reaper and bax initiate two different apoptotic pathways affecting mitochondria and antagonized by...

reaper and bax initiate two different apoptotic pathways affecting

mitochondria and antagonized by bcl-2 in Drosophila

Sylvain Brun1, Vincent Rincheval1, Sebastien Gaumer1, Bernard Mignotte1 and Isabelle Guenal*,1

1Laboratoire de Genetique et Biologie Cellulaire, CNRS UPRES-A 8087, et Laboratoire de Genetique Moleculaire et Physiologiede l’EPHE, Universite de Versailles-St Quentin en Yvelines, 45 avenue des Etats-Unis, F-78035 Versailles cedex, France

bcl-2 was the first regulator of apoptosis shown to beinvolved in oncogenesis. Subsequent studies in mammals,in the nematode and in Drosophila revealed wideevolutionary conservation of the regulation of apoptosis.Although dbok/debcl, a member of the bcl-2 gene familydescribed in Drosophila, shows pro-apoptotic activities,no anti-apoptotic bcl-2 family gene has been studied inDrosophila. We have previously reported that the humananti-apoptotic gene bcl-2 is functional in Drosophila,suggesting that the fruit fly shares regulatory mechan-isms with vertebrates and the nematode, involving anti-apoptotic members of the bcl-2 family. We now reportthat bcl-2 suppresses rpr-induced apoptosis in Droso-phila. Additionally, we have compared features of bax-and rpr-induced apoptosis. Flow cytometry analysis ofwing disc cells demonstrate that both killers triggermitochondrial defects. Interestingly, bcl-2 suppressesboth bax- and rpr-induced mitochondrial defects whilethe caspase-inhibitor p35 is specific to the rpr pathway.Finally, we show that the inhibition of apoptosis by bcl-2is associated with the down-regulation of rpr expression.Oncogene (2002) 21, 6458 – 6470. doi:10.1038/sj.onc.1205839

Keywords: apoptosis; Drosophila; bcl-2; reaper; bax;mitochondria

Introduction

Programmed cell death by apoptosis is essential tometazoan development, metamorphosis and tissuehomeostasis (Vaux and Korsmeyer, 1999). Apoptosisis a powerful biological process that removes poten-tially dangerous cells like self-reactive lymphocytes,virus-infected cells and tumor cells. Genetic andbiochemical studies, in mammals as well as ininvertebrates, reveal a high evolutionary conservationof the regulation of apoptosis. The principal execu-tioners of apoptosis belong to a highly conserved

family of proteases, the cysteinyl-aspartases (caspases).Caspases are constitutively present in cells aszymogenes, and their activation by proteolysis orauto-proteolysis provokes the cleavage of othercaspases in a proteolytic activation cascade thatultimately cleaves and inactivates specific targetsessential for cell structure and DNA integrity (Nunezet al., 1998; Thornberry and Lazebnik, 1998). Geneticstudies in Caenorhabditis elegans identified the genesced-3, ced-4, egl-1, which are essential to trigger celldeath, and ced-9 which inhibits cell death. In cellstriggered to die, the protein EGL-1 inhibits CED-9,which provokes the activation of the caspase CED-3by CED-4 (Conradt and Horvitz, 1998). Although thismodel has been a scaffold for understanding theregulation of caspase activation in other species, theregulation of apoptosis in the fruit fly and inmammals shows an increased complexity comparedto that in the nematode.

First, while only one caspase (CED-3) is involved inapoptosis in the nematode, at least 10 caspases inmammals and seven in Drosophila melanogaster areinvolved in apoptosis (Chen et al., 1998; Dorstyn et al.,1999a,b; Doumanis et al., 2001; Fraser and Evan, 1997;Harvey et al., 2001; Song et al., 1997).

Second, in C. elegans, the regulation of apotosis bythe bcl-2 family is mediated by the anti-apoptotic geneced-9 and the pro-apoptotic BH3-only gene egl-1. InDrosophila as in mammals, pro-apoptotic members ofthe bcl-2 family are not limited to BH3-only genes(Gross et al., 1999). Indeed, both bcl-2 family genesidentified in the fly genome are homologous to themammalian pro-apoptotic gene bok, and presentdifferent BH interaction domains (Brachmann et al.,2000; Colussi et al., 2000; Igaki et al., 2000; Zhang etal., 2000). However, while debcl/drob-1/dborg-1/dbokhas been shown to be pro-apoptotic, whether thesecond predicted gene of the family will be pro- oranti-apoptotic remains to be addressed.

Third, in contrast with CED-4 which constitutivelyactivates the caspase CED-3, Apaf-1 in mammals aswell as DAPAF-1/DARK/HAC-1 in the fruit fly bothneed ATP and cytochrome c (cyt c) as co-factors toactivate caspases (Kanuka et al., 1999; Li et al., 1997;Rodriguez et al., 1999; Zhou et al., 1999). Inmammals, Bax triggers the release of cyt c and otherkey pro-apoptotic factors from the inter-membranespace of mitochondria like the Apoptosis InducingReceived 31 July 2001; revised 7 June 2002; accepted 28 June 2002

*Correspondence: I Guenal, Laboratoire de Genetique et BiologieCellulaire, UPRES-A 8087, Universite de Versailles-St Quentin enYvelines, 45 avenue des Etats-Unis, F-78035 Versailles cedex, France;E-mail: [email protected]

Oncogene (2002) 21, 6458 – 6470ª 2002 Nature Publishing Group All rights reserved 0950 – 9232/02 $25.00

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Factor (AIF) and several caspases (Zamzami et al.,1998), while Bcl-2 inhibits this release and apoptosisinduced by Bax or many other death stimuli (Gross etal., 1999). In Drosophila, cyt c may regulate apoptosiswithout being released (Dorstyn et al., 2002; Varkey etal., 1999; Zimmermann et al., 2002). Indeed, DAPAF-1 and cyt c seem to cooperate with DRONC andDRICE in an apoptosome-like complex localized tomitochondria and responsible for the processing ofboth caspases DRONC and DRICE in Drosophilacells (Dorstyn et al., 2002; Kanuka et al., 1999; Li etal., 1997; Rodriguez et al., 1999; Zhou et al., 1999).Interestingly, dBOK/DEBCL, which localizes tomitochondria, triggers cyt c release and apoptosiswhen expressed in mammalian cells, while in Droso-phila cells, it triggers the translocation of DRONC tomitochondria and caspase activation without cyt crelease.

In Drosophila, mitochondria might be targets of thethree RHG domain protein RPR, HID and GRIM.Although the three genes reaper (rpr), head involutiondefective (hid) and grim are unknown in mammals,they are functional in vertebrates, and HID andGRIM localize to mitochondria during apoptosis inmammalian cells (Claveria et al., 1998; Evans et al.,1997; Haining et al., 1999). Furthermore, in Xenopuscells extracts, rpr induces cyt c release frommitochondria (Thress et al., 1999), and bcl-2 inhibitsrpr-dependent caspase-activation (Evans et al., 1997).Zimmermann et al. (2002) show that rpr-dependentapoptosis can be independent of dapaf-1 and cyt c invitro. However, although rpr-dependent cyt c release isnot detected in Drosophila cells, dapaf-1 mutantspartially suppress rpr-induced apoptosis, suggesting arequirement for active DAPAF-1 in the rpr apoptoticpathway in vivo.

