To test this further, embryos that were mutant for both mapk and hid were examined. In early stage mapk;hid double mutant embryos, the initial generation of the MG appears to be normal. However, in contrast to mapk mutants alone, the MG is rescued in mapk;hid double mutant embryos although the survival function of MAPK is missing in these embryos. Dissection revealed that the MG are located directly at the cuticle of the embryos. Because segmental fusions occur in these embryos, some of the MG cluster in groups of up to 20 cells. In individual segments, five to six MG are visible. This number is larger compared to wild-type (three MG per segment), and is remarkably similar to the number of surviving MG in hid mutant embryos alone, indicating that MAPK promotes MG survival largely through inhibition of HID (Bergmann, 2002).

The mutant analysis revealed that MAPK is required to suppress the activity of HID for MG survival. If hid is mutant in mapk-deficient embryos (i.e., in mapk; hid double mutants), MAPK is no longer needed for the survival of the MG. Thus, MAPK-mediated survival of the MG functions through inhibition of HID (Bergmann, 2002).

In hid mutant embryos there is a 2-fold increase of the MG compared to wild-type. Approximately six MG per segment survive in hid embryos compared to the 2.8 MG per segment in wild-type, indicating that hid is genetically required for MG cell death. MAPK activity is required for MG survival. Does the level of MAPK activity determine the final number of surviving MG cells? Mutational activation of MAPK, using a dominant allele of MAPK termed Sevenmaker or mapk^Sem, promotes survival of extra MG. About 6.0 MG per segment survive in stage 17 mapk^Sem embryos, providing additional evidence that MAPK is required for MG survival. Remarkably, the number of surviving MG in mapk^Sem embryos is very similar to the number of surviving MG in hid mutant embryos. In both cases, approximately six MG survive per segment. Therefore, it was determined whether the six surviving MG in mapk^Sem embryos correspond to the same MG that survive in hid mutant embryos by double mutant analysis. Stage 17 mapk^Sem; hid double mutant embryos contain on average 6.6 MG, or slightly more than the single mutants alone. This result strongly suggests that hid expression and MAPK activation occur in largely the same set of MG, that is, in a group of about six MG. If MAPK activation and hid expression would occur in different MG independently of each other, then the mapk^Sem;hid double mutant would be expected to be the composite of the individual mutants and a total of about ten to twelve MG would survive in the double mutant, similar to what has been observed in H99 mutant embryos. It is inferred from the double mutant analysis that the survival of approximately six MG is regulated by MAPK-dependent inhibition of HID. As long as MAPK is activated, these MG survive (as seen in the activated mapk^Sem mutant). However, MG in this group that does not maintain activated MAPK are eliminated by HID-induced apoptosis. Thus, the coordinated expression of HID and activation of MAPK regulate the final MG cell number (Bergmann, 2002).

MAPK suppresses hid activity in two ways: via downregulation of its transcription and via phosphorylation of HID protein. However, hid mRNA and protein are readily detectable in the surviving MG of wild-type embryos. Therefore, transcriptional downregulation of hid does not account for MG survival. This prompted a test to see whether inhibitory phosphorylation of HID by MAPK might be critical for MG survival. For this purpose, advantage was taken of an observation that overexpression of HID in the MG using the MG-specific sli-GAL4 driver and UAS-hid transgenes is not sufficient to induce MG apoptosis. Even two copies of the UAS-hid transgenes are not able to ablate the MG. This is contrary to findings in other tissues in which expression of hid induces cell death very well. However, since MAPK is activated in the MG and required for MG survival, it was hypothesized that even overexpressed HID might be inactivated via MAPK phosphorylation (Bergmann, 2002).

To examine this further, UAS-hid transgenes were generated that alter the five phosphoacceptor residues of the MAPK phosphorylation sites to nonphosphorylatable Ala residues (UAS-hid^Ala5). The UAS-hid^Ala5 transgenes driven by sli-GAL4 induce apoptosis in the MG very efficiently. One copy of a UAS-hid^Ala5 transgene is sufficient for the ablation of the MG. Occasionally, some embryos are recovered in which the ablation of the MG is incomplete. However, nerve cord preparations reveal that in these embryos, only a small fraction of the MG survives compared to wild-type. Some segments completely lack MG cells, while others just contain one remaining MG. The MG is required for separation of the commissural axon tracts of the CNS. Consistently, expression of the UAS-hid^Ala5 transgenes and consequently ablation of the MG causes a fused commissure phenotype. In summary, this analysis demonstrates that MG survival requires suppression of HID activity by MAPK. The MAPK phosphorylation sites in HID are critical for this response, providing an important mechanism for the regulation of MG number (Bergmann, 2002).

Activation of MAPK usually requires activation of RAS, which in turn is activated by receptor tyrosine kinase (RTK) signaling: this demonstrates that MG survival depends on RAS, which is consistent with the model. Within the embryonic CNS, the Drosophila homolog of the Epidermal growth factor receptor (Egfr) is specifically expressed and required for MG differentiation. The requirement of Egfr signaling for MG survival was examined (Bergmann, 2002).

Due to severe developmental defects in egfr mutants, only a few MG start forming at stage 11, and none of them survive. Thus, it is difficult to directly study the requirement of the Egfr for MG survival. To overcome this problem, a dominant-negative mutant of the Egfr (UAS-Egfr^DN) was expressed in the MG using the sli-GAL4 driver in otherwise wild-type embryos. In this way, Egfr activity is specifically diminished in the MG after their generation. As expected, the MG form normally in these embryos. However, most of the MG die during subsequent developmental stages and only a few survive to the end of embryogenesis, indicating a direct requirement of the Egfr for MG survival. To determine whether the MG death in this experimental condition is due to failure to inhibit HID, Egfr^DN was expressed in the MG of hid mutants. In this genetic background, on average 6.1 MG cells survive, demonstrating that MG survival requires functional Egfr signaling to suppress HID activity (Bergmann, 2002).

The spi gene is required for MG survival and encodes a candidate trophic factor for MG survival. To prove that spi function is required to suppress hid activity, the fate of the MG in spi;hid double mutant embryos was determined. The MG survive in spi embryos if hid is removed as well, and it is concluded that the survival function of spi is mediated through suppression of hid-induced apoptosis (Bergmann, 2002).

The question arises as to which cells process mSPI and provide a source of sSPI for MG survival. Since spi is ubiquitously expressed, it is difficult to determine histochemically where sSPI, the active ligand, is generated. Therefore, a genetic approach was used; whether the loss of MG in spi mutant embryos can be rescued by expression of the membrane bound inactive precursor (UAS-mSPI) either in the MG (using the sim-GAL4 driver) or in neuronal axons (using the elav-GAL4 driver) was examined. It was reasoned that the MG would be rescued in spi mutant embryos only if mSPI is presented in the location where it is normally processed for MG survival in wild-type embryos. Presentation of mSPI by the MG itself does not result in rescue of the MG in spi mutant embryos, ruling out an autocrine mechanism. In contrast, expression of mSPI in neuronal axons appears to be sufficient for MG survival in spi embryos. This argues in favor of a paracrine mechanism. In control experiments, sSPI was examined using these two Gal4 drivers in wild-type embryos. With both GAL4 drivers an increase in the number of MG cells is detected, indicating that they are expressed at the right time and that the MG does not fail to secrete Spi once it has been processed (Bergmann, 2002).

A key regulator of Spi activation is rhomboid, a gene encoding a cell surface, seven-pass transmembrane protein that appears to function as a serine protease directly cleaving mSPI. rhomboid has been implicated in suppression of MG apoptosis. Ectopic expression of Rho in neurons (elav-Gal4/UAS-Rhomboid) promotes an excess of MG, suggesting that neurons have the capacity to process endogenous mSPI. Another essential protein for Spi processing is Star. Star mutants display an MG phenotype similar to spi. STAR regulates intracellular trafficking of mSPI. Expression of Star from the neurons but not from the MG rescues the Star phenotype in the MG. Thus, this analysis clearly demonstrates that the sSPI signal for MG survival is generated and secreted by neurons (Bergmann, 2002).

The surviving MG in late stage embryos are in close contact to commissural axons. In embryos lacking the commissureless (comm) gene, the commissural axons are absent. In comm embryos the MG die prematurely, and some survivors become misplaced laterally along the longitudinal axon tracts. The location of the MG along the longitudinal axons in comm mutant embryos as well as their close contact to commissural axons in wild-type embryos has prompted the suggestion that axon contact is required for MG survival. Axon contact appears to permit the MG to respond to trophic signaling, which is necessary for its survival. Consistent with this notion trophic signaling provided by sSPI/Egfr is present only in MG associated with longitudinal axons in comm;hid double mutants. Thus, it was asked whether axon contact-mediated Egfr signaling in the MG is required to activate MAPK, which in turn suppresses the cell death-inducing ability of HID (Bergmann, 2002).

To address this question, the fate of the MG was examined in comm mutant embryos which are at the same time mutant for hid (comm;hid double mutants) or carry the dominant active mapk allele, mapk^Sem (mapk^Sem;comm double mutants). Strikingly, a substantial number of the MG survive even in the absence of axonal contact if hid function is removed or if MAPK is activated. This strongly suggests that axon contact is necessary to suppress HID via MAPK. Only MG in proximity to neurons undergo Egfr signaling. The additional MG that survive along the midline in comm;hid mutants do not express spry, that is, do not receive an Egfr signal, and survive only because hid is absent in this experimental condition (Bergmann, 2002).

It is noted that active MAPK is capable of rescuing a total of six MG based on analysis of mapk^Sem embryos. Presumably, this MAPK activation in mapk^Sem embryos is inherited from the differentiation period of the MG. However, only three MG survive by stage 17 in wild-type embryos. It is proposed that of the group of six MG that require MAPK for survival, only the three surviving cells make adequate axon contacts necessary to receive sufficient quantities of the survival factor sSPI. According to this model, the remaining three MG die because they lose the competition for axon contact and do not receive levels of sSPI that are high enough to inactivate HID via phosphorylation by MAPK. If additional sSPI is provided in the midline, additional MG can be rescued. The limited availability of axon-derived sSPI would serve to match the number of MG to the length of commissural axons requiring ensheathment. Thus, the regulation of MG number and survival represents a genetically defined example of the classical trophic theory of cell survival (Bergmann, 2002).

The regulation of MG apoptosis in Drosophila bears striking overall similarity to the regulation of glial cell death in the rat optic nerve. There is an early dependence of the oligodendroglia in the rat optic nerve on growth factors for differentiation followed by a dependence on axon contact for survival. However, it is not clear how the oligodendroglia in the rat optic nerve survive upon axon contact. Since mammalian homologs for many of the components in the apoptotic pathway both upstream and downstream of Drosophila HID are known, it will be interesting to analyze whether similar molecules regulate apoptosis and cell number in the mammalian nervous system. Therefore, molecular genetic studies in Drosophila promise considerable insight for advancing an understanding of the basic control mechanisms involved in the regulation of apoptosis in the context of a developing organism in vivo (Bergmann, 2002).

Drosophila E2F1 has context-specific pro- and antiapoptotic properties during development; E2F1 regulates Hid

E2F transcription factors are generally believed to be positive regulators of apoptosis. This study shows that dE2F1 and dDP are important for the normal pattern of DNA damage-induced apoptosis in Drosophila wing discs. Unexpectedly, the role played by E2F varies depending on the position of the cells within the disc. In irradiated wild-type discs, intervein cells show a high level of DNA damage-induced apoptosis, while cells within the D/V boundary are protected. In irradiated discs lacking E2F regulation, intervein cells are largely protected, but apoptotic cells are found at the D/V boundary. The protective effect of E2F at the D/V boundary is due to a spatially restricted role in the repression of hid. These loss-of-function experiments demonstrate that E2F cannot be classified simply as a pro- or anti-apoptotic factor. Instead, the overall role of E2F in the damage response varies greatly and depends on the cellular context (Moon, 2005).

One of the major difficulties in studying the biological functions of E2F is that E2F complexes affect the expression of a large number of genes and can act in a variety of different ways. It is difficult to assess the overall role of E2F regulation in a given process by studying an individual E2F gene, or a single E2F target. The rate-limiting targets for E2F function most likely vary from context to context, and they may not always be the usual suspects. In the D/V boundary of the developing wing disc, in which E2F/DP complexes protect from DNA damage-induced apoptosis, E2F/DP proteins are needed specifically to limit the expression of hid. Remarkably, the loss of E2F/DP leads to an upregulation of hid in this one part of the disc. This change occurs prior to irradiation, and it alters the cellular sensitivity to DNA damage. No apoptosis was found in unirradiated dDP mutant wing discs, implying that the change in hid expression in the dDP mutant wing disc is not, by itself, sufficient to induce apoptosis. The elevated hid expression is clearly important, because reducing the gene dosage of hid almost completely eliminated DNA damage-induced apoptosis in dDP mutant discs, but not in wild-type discs (Moon, 2005).

What is the connection between dE2F1 and hid? Since dE2F1 binds to sequences upstream of the hid transcription start site, the transcription of hid is most likely reduced by the direct action of E2F complexes. Previous studies in mammalian cells have shown that E2F1 can directly repress transcription of some E2F1-specific targets, although the mechanisms underlying these effects are not well understood. The dE2F1 binding site upstream of hid has two interesting features that may be significant. (1) Unlike most dE2F-regulated promoters that have been examined to date, this binding site is bound specifically by dE2F1, but not by dE2F2. This specificity fits with the genetic evidence that de2f1, rather than de2f2, is important for protection from DNA damage-induced apoptosis, and it may explain why hid is not generally repressed by dE2F2 complexes. (2) Another curious feature is that the dE2F1 binding site upstream of hid is surprisingly distal from the transcription start site. In most E2F-induced promoters, E2F binding sites are typically within 500 bp of the transcriptional start site. The position of the E2F1 binding site in the hid promoter, at −1.4 kb, may be part of the reason why hid differs from other dE2F1 targets and is not activated in a cell cycle-dependent manner. It is noted that the pattern of hid expression that sensitizes cells to apoptosis in dDP mutants occurs in the absence of E2F regulation; therefore, dE2F1 does not directly contribute to the pattern itself, but it presumably serves to interfere with another transcription factor (Factor X) that is specifically active within this region. As the hid promoter is largely uncharacterized, the possibility that dE2F1 may act through additional sites or that it may also repress hid expression via an indirect mechanism cannot be excluded. In order to test this model, it will be necessary to identify the factors that control hid expression in vivo (Moon, 2005).

A simple model is presented for the context-specific effects of dE2F1. In the intervein region, dE2F1 increases the expression of proapoptotic genes. In doing so, dE2F1 helps set a level of sensitivity for DNA damage-induced apoptosis, and this threshold is reduced when dE2F1 or dDP are removed. At the D/V boundary, dE2F1/dDP complexes are also needed, most likely in conjunction with RBF, to limit the expression of hid. When E2F regulation is removed, the increase in hid expression outweighs the changes in expression of other E2F targets, making cells more sensitive to apoptosis (Moon, 2005).

If E2F's contribution to the DNA damage response varies in mammalian cells as much as it does in Drosophila, then this would have implications for the use of general E2F inhibitors that are currently under development. These results suggest that a global inhibitor of E2F activity, or even a specific inhibitor of activator E2Fs, may have the unintended consequence of making some normal cell types very sensitive to DNA damage-induced apoptosis (Moon, 2005).

Post-transcriptional regulation of Hid by Bantam

Growth of tissues and organs during animal development involves careful coordination of the rates of cell proliferation and cell death. Cell proliferation depends on signals to stimulate cell growth and cell division. In addition, cells compete for intercellular survival signals which are required to prevent them from undergoing apoptosis in response to growth stimuli. How these cellular processes are coordinated with pattern formation during animal development is a challenging question in developmental biology. The bantam gene of Drosophila has been found to encode a 21 nucleotide microRNA (miRNA) that promotes tissue growth. bantam expression is temporally and spatially regulated in response to patterning cues. bantam microRNA simultaneously stimulates cell proliferation and prevents apoptosis. The pro-apoptotic gene hid has been identified as a target for regulation by bantam miRNA, providing an explanation for bantam's anti-apoptotic activity (Brennecke 2003).

The three proapoptotic genes hid, reaper, and grim downregulate levels of the IAP proteins in Drosophila, thereby preventing caspase activation. Unlike reaper and grim, whose activity appears to be regulated primarily at the transcriptional level, hid mRNA is also detected in cells that do not undergo apoptosis. Evidence has been presented for transcriptional regulation of hid and for posttranslational regulation of Hid activity by the MAPK signaling pathway. By showing that bantam blocks the activity of Hid(Ala5), which is insensitive to MAPK regulation, an indirect effect of bantam mediated by regulation of the MAPK pathway is excluded. The hid 3'UTR confers bantam-mediated regulation on a heterologous reporter. These findings provide evidence that hid is subject to translational regulation in vivo by the bantam miRNA (Brennecke 2003).

hid is known to play an important role in regulating apoptosis in eye development. Removing one copy of the endogenous bantam gene enhances the severity of the Hid-induced apoptosis phenotype in the eye, whereas the severity of the reaper-induced apoptosis phenotype is affected much less strongly. Similarly, overexpression of bantam suppresses both the GMR-hid and GMR-reaper phenotypes, but has a stronger effect on hid. The severity of the GMR-reaper phenotype is sensitive to the levels of hid activity. By overexpressing bantam, Hid levels are reduced, providing an explanation for the observed suppression of the GMR-reaper phenotype. Similarly, by removing one copy of bantam an increase in endogenous hid activity in the eye would be expected. By altering the level of Hid, bantam can indirectly alter the threshold for reaper-induced apoptosis. This provides an explanation for the slight increase in severity of the GMR-reaper phenotype observed. There are no bantam target sites in the reaper gene, suggesting that bantam's effect on the GMR-reaper phenotype must be indirect. Finally, no increase in apoptosis was observed in bantam mutant clones in the wing disc. Endogenous hid has not been implicated in developmental control of cell death in the wing (Brennecke 2003).

Combinatorial use of translational co-factors for cell type-specific regulation during neuronal morphogenesis in Drosophila: Nos and Pum regulate the expression of cut and head involution defective

The translational regulators Nanos (Nos) and Pumilio (Pum) work together to regulate the morphogenesis of dendritic arborization (da) neurons of the Drosophila larval peripheral nervous system. In contrast, Nos and Pum function in opposition to one another in the neuromuscular junction to regulate the morphogenesis and the electrophysiological properties of synaptic boutons. Neither the cellular functions of Nos and Pum nor their regulatory targets in neuronal morphogenesis are known. This study shows that Nos and Pum are required to maintain the dendritic complexity of da neurons during larval growth by promoting the outgrowth of new dendritic branches and the stabilization of existing dendritic branches, in part by regulating the expression of cut and head involution defective. Through an RNA interference screen a role was uncovered for the translational co-factor Brain Tumor (Brat) in dendrite morphogenesis of da neurons, and it was demonstrated that Nos, Pum, and Brat interact genetically to regulate dendrite morphogenesis. In the neuromuscular junction, Brat function is most likely specific for Pum in the presynaptic regulation of bouton morphogenesis. Thess results reveal how the combinatorial use of co-regulators like Nos, Pum and Brat can diversify their roles in post-transcriptional regulation of gene expression for neuronal morphogenesis (Olesnicky, 2012).

Post-transcriptional mechanisms of gene regulation such as translational control play a fundamental role in the development and function of the nervous system. Genetic studies have identified roles for the translational repressors Nos and Pum in sensory neuron and NMJ morphogenesis, NMJ function, and motor neuron excitability, and Pum has been implicated in long-term memory. Understanding the selectivity of these regulators for different mRNA targets is essential to identify the cellular processes they regulate for neuronal morphogenesis and neural function. This study shows that different combinations of Nos, Pum, and the co-factor Brat confer cell type-specific regulation during morphogenesis of Drosophila da sensory neurons and the NMJ (Olesnicky, 2012).

In Drosophila class IV da neurons, dendritic arbors grow rapidly during the first larval instar to establish nonredundant territories that cover the larval body wall. During the second and third larval instars, da neuron dendrites add and lengthen higher order branches to maintain body wall coverage as the larva undergoes dramatic growth. Results from live imaging analysis place the requirement for Nos and Pum during the third larval instar, indicating that Nos and Pum are not involved in the establishment of dendritic territories but rather in maintaining the density of terminal branches during late larval growth by promoting branch extension and preventing branch retraction. The possibility cannot be ruled that branch stabilization depends on Nos and Pum activity earlier during larval development. Evidence is provided that this maintenance function of Nos and Pum depends on their regulation of the proapoptotic protein Hid. Nos has previously been proposed to repress hid mRNA translation in developing germ cells to suppress apoptosis, although requirements for Pum and Brat were not tested. Together, these data showing that Hid is elevated in nos and pum mutant da neurons and that both the upregulation of Hid and the loss of terminal branches in nos mutants are suppressed by reduction of hid gene dosage suggest that repression of hid mRNA translation by Nos and Pum is also crucial for dendrite morphogenesis. Biochemical analysis will be required to test this model directly (Olesnicky, 2012).

In cultured Drosophila cells, Hid localizes to mitochondria and this localization is required for full caspase activation. By contrast, Hid protein is detected in the nucleus in nos and pum mutants. A similar nuclear accumulation has been proposed to sequester Hid in larval malphigian tubules and prevent apoptosis of this tissue during metamorphosis (Shukla, 2011). The nuclear accumulation of Hid may indeed explain why upregulation of Hid in nos and pum da mutants does not cause cell death. Nuclear Hid sequestration in nos and pum mutant neurons is also consistent with the apparent absence of activated caspase. How Hid causes dendrite loss in nos and pum mutant neurons remains to be determined but could involve activation of a pathway similar to injury induced dendrite degeneration, which resembles pruning but is caspase-independent (Olesnicky, 2012).

Nos and Pum were initially identified because of their role in translational repression of hb mRNA in the posterior region of the early embryo. There, the two proteins form an obligate repression complex, with Pum conferring the RNA-binding specificity and Nos, which is synthesized only at the posterior pole of the embryo, providing the spatial specificity. More recent studies have shown that Nos and Pum are not obligate partners, however. In the ovary, Pum functions together with Nos in germline stem cells to promote their self-renewal, while Pum acts independently of Nos in progeny cystoblasts to promote their differentiation (Harris, 2011). In the NMJ, Pum and Nos work in opposition to one another to regulate both morphological and electrophysiological characteristics of synaptic boutons. While Hid levels are similarly elevated in nos and pum mutant da neurons, the differential effects on cut expression observed in the two mutants suggest that in addition to working together, Nos and Pum participate in separate complexes that target different mRNAs even within the same cell type. Presumably, additional factors that associate selectively with Nos or Pum drive the formation of distinct complexes with different binding specificities. Pum represses eIF4E translation in the post-synaptic NMJ independently of Nos, suggesting that some of Pum's effects in da neurons could be through more global effects on translation (Olesnicky, 2012).

A third cofactor, Brat, is required for Nos/Pum-dependent repression of hb mRNA in the early embryo and paralytic mRNA in motorneurons. However, Brat is not required for Nos/Pum-mediated repression of cyclin B mRNA in primordial germ cells or for Nos/Pum function in germline stem-cell maintenance. Structural and molecular analyses have shown that Brat is recruited to the Nos/Pum/NRE ternary complexes through an interaction between its conserved NHL (NCL-1, HT2A, and LIN-41) domain and Pum. The Brat NHL domain also mediates interaction of Brat with the eIF4E-binding protein d4EHP and mutations in Brat that abrogate this interaction partially disrupt translational repression of hb, suggesting a mechanism by which the Pum/Nos/Brat/NRE complex could repress cap-dependent initiation. The results indicate that Brat also collaborates with Nos and Pum to regulate dendrite morphogenesis by a mechanism involving d4EHP interaction and that this requirement is cell type-specific. While genetic analysis suggests that Brat is required for Nos/Pum-mediated regulation of dendrite complexity and Hid expression in class IV da neurons, it is dispensible for Nos and Pum functions in class III da neurons. A similar cell type-specific requirement for Brat function in Nos/Pum-mediated repression within the CNS has been proposed based on the ability of brat mutants to counteract repression of paralytic mRNA due to Pum overexpression. Since Brat is expressed throughout the dorsal cluster of larval sensory neurons and CNS, it is unclear whether the recruitment of Brat to the complex occurs only in certain cell types or whether its function in the complex is target dependent. In contrast to nos and pum mutants, however, brat mutants have no effect on cut expression, suggesting that Brat's role in translational regulation is in fact limited to a subset of Nos/Pum-dependent processes (Olesnicky, 2012).

The findings that Brat functions presynaptically in bouton formation and that brat and pum mutant NMJs exhibit similar defects in bouton formation suggest that Brat is selectively recruited by Pum, but not by Nos, to regulate distinct target mRNAs in bouton development. Similarly, Brat functions selectively with Pum in ovarian cystoblasts to promote differentiation, suggesting that a Pum/Nos/NRE ternary complex is not essential for recruitment of Brat. Pum and many of its homologs in other organisms, members of the large Puf (Pum/FBF) protein family, typically recognize sequences that contain a core UGUA motif, although features beyond the core element also influence target mRNA recognition. Pum has been shown to also recognize a UGUG motif that is found in binding sites for the C. elegans Puf protein FBF (Menon, 2009). Thus, it is possible that the interaction of Pum with different binding sites dictates the assembly of the particular repression complex. Interactors like Brat might add an additional layer of regulation by altering the specificity or affinity of Pum for particular targets, thereby generating diverse cellular and morphological outputs within a particular cell type (Olesnicky, 2012).

Antisense-mediated depletion reveals essential and specific functions of microRNAs in Drosophila development: Differential posttranscriptional repression of the proapoptotic factors hid, grim, reaper, and sickle

MicroRNAs are small noncoding RNAs that control gene function posttranscriptionally through mRNA degradation or translational inhibition. Much has been learned about the processing and mechanism of action of microRNAs, but little is known about their biological function. Injection of 2′O-methyl antisense oligoribonucleotides (2'OM-ORNs) into early Drosophila embryos leads to specific and efficient depletion of microRNAs and thus permits systematic loss-of-function analysis in vivo. Twenty-five of the forty-six embryonically expressed microRNAs show readily discernible defects; pleiotropy is moderate and family members display similar yet distinct phenotypes. Processes under microRNA regulation include cellularization and patterning in the blastoderm, morphogenesis, and cell survival. The largest microRNA family in Drosophila (miR-2/6/11/13/308) is required for suppressing embryonic apoptosis; this is achieved by differential posttranscriptional repression of the proapoptotic factors hid, grim, reaper, and sickle. These findings demonstrate that microRNAs act as specific and essential regulators in a wide range of developmental processes (Leaman, 2005).

miR-2/13 and miR-6 depletion results in catastrophic apoptosis: Embryos injected with miR-2/13 and miR-6 antisense 2′OM-ORNs fail to differentiate normal internal and external structures. At the end of embryogenesis, the embryos fall apart on touch, and no cuticle is recovered. To determine the onset of these problems, blastoderm embryos were examined, and it was found that cellularization and early pattern formation along the anteroposterior axis occur normally for both miRNAs, indicating that early fating and morphogenesis are intact. Interestingly, in miR-6, but not miR-2/13 depleted embryos, pole cell formation at the posterior end is disrupted (Leaman, 2005).

