REVIEWSA PEER REVIEWED FORUM

Regulation and Execution of Apoptosis DuringDrosophila DevelopmentPETER BANGS AND KRISTIN WHITECutaneous Biology Research Center, Massachusetts General Hospital, Harvard Medical School. Charlestown, Massachusetts

ABSTRACT The development of the Dro-sophila embryo into an adult fly is a process thatintegrates cell proliferation and differentiationwith programmed cell death, or apoptosis. Apo-ptosis is an evolutionarily conserved processthat is controlled in the developing fly by theproducts of the genes reaper, grim, and hid. Wediscuss the role of programmed cell death in theestablishment and maintenance of correct pat-terning in the embryo, and examine the coordi-nation of apoptosis with the hormonally con-trolled degeneration of larval tissues duringmetamorphosis. Finally, we address the architec-ture of the adult eye as an example of how pro-grammed cell death plays a key role in the devel-opment of many adult structures. Dev Dyn 2000;218:68–79. © 2000 Wiley-Liss, Inc.

Key words: apoptosis; Drosophila; development

INTRODUCTION

The fact that programmed cell death plays a signif-icant role in animal development has been known for along time (Saunders and Fallon, 1967). Originally ob-served in insect morphogenesis (Lockshin, 1969), celldeath has been recognized in both vertebrate and in-vertebrate development as necessary for the elimina-tion of superfluous cells, morphogenetic changes, andthe hollowing out of solid structures to form cavities ortubes (Coucouvanis and Martin, 1995). In many in-stances, cell death is observed in developing tissues butits function is not known, and it is unclear whetherapoptosis is necessary for these processes to occur or ifit is simply coincident with them (Glucksmann, 1951).The study of Drosophila and C. elegans mutants thatcompletely lack developmental apoptosis has allowedthis question to be addressed. These studies indicatethat in these organisms apoptosis is not necessary forsome aspects of normal development (White et al.,1994; Metzstein et al., 1998). However, as discussedbelow, apoptosis provides developmental flexibility andis required for the maturation of the Drosophila ner-vous system and for metamorphosis.

The past few years have seen tremendous progress inthe identification of the mechanisms by which cells

undergo apoptosis (for reviews, see Bergmann et al.,1998; Newton and Strasser, 1998; Vaux and Kors-meyer, 1999). It is now clear that the components of theapoptotic machinery are constitutively expressed invirtually all nucleated animal cells and that the acti-vation of this machinery is controlled by a set of intra-cellular regulatory proteins that transduce signalsfrom both inside and outside the cell. In most cases,cells require specific survival signals from their localenvironment to prevent the activation of apoptosis(Raff, 1992). For example, when epithelial cells areremoved from the extracellular matrix and no longerhave ligated integrin receptors, they respond by under-going programmed cell death (reviewed in Frisch andRuoslahti, 1997). Cell death can also be induced byspecific signals from neighboring or nearby cells, asexemplified by the death ligands TNF and FasL (re-viewed in Nagata, 1997).

Apoptosis is mediated by the activation of a specificclass of proteases known as caspases. This name re-flects the conserved cysteine in the active site and therequirement for an aspartic acid at the cleavage site oftheir target proteins. Caspases are constitutivelypresent in cells as inactive zymogens with variable-length amino-terminal prodomains (Cryns and Yuan,1998). Caspases themselves have caspase cleavagesites separating each domain, and activation of thepro-caspases requires proteolysis at these sites.Caspases can be divided into two classes based on theirprodomains and roles in cell death. The upstream, orinitiator, caspases have longer prodomains that medi-ate the transduction of death signals and the assemblyof activating complexes. When initiator caspases arebrought into proximity with one another by assemblyinto an activating complex, autoprocessing of thecaspases occurs through an intrinsic proteolytic activ-ity (Salvesen and Dixit, 1999). The newly activatedinitiator caspases are then free to proteolytically acti-

Grant sponsor: National Institutes of Health; Grant sponsor: Shi-seido Company of Japan.

*Correspondence to: Kristin White, Cutaneous Biology Research Cen-ter, Massachusetts General Hospital, Harvard Medical School, Charles-town, MA02129. E-mail: kristin.white@CBRC2.MGH.Harvard.edu

Received 10 January 2000; Accepted 8 February 2000

DEVELOPMENTAL DYNAMICS 218:68–79 (2000)

© 2000 WILEY-LISS, INC.

vate the second class of caspases, the downstream oreffector caspases, which are characterized by shorterprodomains. The resulting protease cascade ends withthe cleavage of a multitude of cellular targets includingstructural proteins and enzymes involved in gene ex-pression, DNA replication and metabolic activities ofthe cell. The dying cell eventually fragments into apo-ptotic bodies which are subsequently engulfed byneighboring cells or macrophages.

Many of the proteins known to be important in apo-ptosis were first identified genetically in C. elegans andhuman cancer mutations (reviewed in Hunter, 1997;Metzstein et al., 1998). Conservation across species isvery high, indicating that the core apoptotic machineryand many regulatory features of apoptosis have beenpreserved through evolution. This has allowed the useof the genetic tools available in invertebrate systems toidentify many of the key proteins in apoptosis and toestablish the pathways involved in its initiation andexecution.