Whether an anti-apoptotic bcl-2 family memberacts on mitochondria and cyt c to protect cells fromapoptosis in Drosophila remains to be addressed. Toour knowledge, no anti-apoptotic member of the bcl-2 gene family has been found in the fruit fly.Nevertheless, we have shown in our laboratory thatthe murine pro-apoptotic gene bax and the humananti-apoptotic gene bcl-2 are functional in Drosophilamelanogaster (Gaumer et al., 2000). Our currentstudy demonstrates that bax and rpr induce twodifferent apoptotic pathways, both affectingmitochondria. We show here that bcl-2 suppressesrpr- and bax-induced cell death as well as rpr- andbax-induced mitochondrial defects, while the protec-tive effect of the caspase-inhibitor p35 is specific tothe rpr death pathway.

Although in mammals most of the components ofthe cell death program are constitutively present,many apoptosis regulators in Drosophila are controlledat the transcriptional level. Among others, rpr istranscriptionally induced to trigger apoptosis duringembryonic development, metamorphosis and after X-ray irradiation (Baehrecke, 2000; Nordstrom et al.,1996). In particular, after X-ray irradiation rprexpression is up-regulated by dmp53, the homologue

of the mammalian oncosuppressor p53. Additionally,the imd/rip pathway, which is involved in the immuneresponse to bacterial infection (Lemaitre et al., 1995),can also trigger rpr expression (Georgel et al., 2001).Strikingly, we also show in this study that inhibitionof apoptosis by expression of bcl-2 during embryonicdevelopment (Gaumer et al., 2000) is correlated withthe down-regulation of rpr expression, suggesting theexistence of an amplification loop in rpr-inducedapoptosis.

Results

A semi-quantitative approach to detect the geneticsuppression of wing phenotypes

We have used the vg-GAL4 transgenic Drosophilastrain to drive the expression of UAS-rpr, UAS-baxand several other transgenes in the wing margin duringlarval development. Flies expressing rpr or bax in thewing show two principal phenotypes: notches and wingfusion defects. We have focused on the notchphenotype to screen the flies. Surprisingly, expressivityand penetrance of the notch phenotype are variable ina population of flies of the same genotype. At 258C,flies expressing rpr in the wing exhibit a distribution ofphenotypes that can be classified into four categoriesaccording to their strength: strong, intermediate, weakand wild type (Figure 1). At 188C, flies expressing baxin the wing exhibit a distribution of phenotypes thatcan be classified into three categories: strong, inter-mediate and weak (Figure 2). Therefore, we have set asemi-quantitative approach to study genetic suppres-sion between flies co-expressing rpr or bax and severalother transgenes. To compare distributions of pheno-types between two different lineages, we have used thestatistical Wilcoxon test (Wilcoxon, 1945). This testdefines a Ws value between two populations and allowsassessment of whether their distributions, whichcorrespond to gradual phenotypes, are significantlydifferent or not, and which population is composed ofstronger phenotypes. We define the statistically signifi-cant limit as a51073.

bcl-2 and p35 suppress rpr-induced cell death in the wing

The Drosophila pro-apoptotic gene rpr plays a key rolein apoptosis. Since bcl-2 and rpr have antagonisticeffects on apoptosis, we tested whether the human bcl-2gene could suppress phenotypes induced by expressionof rpr in the wing. Our results show that thephenotypes induced by rpr are weaker when bcl-2 isco-expressed. Although the suppression effect of bcl-2is partial, distributions of phenotypes clearly shift toweaker phenotypes as the number of UAS-bcl-2 copiesincreases (Figure 3A and Table 1). Indeed, Ws valuesbetween control flies expressing only rpr and flies co-expressing rpr and one to three copies of bcl-2 decreasefrom 74.1 to 79.6 (a51073) as shown in Table 1.Furthermore, almost 60% of flies co-expressing rpr andthree copies of bcl-2 show a wild type phenotype

bcl-2 inhibition of reaper-induced apoptosisS Brun et al

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compared to 4% of control flies. We did not find astatistical difference between distributions of pheno-types observed in flies co-expressing rpr and one or twocopies of bcl-2. However, there is a significantdifference between distributions of flies co-expressingrpr and one or two copies of bcl-2 and the phenotypedistribution of flies co-expressing rpr and three copiesof bcl-2 (Ws3 – 4=76.89; a3 – 4=4.6610711). Theseresults clearly show that bcl-2 suppresses the pheno-types induced by rpr in the wing in a dose-dependentmanner.

Since it has been shown that rpr-induced apoptosis iscaspase-dependent in the developing eye (Hay et al.,1995), we asked whether the caspase-inhibitor p35could inhibit rpr-induced apoptosis in the wing. Ourresults show that expression of p35 partially suppressesthe notches induced by rpr expression (Figure 3B).Phenotypes of flies co-expressing rpr and p35 areweaker than phenotypes of control flies, and almost70% of flies co-expressing rpr and one copy of p35display weak or wild type phenotypes compared to37% of control flies. The Ws1– 5 value between thedistributions is 75.36 and a1 – 551073 (Table 1). Asecond independent p35 strain was also used tosuppress rpr-induced cell death and both p35 strainsdisplay similar protective activity (data not shown).

Since bcl-2 can prevent caspase-independent celldeath (Okuno et al., 1998) in mammals, we havestudied the co-expression of bcl-2 and p35 inDrosophila. The data presented in Figure 3B and Table1 show no statistical difference between phenotypedistributions of flies co-expressing rpr and one copy ofbcl-2, flies co-expressing rpr and one copy of p35 andflies co-expressing rpr and one copy of p35 plus onecopy of bcl-2. Consistent with this observation, we alsoobserve no differences between the effects of two copiesof bcl-2, one copy of p35, two copies of bcl-2 plus onecopy of p35 on rpr-induced phenotypes (Figure 3C andTable 1). Strikingly, while the jump from two to threecopies of UAS-bcl-2 has a great suppressive effect onthe phenotypes induced by rpr, adding a UAS-p35transgene in the same genetic background has no effect.Taken together, these data show that the co-expressionof both inhibitors of apoptosis has no additive effecton the phenotypes induced by rpr, suggesting that theirinhibitory effects are somehow redundant.

bcl-2 down-regulates rpr expression during embryonicdevelopment

Since many death signals transcriptionally induce rpr incells committed to die, we asked whether bcl-2 could

Figure 2 bax induces variable phenotypes in the wing. At 188C,the phenotypes induced by UAS-bax driven by vg-GAL4 are vari-able. Considering the notches appearing on altered wings, we clas-sified flies into three categories (B, C and D) according to thestrength of their wing phenotypes. (A) Wild type Canton Sw1118 fly wing. (B) The size of a wing with a weak phenotype isalmost conserved when compared to wild type, and the notchesare scattered along the margin. (C) The size of a wing with an in-termediate phenotype is reduced, and an important part of themargin has disappeared. (D) The strong phenotype is comparableto the vestigial phenotype. The wing is partially absent or comple-tely absent, sometimes the flies show thorax abnormalities. Scalebar, 500 mm

