Death to flies: Drosophila as a model system to study programmed cell death

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Journal of Immunological Methods 265 (2002) 21 – 38

Death to flies: Drosophila as a model system to study programmed cell death Helena Richardson a,*, Sharad Kumar b,* a

Trescowthick Research Laboratories, Peter MacCallum Cancer Institute, Locked Bag 1, A’Beckett St., Melbourne, Victoria, 8006, Australia Hanson Centre for Cancer Research, Institute of Medical and Veterinary Science, P.O. Box 14, Rundle Mall, Adelaide, SA 5000, Australia


Abstract Programmed cell death (PCD) is essential for the removal of unwanted cells and is critical for both restricting cell numbers and for tissue patterning during development. Components of the cell death machinery are remarkably conserved through evolution, from worms to mammals. Central to the PCD process is the family of cysteine proteases, known as caspases, which are activated by death-inducing signals. Comparisons between C. elegans and mammalian PCD have shown that there is additional complexity in the regulation of PCD in mammals. The fruitfly, Drosophila melanogaster, is proving an ideal genetically tractable model organism, of intermediary complexity between C. elegans and mammals, in which to study the intricacies of PCD. Here, we review the literature on PCD during Drosophila development, highlighting the methods used in these studies. D 2002 Published by Elsevier Science B.V. Keywords: Programmed cell death; Apoptosis; Drosophila; Development; Caspases; RNA interference; In situ TUNEL; Antibody staining; Tyramide amplification; Ectopic expression; Dominant negative mutants; Genetic interactions

1. Introduction: Drosophila as a model to study programmed cell death Programmed cell death (PCD) or apoptosis is essential for life in a multicellular organism. PCD is needed for removal of extraneous cells in tissue

Abbreviations: PCD, programmed cell death; PCR, polymerase chain reaction; TUNEL, TdT-mediated dUTP-Nick-End Labeling; RNAi, RNA interference; GMR, Glass multimer reporter; GAL4(UAS), yeast Saccharomyces cerevisiae GAL4 upstream activator sequence. * Corresponding authors. H. Richardson is to be contacted at Tel.: +61-3-9656-1466; fax: +61-3-9656-1411. S. Kumar, Tel.: +618-8222-3738; fax: +61-8-8222-3139. E-mail addresses: [email protected] (H. Richardson), [email protected] (S. Kumar).

patterning during development and for homeostasis of the adult, where superfluous or damaged cells are eliminated. Failure to remove such cells can lead to developmental disorders or tumorigenesis (reviewed by Vaux and Korsmeyer, 1999; Thompson, 1995; Zheng et al., 1999; Yuan and Yankner, 2000). Developmental- or damage-induced death signals result in the activation of the cysteine proteases, caspases, which orchestrate the events of PCD including membrane blebbing and the degradation of nuclear DNA. Genetic studies in the worm, C. elegans, have revealed the critical components involved in the execution of PCD (reviewed by Hodgkin, 1999; Fraser, 1999). These are, Ced-3 (a cysteine protease/caspase), Ced4 (involved in caspase activation), Ced-9 (an inhibitor of apoptosis) and Egl-1 (a BH3 domain-only protein, which acts by binding to Ced-9 to induce apoptosis).

0022-1759/02/$ - see front matter D 2002 Published by Elsevier Science B.V. PII: S 0 0 2 2 - 1 7 5 9 ( 0 2 ) 0 0 0 6 8 - 6


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Subsequent studies in mammals and in the fly, D. melanogaster, have identified counterparts for these C. elegans genes, demonstrating that the core components of the cell death machinery are conserved through evolution. However, it is clear from these studies that animals with increased complexity contain additional PCD regulators and regulatory mechanisms. For example, there are 14 caspases in humans, 7 caspases in flies and 1 in worms. This extra complexity in PCD components is a reflection of the increased complexity in development and body plan, but in addition, non-PCD roles have been taken on by some caspases (e.g., caspase-1 is involved in processing of the interleukin-1h precursor in the immune response; reviewed by Kumar and Lavin, 1996). This trend towards increased complexity and redundancy is also observed with the death-inducing signaling pathways of mammals when compared with C. elegans. Mammalian cells have two PCD pathways, the intrinsic and the extrinsic (instructive) cell death-inducing pathway (Fig. 1). In the intrinsic pathway, cellular stress leads to the release of mitochondrial cytochrome c, which binds to the adaptor protein Apaf-1 and promotes its oligomerization (reviewed by Budihardjo et al., 1999). Apaf-1 in turn recruits pro-caspase-9 and thereby promotes its proximity-induced autocatalytic activation (Li et al., 1997; Zou et al., 1999). The requirement for cytochrome c in PCD is clearly demonstrated in cytochrome c knockout mice, where activation of the caspase-9/Apaf-1 pathway is partly impaired (Li et al., 2000). In the extrinsic death-inducing pathway, death receptors of the tumor necrosis factor family mediate recruitment of caspases via the adaptor protein Fadd (Nagata, 1997; Ashkenazi and Dixit, 1998). C. elegans has the core components of the intrinsic pathway, but lacks the extrinsic cell death pathway. Another difference is that the intrinsic pathway in C. elegans, unlike mammals, does not require cytochrome c for the activation of Ced-4 (Apaf-1)/Ced-3 (caspase-9). Furthermore, in more complex animals there are both antiapoptotic and pro-apoptotic ced-9-related genes, whilst in C. elegans, ced-9 acts only in a pro-survival manner. Finally, in mammals, a pro-survival role has been adopted by the IAPs (inhibitor of apoptosis, BIR domain-containing proteins), whilst in worms, BIR domain-containing homologs do not function in PCD. Thus, there is considerably more complexity in mammalian cells compared with C. elegans. Understanding

the physiological role of PCD components in mammals requires in vivo studies, best approached by the generation and analysis of gene knockouts in mice. However, these studies are very time-consuming and, due to genetic redundancy, do not always provide simple answers to gene function (see Zheng et al., 1999). Drosophila provides a system of intermediate complexity between worms and mammals in its PCD pathways. Drosophila shares many of the PCD components and pathways found in mammals, but has less redundancy, allowing easier dissection of function. Another distinct advantage is that the recent completion of the Drosophila euchromatic genomic sequence (Rubin et al., 2000), has revealed all of the highly conserved homologs of mammalian PCD genes (Vernooy et al., 2000). In addition, Drosophila is developmentally well characterized and genetically manipulable, proving an ideal system in which to study the role of PCD genes during development. Perhaps the greatest advantage of Drosophila is its powerful genetics, which can be used to generate mutations, generate transgenic lines that ectopically overexpress genes, examine genetic interactions and carry out dominant genetic modifier screens to uncover novel genes (for example Simon et al., 1991; Goyal et al., 2000). All of these genetic approaches have been applied to the study of PCD in Drosophila, as will be described below. This review focuses on the mechanism of PCD during Drosophila development, highlighting the genetic and immunological methods used in these studies.

