Programmed cell death takes flight: genetic and genomic approaches to gene discovery in Drosophila

S. Gorski and M. Marra

Genome Sequence Centre, British Columbia Cancer Agency, Vancouver, British Columbia, Canada V5Z 4E6


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 PCD Function and Dysfunction
 Evolutionary Conservation of...
 PCD in Drosophila
 Genetic Approaches to...
 Drosophila Genome Projects
 Genomic Approaches to...
 Gene Expression Profiling of...
 Concluding Remarks
 REFERENCES
 
Programmed cell death (PCD) is an essential and wide-spread physiological process that results in the elimination of cells. Genes required to carry out this process have been identified, and many of these remain the subjects of intense investigation. Here, we describe PCD, its functions, and some of the consequences when it goes awry. We review PCD in the model system, the fruit fly, Drosophila melanogaster, with a particular emphasis on cell death gene discovery resulting from both genetics and genomics-based approaches.

apoptosis; fruit fly; genome project


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 PCD Function and Dysfunction
 Evolutionary Conservation of...
 PCD in Drosophila
 Genetic Approaches to...
 Drosophila Genome Projects
 Genomic Approaches to...
 Gene Expression Profiling of...
 Concluding Remarks
 REFERENCES
 
PROGRAMMED CELL DEATH (PCD) refers to the induction of physiological cell death by a genetically regulated pathway. Often referred to as an intrinsic cell suicide program, PCD occurs in a defined temporal and spatial manner during the normal development of multicellular eukaryotes. The term "programmed cell death" was used first in 1963 by Lockshin (78) to describe the demise of the intersegmental muscles of the silkmoth (78, 79). The execution of PCD leads to several morphologically distinct forms of cellular breakdown (reviewed in Ref. 104); the one most commonly described is apoptosis, and the two terms, PCD and apoptosis, are often used synonymously. In this review, we will use the term PCD (or simply "cell death") to include apoptosis.

Apoptotic cell death is characterized by the absence of cell swelling and by the lack of an inflammatory response. Features of apoptosis include cell shrinkage, membrane blebbing, morphological conservation of most organelles, nuclear condensation, and phagocytosis of dying cells (reviewed in Ref. 149). These events were described in hepatocytes by Kerr, and the mechanism responsible was named "apoptosis" in 1972 (64). The term "apoptosis" refers to the "falling off" of petals from flowers, or leaves from trees, for the betterment of the organism as a whole. The name is meant to reflect the widespread importance of regulated cell death in homeostasis (64).


    PCD Function and Dysfunction
 TOP
 ABSTRACT
 INTRODUCTION
 PCD Function and Dysfunction
 Evolutionary Conservation of...
 PCD in Drosophila
 Genetic Approaches to...
 Drosophila Genome Projects
 Genomic Approaches to...
 Gene Expression Profiling of...
 Concluding Remarks
 REFERENCES
 
PCD is important not only in homeostasis but also in development and disease pathogenesis. The functions of PCD in animal development are varied and include deletion of unnecessary cells and structures, adjustment of cell numbers, removal of damaged or harmful cells, and sculpting of tissues (reviewed in Refs. 57 and 132). For example, the tadpole tail is deleted by PCD during metamorphosis of the tadpole to an adult frog (65, 126). In developing epithelia, PCD is used to remove interdigital cells during limb pattern formation (92), shape the vertebrate neural tube (140), and deplete specific rhombomeres of neural crest cells in the vertebrate hindbrain (41, 42). During maturation of the vertebrate nervous system, approximately half of the developing neurons die, presumably to match the number of neurons to the number of target cells they innervate (46; reviewed in Refs. 96 and 151). Given the extensive requirement for PCD in normal development, it is not unexpected that developmental abnormalities are some of the manifestations of PCD dysfunction (reviewed in Ref. 105).

