Genetic Mechanism for the Stage- and Tissue-Specific Regulation of Steroid Triggered Programmed Cell Death in Drosophila

Developmental Biology 252, 138–148 (2002)doi:10.1006/dbio.2002.0838

Genetic Mechanism for the Stage- and Tissue-Specific Regulation of Steroid TriggeredProgrammed Cell Death in Drosophila

Cheng-Yu Lee,* ,† Claudio R. Simon,* ,1 Craig T. Woodard,‡ andEric H. Baehrecke* ,2

*Center for Biosystems Research, University of Maryland Biotechnology Institute; †Departmentof Biology, University of Maryland, College Park, Maryland 20742; and ‡Department ofBiological Sciences, Mount Holyoke College, South Hadley, Massachusetts 01075

Steroid hormones trigger a wide variety of cell-specific responses during animal development, but the mechanisms by whichthese systemic signals specify either cell division, differentiation, morphogenesis or death remain uncertain. Here, weanalyze the function of the steroid-regulated genes �FTZ-F1, BR-C, E74A, and E93 during salivary gland programmed celldeath. While mutations in the �FTZ-F1, BR-C, E74A, and E93 genes prevent destruction of salivary glands, only �FTZ-F1is required for DNA fragmentation. Analyses of BR-C, E74A, and E93 loss-of-function mutants indicate that these genesregulate stage-specific transcription of the rpr, hid, ark, dronc, and crq cell death genes. Ectopic expression of �FTZ-F1 issufficient to trigger premature cell death of larval salivary glands and ectopic transcription of the rpr, dronc, and crq celldeath genes that normally precedes salivary gland cell death. The E93 gene is necessary for ectopic salivary gland celldestruction, and ectopic rpr, dronc, and crq transcription, that is induced by expression of �FTZ-F1. Together, theseobservations indicate that �FTZ-F1 regulates the timing of hormone-induced cell responses, while E93 functions to specifyprogrammed cell death. © 2002 Elsevier Science (USA)

Key Words: steroid; ecdysone; programmed cell death; apoptosis; autophagy; development; metamorphosis; Drosophila.

INTRODUCTION

Programmed cell death is an integral component of nor-mal development, maintenance of homeostasis, and hostdefense against pathogens in animals (Jacobson et al., 1997;Vaux and Korsmeyer, 1999). Misregulation of programmedcell death often results in devastating consequences, in-cluding tumorigenesis, autoimmune diseases, and neurode-generative diseases (Krammer, 2000; Thompson, 1995;Yuan and Yanker, 2000).

Programmed cell death is genetically regulated and doesnot lead to an inflammatory response, and is thus distinctfrom necrotic cell death (Lockshin and Zakeri, 1991). Stud-

1 Present address: Department of Cellular Biology and Patho-genic Bioagents, Medical School of Ribeirao Preto, University ofSao Paulo, Av. Bandeirantes 3900, 14090-060, Ribeirao Preto-SP,Brazil.

2
To whom correspondence should be addressed. Fax: (301) 314-9075. E-mail: [email protected].
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ies of developing vertebrate embryos enabled Schweicheland Merker to define apoptotic, autophagic, and nonlysoso-mal programmed cell death on the basis of morphologicalcharacteristics (Schweichel and Merker, 1973). Apoptoticcells exhibit a stereotypical series of morphologicalchanges, including condensation of the nucleus and cyto-plasm, fragmentation of dying cells, and subsequent re-moval of the dying cellular remnants by phagocytes (Kerr etal., 1972). Apoptosis is most frequently observed whenisolated cells die. Unlike apoptosis, autophagic pro-grammed cell death is typically observed when a group ofcells or entire tissues are destroyed. Autophagic cell deathis accompanied by the formation of autophagic vacuolesthat encapsulate cytosolic components for degradation, anddoes not appear to require phagocytes for cell removal.Nonlysosomal cell death is the least prominent and ischaracterized by swelling of cavities with membrane bor-ders followed by degeneration without lysosomal activity.
Although autophagy occurs during development of diverseorganisms and has been linked to cancer, the precise mo-
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lecular mechanisms governing this type of cell death arenot well understood.

Recent studies of programmed cell death in Drosophilalarval salivary glands have suggested that apoptosis andautophagy utilize some common cell-killing components,despite drastic differences in their morphology. Dying lar-val salivary gland cells exhibit morphological characteris-tics of autophagy, including the presence of autophagicvacuoles containing cytoplasmic organelles (Lee and Bae-hrecke, 2001; von Gaudecker and Schmale, 1974). Surpris-ingly, dying salivary gland cells also possess markers thatare typical of apoptosis, including nuclear staining byacridine orange, DNA fragmentation, and phosphatidylser-ine exposure (Jiang et al., 1997; S. van den Eiden and E.H.B.,unpublished observations). In addition, components of theconserved core apoptotic cell death machinery, includingthe caspase dronc and the ced-4/Apaf-1 homolog ark, aretranscribed immediately preceding destruction of salivaryglands, suggesting that caspase activities play a role in thisprocess (Lee et al., 2000). Expression of the baculovirus p35protein, which is a broad-spectrum inhibitor of caspases,inhibits both DNA fragmentation and characteristic cyto-solic changes associated with salivary gland autophagy(Jiang et al., 1997; Lee and Baehrecke, 2001). These datasupport the hypothesis that caspase activities are requiredfor salivary gland autophagy. Thus, studies of the mecha-nisms underlying autophagic cell death will enable us tobetter understand the similarities and differences betweenapoptosis and autophagy.