The fruit fly Drosophila melanogaster has proven tobe a particularly convenient organism for the study ofapoptosis. As will be discussed below, the genes re-quired for the induction of apoptosis in Drosophilahave been cloned, as have numerous other factors in-volved in the initiation and execution of the cell deathpathway (reviewed by Abrams, 1999). The developingorganism is relatively accessible to both observationand dissection, and a veritable treasure chest of genetictools allows for the manipulation of specific genes inspecific tissues at precise timepoints. Additionally, thedevelopment of the fruit fly, from oogenesis throughembryogenesis and metamorphosis into adulthood, hasbeen extremely well characterized and describedthrough decades of work (for example, see Bate andMartinez Arias, 1993).

REGULATION OF CELL DEATH INDROSOPHILA EMBRYOGENESIS

The vital dye acridine orange (AO) specifically stainsapoptotic cells and has been used to examine dyingcells in living Drosophila embryos (Abrams et al.,1993). Microscopic examination of AO stained embryosshows that cell death is prominent and widespreadduring embryogenesis and that it occurs in a relativelypredictable spatial and temporal pattern (Abrams etal., 1993). The first dying cells are invariably detectedin the dorsal region of the head approximately 7 hrafter egg laying, which corresponds to the later part ofthe fully extended germ band stage (stage 11). As de-velopment proceeds, apoptotic cell death becomes moreprominent and widespread throughout the embryo,and corpses are engulfed by circulating macrophages.Time-lapse photography of AO-stained embryos showsthat while cell death occurs in a consistent pattern, theprecise spatial and temporal aspects of this pattern aresomewhat variable, indicating that there is a certaindegree of plasticity in embryonic cell death (Abrams etal., 1993; Pazdera et al., 1998).

Regulators of embryonic cell death in Drosophilawere initially identified by screening for mutants withdisrupted patterns of cell death as assayed by AOstaining. Three overlapping deletions mapping to theleft arm of chromosome 3 result in the complete abro-gation of developmental cell death and lead to embry-onic lethality at a fairly late developmental stage(White et al., 1994). Subsequent analysis of this regionhas identified three novel genes, reaper (rpr), grim, andhead involution defective (hid), whose gene productsare involved in the initiation of all embryonic cell deathin Drosophila (White et al., 1994; Grether et al., 1995;Chen et al., 1996). The apoptotic machinery is intact inembryos deleted for these genes, since apoptosis can beinduced by X-irradiation, with an ultrastructural phe-notype that is indistinguishable from wild-type em-bryos. Therefore, rpr, grim, and hid must play signal-ing roles as opposed to participating in the actualexecution of the apoptotic mechanism.

Sequence analyses of rpr, grim, and hid show themto be unrelated to each other with the exception oflimited homology in the first 15 amino acids of each.When over-expressed in embryos via heat-shock induc-ible transgenes, RPR, GRIM, and HID each induceextensive cell death resulting in the demise of theembryo (Fig. 1). When expressed ectopically in the Dro-sophila eye, each of these genes is sufficient to inducemassive cell death leading to ablation of the eye (Gre-ther et al., 1995; Hay et al., 1995; Chen et al., 1996;White et al., 1996). Over-expression of any of thesegenes in cultured Drosophila cells or other insect celllines also results in the rapid induction of cell death(Chen et al., 1996; Nordstrom et al., 1996; Vucic et al.,1998). Importantly, although vertebrate homologues ofrpr, grim, and hid have yet to be identified, each havebeen shown to induce cell death in a caspase-dependentmanner when expressed in a number of vertebratesystems (Evans et al., 1997; Claveria et al., 1998; Mc-Carthy and Dixit, 1998; Haining et al., 1999). In allcases of over-expression, killing by either of thesegenes is independent of the other two.

In the embryo, rpr and grim expression appears to bepredictive of cells that are fated to die, and transcriptsof these genes can be detected in doomed cells approx-imately 2 hr prior to the onset of morphologically rec-ognizable apoptosis (White et al., 1994; Chen et al.,1996; Robinow et al., 1997) (Fig. 2). In contrast, hidexpression is largely coincident with many cells whichundergo cell death, but this correlation is not absolute.For example, while there is considerable cell deathdetected in the ventral nerve cord during late embryo-genesis, hid expression is not detected in these cells(Abrams et al., 1993; Grether et al., 1995). In addition,hid mRNA can be detected throughout the optic lobeprimordium in stage 12 embryos, but only a fraction ofthese cells die (Grether et al., 1995). The latter obser-vation suggests that HID activity may be regulatedpost-translationally, and it has in fact been shown thatboth hid expression and activity are modulated via the

69APOPTOSIS IN DROSOPHILA DEVELOPMENT

Ras signal transduction pathway (Bergmann et al.,1998; Kurada and White, 1998).