PCD dysfunction is associated also with diseases such as bacterial and viral infections, neurodegenerative disorders, autoimmunity, and cancer (reviewed in Refs. 30 and 128). Bacterial infections are associated with increased cell death, and viral infections are associated with either increased or decreased cell death depending on the particular virus involved. Neurodegenerative disorders are associated with excessive cell death, whereas autoimmunity and cancer are associated with decreased cell death (or increased cell survival). In cancer, for example, inappropriate activation of a negative regulator of PCD, the B-cell lymphoma/leukemia-2 (Bcl-2) gene, is associated with non-Hodgkin’s lymphomas (130). Inactivation of a positive regulator of cell death, Bax, is associated with colon, gastrointestinal, and hematological malignancies (85, 103, 150). In fact, PCD dysfunction has been implicated in multiple stages of cancer including hyperplasia, neoplastic transformation, tumor expansion, neovascularization, and metastasis (reviewed in Ref. 30). In addition, PCD dysfunction is associated with resistance to cancer therapy. The same mutations that inhibit death of a damaged cell and contribute to disease can also interfere with treatment by inhibiting cell death induced by radiation and chemotherapeutic agents (reviewed in Refs. 106 and 128). The many crucial roles of PCD highlight the need to understand fully its mechanisms of action and its modes of regulation. A thorough understanding of these processes will contribute to both a greater knowledge of animal physiology and a more comprehensive array of candidate targets for the development of drugs to help combat PCD-associated diseases.


    Evolutionary Conservation of Death’s Molecular Machinery
 TOP
 ABSTRACT
 INTRODUCTION
 PCD Function and Dysfunction
 Evolutionary Conservation of...
 PCD in Drosophila
 Genetic Approaches to...
 Drosophila Genome Projects
 Genomic Approaches to...
 Gene Expression Profiling of...
 Concluding Remarks
 REFERENCES
 
One of the fastest growing fields in the past decade (86), PCD research has uncovered multiple molecules and pathways important in the death process. Most of the molecules involved in PCD are conserved in organisms as diverse as the nematode Caenorhabditis elegans, the fruit fly Drosophila melanogaster, and humans. Studies in the model organism C. elegans were instrumental in elucidating both the evolutionary conservation of PCD and its genetic basis when mutations in three genes encoding cell death proteins were identified (53; reviewed in Ref. 87). The three proteins, an anti-apoptotic Bcl2-like molecule (CED9), a pro-apoptotic cysteine aspartic acid-specific protease (caspase; CED3), and an apoptosis-protease-activating factor (apaf)-1-like molecule (CED4), form part of what is known as the central " death machinery" that was described subsequently in humans, mouse, and Drosophila (3, 11, 20, 23, 27, 33, 56, 63, 110, 120, 152, 154, 155). Despite a later entry into molecular genetic studies of PCD in the Drosophila model system, it has proved since to be a valuable model for the study of cell death molecules, mechanisms, and pathways. In fact, comparisons of cell death molecules found in the worm, fly, and human indicate that humans and flies share more orthologs than do humans and worms. In addition, humans and flies share a greater complexity in terms of cell death molecules as evidenced by the existence of several multimember cell death gene families (5, 112, 129, 136; see Fig. 1).



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Fig. 1. Model of programmed cell death (PCD) signaling pathways in Drosophila. Cell death-related gene products discovered by genetics approaches are indicated in orange, and those discovered by molecular and genomic based approaches are indicated in blue. Dashed arrows indicate interactions in Drosophila that are speculative. All known members of multigene families are shown, but interaction data and in vivo functions have not been reported in all cases (see text). EcR/USP is the Drosophila ecdysone receptor, which can directly regulate reaper transcription when complexed with the steroid hormone ecdysone (59).

 
While molecules participating in the central death machinery have been identified, new cell death genes are still being discovered (e.g., Refs. 25 and 100). To understand fully PCD-associated physiology and disease and to implement therapeutic strategies most effectively, it is essential to have knowledge of all of the genes involved in PCD. A particular imperative is to understand how these genes and their products are regulated. How does stage-specific, tissue-specific, cell type-specific, and cell position-specific regulation of PCD occur? Also, how are the various cell death and cell survival pathways related functionally and how do they interact? The highly conserved nature of PCD allows exploitation of multiple experimental systems to address these important questions.