Figure 1 rpr induces variable phenotypes in the wing. At 258C,the phenotypes induced by UAS-rpr driven by vg-GAL4 are vari-able. We classified flies into four categories based on the strengthof their wing phenotype: wild type (A), weak (B), intermediate (C)and strong (D) phenotypes. In our experiment, the three mutantcategories are defined as follows: (A) wildtype wings are similarto these of the Canton S w1118 reference strain. Note that the wingin (A) is from the Canton S w1118 strain. (B) Wings with weakphenotypes miss only bristles on the proximal posterior margin(arrow head). (C) Wings with intermediate phenotype bear smallnotches, generally localized on the proximal posterior part of thewing margin. (D) The notches on wings with strong phenotypesare larger extending from the posterior proximal part to the distalpart of the wing. (A) Scale bar, 500 mm


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only inhibit events downstream of rpr, or whether bcl-2also prevents the transcriptional induction of rpr. Inorder to determine if bcl-2 can act upstream of rpr, wehave used the rpr-11-LacZ construct (Nordstrom et al.,1996) to visualize rpr expression during embryonicdevelopment in embryos expressing bcl-2. We haveanalysed the X-gal staining pattern in situ in embryosuntil stage 14 of embryonic development (Figure 4).

In control embryos (rpr-11-LacZ/da-GAL4), LacZstaining patterns are in agreement with those describedby Nordstrom et al. (1996). Interestingly, in embryosco-expressing bcl-2 and the LacZ reporter (UAS-bcl-2/+;rpr-11-LacZ/da-GAL4), rpr-11-LacZ expression doesnot begin until stage 9 (Figure 4c) while it is alreadydetected at stage 8 in control embryos (Figure 4b). Atstage 9, LacZ staining in embryos expressing bcl-2 isweak and only detectable in the posterior end of theextending germ band. In contrast, control embryos at a

similar stage display staining at the posterior end of theextending germ band, dorsally in the region adjacent tothe cephalic furrow, and more anteriorely at theclypeolabrum as well as gnathal buds (Figure 4D).Subsequently, light staining is detected in segmentalstripes in control embryos at stage 9 while thissegmental staining does not appear until stage 10/11in embryos expressing bcl-2 (Figure 4E). Finally, forboth types of embryos, LacZ staining in these zonespersists and intensifies from stage 11 to stage 14(Figure 4E –H), but rpr expression remains weaker inembryos expressing bcl-2.

In order to quantify variations of LacZ stainingobserved in situ, we have measured enzymatic activityof b-galactosidase in embryonic extracts at differenttimes of development for control embryos (rpr-11-LacZ/da-GAL4), embryos expressing bcl-2 (UAS-bcl-2/+;rpr-11-LacZ/da-GAL4) and embryos expressing p35(UAS-p35/+;rpr-11-LacZ/da-GAL4) (Table 2). Activ-ity assays were performed nine times independently,and then analysed with the statistical Student test. Forembryos of the three genotypes analysed, our dataclearly show that b-galactosidase activities are verylow and not statistically different before 3 h AEL(Table 2) (a40.05). Subsequently, b-galactosidase

Figure 3 Analyses of the distributions of the phenotypes inducedby rpr. The percentages of flies listed in each phenotypic category(strong, intermediate, weak and wild type) are reported in (A), (B)and (C). All flies considered express one copy of rpr in the wingas in control (UAS-rpr/X;vg-GAL4/+), and one copy of bcl-2(UAS-rpr/X;vg-GAL4/+;UAS-bcl-2/+), two copies of bcl-2(UAS-rpr/UAS-bcl-2;vg-GAL4/+;UAS-bcl-2/+) or three copiesof bcl-2 (UAS-rpr/UAS-bcl-2;vg-GAL4/UAS-bcl-2;UAS-bcl-2/+)in (A); one copy of p35 (UAS-rpr/X;vg-GAL4/UAS-p35), onecopy of bcl-2 or one copy of bcl-2 plus one copy of p35 (UAS-rpr/X;vg-GAL4/UAS-p35;UAS-bcl-2/+) in (B); two copies ofbcl-2, one copy of p35 or two copies of bcl-2 plus one copy ofp35 (UAS-rpr/UAS-bcl-2;vg-GAL4/UAS-p35;UAS-bcl-2/+) in(C). The statistical Wilcoxon test was used to compare the pheno-type distributions (Table 1). (A) bcl-2 partially suppresses the phe-notypes induced by rpr in a dose-dependent manner. The strengthof the phenotypes induced by rpr using the vg-GAL4 driver de-creases as the number of bcl-2 copies increases. (B and C), p35suppresses rpr-induced phenotypes in the wing, but no additiveeffect on the suppression of rpr-induced phenotypes is observedwhen bcl-2 and p35 are co-expressed

Figure 4 rpr repression during embryonic development. We haveused the rpr-11-LacZ reporter gene (Nordstrom et al., 1996) tostudy rpr expression during embryonic development in embryosexpressing bcl-2. (A, C, E and G) Embryos expressing the UAS-bcl-2 transgene under da-GAL4 control (ubiquitous expression).The approximate times of development and the correspondingstages of development are mentioned on the left and on the rightof the Figure respectively. The b-galactosidase activity in theseembryos is compared to that in control embryos at similar stages(B, D, F and H). The appearance of b-galactosidase activity is de-layed in bcl-2 embryos until stage 9. At stages 11 and 14, the pat-tern of rpr-11-LacZ is comparable in bcl-2 embryos (E and G)and in control embryos (F and H), but the b-galactosidase activityis weaker in embryos expressing bcl-2. All views are sagittal withanterior to the left and dorsal towards the top. Scale bar, 100 mm


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activities slightly increase 3 – 6 h AEL, and becomemuch stronger at later stages (6 – 9 h). Indeed, foreach genotype analysed, activities measured arestatistically different between the three developingtimes (data not shown). Strikingly, as previouslyobserved in situ, b-galactosidase activity detected isstatistically lower in embryos expressing bcl-2 than incontrol embryos at 3 – 6 h and at 6 – 9 h AEL.Furthermore, the activity in 3 – 6 h-embryos expressingbcl-2 is statistically comparable to the activity in 0 –3 h-control embryos (data not shown). Therefore, the

3 – 6 h-embryos expressing bcl-2 (including embryos atstages 8 and 9 of development) exhibit a delay in rprinduction, and at later stages, rpr expression is down-regulated in embryos expressing bcl-2. In contrast,expression of p35 has no effect on rpr expressionduring embryonic development. At all developmentalstages analysed, b-galactosidase activity in embryosexpressing p35 is statistically comparable to that incontrol embryos (Table 2). Taken together, these dataargue that bcl-2 affects the transcriptional induction ofrpr during development, and this activity is specific to

Table 2 Quantification of the b-galactosidase activity during embryonic development

Development b-galactosidase activity Standardtime laps Genotypes analysed (mmolONPG/min/mgprot) deviation a value