Rpr, grim, and hid act cooperatively and combinato-rially in what appears to be a cell lineage-specific man-ner (Robinow et al., 1997; Zhou et al., 1997; Wing et al.,1998). For example, genetic analyses of deletions thatremove hid alone, hid and grim but not rpr, or hid,grim, and rpr indicate that all three death inducers arerequired for the normal pattern of cell death in theembryonic central nervous system (CNS) midline(Zhou et al., 1997; Wing et al., 1998). Generally, each ofthese genes contributes to the overall level of apoptosis(Fig. 3). Ectopic expression of either RPR or HID alonecan induce apoptosis. However, in the midline glia ofthe CNS expression of RPR or HID alone is insufficientto induce apoptosis, whereas ectopic expression of bothRPR and HID together results in the dramatic loss ofthese cells, indicating that RPR and HID function syn-ergistically in the induction of programmed cell death.Interestingly, the ectopic expression of GRIM alone issufficient to induce midline cell death, although GRIMalso acts synergistically with RPR and HID (Wing etal., 1998).

Although vertebrate homologues for rpr, grim, andhid have not yet been identified, a number of key com-

ponents in the mechanism for cell killing in Drosophilahave been evolutionarily conserved (Table 1). The basicapoptotic “machinery” was first characterized in C. el-egans following the identification of ced3, ced4, andced9. ced3 encodes the caspase component of the C.elegans apoptotic program, and numerous caspaseshave been identified in many different organisms in-cluding Drosophila and humans (reviewed in Crynsand Yuan, 1998). ced4 encodes a pro-apoptotic proteinthat binds to, and is necessary for, the activation of thecaspase CED3. Apaf1, the vertebrate homologue ofced4, plays a similar role in that it interacts with andmediates the activation of caspase 9. Apaf1 differs fromCED4 in that Apaf1 contains a regulatory WD-repeatdomain and requires the binding of cytochrome C forcaspase activation. Recently, the Drosophila homo-logue of ced4/Apaf1 has been isolated and is identifiedin the Drosophila database as Ark for apaf1 relatedkiller (Kanuka et al., 1999; Rodriguez et al., 1999; Zhouet al., 1999). ARK binds to the Drosophila caspasesDREDD and DRONC (Kanuka et al., 1999; Rodriguezet al., 1999), indicating that the proapoptotic activity ofARK, like that of CED4 and Apaf1, is likely to mediatethe activation of certain caspases. Like Apaf1, ARKcontains WD repeats and is positively regulated by

Fig. 1. Cell killing by overexpression of rpr. The top row shows AOstained embryos at similar stages. The embryo on the right carries atransgene that expresses rpr ubiquitously from the heat shock promoter.The embryos were heat shocked and stained 1.5 hr later. A substantialincrease in apoptosis can be seen in the hsrpr embryo. The lower twopanels show Drosophila S2 cells that have been transfected with a

constitutively expressed lacZ. The cell on the right also carries a trans-gene in which rpr expression is driven by the metal inducible metallothio-nein promoter. The cells have been induced with copper for 2 hr andstained with lacz to identify transfected cells. When rpr is expressed, itrapidly results in blebbing and apoptosis of the cell.

70 BANGS AND WHITE

cytochrome C. Mutations in Ark greatly suppress kill-ing by ectopically expressed RPR, GRIM, and HID(Kanuka et al., 1999; Rodriguez et al., 1999; Zhou et al.,1999). However, Ark does not appear to be required forviability, as mutants develop into adult flies, albeitwith a number of morphologic abnormalities and asignificant level of sterility in males (Rodriguez et al.,1999). Ark is expressed in many, but not all, cells thatdie during development (Zhou et al., 1999), and it maybe that expression of Ark sensitizes cells to death sig-nals but is not absolutely required for death to occur.

Apoptosis in Drosophila is negatively regulated by aclass of evolutionarily conserved molecules known asinhibitors of apoptosis, or IAPs. Originally identified inbaculoviruses, IAPs have now been characterized froma wide variety of organisms including Drosophila,mice, and humans (reviewed in Deveraux and Reed,1999). IAPs are characterized by a novel domain ofapproximately 70 amino acids called the baculoviralIAP repeat (BIR). A conserved arrangement of cys-teines and histidines in the BIR binds a zinc ion to forma zinc finger-like motif (Hinds et al., 1999). IAPs canhave up to three tandemly repeated BIR domains, andmany of them also have another structural motif, theRING domain, located near the carboxy-terminus.

Two IAPs, dIAP1 and dIAP2, have been identified inDrosophila (Hay et al., 1995). Overexpression of eitherdIAP1 or dIAP2 suppresses normal developmental cell

death as well as killing by ectopically expressed RPR,GRIM, and HID in the eye (Hay et al., 1995). dIAP1binds to a number of Drosophila caspases and inhibitstheir activity (Kaiser et al., 1998; Hawkins et al., 1999;Wang et al., 1999), and null mutations of dIAP1 arelethal very early in embryogenesis (Wang et al., 1999;Lisi et al., 2000), leading to the suggestion that contin-uous levels of dIAP1 are necessary to inactivate consti-tutively expressed caspases. RPR, GRIM, and HIDphysically interact with dIAP1 (Vucic et al., 1997,1998), and binding by HID (presumably RPR andGRIM as well) negatively regulates the capacity ofdIAP1 to inhibit caspase activity (Wang et al., 1999). Amodel for the induction of apoptosis by RPR, GRIM,and HID includes binding of dIAP1 and thereby block-ing the ability of dIAP1 to inhibit caspases, althoughthis may not be the only mechanism by which RPR,GRIM, and HID initiate apoptosis. In addition, RPRhas been shown to bind to and inactivate certain volt-age-gated K1 potassium channels and may initiate ap-optosis in some instances by inducing membrane depo-larization (Avdonin et al., 1998).