    PCD in Drosophila
 TOP
 ABSTRACT
 INTRODUCTION
 PCD Function and Dysfunction
 Evolutionary Conservation of...
 PCD in Drosophila
 Genetic Approaches to...
 Drosophila Genome Projects
 Genomic Approaches to...
 Gene Expression Profiling of...
 Concluding Remarks
 REFERENCES
 
Drosophila is one of the experimental systems well-suited for examining PCD-related questions. In addition to conservation of cell death molecules, many morphological features of apoptosis are conserved between Drosophila and other animals, including humans (1). Furthermore, Drosophila possesses the advantages of a rapid life cycle, sophisticated genetic tools, well-developed misexpression systems, a largely complete and annotated genome sequence, and comprehensive information databases (in 91). Moreover, multiple tissues undergo PCD throughout Drosophila development. Published studies include descriptions of cell death in the Drosophila embryonic central nervous system, embryonic head region, embryonic epidermis, larval salivary glands, larval midgut, larval wing and eye imaginal discs, pupal retina, adult nervous system, and adult female germ line (1, 13, 31, 36, 58, 66, 72, 89, 93, 94, 98, 108, 121, 145, 153). Each of these stages and tissues possesses experimental advantages and lends itself particularly well to the investigation of different aspects of PCD. In the developing embryo, for example, RNA interference has been used to analyze loss-of-function phenotypes of cell death genes (11, 23, 101, 154). In addition to in vivo studies of PCD in Drosophila, there exist Drosophila cell lines (e.g., SL2) that enable the study of expressed gene products, e.g., by interaction studies, caspase assays, and apoptosis assays (18, 144). Studies utilizing various Drosophila developmental stages and tissues are described below. We focus on how both classic genetics approaches and new genomics-based approaches employed in Drosophila have contributed to our knowledge of PCD, particularly with respect to gene discovery, and provided exciting leads for further insights into this fundamental biological phenomenon.


    Genetic Approaches to Identification of Drosophila PCD Genes
 TOP
 ABSTRACT
 INTRODUCTION
 PCD Function and Dysfunction
 Evolutionary Conservation of...
 PCD in Drosophila
 Genetic Approaches to...
 Drosophila Genome Projects
 Genomic Approaches to...
 Gene Expression Profiling of...
 Concluding Remarks
 REFERENCES
 
One of the first genetic screens for PCD-related genes in Drosophila was conducted using the Drosophila retina, a tissue sculpted by PCD. The Drosophila retina is formed by a process of cell proliferation and then successive cell differentiation which yields 750 identical repeating units called ommatidia (reviewed in Ref. 146). Each ommatidium is composed of fourteen cells: eight photoreceptor neurons, four nonneuronal cone cells, and two optically insulating primary pigment cells (Fig. 2). The ommatidia are separated by numerous interommatidial precursor cells; some differentiate to become pigment cells, and the excess cells undergo PCD (13, 145; Fig. 2). The result is a hexagonal cellular lattice, precise with respect to cell number and position, surrounding each ommatidium. Disruptions in the PCD process lead to disorganization of the regular hexagonal array of interommatidial cells, and the effects are detectable in the adult as a "rough eye" phenotype. Wolff and Ready (145) screened a set of existing rough eye mutant adult Drosophila lines for aberrations in the numbers of interommatidial cells and levels of cell death. They discovered two genes, echinus and irreC-rst, that were required for retinal PCD. The echinus gene has not yet been identified molecularly. The irreC-rst gene was cloned and found to be a member of the immunoglobulin superfamily (102), able to mediate homophilic interactions in cell culture (116). Observations of cellular organization in irreC-rst mutant retinas indicate a role for the IrreC-rst protein in the cell rearrangements required for selective cell death in the retina, but the mechanism of IrreC-rst action is undetermined (107).



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Fig. 2. The Drosophila retina is sculpted by PCD. Left: a tracing of a portion of the apical surface of a predeath stage (26 h after puparium formation) retina. In the center of the tracing is a single ommatidium consisting of two primary pigment cells (yellow), four cone cells (orange), and eight photoreceptors (not seen; located below the cone cells). Separating the ommatidia are the interommatidial precursor cells (blue) and bristle cells (black). Right: tracing of a portion of the apical surface of a postdeath stage (40 h after puparium formation) retina. Some of the interommatidial precursor cells have undergone PCD, and the remainder have differentiated to form secondary or tertiary pigment cells (blue). PCD results in the formation of a tightly packed and regular array of ommatidia.