One possible cause of the catastrophic defects observed in miR-2/13 and miR-6 depleted embryos is excessive and widespread apoptosis. In both miR-2/13 and miR-6 antisense injected embryos, the number of apoptotic cells is greatly increased compared to wild-type by stage 13. Notably, the overall morphology of miR-6 depleted embryos is much more affected than that of miR-2/13 depleted embryos. miR-6 depleted embryos are generally smaller in size and have fewer and abnormally large (para-) segments, suggesting greater excess or earlier onset of apoptosis (Leaman, 2005).

To determine the specificity of the effects of miR-6 and miR-2/13 antisense injections, genomic rescue experiments were carried out. Embryos ubiquitously overexpressing mir-6 or mir-2 (Actin-Gal4;UAS-mir6-3/2b-2) show normal cell-death patterns. When injected with miR-6 or miR-2/13 antisense, they show significant rescue of miR-6 antisense by mir-6, with respect to both cell death and morphology, and of miR-2/13 antisense by mir-2. Interestingly, crossrescue of miR-6 antisense by mir-2 overexpression and of miR-2/13 antisense by mir-6 is weak (Leaman, 2005).

The miRNA sequence family miR-6 and miR-2/13 belong to has two additional members, miR-11 and miR-308. Depletion of miR-11 results in a moderate and of miR-308 in a mild increase in apoptosis in midembryogenesis. Thus, for all members of the miR-2 family, antisense-induced depletion results in excess embryonic cell death, but with marked differences in phenotypic strength. This differential could be due to differences in expression level or to sequence divergence and thus differential interaction with target mRNAs (Leaman, 2005).

The miR-2 family regulates cell survival by translational repression of proapoptotic factors: In Drosophila, three pathways are known to control caspase activity. The main control is thought to come from the proapoptotic factors Hid, Grim, and Reaper (Rpr), which are transcriptionally activated in response to a range of natural and toxic conditions; they promote caspase activation through inhibition of the caspase inhibitor Diap1. The three factors appear to act independently, with each being sufficient to drive apoptosis. When miR-2/13 and miR-6 antisense 2′OM-ORNs are injected into embryos deficient for the hid, grim, and rpr genes (H99 deficiency), they are unable to trigger apoptosis, indicating that these miRNAs act through hid, grim, and/or rpr (Leaman, 2005).

To determine whether the regulation of the three proapoptotic factors occurs at the transcriptional or at the posttranscriptional level, their RNA expression was examined in miR-2/13 and miR-6 depleted embryos using in situ hybridization and quantitative PCR. No significant increase was found in the expression level or broadening of the pattern compared to control embryos for any of three transcripts, either at embryonic stage 13 or 1 hr earlier at embryonic stage 12. By contrast, the protein expression of Hid is dramatically increased in miR-6 depleted embryos and modestly in miR-2/13 depleted embryos. These results strongly argue against a transcriptional and in favor of a posttranscriptional regulation of the proapoptotic factors by miR-2/13 and miR-6 (Leaman, 2005).

To test this directly, two existing translation control assays were adapted to the embryonic paradigm. In the first assay, full-length 3′UTRs are fused to a ubiquitously transcribed sensor (tub-GFP); transgenic embryos are injected with sense or antisense 2′OM-ORNs, and GFP fluorescence is measured. The 3′UTRs of hid, grim, rpr, and sickle (skl, a structurally related but less potent proapoptotic factor display marked differences in sensor expression, with rpr showing no expression, hid and skl low uniform expression, and grim strong and spatially modulated expression, indicating that these proapoptotic factors experience quite different levels of translation control. To gauge the efficacy of the assay, hid GFP sensor embryos were injected with bantam antisense 2′OM-ORNs, and mild but statistically significant derepression of GFP expression was found as compared to control, consistent with the weak cell-death phenotype of bantam depleted embryos. Antisense injection of miR-2 family members reveals strong derepression of the hid GFP sensor by miR-6 antisense, but not by miR-2/13, 11, or 308 antisense. Conversely, the grim GFP sensor shows significant derepression as a result of miR-2/13, 11, and 308, but not miR-6 depletion. Finally, the skl GFP sensor shows significant derepression for all four family members (Leaman, 2005).

To assess effects on rpr, a second, more sensitive assay was developed that employs transient expression of a dual-luciferase vector in injected embryos. For initial comparison with the GFP assay, a hid luciferase sensor was tested against the entire miR-2 family and the same profile was found. The rpr luciferase sensor shows strong derepression in miR-6 and 2/13, moderate derepression in miR-11, and no significant effect in miR-308 depleted embryos. Thus, the 3′UTRs of all four proapoptotic factors are subject to translational repression by the miR-2 family, but each miRNA displays a distinct interaction profile. The interaction preferences correlate well with the observed differences in phenotype: miR-6 has the most severe death phenotype and is the only family member to regulate hid, the factor with the broadest expression and the strongest proapoptotic effect. mir-2/13 and miR-11 have the same overall profile, but they differ in the strength of their interaction with rpr and show a corresponding differential in phenotypic strength. Finally, miR-308, which has the mildest death phenotype, interacts only with the weakly proapoptotic skl and with grim (Leaman, 2005).

The differences in target interaction profile between the miR-2 family members are pronounced and do not merely reproduce differences in the strength or onset of miRNA expression. This suggests that differential pairing outside the 5′ core sequence shared by all members has an important role in target selection. Computational predictions indicate that miR-2 family binding sites are present in the 3′UTRs of all four proapoptotic factors: rpr and grim have one, hid and skl two predicted sites. All six miRNA target sites lie in sequence blocks that are conserved between the six sequenced Drosophilid species, spanning an evolutionary distance of 40 Myr. Interestingly, for all sites, absolute conservation extends well beyond the bases complementary to the 5′ core of the miRNA and includes adjacent stretches suitable for pairing with the 3′ end. All but one of the sites show Watson-Crick pairing with miRNA positions 2-7 and variable pairing at the 3′ end. One of the hid sites (hid468) has a mismatch in the core but shows strong pairing with miR-6 at the 3′ end. The rules for 3′ pairing between miRNAs and their targets are not yet well understood, but it is clear that the miR-2 family members differ considerably in their ability to form 3′ matches with the six target sites. Further experimentation will be required to better understand how the observed differences in regulatory effect relate to differences in sequence pairing (Leaman, 2005).

Lack of involvement of mitochondrial factors in caspase activation in a Drosophila cell-free system

Although mitochondrial proteins play well-defined roles in caspase activation in mammalian cells, the role of mitochondrial factors in caspase activation in Drosophila is unclear. Using cell-free extracts, it is demonstrated that mitochondrial factors play no apparent role in Drosophila caspase activation. Cytosolic extract from apoptotic S2 cells, in which caspases were inhibited, induced caspase activation in cytosolic extract from normal S2 cells. Mitochondrial extract does not activate caspases, nor does it influence caspase activation by cytosolic extract. Silencing of Hid, Reaper, or Grim reduces caspase activation by apoptotic cell extract. Furthermore, a peptide representing the amino terminus of Hid is sufficient to activate caspases in cytosolic extract, and this activity is not enhanced by addition of mitochondria or mitochondrial lysate. The Hid peptide also induces apoptosis when introduced into S2 cells. These results suggest that caspase activation in Drosophila is regulated solely by cytoplasmic factors and does not involve any mitochondrial factors (Means, 2006).

The Drosophila homolog of the putative phosphatidylserine receptor functions to inhibit apoptosis, acting upstream of Hid

Exposure of phosphatidylserine is a conserved feature of apoptotic cells and is thought to act as a signal for engulfment of the cell corpse. A putative receptor for phosphatidylserine (PSR) was previously identified in mammalian systems. This receptor is proposed to function in engulfment of apoptotic cells, although gene ablation of PSR has resulted in a variety of phenotypes. The role of the predicted Drosophila homolog of PSR (phosphatidylserine receptor; dPSR) in apoptotic cell engulfment was examined and no obvious role for dPSR in apoptotic cell engulfment by phagocytes was found in the embryo. In addition, dPSR is localized to the nucleus, inconsistent with a role in apoptotic cell recognition. However, it was surprisind to find that overexpression of dPSR protects from apoptosis, while loss of dPSR enhances apoptosis in the developing eye. The increased apoptosis is mediated by the head involution defective (Wrinkled) gene product. In addition, the data suggest that dPSR acts through the c-Jun-NH2 terminal kinase pathway to alter the sensitivity to cell death (Krieser, 2007).

Evidence against a role for PSR in engulfment also comes from two other knockout models and from data on the localization of the protein. One of the reported mouse knockouts showed no difference in engulfment of apoptotic cells by macrophages in the mutant, although PSR^-/- macrophages were generally inhibited in their release of pro- and anti-inflammatory cytokines. In addition, fibroblast lines established from PSR^-/- embryos showed no defect in apoptotic cell engulfment or in their response to apoptotic cells. Zebrafish lacking PSR accumulated dead cells, but were not definitively shown to have defects in apoptotic cell engulfment. Finally, localization data from the current work and from a number of labs strongly supports a nuclear localization for the protein. This is not consistent with a role for PSR as a surface receptor for the recognition for apoptotic cells, although PS could theoretically modulate the activity of this protein within the cell (Krieser, 2007).

The observations support a role for dPSR in cell survival. In zebrafish, reduction of PSR resulted in an increase in the number of apoptotic cells present during development. In particular, the brains of these fish were shrunken and had an increase in apoptotic cells. In two of the mouse knockout models an increase in apoptotic cells was detected. However, all three knockouts resulted in perinatal lethality, with defects in differentiation in a variety of tissues. It is speculated that defects in engulfment detected in some of the gene ablation models could reflect a role for PSR in the proper differentiation of macrophages. Increased apoptosis seen in the current studies and by others might also be due to defects in proper differentiation in the absence of PSR (Krieser, 2007).

What insight can be gained from these studies into the function of dPSR in differentiation and cell survival? Increased dPSR results in a cell survival phenotype that is suppressed by activation of the JNK pathway, while loss of dPSR results in apoptosis, activated by the cell death regulator Hid, a known target of JNK activation in apoptosis. Taken together, these data suggest that dPSR may normally act to suppress JNK activation of Hid-induced apoptosis (Krieser, 2007).

JNK activation is important for many processes in cells, including cell death, proliferation and differentiation. A role for JNK in apoptosis was found in many mammalian cell types. Data from mouse knockouts of JNK also suggest a role for JNK in proliferation and differentiation. In addition, JNK activation in dying cells is required for proliferative signals originating from apoptotic cells in Drosophila. Interestingly, defects in proliferation and differentiation of many tissues were observed in mice that lack PSR. Taken together with observations of increased cell death in dPSR mutant flies, these observations suggest that some of the phenotypes seen in mouse and fish models of PSR gene ablation might be due to inappropriate activation of the JNK pathway (Krieser, 2007).

Based on genetic assays, it is proposed that one function of dPSR is to suppress Hid activation. Flies that lack dPSR show increased apoptosis in the developing pupal eye, which is suppressed in the absence of hid, while overexpression of dPSR results in ectopic cell survival. Hid function is required for the death of the interommatidial cells in the pupal retina. The results also showed that expression of dPSR can inhibit death induced by the expression of Hid- or Grim in the eye, and that loss of dPSR enhances Rpr-, Hid- or Grim-induced death in the eye. Interestingly, loss of one copy of hid can also suppress cell death induced by Rpr or Hid expression in the eye. Therefore alterations of dPSR levels in the eye may be altering Hid activity to modify the Grim- and Rprinduced eye phenotypes (Krieser, 2007).

JNK activation has been shown to increase hid activity. However, Hid activity is also modulated by activation of the Ras/Erk pathway. Ras activation results in the survival of ectopic interommatidial cells, through the downregulation of Hid activity. Ectopic Ras activation also results in genital rotation defects, similar to those seen with dPSR overexpression. This suggests that PSR overexpression might activate the Ras/Erk pathway. Based on the current data, it is not clear whether dPSR might activate Ras and thus suppress JNK activity, whether dPSR could suppress JNK and thus activate Ras, or whether dPSR might act independently in an opposing manner on the JNK and Ras pathways (Krieser, 2007).

By examining the function of dPSR in the Drosophila system, new insight has been provided into the controversy regarding this protein. Although no evidence was found that this protein plays a role in engulfment, it is important in cell survival. This is consistent with phenotypes seen in gene ablation models in other organisms. Furthermore, dPSR affects the JNK pathway, and this may provide a clue as to its diverse functions in mammals (Krieser, 2007).

Epigenetic blocking of an enhancer region controls irradiation-induced proapoptotic gene expression in Drosophila embryos

Drosophila embryos are highly sensitive to gamma-ray-induced apoptosis at early but not later, more differentiated stages during development. Two proapoptotic genes, reaper and hid, are upregulated rapidly following irradiation. However, in post-stage-12 embryos, in which most cells have begun differentiation, neither proapoptotic gene can be induced by high doses of irradiation. The sensitive-to-resistant transition is due to epigenetic blocking of the irradiation-responsive enhancer region (IRER), which is located upstream of reaper but is also required for the induction of hid in response to irradiation. This IRER, but not the transcribed regions of reaper/hid, becomes enriched for trimethylated H3K27/H3K9 and forms a heterochromatin-like structure during the sensitive-to-resistant transition. The functions of histone-modifying enzymes Hdac1(Rpd3) and Su(var)3-9 and PcG proteins Su(z)12 and Polycomb are required for this process. Thus, direct epigenetic regulation of two proapoptotic genes controls cellular sensitivity to cytotoxic stimuli (Zhang, 2008).

Irradiation responsiveness appears to be a highly conserved feature of reaper-like IAP antagonists. A recently identified functional ortholog of reaper in mosquito genomes, michelob_x (mx), is also responsive to irradiation. These results highlighted that stress responsiveness is an essential aspect of functional regulation of upstream proapoptotic genes such as reaper/hid. It is also worth mentioning that several mammalian BH3 domain-only proteins, the upstream proapoptotic regulators of the Bcl-2/Ced-9 pathway, are also regulated at the transcriptional level (Zhang, 2008).

This study shows that the irradiation responsiveness of reaper and hid is subject to epigenetic regulation during development. The epigenetic regulation of the IRER is fundamentally different from the silencing of homeotic genes in that the change of DNA accessibility is limited to the enhancer region while the promoter of the proapoptotic genes remains open. Thus, it seems more appropriate to refer this as the 'blocking' of the enhancer region instead of the 'silencing' of the gene. This region, containing the putative P53RE and other essential enhancer elements, is required for mediating irradiation responsiveness. ChIP analysis indicates that histones in this enhancer region are quickly trimethylated at both H3K9 and H3K27 at the sensitive-to-resistant transition period, accompanied by a significant decrease in DNA accessibility. DNA accessibility in the putative P53RE locus (18,368k), when measured by the DNase I sensitivity assay, did not show significant decrease until sometime after the transition period. It is possible that other enhancer elements, in the core of IRER_left, are also required for radiation responsiveness. An alternative explanation is that the strong and rapid trimethylation of H3K27 and association of PRC1 at 18,366,000-18, 368,000 are sufficient to disrupt DmP53 binding and/or interaction with the Pol II complex even though the region remains relatively sensitive to DNase I. Eventually, the whole IRER is closed with the exception of an open island around 18,387,000 (Zhang, 2008).

The finding that epigenetic regulation of the enhancer region of proapoptotic genes controls sensitivity to irradiation-induced cell death may have implications in clinical applications involving ionizing irradiation. It suggests that applying drugs that modulate epigenetic silencing may help increase the efficacy of radiation therapy. It also remains to be seen whether the hypersensitivity of some tumors to irradiation is due to the dedifferentiation and reversal of epigenetic blocking in cancer cells. In contrast, loss of proper stress response to cellular damage is implicated in tumorigenesis. The fact that the formation of heterochromatin in the sensitizing enhancer region of proapoptotic genes is sufficient to convey resistance to stress-induced cell death suggests it could contribute to tumorigenesis. In addition, it could also be the underlying mechanism of tumor cells' evading irradiation-induced cell death. This is a likely scenario given that it has been well documented that oncogenes such as Rb and PML-RAR fusion protein cause the formation of heterochromatin through recruiting of a human ortholog of Su(v)3-9. In this regard, the reaper locus, especially the IRER, provides an excellent genetic model system for understanding the cis- and trans-acting mechanisms controlling the formation of heterochromatin associated with cellular differentiation and tumorigenesis (Zhang, 2008).

The developmental consequence of epigenetic regulation of the IRER is the tuning down (off) of the responsiveness of the proapoptotic genes, thus decreasing cellular sensitivity to stresses such as DNA damage. Epigenetic blocking of the IRER corresponds to the end of major mitotic waves when most cells begin to differentiate. Similar transitions were noticed in mammalian systems. For instance, proliferating neural precursor cells are extremely sensitive to irradiation-induced cell death while differentiating/differentiated neurons become resistant to γ-ray irradiation, even though the same level of DNA damage was inflicted by the irradiation. These findings here suggest that such a dramatic transition of radiation sensitivity could be achieved by epigenetic blocking of sensitizing enhancers (Zhang, 2008).

Later in Drosophila development, around the time of pupae formation, the organism becomes sensitive to irradiation again, with LD₅₀ values similar to what was observed for the 4–7 hr AEL embryos. Interestingly, it has also been found that during this period, the highly proliferative imaginal discs are sensitive to irradiation-induced apoptosis, which is mediated by the induction of reaper and hid through P53 and Chk2. However, it remains to be studied whether the reemergence of sensitive tissue is due to reversal of the epigenetic blocking in the IRER or the proliferation of undifferentiated stem cells that have an unblocked IRER (Zhang, 2008).

The blocking of the IRER differs fundamentally from the silencing of homeotic genes in several aspects. (1) The change of DNA accessibility and histone modification is largely limited to the enhancer region. The promoter regions of reaper (and hid) remain open, allowing the gene to be responsive to other stimuli. Indeed, there are a few cells in the central nervous system that could be detected as expressing reaper long after the sensitive-to-resistant transition. Even more cells in the late-stage embryo can be found having hid expression. Yet, the irradiation responsiveness of the two genes is completely suppressed in most if not all cells, transforming the tissues into a radiation-resistant state (Zhang, 2008).

(2) The histone modification of the IRER has a mixture of features associated with pericentromeric heterochromatin formation and canonic PcG-mediated silencing. Both H3K9 and H3K27 are trimethylated in the IRER. Both HP1, the signature binding protein of the pericentromeric heterochromatin, and PRC1 are bound to the IRER. As demonstrated by genetic analysis, the functions of both Su(var)3-9 and Su(z)12/Pc are required for the silencing. Preliminary attempts to verify specific binding of PRC2 proteins to this region were unsuccessful. The fact that none of the mutants tested could completely block the transition seems to suggest that there is a redundancy of the two pathways in modifying/blocking the IRER. It is also possible that the genes tested are not the key regulators of IRER blocking but only have participatory roles in the process (Zhang, 2008).

(3) Within the IRER, there is a small region around 18,386,000 to 18,188,000 that remains relatively open until the end of embryogenesis. Interestingly, this open region is flanked by two putative noncoding RNA transcripts represented by EST sequences. If they are indeed transcribed in the embryo as suggested by the mRNA source of the cDNA library, then the 'open island' within the closed IRER will likely be their shared enhancer/promoter region. Sequences of both cDNAs revealed that there is no intron or reputable open reading frame in either sequence. Despite repeated efforts, their expression was not confirmed via ISH or northern analysis. Overexpression of either cDNA using an expression construct also failed to show any effect on reaper/hid-induced cell death in S2 cells. Yet, sections of the two noncoding RNAs are strongly conserved in divergent Drosophila genomes. The potential role of these two noncoding RNAs in mediating reaper/hid expression and/or blocking of the IRER remains to be studied (Zhang, 2008).

Drosophila histone deacetylase-3 controls imaginal disc size through suppression of apoptosis

Histone deacetylases (HDACs) execute biological regulation through post-translational modification of chromatin and other cellular substrates. In humans, there are eleven HDACs, organized into three distinct subfamilies. This large number of HDACs raises questions about functional overlap and division of labor among paralogs. In vivo roles are simpler to address in Drosophila, where there are only five HDAC family members and only two are implicated in transcriptional control. Of these two, HDAC1 has been characterized genetically, but its most closely related paralog, HDAC3, has not. This study describes the isolation and phenotypic characterization of hdac3 mutations. Both hdac3 and hdac1 mutations were found to be dominant suppressors of position effect variegation, suggesting functional overlap in heterochromatin regulation. However, all five hdac3 loss-of-function alleles are recessive lethal during larval/pupal stages, indicating that HDAC3 is essential on its own for Drosophila development. The mutant larvae display small imaginal discs, which result from abnormally elevated levels of apoptosis. This cell death occurs as a cell-autonomous response to HDAC3 loss and is accompanied by increased expression of the pro-apoptotic gene, hid. In contrast, although HDAC1 mutants also display small imaginal discs, this appears to result from reduced proliferation rather than from elevated apoptosis. The connection between HDAC loss and apoptosis is important since HDAC inhibitors show anticancer activities in animal models through mechanisms involving apoptotic induction. However, the specific HDACs implicated in tumor cell killing have not been identified. These results indicate that protein deacetylation by HDAC3 plays a key role in suppression of apoptosis in Drosophila imaginal tissue (Zhu, 2008).

Histone deacetylases (HDACs) are members of an ancient enzyme family that reverses acetylation of protein substrates. The most well-characterized HDAC substrates are the N-terminal tails of the histones. Acetylation of histone tail lysines generally correlates with gene activity, whereas HDAC-sponsored removal of these tail modifications frequently accompanies gene silencing. Histone acetylation state can impact gene expression through recruitment of transcriptional regulatory complexes, such as the SWI/SNF remodelling complex. Changes in charge density resulting from histone acetylation/deacetylation may also affect packaging of nucleosome arrays into higher-order arrangements that can impact transcription rates. A major HDAC regulatory function, then, is to promote gene silencing (Zhu, 2008).

The histone deacetylase HDAC1 has been the most throughly studied HDAC at the biochemical and functional levels. Extensive analysis of HDAC1 in yeast, also known as RPD3, indicates that it can deacetylate all four core histones, that it targets hundreds of genes around the genome, and confirms its major role as a direct transcriptional repressor. Biochemical studies show that HDAC1 is typically assembled into nuclear complexes, such as the SIN3 and NURD complexes. These co-repressor complexes are recruited to target genes through interactions with DNA-binding proteins. A prime example is provided by nuclear hormone receptors such as thyroid hormone receptor; the unliganded receptor recognizes target genes through its zinc finger DNA-binding domain, and it recruits a SIN3/NCoR/HDAC1 complex, which deacetylates target chromatin and leads to gene silencin (Zhu, 2008).

As a consequence of their roles with many co-repressors, HDACs have widespread function around the genome and they participate in many gene regulatory systems. In addition to steroid hormone receptor control in vertebrates and invertebrates, HDACs also function in the TGF-β pathway through Smad-Ski silencing and in repression of neuronal genes in non-neuronal tissues. In the Drosophila system, HDAC1 controls segmentation genes through interaction with the Groucho co-represso, executes Notch signalling readouts through interaction with CSL transcription factors, and HDAC1 has also been linked to silencing by Polycomb repressors. Thus, many endocrine, homeostatic, and developmental pathways employ HDACs in their gene control mechanisms (Zhu, 2008).

There are 11 HDAC family members in humans, defined by an approximately 350 amino acid homology region that encompasses the catalytic domain. These have been classified into three major subfamilies, with class I containing HDACs 1, 2, 3 and 8, class II containing HDACs 4, 5, 6, 7, 9 and 10, and HDAC11 comprising a third distinct subtype. In addition, the sirtuins represent yet another HDAC family, which are distinguished by their NAD-dependent reaction mechanism and are structurally unrelated to the family of 11 human HDACs. The large number of HDACs makes it difficult to determine which functions are shared and which can be uniquely assigned to individual family members. HDAC functional diversity is further complicated by the ability of HDACs to modify many protein substrates besides histones. Indeed, all three major HDAC subtypes are present in bacterial species, indicating that they likely evolved as protein deacetylases that only later acquired ability to act upon histones. In agreement with diversity of function, HDAC1 and 2 are largely nuclear, HDAC6 is cytoplasmic, and still other HDACs, including HDAC3, are found in both nucleus and cytoplasm. Within the nucleus, several transcription factors, including p53, GATA-1, and YY1, are HDAC substrates. In the cytoplasmic compartment, tubulin deacetylation by HDAC6 has been described. The large number of HDAC family members and the diversity of their protein substrates predict a myriad of HDAC regulatory functions in vivo (Zhu, 2008 and references therein).

HDAC functions are simpler to dissect in the Drosophila system, where there are only five HDAC family members. In addition, there are only two HDACs of the class I subtype: HDAC1 (also called DmRpd3) corresponding to the nuclear HDAC1/2 of mammals, and its most closely related fly paralog, HDAC3. Furthermore, HDAC1 and HDAC3 are the only two fly HDACs implicated in transcriptional control. Genetic studies using HDAC1 mutations have identified roles in many processes including heterochromatin silencing, segmentation, and ecdysone receptor function. However, the lack of HDAC3 mutations to date has impeded understanding of its biological functions in the fly system. This study describes isolation of HDAC3 loss-of-function alleles and presents phenotypic characterization. All five HDAC3 mutations are homozygous lethal at late larval or pupal stages. The mutant larvae have abnormally small imaginal discs, which is attributed to cell-autonomous induction of apoptosis rather than defects in cell proliferation (Zhu, 2008).

A recent transcription profile microarray study using cultured fly cells suggests that HDAC1 and HDAC3 are the only two fly HDACs with major functions in transcriptional control. Thus, HDAC3, the closest fly paralog by sequence, is also likely to be the most functionally related fly family member to HDAC1. Isolation of hdac3 mutations has provided the opportunity to begin to assess this HDAC1/HDAC3 relationship in vivo. The results show that both hdac3 and hdac1 mutations can suppress PEV, indicating roles for both HDACs in heterochromatin regulation. The fact that an hdac1; hdac3 double mutant displays an enhanced effect suggests that both HDACs make significant contributions to this process. The simplest molecular explanation is that both enzymes directly deacetylate histone residues that then become methylated in heterochromatin. However, RNA interference experiments using cultured fly S2 cells suggest that HDAC1 is the predominant histone-modifying enzyme, with little unique contribution detected from HDAC3, at least in this cell type. Thus, HDAC3 function at heterochromatin could reflect deacetylation of either histone or non-histone substrates (Zhu, 2008).