2. Cell death during Drosophila development The Drosophila life cycle consists of four stages: embryo, larva (with three larval instars), pupa and adult. During Drosophila development, two types of PCD have been reported: apoptosis and autophagy. Apoptosis is characterized by membrane blebbing, nuclear and cytoplasmic condensation and DNA fragmentation, whereas autophagy is characterized by the destruction of entire tissues and the presence of autophagic vacuoles. Both types of PCD can be detected with vital dyes (such as acridine orange, which, in unfixed tissues, stains dead cells with disrupted membranes; Abrams et al., 1993) or by in

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Fig. 1. PCD pathway in flies compared with mammals. Mammalian cell death components are indicated in red, whilst identified Drosophila counterparts are in black. In mammals, the extrinsic pathway of cell death involves signaling through the tumor necrosis factor receptor (TNFR) death receptor family, via the Fadd adaptor to induce caspase-8. Drosophila contains a homolog of Fadd (dFadd) and caspase-8 (Dredd), but appears to lack any of the classical TNFR family of death receptors. Besides its predicted role in PCD, Dredd is also involved in the innate immunity pathway. In mammals, the intrinsic pathway is induced by cytotoxic agents and stress and involves the activation of the tumour suppressor, p53. The Drosophila homolog of p53, Dmp53, induces expression of the PCD promoter, Rpr. In Drosophila, Rpr, as well as Grim and Hid, are PCD inducers, which lead to the activation of caspases. The mammalian protein, Smac/Diablo may be a functional homolog of Rpr, Hid and Grim. The Iaps (Diaps in Drosophila) act by binding to pro-caspases and prevent their activation. Rpr, Hid and Grim, by binding to Diap1, disrupt IAP-caspase complexes, leading to caspase activation. In mammals, anti-apoptotic (pro-survival) Bcl-2 family members inhibit cell death by binding to the pro-apoptotic Bcl-2s. BH3-only domain proteins induce apoptosis by binding to the pro-survival Bcl-2 family proteins. p53 induces the expression of at least two mammalian BH3-only proteins, Noxa and Puma (Nakano and Vousden, 2001; Yu et al., 2001; Oda et al., 2000), and thereby promotes cell death. Caspase-8 is also linked to activation of at least one BH3-only protein, Bid (not shown; reviewed by Huang and Strasser, 2000). In Drosophila, Debcl acts as a pro-apoptotic Bcl-2 member, while Buffy may be anti-apoptotic. Proapoptotic Bcl-2s promote mitochondrial cytochrome c release, which regulates oligomerization of the adaptor, Apaf-1, followed by caspase-9 recruitment and oligomerization. Cleavage of the Drosophila caspase-9 homolog, Dronc, to its active form is promoted by the Apaf-1 homolog Dark. Active Dronc then mediates activation of the effector caspases, Dcp-1 and Drice. Dredd may also function downstream of Dark. It is unclear at present where the Drosophila caspases, Decay, Damm and Strica fit into the PCD pathway.

situ TUNEL (TdT-mediated dUTP-Nick-End Labeling; Chen et al., 1996) to detect fragmented DNA. By using these methods, the profile of PCD through Drosophila development has been documented

(Abrams et al., 1993). Furthermore, by applying these methods to the phenotypic analysis of mutants and the consequences of altered gene expression, many genes involved in PCD have been characterized (reviewed


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Fig. 2. Ablation of Dronc in embryos using RNAi. RNAi was used to ablate dronc function in embryos. Pre-cellularized embryos injected with double-stranded dronc RNA or uninjected controls were aged to stage 13 before fixation and staining for TUNEL (A, B), with the anti-Dronc antibody (C, D) and with the neural differentiation marker, Mab 22C10 (E, F). (A) TUNEL on an uninjected embryo. (B) TUNEL on dronc double-stranded RNA-injected embryos. (C) Uninjected embryo shown in (A), stained with anti-Dronc antisera. (D) dronc double-stranded RNA-injected embryo shown in (B), stained with anti-Dronc antisera, showing no staining even after long exposure. (E) An uninjected embryo stained with Mab 22C10. (F) dronc double-stranded RNA-injected embryos shown in (B), stained with Mab 22C10.

by Abrams, 1999; Rusconi et al., 2000; Meier et al., 2000). During Drosophila embryogenesis, apoptotic PCD is first observed in a few cells about 6 h after egg deposition (Abrams et al., 1993). As development proceeds, increased numbers of dying cells are observed throughout the embryo, particularly in cells of the nervous system. PCD is also observed in patches of cells during the larval stages. However, during metamorphosis, most larval tissues are destroyed in a controlled manner, regulated by pulses of the steroid hormone ecdysone (reviewed by Baehrecke, 2000). This type of PCD has the characteristics of autophagy, but this pathway utilizes many of the same PCD components that are required for apoptotic PCD (Jiang et al., 1997; Lee et al., 2000; Lee and Baehrecke, 2001). PCD also occurs during oogenesis, where in response to ecdysone signaling, the nurse cells die and discharge their contents into the devel-

oping oocyte (reviewed by Buszczak and Cooley, 2000). In contrast to the PCD that occurs during metamorphosis, nurse cell PCD does not require some of the upstream components of the conserved apoptotic pathway (Foley and Cooley, 1998). Thus, Drosophila presents a number of differently regulated PCD pathways that may provide models for understanding PCD in mammalian development.

3. Drosophila cell death genes and their role in PCD Following the lead of studies in C. elegans, the first Drosophila PCD genes were revealed by genetic analysis. In the first genetic analysis of PCD in Drosophila, a deletion (deficiency H99) was identified that was required for PCD. This deletion removes three genes, reaper (rpr), hid (head involution defect/