 
The retinal PCD screen described above was limited to existing adult mutants with rough eye phenotypes and thus did not involve mutant alleles required also for viability. White et al. (141) took another approach to screen for such essential PCD alleles. They conducted what is termed a "deficiency screen" to identify mutants that altered levels of PCD in the developing embryo. They analyzed a set of existing lines containing deficiencies that together covered over 50% of the Drosophila genome. Homozygous deficiency embryos were analyzed by the vital dye acridine orange (a cell death indicator) for alterations in levels of cell death. This screen identified a single deficiency, H99, required for essentially all cell death in the embryo. Subsequent analyses showed that the deficiency actually contained three genes, reaper, hid, and grim, all required for cell death not only in the embryo but in tissues during later developmental stages as well (16, 43, 141). Expression of Reaper, Hid, or Grim can induce apoptosis in mammalian cells (22, 45, 82), but no mammalian counterparts with extensive sequence similarity have been identified. However, the three gene products share with each other a short region of similarity at their amino termini that mediates interactions with inhibitor of apoptosis proteins (IAPs; see below), and this region is found also at the amino termini of two processed mammalian proteins. The mammalian proteins, Smac/Diablo and the serine protease Omi/HtrA2, bind and regulate IAPs in a manner similar to Drosophila Reaper, Hid, and Grim, indicating that the mammalian and Drosophila proteins function at least somewhat similarly (15, 29, 52, 80, 118, 124, 131, 134, 135, 147, 148).

Other searches for PCD-related genes have utilized another type of genetic screen, called a dominant "enhancer/suppressor" or "second-site modifier" genetic screen, used commonly in Drosophila research. The rationale of dominant enhancer/suppressor screens is that in a sensitized background, the removal of a single copy of a gene will result in the dominant modification of a phenotype (119). A particular advantage of this type of genetic screen is that it can identify homozygous lethal mutations in the F1 generation. In practice, the basic protocol is to start with a phenotype of interest and then screen for second-site mutations that modify the severity of that phenotype, thus identifying genes that participate in the same or related pathway or process. The second-site mutations that are screened can be point mutations or other aberrations that correspond to a single gene, or they can be deficiencies that correspond to one or many genes. Hay et al. (51) used this approach to screen for deficiencies that modified the small, rough eye phenotype resulting from eye-specific misexpression of the reaper gene. Examination of one enhancing deficiency led to the isolation of diap1, a Drosophila homolog of viral and mammalian IAPs. A second IAP homolog, named diap2, was found subsequently by sequence similarity to diap1 (51). Demonstrated to have a PCD role in mammals, IAPs inhibit apoptosis by binding to and preventing the activity of caspases (24). Hay et al. (51) showed that ectopic expression of either the Diap1 protein or the Diap2 protein can block apoptosis induced by ectopic expression of Reaper and Hid. Furthermore, Reaper, Hid, and Grim each can physically interact with Diap1 when overexpressed in lepidopteran SF-21 cells (137, 138). In other screens for modifiers of the small, rough eye phenotype resulting from eye-specific misexpression of reaper (40, 75) or hid (40), several loss-of-function alleles (enhancers) and gain-of-function alleles (suppressors) of diap1 were isolated. Analysis of the various enhancer and suppressor alleles of diap1 supported a model where Reaper, Grim, and Hid interact with Diap1 in vivo (40, 75) and indicated also that Reaper and Grim interact with Diap1 in a different way than Hid (75). Mutant Diap1 proteins, corresponding to the gain-of-function diap1 alleles, displayed reduced binding of Reaper and Hid in vitro (40). As in mammals, Diap1 can also bind directly and inhibit several Drosophila caspases (49, 50, 62; reviewed in Ref. 83). Wang et al. (139) showed that Hid can block the ability of Diap1 to inhibit caspase activity both in vitro and in yeast cells. Together these results support a model where Reaper, Grim, and Hid activate cell death by inactivating Diap1, a negative regulator of caspase activity (see Fig. 1).