In a developmental context, requirements for HDAC3 function distinct from HDAC1 could occur at times or in cell types that accumulate HDAC3 but not HDAC1. However, the spatial distributions of hdac3 and hdac1 mRNAs are both widespread, their temporal profiles during development are similar, and this study has not detected individual tissues or cell types where hdac3 product accumulates without hdac1. In general, hdac1 mRNA levels appear more abundant than those of hdac3 especially in the CNS. This is consistent with the report that the genome-wide transcriptional response to HDAC1 knockdown in cultured fly cells is more robust and involves a larger number of affected genes as compared to HDAC3 knockdown. Thus, HDAC1 may be needed for certain processes that do not require HDAC3. Indeed, both in vivo results, and fly S2 cell studies, support a preferential role for HDAC1, as opposed to HDAC3, in controlling cell proliferation (Zhu, 2008).

The most significant HDAC3 function revealed by the genetic approach is in control of cell death. Since HDAC3 loss is by itself sufficient to trigger ectopic apoptosis, neither HDAC1 nor other fly HDACs can substitute for this requirement, at least in imaginal disc tissue. These results contrast with findings on apoptosis from an HDAC knockdown study using cultured fly cells. Although treatment with a broad-specificity HDAC inhibitor, trichostatin, did induce apoptosis in S2 cells, neither HDAC1 nor HDAC3 knockdown, nor the double knockdown, affected cell viability. One possible explanation for this discrepancy is that the degree of hdac3 loss-of-function produced by mutations in vivo is more severe than the degree achieved by RNA interference. Alternatively, the conflicting results may reflect tissue differences in the response to HDAC loss; S2 cells are derived from embryonic neuronal cells whereas the most dramatic induction of apoptosis is seen in a larval epithelial tissue, imaginal discs. Indeed, it is noted that there is little apoptotic induction in nervous system tissue of the same larvae that display robust induction in discs. Further studies will be needed to determine the mechanisms and pathways by which HDACs control apoptosis in various tissues during normal development as well as in mammalian cell and animal models for cancer (Zhu, 2008).

The bHLH-PAS transcription factor Dysfusion regulates tarsal joint formation in response to Notch activity during Drosophila leg development

A characteristic of all arthropods is the presence of flexible structures called joints that connect all leg segments. Drosophila legs include two types of joints: the proximal or 'true' joints that are motile due to the presence of muscle attachment and the distal joints that lack musculature. These joints are not only morphologically, functionally and evolutionarily different, but also the morphogenetic program that forms them is distinct. Development of both proximal and distal joints requires Notch activity; however, it is still unknown how this pathway can control the development of such homologous although distinct structures. This study shows that the bHLH-PAS transcription factor encoded by the gene dysfusion (dys), is expressed and absolutely required for tarsal joint development while it is dispensable for proximal joints. In the presumptive tarsal joints, Dys regulates the expression of the pro-apoptotic genes reaper and head involution defective> and the expression of the RhoGTPases modulators, RhoGEf2 and RhoGap71E, thus directing key morphogenetic events required for tarsal joint development. When ectopically expressed, dys is able to induce some aspects of the morphogenetic program necessary for distal joint development such as fold formation and programmed cell death. This novel Dys function depends on its obligated partner Tango to activate the transcription of target genes. A dedicated dys cis-regulatory module was identified that regulates dys expression in the tarsal presumptive leg joints through direct Su(H) binding. All these data place dys as a key player downstream of Notch, directing distal versus proximal joint morphogenesis (Cordoba, 2014: PubMed).

Protein Interactions

Drosophila Reaper (Rrp), Head involution defective (Hid), and Grim induce caspase-dependent cell death and physically interact with the cell death inhibitor DIAP1. Hid blocks Diap1's ability to inhibit caspase activity and evidence is provided suggesting that Rpr and Grim can act similarly. Based on these results, it is proposed that Rpr, Hid, and Grim promote apoptosis by disrupting productive IAP-caspase interactions and that Diap1 is required to block apoptosis-inducing caspase activity. Supporting this hypothesis, it is shown that elimination of Diap1 function results in global early embryonic cell death and a large increase in Diap1-inhibitable caspase activity and that Diap1 is still required for cell survival when expression of rpr, hid, and grim is eliminated (Wang, 1999).

Induction of apoptosis in Drosophila requires the activity of three closely linked genes: reaper, hid and grim. The proteins encoded by reaper, hid and grim activate cell death by inhibiting the anti-apoptotic activity of the Drosophila IAP1 (Diap1, also known as Thread) protein. In a genetic modifier screen, both loss-of-function and gain-of-function alleles in the endogenous diap1 gene were obtained, and the mutant proteins were functionally and biochemically characterized. Gain-of-function mutations in diap1 strongly suppress reaper-, hid- and grim-induced apoptosis. Sequence analysis of these diap1 alleles reveals that they are caused by single amino acid changes in the baculovirus IAP repeat domains of Diap1, a domain implicated in binding Reaper, Hid and Grim. Significantly, the corresponding mutant Diap1 proteins display greatly reduced binding of Reaper, Hid and Grim, indicating that Reaper, Hid and Grim kill by forming a complex with Diap1. Collectively, these data provide strong support for the idea that Reaper, Hid and Grim kill by inhibiting DIAP1's ability to antagonize caspase function (Goyal, 2000).

It is thought that the previously proposed function of IAPs upstream of reaper, hid and grim is simply an artifact of unphysiologically high levels of protein expression in heterologous systems. When IAP expression constructs are introduced into cultured cells under the control of strong promoters and at high copy numbers, the levels of proteins expressed far exceed those of the endogenous cellular IAP proteins. Under these unphysiological conditions, cellular IAPs can display properties that do not reflect their normal mechanism of action. In particular, the current results demonstrate that mutant proteins that completely lack anti-apoptotic activity in vivo can still inhibit cell death in vitro as long as they can bind to Reaper, Hid and Grim. Conversely, gain-of-function diap1 alleles that display reduced binding to Reaper, Hid and Grim have strongly increased anti-apoptotic function in vivo, but show reduced protection in heterologous cell transfection assays. These results clearly reveal the limitations of overexpression studies in cultured cells for determining the normal mechanism of action of these proteins in the cell death pathway (Goyal, 2000).

Dronc (Nedd2-like caspase) was isolated through its interaction with the effector caspase drICE. Ectopic expression of Dronc induces cell death in Schizosaccharomyces pombe, mammalian fibroblasts and the developing Drosophila eye. The caspase inhibitor p35 fails to rescue Dronc-induced cell death in vivo and is not cleaved by Dronc in vitro, making Dronc the first identified p35-resistant caspase. The Dronc pro-domain interacts with Drosophila inhibitor of apoptosis protein 1 (Diap1: known as Thread), and co-expression of DIAP1 in the developing Drosophila eye completely reverts the eye ablation phenotype induced by pro-Dronc expression. In contrast, Diap1 fails to rescue eye ablation induced by Dronc lacking the pro-domain, indicating that interaction of Diap1 with the pro-domain of Dronc is required for suppression of Dronc-mediated cell death. Heterozygosity at the Diap1 locus enhances the pro-Dronc eye phenotype, consistent with a role for endogenous Diap1 in suppression of Dronc activation. Both heterozygosity at the Dronc locus and expression of dominant-negative Dronc mutants suppress the eye phenotype caused by Reaper (Rpr) and Head involution defective (Hid), consistent with the idea that Dronc functions in the Rpr and Hid pathway (Meier, 2000).

The finding that Diap1 directly binds to and inhibits cell death caused by ectopic expression of Dronc, as well as by Rpr, Grim and Hid, underscores the key role played by Diap1 in the regulation of apoptosis in D. melanogaster and raises the possibility that Rpr, Hid or Grim may exert some, or all, of their pro-apoptotic action through displacement of Diap1 from the pro-domain of Dronc, thereby allowing activation of the caspase and consequent cell death. This idea is strongly supported by the successful isolation of Diap1 mutants that display greatly reduced binding for Rpr, Hid and Grim and significantly suppress Rpr, Hid and Grim cell killing. According to this model, IAPs function as 'guardians' of the apoptotic machinery: they act to suppress the chance of spontaneous activation of the intrinsic cell death machinery by neutralizing pro-apoptotic caspases, thereby establishing a buffered threshold that must be either exceeded or neutralized in order to initiate the destruction of a cell (Meier, 2000).

The proapoptotic genes reaper (rpr), grim, and head involution defective (hid) are required for virtually all embryonic apoptosis. The proteins encoded by these genes share a short region of homology at their amino termini. The Drosophila IAP homolog Thread/Diap1 (Th/Diap1) negatively regulates apoptosis during development. It has been proposed that Rpr, Grim, and Hid induce apoptosis by binding and inactivating TH/Diap1. The region of homology between the three proapoptotic proteins has been proposed to bind to the conserved BIR2 domain of TH/Diap1. An analysis of loss-of-function and gain-of-function alleles of th indicates that additional domains of Th/Diap1 are necessary to allow th to inhibit death induced by Rpr, Grim, and Hid. In addition, analysis of loss-of-function mutations demonstrates that th is necessary to block apoptosis very early in embryonic development. This may reflect a requirement to block maternally provided Rpr and Hid, or it may indicate another function of the Th/Diap1 protein (Lisi, 2000).

Several mechanisms of action have been suggested for the antiapoptotic properties of the IAP family of proteins. Among these are the binding of the Drosophila IAPs to the proapoptotic proteins Rpr, Grim, and Hid. This interaction has been demonstrated in overexpression systems, and has been proposed to involve the homologous amino-terminal 14 amino acid sequences of the apoptosis initiators with the second BIR domain of the IAPs. The data presented here suggest that this is an oversimplification. Another mechanism that has been proposed for IAP antiapoptotic activity is the direct binding and inhibition of caspases. Th/Diap1 binds to the Drosophila caspases drICE and DCP-1 and functions to inhibit their ability to induce apoptosis. Here again, this binding activity appears to rest within BIR2 (Lisi, 2000).

These physical interactions support a simple model of IAP action. In this model, IAPs act within viable cells to inhibit caspase function. The action of Rpr, Hid, and Grim interferes with the ability of IAPs to inhibit caspases, thus inducing apoptosis. On the basis of the model, the LOF mutations identified in this study would be predicted to interfere with the ability of the Th/Diap1 protein to inhibit caspase function. This is likely to be true for th^109.07, which lacks most of the protein, as well as for th⁵ and th⁴, which affect conserved residues in BIR2. BIR2 is sufficient to inhibit apoptosis induced by the active form of the Drosophila caspase drICE. The th⁹ mutation in BIR1 suggests that this BIR is also important for the full function in caspase inhibition. Alternatively, this change in BIR1 might have long-range effects on BIR2 structure or on protein stability (Lisi, 2000).

It is interesting to note that th⁷, which acts as a very strong LOF mutation and seems to show some dominant-negative properties, has only the BIR1 attached to the spacer and ring domains. Thus, despite the extensive homologies between the two BIR domains of the protein, a single BIR is not sufficient for Th/Diap1 function, at least in the presence of an attached ring domain. BIR2 of Th/Diap1 and Op-IAP, as well as the single BIR of survivin, are able to inhibit apoptosis (Lisi, 2000).

Again, on the basis of the model above, the GOF mutations identified would be predicted to bind to caspases, but not to the inducers. The th^SL mutation maps to a weakly conserved residue in BIR1 and does not result in increased th protein levels. This suggests that BIR1 is important for Rpr and Grim binding, but not for Hid binding, as Hid activity is unaffected in this mutation. Even in the context of overexpression in the eyes of transgenic flies, this mutant IAP retains some specificity for Rpr and Grim killing. This implies that the simple model of BIR2 binding to the conserved NH₂-terminal sequences of Rpr, Grim, and Hid is not accurate, and that other residues in the protein are differentially important for Rpr and Grim, as opposed to Hid binding (Lisi, 2000).

The importance of regions outside of BIR2 for Diap1 activity is supported by the analysis of the GOF1 class of mutations, th^6B and th^81.03. Both of these mutations suppress Hid killing and would be predicted to inhibit Hid binding. These mutations change conserved cysteines in the ring domain to tyrosines. This suggests that the ring is important for Hid/Diap1 interaction. However, the region of Hid binding to Diap1 and Op-IAP has been mapped to BIR2, while the ring does not show any ability to bind to Hid. In addition, mutations in the ring, including those in conserved cysteines, have little effect on the ability of Op-IAP to protect against Hid killing. These data, together with the finding that both GOF1 mutations are cysteine-to-tyrosine changes, suggest that these mutations might have a novel ability to interfere with binding of Hid to BIR2. In addition, the observation that the GOF1 mutations slightly enhance Rpr and Grim killing suggests that these mutants are less potent inhibitors of caspases. This might result from weaker binding to caspases or from proteins that are slightly less stable. This second attribute would be predicted to enhance killing by any inducer that binds the IAP, but not to have an effect on Hid, which is unable to bind (Lisi, 2000 and references therein).

In conclusion, the data support a model where Rpr, Grim, and Hid interact with Th/Diap1 to induce apoptosis. Mutations that affect killing by Rpr and Grim or by Hid can be isolated, indicating that these inducers interact with Th/Diap1 in different ways. The GOF mutations that have been identified also provide useful tools to examine the roles of IAPs, rpr, grim, and hid during Drosophila development. The other Drosophila IAP homolog, DIAP2, has been shown to selectively inhibit Rpr- and Hid-induced but not Grim-induced death (Lisi, 2000).

In LOF th alleles, a developmental arrest occurs at the blastoderm stage and, subsequently, a synchronous apoptosis of all the nuclei. Earlier reports that homozygous th embryos show no ectopic apoptosis probably reflects the very early stage at which this apoptosis occurs. At this time, a direct requirement for th to block apoptosis cannot be distinguised from a requirement for th in another developmental process. This developmental defect could then result in secondary apoptosis. The latter possibility is reasonable, as many failures in development result in ectopic apoptosis. A BIR containing protein from Caenorhabditis elegans is required for cytokinesis in embryos. However, it is also possible that developmental arrest occurs as a result of the initiation of apoptosis, which is manifest only as DNA damage several hours later (Lisi, 2000).

Does this early requirement for th reflect a need to inhibit apoptosis induced by rpr, grim, and hid? Double mutants of th and Df(3L)H99, the deletion that removes rpr, grim, and hid, show a phenotype similar to th alone. This indicates that Th/Diap1 is not required to suppress zygotic Rpr, Grim, and Hid activity. However, hid and rpr mRNA can be seen in a subset of cells in the blastoderm embryo, as judged by in situ analysis. This may indicate that these gene products are supplied maternally. Th/Diap1 may be required to suppress maternally supplied Rpr, Grim, or Hid. Allelic differences in the stage at which apoptosis begins in the th mutants parallel the general ability of the alleles to inhibit apoptosis induced by Rpr, Hid, and Grim. The strong LOF alleles arrest at the blastoderm stage; the GOF1 alleles arrest much later, and the GOF2 allele is completely viable (Lisi, 2000).

Activation of Ras inhibits apoptosis during Drosophila development. Genetic evidence indicates that Ras antiapoptotic activity in the developing eye is regulated by the Drosophila EGF receptor and operates through the Raf/MAPK pathway. Decreased activity of this pathway enhances (and increased activity suppresses) apoptosis induced by ectopic expression of the cell death regulators reaper (rpr) and head involution defective (hid). In addition, ectopic activation of the Ras/MAPK pathway in the developing embryo and in the developing eye suppresses naturally occurring apoptosis and regulates the transcription of the proapoptotic gene hid. Null alleles of hid recapitulate the antiapoptotic activities of Ras/MAPK, providing genetic evidence that downregulation of hid is an important mechanism by which Ras promotes survival (Kurada, 1998).

Extracellular growth factors are required for the survival of most animal cells. They often signal through the activation of the Ras pathway. However, the molecular mechanisms by which Ras signaling inhibits the intrinsic cell death machinery are not well understood. Evidence is presented that in Drosophila, activation of the Ras pathway specifically inhibits the proapoptotic activity of the gene hid. By using transgenic animals and cultured cells, it has been shown that MAPK phosphorylation sites in Hid are critical for this response. These findings define a novel mechanism by which growth factor signaling directly inactivates a critical component of the intrinsic cell death machinery. These studies provide further insight into the function of ras as an oncogene (Bergmenn, 1998).

Reaper (Rpr), Hid, and Grim activate apoptosis in cells programmed to die during Drosophila development. Transient overexpression of Rpr in the lepidopteran SF-21 cell line induces apoptosis. Members of the inhibitor of apoptosis (IAP) family of antiapoptotic proteins can inhibit Rpr-induced apoptosis and physically interact with Rpr through the BIR (baculovirus IAP repeat) motifs of IAP family members. Transient overexpression of HID and GRIM also induces apoptosis in the SF-21 cell line. Baculovirus and Drosophila IAPs block HID- and GRIM-induced apoptosis and also physically interact with them through the BIR motifs of IAP family members. The region of sequence similarity shared by Rpr, Hid, and Grim (the N-terminal 14 amino acids of each protein) is required for the induction of apoptosis by Hid and its binding to IAPs. When stably overexpressed by fusion to an unrelated, nonapoptotic polypeptide, the N-terminal 37 amino acids of Hid and Grim are sufficient to induce apoptosis and confer IAP binding activity. However, Grim is more complex than HID since the C-terminal 124 amino acids of Grim retain apoptosis-inducing and IAP binding activity, suggesting the presence of two independent apoptotic motifs within Grim. Coexpression of IAPs with Hid stabilizes Hid levels and results in the accumulation of Hid in punctate perinuclear locations that coincide with IAP localization. The physical interaction of IAPs with Rpr, Hid, and Grim provides a common molecular mechanism for IAP inhibition of these Drosophila proapoptotic proteins (Vucic, 1998).

Drosophila genes reaper, grim, and head-involution-defective (hid) induce apoptosis in several cellular contexts. N-terminal sequences of these proteins are highly conserved and are similar to N-terminal inactivation domains of voltage-gated potassium (K+) channels. Synthetic Reaper and Grim N terminus peptides induce fast inactivation of Shaker-type K+ channels when applied to the cytoplasmic side of the channel. This inactivation is qualitatively similar to the inactivation produced by other K+ channel inactivation particles. Mutations that reduce the apoptotic activity of Reaper also reduced the synthetic peptide's ability to induce channel inactivation, indicating that K+ channel inactivation correlates with apoptotic activity. Coexpression of Reaper mRNA or direct injection of full length Reaper protein causes near irreversible block of the K+ channels. These results suggest that Reaper and Grim may participate in initiating apoptosis by stably blocking K+ channels (Avdonin, 1998).

The Drosophila reaper, head involution defective (hid), and grim genes play key roles in regulating the activation of programmed cell death. Two useful systems for studying the functions of these genes are the embryonic CNS midline and adult eye. In this study the Gal4/UAS targeted gene expression system was used to demonstrate that unlike reaper or hid, expression of grim alone is sufficient to induce ectopic CNS midline cell death. In both the midline and eye, grim-induced cell death is not blocked by the Drosophila anti-apoptosis protein Diap2, which does block both reaper- and hid-induced cell death. grim can also function synergistically with either reaper or hid to induce higher levels of midline cell death than those observed for any of the genes individually. Analysis was made of the function of a truncated Reaper-C protein, which lacks the NH2-terminal 14 amino acids that are conserved between Reaper, Hid, and Grim. Ectopic expression of Reaper-C reveals cell killing activities distinct from full length Reaper, and indicates that the conserved NH2-terminal domain acts in part to modulate Reaper activity (Wing, 1998).

Reaper, Hid, and Grim are three Drosophila cell death activators that each contain a conserved NH2 -terminal Reaper-Hid-Grim (RHG) motif. The importance of the RHG motifs in Reaper and Grim have been examined for their different abilities to activate cell death during development. Analysis of chimeric R/Grim and G/Reaper proteins indicates that the Reaper and Grim RHG motifs are functionally distinct and help to determine specific cell death activation properties. A truncated GrimC protein lacking the RHG motif retains an ability to induce cell death, and unlike Grim, R/Grim, or G/Reaper, its actions are not efficiently blocked by the cell death inhibitors Diap1, Diap2, p35, or a dominant/negative Dronc caspase. Finally, a second region of sequence similarity was identified in Reaper, Hid, and Grim, that may be important for shared RHG motif-independent activities (Wing, 2001).

While the Grim-Reaper proteins do not contain defined structural domains, they each share sequence similarity in the 14 amino acids at their NH2-termini. This RHG motif is most similar between Reaper and Grim (71.4% identity), and least similar between Hid and Grim (21.4% identity). The RHG motif plays a key role in interactions between Grim-Reaper proteins and members of the Inhibitor-of-Apoptosis-Protein (IAP) family, including Drosophila Diap1 and Diap2. Like other IAPs, Diap1 and Diap2 both contain related baculovirus IAP repeat (BIR) motifs, as well as a Really Interesting New Gene (RING) finger. Diap1 is an essential cell death regulator and diap1 mutants exhibit early embryonic lethality due to massive ectopic cell death. The functions of Diap2 in regulating cell death are less clear; however, it does share a number of functional properties with Diap1. Diap1 can directly bind caspases and repress their proteolytic activities. Significantly, caspase inhibition by Diap1 is antagonized by Hid, suggesting a double-repression model where the Grim-Reaper proteins promote cell death by binding to Diaps, suppressing their ability to inhibit caspases. Recent studies have indicated that the vertebrate Diablo/SMAC protein also promotes cell death activation by binding to IAPs and suppressing their death inhibitory activities. Thus, IAP suppression may be an evolutionarily conserved cell death regulatory mechanism. In this regard, while Grim-Reaper orthologs have not been identified, the expression of each protein can induce vertebrate cells to die, implying that they may suppress vertebrate IAPs (Wing, 2001).

Diap1, like Diap2, exhibits distinct abilities to repress cell death induced by Reaper, Hid, or Grim. In the CNS midline, Diap1 more effectively blocks Grim-induced cell death than cell death induced by Reaper and Hid. In contrast, when examined in the adult eye, Diap1 is most effective at blocking Reaper-induced cell death, moderately effective against Hid, and ineffective against Grim. Similar results were obtained using the th^sl gain-of-function diap1 mutant allele, which represses Reaper-induced eye cell death more effectively than death induced by Hid or Grim. Importantly, these data indicate that Diap1 has distinct, tissue-specific effects on cell death induced by Grim-Reaper proteins, and that these effects differ from those of Diap2. The basis for these functional distinctions are not yet clear. One possibility is that the associations between each Diap and Grim-Reaper protein may differ in strength, or be influenced by specific ancillary factors. Differences have been noted between Diap1 and Diap2 in their ability to bind and repress the actions of certain caspases, and Reaper, Hid, and Grim can act through different downstream caspases. Taken together, these findings suggest potentially complex functional interactions between Grim-Reaper proteins, Diaps, and caspases. It is likely that distinct activities of individual Grim-Reaper and Diap proteins provide enhanced capabilities for regulating cell death processes in different developmental and physiological contexts (Wing, 2001).

Do Reaper, Hid, and Grim share RHG-independent functions? Both truncated ReaperC and GrimC proteins induce cell death in developing tissues, indicating that regions outside the RHG motif also have death-inducing activities. Surprisingly, it was found that cell death induced by GrimC or ReaperC is only partially repressed by p35, suggesting a distinct mode of action compared with native Reaper or Grim. Similar to Reaper, Hid and Grim, GrimC does apparently act through the p35-insensitive caspase, Dronc, as GrimC-induced death is partially suppressed by a dominant/negative Dronc^C318S protein. However, the persistence of some eye cell death in the presence of Dronc^C318S indicates that GrimC and ReaperC also act through alternate pathways. Perhaps GrimC acts through pro-apoptotic Drosophila Bcl-2 orthologs that may induce cell death which is not blocked by p35. Another interesting possibilty is that GrimC might act via a Drosophila ortholog of Scythe, a Xenopus cell death regulator that binds Reaper, Hid, and Grim independently of the RHG motif (Wing, 2001 and references therein).

A second region of sequence similarity, the 30 amino acid Trp-block, has been identified that is present once in Reaper and Grim, and four times in Hid. The Trp-blocks may be important for the cell death activation capabilities of GrimC and ReaperC, as well as for potentially shared RHG motif-independent activities of native Grim-Reaper proteins. This additional sequence similarity also suggests a modular organization of the Grim-Reaper proteins, where distinct functions may be afforded by the RHG motif and Trp-block. Taken together, the sequence similarities of the Grim-Reaper proteins, as well as the organization and chromosomal location of the corresponding genes, imply that the grim-reaper genes arose from duplication of a common ancestor and have diverged to assume overlapping yet distinct cell death activation functions. It will be of interest to determine the representation of grim-reaper orthologs in other species, information that could provide important insights into the evolution of cell death control mechanisms. This is of particular relevance given that inhibition of IAP activity is likely to constitute a conserved mechanism to regulate cell death activation (Wing, 2001).

The inhibitor of apoptosis protein DIAP1 suppresses apoptosis in Drosophila, with the second BIR domain (BIR2) playing an important role. Three proteins, Hid, Grim, and Reaper, promote apoptosis, in part by binding to DIAP1 through their conserved N-terminal sequences. The crystal structures of DIAP1-BIR2 by itself and in complex with the N-terminal peptides from Hid and Grim reveal that these peptides bind a surface groove on DIAP1, with the first four amino acids mimicking the binding of the Smac tetrapeptide to XIAP. The next 3 residues also contribute to binding through hydrophobic interactions. Interestingly, peptide binding induces the formation of an additional alpha helix in DIAP1. This study reveals the structural conservation and diversity necessary for the binding of IAPs by the Drosophila Hid/Grim/Reaper and the mammalian Smac proteins (Wu, 2001).

Many members of the inhibitor of apoptosis (IAP) family of proteins suppress programmed cell death, at least in part, by physically interacting with and inhibiting the catalytic activity of caspases. An important functional unit in all death-inhibiting IAP proteins is the so-called baculoviral IAP repeat (BIR), which contains approximately 80 amino acids folded around a zinc atom. The Drosophila genome contains four genes that encode proteins with BIR domains. The overexpression of two of these, DIAP1 and DIAP2, inhibit both normal developmental cell death and apoptosis induced by expression of proapoptotic genes. In addition, DIAP1 is required for cell survival in the embryo and in a number of adult tissues. These observations, in conjunction with others showing that DIAP1 binds and inactivates several Drosophila caspases and that loss of DIAP1 results in an increase in caspase activity in vivo, argue that DIAP1's function as a caspase inhibitor is required for cell survival. DIAP1 contains two N-terminal BIR repeats and a C-terminal RING domain. DIAP1 fragments containing the BIR2 domain are sufficient to prevent cell death in a number of contexts. Interestingly, fragments consisting of the BIR2 and surrounding linker sequences also bind multiple proapoptotic proteins, including the apical caspase DRONC, and Hid, Grim, and Reaper (Wu, 2001 and references therein).

One mechanism by which Hid, Grim, and Reaper promote cell death is by binding to DIAP1, thereby inhibiting its function as a caspase inhibitor. Although Hid, Grim, and Reaper perform a similar function in promoting cell death, they only share homology in the N-terminal 14 residues of their primary sequences. These N-terminal sequences are sufficient to mediate interactions with DIAP1 and with several mammalian IAPs. In the case of Hid in insects, and Hid and Reaper in mammalian cells, these N-terminal sequences are essential for proapoptotic function (Wu, 2001 and references therein).