Five caspases have thus far been identified and char-acterized in Drosophila (Fraser and Evan, 1997; Songet al., 1997; Chen et al., 1998; Dorstyn et al., 1999a,b).DREDD and DRONC are related to the initiator classof caspases, in that they have relatively long prodo-mains and share substrate specificities with, and ho-

Fig. 2. The patterns of rpr and hid expression in relation to thepatterns of apoptosis. The pattern of apoptosis during various stages ofembryonic development is shown in the upper row. In the second row, rprexpression, as assessed by in situs, can be seen to anticipate the pattern

of apoptosis by 1 to 2 hr. Expression of hid expression correspondsgenerally to the locations of apoptosis, but it is more broadly expressedin some regions. In addition, hid expression is not detected in the CNS ata time when significant numbers of these cells are dying.

71APOPTOSIS IN DROSOPHILA DEVELOPMENT

mology to, several of the mammalian initiator caspases(Chen et al., 1998; Dorstyn et al., 1999a). DCP-1,drICE, and DECAY on the other hand, have the shorterprodomains of the effector caspases and show a closerhomology to this class (Fraser and Evan, 1997; Song etal., 1997; Dorstyn et al., 1999b). DREDD is unique inthat rather than the typical constitutive expressionobserved for other caspases, its expression appears tobe upregulated in dying cells. This upregulation istightly linked to signaling by rpr, grim, and hid (Chenet al., 1998).

To date, only one loss of function mutation has beencharacterized for Drosophila caspases. dcp-1 mutantsappear to have normal embryonic patterns of cell deathsuggesting that zygotically derived dcp-1 is unneces-sary for programmed cell death in the developing em-bryo. This may be because of the abundant maternallyloaded dcp-1 or because other caspases are responsiblefor embryonic deaths (Song et al., 1997). dcp-1 allelesdo cause larval lethality however, with most dcp-1 ho-mozygotes dying before the third instar larval stage.Within those larvae that do reach the third instarlarval stage, several abnormalities are apparent. Al-though their central nervous systems appear to be nor-mal, dcp-1 mutant larvae lack imaginal discs, have no

gonads, and seem to have fragile tracheae. In addition,dcp-1 homozygous larvae have prominent melanotictumors indicative either of over-proliferation of bloodcells (which does not appear to be the case here; Song etal., 1997) or of immune responses toward abnormalcells or tissues within the larva (Watson et al., 1991).dcp-1 is also necessary for proper oogenesis, as germ-line clones homozygous for dcp-1 result in sterile fe-males with arrested oogenesis phenotypes. Analysis ofthe ovaries from these animals showed that nurse celldumping, the process by which nurse cells transfer thecontents of their cytoplasm into the developing oocyte,was severely curtailed (McCall and Steller, 1998).Dumping may be a modified form of apoptosis thatrequires caspase activation.

A reduction in the dosage of dredd, achieved in fliesheterozygous for a small genomic deletion that over-laps the gene, results in the suppression of killinginduced by ectopic expression of either RPR or GRIM inthe eye (Chen et al., 1998). This suggests that dredd isa functional effector of the cell death pathway initiatedby RPR and GRIM. Interestingly, reducing the dosageof dredd does not appear to effect the level of killinginduced by the overexpression of HID in the eye. This

Fig. 3. Apoptosis in embryos mutant for one or more of the proapop-totic regulators in 75C1,2. Embryos of similar stages were stained withAO to visualize apoptosis. When embryos are null for hid, but wild type forgrim and rpr, there is a slight reduction in apoptosis. When both hid and

grim are deleted the number of apoptotic cells is decreased further. Inthe absence of hid, grim, and rpr, there is no apoptosis during embryo-genesis.

72 BANGS AND WHITE

may be an indication that the cell death pathway ini-tiated by HID does not rely on dredd activity.

CELL DEATH DURINGDROSOPHILA DEVELOPMENT

Although cell death has been recognized as a compo-nent of Drosophila development for many years (Lock-shin, 1969; Campos-Ortega and Hartenstein, 1985),specific roles for apoptosis have only recently been rec-ognized. As described below, programmed cell death isa necessary feature of all phases of development, fromearly patterning of the embryo to the removal of obso-lete larval tissues and the final sculpting of adult tis-sues.

Cell Death and Embryonic Pattern Formation

Drosophila development begins in the fertilized eggwith a series of 13 syncytial divisions, followed by threerounds of cellular divisions for most cells. Cells areallocated and positioned along dorsal-ventral and an-terior-posterior axes according to maternally estab-lished morphogen gradients. The fate of these cellsdepends on their positions relative to the axes of theegg. This results in the compartmentalization of thedeveloping embryo into specific domains of cells thatwill further develop into precise regions of the larvaand adult (reviewed in Lawrence and Struhl, 1996).The generation of defined regions within the embryoincludes the establishment of synchronously dividingmitotic zones (Foe, 1989). The varied morphologies and

division patterns of the different mitotic domains con-tribute to the transformation of the monolayered blas-toderm into the multi-layered gastrula and the devel-opment of larval and adult structures.