Tanenbaum et al. (125) conducted another second-site modifier screen using the Drosophila eye, searching for dominant modifiers of the retinal cell death phenotype of irreC-rst. A moderately severe loss-of-function irreC-rst allele was used for the genetic screen, allowing for the isolation of second-site mutations exhibiting more severe phenotypes (i.e., enhancers) and less severe phenotypes (i.e., suppressors) of the rough eye phenotype. Isolated modifiers included mutant alleles of dRas1 and Delta, Drosophila genes demonstrated previously to be required for retinal cell survival and cell death, respectively (90, 97, 114). Delta and its receptor, Notch, were shown to be required also for the correct spatial localization of IrreC-rst in the retina (39, 107). In addition to these known genes, several potentially novel genes with retinal cell death phenotypes were identified, providing further avenues for investigation into cell death in the retina and perhaps additional tissues.

The Drosophila homolog of the mammalian ras gene, dRas1, was discovered as a negative regulator of cell death in another enhancer/suppressor screen, this one designed to identify modifiers of the eye ablation phenotype resulting from the eye-specific misexpression of hid. Mutations in genes that regulate the EGF receptor/Ras1 pathway were recovered as suppressors of Hid-induced apoptosis, and subsequent analyses showed that Hid is a direct target of the Ras/mitogen-activated protein (MAP) kinase pathway (9, 70).

Although genetic approaches have contributed greatly to cell death gene discovery and our knowledge of cell death gene interactions in Drosophila, there are some limitations to a strictly forward genetics approach. One limitation is the design of the genetic screen itself. For example, the diap1 gene is required for viability in the embryo (40, 75, 139) but was missed in the embryonic deficiency screen presumably because diap1 homozygous embryos arrest at a stage earlier than the one examined in the screen (in 7). Further limiting a strictly forward genetics approach is the ability of any given gene to mutate to a readily detectable phenotype. In Drosophila it is estimated that only one-third of all genes fall into this category (88, 123), and the remaining two-thirds possess at least partially redundant functions and could remain obscure or undetected in mutagenesis screens.

Molecular-based gene discovery strategies are not limited by functional redundancy but rely generally on knowledge of a previously existing gene product. For example, Dcp-1, the first described of seven known Drosophila caspases, was recovered via a PCR strategy using degenerate oligonucleotides designed based on knowledge of caspase sequences from other organisms (120). With the advent of Drosophila Genome Projects, direct molecular screening approaches have been replaced largely by screening initially in silico using computer searches of the sequence. Below we describe how a variety of genome-based resources and approaches have simplified and accelerated the discovery of Drosophila cell death genes that share sequence similarity to cell death genes and cell death domain-containing gene products in other organisms.


    Drosophila Genome Projects
 TOP
 ABSTRACT
 INTRODUCTION
 PCD Function and Dysfunction
 Evolutionary Conservation of...
 PCD in Drosophila
 Genetic Approaches to...
 Drosophila Genome Projects
 Genomic Approaches to...
 Gene Expression Profiling of...
 Concluding Remarks
 REFERENCES
 
D. melanogaster was chosen in 1990 to be one of the model organisms studied as part of the Human Genome Project (2). Drosophila Genome Projects, initiated subsequently in Europe (European Drosophila Genome Project, or EDGP, http://edgp.ebi.ac.uk), the United States (Berkeley Drosophila Genome Project, or BDGP, http://www.fruitfly.org/), and Canada, are comprised of several different genomics components, some of which are still ongoing, and include the following: 1) construction of a bacterial artificial chromosome (BAC) physical map (54, 76); 2) sequencing of expressed sequence tags (ESTs) from cDNA clones derived from multiple tissues (113); 3) collection and sequencing of a set of nonredundant full-length cDNA clones, the Drosophila Gene Collection (113); 4) production of gene disruptions and controlled misexpression using P element-mediated mutagenesis (111, 122, 123); and 5) sequencing and biological annotation of the ~120 megabase euchromatic genome in collaboration with Celera Genomics (2). The EST project involved the generation of 5' ESTs from almost 80,000 cDNAs derived from six different cDNA libraries. Subsequently, the ESTs were clustered by sequence comparisons and 3' EST sequencing to yield a set of 5,849 unique clones, representing over 40% of the 13,601 predicted genes (113). Further annotation (38) and additional EST sequencing from tissue-specific cDNA libraries (4, 99; Gorski S and Marra M, unpublished observations) identified additional expressed sequences not contained in the original predicted gene set, indicating that the total number of genes is likely greater than originally predicted. Ongoing projects at the BDGP include additional cDNA sequencing, along with biological annotation and high-quality sequence " finishing" of the euchromatic genome. A new database, Gadfly (Genome Annotation Database of Drosophila), was developed by the BDGP-FlyBase Informatics group to store data from the various components of the Drosophila Genome project, and to make the data readily accessible to the research community. FlyBase is a comprehensive database that also includes, among other items, summaries of molecular, genetic, and publication data relevant to all Drosophila genes (127; http://flybase.bio.indiana.edu/).