Steroid hormones regulate diverse biological responses,including programmed cell death during animal develop-ment (Baehrecke, 2000; Evans-Storm and Cidlowski, 1995).During metamorphosis of Drosophila melanogaster, suc-cessive pulses of the steroid hormone 20-hydroxyecdysone(ecdysone) trigger differentiation and morphogenesis ofimaginal discs to give rise to adult tissues, and programmedcell death of larval cells to eliminate obsolete tissues(Thummel, 1996). An increase in ecdysone titer at the endof the third larval instar triggers puparium formation andmarks the onset of metamorphosis. This ecdysone pulsetriggers programmed cell death of the larval midgut andanterior muscles. Shortly after puparium formation, theecdysone titer decreases. Ten to twelve hours followingpuparium formation, the subsequent ecdysone pulse in-duces adult head eversion. This rise in ecdysone triggerslarval salivary glands to undergo programmed cell death,and these larval cells are destroyed synchronously within4–6 h after the peak of the hormone titer (Jiang et al., 1997;Lee and Baehrecke, 2001).

Ecdysone exerts its effects on developmental processesthrough the heterodimeric receptor complex encoded by theEcdysone Receptor (EcR) and the ultraspiracle (usp) genes(Koelle et al., 1991; Thomas et al., 1993; Yao et al., 1992).The ecdysone receptor complex directly activates transcrip-tion of the early genes, including Broad-Complex (BR-C),E74A, and E75, which all encode transcription regulators(Burtis et al., 1990; DiBello et al., 1991; Segraves and

Hogness, 1990). Subsequently, the early genes amplify ec-dysone signals by activating transcription of the late genes,which are thought to play more direct roles in development(Thummel, 1996). Prior to the increase of ecdysone thattriggers larval salivary glands to die, the orphan nuclearhormone receptor �FTZ-F1 is transcribed (Lavorgna et al.,1993). �FTZ-F1 functions as a competence factor that isboth necessary and sufficient to allow the prepupal pulse ofecdysone to reinduce the BR-C, E74A, and E75 early genes,and activate transcription of the stage-specific E93 earlygene (Broadus et al., 1999; Woodard et al., 1994). The�FTZ-F1, BR-C, E74A and E93 genes are all required forsalivary gland programmed cell death (Broadus et al., 1999;Jiang et al., 2000; Lee and Baehrecke, 2001; Lee et al., 2000;Restifo and White, 1992). While the �FTZ-F1, BR-C, andE74A genes possess pleiotropic functions in diverse biologi-cal responses (Broadus et al., 1999; Fletcher et al., 1995;Restifo and White, 1992), the E93 gene appears to be aspecific regulator of cell death (Lee and Baehrecke, 2001;Lee et al., 2000).

Studies of various animals indicate that the componentsof the apoptotic machinery are conserved (Aravind et al.,2001). The Drosophila genome encodes seven caspases.DREDD, DRONC, and DREAM/STRICA are apicalcaspases, and DCP-1, DRICE, DECAY, and DAYDREAM/DAMM are effector caspases (Chen et al., 1998; Dorstyn etal., 1999a,b; Doumanis et al., 2001; Fraser and Evan, 1997;Harvey et al., 2001; Song et al., 1997). Active caspasestrigger many morphological and biochemical hallmarks ofapoptosis by cleaving proteins, including nuclear laminsand cell death regulatory components such as dCAD (Dro-sophila caspase-activated DNase) (McCall and Steller, 1998;Thornberry and Lazebnik, 1998; Yokoyama et al., 2000).Other conserved components of the core cell death machin-ery in Drosophila include the Ced-4/Apaf-1 homolog ARK(Kanuka et al., 1999; Rodriguez et al., 1999; Zhou et al.,1999) and the Ced-9/Bcl-2 homologs Debcl-1/Drob-1/Dborg-1/Dbok (Baker Brachmann et al., 2000; Colussi et al.,2000; Igaki et al., 2000; Zhang et al., 2000) and Buffy/Dborg-2 (Baker Brachmann et al., 2000; Colussi et al., 2000).Drosophila also possess DIAP-1, DIAP-2, dBRUCE, andDeterin, which are homologs of baculoviral inhibitors ofapoptosis (IAP) (Hay et al., 1995; Jones et al., 2000; Vernooyet al., 2000). In addition, four cell death activators, reaper(rpr), head involution defective (hid), grim, and sickle havebeen shown to activate programmed cell death (Chen et al.,1996; Christich et al., 2002; Grether et al., 1995; Srinivasulaet al., 2002; White et al., 1994; Wing et al., 2002). Geneticstudies indicate that rpr, hid, grim, and sickle activateprogrammed cell death by interacting with DIAP-1 toalleviate its inhibitory effects on caspases (Christich et al.,2002; Goyal et al., 2000; Lisi et al., 2000; Srinivasula et al.,2002; Wang et al., 1999; Wing et al., 2002). In agreementwith this observation, structural studies have shown thatthe amino termini of RPR, HID, and GRIM proteins canbind to a surface groove on DIAP-1 mimicking binding ofSMAC/DIABLO to mammalian XIAP (Wu et al., 2000,

139Salivary Gland Cell Death

© 2002 Elsevier Science (USA). All rights reserved.