Although cell death does not normally occur duringthese early stages of development (Abrams et al.,1993), the stage is set at this point for apoptosis to playa vital role later. A general model for animal develop-ment includes the accumulation of excess cells requiredfor the formation of a viable embryo. Extraneous cellsare then removed at later points by programmed celldeath (reviewed in Jacobson et al., 1997). Developmentin Drosophila is consistent with this model, since earlydivisions produce more cells than are needed for thesubsequent development of specific structures. Theseextra cells are removed apoptotically at precise timesalong the developmental pathway. For instance, whencell death is blocked in the absence of rpr, grim, andhid, the embryonic nervous system contains a largeexcess of cells at the end of embryogenesis (White et al.,1994). Acridine orange staining shows that cell deathoccurs in a consistent spatial and temporal pattern(Abrams et al., 1993; Pazdera et al., 1998). However,the precise locations and numbers of dying cells varies,indicating that the number of cells being eliminateddepends on the developmental circumstances for agiven embryo.

The elimination of excess cells appears to be at leastin part related to intercellular signaling via the seg-ment polarity genes. For instance, cell death observedin the epidermal segments at stage 12–14 occursamong cells expressing the segment polarity gene en-grailed or are immediately adjacent to cells that ex-press engrailed (Pazdera et al., 1998). When cell sig-naling is disrupted by mutations in the winglesssignaling pathway, cell death among the ENGRAILED-expressing cells increases approximately 5-fold. Thisincreased death is seen in cells located approximatelysix rows away from WINGLESS-secreting cells butdoes not occur in the cells that secrete WINGLESS orthose adjacent to them. Mutants in armadillo, an in-tracellular transducer of WINGLESS signaling, alsoshow increased cell death in the same stripe of cellsseen with wingless mutants. A similar situation seemsto exist in the embryonic brain. When the winglessgene is deleted in null mutants, the protocerebrumdevelops initially but is then largely deleted by pro-grammed cell death in later embryonic stages (Richteret al., 1998). Conversely, when WINGLESS is over-expressed, a dramatic enlargement of the CNS is ob-served. These results indicate that one of the roles forthe segment polarity genes is to establish cell survivalthrough a precise signaling mechanism. It may be thatin normal development excess cells are eliminated af-ter pattern formation because they are not in the idealposition to receive the appropriate survival signals atthe appropriate time.

Successful development requires the tight regulationof cell number to assure correct patterning and cell

TABLE 1. Conserved Elements of the ApoptoticMachinery in Nematodes, Mammalian Systems, and

Drosophila

C. elegans Mammals DrosophilaCed 3 Initiator caspases (CP)

DED domain: CP-8, 10 Dredd

CARD domain: CP-2, 9 Dronc

Effector caspases DrICECP-3, 6, 7 Dcp-1

Decay

Ced 4 Apaf-1 Ark

Proapoptotic bcl-2family proteins

Drob-1Others?

Ced 9 Antiapoptotic bcl-2family proteins

?

Bir-1 cIAP-1 dIAP1Bir-2 cIAP-2 dIAP2

NAIPXIAPsurvivin

? ? HidGrimRpr

73APOPTOSIS IN DROSOPHILA DEVELOPMENT

signaling. When the normal regulation of cell numberis disrupted, cell death increases to compensate for theextra cells. This has been examined by using ectopicexpression of CYCLIN E to extend the normal cycles ofcell division by one extra round (Li et al., 1999). Whenthis is done, the cell density in the embryo is nearlydoubled within 1 hr following the expression ofCYCLIN E. Despite the huge excess of cells, the stripedpatterns of ENGRAILED and WINGLESS staining areunaltered when compared to wild-type embryos, andmost of the embryos develop and hatch into larvae,albeit at a slower rate of development. Examination ofcell death in these embryos shows that hyperplasiainduced by CYCLIN E expression is largely compen-sated for by increased apoptosis in the first 4 hr follow-ing the induction of CYCLIN E. The excess cells areremoved in a pattern-specific fashion rather than ran-domly, and the dying cells express rpr prior to theirdemise.

The plasticity of embryogenesis and the role of apo-ptosis in this plasticity are apparent when examiningpattern repair in the Drosophila embryo. It has beenshown that the mis-expression of the anterior morpho-gen bicoid results in the mispatterning of a number ofanterior morphological markers. Eggs containing inap-propriate doses of bicoid have a number of early devel-opmental abnormalities. These include the shifting ofthe cephalic furrow either anteriorly, in embryos withreduced bicoid levels, or posteriorly in embryos withincreased bicoid (Driever and Nusslein-Volhard, 1988).This latter shift results in the enlargement of headstructures in the early embryo (Fig. 4). Despite therather significant departure from the normal pattern ofdevelopment, these embryos frequently develop intohealthy adults, indicating the embryo’s plasticity andcapacity for pattern repair. Analysis of cell death pat-

terns in embryos with inappropriate dosages of bicoidshows that excess cells generated by an expansion ofthe head domain are deleted by apoptosis (Namba etal., 1997) (Fig. 4). These extra cell deaths occur on thesame schedule as seen for wild-type embryos and ap-pear to be by the same mechanism, since increased rprexpression is also observed in this domain (Namba etal., 1997). Interestingly, the compressed posterior do-main is repaired by a down-regulation of cell death,rather than by an increase in cell proliferation. Thisuse of cell death regulation to repair both the expan-sion and compression of fate maps reflects the eleganceof the system.