In mammalian cells, caspase inhibition by IAPs is negatively regulated by a mitochondrial protein Smac/DIABLO, which is released from the mitochondrial intermembrane space into the cytosol upon apoptotic stimuli. Smac/DIABLO physically interacts with multiple IAPs and relieves their inhibitory effect on both initiator and effector caspases. Thus, Smac/DIABLO represents the mammalian functional homolog of the Drosophila Hid, Grim, and Reaper proteins. Recent structural studies reveal that the N-terminal tetrapeptide of Smac/DIABLO binds a surface groove on XIAP-BIR3, thus competitively removing the inhibition of caspase-9 by XIAP. Smac/DIABLO shares sequence homology with Hid, Grim, and Reaper only in the N-terminal 4 residues, prompting the hypothesis that Hid, Grim, and Reaper interact with DIAP1 using similar tetrapeptides and binding to a similar surface groove on DIAP1 (Wu, 2001 and references therein).

There is currently no structural information on DIAP1 or Hid, Grim, or Reaper. To investigate the structural mechanisms of DIAP1 recognition by the Drosophila Hid, Grim, and Reaper proteins, the DIAP1-BIR2 domain was crystalized by itself and in complex with the N-terminal peptides from both Hid and Grim (these structures were determined at 2.7, 2.7, and 1.9 Angstrom resolution, respectively). By analogy to the Smac-XIAP interactions, the first four amino acids of Hid and Grim bind an evolutionarily conserved surface groove on DIAP1-BIR2. The next 3 conserved residues of Hid and Grim also contribute to the interactions with DIAP1 through extensive van der Waals contacts. Interestingly, peptide binding to DIAP1-BIR2 appears to induce the formation of an additional alpha helix, which appears to stabilize peptide binding. In conjunction with biochemical analysis, this structural study reveals a molecular basis for the conservation and diversity necessary for the recognition of IAPs by the Drosophila Hid/Grim/Reaper and the mammalian Smac proteins. These results have important ramifications for the design of IAP inhibitors toward therapeutic applications (Wu, 2001).

It has been suggested that the Drosophila Hid protein interacts with the baculovirus Op-IAP protein in a manner similar to that of human Smac binding to XIAP, based largely on amino acid sequence homology. The interaction between Hid and Op-IAP has been precisely mapped; the biochemical interactions between the amino terminus of Hid and BIR2 of Op-IAP are highly similar to those found between the processed amino terminus of Smac and BIR3 of XIAP. Also similar to Smac, the amino terminus of Hid must be processed to bind Op-IAP. In addition, the data also suggest that a second interaction between Hid and Op-IAP exists that does not involve the amino terminus of Hid. The evolutionary conservation of this mechanism of binding underscores its importance in apoptotic regulation. Nevertheless, interaction with Hid is not sufficient for Op-IAP to inhibit apoptosis induced by Hid overexpression or by treatment with actinomycin D, indicating that additional sequence elements are required for the anti-apoptotic function of Op-IAP (Wright, 2002).

Members of the IAP family block activation of the intrinsic cell death machinery by binding to and neutralizing the activity of pro-apoptotic caspases. In Drosophila melanogaster, the pro-apoptotic proteins Reaper Rpr, Grim and Hid all induce cell death by antagonizing the anti-apoptotic activity of Drosophila IAP1 (DIAP1), thereby liberating caspases. In vivo, the RING finger of DIAP1 is essential for the regulation of apoptosis induced by Rpr, Hid and Dronc. Furthermore, the RING finger of DIAP1 promotes the ubiquitination of both itself and of Dronc. Disruption of the DIAP1 RING finger does not inhibit its binding to Rpr, Hid or Dronc, but completely abrogates ubiquitination of Dronc. These data suggest that IAPs suppress apoptosis by binding to and targeting caspases for ubiquitination (Wilson, 2002).

Drosophila IAP antagonists form multimeric complexes to promote cell death

Apoptosis is a specific form of cell death that is important for normal development and tissue homeostasis. Caspases are critical executioners of apoptosis, and living cells prevent their inappropriate activation through inhibitor of apoptosis proteins (IAPs). In Drosophila, caspase activation depends on the IAP antagonists, Reaper (Rpr), Head involution defective (Hid), and Grim. These proteins share a common motif to bind Drosophila IAP1 (DIAP1) and have partially redundant functions. This study shows that IAP antagonists physically interact with each other. Rpr is able to self-associate and also binds to Hid and Grim. The domain involved in self-association has been defined and it was demonstrated to be critical for cell-killing activity in vivo. In addition, Rpr requires Hid for recruitment to the mitochondrial membrane and for efficient induction of cell death in vivo. Both targeting of Rpr to mitochondria and forced dimerization strongly promotes apoptosis. These results reveal the functional importance of a previously unrecognized multimeric IAP antagonist complex for the induction of apoptosis (Sandu, 2010).

This study shows that IAP antagonists undergo self-association and hetero-association that is essential for their full killing activity. Specifically, the physical association between Rpr, Hid, and Grim involves the central helical domain of Rpr. Disrupting this protein-protein interface leads to a significant loss of Rpr’s ability to induce cell death in vivo. The importance of Rpr self-association was revealed by generating enforced Rpr dimers in which the central helical domain of this protein is replaced by defined dimerization motifs. These experiments revealed that enforced parallel, but not anti-parallel dimerization of Rpr (RprLZ) can induce cell death very efficiently in transgenic Drosophila. The resulting cell death occurred by apoptosis and was rescued by the overexpression of the caspase inhibitor p35, or through Rpr-insensitive diap1 alleles. Furthermore, mutants that inhibit the self-association of Rpr have reduced pro-apoptotic activity, providing independent support for the importance of Rpr multimerization. Because an anti-parallel Rpr dimer (RprProP) was not efficiently inducing cell death in transgenic animals, it appears that the IBM motifs of multimeric Rpr have to be in a specific conformation, or at least in close proximity for efficient DIAP1 inactivation. This may occur, for example, by engaging both BIR domains of one DIAP1 molecule in a similar fashion to how SMAC can engage XIAP (Sandu, 2010).

The association of Rpr with the other IAP antagonists Grim and Hid is also reported. Hid is the only IAP antagonist that has a defined mitochondrial targeting sequence at its C terminus and is targeted to the mitochondria by itself; therefore, focus was placed particularly on the interaction between Rpr and Hid. Consistent with previous reports, it was found that Hid consistently localizes to the mitochondria in both human and Drosophila cells. Although it has been previously reported that Rpr localizes to the mitochondria through the GH3-lipid interaction, the current results support an alternative view that Rpr’s ability to translocate to the mitochondria is an indirect consequence of associating with Hid. Specifically, in support of this model, it was shown that Rpr is uniformly distributed in cells when transfected alone in heterologous cells, translocating to the mitochondria only when cotransfected with Hid. It was further shown that the GH3 mutant F34AL35A, unlike wild-type Rpr, does not coimmunoprecipitate with Hid. This is in agreement with previous observations that a GH3 mutant failed to localize to the mitochondria in Drosophila S2 cells (Sandu, 2010).

Rpr induces ubiquitination of DIAP1 in vitro and in HEK293 cells. Unlike Rpr, Hid is not able to perform this function. Thus, the significance of Rpr-Hid interaction might be to bring Rpr at the mitochondrial surface to degrade DIAP1. Although both Rpr and Hid belong to the IAP antagonists family, share a conserved IBM motif, bind DIAP1, and induce cell death, their role in induction of cell death seems to be distinct. In many paradigms Hid appears to be a more potent inducer of cell death than Rpr. It is possible that the primary role of Hid is to assemble a complex at the mitochondrial membrane that recruits Rpr as one the players. The role of Rpr in this complex is to induce DIAP1 ubiquitination. Inability of Hid itself to induce DIAP1 degradation might be related to its larger size (410 amino acids) as compared with Rpr (64 amino acids) or even Grim (138 amino acids). Potentially, the bulkier Hid might interfere with conformational changes in DIAP1 or with the ubiquitin-related transfer process (Sandu, 2010).

In addition, evidence is provided that Rpr is more potent at inducing apoptosis when present at the mitochondrial membrane. When Rpr was fused to the mitochondrial targeting sequence from Hid and expressed in Drosophila eyes, strong cell killing and pupal lethality were observed. Flies dissected from the pupal cases show severely ablated eyes that are reduced to black spots. Even the inactive GH3 mutant F34AL35A, when artificially targeted to the mitochondria using the Hid MTS, induces significant eye ablation. Therefore, Rpr is more potent when present at the mitochondrial membrane. Two possible explanations are considered for this enhanced pro-apoptotic activity: First, Rpr may be more active at the mitochondrial surface because of increased protein stability. Consistent with this idea, cytoplasmic Rpr is not very stable and it was found that Rpr accumulates to higher protein levels when the presence of Hid permits mitochondrial localization. The resulting high local concentration of Rpr may be critical for DIAP1 ubiquitination. As predicted by this model, it was found that Rpr-induced cell death is less efficient when Hid is depleted by RNA knockdown. The model is also in agreement with several previous observations. For example, it has been reported that Rpr and Hid localize to mitochondria and can induce changes of the mitochondrial ultrastructure. This study also showed that inhibition of Rpr localization to mitochondria significantly inhibits cell killing, and that Rpr and Hid act in concert with caspases to promote mitochondrial disruption and Cyt C release. In addition, overexpression of both rpr and hid is required to induce cell death in midline cells of the nervous system, and neither of them kills well individually. This is consistent with the observation that more than one IAP antagonist is expressed and they act synergistically in the dying midline glia cells. Finally, Drosophila salivary gland cell death is preceded by the expression of both rpr and hid, and RNAi knockdown of hid alone is sufficient to block the death of these cells. The second, and not mutually exclusive explanation is that Rpr may be more active at the mitochondria because of local concentration of apoptosis regulators that operate at this surface. It has been previously shown that Dronc and active Drice are present at the mitochondrial membrane, and more recently that mammalian XIAP can translocate to the mitochondrial surface in response to apoptotic stimuli. In addition, mitochondrial proteins involved in energy metabolism have been recently described to modulate caspase activity and cell death in Drosophila cells. Recently, it was shown by coimmunoprecipitation experiments in fly cell culture that Grim interacts with the Bcl-2 family proteins Debcl and Buffy. Thus, Rpr may be part of a higher-order complex at the mitochondria to locally regulate IAP turnover and caspase activity (Sandu, 2010).

Taken together, this study uncovered the role of the Rpr helical domain in self-association and interaction with Hid and Grim. The mechanism of Rpr recruitment to the mitochondria by interaction with Hid was revealed. Most importantly, this study has provided a new concept with respect to IAP antagonist activity in fly, which acts cooperatively by physical interaction rather than by additive cell death output (Sandu, 2010).

A gain-of-function germline mutation in Drosophila ras1 affects apoptosis and cell fate during development

The RAS/MAPK signal transduction pathway is an intracellular signaling cascade that transmits environmental signals from activated receptor tyrosine kinases (RTKs) on the cell surface and other endomembranes to transcription factors in the nucleus, thereby linking extracellular stimuli to changes in gene expression. Largely as a consequence of its role in oncogenesis, RAS signaling has been the subject of intense research efforts for many years. More recently, it has been shown that milder perturbations in Ras signaling during embryogenesis also contribute to the etiology of a group of human diseases. This study reports the identification and characterization of the first gain-of-function germline mutation in Drosophila ras1 (ras85D), the Drosophila homolog of human K-ras, N-ras and H-ras. A single amino acid substitution (R68Q) in the highly conserved switch II region of Ras causes a defective protein with reduced intrinsic GTPase activity, but with normal sensitivity to GAP stimulation. The ras1^R68Q mutant is homozygous viable but causes various developmental defects associated with elevated Ras signaling, including cell fate changes and ectopic survival of cells in the nervous system. These biochemical and functional properties are reminiscent of germline Ras mutants found in patients afflicted with Noonan, Costello or cardio-facio-cutaneous syndromes. Finally, ras1^R68Q was used to identify novel genes that interact with Ras and suppress cell death (Gafuik, 2011).

Genetic screens were conducted for dominant modifiers of cell death induced by the Drosophila IAP-antagonists, hid and rpr. From over 150 mutants initially isolated, secondary screens allowed identification of 58 cell death specific modifiers. Of these, 40 alleles were placed into six complementation groups that define both known and unknown genes. These include Star, gap and sprouty involved in EGFR/MAPK signaling, the known cell death regulator diap1, the very large BIR and UBC containing dbruce, and an unknown gene, Su(GMRhid)2A that remains unidentified. This study focused on a previously uncharacterized cell death suppressor originally termed Su(21-3s). Using a combination of meiotic and P-element induced male recombination, genetic reversion, biochemistry and in vivo analysis, this mutant was demonstrated to be a gain-of-function mutation in ras1 (ras85D), the Drosophila homolog of human K-ras, N-ras and H-ras. This allele affects cell fate decisions and the pattern of normal, developmental apoptosis in paradigms known to depend on Ras-signaling (Gafuik, 2011).

One important role of Ras signaling during development is the transmission of an anti-apoptotic signal. The pro-apoptotic protein Hid contains 5 potential MAPK phosphorylation sites that are essential for its sensitivity to Ras-mediated inhibition. A Hid protein with either 3/5 or 5/5 mutant MAPK sites (Hid^Ala3 and Hid^Ala5, respectively) was refractory to suppression by the gain-of-function MAPK allele rl^Sem (a very mild suppression by rl^Sem is due to phosphorylation of the endogenous wildype Hid protein). In contrast, there was still some suppression of Hid^Ala3 and Hid^Ala5 by RasV12. It was postulated that this might be due to the ability of Ras, unlike MAPK, to exert additional anti-apoptotic effects through activation of the PI3-K/Akt-kinase effector branch. The current study found that ras^R68Q was able to partially suppress Hid^Ala3 but not Hid^Ala5. Because Hid^Ala3 retains two phosphorylation sites, it appears that partial phosphorylation of Hid is sufficient for a mild inhibitory effect, and that all five phospho-acceptor sites need to be eliminated in order for Hid to become refractory to inhibition by MAPK. Furthermore, it appears that Ras^R68Q, unlike Ras^V12, is unable to exert an additional suppressive effect via PI3-K/Akt-kinase. Perhaps the enhanced signaling activity of Ras^R68Q is able to activate the MAPK effector branch, but does not reach a required threshold to engage the PI3-K/Akt-kinase pathway. This may also help to explain the organismal viability of Ras^R68Q as compared to Ras^V12. Along the same line, Ras^R68Q was able to suppress Hid-induced cell death of lymphocytes within the protective environs of the lymph gland but not of those that were circulating. In sharp contrast, over-expression of Ras^v12 in hemocytes not only leads to survival of circulating hemocytes but in fact results in a massive overproliferation of hemocytes. These results serve to highlight the exquisite sensitivity of biological systems to the degree of Ras signaling and suggest that between the extremes of wildtype Ras and constitutively active Ras^V12 lies a large spectrum of biological responsiveness (Gafuik, 2011).

Ras is highly conserved among metazoans and a number of Ras structures have been published that make it possible predict how mutations in specific regions might affect function. In the case of Ras^R68Q, it was considered that this change may affect the transition state of Ras. According to the 'arginine-finger hypothesis' GTPase-activating-proteins (GAPs) dramatically accelerate the GTPase reaction of Ras by supplying an arginine side chain (arginine-789 in the case of GAP-334) into the active site of Ras to neutralize developing charges in the transition state. A detailed analysis of the interactions between Ras and GAP-334 showed no role for R68 of Ras, explaining why Ras^R68Q can be stimulated by GAP. However, a close inspection of the Ras catalytic site shows that R68 extends its side chain towards the catalytic center. Mutating R68 to glutamine removes a stabilizing positive charge from the transition state and, according to the arginine-finger hypothesis, would be expected to result in less efficient hydrolysis of GTP. This prediction was tested biochemically and indeed it was found that Ras^R68Q hydrolyzes GTP intrinsically at a reduced rate, approximately 30% of that of wild type GTP (Gafuik, 2011).

Oncogenic mutations in Ras occur most frequently at codons 12,13 or 61 and result in an enzyme with deficient GTPase activity. This renders Ras inactive because Ras is 'on' when bound to GTP and switches 'off' by hydrolyzing bound GTP to GDP. Inhibition of Ras GTPase activity therefore stabilizes Ras in its active conformation, prolonging its recruitment and activation of downstream signaling components. The reduced GTPase activity of Ras^R68Q means that it would remain in its active GTP-bound conformation for longer periods of time allowing for enhanced signaling to downstream effector pathways. As noted above, however, Ras^R68Q may not remain in an active state sufficiently long to engage the catalytic p110 subunit of PI3K. An interesting alternative possibility however may be that R68 is directly involved in an interaction with PI3K and a mutation in R68 negatively affects this interaction. This raises the intriguing possibility that some of the phenotypes described for Ras^R68Q may actually be due to a loss, rather than a gain of PI3K activity (Gafuik, 2011).

During the initial mapping and characterization of ras1^R68Q, a reversion screen was conducted in order to provide genetic evidence for the hypothesis that a rare gain-of-function allele in ras85D was identified. While searching for revertants, several mutants were recovered that were strong suppressors of GMR-hid. Recognizing that these mutants might be synergizing with ras1^R68Q to produce such a strong suppression, 14 of these suppressors were successfully recovered and mapped. Most were mapped to a single candidate gene. Since these mutants were essentially derived from a dominant modifier screen for suppression of GMR-hid induced cell death, but within a sensitized ras1^R68Q background, the mutational spectrum was expected to be overlapping, yet distinct from that of previous GMR-hid or UAS-Ras^V12 based screens. Indeed several suppressors turned out to overlap with ones identified previous screens. However, two novel interactors were also isolated: one allele of notum and four alleles of Su(Tpl). This demonstrates the utility of ras1^R68Q to identify novel genetic interactions. While notum affects the Wnt/Wingless signaling pathway, Su(Tpl) is thought to function in the regulation of transcription in response to stress (Gafuik, 2011).

Much of the understanding of Ras-mediated signaling is derived from a combination of biochemical experiments conducted in mammalian tissue culture, and genetic studies in model organisms. For example, Ras-mediated signaling regulates the specification and differentiation of R7 photoreceptors in the Drosophila eye. However, until now, studies on the physiological consequences of elevated Ras in Drosophila have relied on overexpression of the activated ras1^v12 allele. The viable hypermorphic ras1 allele described in this study, ras1^R68Q, represents the first endogenous gain-of-function mutation in Drosophila Ras and hence offers a new tool for the analysis of Ras biology in situ. In particular, certain aspects of Ras biology have remained largely inaccessible to the use of constitutively active versions of this protein. This is because mutants, such as ras1^v12, do not cycle normally between off and on states, are insensitive to regulatory circuits and are generally not compatible with organismal development. As a consequence, in certain paradigms and contexts, ras1^v12 actually behaves as a loss-of-function mutant rather than a hypermorph, occluding the biological interpretation of Ras function in vivo. Therefore, the use of milder, viable hypermorphs of Ras, such as ras1^R68Q, offers the potential for a refined understanding of the normal physiological roles of this important protein. Significantly, the ras1^R68Q allele described in this study shares overall biochemical properties with recently discovered mutations in k-ras and h-ras that underlie human developmental disorders, such as Noonan, Costello and CFC syndromes (Gafuik, 2011).

CDK7 regulates the mitochondrial localization of a tail-anchored proapoptotic protein, Hid

The mitochondrial outer membrane is a major site of apoptosis regulation across phyla. Human and C. elegans Bcl-2 family proteins and Drosophila Hid require the C-terminal tail-anchored (TA) sequence in order to insert into the mitochondrial membrane, but it remains unclear whether cytosolic proteins actively regulate the mitochondrial localization of these proteins. This study reports that the cdk7 complex regulates the mitochondrial localization of Hid and its ability to induce apoptosis. cdk7 was identified through an in vivo RNAi screen of genes required for cell death. Although CDK7 is best known for its role in transcription and cell-cycle progression, a hypomorphic cdk7 mutant suppresses apoptosis without impairing these other known functions. In this cdk7 mutant background, Hid fails to localize to the mitochondria and fails to bind to recombinant inhibitors of apoptosis (IAPs). These findings indicate that apoptosis is promoted by a newly identified function of CDK7, which couples the mitochondrial localization and IAP binding of Hid (Morishita, 2013).

This study reports a mechanism of cell death regulation in Drosophila in which the mitochondrial localization of a proapoptotic TA protein is regulated by CDK7. Moreover, the mitochondrial localization of Hid is coupled with its ability to bind to DIAP1. These finding provides an explanation for the mitochondrial requirement of IAP antagonists (Morishita, 2013).

Future studies are required to elucidate the structural nature of these Hid subspecies, and how they can be generated in a CDK7-dependent manner. Since only the faster-migrating form binds to DIAP1, the idea is favored that the two isoforms differ in their N terminus. In one speculative model, the faster-migrating form represents the proteolytically processed form that exposes the critical N-terminal alanine, which is responsible for DIAP1 binding. Alternatively, it is also possible that the slower-migrating form undergoes a modification that inhibits DIAP1 binding (Morishita, 2013).

Recent studies indicated that dedicated trafficking machinery exists for other TA proteins destined for the endoplasmic reticulum. However, the equivalent trafficking factors for mitochondria-destined TA proteins have not yet been found, and it is widely assumed that these TA proteins insert into the mitochondrial outer membrane without active assistance. By contrast, the finding of this study indicates that Hid's mitochondrial localization can be regulated in cells, suggesting the existence of an active trafficking machinery for the mitochondrial TA protein (Morishita, 2013).

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).

Sex-specific apoptosis regulates sexual dimorphism in the Drosophila embryonic gonad

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).

Temporal patterning of neuroblasts controls Notch-mediated cell survival through regulation of Hid or Reaper

Temporal patterning of neural progenitors is one of the core mechanisms generating neuronal diversity in the central nervous system. This study shows that, in the tips of the outer proliferation center (tOPC) of the developing Drosophila optic lobes, a unique temporal series of transcription factors not only governs the sequential production of distinct neuronal subtypes but also controls the mode of progenitor division, as well as the selective apoptosis of Notch^OFF or Notch^ON neurons during binary cell fate decisions. Within a single lineage, intermediate precursors initially do not divide and generate only one neuron; subsequently, precursors divide, but their Notch^ON progeny systematically die through Reaper activity, whereas later, their Notch^OFF progeny die through Hid activity. These mechanisms dictate how the tOPC produces neurons for three different optic ganglia. It is concluded that temporal patterning generates neuronal diversity by specifying both the identity and survival/death of each unique neuronal subtype (Bertet, 2014).

Although apoptosis is a common feature of neurogenesis in both vertebrates and Drosophila, the mechanisms controlling this process are still poorly understood. For instance, several studies in Drosophila have shown that, depending on the context, Notch can either induce neurons to die or allow them to survive during binary cell fate decisions. This is the case in the antennal lobes where Notch induces apoptosis in the antero-dorsal projecting neurons lineage (adpn), whereas it promotes survival in the ventral projecting neurons lineage (vPN). In both of these cases, the entire lineage makes the same decision whether the Notch^ON or Notch^OFF cells survive or die. This suggests that, in this system, Notch integrates spatial signals to specify neuronal survival or apoptosis (Bertet, 2014).

This study shows that, during tOPC neurogenesis, neuronal survival is determined by the interplay between Notch and temporal patterning of progenitors. Indeed, within the same lineage, Notch signaling leads to two different fates: it first induces neurons to die, whereas later, it allows them to survive. This switch is due to the sequential expression of three highly conserved transcription factors-Dll/Dlx, Ey/Pax-6, and Slp/Fkh-in neural progenitors. These three factors have distinct functions, with Dll promoting survival of Notch^OFF neurons, Ey inducing apoptosis of Notch^OFF neurons, and Slp promoting survival of Notch^ON neurons. These data suggest that Ey induces death of Notch^OFF neurons by activating the proapoptotic factor hid. Thus, Dll probably antagonizes Ey activity by preventing Ey from activating hid. The data also suggest that Notch signaling induces neuronal death by activating the proapoptotic gene rpr. Thus, Slp might promote survival of Notch^ON neurons by directly repressing rpr expression or by preventing Notch from activating it. In both cases, the interplay between Notch and Slp modifies the default fate of Notch^ON neurons, allowing them to survive. Further investigations will test these hypotheses and determine how Dll, Ey, Slp, and Notch differentially activate/repress hid and rpr (Bertet, 2014).

Although the tOPC and the main OPC have related temporal sequences, their neurogenesis is very different. This difference is in part due to the fact that newly specified tOPC neuroblasts express Dll, which controls neuronal survival, instead of Hth. Why do tOPC neuroblasts express Dll? The tOPC, which is defined by Wg expression in the neuroepithelium, is flanked by a region expressing Dpp. Previous studies have shown that high levels of Wg and Dpp activate Dll expression in the distal cells of the Drosophila leg disc. Wg and Dpp could therefore also activate Dll in the neuroepithelium and at the beginning of the temporal series in tOPC progenitors. Another difference between the main OPC and tOPC neurogenesis is that Ey and Slp have completely different functions in these regions. Indeed, unlike in the main OPC, Ey and Slp control the survival of tOPC neurons. This suggests that autonomous and/or nonautonomous signals interact with these temporal factors and modify their function in the tOPC (Bertet, 2014).

Finally, tOPC neuroblasts produce neurons for three different neuropils of the adult visual system, the medulla, the lobula, and the lobula plate. This ability could be due to the particular location of this region in the larval optic lobes. Indeed, the tOPC is very close to the two larval structures giving rise to the lobula and lobula plate neuropils-Dll-expressing neuroblasts are located next to the lobula plug, whereas D-expressing neuroblasts are close to the IPC. Interestingly, Dll and D neuroblasts specifically produce lobula plate neurons. This raises the possibility that these neuroblasts and/or the neurons produced by these neuroblasts receive signals from the lobula plug and the IPC, which instruct them to specifically produce lobula plate neurons. These nonautonomous signals could also modify the function of Ey and Slp in the tOPC (Bertet, 2014).

In summary, this study demonstrates that temporal patterning of progenitors, a well-conserved mechanism from Drosophila to vertebrates, generates neural cell diversity by controlling multiple aspects of neurogenesis, including neuronal identity, Notch-mediated cell survival decisions, and the mode of intermediate precursor division. In the tOPC temporal series, some factors control two of these aspects (Ey), whereas others have a specialized function (Dll, Slp, and D). This suggests that temporal patterning does not consist of a unique series of transcription factors controlling all aspects of neurogenesis but instead consists of multiple superimposed series, each with distinct functions (Bertet, 2014).

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).

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).