2001). Thus, Drosophila provides an ideal genetic systemfor dissecting the mechanisms that underlie programmedcell death.

In this paper, we investigate the molecular mechanismthat allows the steroid ecdysone to trigger the stage-specificprogrammed cell death of larval salivary glands. While the�FTZ-F1, BR-C, E74A, and E93 genes are required fordestruction of salivary glands, only �FTZ-F1 is required forDNA fragmentation. These ecdysone-regulated genes acti-vate transcription of a complex network of genes, includingthe BR-C, E74A, and E93 early genes themselves and thelate cell death genes rpr, hid, ark, dronc, and crq. Inaddition, we demonstrate that ectopic expression of�FTZ-F1 is sufficient to induce transcription of the late celldeath genes and to trigger ectopic salivary gland cell death.The E93 gene is necessary for �FTZ-F1-regulation of sali-vary gland cell death and induction of the late cell deathgenes rpr, dronc, and crq. These studies indicate that�FTZ-F1 defines the timing of ecdysone-triggered cell re-sponses, and E93 is critical to refine the global ecdysonesignal into a cell death response.

MATERIALS AND METHODS

Impact of �FTZ-F1, BR-C, E74A, and E93 Mutantson DNA Fragmentation

Wild-type Canton S and �FTZ-F1 (�FTZ-F117/�FTZ-F119), BR-C(rbp5/Y), E74A (E74AP[neo]/Df(3L)st-81k-19), and E93 (E931/Df(3R)93Fx2) mutant larvae were staged at puparium formation,and aged for 6, 12, or 24 h after puparium formation (APF) at 25°C.Animals were fixed, embedded in paraffin, sectioned, and analyzedfor DNA fragmentation by the TUNEL method as previouslydescribed (Lee and Baehrecke, 2001; Restifo and White, 1992).

Northern Blot Analyses of Ecdysone MutantSalivary Glands

Control (E74AP[neo]/wild-type Canton S), BR-C (rbp5/Y), E74A(E74AP[neo]/Df(3L)st-81k-19), and E93 (E931/Df(3R)93Fx2) animalswere raised under standard conditions. White prepupae of each ofthe genotypes were collected and raised at 25°C, and the timing ofadult head eversion was determined empirically by using a stereomicroscope. Head eversion occurs 12 h APF in controls, 13 h APFin BR-C and E7A mutants, and 16 h APF in E93 mutants. Subse-quently, animals of each of the genotypes were aged for �6, �4, �2,0, �2, �4, �6, or �8 h relative to the timing of adult head eversion.RNA was extracted from salivary glands dissected from stagedanimals, electrophoresed, and transferred to nylon membranes.Control and mutant blots were cohybridized with radiolabeledprobes to detect BR-C, E74A, E93, rpr, hid, ark, dronc, and crqmRNA. Exposures of the Northern blots were normalized based onthe time required for equal detection of rp49.

Activation of Premature Salivary Gland Cell Deathby Expression of �FTZ-F1

Transgenic hs-�FTZ-F1 (P[F-F1]) flies have been previously de-scribed (Woodard et al., 1994). To assess whether expression of

�FTZ-F1 is sufficient to allow ecdysone to induce the prematuresalivary gland cell death response, wild-type Canton S and w;hs-�FTZ-F1/hs-�FTZ-F1 third instar larvae were maintained oncornmeal–molasses–agar food containing 0.05% bromophenol blue(Maroni and Stamey, 1983). While Canton S and hs-�FTZ-F1 cleargut larvae were heat-shocked in a 37°C water bath for 1 h, controlnon-heat-shocked hs-�FTZ-F1 clear gut larvae were maintained at25°C. Both the heat-shocked and control animals were allowed torecover at 25°C for 2 h (Woodard et al., 1994). White prepupae werethen selected, aged at 25°C for 6 h, and processed for analyses ofparaffin sections for the presence or absence of salivary glands andfor DNA fragmentation by the TUNEL method as described (Leeand Baehrecke, 2001).

To investigate whether expression of �FTZ-F1 is sufficient toinduce premature transcription of cell death genes, wild-typeCanton S and w; hs-�FTZ-F1/hs-�FTZ-F1 larvae were raised andtreated under the same condition as described above. RNA wasextracted from salivary glands dissected from prepupae that wereaged at 25°C for 0, 2, and 4 h APF, electrophoresed, and transferredto nylon membranes. Northern blots containing salivary glandRNA isolated from the heat-shocked and the control animals weresequentially hybridized with radiolabeled probes to detect rpr, hid,ark, dronc, crq, and rp49, which serves as a loading and transfercontrol (Lee et al., 2000). Exposures of the Northern blots werenormalized based on the time required for equal detection of rp49.