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Wrinkled) and grim, which cooperate to mediate PCD in the embryo (White et al., 1994; Chen et al., 1996; Grether et al., 1995). Interestingly, these genes are not required for nurse cell PCD during oogenesis (Foley and Cooley, 1998). Unlike other components of the apoptotic machinery mentioned above (see Fig. 1), the recent analysis of the almost complete human genome sequence shows that there are no strongly related homologs of rpr, hid and grim (Aravind et al., 2001). However, the recently discovered mammalian apoptosis inducer Smac/Diablo (Du et al., 2000; Verhagen et al., 2000) appears to act as a functional homolog of Rpr, Hid or Grim. From PCR cloning studies and from analysing the complete Drosophila euchromatic genomic sequence (Rubin et al., 2000; Vernooy et al., 2000), we know that there are Drosophila homologs of many of the PCD genes, defined by studies in mammalian cells (Fig. 1). The advantage of Drosophila is that the function of these PCD genes in vivo can be readily investigated. To understand the function of a gene in vivo, knowledge is required of its expression pattern during development, the effect of ectopic expression and most importantly, the effect of loss-of-function on the animal. The developmental expression profile of the gene can be obtained by in situ hybridization to mRNA or by in situ antibody staining. This expression pattern can then be compared with the pattern of PCD, as revealed by TUNEL or acridine orange staining (Chen et al., 1996; Abrams et al., 1993; see Figs. 2 and 3). Ectopic overexpression studies can be carried out in transgenic flies in order to determine whether the gene is capable of inducing or preventing PCD in different tissues and stages during development. Analysis of the phenotypic consequences of loss-of-function of a gene is the definitive means for determining the importance of the gene in vivo. This relies on generating specific loss-of-function (preferably null) mutants in the gene. Since null alleles only exist in a few PCD genes and it is not always straightforward to generate null mutants, another approach to ablate the gene product is to use antisense technology (such as RNA interference, Hunter, 1999, 2000; Sharp, 2001). Alternatively, dominant negative constructs (which act by sequestering interacting proteins from the endogenous wild type protein and thereby its function) can be used to generate loss-offunction phenotypes. The existence of mutants in


Fig. 3. Genetic interactions of dronc with rpr, hid and grim. GMR – rpr-, – hid- and – grim-mediated cell death is suppressed when the dosage of dronc is halved using a deficiency, showing that Dronc acts downstream of Rpr, Hid and Grim. (A) GMR – hid/+, (B) GMR – hid/ + Df(3L)AC1 (deficiency of dronc), (C) GMR – rpr/+, (D) GMR – rpr/ + Df(3L)AC1 (deficiency of dronc)/+, (E) GMR – grim/+( F) GMR – grim/ + Df(3L)AC1 (deficiency of dronc)/+. The GMR (Glass multimer reporter) drives expression in the posterior part of the eye disc (see Fig. 4).

PCD genes or phenotypes generated by ectopic overexpression of a gene also allows the analysis of genetic interactions between different genes, which is important in dissecting functional relationships in PCD pathways. Use of the genetic approaches described above has been carried out, to some extent, on several Drosophila PCD genes. These analyses have provided valuable information on the in vivo function of PCD


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genes and their genetic interactions with other PCD components. Since this analysis has only been done thoroughly on a few PCD genes, at present, we do not have a complete picture of the in vivo function of many PCD genes, and our understanding of PCD in

Drosophila is far from complete. For the remainder of this review, we will describe the current understanding of PCD genes in Drosophila development and present examples of approaches used to analyze gene function in vivo (see Table 1).

Table 1 Summary of approaches and results of in vivo gene analysis in Drosophila Gene


Ectopic overexpression

Rpr Grim Hid

GMR,a hsp70 transgene—induced cell death GMR transgene—induced cell death GMR, hsp70 transgene—induced cell death


H99 deficiency—prevents embryonic cell death H99 deficiency—prevents embryonic cell death H99 and other deficiencies—prevent embryonic cell death and results in a head involution defect Wrinkled-hypomorphic allele—defect in wing development Mutants—defects in the immune response Mutants—genetically interacts with rpr and grim RNAib—prevents PCD in the embryo Dronc deficiency and Dronc dominant negative transgene—genetically interacts with rpr, hid and grim Null allele—third instar larval lethal—no imaginal discs or gonads, melanotic tumors Germ line clones—nurse cell dumping defect – – Damm dominant negative transgene—genetically interacts with hid – Mutant—viable with hyperplasia of the nervous system, melanotic tumors, defective wings. Mutants prevent caspase activation and PCD Mutants—genetically interacts with dronc, dcp-1, rpr, hid and grim RNAi—prevents PCD in the embryo



thread loss-of-function—Increased cell death in the embryo—embryonically lethal. Epistatic to rpr, hid and grim —genetically interacts with Dronc and Debcl Gain-of-function—prevents rpr-, hid- and grim-induced cell death Deficiency—does not genetically interact with Dronc or Debcl – —Dmp53 dominant negative transgene blocks DNA damage-induced cell death in the wing disc

Dredd Dronc


Drice Decay Damm Strica Dark

Diap2 dFadd Dmp53 a b c

– GAL4(UAS)c transgene—expression via GMR, hsp70 and other drivers—induces cell death

GMR transgene—promotes cell death

GMR transgene—no effect – GMR transgene—promotes cell death – –

GAL4(UAS) transgene—expression via GMR, hsp70 and other drivers—induces cell death GAL4(UAS) transgene—expression via GMR, hsp70 and other drivers—does not induce cell death GMR transgene—no effect. Inhibits Dronc- or Debcl- induced cell death

GMR transgene—no effect. Inhibits Dronc- or Debcl- induced cell death – GMR transgene—induces cell death

GMR = Glass multimer reporter (expressed in the posterior part of the eye imaginal disc during eye development). RNAi = RNA interference. GAL4(UAS) = Yeast, S cerevisiae, GAL4 upstream activator sequence.

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4. Analysis of caspase function in Drosophila PCD Caspases are cysteine proteases, which cleave their substrates after an aspartate residue (Kumar and Lavin, 1996; Thornberry and Lazebnik, 1998; Cryns and Yuan, 1999; Nicholson, 1999). Caspases are present as inactive precursors (zymogens/pro-caspase) in cells, but upon receiving an apoptotic signal, the pro-caspases undergo proteolytic processing to generate active enzyme. There are seven caspases in D. melanogaster: Dcp-1, Dredd/Dcp-2, Drice, Dronc, Decay, Strica/Dream and Damm/Daydream (Song et al., 1997; Fraser and Evan, 1997; Chen et al., 1998; Inohara et al., 1997; Dorstyn et al., 1999a,b; Doumanis et al., 2001; Harvey et al., 2001; reviewed by Kumar and Doumanis, 2000). Dredd, Dronc and Strica are class I caspases which contain long prodomains. Two types of domains have been defined in the mammalian prodomain regions, the caspase recruitment domain, CARD, and death effector domain, DED (reviewed by Aravind et al., 1999). These domains are required for the recruitment of procaspase molecules by their respective adaptors that are required to promote their autocatalytic activation. The CARD of caspase-9 is required for its recruitment by the adaptor Apaf-1 via the CARD at the N terminus of Apaf-1 (Li et al., 1997; Zou et al., 1999). Similarly, in the extrinsic apoptotic pathway, one of the two DEDs of caspase-8 mediates recruitment to the DED in the adaptor Fadd (Aravind et al., 1999). Drosophila Dredd contains two DEDs in its prodomain region, whereas Dronc has a CARD (Chen et al., 1998; Inohara et al., 1997; Dorstyn et al., 1999a). Interestingly, Strica contains neither a DED nor CARD, but a unique Ser/Thr-rich domain at its N terminus, the function of which is as yet unknown (Doumanis et al., 2001). Dcp-1, Drice, Decay and Damm lack long prodomains and are thus similar to class II effector caspases in mammals (reviewed by Kumar, 1999). Specific mutations are available in two of the fly caspases, dcp-1 and dredd (Table 1). dcp-1 null mutations are third instar larval lethal and do not show any abnormalities in cell death in the embryo, most likely due to maternal contribution of the protein (Song et al., 1997). The most striking defect of dcp-1 null mutant larvae is that they lack imaginal discs (groups of cells that are progenitors of adult struc-