    Genomic Approaches to Identification of Drosophila PCD Genes
 TOP
 ABSTRACT
 INTRODUCTION
 PCD Function and Dysfunction
 Evolutionary Conservation of...
 PCD in Drosophila
 Genetic Approaches to...
 Drosophila Genome Projects
 Genomic Approaches to...
 Gene Expression Profiling of...
 Concluding Remarks
 REFERENCES
 
Fly genomics projects have accelerated substantially the recovery of Drosophila cell death genes. For example, at least seven functional caspases, the main effectors of apoptosis, have been described in Drosophila. With the exception of Dcp-1 mentioned above, the other six Drosophila caspases were identified initially through database searches of either Drosophila EST or genomic DNA sequence. Two Drosophila caspases, Dronc and Decay, were identified in the EST database, while four others, Dredd, Strica/Dream, Damm, and Drice, were all identified initially by searches of genomic DNA sequence (17, 2628, 33, 48). Caspases are produced as precursor procaspases that are activated during cell death by proteolytic processing (reviewed in Ref. 68). Based on the length of their prodomains, caspases can be divided into upstream or initiator (long prodomain) caspases and downstream or effector (short prodomain) caspases. Dredd, Dronc, and Strica fall into the initiator class with long prodomains containing protein-protein interaction motifs. Dredd contains some similarity to two death effector domains (DEDs), Dronc contains similarity to a caspase recruitment domain (CARD), and Strica contains a Ser/Thr-rich domain not described in any other caspase (17, 26, 28). The effector caspase Damm has a caspase domain similar to Strica, whereas the other three effectors, Dcp-1, Drice, and Decay, are most similar to mammalian caspase-3 (69) and can cleave some of the same substrates (in 69).

Drosophila genomics resources were also instrumental in the discovery of molecules that function to activate initiator caspases, a key step in cell death. In mammals, this event is mediated by the prodeath adapter protein FADD (19) and the apoptosis-protease-activating factor, Apaf-1 (reviewed in Ref. 14; 155). A Drosophila homolog of FADD, dFADD, was identified by sequence similarity to a Drosophila EST and found to physically interact with and activate the procaspase Dredd (55). In mammals, FADD links caspases to the cytoplasmic tail of transmembrane death receptors of the tumor necrosis factor (TNF) receptor superfamily (reviewed in Ref. 6), but a TNF receptor functioning in Drosophila cell death has not yet been described. A Drosophila apoptosis-protease-activating factor (Apaf-1)-like protein, Ark/ Hac-1/dApaf-1, identified by searches of Drosophila genomic DNA sequence, functions like its mammalian and C. elegans counterparts to induce cell death (63, 110, 154; reviewed in Ref. 143). Ark contains an NH2-terminal CARD-like domain that is required for its interaction with the upstream caspase, Dredd (110). As in mammals, a COOH-terminal WD domain in Ark can bind cytochrome c (63, 110), and there is some evidence that this interaction can trigger caspase activation (63). Loss-of-function mutations indicate a role for ark in PCD during development, and expression studies indicate that its transcription can be induced by exogenous sources such as X-ray and ultraviolet (UV) irradiation, properties which suggest it functions in the DNA damage response pathway (110, 154).