To assess the effect of ectopic expression of �FTZF1 in E93mutants, clear gut larvae with the genotype of w; hs-�FTZ-F1/hs-�FTZ-F1; E931/TM6B and w; hs-�FTZ-F1/hs-�FTZ-F1; E931/Df(3R)93Fx2 were treated and aged with the same regimen asdescribed above. White prepupae were collected, aged for 6 h at25°C, fixed, embedded in paraffin, sectioned, and processed for theTUNEL assay (Lee and Baehrecke, 2001). To determine how E93mutants impact cell death gene transcription induced by ectopic�FTZF1 expression, white prepupae of w; hs-�FTZ-F1/hs-�FTZ-F1;E931/TM6B and w; hs-�FTZ-F1/hs-�FTZF1; E931/Df(3R)93Fx2 wereheat-shocked, and aged for 0, 2, and 4 h at 25°C. RNA was extractedfrom salivary glands dissected from these staged animals, electro-phoresed, transferred, and hybridized with radiolabeled probes toreveal the transcription patterns of rpr, ark, dronc, crq, and rp49genes.

RESULTS

�FTZ-F1 Is Required for DNA Fragmentationduring Salivary Gland Programmed Cell Death

Genetic analyses indicate that the �FTZ-F1, BR-C, E74A,and E93 genes are required for destruction of salivaryglands, but the degradation of the cytoplasm by autophagydiffers in these mutants (Lee and Baehrecke, 2001). Tofurther investigate the function of these ecdysone-regulatedgenes in nuclear changes during programmed cell death,wild-type Canton S and homozygous �FTZ-F1, BR-C, E74A,and E93 mutant animals were aged for 6, 12, or 24 h APF,fixed, embedded in paraffin, sectioned, and processed for theTUNEL assay to detect DNA fragmentation. Salivary glandnuclei of 6-h wild-type prepupae are not stained by theTUNEL assay, indicative of a lack of DNA fragmentation,and serve as a negative control (Fig. 1A; n � many cells from10 salivary glands). Following the rise of the prepupal pulse

140 Lee et al.


of ecdysone 12 h APF, salivary gland polytene chromo-somes of wild-type pupae are positive for DNA fragmenta-tion (Fig. 1B; n � many cells from 12 salivary glands).Salivary gland nuclei of �FTZ-F1 homozygous mutants aged

for 24 h APF are not stained by TUNEL and possess largecytoplasmic vacuoles reminiscent of the cell morphology ofwild-type 12 h salivary glands (Fig. 1C; n � many cells from10 salivary glands). In contrast, BR-C, E74A, and E93mutant salivary gland nuclei are all positively stainedfollowing the TUNEL assay (Figs 1D–1F; n � many cellsfrom 10 salivary glands of each genotype). The observationthat DNA degradation is blocked in �FTZ-F1 mutant sali-vary glands (Fig. 1C), while adult head eversion occursnormally (data not presented), indicates that these mutantanimals have progressed through the ecdysone-regulatedprepupal to pupal transition and that the defect in DNAdegradation is not caused by a general developmental delay.

Mutations in Steroid-Regulated Early Genes AffectTranscription of Cell Death Genes

The ecdysone-regulated early genes play critical roles intransducing hormonal signals into stage- and tissue-specificbiological responses by activating transcription of targetgenes. During salivary gland programmed cell death, theBR-C, E74A, and E93 early genes activate transcription ofcell death genes, including rpr, hid, ark, dronc, and crq(Jiang et al., 2000; Lee et al., 2000). However, the relation-ship among the early genes and between the early genes andthe late cell death genes is not clear. Developmental North-ern blots were constructed using RNA extracted fromsalivary glands dissected from either control, or BR-C,E74A, or E93 mutants that were staged in 2-h intervalsrelative to the timing of adult head eversion. Time 0indicates the timing of adult head eversion, which repre-sents the peak of the prepupal ecdysone titer. Cohybridiza-tion of these membranes allows systematic investigation ofhow BR-C, E74A, and E93 might regulate transcription ofthe ecdysone-triggered early genes themselves, and the latecell death genes rpr, hid, ark, dronc, and crq (Fig. 2).Consistent with previous studies, BR-C, E74A, E93, rpr,hid, ark, dronc, and crq are all induced at the time of headeversion in salivary glands of control animals, while rp49 isconstitutively transcribed. While BR-C is transcribed nor-mally in BR-C and E74A mutant salivary glands, BR-CmRNA is submaximally induced in E93 mutants. Thelarger E74A transcript is not detectable in BR-C mutants,while the smaller E74B RNA is transcribed at a higher leveland not properly repressed in BR-C mutants. While theE74A transcript is not detectable in E74A mutants, E74BRNA appears to be transcribed at a very low level. Both theE74A and E74B RNA are transcribed in E93 mutants, butthe levels of transcription are dramatically reduced. E93RNA appears to be submaximally transcribed in BR-Cmutants, even though it is induced prematurely, is tran-scribed at normal levels in E74A mutant salivary glands,and is not detectable in E93 mutants.