tures) and gonads. In addition, these larvae have melanotic masses (tumors), which, contrary to expectations, do not arise as a consequence of over-proliferation of the blood cells, but rather are thought to be an immune response to persisting ‘‘undead’’ cells that were not eliminated by PCD or due to improper differentiation (Song et al., 1997). To analyze the role of Dcp-1 in oogenesis, germ line dcp-1 mutant clones were generated by the established technique of mitotic recombination using the yeast FLP recombinase/FRT site system (McCall and Steller, 1998; Xu and Rubin, 1993; Chou and Perrimon, 1996). This technique allows removal of the maternally supplied product so that the function of a gene in oogenesis or in the early embryo can be analyzed. Using this method, it was discovered that female flies carrying the dcp-1 germ line clones are sterile due to a defect in the transfer of the nurse cell cytoplasmic contents to the developing oocytes as a consequence of a failure in nurse cell PCD (McCall and Steller, 1998). These studies showed that Dcp-1 has a specific role during development, since only particular tissues, but not others (e.g., the larval neural cells), were affected in the dcp-1 mutant. Studies using transgenic expression of truncated Dcp-1 in the developing Drosophila eye showed that Dcp-1 promotes cell death leading to a small rough eye phenotype (Song et al., 2000). Ectopic expression of rpr or grim, but not hid, enhanced this phenotype, suggesting that Dcp-1 may act downstream of Rpr and Grim (Song et al., 2000). dredd mRNA expression correlates with ‘‘doomed’’ cells, destined to undergo PCD, and its accumulation is dependent on rpr, hid and grim (Chen et al., 1998), leading to the hypothesis that Dredd acts downstream of these apoptotic inducers. Furthermore, heterozygosity at the dredd locus suppresses cell death induced by the ectopic expression of rpr and grim, showing that Dredd is rate limiting for apoptosis mediated by rpr and grim. Consistent with this observation is that Rpr, Hid and Grim expressions result in the processing of Dredd to its active form in transfected Drosophila S2 tissue culture cells. Given the evidence for Dredd playing an important role in PCD, it was surprising that a dredd mutant was identified in a screen for mutants affecting the immune response in Drosophila (Elrod-Erickson et al., 2000). Flies mutated at the dredd locus fail to induce the synthesis of anti-microbial peptides and are highly susceptible to bacterial


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infection. In this pathway, signaling via the Toll receptor activates Dredd, which in turn plays a role in the proteolytic processing and activation of Relish, a member of the NFnB family of transcription factors (Leulier et al., 2000; Stoven et al., 2000). Thus, Dredd appears to have a function in the PCD pathway as well as in the immune response. This example indicates the value of whole animal genetic analyses to determine the in vivo function of a gene. No specific mutations have been isolated in the other Drosophila caspases. However, in vivo functional analyses have been carried out on dronc using the RNA interference (RNAi) technique (Hunter, 1999, 2000; Sharp, 2001) and on Dronc, Damm and Drice by ectopic expression studies in transgenic flies (Table 1). Studies in transgenic flies using a catalytically inactive dominant negative Dronc mutant have been informative in analyzing the in vivo function of Dronc (Meier et al., 2000). The RNAi technique was developed in C. elegans and is proving to be very powerful in specifically ablating gene function in Drosophila as well as in mammalian cells (Hunter, 1999, 2000; Sharp, 2001; Misquitta and Paterson, 1999; Wianny and ZernickaGoetz, 2000). In this procedure, double-stranded RNA is injected into syncitial stage Drosophila embryos and leads to the rapid ablation of the endogenous mRNA and eventually the protein (e.g., Kennerdell and Carthew, 1998; Bhat et al., 1999). RNAi works by utilizing an evolutionarily conserved antiviral mechanism, which leads to the degradation of the endogenous mRNA (Hunter, 1999, 2000; Sharp, 2001). The RNAi technique was used to ablate Dronc function in early Drosophila embryos by the injection of in vitro prepared double-stranded Dronc RNA (Quinn et al., 2000). This procedure results in a significant decrease in apoptosis in embryos, but did not affect other aspects of development, such as neural differentiation (Fig. 2). These data demonstrate that Dronc is required for PCD during embryogenesis. Despite the absence of a specific mutation in Dronc, the existence of deficiencies (deletions) covering the dronc locus enables genetic interactions to be explored. When the dosage of dronc is halved, using a deficiency, the ablated eye phenotype of transgenic flies overexpressing rpr, hid or grim is suppressed (Fig. 3; Quinn et al., 2000). This result was confirmed by using transgenic expression of a catalytically inactive dronc mutant,