There are limits to the extent of expansion and com-pression that the embryo can tolerate. Tumorous struc-tures and breaches in the epithelium are observed indomains that have expanded to the point where celldeath can no longer completely eliminate the excess.Whether this is due to difficulties with signaling withinan overly large cell mass or simply a matter of timerunning out before all of the extra deaths can be initi-ated remains unclear. Conversely, if the compression ofa developmental domain drops the cell number below acertain threshold, structural defects occur or particularorgans fail to develop (Namba et al., 1997).

Cell Death During Metamorphosis

The transition from larval to adult life in Drosophilais regulated by the steroid hormone 20-hydroxyecdys-one (ecdysone). The onset of metamorphosis is signaledby a sharp rise in the ecdysone titer that initiatespuparium formation. During the next several hours,most larval tissues undergo programmed cell death,adult tissues begin to differentiate from imaginal pre-cursor cells, and the ecdysone titer returns to basallevels (reviewed in Thummel, 1996). A second major

Fig. 4. Apoptosis can contribute to pattern repair. Multiple copies ofthe anterior morphogen bicoid lead to an enlarged head in young em-bryos. Increased apoptosis (arrow) can be seen in the head of theseembryos (63 bcd, B), when compared to the wild-type levels seen in A.This contributes to the normal appearance and viability of the embryos bythe end of embryogenesis (63 bcd, C). If apoptosis is blocked by putting

extra copies of bicoid into a background where hid, grim, and rpr aredeleted (63 bcd; H99/H99, D), then the head is greatly enlarged at theend of embryogenesis (63 bcd; H99/H99, F) when compared to thephenotype of the deletion alone (H99/H99, E). Brackets denote the headregion.

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ecdysone pulse initiates the start of pupation, duringwhich the pupal cuticle degenerates and the adult cu-ticle forms. As the pupal stage ends, the ecdysone titeragain returns to basal levels and the adult fly emerges.

Larval tissue degeneration occurs in a stage- andtissue-specific manner that follows each ecdysonepulse. For instance, the degeneration of the anteriorlarval muscles and the larval midgut follows the firstpupal ecdysone pulse, while histolysis of some abdom-inal muscles and the larval salivary glands occursshortly after the second major ecdysone pulse duringpupation. Acridine orange staining and TUNEL assaysshow that cells in these tissues initiate apoptosis justprior to the first morphological indications of histolysis(Jiang et al., 1997). Expression of the caspase inhibitorp35 blocks histolysis in these tissues, further confirm-ing that the loss of larval tissue is through apoptosis(Jiang et al., 1997).

The response of tissues to ecdysone is mediatedthrough the ecdysone receptor which is encoded by theEcR gene (Koelle et al., 1991). Like steroid hormonereceptors in other organisms, EcR is a nuclear receptorthat must heterodimerize with a retinoid X receptor(RXR) in order to exert its regulatory functions (Oro etal., 1990; Yao et al., 1992; Hall and Thummel, 1998).The EcR gene encodes at least three protein isoforms(EcR-A, EcR-B1, and EcR-B2), which differ in theirN-terminal sequences (Talbot et al., 1993). The obser-vation that cells expressing different forms of the EcRrespond differently to ecdysone has led to the hypoth-esis that the ratio of distinct EcR isoforms within cellsdictates the specificity of the response to ecdysone(Robinow et al., 1993). This model is supported byexperiments that show that the metamorphic re-sponses of various tissues are differentially effected inlarvae mutant for the receptor EcR-B1 (Schubiger etal., 1998). Cells in which EcR-B1 is normally the pre-dominant form fail to respond to ecdysone pulses, whilecells that typically express larger amounts of EcR-Aundergo the morphogenetic changes expected of them(Bender et al., 1997; Schubiger et al., 1998).

Programmed cell death in larval tissues appears tobe transcriptionally regulated in response to ecdysonelevels. In both the larval midgut and the larval salivarygland, ecdysone pulses are followed promptly by theexpression of the cell-death genes rpr and hid and thesubsequent destruction of these tissues (Jiang et al.,1997). Ecdysone treatment of isolated midgut and sal-ivary gland tissue has also been shown to induce thetranscription of the caspase dronc (Dorstyn et al.,1999). Furthermore, the anti-apoptotic diap 2 gene istranscriptionally down-regulated following the steroidpulses (Jiang et al., 1997). Following adult eclosion,and a corresponding drop in ecdysone levels, certainlarval neurons degenerate coincidentally with the eclo-sion-specific muscles they innervate (Robinow et al.,1993). These neurons accumulate rpr and grim tran-scripts in response to the drop in ecdysone levels, andthe accumulation of these transcripts, as well as the

subsequent deaths of the neurons, can be suppressedby treatment with ecdysone (Robinow et al., 1997).These observations demonstrate the complexity of thecell death response as regulated by steroid hormonelevels. On the one hand, certain tissues respond toincreasing levels of ecdysone by undergoing apoptosis,as seen with the larval midgut and salivary glands.Other tissues, for instance some larval neurons, re-spond in the opposite manner and die due to decreasinglevels of ecdysone. These differential responses are atleast in part due to the ratio of ecdysone receptor iso-forms expressed in these cells. Those cells that diewhen the ecdysone titer increases express mainly theEcR-B isoforms, whereas those cells dying in responseto a drop in ecdysone levels express primarily theEcR-A isoform.