We also hybridized the control, BR-C, E74A, and E93mutant salivary gland Northern blots with probes that arespecific for rpr, hid, ark, dronc, and crq to examine celldeath gene transcription profiles (Fig. 2). Transcription of

FIG. 1. �FTZ-F1, but not BR-C, E74A, or E93, is required for DNAfragmentation during salivary gland programmed cell death. (A)Salivary gland cell nuclei of wild-type animals that were staged 6 hAPF lack DNA fragmentation (arrows indicate unstained nuclei)and served as a negative control. (B) Salivary gland cell nuclei ofwild-type animals that were staged 12 h APF, serving as thepositive control, exhibit DNA fragmentation (arrows indicatedarkly stained nuclei). Salivary gland nuclei of �FTZ-F1 mutantanimals (C) staged 24 h APF lack DNA fragmentation (arrowsindicate unstained nuclei), while salivary gland nuclei of similarlystaged BR-C (D), E74A (E), and E93 (F) mutant animals all exhibitedDNA fragmentation (arrows indicate darkly stained nuclei).



rpr is dramatically reduced in BR-C mutant salivary glands,unaffected in E74A mutant salivary glands, and abolished inE93 mutant salivary glands. hid mRNA is not detectable inBR-C mutants, slightly reduced in E74A mutants, and isabolished in E93 mutant salivary glands. Transcription ofark appears to be normal in salivary glands of all threemutants. The level of dronc RNA is reduced in BR-Cmutant salivary glands, normal in E74A mutant salivaryglands, and is dramatically reduced in E93 mutant salivaryglands. crq is transcribed at a lower level in BR-C and E74Amutant salivary glands, and is not expressed in E93 mutantsalivary glands. Thus, Northern blot analyses of gene tran-scription reveal a complex interaction between theecdysone-regulated early genes and late cell death genes.

Ectopic Expression of �FTZ-F1 Is Sufficient toPrematurely Induce Salivary Gland Cell Death

�FTZ-F1 is expressed immediately preceding the rise ofecdysone that triggers larval salivary gland cell death (La-vorgna et al., 1993), and �FTZ-F1 mutant salivary glandsfail to die (Broadus et al., 1999). Furthermore, ectopicexpression of a �FTZ-F1 transgene in late third instar larvaeis sufficient to allow ecdysone to prematurely activatetranscription of the pupal-specific E93 early gene (Woodardet al., 1994). While ectopic expression of �FTZ-F1 is suffi-cient to prematurely induce E93 transcription, it is not clearif ectopic expression of �FTZ-F1 is sufficient to activatepremature cell death of larval salivary glands. We tested

FIG. 2. Mutations in ecdysone-regulated genes impact transcription of the early and late cell death genes. Larval salivary glands weredissected from control, and BR-C, E74A, and E93 homozygous mutant animals that had been staged in 2-h intervals from �6 to �8 hrelative to the timing of adult head eversion. Total RNA was extracted from salivary glands and analyzed by Northern blot hybridization.Blots of each genotype were cohybridized with the same probe to reveal the relative transcription profile of genes in these mutants.Exposure of these blots was normalized based on the detection of rp49 as a control.

142 Lee et al.


whether ectopic expression of �FTZ-F1 immediately pre-ceding the increase of ecdysone in late third larval instarlarvae would be sufficient to induce larval salivary glandcell death using a heat-inducible hs-�FTZ-F1 (P[F-F1]) trans-gene (Woodard et al., 1994). Wild-type and transgenic ani-mals that possess the hs-�FTZ-F1 transgene were stagedand either heat-shocked at 37°C or held at room tempera-ture. Subsequently, these animals were allowed to recoverat room temperature. White prepupae were collected, agedfor 6 h, and analyzed for cell death of salivary glands byinspection of sections that were subjected to the TUNELassay. Heat-shocked wild-type animals possess salivaryglands, and the nuclei are not stained by TUNEL, indicatingan absence of DNA fragmentation 6 h APF (Fig. 3A; n �many cells from 10 salivary glands). Similarly, controlP[F-F1] prepupae that were held at room temperature pos-sess intact salivary glands and do not exhibit DNA fragmen-tation 6 h APF (Fig. 3B; n � many cells from 10 salivaryglands). In contrast, heat-shocked P[F-F1] animals do nothave salivary glands, and remnants of salivary gland nucleiare positively stained with TUNEL 6 h APF (Fig. 3C; n �many cells from 10 salivary glands).

To assess whether expression of �FTZ-F1 is sufficient totrigger transcription of cell death genes, RNA was extractedfrom salivary glands dissected from staged heat-shockedwild-type, non-heat-shocked P[F-F1] animals and heat-shocked P[F-F1] animals, and analyzed by Northern blothybridization (Fig. 4). The levels of rpr, ark, and dronctranscription are extremely low in salivary glands of theheat-shocked wild-type and non-heat-shocked P[F-F1] ani-mals. Following the heat-induction of �FTZ-F1 expression,rpr, ark, and dronc mRNA is ectopically induced. crq istranscribed in salivary glands of heat-shocked wild-type andP[F-F1] animals held at room temperature. However, ec-topic expression of �FTZ-F1 appears to enhance inductionof crq. We could not detect transcription of hid in eitherheat-shocked wild-type, non-heat-shocked P[F-F1] animals,or heat-shocked P[F-F1] animals (data not presented). Thesedata indicate that ectopic expression of �FTZ-F1 is suffi-cient to induce ectopic salivary gland cell death and thatthis ectopic death involves the induction of genes thatfunction in apoptosis.