Other important regulators of cell death, similar to mammalian survivin (67, 74), Bcl-2 family members (reviewed in Ref. 105), and p53 (reviewed in Ref. 81), were discovered recently in Drosophila due in part to the availability of the Drosophila EST and genomic sequence. A negative regulator of cell death in mammals, survivin is a member of the IAP family. Although most IAP family members, including Drosophila Diap1 and Diap2 mentioned above, contain several BIR (baculovirus IAP repeat) domains and a RING domain, survivin contains only a single BIR domain (74). Similarly, the Drosophila Deterin protein contains a single BIR domain; it has been shown to suppress cell death induced by reaper or cytotoxicants in cultured cells (60). The Bcl-2 family of proteins, consisting of both positive and negative regulators of PCD in mammals, has at least two conserved relatives in Drosophila: dBorg-1/Drob-1/Debcl/DBok and dBorg-2/Buffy. Functional studies indicate that dBorg-1 acts primarily in a prodeath manner in vivo and in insect and human cultured cells (11, 23, 56, 152). Ectopic expression of dBorg-1 in the developing eye appears to result in low levels of ectopic cell death that are dramatically potentiated by UV irradiation, indicating that dBorg-1 may be activated by the DNA damage response pathway (11). A gene known to function in that pathway is the Drosophila homolog of the mammalian tumor suppressor gene p53, Dmp53. Like its mammalian counterpart, inhibition of Dmp53 function makes cells resistant to irradiation-induced cell death, and overexpression of Dmp53 induces cell death (12, 95). These findings further indicate that Drosophila may be a useful model system for understanding not only developmental cell death but also irradiation-induced cell death and p53-mediated PCD signaling pathways.


    Gene Expression Profiling of PCD
 TOP
 ABSTRACT
 INTRODUCTION
 PCD Function and Dysfunction
 Evolutionary Conservation of...
 PCD in Drosophila
 Genetic Approaches to...
 Drosophila Genome Projects
 Genomic Approaches to...
 Gene Expression Profiling of...
 Concluding Remarks
 REFERENCES
 
As summarized above, discovery of Drosophila cell death genes by sequence similarity to known cell death genes and/or cell death domains in other organisms was simplified and made more rapid by the advent of the Drosophila genome projects. Although these genes had been described previously in other organisms, Drosophila provides a powerful in vivo system for further studies of gene function, gene interactions, and elucidation of genetic pathways. But what about new genes? Can the genome sequence and the new genomic methodologies be used to identify novel PCD-related genes? Genome-wide investigations, now possible at the level of both proteins (proteome analysis) and mRNAs (transcriptome analysis), indicate that the answer is likely to be an affirmative one. Here we describe transcriptome or gene expression analysis, an approach utilized in our laboratory (61) and one that is particularly powerful in the context of a fully sequenced genome like that of Drosophila.

Gene expression profiling refers to the identification and quantitation of mRNAs present in a particular cell population. Implicit in this definition is that gene expression profiling is limited to transcriptional events. These are not always an accurate reflection of corresponding protein activity (44), but nevertheless, gene expression profiling is contributing to our understanding of various processes including cell death (10, 21, 25, 32). While clearly not all cell death molecules are regulated transcriptionally, there are multiple examples of cell death molecules that are, at least in some contexts (8, 21, 25, 32, 37, 47, 117). In addition, some cell death molecules are regulated at both transcriptional and posttranslational levels. For example, vertebrate Apaf1 is transcriptionally regulated in p53-mediated neuronal cell death (32) and is also regulated at the protein level by interaction with cytochrome c (156). In Drosophila, the expression of reaper, hid, and grim are all regulated transcriptionally during development (16, 43, 58, 71, 109, 141). Reaper and grim transcripts accumulate specifically in cells destined to die, whereas hid is expressed more broadly, in cells that die and in some cells that live. The expression of ark, dredd, dronc, and diap2 is also regulated transcriptionally, at least in some tissues (17, 58, 59, 71, 154). Given the existence of transcriptional regulation of cell death genes, at least in some contexts, it is likely that comprehensive transcriptome analyses will yield additional PCD genes similarly regulated.

