death to flies: drosophila as a model system to study programmed cell death

Death to f lies: Drosophila as a model system to study

programmed cell death

Helena Richardson a,*, Sharad Kumar b,*

aTrescowthick Research Laboratories, Peter MacCallum Cancer Institute, Locked Bag 1, A’Beckett St., Melbourne, Victoria, 8006, AustraliabHanson 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

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). Devel-

opmental- or damage-induced death signals result in

the activation of the cysteine proteases, caspases,

which orchestrate the events of PCD including mem-

brane 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), Ced-

4 (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: S0022 -1759 (02 )00068 -6

Abbreviations: PCD, programmed cell death; PCR, polymerase

chain reaction; TUNEL, TdT-mediated dUTP-Nick-End Labeling;

RNAi, RNA interference; GMR, Glass multimer reporter; GA-

L4(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.: +61-

8-8222-3738; fax: +61-8-8222-3139.

E-mail addresses: [email protected]

(H. Richardson), [email protected] (S. Kumar).

www.elsevier.com/locate/jim

Journal of Immunological Methods 265 (2002) 21–38

Subsequent studies in mammals and in the fly, D.

melanogaster, have identified counterparts for these C.

elegans genes, demonstrating that the core compo-

nents 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 complex-

ity 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. Mam-

malian 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 activa-

tion (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 differ-

ence 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). Fur-

thermore, in more complex animals there are both anti-

apoptotic 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 mam-

malian cells compared with C. elegans. Understanding

the physiological role of PCD components in mam-

mals 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 com-

plexity between worms and mammals in its PCD

pathways. Drosophila shares many of the PCD com-

ponents and pathways found in mammals, but has less

redundancy, allowing easier dissection of function.

Another distinct advantage is that the recent comple-

tion of the Drosophila euchromatic genomic sequence

(Rubin et al., 2000), has revealed all of the highly

conserved homologs of mammalian PCD genes (Ver-

nooy et al., 2000). In addition, Drosophila is devel-

opmentally 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 immuno-

logical 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 frag-

mentation, 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

H. Richardson, S. Kumar / Journal of Immunological Methods 265 (2002) 21–3822

situ TUNEL (TdT-mediated dUTP-Nick-End Label-

ing; 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

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. Pro-

apoptotic 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.

H. Richardson, S. Kumar / Journal of Immunological Methods 265 (2002) 21–38 23

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 Baeh-

recke, 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 apop-

totic pathway (Foley and Cooley, 1998). Thus, Dro-

sophila presents a number of differently regulated

PCD pathways that may provide models for under-

standing 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 identi-

fied that was required for PCD. This deletion removes

three genes, reaper (rpr), hid (head involution defect/

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.


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 expres-

sion 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 develop-

ment. 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 (prefera-

bly 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-of-

function phenotypes. The existence of mutants in

PCD genes or phenotypes generated by ectopic over-

expression 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 Droso-

phila PCD genes. These analyses have provided

valuable information on the in vivo function of PCD

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).


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 Loss-of-function Ectopic overexpression

Rpr H99 deficiency—prevents embryonic cell death GMR,a hsp70 transgene—induced cell death

Grim H99 deficiency—prevents embryonic cell death GMR transgene—induced cell death