E93 Is Required for Premature Death of SalivaryGlands Induced by Ectopic Expression of �FTZ-F1

Previous studies indicate that �FTZ-F1 serves as a com-petence factor that regulates the timing of ecdysone-induced gene transcription (Woodard et al., 1994) and thatthe E93 early gene is a regulatory target of �FTZ-F1 thatfunctions to specify ecdysone-triggered programmed celldeath of larval salivary glands (Lee et al., 2000). In supportof this hypothesis, �FTZ-F1 is expressed just before the riseof ecdysone that induces salivary gland cell death, �FTZ-F1binds to the E93 locus in salivary gland polytene chromo-somes (Lavorgna et al., 1993), and �FTZ-F1 mutants havedecreased levels of E93 RNA (Broadus et al., 1999). How-

ever, it is possible that �FTZ-F1 and E93 could function inparallel genetic pathways. To test these hypotheses,�FTZ-F1 was ectopically expressed in E93 homozygousmutants in the late third instar larvae. Salivary glands of theheat-shocked P[F-F1] prepupae that are E93 heterozygousmutants undergo premature cell death, and the nucleiexhibit DNA fragmentation 6 h APF (Fig. 5A; n � manycells from 10 salivary glands). In contrast, ectopic expres-sion of �FTZ-F1 in identically staged siblings that are E93homozygous mutants fail to elicit premature cell death oflarval salivary glands (Fig. 5B; n � many cells from 10salivary glands). Interestingly, the salivary gland nuclei ofthese animals also exhibit DNA fragmentation, while thecytoplasm possesses a large number of vacuoles. Thissalivary gland phenotype that is induced by expression of�FTZ-F1 in E93 homozygous mutant animals is identical tothe salivary gland phenotype of E93 homozygous mutants24 h APF (Fig. 1).

E93 function is required for ectopic activation of salivarygland cell death by �FTZ-F1. Thus, we examined whether

FIG. 3. Expression of �FTZ-F1 induces premature cell death ofsalivary glands. Animals were heat-shocked, staged 6 h APF, fixed,embedded in paraffin, sectioned, and processed for the TUNELprocedure to detect DNA fragmentation. (A) Salivary glands ofheat-treated wild-type control animals appear normal and do notexhibit DNA fragmentation (arrows indicate unstained nuclei). (B)Salivary glands of transgenic P[F-F1] control animals that were notheat-shocked also appear to be normal, and are negative for DNAfragmentation (arrows indicate unstained nuclei). (C) Ectopic ex-pression of �FTZ-F1 by heat-shock of P[F-F1] animals is sufficientto trigger premature cell death of larval salivary glands 6 h APF, andnuclei of these dying cells exhibit DNA fragmentation (arrowsindicate darkly stained nuclei).



E93 function is required for the ectopic induction of celldeath gene transcription following ectopic expression of�FTZ-F1 in salivary glands. RNA was extracted from stagedsalivary glands of heat-shocked P[F-F1] in E93 heterozygousand E93 homozygous mutant animals (Fig. 6). Ectopicinduction of rpr transcription by �FTZ-F1 in E93 homozy-gous mutant salivary glands is greatly reduced comparedwith the level of rpr transcription in salivary glands of E93heterozygous animals. Similarly, the levels of dronc and crqtranscription induced by �FTZ-F1 are reduced in E93 ho-mozygous mutant salivary glands. In contrast, ark tran-scription that is induced by �FTZ-F1 does not seem to beaffected in salivary glands of E93 homozygous mutants.Combined, these data indicate that E93 is a critical down-stream target of �FTZ-F1, and functions to mediate stage-specific programmed cell death gene transcription that isinduced by ecdysone.

DISCUSSION

The steroid hormone ecdysone regulates a wide variety oftemporally and spatially coordinated biological changes,including programmed cell death during development. Dur-ing programmed cell death, both the nucleus and thecytoplasm undergo a series of stereotypical changes. How-ever, the precise molecular mechanisms by which these celldeath changes are regulated are not well understood. Dro-sophila larval salivary glands undergo rapid and synchro-nous hormone-dependent programmed cell death duringpupal development. The orphan nuclear hormone receptor�FTZ-F1 is expressed immediately preceding the increase ofecdysone that triggers larval salivary gland cell death. Here,we demonstrate that �FTZ-F1 is necessary and sufficient forthe stage-specific programmed cell death of larval salivaryglands. �FTZ-F1, but not BR-C, E74A, or E93, is responsiblefor nuclear DNA fragmentation (Fig. 1). �FTZ-F1 regulatesstage-specific expression of the BR-C, E74A, and E93 earlygenes in larval salivary glands (Broadus et al., 1999; Wood-ard et al., 1994). The BR-C, E74A, and E93 early genesregulate proper transcription of a complex network ofgenes, including the ecdysone-regulated transcription fac-tors and the late cell death genes rpr, hid, ark, dronc, andcrq (Fig. 2). Furthermore, the pupal-specific E93 gene iscritical for ectopic �FTZ-F1-activated salivary gland celldeath.