Distinct mechanisms of apoptosis-induced compensatory proliferation in proliferating and differentiating tissues in the Drosophila eye

In multicellular organisms, apoptotic cells induce compensatory proliferation of neighboring cells to maintain tissue homeostasis. In the Drosophila wing imaginal disc, dying cells trigger compensatory proliferation through secretion of the mitogens Decapentaplegic (Dpp) and Wingless (Wg). This process is under control of the initiator caspase Dronc, but not effector caspases. This study shows that a second mechanism of apoptosis-induced compensatory proliferation exists. This mechanism is dependent on effector caspases which trigger the activation of Hedgehog (Hh) signaling for compensatory proliferation. Furthermore, whereas Dpp and Wg signaling is preferentially employed in apoptotic proliferating tissues, Hh signaling is activated in differentiating eye tissues. Interestingly, effector caspases in photoreceptor neurons stimulate Hh signaling which triggers cell-cycle reentry of cells that had previously exited the cell cycle. In summary, dependent on the developmental potential of the affected tissue, different caspases trigger distinct forms of compensatory proliferation in an apparent nonapoptotic function (Fan, 2008).

In developing wing discs in which apoptosis was induced by expression of the pro-apoptotic gene hid, loss of the caspase inhibitor DIAP1, or by X-ray treatment, the accumulation of two major mitogens, Dpp and Wg, has been observed in dying cells. Key for this finding is the simultaneous expression of the caspase inhibitor P35. Under these conditions, the dying cells were kept alive ('undead'), allowing accumulation of Dpp and Wg. This accumulation appears to be dependent on the initiator caspase Dronc, because it cannot be blocked by expression of P35 which inhibits effector caspases but not Dronc. In addition, the Drosophila homolog of the tumor suppressor p53, Dp53, has been implicated downstream of Dronc for compensatory proliferation. Notably, these studies on mechanisms of compensatory proliferation were carried out in developing larval wing imaginal discs in Drosophila. Cells in wing discs proliferate extensively during larval stages, and the majority of these cells does not differentiate before they reach pupal development. Hence, the mechanisms of compensatory proliferation have so far only been investigated in situations where most cells are proliferating. Interestingly, apoptosis-induced compensatory proliferation in differentiating eye tissue of third-instar larvae. However, it is unclear whether this form of compensatory proliferation is controlled by a similar mechanism as reported for larval proliferating wing discs (Fan, 2008).

This study revealed that there are at least two distinct mechanisms that promote compensatory proliferation in response to apoptotic activity. The general difference between these two mechanisms lies in the developmental context of the tissue in which compensatory proliferation occurs. In proliferating wing and eye tissues, compensatory proliferation induced by extensive apoptosis is dependent on Dronc and Dp53, which induce Dpp and Wg expression. In contrast, in differentiating eye tissue, apoptosis induces compensatory proliferation through a novel mechanism requiring the effector caspases DrICE and Dcp-1, which induce Hh signaling in a nonapoptotic function (Fan, 2008).

When cells stop proliferating and become committed to adopt cell fate, dramatic changes in gene expression are occurring. Given these changes in developmental plasticity, it is not surprising that distinct mechanisms of apoptosis-induced compensatory proliferation are employed in proliferating versus differentiating tissues. However, it should be noted that the proliferating capacity of differentiating tissues is rather restricted. In GMR-hid eye discs, although hid is expressed in all cells posterior to the MF, compensatory proliferation occurs only in cells that are still undifferentiated. Yet, even though they are undifferentiated they have withdrawn from the cell cycle and, under normal developmental conditions (i.e., without GMR-hid), they would soon be recruited to adopt cell fate. However, the apoptotic environment causing increased Hh signaling appears to be able to trigger reentry of these cells into the cell cycle (Fradkin, 2008).

Interestingly, the Hh signal is specifically increased in photoreceptor neurons requiring a nonapoptotic activity of effector caspases. Hh signaling can then nonautonomously induce proliferation of undifferentiated cells at the basal side of the eye disc. However, overexpression of Hh posterior to the MF in wild-type eye discs alone is not sufficient to induce a comparable wave of compensatory proliferation as in GMR-hid eye discs. This suggests that cell-cycle reentry requires activation of additional factors/pathways stimulated in apoptotic cells (Fradkin, 2008).

Although hid can stimulate increased Hh expression in photoreceptor neurons throughout the posterior half of the eye disc, compensatory proliferation is restricted to a certain distance (six to ten ommatidial columns) from the MF. This corresponds to approximately 6-15 hr of developmental time, and might be the time required for cell-cycle reentry. Similarly, when mammalian cells that have exited the cell cycle are stimulated to reenter the cell cycle, they need about 8 hr to do this. The reason for this delay is unknown. Studying compensatory proliferation in GMR-hid eye discs might provide a genetic model to address this interesting problem (Fradkin, 2008).

It is not clear whether this novel effector caspase-, Hh-dependent pathway of compensatory proliferation also applies to other, or even all, differentiating tissues. However, what this study shows is that there are at least two distinct mechanisms of apoptosis-induced compensatory proliferation. It is also possible that other mechanisms of compensatory proliferation in different developmental contexts are going to be uncovered in the future. Interestingly, in developing larval wing discs, P35-dependent compensatory proliferation has been implicated in cell competition. This suggests that, even in tissue with the same developmental potential, compensatory proliferation can occur with distinct mechanisms (Fradkin, 2008).

How cells sense different developmental contexts and operate distinct proliferating mechanisms in response to apoptotic stress is unknown. Specifically, where is the specificity and selectivity for distinct caspases coming from in tissues of different developmental potential? What are the mechanisms engaged by these caspases to trigger secretion of either Dpp and Wg or Hh? These are questions which need to be addressed in the future (Fan, 2008).

This study has several implications for tumorigenesis. First, many tumors develop when quiescent cells reenter the cell cycle. The mechanisms for cell-cycle reentry are largely unknown. Second, evasion from apoptosis is a hallmark of cancer. Many tumor cells are induced to undergo apoptosis. However, they do not die, because they downregulate essential components of the apoptotic pathway such as Apaf-1 and caspases. Thus, these undead tumor cells might secrete mitogens which might induce compensatory proliferation similar to the Drosophila case. In this way, undead cells might contribute to the growth of the tumor. A similar argument can be made for chemotherapy, which in many cases attempts to activate the apoptotic program in a tumor cell. If the death of the tumor cell is blocked, or slow, mitogens might be produced and the tumor growth could be even more severe. This is very obvious in the apoptotic wing or anterior eye discs in Drosophila when apoptosis is blocked by P35. Under these conditions, overgrown wing and eye tissues are observed. Thus, evasion of apoptosis might directly contribute to tumor growth. Finally, although increased Hh signaling can lead to various cancers, how Hh induces cellular proliferation and tissue overgrowth is not well understood. Mutations in Patched1, a negative regulator of sonic Hh, frequently give rise to human tumors. The exact cause is unknown. These data imply that Hh signaling might be involved in cell-cycle reentry allowing cells to resume proliferation (Fan, 2008).

Coupling of apoptosis and L/R patterning controls stepwise organ looping

Handed asymmetry in organ shape and positioning is a common feature among bilateria (for a review see Huber, 2007), yet little is known about the morphogenetic mechanisms underlying left-right (LR) organogenesis. This study utilized the directional 360° clockwise rotation of genitalia in Drosophila to study LR-dependent organ looping. Using time-lapse imaging, it was shown that rotation of genitalia by 360° results from an additive process involving two ring-shaped domains, each undergoing 180° rotation. The results show that the direction of rotation for each ring is autonomous and strictly depends on the LR determinant myosin ID (MyoID: Myo31DF). Specific inactivation of MyoID in one domain causes rings to rotate in opposite directions and thereby cancels out the overall movement. A specific pattern of apoptosis at the ring boundaries is revealed, and this study also shows that local cell death is required for the movement of each domain, acting as a brake-releaser. These data indicate that organ looping can proceed through an incremental mechanism coupling LR determination and apoptosis. Furthermore, they suggest a model for the stepwise evolution of genitalia posture in Diptera, through the emergence and duplication of a 180° LR module (Suzanne, 2010).

Left-right (LR) asymmetric development is essential to the morphogenesis of many vital organs, such as the heart. Directional looping of LR organs is a complex morphogenetic process relying on proper coordination of early LR patterning events with late cell-tissue behaviors. In vertebrates, several developmental models have been proposed for gut coiling downstream of the Nodal-Pitx2 regulatory pathway, including intrinsic asymmetric elongation of the gut in Xenopus or extrinsic force generation by mesenchymal tissue in Zebrafish and by dorsal mesentery in the chick and mouse embryos. However, the cellular mechanisms underlying LR organ morphogenesis are mostly unknown (Suzanne, 2010).

In Drosophila, directional clockwise (or dextral) rotation of the genital plate and gut has been shown only recently to be controlled by the LR determinant myosin ID (MyoID). In myoID mutant flies, LR morphological markers are inverted, leading to counterclockwise (or sinistral) looping of the genital plate, spermiduct, gut, and testis. This indicates that myoID is a unique situs inversus gene in Drosophila. Intriguingly, the expression of MyoID is restricted to two rows of cells within the A8 segment of the genital disc (the analia and genitalia precursor), with one row of expression in the anterior compartment (A8a) and the other in the posterior compartment (A8p) (Suzanne, 2010).

Removal of myoID function specifically in the A8 segment is sufficient to provoke the complete inversion of rotation (360° counterclockwise) of the genitalia and sinistral looping of the spermiduct to which it is attached. The A8 segment therefore represents a LR organizer controlling the directional rotation of the whole genitalia in Drosophila (Suzanne, 2010).

Because circumrotation (the process of 360° rotation) may result from a number of different morphogenetic processes, not deducible from the simple observation of the final adult phenotype, a new and innocuous imaging method was developed to follow the rotation in living pupae (Suzanne, 2010).

To be able to analyze the movement of distinct domains in live developing genitalia, time-lapse imaging was coupled with genital disc 'painting' by expressing different fluorophores in various regions of the genitalia precursor. Analysis of wildtype live genitalia through this method revealed their spatial and temporal organization during rotation. It was first determined that rotation begins at around 25 hr after puparium formation (APF) and lasts 12-15 hr. At 25 hr APF, the genital disc is organized into concentric rings, which, from anterior to posterior, include an A8a ring, an A8p ring, and a large central disc composed of A9-A10 tissues. The analysis of rotation in live pupae coupled to manual tracking allowed the identification of two distinct moving domains: a large posterior domain comprising A8p-A9-A10 (hereafter referred to as A8p) and a smaller anterior domain made of A8a. The A8p domain moves first and is followed by A8a, which starts moving later on. During the entire process, cells from the abdomen, to which the genital disc is connected, remain immobile. The finding of two rotating domains, A8a and A8p, was unexpected. It reveals a complex rotational activity of the genitalia and rules out a simple model in which the genital plate would rotate by 360° as a whole. To further understand how rotation occurs, timelapse imaging of the full, 15-hr-long rotation was performed. This analysis revealed that each ring had a different rotational activity. When viewed from the posterior pole, the A8p ring undergoes 360° clockwise rotation, while the A8a ring makes a 180° clockwise rotation. Whereas the rotation of the central part (A8p-A10) of the disc was inferred from the looping of the spermiduct around the gut, the 180° rotation of A8a was not predicted and could only be revealed by time-lapse analysis because this compartment solely gives rise to a tiny and colorless part of the cuticle. Altogether, these in vivo analyses show that rotation of genitalia in Drosophila is a composite process involving two compartments of the A8 segment, A8a and A8p, each expressing a row of MyoID at its anterior boundary and having its own rotational behavior (Suzanne, 2010).

These findings raise the questions of the contribution of each of the two rings to the entire rotation and of how they interact during rotation. In order to address this question, the intrinsic or real rotational activity of A8a and A8p was determined. So far, each ring movement was analyzed relative to the same immobile referential: the abdomen. Although this referential allows the real movement of A8a to be determined, it cannot be used to determine that of A8p, because A8p moves relatively to a mobile referential, i.e., A8a, to which it is attached. To determine the real movement of A8p, it is thus essential to analyze its angular movement relative to A8a, in other words A8a contribution to motion must be subtracted from the apparent A8p movement. To do so, movies were analyzed by setting A8a as a referential and by determining the angular movement of A8p. Reassessing A8p movement through this approach revealed that A8p rotates clockwise only by 180° relative to A8a. The new angular velocity curve of A8p fits almost perfectly with that of A8a, indicating that both movements have similar features. Importantly, these data also indicate that the observed 360° clockwise rotation is the result of a composite process involving two additive 180° clockwise components: a 180° rotation of the A8a relative to the abdomen and an 180° rotation of A8p relative to A8a (Suzanne, 2010).

To further determine the autonomy of each ring relatively to the other, the role of the LR determinant MyoID in this process was dissected by specifically inactivating myoID in either A8a or A8p or in both. By convention, the presence or absence of myoID is represented by a + or - sign, respectively. Accordingly, the wild-type context is noted 'A8a+A8p+' and the myoID mutant 'A8a-A8p-.' Upon specific inactivation of myoID in the A8a domain (A8a-A8p+ context), the adults showed an apparent 'nonrotation phenotype' (0°, no spermiduct looping and genitalia correctly oriented). However, time-lapse imaging revealed that both rings were spinning, although in opposite directions: the A8a domain rotated counterclockwise by 180° (-180°), whereas the A8p domain rotated clockwise by 180° (+180°, real movement). Reciprocally, the inactivation of myoID in the A8p domain (A8a+A8p- context) also led to an apparent nonrotation phenotype. In this context, the behavior of each domain was inverted compared to the previous condition, with the A8a domain rotating clockwise by 180° (+180°) whereas the A8p domain rotated anticlockwise by 180° (-180°, relative or real movement). In both cases, the movement of each ring is consistent with its myoID genotype and the 'dextralizing' activity of this gene. The strict dependence on MyoID for the direction of the rotation is further confirmed in flies where both A8a and A8p were mutants for myoID (myoID^k1). The rotation is often incomplete in this genotype because of the hypomorphic nature of the myoID^k1 allele analyzed; however, both domains show an anticlockwise movement. Therefore, in all genetic contexts analyzed, all parameters of the rotation remain unaffected except the direction of rotation, as illustrated by the perfect mirror image of the angular velocity curves (Suzanne, 2010).

These experiments reveal that each ring adopts an independent 180° movement relative to more anterior structures (A8a relative to the abdomen and A8p relative to A8a): clockwise in the presence of MyoID, anticlockwise in its absence. When both movements are unidirectional, the net rotation is circumrotation (± 360°), whereas upon opposite movements of A8a and A8p, the net rotation is zero (0°), leading to an apparent nonrotation phenotype. Therefore, the net rotation (or apparent rotation = R) can be modeled through a simple equation in which R equals the addition of A8a and A8p movements, with MyoID acting as a sign function (Suzanne, 2010).

It was next of interest to characterize potential cellular mechanisms acting downstream of LR determination during genitalia rotation. In particular, the cellular events responsible for uncoupling rings at the onset of their rotation was determined. Initial insights came from blocking apoptosis, which leads to genitalia rotation defects, but the role of apoptosis in the process is not completely understood. To determine the morphogenetic function of the apoptotic pathway during genitalia rotation, the spatial and temporal requirements for apoptosis were first characterized by analyzing the expression pattern of hid and reaper (rpr) in the genital disc, using two reporter lines. Both reporters were strongly expressed in the A9 and A10 segments. However, in the A8 segment, only hid expression is observed. This coincides with the phenotype of misrotated genitalia observed specifically when hid function is altered but not in rpr mutants. Then the pattern and timing of cell death was determined in the genital disc. To do so, nuclear fragmentation was followed, and an in vivo reporter of caspase activation (the apoliner construct) was used. At the onset of rotation, a large number of apoptotic cells was detected on the most ventral part of the genital disc, first within the A8p ring bordering A8a, coinciding with the beginning of A8p movement. These data indicate an overlap between the apoptotic field and the domain of MyoID expression. These results have been further confirmed by the detection of apoptotic cells by TUNEL staining of fixed pupal genital discs. Later on, a new wave of apoptosis was detected in the most anterior part of the A8a ring, at the junction between A8a and the abdomen. In contrast, only marginal if any apoptosis was detected before and at the end of rotation. Therefore, two waves of cell death are taking place in the A8 segment, coinciding spatially and temporally with the rotation of A8a and A8p rings (Suzanne, 2010).

Given that rings are initially part of the same epithelium and move independently later, it was reasoned that local cell death may be a mechanism to provide the degree of liberty necessary for proper movement. To test this hypothesis, cell death was inhibited in each compartment separately by expressing the caspase inhibitor p35. Interestingly, inactivation of apoptosis in either A8p or A8a leads to a similar phenotype, with flies showing a high proportion of half-rotated genitalia (180° rotation), suggesting that rotation was blocked in the ring deficient for apoptosis. This has been further demonstrated by following the rotation process in vivo, when apoptosis is specifically blocked in the A8a. In this genetic context, the A8a ring stayed mostly still during the whole process, whereas A8p rotated normally. The resulting 180° rotation is thus exclusively due to the movement of one ring, i.e., A8p, in which apoptosis is unaffected. Inhibiting apoptosis in both domains strongly aggravates the phenotype, with 40% of the flies showing nonrotated genitalia (0°), suggestive of an additive phenotype. The rest of the population had 90° rotated genitalia, which may be due to incomplete inhibition of apoptosis. Alternatively, it is possible that some rotation occurs without apoptosis thanks to tissue elasticity. In any case, the results indicate that cell death is required in each ring for separating them from the neighboring tissues and allowing their free rotation. Consistently, nuclei fragmentation and cell death occur normally in a myoID mutant background. Because local cell death is not likely to provide a direct force for rotation, it is proposed that it contributes to the release of rings from neighboring tissues (Suzanne, 2010).

This study has revealed that organ looping can proceed through discrete steps, breaking down circumrotation into the simple building blocks of 180° each. The incremental nature of genitalia rotation is indeed based upon two 180° LR modules, sharing identical angular velocity and range as well as requirement for MyoID and apoptosis. Modularity in morphogenesis provides interesting control mechanisms (through addition or substraction) and therefore plasticity to the process, both at the organism level and during evolution. Entomologists have described different patterns of genitalia rotation in Diptera, ranging from 0° to 360°, that evolved together with changes in mating position. Interestingly, in the Brachycera suborder, to which Drosophilidae belong, we notice that most ancestral species have a nonrotated genitalia (Stratiomyomorpha and Tabanomorpha), whereas 180° and 360° rotation have appeared progressively later in evolution (in Muscomorpha and Cyclorrhapha, respectively). Together with this sequential organization of rotation amplitude in the phylogenetic tree, these data strongly support a model by which the 360° rotation observed in Brachycera ('modern Diptera') would result from the emergence (transition from 0
to 180°) and duplication (transition from 180° to 360°) of a 180° L/R module (Figure S3), thus providing a simple additive model for both the origin of circumrotation and the evolution of genitalia rotation and mating position. However, it should be noted that alternative mechanisms maylead to a similar pattern of genitalia rotation among Diptera (Suzanne, 2010).

The incremental model presented here also offers a solution to the apparent paradox of circumrotation and the question of its elusive utility, illustrated by the fact that both 360° rotation and the absence of rotation lead to the same final posture of genitalia. A facultative role of 360° rotation is further supported by the finding that D. melanogaster males with nonrotating genitalia (A8a-A8p+ or A8a+A8p-) are normally fertile (data not shown). An incremental origin of 360° rotation in which a second half-turn would be added to the existing 180° rotation would well explain this paradox. Thus, circumrotation can be viewed as recapitulating the evolutionary history of genitalia rotation in Brachycera, and its logic would reveal a case of 'retrograde evolution,' in which duplication of a functional module is used to revoke a previous evolutionary step (Suzanne, 2010).

Finally, this analysis of genitalia rotation highlights a new mechanism of morphogenesis relying on a combination of LR patterning and apoptosis. In this process, a new role for apoptosis is revealed as a releasing mechanism allowing the sliding of two parts of an organ. It will be interesting to test in the future whether this releasing role of apoptosis is used more generally, in other morphogenetic movements requiring important cellular rearrangement (Suzanne, 2010).

EFFECTS OF MUTATION

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 hid expression to develop the final phenotypes. Moreover, many gene disruptions in mice result in inappropriate cell death in the tissue that requires the function of the disrupted gene, suggesting that similar mechanisms might exist in mammalian development. Finally, cell death may be an important contributing factor to human congenital birth defects. Thus, an understanding of the underlying mechanisms is of general interest (Werz, 2005).

Interestingly, not all segment polarity mutants analyzed induce hid expression and cell death. Embryos mutant for patched, which encodes the hedgehog receptor, were not found to express hid and do not exhibit increased cell death, although hedgehog mutants both upregulate hid and contain increased amounts of cell death. The reasons for these differences are not known, but partial redundancy might account for lack of hid expression in patched mutants. The Drosophila genome encodes another patched homolog, patched-related, which might provide the survival requirement for mis-specified cells in patched mutants (Werz, 2005).

Mis-specified cells in bicoid and oskar mutants induce expression of hid. No increased reaper or grim expression was observed in these mutants. However, expression of reaper has been reported in crumbs mutants, which affect epithelial integrity. X-ray-treated embryos also preferentially respond by upregulation of reaper, rather than hid. Although crumbs mutants were not analyzed for hid expression, it appears that cells contain several developmental checkpoints, which activate different cell death-inducing regulators depending on the type of abnormal cellular development (Werz, 2005 and references therein).

Mis-specified cells can survive if an alternative survival pathway is provided. The example presented here is the acron into telson transformation in bicoid mutants, which is mediated by the torso signaling pathway. Although the cells giving rise to telson structures at the anterior tip are mis-specified based on Abd-B-labeling experiments, they survive because they receive a survival signal from the torso signaling system. In this case, transformation rather than cell death is favored. It has been shown that activation of the Ras/Mapk pathway protects cells from hid-induced apoptosis, both by transcriptional repression of hid and by phosphorylation of Hid protein by Mapk. Because Torso, which encodes a receptor tyrosine kinase (RTK), is known to activate Ras and Mapk, tests were performed to see whether manipulation of active Mapk levels using a gain-of-function allele, Mapk^Sem, can suppress hid expression and cell death in bicoid mutants. However, this was found not to be the case. Thus, torso appears to protect mis-specified cells independently of Mapk activation (Werz, 2005).

The hid bicoid double mutant analysis reveals that the transformation of anterior into posterior identity expands beyond the telson, and that this expansion undergoes hid-induced cell death in bicoid single mutants. The rescued cells secrete larval cuticle elements, suggesting that mis-specified cells have the developmental capacity to terminally differentiate. However, in hid⁺ background, they instead die, presumably because equivalent survival signals are lacking. It is proposed that mis-specified cells undergo cell death if no alternative survival pathway is provided to protect them (Werz, 2005).

An alternative survival mechanism might also operate in other developmental mutants where transformation rather than cell death occurs. Mutations in the sev RTK and its ligand boss result in transformation of the R7 photoreceptor cell into a non-neuronal cone cell. Survival of this cell could be mediated by the Drosophila Egf receptor (Egfr), another RTK, which is required to maintain cell survival in the developing eye disc. Accordingly, activation of the Ras/Mapk pathway by Egfr would inhibit hid expression and support survival of the presumptive R7 photoreceptor cell. This interpretation is also consistent with observations that egfr^- clones are small and undergo cell death, and that this death can be suppressed in hid mutants. Thus, transformation of the R7 photoreceptor to a cone cell rather than R7 cell death in sev and boss mutants could occur because of survival signaling by the Egfr (Werz, 2005).

The hid bicoid double mutant analysis suggests that mis-specified cells can continue to develop and differentiate. Yet, they die. Presumably, this cell death protects the organism from potentially dangerous cells. For example, it is conceivable that in mammals, surviving mis-specified cells might lie dormant in the host organism for years. During this time, they might acquire additional genetic alterations that could drive the progressive transformation of these cells into malignant cancer. In wild-type embryos, mis-specification probably occurs in cells in isolation, and elimination of these cells does not interfere with development and survival of the organism. Only in extreme situations, such as the patterning mutants analyzed in this study, is the mis-specification caused by aberrant development so severe that the affected organism dies (Werz, 2005).

The cause of mis-specification in each segmentation mutant is different. Usually, the expression of other segmentation genes is shifted and expanded, resulting in flattened gradients. Yet, irrespective of the cause of mis-specification, most of these mutants have in common that they induce hid expression. It is currently unknown how the mis-specified fate of cells is recognized, and how hid expression is induced. One possibility might be that the protein gradients established by bicoid⁺ and oskar⁺, as well as other segmentation genes are used as readout for proper cellular specification. The steepness of protein gradients as a means to determine life or death decisions has recently been proposed. Such a model would imply that cells are able to determine their position in a graded field and compare this readout with their neighbors. Because in bicoid and oskar mutants these gradients do not form, the concentration difference between neighboring cells would be zero. If the concentration difference between two neighboring cells is below a crucial threshold, they induce the expression of hid and undergo cell death. This model could also explain embryonic pattern repair, which was described in embryos that express six copies of the bicoid gene. In these embryos, the head and thorax primordia are expanded because of the presence of six copies of bicoid. However, this expansion is corrected for by induction of cell death, and relatively normal larvae develop. In this case, the Bicoid protein gradient does form, but would be flatter compared with wild type. Thus, the concentration difference between neighboring cells would be below a critical threshold, sufficient to induce hid-dependent cell death. However, it is largely unknown how cells compare their position in a graded field with those of their neighbors. It has been proposed that short-range cell interactions mediated via the cell-surface proteins Capricious and Tartan provide cues that support cell survival during wing development. Cells unable to participate in these interactions are eliminated by cell death. It is unclear, however, whether short-range interactions are sufficient to explain the cell death phenotype in bicoid and oskar mutants (Werz, 2005).

Irrespective of the underlying mechanism for sensing mis-specification, the current results highlight the role of an active gene-directed process that removes mis-specified cells during development. However, if a survival mechanism is provided, mis-specified cells can survive and adopt a different fate. In wild-type embryos, mis-specification probably occurs in cells in isolation, and hence is difficult to study. However, in bicoid and oskar mutants, large regions of neighboring cells are mis-specified and undergo cell death simultaneously, providing a unique opportunity to clarify the signals that initiate cell death in situations where cells are developmentally mis-specified (Werz, 2005).

Ionizing radiation induces caspase-dependent but Chk2- and p53-independent cell death in Drosophila melanogaster

Ionizing radiation (IR) can induce apoptosis via p53, which is the most commonly mutated gene in human cancers. Loss of p53, however, can render cancer cells refractory to therapeutic effects of IR. Alternate p53-independent pathways exist but are not as well understood as p53-dependent apoptosis. Studies of how IR induces p53-independent cell death could benefit from the existence of a genetically tractable model. In Drosophila, IR induces apoptosis in the imaginal discs of larvae, typically assayed at 4-6 hr after exposure to a LD50 dose. In mutants of Drosophila Chk2 or p53 homologs, apoptosis is severely diminished in these assays, leading to the widely held belief that IR-induced apoptosis depends on these genes. This study shows that IR-induced apoptosis still occurs in the imaginal discs of chk2 and p53 mutant larvae, albeit with a delay. This phenomenon is a true apoptotic response because it requires caspase activity and the chromosomal locus that encodes the pro-apoptotic genes reaper, hid, and grim. Chk2- and p53-independent apoptosis is IR dose-dependent and is therefore probably triggered by a DNA damage signal. It is concluded that Drosophila has Chk2- and p53-independent pathways to activate caspases and induce apoptosis in response to IR. This work establishes Drosophila as a model for p53-independent apoptosis, which is of potential therapeutic importance for inducing cell death in p53-deficient cancer cells (Wichmann, 2006).