which acts as a dominant negative allele (Meier et al., 2000; Hawkins et al., 2000). Expression of this catalytically inactive dominant negative dronc mutant resulted in suppression of the Rpr or Hid ablated eye phenotypes. These results show that Dronc acts downstream of Rpr-, Hid- and Grim-mediated cell death. Consistent with this idea, transgenic overexpression of Dronc in developing Drosophila tissues leads to ectopic cell death and to an ablated eye phenotype (Meier et al., 2000; Hawkins et al., 2000; Quinn et al., 2000). This ablated eye phenotype has been used to examine genetic interactions between dronc and other PCD genes (Quinn et al., 2000; see below). Dronc is also subjected to another mode of PCD regulation, in that dronc transcription is developmentally upregulated in the larval salivary gland and gut tissues by the steroid hormone, ecdysone, which is essential for inducing PCD in these tissues (Dorstyn et al., 1999a; Lee et al., 2000; Lee and Baehrecke, 2001). Dissection of the dronc promoter is in progress and will permit the identification of the ecdysone-responsible elements (T. Daish and S. Kumar, unpublished data). Only limited in vivo analysis has been carried out with the remaining Drosophila caspases. Expression of full-length or an N-terminal truncation of Drice in the Drosophila eye shows no overt effects (Song et al., 2000). However, in Drosophila S2 tissue culture cells, expression of Drice induces apoptosis and Drice is proteolytically processed after induction of apoptosis by rpr overexpression or by cytotoxic agents (Fraser and Evan, 1997; Fraser et al., 1997). Furthermore, immunodepletion of Drice from Drosophila S2 cell lysates, using Drice-specific antibodies, considerably reduces in vitro apoptotic activity (Fraser et al., 1997). Thus, in S2 hemocyte-derived cells, Drice plays an essential role in apoptosis. The absence of a measurable effect of ectopic Drice expression in the Drosophila eye suggests that Drice may be a tissuespecific apoptotic factor. The identification of a Drice mutant or RNAi studies will help resolve this issue. Damm overexpression in the developing eye leads to an ablated eye phenotype, although this effect is mild compared with other PCD genes (Harvey et al., 2001). Interestingly, expression of a transgenic Damm catalytically inactive, dominant negative mutant in the eye suppressed the ablated eye phenotype due to overexpression of hid, but not rpr (Harvey et al., 2001).

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This suggests that Damm may function specifically downstream of hid-induced apoptosis. Ectopic expression of Strica or Decay in mammalian cells or Drosophila S2 tissue culture cells has a weak apoptotic effect (Doumanis et al., 2001; Dorstyn et al., 1999b), but their specific function in Drosophila cell death at present is not known. Clearly, the understanding of the in vivo function of Damm, Strica and Decay will be facilitated by loss-of-function analysis.

5. The in vivo function of Drosophila PCD regulators 5.1. The Drosophila Apaf1/Ced-4 homolog The apoptosis adaptor protein, Ced-4/Apaf-1, is required in C. elegans and mammalian cells for the activation of Ced-3/caspase-9 (Yang et al., 1998; Li et al., 1997; Zou et al., 1999). Similarly, the Drosophila homolog, Dark/Dapaf-1/Hac-1, mediates caspase activation in vitro and binds to the initiator caspases Dronc and Dredd, (Kanuka et al., 1999; Rodriguez et al., 1999; Zhou et al., 1999). A loss-of-function allele of dark was identified that was due to the insertion of a P element transposon (Kanuka et al., 1999; Zhou et al., 1999; Rodriguez et al., 1999). Rodriguez et al. (1999) used the technique of local P element jumping followed by PCR screening (Zhang and Spradling, 1993; Tower et al., 1993; Dalby et al., 1995), to identify new mutations in dark where an additional P element had inserted into the first intron of the gene. Based on Northern analyses, these new alleles were expected to be null mutants, since no mRNA could be detected (Rodriguez et al., 1999). Mutants in dark are homozygous viable, but show hyperplasia of the central nervous system, melanotic tumors and defective wings, consistent with a decrease in PCD (Kanuka et al., 1999; Zhou et al., 1999; Rodriguez et al., 1999). Furthermore, decreased TUNEL-positive cells were observed in dark mutant embryos and larval brains, as well as decreased caspase activity in dark mutant embryonic or adult extracts (Kanuka et al., 1999; Zhou et al., 1999; Rodriguez et al., 1999; Quinn et al., 2000). The technique of RNAi with double-stranded dark cRNA was also used to show that Dark is required for PCD in embryos using acridine orange staining (Zhou et


al., 1999). Increased neuron numbers were observed in the central nervous system of dark mutant embryos, by monitoring nervous system development using in situ antibody staining with an anti-Elav antibody that stains differentiated neural cells (Zhou et al., 1999). These results show that Dark has an important role in PCD in vivo, but it is not essential for viability, since flies carrying the null allele survive. Various biochemical and genetic interaction studies using dark mutants have shown that Dark interacts with the caspases Dredd and Dronc. Dredd forms a complex with Dark through its N-terminal domain, and a catalytically inactive dominant negative version of Dredd suppresses Dark-induced cell death in SL2 tissue culture cells (Rodriguez et al., 1999). In vivo, heterozygosity for dark results in suppression of the eye ablation phenotype caused by the overexpression of dronc (Quinn et al., 2000). Dronc has also been shown to form a complex with the N-terminal region of Dark in SL2 cells (Quinn et al., 2000). Furthermore, dark mutant extracts have considerably reduced ability to cleave Dronc to its active form, showing that Dark is important for Dronc activation (Quinn et al., 2000). There are conflicting reports as to whether Dark can form a complex with the executioner caspase, Drice. Kanuka et al. (1999) observed a complex with the N-terminal region of Dark, but not full-length Dark, and Drice in transfected Drosophila S2 cells, whereas Rodriguez et al. (1999) failed to observe such an interaction in SL2 cells. Dark also genetically interacts with Dcp-1, as evidenced by the suppression of the ablated-eye phenotype, due to overexpression of dcp-1, by halving the dosage of dark (Zhou et al., 1999). Interestingly, co-immunoprecipitation experiments in Drosophila tissue culture cells demonstrated that Dark binds to cytochrome c (Kanuka et al., 1999; Rodriguez et al., 1999). The mammalian, but not the C. elegans, homolog also binds to cytochrome c. Structurally, Dark is more similar to its mammalian counterpart Apaf-1 than to C. elegans Ced-4 in that it has several WD40 repeats at its C terminus not found in Ced-4. Indeed, cytochrome c binds to the Cterminal WD40 domain region of Dark (Rodriguez et al., 1999), but it is not known whether Dark requires cytochrome c for its oligomerization. This area of research will be aided by the isolation of Drosophila cytochrome c mutants or RNA ablation


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studies to determine the in vivo function of cytochrome c in PCD during development. Genetic interaction studies of Dark in Rpr-, Hidor Grim-mediated cell death gave conflicting results (Rodriguez et al., 1999; Kanuka et al., 1999; Zhou et al., 1999). Rodriguez et al. (1999) showed that cell death induced by the ectopic expression of rpr, hid or grim was substantially reduced when both copies of dark were mutated. Kanuka et al. (1999) found that while rpr-induced cell death in the eye was substantially inhibited in homozygous dark mutants, hidinduced apoptosis was not affected. In contrast, Zhou et al. (1999) failed to see any significant effect on killing by rpr, hid or grim when the dosage of dark was halved. It should be noted that Kanuka et al. (1999) and Zhou et al. (1999) used a relatively weak allele of dark in their experiments, and Zhou et al. (1999) only examined the effect of halving the dosage of dark. The failure to see a suppression of the Rpr-, Hid- or Grim-ablated eye phenotype when the dosage of Dark is halved (Zhou et al., 1999) suggests that Dark is not rate limiting for Rpr, Hid or Grim PCD. In contrast, halving the dosage of other downstream PCD genes, e.g. Dronc (Quinn et al., 2000; Fig. 3) clearly suppressed Rpr-, Hid- or Griminduced PCD. However, Dark appears to facilitate Rpr-, Hid- and Grim-mediated PCD, since in dark null homozygous mutants, PCD induced by these genes is strongly suppressed (Rodriguez et al., 1999). Nevertheless, further experiments are necessary to confirm the role of Dark in Rpr-, Hid- or Grimmediated PCD.