�FTZ-F1 Provides Competence for Stage-SpecificProgrammed Cell Death of Larval Salivary Glands

The orphan nuclear hormone receptor �FTZ-F1 functionsas a competence factor for pupal-specific responses toecdysone (Broadus et al., 1999; Woodard et al., 1994).Homozygous �FTZ-F1 mutants exhibit pupal-specific de-velopmental defects in adult leg morphogenesis and larvalsalivary gland programmed cell death. Pupal-specific induc-tion of the BR-C, E74A, and E93 early genes by ecdysone is

dramatically reduced in homozygous �FTZ-F1 mutant ani-mals (Broadus et al., 1999). Furthermore, expression of�FTZ-F1 allows ecdysone to prematurely induce transcrip-tion of the pupal-specific E93 gene at the end of third larvalinstar (Woodard et al., 1994). These studies have provided aframework to further dissect how expression of theseecdysone-regulated genes can propagate the hormonal sig-nal into different developmental responses in a temporal-and spatial-specific manner.

�FTZ-F1 function is required for the destruction of thecytoplasm (Lee and Baehrecke, 2001) and nuclear DNAfragmentation during salivary gland programmed cell death(Fig. 1). Ectopic expression of �FTZ-F1 using a heat-inducible transgene allows ecdysone to prematurely inducecell death of larval salivary glands (Fig. 3). Prematureinduction of salivary gland cell death by �FTZ-F1 is accom-panied by ectopic induction of the late cell death genes rpr,ark, dronc, and crq that normally precede the death of thistissue during pupal development (Fig. 4). These series ofexperiments indicate that �FTZ-F1 is necessary and suffi-cient to provide the stage specificity for larval salivary gland

FIG. 4. Ectopic expression of �FTZ-FI is sufficient to triggerectopic transcription of cell death genes. RNA was extracted fromsalivary glands dissected from staged heat-shocked (�hs) wild-type(control), non-heat-shocked (�hs) P[F-F1] transgenic animals (con-trol), and heat-shocked (�hs) P[F-F1] transgenic animals, andanalyzed by Northern blot hybridization. While rpr, ark, dronc, andcrq are transcribed at low levels in heat-shocked wild-type andnon-heat-shocked P[F-F1] control animals, ectopic expression of�FTZ-F1 induces elevated RNA levels of these cell death genes.

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programmed cell death. However, ectopic expression of�FTZ-F1 did not substantially increase the overall amountof cell death in tissues other than larval salivary glands(C-Y.L and E.H.B.; data not presented), arguing that theacquisition of competence to undergo a stage-specific celldeath response occurs in a spatially restricted fashion.�FTZ-F1 allows ecdysone to activate the stage-specific E93gene (Woodard et al., 1994), which refines the temporal andspatial pattern of the global hormonal signal into thespecific cell death response. Furthermore, ectopic activa-tion of salivary gland cell death by expression of �FTZ-F1requires E93 function (Fig. 5). Thus, E93 appears to be acritical regulator of the �FTZ-F1-mediated cell death signal-ing pathway, and E93 specifies the death response.

Regulation of Cellular Changes Associated withAutophagic Programmed Cell Death

Salivary gland destruction was previously shown to occurby autophagic programmed cell death, which includes theformation of autophagic vacuoles in the cytoplasm andDNA fragmentation in the nucleus (Lee and Baehrecke,2001). Ectopic expression of the broad-spectrum caspaseinhibitor p35 completely inhibits DNA fragmentation dur-ing salivary gland cell death, indicating that p35-sensitivecaspases are required for this process. The Drosophilahomolog of mammalian caspase-activated DNase dCAD,requires proteolytic cleavage by caspases such as the mam-malian Caspase 3 or fly caspase DrICE to form a functional

heterotetramer to induce DNA fragmentation (Yokoyamaet al., 2000). p35 effectively inhibits the activities ofCaspase 3 and DrICE (Fraser and Evan, 1997; Zhou et al.,1998). Thus, p35 might inhibit nuclear DNA fragmentationduring salivary gland cell death by hindering the proteolyticactivities of caspases such as DrICE and prevent activationof dCAD. Interestingly, we detected increased transcriptionof drICE in dying salivary, glands lending support to thishypothesis (C-Y.L., Clough, and E.H.B., unpublished obser-vations). The nuclei of �FTZ-F1 mutant salivary gland cellsdo not exhibit DNA fragmentation (Fig. 1), indicating thatthis process might be regulated by �FTZ-F1 at the transcrip-tional level with drICE or other caspases being potentialtargets. Future analyses of �FTZ-F1 targets will result in theidentification of genes that function to regulate DNAfragmentation.

In contrast, expression of p35 has limited impact ondestruction of the salivary gland cytoplasm by autophagy,suggesting that cell death changes associated with thecytosol might occur through a caspase-independent mecha-

FIG. 5. E93 function is required for �FTZ-F1-induced pro-grammed cell death of salivary glands. (A) Salivary glands ofheat-shocked control animals that possess the P[F-F1] transgeneand are heterozygous for E93 (E931/�) undergo premature celldeath, and their nuclei exhibit DNA fragmentation (arrows indi-cate darkly stained nuclei). (B) Salivary glands of similarly stagedheat-shocked transgenic animals that are E93 homozygous mu-tants (E931/Df) remain intact although their nuclei are positive forDNA fragmentation (arrows indicate darkly stained nuclei).