0 – 3 h Control (rpr-11-lacZ/da-GAL4) 0.9 0.8 1a

One copy p35 (UAS-p35/+; rpr-11-lacZ/da-GAL4) 0.7 0.8 0.48a

One copy bcl-2 (UAS-bcl-2/+; rpr-11-lacZ/da-GAL4) 0.6 0.5 0.31a

3 – 6 h Control 2.8 0.9 1a

One copy p35 3.2 1.4 0.46a

One copy bcl-2 1.6 0.7 0.01

6 – 9 h Control 17.4 6.4 1a

One copy p35 18.1 8.3 0.85a

One copy bcl-2 7.5 5.7 3.261073

The b-galactosidase activity has been measured nine times independently (n=9). a values presented here are given by the comparison of the b-galactosidase activity of each genotype analysed with the activity of the control genotype of the same development time laps. aa values aresuperior to the statistical significant limit fixed at 0.05

Table 1 Ws values and a values of the Wilcoxon test in the rpr suppression study

Genotypes analysed G 1 2 3 4 5 6

Control (n=217)UAS-rpr/X;vg-GAL4/+ 1

One copy bcl-2 (n=210) Ws1– 2=74.1UAS-rpr/X;vg-GAL4/+; 2 a1 – 2=4.961075

UAS-bcl-2/+

Two copies bcl-2 (n=176) Ws1– 3=75.3 Ws2– 3=71.0UAS-rpr/UAS-bcl2; 3 a1 – 3=1.561077 a2 – 3=0.34a

vg-GAL4/+;UAS-bcl-2/+

Three copies bcl-2 (n=104) Ws1– 4=79.6 Ws2– 4=77.0 Ws3– 4=76.8UAS-rpr/UAS-bcl-2; 4 a1 – 451610713 a2 – 4=1.7610711 a3 – 4=4.6610711

vg-GAL4/UAS-bcl-2;UAS-bcl-2/+

One copy p35 (n=247) Ws1– 5=75.4 Ws2– 5=73.0 Ws3– 5=72.2 Ws4– 5=5.5UAS-rpr/X; vg-GAL4/ 5 a1 – 5=2.4610712 a2 – 5=3.061073 a a3 – 5=0.03a a4 – 5=6.361078

UAS-p35

One copy bcl-2+1 copy p35 Ws1–6=75.3 Ws2– 6=71.1 Ws3– 6=70.2 Ws4– 6=6.5 Ws5 – 6=1.9(n=207)

UAS-rpr/X; vg-GAL4/ 6 a1 – 6=1.961077 a2 – 6=0.28a a3 – 6=0.82a a4 – 6=2.6610710 a5 – 6=0.06a

UAS-p35;

Two copies bcl-2+1 copy p35 Ws1–7=77.7 Ws2– 7=73.5 Ws3– 7=72.7 Ws4– 7=4.9 Ws5 – 7=70.7 Ws6– 7=72.5(n=226)

UAS-rpr/UAS-bcl-2; vg-GAL4/ 7 a1 – 7=1.6610713 a2 – 7=4.361074 a3 – 7=5.3610710 a4 – 7=9.961077 a5 – 7=0.49a a6 – 7=1.3610712 a

UAS-p35; UAS-bcl-2/+

aa values are superior to the significant limit fixed at 161073. The genotypes of the considered distributions are numbered from 1 to 6 (G).n specifies the number of flies analysed


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bcl-2 since the expression of p35 has no effect on therpr-11-LacZ transgene.

bax-induced cell death is sensitive to bcl-2 level butinsensitive to p35

Since p35 inhibits many Drosophila cell death path-ways, we have tested whether p35 could also suppressbax-induced phenotypes. As previously described, bax-induced phenotypes are variable at 188C. The severityof wing notches ranges from strong to weakphenotypes (Figure 2). To study genetic interactionsbetween bax and bcl-2 or p35, females carrying boththe vg-GAL4 driver and UAS-bax (vg-GAL4,UAS-bax/CyO-gfp) were crossed to males carrying copies of theUAS-bcl-2 or UAS-p35 transgenes. Results arepresented in Figure 5, and distributions of phenotypesare compared using the statistical Wilcoxon test(Tables 3 and 4).

Distributions of phenotypes between females co-expressing bax and p35 and females expressing bax butnot p35 are not statistically different (Ws1 – 2=70.79;a1 – 2=0.43). This result is comparable to that in malelineages: Ws5 – 6=2.55; a5 – 6=0.01. A similar result wasobtained using a different UAS-p35 transgenic strain aswell as a third strain carrying two copies of UAS-p35(data not shown). Note that the different p35 strains

suppress rpr-induced cell death in the wing (Figure 3and data not shown). In contrast, bax is sensitive tobcl-2 level and the strength of bax-induced phenotypesdecreases as the number of bcl-2 copies increases.Indeed, 73% of females and 41% of males co-expressing bax and one copy of bcl-2 have weakphenotypes compared respectively to 10% of femalesand 15% of males expressing only bax. Moreover,females co-expressing bax and three copies of bcl-2have weaker phenotypes than those co-expressing asingle copy of bcl-2 (Ws3– 4=73.76; a3 – 451073).Similarly, two copies of bcl-2 in male lineage suppressphenotypes induced by bax more effectively than onecopy (Ws7 – 8=75.97; a7 – 851073). In these experi-ments, bcl-2 suppresses phenotypes induced by bax in adose-dependent manner while p35 has no effect. Thesedata suggest that bcl-2 inhibits apoptosis triggeredthrough a p35-insensitive pathway in the fly.

Wing discs are differently affected by bax and rprexpression

As shown in Figure 6, acridine orange (AO) staining ofwing discs of third instar larvae expressing rpr or baxdriven by vg-GAL4 shows prominent apoptosis, whilein wild type discs, apoptosis is very low and appearspreferentially in the notum (Figure 6). Staining in discs

Table 3 Ws values and a values of the Wilcoxon test for the female lineages in the bax suppression study

Females analysed G 1 2 3

Control (n=101)vg-GAL4,UAS-bax/+ 1

One copy p35 (n=111) Ws1– 2=70.8vg-GAL4,UAS-bax/UAS-p53 2 a1 – 2=0.43a

One copy bcl-2 (n=93) Ws1– 3=79.1 Ws2– 3=79.3vg-GAL4,UAS-bax/UAS-bcl-2 3 a1 – 351610713 a2 – 351610713

Three copies bcl-2 (n=94) 4 Ws1 – 4=711.4 Ws2– 4=711.7 Ws3– 4=73.8UAS-bcl-2/X; vg-GAL4, UAS-bax/UAS-bcl-2; a1 – 4=51610713 a2 – 4=51610713 a3 – 4=1.761074

UAS-bcl2/+


Table 4 Ws values and a values of the Wilcoxon test for the male lineages in the bax suppression study

Males analysed G 5 6 7

Control (n=67)vg-GAL4,UAS-bax/+ 5

One copy p35 (n=64) Ws5– 6=2.6vg-GAL4,UAS-bax/UAS-p35 6 a5 – 6=0.01a

One copy bcl-2 (n=88) Ws5– 7=74.4 Ws6– 7=76.6vg-GAL4,UAS-bax/UAS-bcl-2 7 a5 – 7=1.361075 a6 – 7=1.6610710

Two copies bcl-2 (n=117) Ws5– 8=79.0 Ws6– 8=710.3 Ws7– 8=76.0vg-GAL4,UAS-bax/UAS-bcl-2; 8 a5 – 851610713 a6 – 851610713 a7 – 8=4.961079