The Drosophila homologs of Chk2 and p53 are required, not for induction of apoptosis, but for timely induction of apoptosis in response to irradiation. Radiation-induced cell death still occurs in chk2 and p53 mutants, albeit with a delay. Four lines of evidence support the idea that this delayed cell death is apoptosis rather than necrosis: (1) it is detected by staining with AO, which has been shown to stain apoptotic but not necrotic cells; (2) it accompanies activation of caspases, a hallmark of apoptosis but not necrosis; (3) it requires caspase activity, which is required for apoptosis but not necrosis, and (4) it requires the chromosomal locus encoding the proapoptosis genes rpr, hid, and grim, whose protein products are required to inhibit DIAP1 and activate caspases. These results indicate that there is a Chk2-/p53-independent pathway that commits damaged cells to apoptosis and utilizes many of the same downstream components as the Chk2-/p53-dependent apoptosis pathway (Wichmann, 2006).

Two lines of evidence support the idea that DNA damage is the signal that induces Chk2-/p53-independent apoptosis after exposure to ionizing radiation. First, the amount of Chk2-/p53-independent apoptosis appears to increase with IR dose. This dose dependence suggests that the amount of DNA damage is what induces Chk2-/p53-independent apoptosis but does not rule out the contribution of other damages that result from IR. Second, higher levels of Chk2-/p53-independent apoptosis are observed when the ability to repair DNA is compromised, as in mei-41, p53 double mutants. Collectively, these data suggest that DNA damage caused by x-rays induces Chk2-/p53-independent apoptosis (Wichmann, 2006).

IR-induced apoptosis in chk2 and p53 mutants shows a temporal delay. IR-induced apoptosis is also delayed in H99 heterozygotes, possibly because H99 heterozygotes contain half the gene dose of the proapoptotic Smac/Diablo orthologs and it may take longer for the proapoptotic gene products to accumulate to the point of an apoptosis-stimulating threshold. IR induced increase in the transcripts of rpr and hid, two of the H99-encoded genes, still occurred in chk2 (rpr and hid) and p53 (hid) mutants, but to lower levels (for rpr) and after a delay. Therefore, apoptosis may be delayed in chk2 and p53 mutants because proapoptotic gene products take longer to accumulate to a threshold in the absence of Chk2 or p53 regulation. The data showing that IR-induced apoptosis is further delayed in a chk2, H99/+ double mutant, compared with a chk2 single mutant, support this claim. Furthermore, the results suggest the existence of at least another signaling pathway that does not operate through Chk2 or p53, but nonetheless links the same signal (DNA damage) to a similar outcome (accumulation of H99-encoded gene products) (Wichmann, 2006).

RT-PCR experiments revealed interesting differences in the identity and onset of induction of proapoptotic genes in chk2 and p53 mutants. rpr and hid are induced at 4 hr after irradiation in chk2 mutants, whereas hid and skl are induced between 12 and 18 hr after irradiation in p53 mutants. The basis for these differences is not understood. More detailed time courses as well as deletion analysis of the H99 locus to determine the contribution of each proapoptotic gene to Chk2-/p53-independent apoptosis needs to be performed to address these issues (Wichmann, 2006).

The data presented in this study establish Drosophila as a model for studying p53-independent apoptosis. p53 is the most commonly mutated gene in human cancers. Loss of p53 poses an immense clinical problem because p53-deficient cancer cells no longer stimulate p53-dependent apoptosis in response to radiation or genotoxic chemotherapy drugs. In this scenario, p53-independent apoptotic pathways become key for inducing cancerous cells to die because they provide potential therapeutic targets. In mammals, a p53-independent apoptosis pathway that is mediated by p73, another member of the p53 family, has been identified. In Drosophila, Dmp53 is the only known p53 family member. Therefore, the p53-independent apoptosis that was identified and characterized in this article is likely to represent a previously unknown process. An important goal in the future will be to dissect the Chk2-/p53-independent pathway that links DNA damage to the proapoptotic genes of the H99 locus (Wichmann, 2006).

Several candidates were tested and eliminated as regulators of Chk2-/p53-independent cell death. Mei-41 (ATR) is not required for Chk2-/p53-independent cell death because mei-41, p53 double mutants actually exhibit more cell death than p53 alone. Recent work showed that ectopic induction of eiger, a TNF ligand homolog, can induce apoptosis in Drosophila. Chk2-/p53-independent cell death still occurs in p53, eiger double mutants, suggesting that the TNF pathway is not involved in the induction of cell death characterized in this study. Work in mammalian cells showed that overexpression of c-Myc can induce p53-independent apoptosis. Chk2-/p53-independent apoptosis still occurs in Dmyc, p53 double mutants, indicating that Dmyc is not required for this response (Wichmann, 2006).

A classical genetic screen may identify components of the Chk2-/p53-independent apoptosis pathway, as well as testing more candidates, such as the transcription factor de2f1, grapes (DmChk1), DmATM, and genes required for autophagy. Autophagic cell death, in which a cell lyses itself, occurs during Drosophila metamorphosis to lyse polyploid tissues such as the salivary glands and the fat body and provide nutrients for diploid cells of the imaginal discs; autophagy has been described in larvae only in the polyploid cells and only in response to starvation. Nonetheless, autophagy shares characteristics with apoptosis, including being detectable by AO staining and being dependent on caspases and the H99 locus, and for this reason remains a formal possibility (Wichmann, 2006).

In conclusion, studies have shown that IR-induced apoptosis in two key models for apoptosis, C. elegans and Drosophila, depends on p53. This study has provided evidence that, contrary to the accepted view, Chk2 and p53 are not required for radiation-induced cell death in Drosophila. Furthermore, normal timing of apoptosis that depends on Chk2 and p53 is also not required for ensuring survival after irradiation. Radiation-induced cell death that is independent of Chk2 and p53 depends on radiation dose, has characteristics of apoptosis and is likely to rely on a novel mechanism(s) because no other members of the p53-family are known in Drosophila. This work is the first to establish Drosophila as a model for p53-independent apoptosis. Identification of genes required for Chk2-/p53-independent cell death in Drosophila is of potential therapeutic value because protein products of their human homologs may represent novel targets that can be activated clinically to eliminate p53-deficient cancer cells (Wichmann, 2006).

vps25 mosaics display non-autonomous cell survival and overgrowth, and autonomous apoptosis

Appropriate cell-cell signaling is crucial for proper tissue homeostasis. Protein sorting of cell surface receptors at the early endosome is important for both the delivery of the signal and the inactivation of the receptor, and its alteration can cause malignancies including cancer. In a genetic screen for suppressors of the pro-apoptotic gene hid in Drosophila, two alleles of vps25, a component of the ESCRT machinery required for protein sorting at the early endosome, were identified. Paradoxically, although vps25 mosaics were identified as suppressors of hid-induced apoptosis, vps25 mutant cells die. However, evidence is provided that a non-autonomous increase of Diap1 protein levels, an inhibitor of apoptosis, accounts for the suppression of hid. Furthermore, before they die, vps25 mutant clones trigger non-autonomous proliferation through a failure to downregulate Notch signaling, which activates the mitogenic JAK/STAT pathway. Hid and JNK contribute to apoptosis of vps25 mutant cells. Inhibition of cell death in vps25 clones causes dramatic overgrowth phenotypes. In addition, Hippo signaling is increased in vps25 clones, and hippo mutants block apoptosis in vps25 clones. In summary, the phenotypic analysis of vps25 mutants highlights the importance of receptor downregulation by endosomal protein sorting for appropriate tissue homeostasis, and may serve as a model for human cancer (Herz, 2006).

The inactivation of signaling pathways is as important for appropriate tissue homeostasis as its activation. Interference with the inactivation process often gives rise to malignant phenotypes, including cancer. Several strategies to restrict signaling exist, including receptor sequestration, receptor inactivation, production of inhibitory signaling proteins and inactivation of intracellular signaling proteins. The phenotypic analysis of vps25 mutants highlights the importance of receptor downregulation by endosomal protein sorting. Lack of vps25 function causes at least three phenotypes: non-autonomous proliferation, non-autonomous resistance to cell death and autonomous apoptosis. The cause of these phenotypes and the potential role of class E Vps proteins for tumorigenesis will be discussed (Herz, 2006).

Vps25 is a component of the ESCRT-II complex required for internalization of cell surface receptors into MVBs at the early endosome. The signal for protein sorting into MVBs is provided by mono-ubiquitylation. In yeast, vps25 mutants cause aberrant endosomal structures and the accumulation of ubiquitylated proteins. A similar phenotype in vps25 clones in Drosophila, suggesting the conserved function of vps25 (Herz, 2006).

The lack of appropriate protein sorting at early endosomes in vps25 clones causes the accumulation of cell surface receptors including N and Dl. Genetic analysis using a dominant-negative N transgene (N^DN) suggests that the strong overgrowth phenotype of vps25 mosaics is largely due to inappropriate N signaling, which is known to induce proliferation non-autonomously through activation of the JAK/STAT pathway (Herz, 2006).

It is unclear whether N exerts this function in a ligand-dependent manner. Dl protein also accumulates in vps25 clones, and endocytosis of Dl is required for N activation. Thus, blocking MVB formation in vps25 clones may lead to the accumulation of active Dl, resulting in increased N activity. However, it was also shown that N is required for Dl accumulation in vps25 clones. Therefore, Dl accumulation is either directly or indirectly the result of increased N activity in vps25 clones. This conclusion infers that N activation occurs before Dl accumulation and would argue in favor of a ligand-independent mechanism for N activation in vps25 clones, although Dl may be required for maintaining N activity. N activity is also controlled by several proteolytic cleavages, which lead to translocation of the intracellular domain of N to the nucleus where it regulates the expression of target genes. Thus, a potential ligand-independent mode of N activation may include inappropriate cleavage of N at the vps25 endosome. Further studies are needed to clarify this point (Herz, 2006).

Mutations in erupted, the vps23 homolog that encodes a component of ESCRT-I, give rise to similar phenotypes to those observed for vps25. However, in hrs mosaics in Drosophila, non-autonomous cell proliferation has not been observed, although signaling receptors including N and Dl accumulate in hrs clones. This is a puzzling observation as hrs encodes a class E Vps protein acting immediately upstream of the ESCRT complexes. It is possible that N and Dl are not in an environment in the hrs endosome that permits signaling. Alternatively, it has been shown showed that hrs controls the steady-state levels of non-activated receptors at the plasma membrane. Although this function may apply to vps25, it may also indicate that there are inherent differences between the different class E proteins regarding protein sorting at the early endosome (Herz, 2006).

Paradoxically, although vps25 clones die by apoptosis, the vps25 alleles were identified as being recessive suppressors of GMR-hid-induced cell death. This analysis demonstrates that the wild-type tissue accounts for this suppression even though these cells are exposed to GMR-hid. The initial explanation for this observation was that non-autonomous proliferation mediated by JAK/STAT signaling in vps25 mosaics overrides the apoptotic activity of GMR-hid. However, overexpression of Upd, the ligand of the JAK/STAT pathway, does not significantly suppress GMR-hid, although GMR-upd flies have a similar overgrowth phenotype to vps25 mosaics. This finding excludes non-autonomous proliferation for the suppression of GMR-hid by vps25. However, Diap1 protein levels are increased in tissue abutting vps25 clones. GMR-hid is sensitive to altered levels of Diap1, suggesting that the increase of Diap1 outside of vps25 clones may account for the suppression of GMR-hid. Thus, in addition to non-autonomous proliferation, vps25 clones also increase the apoptotic resistance of adjacent wild-type tissue in a non-autonomous manner. The signaling pathway that can induce non-autonomous survival by increasing Diap1 protein levels is currently unknown (Herz, 2006).

The data suggest that apoptosis in vps25 mutant tissue is not only executed via the Hid/Diap1/Dronc/Ark pathway. vps25 ark clones still died, suggesting that in addition to Ark at least one other cell death pathway is activated in vps25 clones. It has been shown that a Dronc/Ark-independent cell death pathway exists in Drosophila, but this pathway has not been identified. The data in this study implicates JNK as potential mediator of the alternative cell death pathway. vps25 ark/Puc mosaic eye discs are extremely overgrown and the clones occupy a large area of the disc. Anti-cleaved Caspase-3 ^*-dependent apoptosis is blocked in these clones. Only at the clonal boundaries is Caspase-3^* activity still detectable, suggesting that at the interface between vps25 clones and wild-type tissue a third potential apoptotic pathway is activated (Herz, 2006).

The data show that cell competition is not sufficient to induce cell death in vps25 clones. By contrast, given the extremely large size of cell death-inhibited vps25 clones, it appears that vps25 clones have no intrinsic growth disadvantage, and have the capability to overgrow and outcompete the surrounding wild-type tissue if cell death is blocked. Thus, cell competition does not contribute significantly to the apoptotic phenotype of vps25 clones (Herz, 2006).

Hippo signaling is increased in vps25 clones. Hippo signaling can induce cell death, and, consistently, hippo mutants block cell death in vps25 clones. It is unknown how Hippo signaling is activated in vps25 clones. However, in analogy to N, a putative receptor that controls Hippo signaling may be deregulated in vps25 clones and triggers Hippo signaling. This receptor is currently unknown, but has been postulated previously. However, it should be pointed out that ESCRT components have additional functions outside of MVB protein sorting. Certain ESCRT-II members have been shown to bind to the transcriptional elongation factor ELL in order to derepress transcription by RNA polymerase II. Thus, in the absence of Vps25, transcriptional control of components of the Hippo pathway may be deregulated and contribute to cell death (Herz, 2006).

In summary, the data suggest that impaired ESCRT function leads to the accumulation of N and Dl, and possibly of a receptor controlling the Hippo pathway. These receptors control non-autonomous proliferation and autonomous apoptosis, respectively. In addition, a signaling pathway is postulated that induces non-autonomous cell survival by controlling Diap1 protein levels. Further characterization of the vps25 mutant phenotype may help to identify the postulated receptor of the Hippo pathway and the cell survival signaling pathway (Herz, 2006).

Human ESCRT components, most notably TSG101 (Vps23p), have been implicated in tumor suppression. NIH3T3 cells, depleted of Tsg101 by an antisense approach, formed colonies on soft agar and produced metastatic tumors in nude mice. However, the conditional Tsg101 knockout in mouse mammary glands did not cause the formation of tumors over a period of two years, making a role of TSG101 as tumor suppressor controversial. However, Tsg101 mutant cells are very sensitive to apoptotic death, implying that they die before they become harmful to the organism (Herz, 2006).

The phenotypical characterization of vps25 mutants in Drosophila provides an explanation for the failure to confirm TSG101 as tumor suppressor. vps25 clones need to survive over extended periods of time in order to sustain growth. Even though they induce non-autonomous proliferation, after they have died, N signaling is turned off and proliferation stops. Furthermore, the size of the adult eye of vps25 mosaics is only slightly increased when compared with wild type, and does not match the strong overgrowth phenotype of larval imaginal discs, which can be twice as large as wild-type discs. Thus, as long as vps25 clones are not resistant to their own apoptotic death, tissue repair during pupal stages may partially regress the size of the imaginal disc back to almost normal. Instead, it appears that inhibition of cell death is the triggering event for a tumorous phenotype of vps25 clones. vps25/Diap1 and vps25 ark/Puc clones can make up a large fraction of the tissue of imaginal discs, and the entire discs can be five times as large as wild-type discs (Herz, 2006).

Tumorigenesis requires multiple genetic alterations that transform normal cells progressively into malignant cancer cells. Thus, additional genetic 'hits' may be necessary to inhibit apoptosis of Tsg101 mutant cells, which may then be able to induce a similar growth phenotype to that observed for vps25. Thus, although a tumor suppressor function for Tsg101 was not confirmed in a mouse model, it still is possible that Tsg101 and other mammalian ESCRT members have tumor suppressor properties (Herz, 2006).

Programmed cell death in the embryonic central nervous system of Drosophila

Although programmed cell death (PCD) plays a crucial role throughout Drosophila CNS development, its pattern and incidence remain largely uninvestigated. This study provides a detailed analysis of the occurrence of PCD in the embryonic ventral nerve cord (VNC). The spatio-temporal pattern of PCD was traced and the appearance of, and total cell numbers in, thoracic and abdominal neuromeres of wild-type and PCD-deficient H99 mutant embryos were compared. Furthermore, the clonal origin and fate of superfluous cells in H99 mutants was examined by DiI labeling almost all neuroblasts, with special attention to segment-specific differences within the individually identified neuroblast lineages. These data reveal that although PCD-deficient mutants appear morphologically well-structured, there is significant hyperplasia in the VNC. The majority of neuroblast lineages comprise superfluous cells, and a specific set of these lineages shows segment-specific characteristics. The superfluous cells can be specified as neurons with extended wild-type-like or abnormal axonal projections, but not as glia. The lineage data also provide indications towards the identities of neuroblasts that normally die in the late embryo and of those that become postembryonic and resume proliferation in the larva. Using cell-specific markers it was possible to precisely identify some of the progeny cells, including the GW neuron, the U motoneurons and one of the RP motoneurons, all of which undergo segment-specific cell death. The data obtained in this analysis form the basis for further investigations into the mechanisms involved in the regulation of PCD and its role in segmental patterning in the embryonic CNS (Rogulja-Ortmann. 2007).

In this analysis of PCD distribution it was found that, macroscopically, the CNS of wt and PCD-deficient (H99) embryos do not show large differences. These observations indicate that the supernumerary cells do not disturb developmental events in the CNS of H99 embryos, such as cell migration and axonal pathfinding. The glial cells mostly find their appropriate positions accurately. The DiI-labeled NB lineages were, in the majority of cases, easily identifiable based on their shape, position and axonal pattern, despite the supernumerary cells. The FasII pattern showed that the axonal projections form and extend along their usual paths. In fact, the supernumerary cells themselves are capable of differentiating i.e. expressing marker genes and extending axons, as shown by clones of several NBs and by cell marker expression analysis in H99 (e.g. NB7-3) (Rogulja-Ortmann. 2007).

It has been shown that a large number of CNS cells undergo PCD during embryonic development. The distribution of activated Caspase-3-positive cells in wt embryos suggests that the death of some cells is under tight spatial and temporal control, as revealed by their regular, segmentally repeated occurrence. Other dying cells were rather randomly distributed, suggesting a certain amount of developmental plasticity. The overall counts of Caspase-3-positive cells give an estimate of the numbers of dying cells at a given time. They indicate that PCD becomes evident in the CNS at stage 11 and is most abundant in the late embryo (from stage 14). It is however difficult to estimate the total number of apoptotic cells throughout CNS development by anti-Caspase-3 labeling, because the cell corpses are removed fairly quickly. Therefore the total number of cells were counted per thoracic and abdominal hemineuromere in the late embryo. Comparison between stage 16 and stage 17 wt embryos indicates that 25-30 % of all cells are removed in both tagmata after stage 16, which in turn suggests that the total percentage of removed cells must be high, since PCD occurs at high levels already from stage 14 on. In comparison to the developing nervous system of C. elegans, where PCD removes about 10% of cells, and of mammals, where this number can be as high as 50-90%, PCD in the fly CNS appears to show an intermediate prevalence. This lends support to the hypothesis of an increasing contribution of PCD in shaping more advanced nervous systems during evolution (Rogulja-Ortmann. 2007).

Comparisons between wt and H99 reveal, as expected, a greater number of cells in both tagmata of H99 embryos (151% increase in the thorax and 162% in the abdomen at stage 17). These additional cells in H99 may reflect the total number of cells normally undergoing cell death until stage 17. However, there is a large variability in the total number of cells, especially within the H99 strain. In wt embryos, it seems to be more pronounced in the thorax and at stage 17, which might be a consequence of variable amounts of PCD occurring until this stage. The even higher variability within the H99 strain (both in thorax and abdomen) is likely to reflect variable numbers of additional cell divisions. The great majority of abdominal NBs are normally removed by PCD after they have generated their embryonic progeny, whereas in the thoracic neuromeres most of the NBs enter quiescence at the end of embryogenesis and continue dividing as postembryonic NBs in larval stages. Thus, there are few mitoses occurring in the wt CNS from stage 16 onwards. BrdU labeling experiments revealed a high number of BrdU-positive cells in some H99 embryos injected at early stage 17. It is assumed that these are progeny of mitotic NBs and/or GMCs that survive and continue dividing, generating cells that do not exist in wt. Clones obtained by DiI labeling in H99 confirm this conclusion. The finding that surviving cells divide already in the embryo complement results that showed that, in reaper mutants, NBs in the abdominal neuromeres survive and generate progeny in larval stages (Rogulja-Ortmann. 2007).

Among the DiI-labeled clones in H99 embryos, very few NB lineages were obtained which did not differ from their wt counterparts. The majority contained, as expected, supernumerary cells. In some cases axons projected by these cells could be identified, showing that they are specified as neurons. In fact, in three cases (NB4-2, NB5-3 and NB7-3), these additional cells were found to be specified as motoneurons. As additional axons within a fascicle were generally difficult to identify, it is possible that these are not the only lineages which make additional motoneurons in H99. Whether these cells are normally born and apoptose, or originate from additional divisions of surviving NBs or GMCs, cannot be determined from these experiments, but similar observations have been made for both cases. It is interesting that none of these cells, regardless of their origin, are specified as glia. No additional glia were observed in the NB clones in H99 embryos, and equal numbers of Repo-expressing glial cells were found in wt and H99. It is concluded that PCD occurs almost exclusively in neurons and/or undifferentiated cells, and that lateral glia are not produced in excess numbers in the embryo. Furthermore, because it is likely that NBs, which normally die, stay in a late temporal window in H99, one could speculate that NBs in this window normally do not give rise to glia. These results are not in agreement with the notion that LG are overproduced, and their numbers adjusted through axon contact. Occasional apoptotic LG have been observed and it is possible that the current method of counting does not allow a resolution fine enough to account for an occasional additional Repo-positive cell in H99 embryos. However, if LG were consistently overproduced, a higher number of glia in would be expected H99 embryos. It is assumed that LG cell death may reflect a small variability in the number of cells needed, and not a general mechanism for adjusting glial cell numbers (Rogulja-Ortmann. 2007).

Generally, no difference was found between Repo-expressing glia numbers in wt and H99. However, a small difference does become apparent when one separates the total cell counts into those in the CNS and those in the periphery: 25.67±0.45 cells/hs and 28.42±0.64 cells/hs for wt and H99, respectively, were counted in the CNS, whereas 8.50±0.28 cells/hs and 6.35±0.82 cells/hs for wt and H99, respectively, were found in the periphery. The reasons for this difference might be the greater width of the CNS in H99 embryos, and that the cues required for proper migration of the peripheral glia are disturbed by additional cells. Alternatively, the difference might be due to differentiation defects in these cells (Rogulja-Ortmann. 2007).

In addition to NB clones with too many cells and wild-type-like axon projections in H99, some lineages were obtained whose clones exhibited atypical projection patterns. These projections were found to belong both to motoneurons (e.g. in NB4-2) and interneurons (e.g. NB5-3, NB7-2 and NB-7-4). NB4-2 normally produces two motoneurons (RP2 and 4-2Mar) and 8-14 interneurons. In two out of three NB4-2 clones in H99 two additional motoneurons that project anteriorly were found, similar to RP2. One of the two clones was found in the thorax and had a normal cell number (16), whereas the other was abdominal and had too many cells (25). Thus, the two additional motoneurons are likely to be the progeny of divisions occurring in the wt, and not of an additional NB or GMC mitosis. The fact that the third NB4-2 clone (found in the abdomen and comprising 17 cells) did not show the same motoneuronal projections could be due to these cells not being differentiated at the time of fixation (clones of different ages were occasionally observed in the same embryo), or they may not have differentiated at all. It would be interesting to determine the target(s) of these additional motoneurons and thereby perhaps gain insight into physiological reasons for their death. However, such an experiment has to await tools that allow specifically labeling of the NB4-2 lineage, or these motoneurons, in the H99 mutant background (Rogulja-Ortmann. 2007).

The other three lineages (NB5-3, NB7-2 and NB7-4) all have atypical interneuronal projections. The cells which these atypical axons belong to may represent evolutionary remnants that are not needed in the Drosophila CNS. Alternatively, they might have a function earlier in development and be removed when this function is fulfilled. Such a role has been shown for the dMP2 and MP1 neurons, which are born in all segments and pioneer the longitudinal axon tracts. At the end of embryogenesis these neurons undergo PCD in all segments except A6 to A8, where their axons innervate the hindgut. It is known that some cells of the NB5-3 lineage express the transcription factor Lbe, and that H99 mutants show about three additional Lbe-positive neurons per hemisegment, which mostly likely belong to NB5-3. The DiI-labeling results complement this finding in that four or more additional neurons were also found in H99 clones. The supernumerary Lbe-positive neurons in H99 could possibly be the ones producing the atypical axonal projections (Rogulja-Ortmann. 2007).

In the wt embryo, only eight NB lineages show obvious tagma-specific differences in cell number and composition. Tagma-specific differences among serially homologous CNS lineages have been shown to be controlled by homeotic genes. Therefore, these lineages provide useful models for studying homeotic gene function on segment-specific PCD. In H99 embryos, further lineages were observed that were differently affected in the thorax and abdomen. How these tagma-specific differences arise in a PCD-deficient background is an interesting question. For example, NB4-3 shows a wild-type cell number in the thorax (8 and 12-13), but has too many cells in the abdomen (15, 15 and 22). There are a couple of plausible scenarios to explain this observation. (1) The development of the NB4-3 lineage, including the involvement of PCD, could actually differ in the thorax and abdomen of wt embryos, with the final cell number being similar by chance. The DiI-labeled clones allow determination of the final cell number, but do not reveal how this number is achieved. The difference would become obvious in an H99 mutant background, at least regarding the involvement of PCD. (2) This possibility does not exclude the first one, the thoracic NB4-3 could become a postembryonic NB (pNB) and the abdominal NB4-3 might undergo PCD after generating the embryonic lineage. In H99, the abdominal NB would be capable of undergoing a variable number of additional divisions to generate a variable number of progeny. This would easily explain larger discrepancies in cell number between individual clones in H99 (e.g. the abdominal NB4-3 clone with 22 cells), and is in agreement with occasional observations of H99 embryos with a very high CNS cell number per segment, and with the two observed classes of H99 embryos with high and low numbers of BrdU-positive cells (Rogulja-Ortmann. 2007).