Two methods used for the large-scale profiling of gene expression are microarrays, using either cDNAs (115) or oligonucleotides (i.e., Affymetrix GeneChips; 77), and serial analysis of gene expression (SAGE) (133). Expression profiling with a cDNA microarray involves hybridization of mRNAs, labeled with radioactivity or fluorescence, to a defined set of cDNAs fixed to a solid support, followed by quantitation of mRNA levels based on relative hybridization signals. Oligonucleotide arrays involve hybridization of fluorescently labeled mRNAs to a series of oligonucleotide probes synthesized on a glass substrate, followed by detection and quantitation of the fluorescent signals. An oligonucleotide array representing greater than 13,500 Drosophila sequences is available commercially (GeneChip Drosophila Genome Array from Affymetrix), as are custom-designed arrays representing a subset of Drosophila sequences (e.g., 73). Drosophila cDNA microarrays have been used to confirm gene expression in the testes (4) and to investigate transcriptional changes during mesoderm development (34), aging (157), transcriptional coactivation (35), and metamorphosis (142). In the latter report, the microarray contained more than 4,500 unique Drosophila cDNA EST clones, representing approximately one-third of all predicted Drosophila expressed sequences, along with several controls. During metamorphosis, several larval tissues, including the midgut and salivary gland, undergo steroid hormone-regulated PCD. White et al. (142) found that genes implicated previously in PCD were expressed differentially during metamorphosis but, with respect to PCD, the experimental conditions were not ideal. Approaches focused on monitoring tissue-specific gene expression are underway in our laboratory (Gorski S and Marra M, unpublished observations). These are being performed using the SAGE technology. Essentially a highly efficient EST-like approach, SAGE produces data that are digital and absolute. Possibly the most important property of the technique is that, unlike microarray technologies, it allows the identification of previously undiscovered genes. This, and the limited amount of tissue required for analysis, make SAGE nearly ideal for tissue-specific mRNA expression analysis.

The output of gene expression profiling is generally a long list of differentially expressed genes associated with a particular disease state or biological process. In some ways analogous to the numerous mutants often recovered in a genetic screen, the list of genes must be subjected to secondary screening methods to identify those of interest and prioritize them for further study. This is a major challenge in any gene expression profiling study and the secondary screens employed depend on the specific problems under study. For example, gene expression data can be combined with sequence similarity searches (e.g., for death domains) and with functional data derived from gene knockouts and/or misexpression studies. In addition, gene expression data from multiple sources can be combined. For example, gene expression in predeath Drosophila salivary glands could be compared with that in predeath Drosophila midguts or to that in mammalian cells induced to die by various stimuli. In the context of PCD studies in Drosophila, the most informative approach perhaps will be to combine comprehensive gene expression data with the phenotypic information derived from Drosophila genetic and molecular analyses.


    Concluding Remarks
 TOP
 ABSTRACT
 INTRODUCTION
 PCD Function and Dysfunction
 Evolutionary Conservation of...
 PCD in Drosophila
 Genetic Approaches to...
 Drosophila Genome Projects
 Genomic Approaches to...
 Gene Expression Profiling of...
 Concluding Remarks
 REFERENCES
 
PCD is a complex process involving a multitude of gene products. Here we have discussed the role of many of the central regulators of cell death in Drosophila and presented evidence that they are conserved across evolutionary boundaries. We have reviewed how Drosophila genetics played a key role in cell death gene discovery and how Drosophila genome projects accelerated the discovery of Drosophila homologs of mammalian cell death genes. The conservation of PCD genes in Drosophila provides an important opportunity to study PCD gene function in vivo, using the powerful molecular and genetic tools available in this model system. With the complete Drosophila genome sequence now in hand, along with emerging gene expression and proteomic technologies, we look forward to the challenge of identifying and characterizing novel genes that participate in this fundamental biological process.


    ACKNOWLEDGMENTS
 
We gratefully acknowledge the support of the British Columbia Cancer Agency and the British Columbia Cancer Foundation.

S. Gorski is a Research Fellow of the National Cancer Institute of Canada supported with funds provided by the Terry Fox Run. M. Marra is a Michael Smith Foundation for Health Research Scholar.


    FOOTNOTES
 
Article published online before print. See web site for date of publication (http://physiolgenomics.physiology.org).

Address for reprint requests and other correspondence: S. Gorski, Genome Sequence Centre, British Columbia Cancer Agency, 600 West 10th Ave., Rm. 3427, Vancouver, BC, Canada V5Z 4E6 (E-mail: sgorski{at}bcgsc.bc.ca)

10.1152/physiolgenomics.00114.2001.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 PCD Function and Dysfunction
 Evolutionary Conservation of...
 PCD in Drosophila
 Genetic Approaches to...
 Drosophila Genome Projects
 Genomic Approaches to...
 Gene Expression Profiling of...
 Concluding Remarks
 REFERENCES
 

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