5.2. Drosophila Bcl-2/Ced-9 homologs From the Drosophila genomic sequence (Rubin et al., 2000), two homologs of the Bcl-2/Ced-9 family of PCD proteins, Debcl/dBorg-1/dRob-1 and Buffy/ dBorg-2, were identified (Igaki et al., 2000; Colussi et al., 2000; Brachmann et al., 2000; Zhang et al., 2000; reviewed by Chen and Abrams, 2000). Both Debcl and Buffy share the BH1, BH2, BH3 and C-terminal transmembrane domains of the Bcl-2 family of proteins, but appear to lack the N-terminal BH4 domain. The BH4 domain distinguishes the pro-apoptotic Bcl2 family members, e.g. Bax and Bok, from the antiapoptotic members, e.g. Bcl-2, Bcl-xL and Bcl-w (Adams and Cory, 1998), and based on this, both Debcl and Buffy may be expected to be pro-apoptotic. Indeed, both Debcl and Buffy are most closely related to the mammalian Bok pro-apoptotic protein. Consistent with this notion, ectopic overexpression of Debcl in transgenic flies results in ectopic PCD in various Drosophila tissues and, when expressed in the eye, leads to an ablated eye phenotype (Igaki et al., 2000; Colussi et al., 2000; Brachmann et al., 2000; Fig. 4). In the studies of Igaki et al. (2000) and Brachmann et al. (2000), transgenic constructs were generated by cloning the Debcl cDNA downstream of the GMR promoter, thereby permitting expression in the posterior part of the developing eye (Ellis et al., 1993). The study of Colussi et al. (2000) was different from the others in that the transgenic construct was made using the yeast GAL4(UAS) system, which contains the yeast Saccharomyces cerevisiae Gal4

Fig. 4. Ectopic overexpression of Debcl in transgenic flies induced cell death and resulted in an ablated eye phenotype. (A) Eye imaginal disc showing the region of expression of the GMR – GAL4 driver (dark blue). (B) Acridine orange-stained wild type eye disc (C) Acridine orangestained eye disc from a GMR – GAL4 UAS – debcl larva, showing increased staining in the posterior region. (D) Wild type adult eye (E) GMR – GAL4 UAS – debcl adult eye showing severe ablation.

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DNA-binding sites (Brand and Perrimon, 1993). The advantage of this system is that it permits greater flexibility, since one transgene under GAL4(UAS) control can be expressed in many different spatiotemporal developmental patterns by crossing flies to different GAL4 drivers (Brand and Perrimon, 1993). For example, Colussi et al. (2000) ectopically overexpressed Debcl at different developmental stages and in various tissues using either the GMR – GAL4, a salivary gland GAL4 driver or hsp70 – GAL4 (which allows a burst of ubiquitous expression following heat shock induction). In all these cases, Debcl expression promoted increased apoptosis, showing that Debcl was able to function as a pro-apoptotic factor in different developmental stages (Colussi et al., 2000). In contrast, using this GAL4(UAS) system, ectopic overexpression of Buffy does not result in increased cell death in any tissue examined, but rather gives rise to phenotypes suggestive of an anti-apoptotic effect (L. Quinn, S. Kumar and H. Richardson, unpublished results). This result suggests that despite the absence of a BH4 domain, Buffy may function as an antiapoptotic factor. In the studies of Brachmann et al. (2000) and Colussi et al. (2000), RNAi, using a double-stranded debcl cDNA, was used to ablate Debcl from embryos. debcl RNA-ablated embryos, analyzed by TUNEL, showed considerably less TUNEL-positive cells compared with buffer-injected controls (Colussi et al., 2000; Brachmann et al., 2000). In addition, Brachmann et al. (2000) demonstrated that Debcl-ablated embryos contained additional cells by staining debcl RNA-ablated embryos with an anti-glial cell antibody (anti-Repo). These data demonstrate that Debcl is required for PCD during embryogenesis. Debcl mRNA is expressed at very low levels, and it is difficult to visualize using the standard immunological staining methods. In order to amplify the signal, Colussi et al. (2000) used the indirect tyramide amplification (TSAk) system (New England Nuclear Life Science Products). This system allows four steps of amplification and thereby achieving greater sensitivity in detection. It relies upon detection of the primary antibody with a horseradish peroxidase (HRP)-conjugated secondary antibody followed by the HRP-mediated deposition of the tyramide conjugate (reviewed by Raap, 1998). The tyramide, which is conjugated with biotin, can then be detected using a


streptavidin-conjugated HRP or alkaline phosphatase, followed by standard colorimetric staining methods. This method allowed much greater sensitivity in detecting debcl mRNA distribution (Colussi et al., 2000) compared with standard approaches (L. Quinn, S. Kumar and H. Richardson, unpublished data; Igaki et al., 2000; Brachmann et al., 2000). The eye ablation phenotype due to overexpression of Debcl from the GMR driver (see Fig. 4) has been used to examine genetic interactions with other PCD genes (Colussi et al., 2000). By halving the dosage of PCD genes in a GMR – GAL4 UAS – debcl background, it was shown that Debcl genetically interacts with Dark and with the apoptosis inhibitor Diap1/ thread, but not with Diap2 (Colussi et al., 2000). Brachmann et al. (2000) used the rough eye phenotype of GMR –Debcl flies to examine the role of Debcl in the DNA damage response to UV irradiation and showed that mild overexpression of Debcl sensitized cells to undergo PCD in response to UV irradiation. These data demonstrate that Debcl plays a proapoptotic role upstream of Dark and Diap1 during Drosophila development.