FIG. 6. E93 function is required for �FTZ-F1 induction of celldeath gene transcription. RNA was extracted from salivary glandsdissected from staged heat-shocked P[F-F1] animals in either E93heterozygous control (E931/�) or E93 homozygous mutant (E931/Df) animals and analyzed by Northern blot hybridization. Whileectopic ark transcription appears unaffected in E93 homozygousmutants, E93 function is required for �FTZ-F1 induction of rpr,dronc, and crq.



nism (Lee and Baehrecke, 2001). Recent studies have led tothe observation that other types of proteases might playroles in programmed cell death (Leist and Jaatela, 2001).Genomic analyses of ecdysone-induced autophagy also in-dicate that families of proteases other than caspases exhibitincreased levels of transcription prior to salivary gland celldeath (C-Y.L., Clough, and E.H.B., unpublished observa-tions). These proteases are likely to contribute to thecharacteristic cellular changes associated with salivarygland autophagic cell death, but more studies are needed toempirically test this hypothesis. Alternatively, destructionof the cytoplasm may utilize p35-insensitive caspases.Several pieces of evidence appear to provide support for thishypothesis. The Drosophila apical caspase dronc is ex-pressed immediately prior to salivary gland cell death, andhas previously been shown to be p35-insensitive (Hawkinset al., 2000; Lee et al., 2000; Meier et al., 2000). Expressionof dronc is dramatically reduced in E93 mutants (Fig. 2),which exhibit defects in the destruction of the cytoplasm.In contrast, the dronc mRNA level is only slightly reducedin E74A mutants (Fig. 2), and the cytoplasm of the E74Amutant salivary gland cells seem to have progressed to thelate stage of destruction. In addition, ectopic expression ofp35 does not prevent destruction of the salivary glandcytoplasm. Together, these data suggest that dronc mightfunction in the destruction of the cytoplasm.

Apoptosis and Autophagy Are Distinct Forms ofCell Death with Some Common Regulatory Factors

Studies of salivary gland programmed cell death haveprovided evidence suggesting that autophagy and apoptosisshare many characteristics, even though the morphology ofthese two types of cell death are distinct. Genes thatfunction in apoptosis, including rpr, hid, ark, dronc, andcrq, appear to function in salivary gland autophagic celldeath. The ecdysone-regulated early genes BR-C, E74A, andE93 are required for salivary gland cell death and regulateproper induction of these cell death genes (Fig. 2). Theseobservations lead to the hypothesis that apoptosis andautophagy share some cell death regulatory components,despite the drastic morphological differences that existbetween these two types of cell death.

Do these cell death regulators possess the same functionin both apoptotic and autophagic cell death? Ectopic expres-sion of E93 in Drosophila embryos was able to triggerapoptotic cell death responses, including DNA fragmenta-tion and phagocytosis (Lee and Baehrecke, 2001). The in-duction of DNA fragmentation required the function of theH99 genetic region, which contains rpr, hid, and grimgenes. These three genes, thus, appear to function in regu-lating nuclear DNA fragmentation. rpr and hid are ex-pressed prior to salivary gland autophagic cell death. Ex-pression of these two cell death genes is abolished in E93mutant salivary gland cells (Fig. 2), which exhibit nuclearDNA fragmentation (Fig. 1). Thus, rpr and hid functionmight not be required for nuclear DNA fragmentation

during salivary gland autophagic cell death. This observa-tion raises the possibility that the molecular mechanismsutilized in apoptosis and autophagy are different despitesharing common cell death hallmarks and some cell deathcomponents. During apoptosis, the intrinsic cell killingmachinery of dying cells becomes activated and triggersdestruction of many cellular components. Following phago-cytosis of apoptotic cells by macrophages, the degradationmachinery inside phagocytic cells further breaks downdying cellular debris and clears the remnants (Ferri andKroemer, 2000). Thus, the cell death machinery of thephagocyte plays an important role in apoptosis. In contrast,autophagic cell death of larval salivary glands appears toinvolve only the dying cells—no phagocytes seem to beassociated with these dying cells (Martin and E.H.B. unpub-lished observations). Therefore, the cell-killing machineryinside salivary gland cells is not only responsible for degrad-ing its own cellular contents, but also for clearing the dyingcellular debris. Thus, autophagic cells might possess thecell death machinery of both the dying cells and phagocyteswhen considered in the context of apoptosis. Therefore,DNA fragmentation might occur by an alternative mecha-nism in mutant salivary gland cells that lack rpr and hidfunction. Further analyses of the targets of differentecdysone-regulated genes will enable the identification ofnew components of the pathways that regulate autophagiccell death and further establish the genetic mechanism thatregulates this understudied form of programmed cell death.

ACKNOWLEDGMENTS

We thank members of the Baehrecke lab for helpful discussions.This work was supported by FAPESP (to C.R.S), NSF Career AwardMCB-9722205 and Grant MCB-0110238 (to C.T.W.) and NSF GrantBES-9908942 and NIH Grant GM59136 (to E.H.B).

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Received for publication June 5, 2002Revised August 16, 2002

Accepted September 3, 2002Published online November 7, 2002

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Genetic Mechanism for the Stage- and Tissue-Specific Regulation of Steroid Triggered Programmed Cell Death in Drosophila

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