UAS-bcl-2/+



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expressing rpr is intense and foreshadows vg-GAL4expression. More central labeling is detectable in discsexpressing bax, though most of AO staining is alsofound in the area of vg-GAL4 expression. Thisdiscrepancy of staining pattern suggests that althoughmassive apoptosis occurs in discs expressing rpr like indiscs expressing bax, the wing pouch of the disc ismore affected by bax expression. Additionally, expres-sion of bax or rpr triggers a dramatic decrease of discsize. Thus, both reduced size of discs and AO stainingpattern show that rpr- and bax-induced apoptosisoccur at least before as well as within the third instarlarval stage of development.

bax and rpr induce mitochondrial membrane potentialcollapse, cell shrinkage but no necrosis

It has been shown in mammalian cells and recently inDrosophila cells that mitochondrial membrane poten-tial (DCm) drop is an early event in apoptosis whencompared to DNA fragmentation and plasmamembrane injuries (Vayssiere et al., 1994; Zimmer-mann et al., 2002). Therefore, we asked whether rpr-and bax-induced cell death in vivo in Drosophilaprovoke DCm collapse. Flow cytometry analyses werecarried out to study the integrity of the plasmamembrane, variations of DCm and cell shrinkage in

third instar larvae wing discs. We have used PropidiumIodide (PI) which penetrates only cells with a loss ofmembrane integrity, like necrotic cells or late apoptoticcells, as a specific indicator of cell membrane integrity.We have assessed mitochondrial membrane potentialusing DiOC6(3), a lipophilic cation taken up bymitochondria in proportion to DCm, and cell shrinkagewas analysed by observing cell size reduction. Resultspresented for the study of bax-induced apoptosis andfor the study of rpr-induced apoptosis are taken fromindependent experiments. In both studies, assays oncells isolated from discs carrying the death inducerwithout a driver (control discs) or from discsexpressing the death inducer alone or co-expressingthe death inducer and p35 or bcl-2 have beenperformed in parallel. Only cytograms correspondingto cells of genotype (UAS-rpr/X) and (UAS-rpr/X;vg-GAL4/+) are presented (Figure 7A). For each sampleanalysed, cell debris and aggregates as well as PI-positive cells are excluded from the DiOC6(3) analysis.

In rpr control discs (Figure 7Aa), 6% of cells showhigh PI fluorescence (window P) and PI-negative cellswith no major membrane injuries split into two distinctpopulations based on their DiOC6(3) fluorescence andtheir size (Figure 7Ab): 84% of cells have a large sizeand high DiOC6(3) fluorescence (Figure 7Ab, windowH), while other dots in window L characterize cellswith a reduced size and DCm drop. These 16% of cellsin window L exhibit characteristics of apoptotis: theyundergo DCm collapse and cell shrinkage while theirplasma membrane is not permeable. Moreover, asdemonstrated below, expression of each death inducerrpr or bax increases this apoptotic population. Thus,we observe a background level of apoptotic cells incontrol discs, which is in agreement with in situobservation of normal developmental apoptosis by

Figure 5 Analyses of the distributions of the phenotypes inducedby bax. The percentages of flies listed in each phenotypic category(strong, intermediate and weak) are reported in (A) and (B). Allflies considered express one copy of bax in the wing as in control(vg-GAL4,UAS-bax/+) and one copy of bcl-2 (vg-GAL4,UAS-bax/UAS-bcl-2), two copies of bcl-2 in males (Y/+;vg-GA-L4,UAS-bax/UAS-bcl-2;UAS-bcl-2/+), three copies of bcl-2 in fe-males (UAS-bcl-2/X;vg-GAL4,UAS-bax/UAS-bcl-2;UAS-bcl-2/+)or one copy of p35 (vg-GAL4,UAS-bax/UAS-p35). The phenotypedistributions for the different lineages are compared to each otherwith the statistical Wilcoxon test (Tables 3 and 4). (A) In females,bcl-2 partially suppresses the phenotypes induced by bax in adose-dependent manner while p35 has no effect. (B) Similarly,in males, bcl-2 suppresses the phenotypes induced by bax in adose-dependent manner while p35 has no effect

Figure 6 Apoptosis induced by bax and rpr in wing discs.(A) vg-GAL4 drives the expression of GFP in an extended zoneof (vg-GAL4/UAS-gfp) wing disc that corresponds essentially tothe future hinge and the future margin of the wing. (B, C, D)AO staining of wing discs of third instar larvae. (B) Low apopto-sis is observed in (w1118) wild type discs, and most of this apop-tosis is localized in the notum. (C) Massive apoptosis is inducedin the area of vg-GAL4 expression in (UAS-rpr/+;vg-GAL4/+)wing discs, and (D) in (UAS-bax,vg-GAL4/+) wing discs. How-ever, cells in the wing pouch are differently affected by bax andrpr. All discs are shown with anterior to the top. Scale bar,100 mm


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AO staining of wild type wing discs (Figure 6B). Inaddition, the dissociation process, performed to obtainindividual disc cells, probably deprives cells of survivalsignals, thus accelerating commitment to apoptosis (seeMaterials and methods). Therefore, since experimentalprocedures as well as markers used for in situ labelingand in vitro measurements are different, the resultsfrom both types of experiments are not quantitativelycomparable.

However, it is clear that discs expressing rpr or baxexhibit a significant increase in the portion of apoptoticcells. Thus, in order to estimate apoptosis triggered byexpression of rpr or bax, we have subtracted thebackground level of apoptosis occurring in controldiscs (window L) from apoptosis measured in discsexpressing rpr or bax (Figure 7C). This analysis showsthat expression of rpr induces DCm drop and cellshrinkage. Indeed, only 63% of cells in discs expressingrpr maintain high DiOC6(3) fluorescence (window H),versus 84% in control discs (Figure 7Ab and d). Indiscs expressing rpr, 21% more cells undergo apoptosisthan in control discs (Figure 7C), while almost nonebecome PI-positive (Figure 7Ac). Consistent with the insitu observations, equivalent results are obtained whenbax is expressed (Figure 7B, C). We observe 8% of PIpositive cells in bax expressing discs and 7% in baxcontrol discs, showing that bax expression does notinduce necrosis. Moreover, bax induces 22% moreapoptosis than in control discs (Figure 7C). This quitelow apoptogenic effect of rpr and bax is likely due tothe restricted vg-GAL4 expression in the wing disc(Figure 6A). Indeed, flow cytometry analysis of (vg-GAL4/UAS-gfp) third instar larvae shows that only30% of cells in wing discs are positive for GreenFluorescent Protein (data not shown).

bcl-2 suppresses DCm collapse induced by bax and rpr,while p35 activity is specific to rpr