NB6-2 is another lineage whose clones differ in the two tagmata of H99 embryos. In this case, the abdominal clones showed no difference to their wt counterparts, whereas the thoracic clones did (18 and 19 cells). Although no difference in cell number between thoracic and abdominal clones was reported for this lineage, a rather large count range (8-16 cells) was given, which would allow for a thorax-specific PCD of two to three postmitotic progeny. Alternatively, the thoracic NB6-2 might undergo cell death upon generating its progeny, which would make it the first identified apoptotic NB in the thorax. When PCD is prevented, this NB may undergo a few additional rounds of division. The data obtained in these experiments do not counter this notion, but the number of clones obtained in the thorax was not sufficient to draw a definite conclusion. As the abdominal NB6-2 lineage in H99 did not differ from the one in wt, its NB may be one of the few abdominal postembryonic NBs (Rogulja-Ortmann. 2007).

A specific set of NBs undergoes PCD in the late embryo, whereas surviving NBs resume proliferation in the larva as pNBs, after a period of mitotic quiescence. The identities of the individual NBs undergoing PCD versus those surviving as pNBs are still unknown. The sizes of NB lineages obtained in H99 embryos may provide hints for identifying candidate pNBs in the abdomen [12 NBs/hs in A1, four in A2 and three in A3 to A7, and NBs that undergo PCD in the thorax at the end of embryogenesis [seven NBs/hs in T1 to T3. In the abdomen, NB1-1a and NB6-2 are obvious candidates for pNBs, as they remained consistently unchanged in H99 embryos. Two other NBs, NB1-2 and NB3-2, are also potential abdominal pNBs as they mostly did not differ from their wt counterparts, and only occasionally contained one additional cell. On the other hand, clones which showed more than twice the cell number in H99 (NB2-1, NB5-4a and NB7-3) than in wt, strongly suggest that these NBs normally undergo PCD in the abdomen (but perform additional divisions in H99), because, even if one daughter cell of each GMC undergoes PCD, they still cannot account for all cells found in H99 clones (Rogulja-Ortmann. 2007).

Regarding thoracic NBs, it can only be speculated on account of low sample numbers. NBs which seem to become pNBs in the thorax, as they showed no difference between wt and H99 clones, are NB3-2, NB4-3 and NB4-4. Potential candidates for NBs which do not become pNBs, but undergo PCD in the thorax, are expected to consistently have a significant increase in cell number in H99. These are NB5-1 and NB5-5. In addition, lineages for which one clone was obtained in H99 but which also showed many more cells in the thorax than normal are NB2-2t, NB5-4t and NB7-3 (Rogulja-Ortmann. 2007).

In order to investigate the developmental signals and mechanisms involved in the regulation of PCD in the embryonic CNS, some of the apoptotic cells were identified which will be used as single-cell PCD models. These are the dHb9-positive RP neuron from NB3-1, Lbe-positive neurons from NB5-3, the Eg-positive GW neuron from NB7-3 and the Eve-positive U neurons from NB7-1. As not much is known about the dying RP motoneuron or the Lbe-positive neurons, the first goal will be to characterize each of these cells more closely, based on the combination of expressed molecular markers (Rogulja-Ortmann. 2007).

Some of the dying NB7-3 cells are already known to be undifferentiated daughter cells of the second and third GMC, which undergo PCD shortly after birth. Notch has been identified as the signal initiating PCD. The surviving daughters receive the asymmetrically distributed protein Numb, which counteracts the PCD-inducing Notch signal. The same had been shown in a sensory organ lineage of the embryonic peripheral nervous system, where cells produced in two subsequent divisions undergo Notch-dependent PCD. Both the PCD in the NB7-3 lineage and in the sensory organ lineage require the Hid, rpr and grim genes. It will be interesting to see whether the Notch-Numb interaction also plays a role in the segment-specific PCD of the differentiated GW motoneuron, or if another signal is used for the removal of this, and possibly other, differentiated cells (Rogulja-Ortmann. 2007).

The U motoneurons also show a segment-specific cell death pattern (they apoptose in A6 to A8), thus somewhat resembling the MP1 and dMP2 neurons. However, in contrast to MP1 and dMP2, the U neurons survive in the anterior segments and undergo PCD in the posterior ones. Whether homeotic genes play any role in the survival or death of these cells remains to be investigated (Rogulja-Ortmann. 2007).

In summary, this study has presented descriptions of PCD in the developing CNS of the wt Drosophila embryo, and of the CNS of PCD-deficient embryos. The pattern of Caspase-dependent PCD is partly very orderly, suggesting tight spatio-temporal control of cell death, and partly random, which suggests a certain amount of plasticity already in the embryo. The CNS of PCD-deficient embryos is nevertheless well organized, despite the presence of too many cells. These superfluous cells come from both a block in PCD and from additional divisions that surviving NBs go through. It was possible to link the occurence of cell death to identified NB lineages by clonal analysis in PCD-deficient embryos, to uncover segment-specific differences, and to establish single-cell PCD models that will be used in further studies to investigate mechanisms responsible for controlling PCD in the embryonic CNS (Rogulja-Ortmann. 2007).

Lineage-specific cell death in postembryonic brain development of Drosophila

The Drosophila central brain is composed of thousands of neurons that derive from approximately 100 neuroblasts per hemisphere. Functional circuits in the brain require precise neuronal wiring and tight control of neuronal numbers. How this accurate control of neuronal numbers is achieved during neural development is largely unclear. Specifically, the role of programmed cell death in control of cell numbers has not been studied in the central brain neuroblast lineages. This study focusses on four postembryonic neuroblast lineages in the central brain identified on the basis that they express the homeobox gene engrailed (en). For each lineage, the total number of adult-specific neurons generated was determined, as well as number and pattern of en-expressing cells. Programmed cell death has a pronounced effect on the number of cells in the four lineages; approximately half of the immature adult-specific neurons in three of the four lineages are eliminated by cell death during postembryonic development. Moreover, programmed cell death selectively affects en-positive versus en-negative cells in a lineage-specific manner and, thus, controls the relative number of en-expressing neurons in each lineage. Furthermore, evidence is provided that Notch signaling is involved in the regulation of en expression. Based on these findings, it is concluded that lineage-specific programmed cell death plays a prominent role in the generation of neuronal number and lineage diversity in the Drosophila brain (Kumar, 2009).

In postembryonic CNS development of holometabolous insects such as flies, a combination of programmed cell death and neuronal process re-innervation allows the larval nervous system to reorganize and innervate new body structures. During metamorphosis many adult-specific neurons in the ventral ganglia are targeted by programmed cell death, particularly in abdominal segments. Furthermore, extensive cell death occurs during postembryonic development in the insect visual system, where cells are overproduced and those that do not make the appropriate targets are eliminated by apoptosis. By contrast, very little is currently known about the prevalence and functional roles of programmed cell death in development of the insect adult central brain (Kumar, 2009).

This report identifies four neuroblast lineages in the postembryonic central brain and finds that programmed cell death occurs in all four lineages, albeit to different extents. Whereas cell death plays only a minor role in the medial cluster MC1 lineage, it has dramatic effects in anterior cluster (AC), posterior cluster (PC) and medial cluster MC2 lineages, in which nearly half of the adult-specific neuronal progeny are programmed to die during larval development. It is noteworthy that the adult-specific neurons targeted by cell death are generated during larval development and are eliminated before their respective neuroblasts stop proliferating (12-24 hours after pupal formation). Because the cell death reported here occurs before neuronal differentiation, it is probably not involved in events of brain reorganization that take place during metamorphosis (Kumar, 2009).

Another central feature of the cell death events demonstrated here is that none of the four lineages is completely eliminated by cell death; all four neuroblasts and a significant number of their neuronal progeny survive at the end of larval development, and these neuronal progeny are largely present in the adult. In this sense, the programmed cell death reported here is likely to be functionally different from the cell death observed in the ventral ganglia, where the neuroblast itself undergoes apoptosis, regulating neuronal numbers in the abdominal segments (Kumar, 2009)

These experiments indicate that programmed cell death plays a prominent role in determining lineage-specific features; if cell death is blocked the total neuronal number increases in all four lineages and the number of en-expressing neurons increases in AC, PC and MC2. Furthermore, the axonal projection pattern of H99 mutant (deleting rpr, hid and grim) and Notch mutant en-expressing lineages was examined, comparing them to wild type. Both cell death defective H99 and Notch mutant PC lineages showed an additional projection that was not present in the wild type, whereas the other three H99 lineages did not appear to change drastically in their projection patterns. In conclusion, programmed cell death appears to contribute to the cellular diversity of neuronal lineages in the central brain (Kumar, 2009).

Studies on neuroblast lineages in the developing ventral ganglia indicate that proliferating neuroblasts generate a largely invariant clone of neural cells. In general, each neuroblast division produces a distinctly fated GMC, and each GMC division produces two sibling progeny of different fates. There is some evidence that the fate of these progeny is controlled by the parental GMC; the two siblings are restricted to a pair of different cell fates, with one sibling adopting an 'A' fate and the other adopting a 'B' fate. This, in turn, has led to a model in which a neuroblast lineage can be thought of as composed of two hemilineages, with one hemilineage comprising 'A'-fate cells and the other hemilineage comprising 'B'-fate cells. It is thought that an interaction between Notch and Numb is responsible for generating distinct neural fates of the two GMC daughter cells, with a loss of Notch or Numb resulting in reciprocal cell-fate duplication. However, Notch signaling does not appear to confer a particular fate; rather, it acts generically as a mechanism to enable two siblings to acquire different fates, and other developmental control genes that are inherited from the specific parental GMC are thought to be instrumental in determining the final identity of each progeny (Kumar, 2009).

Findings on lineage-specific cell death support a comparable model in which all four brain neuroblasts can generate one en-positive hemilineage and one en-negative hemilineage. In this model, programmed cell death is then targeted in a lineage-specific manner to either the en-negative hemilineage (AC, PC), or the en-positive hemilineage (MC2), or neither hemilineage (MC1). Alternatively, en-positive and en-negative neurons in the lineages could be generated in a temporal fashion and subsequently en-positive or en-negative neurons could be eliminated in a lineage-specific manner. However, the results suggest that this is unlikely. In particular, in the PC lineage, more than 80% of the two-cell clones examined were composed of one en-positive and one en-negative cell. If the above did occur, a significant number of two-cell en-positive clones should have been obtained along with two-cell clones comprising one en-positive and one en-negative neuron. Similar analysis of single and two cell clones in the other three en lineages is further required to confirm the occurrence of hemilineage-specific programmed cell death (Kumar, 2009).

Based on these experimental results, it was postulate that Notch signaling is an important generic mechanism underlying generation of the two different hemilineages, as in the absence of Notch signaling, cell-fate duplication of GMC siblings occurs. Indeed, analysis of Notch loss-of-function neuroblast clones suggests that in the absence of Notch signaling most of the neurons in the four lineages acquire an en-positive cell fate. Alternatively, in the four lineages examined, being positive for en may be the default state of the cells, and Notch induces secondary fate by repressing en in subsets of cells in each lineage. These en-positive neurons then appear to survive or undergo programmed cell death depending on the lineage-specific context. However, it remains to be seen whether Notch itself acts on the apoptotic machinery, independent of en (Kumar, 2009).

This study, used en as a molecular marker to identify four lineages in the postembryonic central brain. Might en itself be functionally involved in regulating programmed cell death in these lineages? For the PC lineage, there is some indication that the total clone size is reduced by approximately half in en loss-of-function mutants, compared with wild type. Although this suggests that en may be involved in promoting survival of en-positive neurons in PC (and probably AC), it does not explain the role of en in the MC2 lineage, where it would have to play an opposing role, as en-positive neurons die in this lineage. Thus, en could act either as a pro-apoptotic or an anti-apoptotic factor, depending on the lineage-specific context. Moreover, a direct genetic interaction between en and the apoptotic machinery remains to be investigated. As en is known to have multiple interactions with other proteins, a complex regulatory network involving target proteins of en may be responsible for regulating apoptosis in a lineage-specific manner. Further analysis of interactions with such target proteins is necessary to reveal the full regulatory network in more detail (Kumar, 2009).

The lineage-specific effects of cell death and of Notch signaling in AC and PC are distinctly different from those observed in MC1 or MC2 lineages. However, when compared with each other, many aspects of AC and PC lineages are similar. In wild type, both lineages consist of similar numbers of adult-specific neurons, and the majority (approximately 80%) of these neurons are positive for en, whereas neuroblasts and GMCs are negative for en in both lineages. Blocking cell death results in a substantial (approximately double) increase in total cell number in both lineages, and this increase is almost exclusively due to an increase in the number of surviving en-negative neurons in both lineages. Moreover, loss of Notch function causes a marked increase in the number of surviving en-positive neurons without affecting the number of en-negative neurons in both lineages. The only significant difference between AC and PC lineages observed in this study is that the AC lineage is located in the protocerebrum, whereas the PC lineage is located in the deutocerebrum (Kumar, 2009).

What might be responsible for these similarities in the AC and PC neuroblast lineages? There is some evidence for the existence of serially homologous neuroblasts in the fly brain and VNC. In the VNC, serially homologous neuroblasts, defined by comparable time of formation, similar positions in the neuromeric progenitor array and similar expression of developmental control genes, such as segment polarity genes, dorsoventral patterning genes and other molecular markers, can give rise to almost identical cell lineages. This suggests that similar regulatory interactions take place during development of serially homologous neuroblasts and their neural lineages. A comparison of molecular expression patterns in neuroblasts from different neuromeres of the brain and ventral ganglia suggests that several of them might be serial homologs of each other. For example, neuroblasts NB5-6 in the abdominal, thoracic and subesophageal ganglia have been proposed to be homologous to NBDd7 in the deutocerebrum and NBTd4 in the tritocerebrum (Kumar, 2009).

Given the remarkable similarities in AC and PC neuroblast lineages, it is possible that the protocerebral AC lineage and the deutocerebral PC lineages represent serial homologs. If this is the case, then investigations of the cellular and molecular mechanisms that control their lineage-specific development should be useful for understanding of how regionalized neural diversity in the brain evolves from a basic metameric ground state. However, as neither the combination of developmental control genes expressed in AC and PC neuroblasts nor the position of the two brain neuroblasts in their neuromeres of origin are currently known in sufficient detail, further experiments are needed before the issue of serial homology can be resolved for these brain neuroblast lineages (Kumar, 2009).

The role of apoptosis in shaping the tracheal system in the Drosophila embryo

The tubular network of the tracheal system in the Drosophila embryo is created from a set of epithelial placodes by cell migration, rearrangements, fusions and shape changes. A designated number of cells is initially allocated to each branch of the system. The final cell number in the dorsal branches is not only determined by early patterning events and subsequent cell rearrangements but also by elimination of cells from the developing branch. Extruded cells die and are engulfed by macrophages. These results suggest that the pattern of cell extrusion and death is not hard-wired, but is determined by environmental cues (Baer, 2010).

In live studies of the tracheal system using embryos expressing GFP under the control of the breathless (btl) promoter GFP-expressing motile cells were observed that were not attached to the tracheal system. The btl gene, which encodes an FGF receptor homolog, is mainly expressed in the tracheal system, but also in glial cells and a few other cell type. However, it had not previously been described as being expressed in individual cells that were dispersed in the embryo. Since it was not clear what the individual cells outside the tracheal system were, other tracheal markers were used to determine whether they indeed derived from the tracheal system, or rather represented an as yet undiscovered cell type in which the btl promoter is active. When the tracheal system was marked with lacZ expressed under the control of the promoter of another tracheal gene, trachealess (trh-lacZ), it was observed that lacZ was also expressed not only in the tubular tracheal epithelia but also in single cells detached from the tracheal system (Baer, 2010).

In the live observations using GFP it was noticed that these cells appeared to be moving around in the embryo, preferentially along the dorsal trunk of the trachea. Previously hemocytes had been described to move along the tracheal system. Thus it was asked whether these cells might be a subpopulation of hemocytes that shared expression of some genes with the tracheal system. To assess their cell type, the cells were analyzed in embryos in which markers for the tracheal system and for hemocytes were simultaneously visualized. Hemocytes were visualized by using a croquemort-GAL4 transgene to control the expression of GFP. Croquemort is expressed in a subpopulation of hemocytes, the macrophages. For the tracheal system trh-lacZ was used (Baer, 2010).

It was found that the individual lacZ-expressing cells that are detached from the tracheal system also expressed the crq-controlled GFP. In addition, there were also many cells that showed only crq-GFP staining and no lacZ. There are two possible explanations for these findings. Either a subset of macrophages can activate transcription of the btl and trh promoters, or the doubly-stained objects are not single cells. Consistent with the latter explanation, it was noticed that the two fluorescent markers were often localized in non-overlapping parts of the cells, suggesting that lacZ-expressing cells or cell fragments might have been engulfed by macrophages. If this were the case, then the doubly-stained objects should contain two nuclei. Indeed, in triple stainings with the nuclear marker TOTO-3 two TOTO-3 signals were observed in the majority of the doubly-stained objects, indicating the presence of two nuclei. It is concluded therefore that the objects are hemocytes that have engulfed tracheal cells that have left the tracheal epithelium (Baer, 2010).

To test directly whether these single cells originate from the developing tracheal tree, more detailed live observations were performed. In previous experiments it was noticed that the disconnected cells can be preferentially found near the branches that undergo cellular rearrangements, for example, at the bases of dorsal branches. The areas around these sites were chosen for further studies. Live imaging of dorsal branches of the embryos expressing α-cat-GFP or sqh-GFP revealed that during the outgrowth of the dorsal branches individual tracheal cells detach from the tracheal system at, or near the site where the dorsal branches emerge from the dorsal trunk. The numbers of cells leaving the system from the base of branches and from the interbranch-regions of the dorsal trunk were compared in 12 videos of wildtype embryos. The 'base of the branch' is described as those cells that bordered at least on one side on a cell that was part of the dorsal branch. 42 branches were evaluated and cells were found leaving from the base in 20 cases, whereas in 40 interbranch-regions seven cells leaving the system were found. Thus, cells are almost threefold less likely to be extruded in the large interbranch area than from the small area at the base of the branch (Baer, 2010).

Since the detached cells appeared to be engulfed by macrophages and one function of macrophages is to clear up apoptotic cells, whether the trh-lacZ positive cells seen inside the hemocytes displayed markers for apoptosis was tested. It was found that some but not all of the trh-lacZ cells that were detached from the tracheal system gave a positive signal in a TUNEL reaction. Since the TUNEL assay only detects cells in a brief phase of apoptosis it was not surprising that only a subset of the detached cells were labelled. Thus this result is consistent with the notion that the engulfed cells are dying or dead cells. A further hallmark of apoptosis is the activation of caspase 3, which can be detected with an antibody that specifically recognizes the activated form. Trh-lacZ-expressing cells that were detached from the tracheal system also gave a positive signal when stained with antibodies against activated caspase3 (Baer, 2010).

Next, it was asked whether the detached cells undergo apoptosis because they have lost contact with the tracheal epithelium, or, conversely, whether they die within the epithelium and are subsequently extruded. This question was addressed by two approaches: tracing caspase activity in vivo and blocking apoptosis. To examine when the apoptotic program is induced in tracheal cells leaving the system an in vivo fluorescent sensor of caspase activity, the 'Apoliner' transgene (Bardet, 2008), was used. The Apoliner consists of two fluorophores, a membrane-anchored RFP and a GFP with nuclear localisation signal (NLS), which are linked by a caspase sensitive fragment of the DIAP1 protein. Upon caspase activation within the cell, the sensor is cleaved, resulting in translocation of GFP into the nucleus, whereas RFP stays at the membrane. The Apoliner was expressed using btl-GAL4 and the development of dorsal branches was followed in vivo. Nuclear GFP can be observed in individual cells in the tracheal system. Shortly after they begin to accumulate nuclear GFP these cells detach from the system. Thus apoptosis precedes cell removal from the tissue (Baer, 2010).

If death is a prerequisite for expulsion, then suppressing apoptosis should reduce the number of detached cells. To test this, the tracheal system was examined in embryos homozygous for a deficiency Df(3L)H99, which removes the three pro-apoptotic genes, grim, reaper and hid, and in which apoptosis therefore cannot occur. Since homozygous Df(3L)H99 embryos show strong overall developmental defects, the analysis was restricted to the dorsal branches in segments that do not show gross morphological abnormalities, i.e. segments 3-7. Whereas wild type stage 14/15 embryos show approximately 2-4 detached cells in segments 3-7 on each side, no detached cells were observed in embryos that were homozygous for Df(3L)H99. To exclude the possibility that this was due to a non-autonomous effect of the mutant phenotype of surrounding tissues in Df(3L)H99 mutant embryos, an alternative way of blocking apoptosis only in the tracheal system was used. The baculoviral inhibitor of apoptosis p35 was expressed in tracheal cells using the btl-GAL4 driver line. To confirm that apoptosis was blocked efficiently, the Apoliner was co-expressed together with p35. In these embryos no Apoliner signal was detected, indicating the absence of caspase activity, and no cells were seen to leave the tracheal system. This shows that the completion of the apoptotic program is necessary for detachment of the tracheal cells (Baer, 2010).

The fact that the detached cells were frequently found near the base of the dorsal branches of the tracheal system raises the question whether their leaving the epithelium might be associated with a specific developmental programme, such as the morphogenesis of the dorsal branches. Since each dorsal branch arises from six cells, but the final branch typically consists of only five cells, it has been suggested that the sixth cell migrates back to the dorsal trunk. The results point to an alternative possibility suggesting that apoptosis might be a mechanism that contributes to the determination of cell number in the dorsal branches. If the assumption that cell death is involved in determining the number of cells is correct, then dorsal branches should contain more cells if apoptosis cannot occur. To test this, cell numbers were counted in the dorsal branches of Df(3L)H99 mutant embryos and in embryos expressing p35 in the tracheal system. The embryos were stained with an antibody against Trachealess to mark specifically the nuclei of tracheal cells. The nuclei in dorsal branches were then counted under the microscope by focusing through the entire depth of the dorsal branch. Branches with an unclear course were not included in the analysis (Baer, 2010).

All tested genotypes showed variability in cell number in the dorsal branches, but there were clear differences between the mutant and the wild type embryos. 76.3% of the 93 dorsal branches evaluated in 10 wild type embryos consisted of 5 cells, 8.6% consisted of six cells and 15.1% consisted of 3 or 4 cells. This shows that branches with six cells are possible, but 5 cells are preferred. By contrast, embryos with blocked apoptosis had 51.6% (btl-Gal4, UAS-p35 embryos) or 56.6% (Df(3L)H99 mutant embryos) dorsal branches with six cells. These results indicate that removal of the cells by apoptosis indeed contributes to the determination of the final cell number in the dorsal branches (Baer, 2010).

It is unclear how the emigrating cell is determined, and why it is not determined in every one of the branches. The fact that this is so shows that, in contrast to many other situations in which apoptosis eliminates unwanted cells during development, cell death in this case is not the result of a hard-wired developmental programme. Instead it is more likely to be a response to unfavourable conditions in the cell's environment. Changes in the microenvironment have been shown to induce cell death in mammalian endothelial cells, with signals from the extracellular matrix influencing the balance between cell survival and apoptosis. Adhesion via integrins can protect cells from FAS mediated apoptosis, and integrins may act as mechano-transducers in this context. Thus it is possible that weakening of cell-cell junctions resulting from tissue remodelling in the tracheal system could trigger the apoptotic pathway. Junction rearrangement also affects cell death in the wing disc, where an imbalance in the junctional forces within the epithelium was found to be rebalanced by the elimination of cells. In this system, the initial irregularity arose through cell proliferation, but it is imaginable that in the tracheal system cell intercalation might affect local junctional forces, and cell extrusion can be triggered to obtain a geometrically optimal structure (Baer, 2010).

The fact that only half of the dorsal branches in the mutant embryos had six cells shows that apoptosis cannot be the only mechanism for the removal of supernumerary cells. There must be other ways of losing cells, perhaps by re-integration into the dorsal trunk, as previously suggested. Such cases were indeed observed. More stringently, even embryos in which a larger number of branches have six cells, namely btl-Gal4, UAS-p35 embryos, can develop normally and hatch. It is of course possible that subtle defects, for example a reduced efficiency in oxygen delivery, would not manifest themselves in easily measurable phenotypes in a laboratory setting (Baer, 2010).

In summary, the results are consistent with a scenario in which cells that find themselves in a sub-optimal epithelial context either move away, or die and leave the epithelium. If the apoptotic pathway is blocked, they may be forced to use the option of moving elsewhere, or induce neighbouring cells to rearrange further, or they remain in place, and the system is able to tolerate a sub-optimal structure (Baer, 2010).

temporal and molecular hierarchies in the link between death, delamination and dorsal closure

One process that occurs during dorsal closure is cell delamination, the seemingly stochastic, rapid apical constriction of cells that culminates in their extrusion from the ectodermal layer. Their number (between 10% and 30%) and position is variable and unpredictable. Extruded cells are engulfed by hemocytes. This behaviour is thought to contribute to up to one-third of the force generated in the amnioserosa for dorsal closure and exhibits a preferential occurrence at the anterior canthus. Its suppression by caspase inhibition has led to the suggestion that ‘apoptosis’ triggers delamination. This study explored whether cell delamination in the amnioserosa, a seemingly stochastic event that results in the extrusion of a small fraction of cells and known to provide a force for dorsal closure, is contingent upon the receipt of an apoptotic signal. Through the analysis of mutant combinations and the profiling of apoptotic signals in situ, spatial, temporal and molecular hierarchies were establish in the link between death and delamination. Although an apoptotic signal is necessary and sufficient to provide cell-autonomous instructions for delamination, its induction during natural delamination occurs downstream of mitochondrial fragmentation. It was further shown that apoptotic regulators can influence both delamination and dorsal closure cell non-autonomously, presumably by influencing tissue mechanics. The spatial heterogeneities in delamination frequency and mitochondrial morphology suggest that mechanical stresses may underlie the activation of the apoptotic cascade through their influence on mitochondrial dynamics. These results document the temporal propagation of an apoptotic signal in the context of cell behaviours that accomplish morphogenesis during development. They highlight the importance of mitochondrial dynamics and tissue mechanics in its regulation. Together, they provide novel insights into how apoptotic signals can be deployed to pattern tissues (Muliyil, 2011).