Hid H99 and other deficiencies—prevent embryonic

cell death and results in a head involution defect

GMR, hsp70 transgene—induced cell death

Wrinkled-hypomorphic allele—defect in

wing development

Dredd Mutants—defects in the immune response –

Mutants—genetically interacts with rpr and grim

Dronc RNAib—prevents PCD in the embryo

Dronc deficiency and Dronc dominant negative

transgene—genetically interacts with rpr,

hid and grim

GAL4(UAS)c transgene—expression via GMR,

hsp70 and other drivers—induces cell death

Dcp-1 Null allele—third instar larval lethal—no imaginal

discs or gonads, melanotic tumors

GMR transgene—promotes cell death

Germ line clones—nurse cell dumping defect

Drice – GMR transgene—no effect

Decay – –

Damm Damm dominant negative transgene—genetically

interacts with hid

GMR transgene—promotes cell death

Strica – –

Dark 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

Debcl RNAi—prevents PCD in the embryo GAL4(UAS) transgene—expression via GMR,

hsp70 and other drivers—induces cell death

Buffy – GAL4(UAS) transgene—expression via GMR,

hsp70 and other drivers—does not induce

cell death

Diap1 thread loss-of-function—Increased cell death in

the embryo—embryonically lethal. Epistatic

to rpr, hid and grim

GMR transgene—no effect. Inhibits Dronc- or

Debcl- induced cell death

—genetically interacts with Dronc and Debcl

Gain-of-function—prevents rpr-, hid- and

grim-induced cell death

Diap2 Deficiency—does not genetically interact with

Dronc or Debcl

GMR transgene—no effect. Inhibits Dronc- or

Debcl- induced cell death

dFadd – –

Dmp53 —Dmp53 dominant negative transgene blocks

DNA damage-induced cell death in the wing disc

GMR transgene—induces cell death

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


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 gen-

erate 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; Douma-

nis et al., 2001; Harvey et al., 2001; reviewed by

Kumar and Doumanis, 2000). Dredd, Dronc and

Strica are class I caspases which contain long prodo-

mains. Two types of domains have been defined in the

mammalian prodomain regions, the caspase recruit-

ment domain, CARD, and death effector domain,

DED (reviewed by Aravind et al., 1999). These

domains are required for the recruitment of pro-

caspase 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 recruit-

ment 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 expect-

ations, do not arise as a consequence of over-prolif-

eration 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).

dreddmRNA 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, heterozygos-

ity 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-micro-

bial peptides and are highly susceptible to bacterial


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 func-

tional 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 catalyti-

cally 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 Zernicka-

Goetz, 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 mecha-

nism, which leads to the degradation of the endoge-

nous 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 overexpress-

ing 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 down-

stream 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 upregu-

lated 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-respon-

sible 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 apopto-

sis by rpr overexpression or by cytotoxic agents

(Fraser and Evan, 1997; Fraser et al., 1997). Further-

more, immunodepletion of Drice from Drosophila S2

cell lysates, using Drice-specific antibodies, consid-

erably 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 tissue-

specific 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 cata-

lytically inactive, dominant negative mutant in the eye

suppressed the ablated eye phenotype due to over-

expression of hid, but not rpr (Harvey et al., 2001).


This suggests that Damm may function specifically

downstream of hid-induced apoptosis. Ectopic expres-

sion of Strica or Decay in mammalian cells or

Drosophila S2 tissue culture cells has a weak apop-

totic effect (Doumanis et al., 2001; Dorstyn et al.,

1999b), but their specific function in Drosophila cell

death at present is not known. Clearly, the under-

standing 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 acti-

vation 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). Further-

more, 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 cas-

pase, 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 C-

terminal 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


studies to determine the in vivo function of cyto-

chrome c in PCD during development.

Genetic interaction studies of Dark in Rpr-, Hid-

or 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 substan-

tially inhibited in homozygous dark mutants, hid-

induced 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 Grim-

induced 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 Grim-

mediated 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 pro-

teins, but appear to lack the N-terminal BH4 domain.

The BH4 domain distinguishes the pro-apoptotic Bcl-

2 family members, e.g. Bax and Bok, from the anti-

apoptotic 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-apop-

totic. 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 orange-

stained 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.


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 spatiotem-

poral developmental patterns by crossing flies to

different GAL4 drivers (Brand and Perrimon, 1993).

For example, Colussi et al. (2000) ectopically over-

expressed 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 anti-

apoptotic 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) demon-

strated that Debcl-ablated embryos contained addi-

tional 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 immuno-

logical 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 sensi-

tivity 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 conju-

gate (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 back-

ground, 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 pheno-

type 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 sensi-

tized cells to undergo PCD in response to UV irradi-

ation. These data demonstrate that Debcl plays a pro-

apoptotic role upstream of Dark and Diap1 during

Drosophila development.

6. Drosophila Iap homologs

Apoptosis is negatively regulated by the Iap (inhib-

itor 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 func-

tion in a similar manner to Rpr, Hid and Grim. In

conflict to this idea, however, a recent study showed


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 Diap-

binding 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 domi-

nant 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 overexpres-

sion of rpr or hid during eye development, both loss-

of-function and gain-of-function alleles of diap1/

thread were isolated (Goyal et al., 2000). Gain-of-

function 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 over-

expression 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, con-

sistent 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 signal-

ing 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 mamma-

lian 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 Droso-

phila-cultured cells (Kondo et al., 1997). In vivo


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; Oll-

mann et al., 2000; reviewed by Nordstrom and

Abrams, 2000). Overexpression of mutated versions

of Dmp53, disrupted for DNA binding, acts as dom-

inant-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 apoptosis-

inducing 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 demon-

strated 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 func-

tion 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 character-

ized, 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 ele-

ment 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 compo-

nents are present in Drosophila (Vernooy et al., 2000),


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 trans-

genic 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 devel-

opmental signals, ecdysone signaling, growth factors,

the Dmp53-mediated DNA damage response and cell

cycle perturbations regulate the central PCD compo-

nents. 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 under-

standing 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 develop-

ment, 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 pro-

survival role and identifying BH3-only domain pro-

teins, as well as elucidating PCD signaling pathways.

The recent innovations of novel techniques and whole

animal genetic approaches (as discussed above) pro-

vides 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.

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death to flies: drosophila as a model system to study programmed cell death

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