In order to characterize the effect of bcl-2 and p35 onDCm collapse specifically triggered by bax and rpr, wehave studied the co-expression of bax or rpr with p35or bcl-2 (Figure 7B,C). Control discs and discs co-expressing rpr and bcl-2 display similar numbers ofapoptotic cells (i.e. cells with a low DCm and adecrease in size) (16% and 15% respectively) (FigureFigure 7 p35 and bcl-2 affect mitochondrial changes induced by

bax or rpr expression in wing discs. (A) Multiparametric analysisby flow cytometry of cells extracted from third instar larvae discs.Each dot represents one cell. The data shown are relative to cellsextracted from rpr control (UAS-rpr/X) larvae (a,b) or from(UAS-rpr/X;vg-GAL4/+) larvae (c,d). For each sample analysed,size and membrane integrity, as measured by PI fluorescence(reported in cytograms PI versus size) as well as the mitochondrialmembrane potential, as measured by DiOC6(3) fluorescence(reported in cytogram DiOC6(3) versus size) were recorded. PIcytograms are separated into two windows, P (upper window)and N which designate PI-positive and PI-negative fluorescentcells respectively. For analysis of DiOC6(3) fluorescence, onlyPI-negative cells are considered in multiparametric cytograms,and cells can be separated into two populations: one with bothhigh fluorescence and large cell size (noted H) and the other withlower fluorescence and reduced cell size (noted L). The percentageof cells in each population is specified. Note that while rpr expression induces a loss of mitochondrial membrane potential

and a reduction of cell size, PI fluorescence is not affected. (B)The same analyses have been performed with (UAS-rpr/X;vg-GAL4/+;UAS-p35/+) and (UAS-rpr/UAS-bcl-2;vg-GAL4/+;UAS-bcl-2/+), bax control (UAS-bax/+), (UAS-bax/vg-GAL4), (UAS-bax/vg-GAL4;UAS-p35/+) and (UAS-bcl-2/X;UAS-bax/vg-GAL4;UAS-bcl-2/+) larvae. The percentage ofcells in populations described in (A) are presented in the tablein (B) for the different cell samples. (C) The histograms presentthe percentage of cells showing a mitochondrial membrane poten-tial decay in window L (low DiOC6(3) fluorescence) after percen-tage of the control has been deducted. In these experiments, bcl-2can clearly block loss of mitochondrial membrane potential in-duced by both rpr and bax whereas p35 only affects changes inmitochondrial potential triggered by rpr. The results obtainedhave been successfully reproduced


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7B). When rpr and p35 are co-expressed, only 13%more cells than in controls undergo apoptosis (Figure7C) compared to 21% more when only rpr isexpressed. These data argue that p35 protects morethan half of the cells triggered to die by rpr, and bcl-2completely restores wild type mitochondrial phenotype.

In wing discs over-expressing bax, 22% more cellsundergo apoptosis than in bax control discs. When bcl-2 and bax are co-expressed, only 6% more cellsundergo apoptosis than in controls (Figure 7C). Incontrast, the percentage of cells undergoing apoptosisin discs co-expressing bax and p35 remains similar todiscs expressing bax but not p35 (38 and 40%respectively) (Figure 7B). Therefore, p35 has no effecton bax-induced cell shrinkage and DCm drop. Takentogether these results demonstrate that both bcl-2 andp35 can protect cells from DCm drop and cellshrinkage, but while bcl-2 is effective against bothbax- and rpr-induced defects, p35 protection is specificto rpr-induced effect.

Discussion

To further characterize the genetic regulation ofapoptosis, we have used the UAS/GAL4 system toexpress mammalian regulators of apoptosis in Droso-phila. Specifically, we have studied the geneticinteractions between the human anti-apoptotic genebcl-2 with the Drosophila death pathway controlled byrpr. In addition, we have compared apoptosis inducedby the Drosophila gene rpr and the murine gene bax inthe wing margin during development. Since both killersinduce a notch phenotype with variable expressivity,we have set a semi-quantitative approach to comparedistributions of gradual phenotypes between differentgenotypes. As the classification of the phenotypesmight depend on the observer, the suppression analysishas only been performed on progeny from crossesperformed in parallel and carefully analysed by a singleblind observer. Therefore, we have not compareddistributions from independent sets of experiments.However, we have analysed the distributions of wingphenotypes of flies expressing bax in six independentexperiments, and 5/6 distributions compared using theWilcoxon test were statistically similar. Only onedistribution analysed was statistically different fromfour others (data not shown). Additionally, the effectsobserved in our experiments are not likely to bemediated by the titration of the GAL4 activator byUAS elements. Indeed, we show that there is noadditive effect on the phenotypes induced by UAS-rprwhen the UAS-p35 transgene is added to one or twoUAS-bcl-2 transgenes. Furthermore, two independentUAS-p35 insertions, alone or together, have no effecton the phenotypes induced by UAS-bax (data notshown). This method has proven to be very powerful indetecting gradual suppression effects that a classicaldichotomy screen can not detect.

In this study, we have focused on the involvement ofmitochondria as well as the bcl-2 gene family in

apoptosis in Drosophila melanogaster. In mammals,bax triggers apoptosis by permeabilizing the outermitochondrial membrane. This permeabilization iscorrelated with mitochondrial membrane potential(DCm) drop and cyt c release (Desagher and Martinou,2000). rpr is also thought to act on mitochondria.Indeed, in Xenopus cells extracts, RPR binds to Scytheto induce cyt c release from purified mitochondria(Thress et al., 1999). We report here that expression ofbax or rpr in the wing margin during developmentinduces apoptosis, and flow cytometry experimentsperformed on dissociated wing disc cells show that baxand rpr trigger DCm drop and cell shrinkage in vivo inDrosophila. These results point to mitochondrialchanges as common events in bax and rpr deathpathways in Drosophila (Figure 8). Moreover, bax andrpr do not induce necrosis and late apoptotic cells orbodies are likely phagocytosed in vivo as previouslyreported (Milan et al., 1997). Nevertheless, severalfeatures differentiate bax- and rpr-induced apoptosis.Although bax and rpr induce comparable decrease inwing disc size, massive AO staining, as well ascomparable apoptogenic effect (about 20%), adultphenotypes are much stronger after bax expression.One explanation could be that bax-induced apoptosis isirreversible while some of the cells initially triggered byrpr and showing DCm collapse ultimately do notundergo apoptosis. Since AO staining is prominent inrpr as well as bax expressing discs, and given that AOlikely stains cells which are committed to apoptosis,this hypothesis appears unlikely. In addition, as shown

Figure 8 Hypothetical model of apoptosis control in Drosophila.This model takes into account the following points: (1) mitochon-dria are a target of Bax and RPR in Drosophila; (2) unlike rpr,bax induces apoptosis through a p35-insensitive pathway; (3)Bcl-2 probably acts by counteracting mitochondrial defects trig-gered by bax and rpr; (4) a pathway homologous to the TNFadeath pathway may exist in the fly (Georgel et al., 2001; Huand Yang, 2000); and (5) Bcl-2 down-regulates rpr expression.This last effect of Bcl-2 might be mediated through the indirectinhibition of Rel/NF-kB


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by AO staining in situ, the wing pouch of third instarlarvae is more affected by bax expression than by rprexpression. This result suggests that earlier duringdevelopment, cells in the wing disc respond differentlyto both death inducers. Since cells of the wing pouchgive rise to the adult wing blade (Bate and MartinezArias, 1993), it is likely that adult flies expressing baxpresent stronger wing phenotypes. Finally, when celldeath is induced in the wing during larval and pupaldevelopment, the adult phenotype is also dependent oncompensation mechanisms triggered in order to recovernormal wing (Milan et al., 1997). Therefore, we cannot exclude that this process respond differently to bax-and rpr-induced apoptosis.