These results establish the necessity and utility of apoptotic signals in driving cellular delamination in the amnioserosa and in patterning the spatiotemporal dynamics of closure. They invoke the induction of pro-apoptotic genes and thus go beyond earlier observations that inferred the role of an apoptotic cascade through the effects of caspase suppression. The results also provide mechanistic insights into the mode of action of the apoptotic cascade by demonstrating cell-autonomous effects of pro-apoptotic genes and caspase activity (DIAP1 overexpression) on the rates of apical constriction. This suggests that apoptotic regulators must regulate cytoskeletal organisation and cell mechanics. A question that arises is whether both classes of regulators function in the linear hierarchy that was delineated or whether functions independent of the apoptotic cascade contribute to their role in driving delamination. The analysis of the molecular hierarchy shows that caspase activation induced by reaper upregulation is a necessary downstream event. Its late activation in delaminating cells, however, raises the issue of whether it is necessary for apical constriction or just for cell extrusion. Although the complete suppression of delamination by p35 overexpression precludes the analysis of constriction rates, this analysis reveals an absence of rosette patterns that characterise delamination rather than the presence of constricted cells that fail to extrude. This suggests that caspase activation must also be necessary for apical constriction. One explanation is that this marked upregulation of the cascade triggers the almost abrupt transition in cell behaviour, characterised by the rapid fall in cell area in a delaminating cell. This is consistent with the higher rates of decrease in cell area with increases in the amounts of caspases/Reaper. Although the phenotypes associated with DIAP1 overexpression also support a role for caspases in cell constriction, caspase-independent functions of DIAP1 have been reported to influence actin organisation in Drosophila border cells. Thus, apoptotic signals must impinge on a distinct set of regulators of the actin cytoskeleton to facilitate apical constriction and tissue contraction. Caspase activation may also regulate adhesion to facilitate extrusion. Indeed, the adherens junction component armadillo/β-catenin is a caspase substrate during cell death in Drosophila and mammals (Muliyil, 2011).

The results also provide evidence for cell non-autonomous regulation of delamination by components of the apoptotic cascade. Further support for this comes from ongoing observations that caspase inhibition influences actin organisation in the entire amnioserosa. It is speculated that the influence of low undetectable levels of caspase activation not restricted to delaminating cells, regulates tissue mechanics in the amnioserosa and through it also influences cell delamination. The results also show that non-autonomous influences on delamination can originate in the epidermis. Uncovering the molecular players that underlie both autonomous and non-autonomous effects of apoptotic signals on cell behaviour will be interesting avenues to pursue (Muliyil, 2011).

Temporal and epistatic analysis position mitochondrial fragmentation upstream of the induction of pro-apoptotic genes and caspase activation both during delamination and degeneration. This is the first time that the sequence of propagation of an apoptotic signal has been elucidated in the context of cell behaviour in vivo. Mitochondrial fragmentation is thus the earliest indicator of the cellular commitment to delamination. Other studies have placed mitochondrial fragmentation downstream of the pro-apoptotic genes reaper and hid. What, if not the pro-apoptotic genes, then triggers mitochondrial fragmentation in the amnioserosa? Two recent reports have documented the ability of chemical and radiation injuries to trigger changes in mitochondrial morphology and lead to the induction of apoptosis. An attractive candidate for the trigger in the amnioserosa, consistent with the spatial heterogeneities in delamination frequency and mitochondrial morphology observed, is mechanical stress. Two sets of observations support this. First, not all cells that overexpress pro-apoptotic genes delaminate, and the anterior predominance of such events is maintained. This suggests that although an apoptotic signal is necessary, it must cooperate with other permissive signals to accomplish delamination. Second, the studies of native dorsal closure uncover spatial heterogeneities in mitochondrial morphology. Two features characterise this heterogeneity: (1) the early abundance of cells with predominantly fragmented mitochondria in the anterior AS and (2) their delayed transition to tubular/hyperfused morphologies prior to degeneration compared with the posterior. It has been suggested in a different context that low levels of chemical stress can induce hyperfusion as a means of countering stress (through optimisation of mitochondrial ATP production), whereas higher magnitudes of stress lead to fragmentation and apoptosis. A similar reasoning (with the substitution of chemical stresses by mechanical stresses) might underlie the spatial heterogeneities in delamination frequency. Specifically, high magnitudes of stress locally (from head involution) might be responsible for increased mitochondrial fragmentation and subsequent delamination, whereas prolonged lower levels of stresses (from the leading edge) may drive hyperfusion and subsequent degeneration of the amnioserosa. Adhesion anisotropies resulting from differences in the substrate (yolk anteriorly and hindgut posteriorly) could additionally contribute to the force anisotropies between the anterior and posterior amnioserosa (Muliyil, 2011).

Taken together, the results reveal that apoptotic regulators contribute multiple forces to dorsal closure. In the amnioserosa, they act locally to drive delamination but also globally to maintain tissue tension. The latter is attributed to the low levels of caspase activation and pro-apoptotic gene induction. This provides a permissive environment for mitochondrial fragmentation and the subsequent marked upregulation of the cascade in delaminating cells. Additionally, they contribute to forces generated in the epidermis. This is best inferred from anti-apoptotic perturbations. In hid mutants, the rates of dorsal closure are higher despite the absence of delaminations in the amnioserosa. Conversely, delamination in the amnioserosa is 'upregulated' when either caspases or hid is downregulated in the epidermis, but their effects on closure rates are different. These non-autonomous effects must reflect the feedback regulation of forces generated in the epidermis and in the amnioserosa. That multiple forces contribute to dorsal closure and can feedback regulate each other has been long appreciated. These studies identify apoptotic signals as crucial regulators of the balance of forces that drive dorsal closure. Uncovering the basis for feedback regulation and the force hierarchies that lend dorsal closure resilience will be interesting. A recent study reported on a novel, non-apoptotic role for an epidermal caspase, caspase 8: its effect on interleukin signalling resulted in the recapitulation of a wound healing response when deleted in the skin. In light of the above observations, it is interesting that in this analysis of dorsal closure, which recapitulates wound closure, some perturbations that suppress apoptosis also resulted in accelerated closure (Muliyil, 2011).

These explorations demonstrate the primacy of mitochondrial fragmentation in the induction of apoptotic signalling and uncover the complex relationships between death signals, delamination and dorsal closure. Furthermore, they illustrate how an apoptotic signal is deployed multiple times in the same tissue to accomplish heterogeneity in cell behaviour and have helped identify some of the cellular properties they modulate. Understanding the triggers for mitochondrial fragmentation and the precise outcomes and mechanisms of apoptotic signals on cell biological attributes of delaminating cells will be interesting avenues to explore (Muliyil, 2011).

REFERENCES

Search PubMed for articles about Drosophila Head involution defective

Abbott, M. K. and Lengyel, J. A. (1991). Embryonic head involution and rotation of male terminalia require the Drosophila locus head involution defective. Genetics 129(3): 783-9.

Avdonin, V., et al. (1998). Apoptotic proteins Reaper and Grim induce stable inactivation in voltage-gated K+ channels. Proc. Natl. Acad. Sci. 95(20): 11703-8.

Baer, M. M., Bilstein, A., Caussinus, E., Csiszar, A., Affolter, M. and Leptin, M. (2010). The role of apoptosis in shaping the tracheal system in the Drosophila embryo. Mech. Dev. 127(1-2): 28-35. PubMed Citation: 19995601

Bardet, P. L. et al. (2008). A fluorescent reporter of caspase activity for live imaging. Proc. Natl. Acad. Sci. 105: 13901-13905. PubMed Citation: 18779587

Bergmann, A., et al. (1998). The Drosophila gene hid is a direct molecular target of Ras-dependent survival signaling. Cell 95(3): 331-41. PubMed Citation: 9814704

Bergmann, A., Tugentman, M., Shilo, B.-Z. and Steller, H. (2002). Regulation of cell number by MAPK-dependent control of apoptosis: A mechanism for trophic survival signaling. Dev. Cell 2: 159-170. 11832242

Bertet, C., Li, X., Erclik, T., Cavey, M., Wells, B., Desplan, C. (2014) Temporal patterning of neuroblasts controls Notch-mediated cell survival through regulation of Hid or Reaper. Cell 158: 1173-1186. PubMed ID: 25171415

Bhaskar, P. K., Surabhi, S., Tripathi, B. K., Mukherjee, A. and Mutsuddi, M. (2014). dLin52 is crucial for dE2F and dRBF mediated transcriptional regulation of pro-apoptotic gene hid. Biochim Biophys Acta. PubMed ID: 24863159

Bhaskar, P. K., Mukherjee, A. and Mutsuddi, M. (2012). Dynamic pattern of expression of dlin52, a member of the Myb/MuvB complex, during Drosophila development. Gene Expr Patterns 12: 77-84. PubMed ID: 22178095

Boichuk, S., Parry, J. A., Makielski, K. R., Litovchick, L., Baron, J. L., Zewe, J. P., Wozniak, A., Mehalek, K. R., Korzeniewski, N., Seneviratne, D. S., Schoffski, P., Debiec-Rychter, M., DeCaprio, J. A. and Duensing, A. (2013). The DREAM complex mediates GIST cell quiescence and is a novel therapeutic target to enhance imatinib-induced apoptosis. Cancer Res 73: 5120-5129. PubMed ID: 23786773

Borensztejn, A., Boissoneau, E., Fernandez, G., Agnes, F. and Pret, A. M. (2013). JAK/STAT autocontrol of ligand-producing cell number through apoptosis. Development 140: 195-204. PubMed ID: 23222440

Brodsky, M. H., et al. (2004). Drosophila melanogaster MNK/Chk2 and p53 regulate multiple DNA repair and apoptotic pathways following DNA damage. Mol. Cell. Biol. 24: 1219-1231. 1472996

Busto, M., Iyengar, B. and Campos, A. R. (1999). Genetic dissection of behavior: modulation of locomotion by light in the Drosophila melanogaster larva requires genetically distinct visual system functions. J. Neurosci. 19(9): 3337-44. PubMed Citation: 10212293

Chen, P., et al. (1996). grim, a novel cell death gene in Drosophila. Genes Dev. 10: 1773-1782. PubMed Citation: 8698237

Chen, P., et al. (1998b). Dredd, a novel effector of the apoptosis activators Reaper, Grim, and Hid in Drosophila. Dev. Biol. 201(2): 202-16. PubMed Citation: 9740659

Chai, J., et al. (2000). Structural and biochemical basis of apoptotic activation by Smac/DIABLO. Nature 406(6798): 855-62. 10972280

Chai, J., Yan, N., Huh, J. R., Wu, J. W., Li, W., Hay, B. A. and Shi, Y. (2003). Molecular mechanism of Reaper-Grim-Hid-mediated suppression of DIAP1-dependent Dronc ubiquitination. Nat Struct Biol 10: 892-898. PubMed ID: 14517550

Cordero, J., Jassim, O., Bao, S. and Cagan, R. (2004). A role for wingless in an early pupal cell death event that contributes to patterning the Drosophila eye. Mech. Dev. 121: 1523-1530. 15511643

Cordoba, S. and Estella, C. (2014). The bHLH-PAS transcription factor Dysfusion regulates tarsal joint formation in response to Notch activity during Drosophila leg development. PLoS Genet 10: e1004621. PubMed ID: 25329825

Cox, R. T., et al. (2000). A screen for mutations that suppress the phenotype of Drosophila armadillo, the ß-catenin homolog. Genetics 155: 1725-1740. PubMed Citation: 10924470

Cullen, K. and McCall, K. (2004). Role of programmed cell death in patterning the Drosophila antennal arista. Dev. Biol. 275: 82-92. 15464574

Davidson, J. M. and Duronio, R. J. (2012). S phase-coupled E2f1 destruction ensures homeostasis in proliferating tissues. PLoS Genet. 8(8): e1002831. PubMed Citation: 22916021

DeFalco, T. J., et al. (2003), Sex-specific apoptosis regulates sexual dimorphism in the Drosophila embryonic gonad. Dev. Cell 5: 205-216. 12919673

de la Cova, C., et al. (2004). Drosophila Myc regulates organ size by inducing cell competition. Cell 117: 107-116. 15066286

Downward, J. (1998). Ras signaling and apoptosis. Curr. Opin. Genet. Dev. 8, 49-54. PubMed Citation: 9529605

Draizen, T. A., Ewer, J. and Robinow, S. (1999). Genetic and hormonal regulation of the death of peptidergic neurons in the Drosophila central nervous system. J. Neurobiol. 38(4): 455-65. PubMed Citation: 10084681

Fan, Y, and Bergmann, A. (2008). Distinct mechanisms of apoptosis-induced compensatory proliferation in proliferating and differentiating tissues in the Drosophila eye. Dev. Cell 14: 399-410. PubMed Citation: 18331718

Fichelson, P. and Gho, M. (2003). The glial cell undergoes apoptosis in the microchaete lineage of Drosophila. Development 130: 123-133. 12441297

Gafuik, C. and Steller, H. (2011). A gain-of-function germline mutation in Drosophila ras1 affects apoptosis and cell fate during development. PLoS One 6(8): e23535. PubMed Citation: 21858158

Goyal, L., et al. (2000). Induction of apoptosis by Drosophila reaper, hid and grim through inhibition of IAP function. EMBO J. 19(4): 589-597. PubMed Citation: 10675328

Grether, M. E., et al. (1995). The head involution defective gene of Drosophila melanogaster functions in programmed cell death. Genes Dev. 9(14): 1694-708. PubMed Citation: 7622034

Haining, W. N., et al. (1999). The proapoptotic function of Drosophila Hid is conserved in mammalian cells. Proc. Natl. Acad. Sci. 96(9): 4936-41. PubMed Citation: 10220397

Hawkins, C. J., et al. (2000). The Drosophila caspase DRONC cleaves following glutamate or aspartate and is regulated by DIAP1, HID, and GRIM. J. Biol. Chem. 275: 27084-27093. 10825159

Hay, B. A., Wassarman, D. A. and Rubin, G. M. (1995). Drosophila homologs of baculovirus inhibitor of apoptosis proteins function to block cell death. Cell 83: 1253-1262. PubMed Citation: 8548811

Herz, H. M., et al. (2006). vps25 mosaics display non-autonomous cell survival and overgrowth, and autonomous apoptosis. Development 133(10): 1871-80. PubMed Citation: 16611691

Holley, C. L., Olson, M. R., Colon-Ramos, D. A. and Kornbluth, S. (2002). Reaper eliminates IAP proteins through stimulated IAP degradation and generalized translational inhibition. Nat. Cell Biol. 4: 439-444. 12021770

Huber. B. A., Sinclair, B. J. and Schmitt, M. (2007). The evolution of asymmetric genitalia in spiders and insects. Biol. Rev. Camb. Philos. Soc. 82(4): 647-98. PubMed Citation: 17944621

Huh, J. R., et al. (2004). Multiple apoptotic caspase cascades are required in nonapoptotic roles for Drosophila spermatid individualization. PLoS Biol. 2: E15. Medline abstract: 14737191

Jassim, O. W., Fink, J. L. and Cagan, R. L. (2003). Dmp53 protects the Drosophila retina during a developmentally regulated DNA damage response. EMBO J. 22: 5622-5632. 14532134

Jiang, C., et al. (2000). A steroid-triggered transcriptional hierarchy controls salivary gland cell death during Drosophila metamorphosis. Molec. Cell 5: 445-455

Jollos, V., (1936). Mutations observed in Drosophila stocks taken up into the stratosphere. Natn. Geogr. Soc. Tech. Pap. Stratosphere 2: 153-157

Krieser, R. J., (2007). The Drosophila homolog of the putative phosphatidylserine receptor functions to inhibit apoptosis. Development 134(13): 2407-14. Medline abstract: 17522160

Kumar, A., Bello, B. and Reichert, H. (2009). Lineage-specific cell death in postembryonic brain development of Drosophila. Development 136(20): 3433-42. PubMed Citation: 19762424

Kurada, P. and White, K. (1998). Ras promotes cell survival in Drosophila by downregulating hid expression. Cell 95(3): 319-29. PubMed Citation: 9814703

Leaman, D., et al. (2005). Antisense-mediated depletion reveals essential and specific functions of microRNAs in Drosophila development. Cell 121: 1097-1108. 15989958

Lee, C.-Y., Cooksey, B. A. and Baehrecke, E. H. (2002). Steroid regulation of midgut cell death during Drosophila development. Dev. Bio. 250: 101-111. 12297099

Leevers, S.J., Weinkove, D., MacDougall, L.K., Hafen, E., and Waterfield, M.D. (1996). The Drosophila phosphoinositide 3-kinase Dp110 promotes cell growth. EMBO J. 15: 6584-6594. PubMed Citation: 8978685

Levine, A. J., Hu, W. and Feng, Z. (2006). The P53 pathway: what questions remain to be explored? Cell Death Differ. 13: 1027-1036. Medline abstract: 16557269

Lewis, P. W., Sahoo, D., Geng, C., Bell, M., Lipsick, J. S. and Botchan, M. R. (2012). Drosophila lin-52 acts in opposition to repressive components of the Myb-MuvB/dREAM complex. Mol Cell Biol 32: 3218-3227. PubMed ID: 22688510

Lin, H. V., Rogulja, A. and Cadigan, K. M. (2004). Wingless eliminates ommatidia from the edge of the developing eye through activation of apoptosis. Development 131: 2409-2418. 15128670

Lisi, A., Mazzon, I. and White, K. (2000). Diverse domains of THREAD/DIAP1 are required to inhibit apoptosis induced by REAPER and HID in Drosophila. Genetics 154: 669-678. PubMed Citation: 10655220

Loh, S. H. Y. and Russell, S. (2000). A Drosophila group E Sox gene is dynamically expressed in the embryonic alimentary canal. Mech. Dev. 93: 185-188. 10781954

McNabb, S. L., et al. (1997). Disruption of a behavioral sequence by targeted death of peptidergic neurons in Drosophila. Neuron 19(4): 813-23

McNamee, L. M. and Brodsky, M. H. (2009). p53-independent apoptosis limits DNA damage-induced aneuploidy. Genetics 182: 423-435. PubMed Citation: 19364807

Means, J. C., Muro, I. and Clem, R. J. (2006). Lack of involvement of mitochondrial factors in caspase activation in a Drosophila cell-free system. Cell Death Differ. 13: 1222-1234. PubMed citation: 16322754

Meier, P., et al. (2000). The Drosophila caspase Dronc is regulated by DIAP1. EMBO J. 19: 598-611

Menon, K. P., et al.,(2009). The translational repressors Nanos and Pumilio have divergent effects on presynaptic terminal growth and postsynaptic glutamate receptor subunit composition. J. Neurosci., 29:. 5558-5572. PubMed Citation: 19403823

Merino, M.M., Rhiner, C., Lopez-Gay, J.M., Buechel, D., Hauert, B. and Moreno, E. (2015). Elimination of unfit cells maintains tissue health and prolongs lifespan. Cell 160: 461-476. PubMed ID: 25601460

Moon, N. S., et al. (2005). Drosophila E2F1 has context-specific pro- and antiapoptotic properties during development. Dev Cell. 9(4):463-75. 16198289

Moreno, E., Yan, M. and Basler, K. (2002). Evolution of TNF signaling mechanisms: JNK-dependent apoptosis triggered by Eiger, the Drosophila homolog of the TNF superfamily. Curr. Biol. 12: 1263-1268. 12176339

Morishita, J., Kang, M. J., Fidelin, K. and Ryoo, H. D. (2013). CDK7 regulates the mitochondrial localization of a tail-anchored proapoptotic protein, Hid. Cell Rep 5: 1481-1488. PubMed ID: 24360962

Muliyil, S., Krishnakumar, P. and Narasimha, M. (2011). Spatial, temporal and molecular hierarchies in the link between death, delamination and dorsal closure. Development 138(14): 3043-54. PubMed Citation: 21693520

Muraro, N. I. et al., (2008). Pumilio binds para mRNA and requires Nanos and Brat to regulate sodium current in Drosophila motoneurons. J. Neurosci. 28: 2099-2109. PubMed Citation: 18305244

Nagata, R., Akai, N., Kondo, S., Saito, K., Ohsawa, S. and Igaki, T. (2022). Yorkie drives supercompetition by non-autonomous induction of autophagy via bantam microRNA in Drosophila. Curr Biol 32(5): 1064-1076. PubMed ID: 35134324

Olesnicky, E. C., Bhogal, B. and Gavis, E R. (2012). Combinatorial use of translational co-factors for cell type-specific regulation during neuronal morphogenesis in Drosophila. Dev. Biol. 365(1): 208-18. PubMed Citation: 22391052

Orgogozo, V., Schweisguth, F. and Bellaïche, Y. (2002). Binary cell death decision regulated by unequal partitioning of Numb at mitosis. Development 129: 4677-4684. 12361960

Orian, A., van Steensel, B., Delrow, J., Bussemaker, H.J., Li, L., Sawado, T., Williams, E., Loo, L.W., Cowley, S. M., and Yost, C. et al. (2003). Genomic binding by the Drosophila Myc, Max, Mad/Mnt transcription factor network. Genes Dev. 17: 1101-1114. 12695332

Quinn, L. M., et al. (2000). An essential role for the caspase dronc in developmentally programmed cell death in Drosophila. J. Biol. Chem. 275(51): 40416-24. 10984473

Rogulja-Ortmann. A., et al. (2007). Programmed cell death in the embryonic central nervous system of Drosophila melanogaster. Development 134: 105-116. Medline abstract: 17164416

Ryoo, H. D., Gorenc, T. and Steller, H. (2004). Apoptotic cells can induce compensatory cell proliferation through the JNK and the Wingless signaling pathways. Dev. Cell 7: 491-501. 15469838

Sandu, C., Ryoo, H. D. and Steller, H. (2010). Drosophila IAP antagonists form multimeric complexes to promote cell death. J. Cell Biol. 190(6): 1039-52. PubMed Citation: 20837774

Scuderi, A., Simin, K., Kazuko, S. G., Metherall, J. E. and Letsou, A. (2006). scylla and charybde, homologues of the human apoptotic gene RTP801, are required for head involution in Drosophila. Dev. Biol. 291(1): 110-22. 1642334

Sen, A., Kuruvilla, D., Pinto, L., Sarin, A. and Rodrigues, V. (2004). Programmed cell death and context dependent activation of the EGF pathway regulate gliogenesis in the Drosophila olfactory system. Mech. Dev. 121: 65-78. 14706701

Shukla, A. and Tapadi, M. G. (2011). Differential localization and processing of apoptotic proteins in Malpighian tubules of Drosophila during metamorphosis. Eur. J. Cell Biol. 90: 72-80. PubMed Citation: 21035895

Srinivasula, S. M., et al. (2001). A conserved XIAP-interaction motif in caspase-9 and Smac/DIABLO regulates caspase activity and apoptosis. Nature 410(6824): 112-6. 11242052

Staveley, B.E., Ruel, L., Jin, J., Stambolic, V., Mastronardi, F.G., Heitzler, P., Woodgett, J.R., and Manoukian, A.M. (1998). Genetic analysis of protein kinase B (AKT) in Drosophila. Curr. Biol. 8: 599-602

Suzanne, M., et al. (2010). Coupling of apoptosis and L/R patterning controls stepwise organ looping. Curr. Biol. 20(19): 1773-8. PubMed Citation: 20832313

Tanaka-Matakatsu, M., Xu, J., Cheng, L. and Du, W. (2009). Regulation of apoptosis of rbf mutant cells during Drosophila development. Dev. Biol. 326: 347-356. PubMed Citation: 19100727

Tapon, N., et al. (2002). salvador promotes both cell cycle exit and apoptosis in Drosophila and is mutated in human cancer cell lines. Cell 110: 467-478. 12202036

Vucic, D., et al. (1998). Inhibitor of apoptosis proteins physically interact with and block apoptosis induced by Drosophila proteins HID and GRIM. Mol. Cell. Biol. 18(6): 3300-3309

Waddington, C.H. (1940). The genetic control of wing development in Drosophila. J. Genet. 41: 75-139

Wang, S. L., et al. (1999). The Drosophila caspase inhibitor DIAP1 is essential for cell survival and is negatively regulated by HID. Cell 98(4): 453-63

Wells, B. S., Yoshida, E. and Johnston, L. A. (2006). Compensatory proliferation in Drosophila imaginal discs requires Dronc-dependent p53 activity. Curr. Biol. 16(16): 1606-15. Medline abstract: 16920621

Werz, C., et al. (2005). Mis-specified cells die by an active gene-directed process, and inhibition of this death results in cell fate transformation in Drosophila. Development 132(24): 5343-52. 16280349

Wichmann, A., Jaklevic, B. and Su, T. T. (2006). Ionizing radiation induces caspase-dependent but Chk2- and p53-independent cell death in Drosophila melanogaster. Proc. Natl. Acad. Sci. 103(26): 9952-7. 16785441

Wichmann, A., Uyetake, L. and Su, T. T. (2010). E2F1 and E2F2 have opposite effects on radiation-induced p53-independent apoptosis in Drosophila. Dev. Biol. 346(1): 80-9. PubMed Citation: 20659447

Wilson, R., Goyal, L., Ditzel, M., Zachariou, A., Baker, D. A., Agapite, J., Steller, H., and Meier, P. (2002). The DIAP1 RING finger mediates ubiquitination of Dronc and is indispensable for regulating apoptosis. Nat. Cell Biol. 4: 445-450. 12021771

Wing, J. P., et al. (1998). Distinct cell killing properties of the Drosophila reaper, head involution defective, and grim genes. Cell Death Differ. 5(11): 930-9

Wing, J. P., Schwartz, L. M. and Nambu, J. R. (2001). The RHG motifs of Drosophila Reaper and Grim are important for their distinct cell death-inducing abilities. Mech. Dev. 102: 193-203. 11287192

Wright, C. W. and Clem, R. J. (2002). Sequence requirements for Hid binding and apoptosis regulation in the baculovirus inhibitor of apoptosis Op-IAP. Hid binds Op-IAP in a manner similar to Smac binding of XIAP. J. Biol. Chem. 277(4): 2454-62. 11717313

Wu, G., et al. (2000). Structural basis of IAP recognition by Smac/DIABLO. Nature 408(6815): 1008-12. 11140638

Wu, J. W., et al. (2001). Structural analysis of a functional DIAP1 fragment bound to Grim and Hid peptides. Mol. Cell 8: 95-104. 11511363

Xing, Y., Su, T. T. and Ruohola-Baker, H. (2015). Tie-mediated signal from apoptotic cells protects stem cells in Drosophila melanogaster. Nat Commun 6: 7058. PubMed ID: 25959206

Yoo, S. J., et al. (2002). Apoptosis inducers Hid, Rpr and Grim negatively regulate levels of the caspase inhibitor DIAP1 by distinct mechanisms. Nat. Cell Biol. 4: 416-424. 12021767

Yu, S.-Y., et al. (2002). A pathway of signals regulating effector and initiator caspases in the developing Drosophila eye. Development 129: 3269-3278. 12070100

Zhang, Y., et al. (2008). Epigenetic blocking of an enhancer region controls irradiation-induced proapoptotic gene expression in Drosophila embryos. Dev. Cell 14(4): 481-93. PubMed Citation: 18410726

Zhou, L., et al. (1997). Cooperative functions of the reaper and head involution defective genes in the programmed cell death of Drosophila central nervous system midline cells. Proc. Natl. Acad. Sci. 94(10): 5131-6. PubMed Citation: 9144202

Zhu, C. C., Bornemann, D. J., Zhitomirsky, D., Miller, E. L., O'Connor, M. B. and Simon, J. A. (2008). Drosophila histone deacetylase-3 controls imaginal disc size through suppression of apoptosis. PLoS Genet. 4(2): e1000009. PubMed Citation: 18454196