6. Drosophila Iap homologs Apoptosis is negatively regulated by the Iap (inhibitor of apoptosis) family of proteins, which act to inhibit caspase function by directly binding to them (reviewed by Deveraux and Reed, 1999; Goyal, 2001). In Drosophila, two Iap homologs have been reported, Diap1/thread and Diap2 (reviewed by Hay, 2000). Diap1 has been shown to inhibit several caspases, including Dcp-1, Drice and Dronc (Meier et al., 2000; Hawkins et al., 1999; Kaiser et al., 1998; Wang et al., 1999; Goyal et al., 2000). Genetic and biochemical studies have shown that Diap1 functions by blocking the activation of caspases and that Rpr, Hid and Grim promote apoptosis by disrupting the Diap1 – caspase interaction (Wang et al., 1999; Goyal et al., 2000). Rpr, Hid and Grim share a small region of homology in their N-terminal regions that is important for binding of Diaps (reviewed by Goyal, 2001). This region of homology is also shared by mammalian Smac/Diablo, which are thought to function in a similar manner to Rpr, Hid and Grim. In conflict to this idea, however, a recent study showed


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that the Iap-binding domain of Smac/Diablo is not necessary for its pro-apoptotic activity (Roberts et al., 2001). This data suggest that Smac/Diablo induces cell death by a mechanism that is independent of Iaps. Similarly, N-terminal deletions, removing the Diapbinding domain of Rpr and Grim, but not Hid, are also able to promote cell death, although in these cases, complexes with Diaps are still observed (Wing et al., 1998; Chen et al., 1996; Vucic et al., 1997, 1998). However, an N-terminal truncated version of Grim can promote cell death, independent of co-expression of Diap1, Diap2, the caspase inhibitor p35 or dominant negative Dronc (Wing et al., 2001). This suggests that Grim, at least, can induce apoptosis independent of Diaps and caspases. Clearly, more studies are required to further elucidate the cell death-inducing mechanisms of Rpr, Hid and Grim. Mutant alleles in diap1/thread are embryonically lethal and result in increased caspase activity and increased TUNEL-positive cells in the embryo (Wang et al., 1999). By genetic analyses Wang et al. (1999) showed that diap1/thread is epistatic to rpr, hid and grim. A deficiency (deletion) that removes rpr, hid and grim (H99) prevents apoptosis in embryos (White et al., 1994), but when combined with a diap1/ thread mutant, apoptosis increases in embryos. This suggested that Diap1 functions to inhibit caspases and that Rpr, Hid and Grim oppose this. In a dominant genetic modifier screen for suppressors and enhancers of the eye phenotype caused by ectopic overexpression of rpr or hid during eye development, both lossof-function and gain-of-function alleles of diap1/ thread were isolated (Goyal et al., 2000). Gain-offunction alleles of diap1/thread strongly suppress rpr-, hid- and grim-induced apoptosis by disrupting the ability of Rpr, Hid or Grim to bind the mutant Diap1 protein (Goyal et al., 2000). These studies have been instrumental in elucidating the mechanism of Diap function in vivo (see Fig. 1). Genetic interaction studies using phenotypes generated by ectopic overexpression of PCD genes in transgenic flies have provided evidence that supports the role of Diap1 in PCD. For example, the eye ablation phenotype induced by Dronc overexpression is suppressed by co-expression of Diap1, whereas heterozygosity at the diap1 locus enhances the Dronc eye phenotype, consistent with a role for Diap1 in inhibiting Dronc activation (Meier et al., 2000; Hawkins et al., 2000;

Quinn et al., 2000). Although Diap2 co-expression can also suppress the Dronc-induced eye phenotype, it is interesting to note that a deficiency removing diap2 does not interact with Dronc, suggesting that Diap2 is not a rate-limiting inhibitor of Dronc activation (Quinn et al., 2000). Consistent with this notion is the fact that Diap2 was unable to form a complex with Dronc in tissue culture cells (Quinn et al., 2000). Further in vivo analysis using specific mutants of diap2 or diap2 RNA ablation is required to further explore the role of Diap2 in PCD.

7. PCD signaling pathways In contrast to our understanding of the core PCD machinery in flies, the characterization of PCD signaling pathways in Drosophila is at a relatively nascent stage. The recent identification of a Drosophila Fadd homolog (Hu and Yang, 2000), a component of the extrinsic PCD pathway in mammalian cells, has demonstrated that at least part of this pathway is present in Drosophila (see Fig. 1). Drosophila Fadd (dFadd) contains a death domain that is highly related to the mammalian Fadd death domain, and it also shares a novel domain with the caspase Dredd, termed the death-inducing domain (Hu and Yang, 2000). In vitro studies and studies in mammalian tissue culture cells have shown that dFadd binds to Dredd through the death-inducing domain and activates proteolytic processing of Dredd and its death-inducing activity (Hu and Yang, 2000). As yet, no in vivo studies have been reported with dFadd. Furthermore, no death receptors (tumor necrosis factor receptor [TNFR] family) have been reported in Drosophila, and an analysis of the Drosophila genomic sequence has not revealed any sequences that have both a death domain and a death effector domain characteristic of mammalian death receptors (Vernooy et al., 2000; Aravind et al., 2001). From the available data, it seems that the role of dFadd is to activate Dredd, and since Dredd has been shown to play a major role in the innate immune response, it is possible that dFadd also functions in this pathway. However, the expression of the Fas cytoplasmic domain in Drosophila cells resulted in the induction of apoptosis, suggesting that death receptor signaling can occur at least in Drosophila-cultured cells (Kondo et al., 1997). In vivo

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studies and genetic analysis are needed to dissect the role of dFadd and further elucidate the components of the extrinsic PCD pathway in Drosophila. The p53 transcription factor tumor suppressor acts as a ‘‘guardian of the genome’’, responding to DNA damage and cell cycle perturbations to mediate PCD or cell cycle arrest and DNA repair (reviewed by Levine, 1997; May and May, 1999). The recent discovery of a Drosophila p53 homolog (Dmp53) and the finding that ectopic overexpression of Dmp53 in transgenic flies induces PCD demonstrates that this DNA damage response pathway is also conserved in Drosophila (Brodsky et al., 2000; Ollmann et al., 2000; reviewed by Nordstrom and Abrams, 2000). Overexpression of mutated versions of Dmp53, disrupted for DNA binding, acts as dominant-negative alleles and block radiation-induced apoptosis in the developing wing disc (Brodsky et al., 2000; Ollmann et al., 2000). In the study of Brodsky et al. (2000), an important connection was established between Dmp53 and the PCD-inducing gene rpr that is specifically expressed in ‘‘doomed’’ cells. Dmp53 was shown to induce expression of rpr through a Dmp53 radiation-inducible element (p53RE) located ~5 kb upstream of the rpr start codon. By constructing transgenic flies containing the p53RE upstream of a lacZ reporter construct, Brodsky et al. (2000) showed that in response to irradiation, lacZ expression was induced in many cells throughout the embryo. The p53RE was also shown to respond specifically to radiation-induced apoptosis, since crumbs mutant embryos, in which there is widespread apoptosis, did not show induction of p53RE – lacZ expression, although expression was induced from a 2-kb rpr promoter –lacZ reporter. This study provided the first direct connection between a central apoptosisinducing gene and an apoptotic signal. Rpr expression is also regulated by the steroid hormone ecdysone and by developmental signals (Jiang et al., 2000; Nordstrom et al., 1996). grim, like rpr, is also expressed specifically in ‘‘doomed’’ cells, while hid has a more general expression pattern (White et al., 1994; Chen et al., 1996; Grether et al., 1995). In contrast, Hid is negatively regulated at the transcriptional and posttranscriptional levels by the epidermal growth factor receptor (EGFR) – Ras –Raf – MAPK signaling pathway (Kurada and White, 1998; Bergmann et al., 1998). By carrying out genetic