It has already been shown that rpr-inducedapoptosis in the eye is caspase-dependent (Hay et al.,1995). To test the requirement of caspases in bax andrpr apoptotic pathways in the wing, we have studiedtheir genetic interactions with the caspase-inhibitorp35. We show here that cell death triggered by rpr inthe wing involves cell shrinkage and mitochondrialdefects, and that this process is caspase-dependent. Incontrast, p35 has no effect on mitochondrial changestriggered by bax. Moreover, we have never observedany inhibitory effect of p35 on bax-induced apoptosisin the wing by using two independent p35-transgeniclines, and a third line carrying two copies of p35. Theinability of p35 to suppress the bax death pathway maybe due to the activation of p35-insensitive caspases orto the activation of caspase-independent events. Inmammals, it remains controversial whether bax-induced cell death is caspase-dependent (Okuno etal., 1998; Vekrellis et al., 1997; Xiang et al., 1996). InDrosophila, it is also not clear whether the pro-apoptotic bcl-2 family gene dbok/debcl acts in acaspase-dependent death pathway (Chen and Abrams,2000; Zimmermann et al., 2002). In our experiments,bcl-2 inhibits bax- as well as rpr-induced phenotypes ina dose-dependent manner, and bcl-2 inhibits DCm dropand cell shrinkage induced by bax and rpr in vivo.Taken together, our data suggest that bax and rprinduce two different death pathways, though mito-chondrial defects as well as the inhibition by bcl-2constitute two common features of rpr- and bax-induced apoptosis (Figure 8).

Taking into account what occurs in vitro, wehypothesize that bcl-2 can inhibit rpr- and bax-inducedapoptosis by regulating cyt c-dependent DAPAF-1activation. But in this case, since we have shown thatp35 does not counteract bax, the caspases regulated byDAPAF-1 would be p35-insensitive. DREDD cleavage(possibly by auto-activation), DRONC activity in theeye as well as STRICA-induced cell death are notinhibited by p35 (Chen et al., 1998; Doumanis et al.,2001; Hawkins et al., 2000). Therefore, these caspasesare good candidates for potential members of a bax-induced cell death pathway. In addition, consistentwith what occurs in mammals (Okuno et al., 1998), weshow that in Drosophila, bcl-2 suppresses caspase-dependent and -independent apoptotic mitochondrialevents and apoptosis.

Our results are the first evidence of a protectiveactivity of bcl-2 against rpr-induced apoptosis in vivo inDrosophila. Since bcl-2 can prevent caspase-indepen-dent cell death in mammals (Okuno et al., 1998), wehave studied the effect of the co-expression of bcl-2 andp35 on rpr-induced adult wing phenotypes. We showthat there is no additive effect of p35 and bcl-2 whenthey are co-expressed. This data suggests that bcl-2 andp35 do not inhibit completely different pathways, andthat bcl-2 could affect the activation of p35-sensitivecaspases downstream of rpr expression. Furthermore,we show that p35 suppresses DCm collapse induced byrpr, which suggests that p35-sensitive caspases act onmitochondria (Figure 8). This data is in agreement withVarkey et al. (1999) who have shown that rpr-inducedcyt c modifications are caspase-dependent. Neverthe-less, we do not know whether the action of these p35-sensitive caspases initiates a mitochondrial apoptoticpathway, or whether it is the result of positive feedbackinvolving these caspases, which would acceleratemitochondrial dysfunction.

Finally, while bcl-2 displays anti-apoptotic proper-ties related to an activity downstream of rpr, we alsoshow here that bcl-2 can act by regulating rprtranscriptional induction. This is the first evidence ofthe action of the bcl-2 gene family on the expressionof rpr. In regards to the current knowledge of thegenetic regulation of apoptosis, and considering ourresults, it is likely that the human gene bcl-2 acts onmitochondria in Drosophila to inhibit apoptosistriggered by rpr or by bax (Figure 8). However,exactly how bcl-2 down-regulates rpr expressionremains to be addressed. One can ask whether thiseffect is cell-autonomous. Indeed, in embryos, cellscommitted to die may sensitize neighbor cells andincrease their level of rpr product. By decreasing thenumber of apoptotic cells, bcl-2 would reduce rprexpression. Nevertheless, since p35 has no effect on rprexpression during embryonic development, it isunlikely that the effect of bcl-2 is due to such a noncell-autonomous activity.

Until recently, two death signals were known to beinvolved in the regulation of rpr expression. First, rpris a transcriptional target of the steroid hormoneecdysone during metamorphosis (Baehrecke, 2000),but to our knowledge, no interaction between bcl-2and steroid-induced gene expression has beendescribed. Second, it has been shown that rpr is atranscriptional target of dmp53 after X-ray irradiation(Brodsky et al., 2000). Similarly, in mammals, thetranscription factor NF-kB is involved in cellproliferation as well as in oncogenesis (Rayet andGelinas, 1999), and among the many target genes ofNF-kB are p53 and c-myc. In Drosophila, a deathdomain protein that promotes apoptosis, imd/rip, anda homologue of a mammalian member of the deathdomain receptor pathway, dfadd, have been recentlycharacterized (Georgel et al., 2001; Hu and Yang,2000). Both of them interact with dredd/caspase-8,which suggests that a death domain receptor pathwaythat utilizes caspase-mediated NF-kB activation of rpr


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transcription could regulate apoptosis (Figure 7). Ourresults suggest that this feedback control of rprexpression should be sensitive to bcl-2 but not top35. Whether bcl-2 activates or inhibits NF-kB toregulate apoptosis remains controversial (de Moissacet al., 1998; Grimm et al., 1996; Hour et al., 2000).Since rpr expression is affected by imd/rip, dmp53 andbcl-2, genetic interaction studies between these geneswill be of great interest to better understand the linkbetween the rel/NF-kB gene family and the regulationof apoptosis by p53, bcl-2 and death domain receptorssuch as the TNFa-receptors in Drosophila, and inmammals. More generally, since no anti-apoptotic bcl-2 has been studied in Drosophila, further studies of thehuman anti-apoptotic bcl-2 gene in the fly will givenew insights into the regulation of apoptosis by thebcl-2 family, and delineate new strategies for thedesign of chemotherapeutic apoptosis inducers whichare insensitive to the bcl-2 oncogene.

Materials and methods

Fly stocks

Flies were raised on standard medium. Both driver strainsused in this study, vestigial-GAL4 (vg-GAL4) and daughter-less-GAL4 (da-GAL4) as well as the (UAS-gfp) strain (UAS:Upstream Activating Sequence) were described previously(Gaumer et al., 2000). The strains carrying UAS-p35, UAS-bax and UAS-bcl-2 transgenes have been generated in ourlaboratory and were described previously (Gaumer et al.,2000). The rpr-11-LacZ construct was first described byNordstrom et al. (1996). The fly stock carrying the UAS-rprtransgene was generated by recombination of the X-linked(UAS-rpr, UAS-hid) strain described previously (Zhou et al.,1997). We used w1118 Canton S fly stock as the referencestrain.

rpr suppression study

Males (UAS-rpr/Y), (UAS-rpr/Y;vg-GAL4/CyO) or (UAS-rpr/Y;UAS-p35) were crossed at 258C to females of thefollowing strains: (vg-GAL4), (vg-GAL4;UAS-bcl-2), (UAS-bcl-2;vg-GAL4;UAS-bcl-2), (UAS-bcl-2;UAS-bcl-2;UAS-bcl-2). The progeny were then raised at 258C and femalesexpressing UAS-rpr were analysed within the first day aftereclosion to prevent wings from being damaged.

bax suppression study

Females (vg-GAL4,UAS-bax/CyO-gfp) (GFP: Green fluores-cent protein) were crossed at 188C to males of the followingstrains: (w1118), (UAS-bcl-2), (UAS-p35), (UAS-bcl-2/Y;UAS-bcl-2;UAS-bcl-2). The progeny were raised at 188C andanalysed every day.