CELL DEATH IN DEVELOPINGADULT STRUCTURES

Adult structures in Drosophila develop from imagi-nal discs of primordial cells. As in the embryo, cell fatesin the imaginal discs are determined by positional pa-rameters that relate to the numbers of cells in partic-ular domains and cell signaling within and betweendomains. The development of a properly proportionedstructure requires the strict regulation of cell numbers,and programmed cell death plays a key role. In thedeveloping wing, a small number of epithelial cells areundergoing apoptosis at any given time during thesecond and third larval instars and during pupal de-velopment (Milan et al., 1997). Cell death during thisperiod is seen in isolated single cells or in small clus-ters of synchronously dying cells. This pattern is con-sistent with the pattern of rpr expression (Nordstromet al., 1996; Milan et al., 1997) and indicates that thecells die in a coordinated, non-random way. When thenormal regulation of cell death is disrupted locally, theresult is an altered cell death pattern in other parts ofthe wing to compensate for disproportionate cell num-bers. For example, when the expression of a ricin toxinis targeted to the posterior compartment of the devel-oping wing, cell death in this compartment is increasedduring the period of ricin expression. Cell death in theposterior decreases following the inactivation of ricin,but an increase in cell death is then observed in theanterior compartment (Milan et al., 1997). This ectopiccell death is not due to diffusible pro-apoptotic factorsreleased from dying cells but instead seems to be inresponse to the change in positional values caused bythe abnormal loss of cells in the posterior domain.Direct disruption of positional values by inappropriatelevels of morphogens can also result in apoptosis (forexample Bryant, 1988; Adachi-Yamada et al., 1999).

Imaginal discs also accommodate for localized in-creases in cell death by modulating cell proliferation.Thus, an increase in cell proliferation follows ricin-induced cell death in the posterior domain, while adecrease in normal cell proliferation is observed con-comitantly with increased cell death in the anterior

75APOPTOSIS IN DROSOPHILA DEVELOPMENT

compartment. If the expression of ricin is temporary,the wing eventually develops into a correctly propor-tioned adult-sized wing. Interestingly, when increasedcell death is chronically maintained by continuous ricinexpression, the wing develops with up to 40% fewercells, but with the correct wing shape, proportions, andvein patterning (Milan et al., 1997). Correct propor-tions in developing adult structures are thereby main-tained through a combination of cell proliferation andcell death. As in embryos, if the number of cells in adeveloping disc is reduced below a threshold, for exam-ple, by a cell cycle block, the ability of the tissue tocompensate is overwhelmed, and defects in adult struc-tures occur (de Nooij and Hariharan, 1995).

PATTERNING IN THE EYE ANDEGFR SIGNALING

The compound eye of the adult fly is a highly orderedarray of approximately 750 ommatidia. Each ommatid-ium is composed of an invariant arrangement of cellsconsisting of eight photoreceptor neurons, four lens-secreting cone cells, and two primary pigment cells.Ommatidia are arrayed within a hexagonal latticecomposed of secondary and tertiary pigment cells. Thecomponents of the ommatidia are specified in a stereo-typical sequential order by the recruitment of undiffer-entiated cells via signaling from their differentiatedneighbors. Thus the photoreceptors are specified in theorder R8, then pairwise R2 and R5, R3 and R4, and R1and R6, followed at the end by the addition of R7. Thecone cells are recruited next and then the primary,secondary, and tertiary pigment cells in that order.Excess cells are subsequently removed by apoptosis,resulting in the precise architecture of the adult eye(reviewed in Wolff and Ready, 1993).

Cell survival in the developing eye is regulated bycell-cell signaling through EGFR, the Drosophila ho-mologue of the epidermal growth factor (EGF) receptor.

Evidence for this comes from the observation that ex-pression of a dominant-negative form of EGFR in thedeveloping eye results in massive apoptosis (Freeman,1996) (Fig. 5), as does the ectopic expression of argos, adiffusible inhibitor of the EGFR (Sawamoto et al.,1998). Furthermore, when EGFR signaling is main-tained by ectopic expression of a constitutively acti-vated form of the receptor, cell death is dramaticallyreduced in the eye, resulting in excess cells in theommatidial lattice (Miller and Cagan, 1998).

The signal mediated by EGFR activation is trans-duced through the Ras/Raf/MAPK pathway (Diaz-Ben-jumea and Hafen, 1994; Freeman, 1997), suggestingthat the anti-apoptotic activity of EGFR is dependenton Ras signaling. This has been confirmed by the dem-onstration that the eye ablation phenotype resultingfrom the ectopic expression of the death-inducing genesrpr and hid is enhanced by a reduction in Ras activity,and suppressed by constitutively activated Ras or mu-tations that increase the signaling strength of the Raspathway (Bergmann et al., 1998; Kurada and White,1998). Ras signaling is thought to result in the phos-phorylation of HID, which apparently downregulatesits cell killing activity (Bergmann et al., 1998). A sig-nificant reduction in the transcription of hid is alsoobserved following increased activity of the Ras path-way (Kurada and White, 1998). Thus, cell survival inthe developing eye is mediated by signaling throughthe EGFR, resulting in the activation of the Ras/Raf/MAPK pathway and a downregulation in both the lev-els and activity of the death-inducing HID.