interaction studies in transgenic flies, it was demonstrated that the EGFR pathway promotes cell survival and that the MAPK phosphorylation sites in Hid are critical for this response. Furthermore, the Pointed and Yan transcription factors, which act downstream of the EGFR –Ras signaling pathway, regulate hid mRNA expression. Additional in vivo analysis involving promoter dissection of rpr, grim and hid genes as well as mutant analysis are required to discover how the steroid hormone ecdysone, developmental signals, cell cycle perturbations and growth factors induce expression of these genes.

8. Future genetic approaches to elucidating PCD pathways in Drosophila Drosophila is a powerful system to study the function of individual genes and to define interactions between different molecules of a pathway. Although the analysis of PCD in flies is a relatively new field of research, the in vivo studies described above have revealed the in vivo roles for several Drosophila PCD genes during development. Further in vivo analysis is needed for many of the PCD genes, as discussed above. The targeted gene knockout technique that has been described in Drosophila has been described (Rong and Golic, 2001), and although labor-intensive, it is now possible to generate specific mutations in any gene of interest. For a number of the genes already characterized, however, more straightforward approaches can be used to create mutations. For example, P element mutations exist near Debcl and Buffy, and by imprecise excision of the P alleles, it should be possible to create deletions covering these genes. Alternatively, P element mutations in these genes may be obtained using the method of local P element transposition (Dalby et al., 1995; Tower et al., 1993; Zhang and Spradling, 1993). For example, this method was used successfully to generate dark null mutants (Rodriguez et al., 1999). The generation of transgenic flies containing constructs capable of forming double-stranded RNA in vivo allows the RNAi technique to be used to ablate gene function in somatic tissues in order to assess the role of PCD genes at later stages of development (Kennerdell and Carthew, 2000). Additional homologs of mammalian PCD components are present in Drosophila (Vernooy et al., 2000),


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although their function is yet to be analyzed. For example, Scythe, which was identified in Xenopus as a protein that binds to Drosophila Rpr and is involved in the release of cytochrome c from the mitochondria (Thress et al., 1998, 1999), is present in flies but has not yet been analyzed. There are also fly homologs of the nuclease Cad and the nuclease inhibitor Icad (Mukae et al., 2000; Yokoyama et al., 2000), but these homologs have not yet been characterized in flies. There are also fly homologs of Aif and Acinus, which promote nuclear events of apoptosis, DNA fragmentation and/or DNA condensation (Susin et al., 1999; Sahara et al., 1999), which are yet to be examined. Clearly, further in vivo studies using transgenic flies and the generation and analysis of mutants, or ablation of gene function with RNAi, are required to understand the developmental role of these PCD genes. Since the intrinsic PCD pathway is present in flies, homologs of the BH3-only domain protein (C. elegans Egl-1 homologs) family (reviewed by Huang and Strasser, 2000; Lutz, 2000) are expected to exist. Since some components of the extrinsic PCD pathway exist in flies, it is possible that functional homologs of mammalian death receptors are also present. Genetic modifier screens in flies or yeast with two hybrid screens will undoubtedly be important in identifying such PCD components. Another primary goal is to understand how developmental signals, ecdysone signaling, growth factors, the Dmp53-mediated DNA damage response and cell cycle perturbations regulate the central PCD components. The Dmp53-mediated DNA damage response mechanism and ecdysone signaling have been shown to directly induce rpr gene expression, but it is likely that they act upon other PCD genes as well. For example, the expression of the apical caspase Dronc is likely to be directly or indirectly regulated by the ecdysone-signaling pathway (Dorstyn et al., 1999a; Lee et al., 2000; Lee and Baehrecke, 2001). Perhaps the most powerful approach to the understanding of PCD in Drosophila is the ability to carry out unbiased dominant genetic interaction screens. Such screens have already been carried out for the GMR – rpr and GMR – hid eye ablation phenotypes and have been invaluable in identifying new alleles of diap1 (Goyal et al., 2000), as described above. Furthermore, a dominant modifier screen using the irregular ChiasmC – roughest mutant phenotype,

which specifically prevents PCD during eye development, has revealed a number of potential novel PCD genes (Tanenbaum et al., 2000). Similar screens using the eye ablation phenotypes of Grim, Dronc, Debcl and other PCD genes should reveal novel PCD genes. The generation of modified P elements (EP and GS elements) that carry the GAL4(UAS) have been used to create banks of flies capable of overexpressing genes (Rorth, 1996; Toba et al., 1999). This provides an alternative way in which to carry out genetic modifier screens. An advantage of carrying out an overexpression screen is that it can identify interacting genes that are not rate limiting, as is required in dominant loss-of-function screens (e.g., Simon et al., 1991; Tanenbaum et al., 2000). Furthermore, the interacting gene affected by the P allele can be readily identified, using a reverse transcriptase (RT)-PCR approach. The insertion of the EP or GS element can also inactivate the gene in some instances and therefore can be used to examine the consequences of gene loss-of-function on PCD. In conclusion, the application of the genetically manipulable Drosophila animal model system to study PCD has already proved to be very valuable in revealing the in vivo function of PCD genes. Perhaps the most pressing current issues in the understanding of PCD in flies is defining the role of Buffy in a prosurvival role and identifying BH3-only domain proteins, as well as elucidating PCD signaling pathways. The recent innovations of novel techniques and whole animal genetic approaches (as discussed above) provides exciting new possibilities for elucidating the intricacies of PCD pathways within a whole animal. Acknowledgements We thank Bill Kalionis and Leonie Quinn for comments on this article. Work on this subject has been supported by the Wellcome Trust and by the National Health and Medical Research Council. Helena Richardson and Sharad Kumar are Wellcome Senior Research Fellows in Medical Science. References Abrams, J.M., 1999. An emerging blueprint for apoptosis in Drosophila. Trends Cell Biol. 9, 435 – 440.

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