Classification of the phenotypes and the Wilcoxon test

The principle of the phenotype classification was different inthe rpr suppression study and in the bax suppression study.Note that in both sets of experiments, all the lineages wereanalysed in parallel and by a blind observer. In the rprsuppression study, flies were classified in four differentphenotypic categories according to their wing phenotype:strong, intermediate, weak and wild type (Figure 1). For flies

showing two different wings, the strongest phenotype of bothwings was used for the classification. In the bax suppressionstudy, flies were classified in the ‘strong’ category when bothwings had strong phenotypes, in the ‘weak’ category whenboth wings had weak phenotypes and in the ‘intermediate’category when it did not belong to either the ‘strong’ or‘weak’ category (Figure 2).

In our experiments we have analysed the distribution ofgraded phenotypes based on their expressivity. The Wilcoxontest was used to compare the distributions of the phenotypesbetween two lineages A and B (Wilcoxon, 1945) (Tables 1, 3and 4). The sign of WsA-B value determines whether thedistribution A is stronger than B (Ws50) or whether thedistribution B is stronger than A (Ws40). We considered thedifference between A and B significant when aA-B51073.

Staining of embryos with X-gal

Females (UAS-bcl-2;da-GAL4) or (da-GAL4) were crossed tomales (rpr-11-LacZ) at 258C. Embryos were dechorionated5 min in 16bleach and rinsed in water. Embryos were fixed10 min in 16PBS pH 7.6, 3.7% formaldehyde/heptane (1 : 2)and washed four times in 16PBS pH 7.6, 0.3% triton X-100.The embryos were stained for 15 to 25 min in 16PBSpH 7.6, K4Fe2 4 mM, K3Fe3 4 mM, MgCl2 1 mM, 1% TritonX-100, X-gal 560 mg ml71 (Eurogentec), fixed 3 min in16PBS pH 7.6, 3.7% formaldehyde and washed three timesin 1 PBS pH 7.6, 0.05% triton X-100. The embryos wereswelled over night in 16PBS pH 7.6, 0.05% Triton X-100/glycerol (40 : 60) and finally mounted in glycerol. Embryoswere staged according to Campos-Ortega and Hartenstein,1985). Samples were observed with a conventional researchmicroscope DMRHC Leica.

b-galactosidase activity assay

Females (UAS-bcl-2;da-GAL4), (UAS-p35;da-GAL4) or (da-GAL4) were crossed to males (rpr-11-LacZ) at 258C.Embryos were collected at different development timesAEL and harvested in 500 ml buffer Z (described by Tzouet al., 2002). The final concentration of proteins in extractswas 138+87 mg ml71 (n=81), and the final concentrationof ONPG (Sigma) for the b-galactosidase activity assay was0.35 mg ml71. The kinetics of ONPG hydrolysis weremeasured over 6 h. The activity assays for each genotypeanalysed and at all development time segments wereperformed nine times independently (Table 2). Themeasured activities were compared using the statisticalStudent t-test.

Detection of vg-GAL4 expression in wing discs

Females (vg-GAL4) were crossed to (UAS-gfp) males. vg-GAL4/UAS-gfp lineage was raised at 258C and third instarlarvae were dissected and mounted in 16PBS pH 7.6. Discswere then observed with a conventional research microscopeDMRHC Leica using the L5 filter to detect greenfluorescence.

AO staining of wing discs

Females (UAS-rpr) or (UAS-bax,vg-GAL4/CyO-gfp) werecrossed respectively to (vg-GAL4) or (w1118) males. UAS-rpr/+;vg-GAL4/+ lineage was raised at 258C and (UAS-bax,vg-GAL4/+) as well as (w1118) lineage were raised at218C. Third instar larvae were dissected in 16PBS pH 7.6and stained for 2 min in (100 ng ml71) Acridine Orange


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(Molecular Probes). Discs were then mounted in PBS andobserved with a conventional research microscope DMRHCLeica using the I3 filter to detect green fluorescence.

Flow cytometry analysis

Males (UAS-rpr/Y) or (UAS-bax/CyO-gfp) were crossedrespectively at 258C and 218C to females of the followingstrains: (w1118), (vg-GAL4), (UAS-bcl-2;vg-GAL4;UAS-bcl-2),(vg-GAL4;UAS-p35). Progeny were raised at the sametemperatures. Wing discs from third instar female larvaewere dissected in 16PBS pH 7.6, and pooled in Schneider’sDrosophila medium (Gibco –BRL). Six discs were pooled persample. The discs were then incubated in 150 ml Trypsine(Gibco –BRL) for 1 h 30 min at 288C. The reaction wasstopped by adding 10 ml Fetal Calf Serum (FCS). Thesamples were stained at room temperature in Schneider’sDrosophila medium for 30 min in 0.1 mM DiOC6(3), and for5 min in 50 mg ml71 Propidium Iodide (PI) (final volume of500 ml). Samples were preserved on ice before analysis. Thecationic lipophilic fluorochrome DiOC6(3) (MolecularProbes) is a cell permeable marker which at low doses(0.1 mM), specifically accumulates in mitochondria inmammalian cells in proportion to DCm (Petit et al., 1990).PI (Sigma) is a non specific DNA intercalating agent which isexcluded by the plasma membrane of living cells.

Flow cytometry measurements were performed on a XL3Cflow cytometer (Beckman-Coulter France). Fluorescenceexcitation was obtained using the blue wavelength (488 nm)of an argon ion laser operating at 15 mW. Green fluorescenceof DiOC6(3) was collected with a 525 nm band pass filter andred fluorescence of PI with a 620 nm band pass filter.Analyses were performed on 104 cells and data were stored inlistmode. Light scattering values were measured on a linearscale of 1024 channels and fluorescence intensities on alogarithmic scale of fluorescence.

Acknowledgements

We are grateful to Dr G Lecellier for expert help withstatistics. We also thank E Blanchardon who provided usthe (UAS-rpr) strain, Y Risler and I Seminet for their helpduring some experiments, Dr L Theodore for helpfuldiscussions and MR Watson for her critical reading ofthe manuscript. This work was supported in part by grantsfrom the Association pour la Recherche contre le Cancer(no. 4480) and from the Ligue Contre le Cancer (Comitedes Yvelines). S Brun was a recipient of a fellowship fromthe Ministere de l’Education Nationale et de l’Enseigne-ment Superieur et de la Recherche. S Gaumer wassupported by a fellowship from the Association pour laRecherche contre le Cancer.

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