One model for the development of the adult eye in-vokes the progressive recruitment and differentiationof the various cell types of the ommatidia by reiterativesignaling by the EGFR, modulated by secretion of theactivating and inactivating ligands SPITZ and AR-GOS, respectively (Freeman, 1997). Multiple functionsfor the EGFR are suggested for this model including

Fig. 5. Inhibition of EGF receptor signaling results in massive apo-ptosis in the developing eye. Third instar eye discs were labeled with anantibody to a nuclear neuronal antigen (elav), in green. The nuclearmaterial of apoptotic cells is labeled with the TUNEL technique, shown in

red. In the wild-type disc very few cells undergo apoptosis during devel-opment. If a dominant negative version of the EGFR is expressed inthese cells, massive apoptosis is detected, and the regular array ofdeveloping photoreceptors is disrupted.

76 BANGS AND WHITE

roles in cell proliferation, cell survival and cell differ-entiation (Dominguez et al., 1998). However, it is pos-sible that the role of EGFR signaling may be to specifywhich of the cells in the developing retina survive, andthus differentiate, and which cells are not needed, andthus die. While differentiation is aborted in the absenceof EGFR, it may be that the failure to differentiatereflects the fact that the cells are dying rather than theconverse view that the cells are dying because they failto differentiate. This can only be addressed by exam-ining the consequences of the loss of EGFR in theabsence of cell death. Considering the fact that EGFRsignaling protects against cell death by downregulat-ing hid, it is likely that this question can be addressedby eliminating EGFR function in a hid null back-ground.

Cell death regulation by the EGFR may occur in anumber of developing tissues. For instance, mutationsin the EGFR ligand spitz disrupt embryonic musclepatterning, and targeted expression of a dominant neg-ative EGFR in developing mesoderm results in the lossof approximately half of the normal complement ofmyofibrils. Conversely, upregulation of EGFR activityin the developing mesoderm leads to an increase in thenumber of myogenic founder cells and the duplicationof some adult muscle types (Buff et al., 1998). Theseobservations suggest that, as has been proposed forthe developing eye, cell numbers are regulated byEGFR signaling and that this regulation is necessaryfor normal muscle development. In fact, the regulationof cell number may prove to be a general role of EGFRsignaling.

CONCLUSIONS AND FUTURE DIRECTIONS

Drosophila provides a powerful model system to in-vestigate both the mechanisms and roles of apoptosisduring animal development. Genetic manipulation al-lows the study of apoptotic regulators and effectors inthe context of the whole animal. In addition this worktakes place in the context of a huge body of knowledgeon other aspects of Drosophila development. This pro-vides the opportunity to understand how different cel-lular processes are coordinately regulated to form thedeveloped organism. For example, apoptosis is neces-sary to establish and maintain the correct cell densitiesrequired for efficient and precise patterning mecha-nisms. Coupled with cell proliferation, cell death en-sures a plasticity in developmental processes that al-lows an embryo to develop dynamically according tocircumstances. The investigation of how cell divisionand apoptosis are coupled is likely to reveal fundamen-tal mechanisms of organogenesis.

The study of apoptosis during metamorphosis pro-vides a fertile area for the investigation of how differ-ent tissues respond differently to the same hormonalsignal. The use of alternate isoforms of the ecdysonereceptor coordinates the transcriptional regulation ofapoptotic effectors in some cells with proliferation anddifferentiation in others. This results in the timely

degeneration of obsolete larval tissues coincidentallywith the development of adult structures. This workprovides an excellent opportunity to understand differ-ential sensitivity to death-inducing signals.

Finally, it is likely that the regulation of apoptosisin a wide variety of developmental processes is morecomplex and important than has been previously rec-ognized. Recent work has suggested that survival sig-naling may play a significant role throughout develop-ment. For example, reiterative signaling through theEGF receptor integrates differentiation with cell deathto finely sculpt the compound eye of the fly. The con-tribution of apoptosis must be considered to fully un-derstand the phenotypes of many genes important indevelopment.

Our understanding of apoptosis during animal devel-opment is still in its infancy. It is apparent that celldeath occurs throughout development, and rudimen-tary maps detailing the spatial and temporal occur-rence of cell death allow us to examine its conse-quences, and thus gain insight about specific functions.The identification of many of the central genes requiredto execute apoptosis provide the tools for the explora-tion of how this process is integrated with signalingpathways that specify proliferation or differentiationduring development.

ACKNOWLEDGMENTS

We thank Christian Peterson for the data shown inFigure 3 and Phani Kurada for the data in Figure 5. PBis sponsored by an NIH training grant to MGH. KW’sresearch is sponsored by a grant from the NIH and agrant from the Shiseido Company of Japan to Massa-chusetts General Hospital/Harvard Medical School.

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