QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 3 August 2006
doi: 10.1242/dev.02495

This Article Summary Figures Only Full Text (PDF) Supplementary Material OA All Versions of this Article: dev.02495v1 133/17/3305    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Muro, I. Articles by Hay, B. A. Search for Related Content PubMed PubMed Citation Articles by Muro, I. Articles by Hay, B. A.

The Drosophila caspase Ice is important for many apoptotic cell deaths and for spermatid individualization, a nonapoptotic process

¹ Division of Biology, MC 156-29, California Institute of Technology, Pasadena, CA 91125, USA.
² Center for Biosystems Research, University of Maryland Biotechnology Institute, College Park, MD 20742, USA.
³ Department of Biology, Kyung Hee Institute of Age-related and Brain Diseases, Kyung Hee University, Seoul, 130-701, Korea.
⁴ Department of Neurology, Brain Research Institute, The David Geffen School of Medicine at UCLA, Los Angeles, CA 90095, USA.

^* Author for correspondence (e-mail: imuro{at}caltech.edu)

Accepted 5 June 2006

	SUMMARY

TOP SUMMARY INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Caspase family proteases play important roles in the regulation of apoptotic cell death. Initiator caspases are activated in response to death stimuli, and they transduce and amplify these signals by cleaving and thereby activating effector caspases. In Drosophila, the initiator caspase Nc (previously Dronc) cleaves and activates two short-prodomain caspases, Dcp-1 and Ice (previously Drice), suggesting these as candidate effectors of Nc killing activity. dcp-1-null mutants are healthy and possess few defects in normally occurring cell death. To explore roles for Ice in cell death, we generated and characterized an Ice null mutant. Animals lacking Ice show a number of defects in cell death, including those that occur during embryonic development, as well as during formation of adult eyes, arista and wings. Ice mutants exhibit subtle defects in the destruction of larval tissues, and do not prevent destruction of salivary glands during metamorphosis. Cells from Ice animals are also markedly resistant to several stresses, including X-irradiation and inhibition of protein synthesis. Mutations in Ice also suppress cell death that is induced by expression of Rpr, Wrinkled (previously Hid) and Grim. These observations demonstrate that Ice plays an important non-redundant role as a cell death effector. Finally, we demonstrate that Ice participates in, but is not absolutely required for, the non-apoptotic process of spermatid differentiation.

Key words: Drosophila, Ice, Apoptosis

	INTRODUCTION

TOP SUMMARY INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Programmed cell death, or apoptosis, is a genetically encoded form of cell elimination that results in the orderly death and phagocytic removal of excess, damaged or dangerous cells during development and in the adult (Baehrecke, 2002; Benedict et al., 2002; Green and Evan, 2002; James and Green, 2004; Opferman and Korsmeyer, 2003). Caspase family proteases are the central executioners for most genetically encoded cell deaths in animals (Degterev et al., 2003). In caspase-dependent death, signals arising from specific cellular compartments, such as the plasma membrane or mitochondria, promote the activation of adaptor proteins that recruit long N-terminal prodomain-containing initiator caspases into macromolecular complexes in which caspase activation occurs. Once activated, initiator caspases can cleave and thereby activate short prodomain effector caspases. These cleave a number of target proteins, bringing about death and ultimately phagocytosis of the cell (Degterev et al., 2003; Hay et al., 2004). The Drosophila genome encodes seven caspases. Three of these, Nc (Dorstyn et al., 1999a), Strica (Doumanis et al., 2001) and Dredd (Chen et al., 1998), contain long prodomains characteristic of initiator caspases. A large body of evidence implicates Nc as an important cell death activator (reviewed by Hay and Guo, 2006).

Nc contains an N-terminal CARD (caspase recruitment domain) motif, as does the mammalian cell death caspase caspase 9, and the C. elegans caspase CED-3. Homotypic interactions between the CARD in caspase 9 and a similar motif in the cytoplasmic adaptor Apaf1, in the presence of cytoplasmic cytochrome c and dATP, results in caspase 9 recruitment into a multimeric complex known as the apoptosome. Caspase activation occurs in the apoptosome and activated caspase 9 then cleaves and activates effector caspases such as caspase 3. CED-3 activation in C. elegans is also mediated by interactions with an Apaf1-like molecule known as CED-4 (reviewed by Yan and Shi, 2005). Drosophila encodes one Apaf1 homolog, Ark (also known as Hac-1 or dAPAF-1) (Kanuka et al., 1999; Rodriguez et al., 1999; Zhou et al., 1999). Ark binds Nc, and decreasing or eliminating ark expression results in defects in cell death that are thought to be Nc-dependent (Kanuka et al., 1999; Rodriguez et al., 1999; Zhou et al., 1999; Igaki et al., 2002; Muro et al., 2002; Zimmermann et al., 2002; Muro et al., 2004; Akdemir et al., 2006; Mills et al., 2006; Srivastava et al., 2006).

Caspase activity is inhibited by members of the inhibitor of apoptosis (IAP) family of proteins through several different mechanisms (reviewed by Clem, 2001; Hay, 2000; Vaux and Silke, 2005; Hay and Guo, 2006). Expression of the Drosophila IAP Thread (Th; previously Diap1) inhibits caspase-dependent cell death (Hay et al., 1995), and is essential for the survival of many otherwise healthy cells (Goyal et al., 2000; Hay et al., 1995; Lisi et al., 2000; Wang et al., 1999). Importantly, the death of healthy cells in response to loss of Th can be inhibited by removal of Ark or Nc (Igaki et al., 2002; Muro et al., 2002; Rodriguez et al., 2002; Zimmermann et al., 2002). These observations suggest that Ark-dependent activation of Nc occurs constitutively, and that Th is required continuously to inhibit this activity and the activity of caspases activated by Nc. In one major pathway, caspase-dependent cell death is initiated by increased expression or release from a sequestering environment of proteins such as Reaper (Rpr) (White et al., 1994), Head involution defective (W; previously Hid) (Grether et al., 1995), Grim (Chen et al., 1996), Sickle (Skl) (Christich et al., 2002; Srinivasula et al., 2002; Wing et al., 2002) and Jafrac2 (Tenev et al., 2002). These proteins disrupt Th-caspase interactions, liberating Nc and Nc target caspases from inhibition by Th, thereby initiating apoptosis.

An important unresolved issue in the context of the above-described pathway is the identity of the effector caspase(s) that cleaves the cellular substrates that actually bring about cell destruction. That such caspases must exist is indicated by the fact that although expression of the baculovirus caspase inhibitor p35 inhibits many cell deaths in Drosophila, at least some of which are known to be Nc-dependent, it does not inhibit Nc (Hay et al., 1994; Hawkins et al., 2000; Meier et al., 2000; Martin and Baehrecke, 2004). Thus, the activation of Nc, in the absence of other p35-sensitive caspases, is insufficient to bring about cell death. The Drosophila genome encodes four short prodomain caspases that are candidate effectors: Damm (Harvey et al., 2001), Decay (Dorstyn et al., 1999b), Dcp-1 (Song et al., 1997) and Ice (Fraser and Evan, 1997). Little is known about the roles that Damm and Decay play in cell death. By contrast, Dcp-1 and Ice share a high degree of homology with each other, and are most homologous among the Drosophila caspases to the mammalian death effector caspases caspase 3, caspase 6 and caspase 7. In addition, they are both expressed broadly throughout development (Arbeitman et al., 2002), inhibited by p35 (Hawkins et al., 1999; Wang et al., 1999), and cleaved and activated by Nc (Hawkins et al., 2000; Meier et al., 2000; Muro et al., 2002). However, although dcp-1-null mutants do show mild defects in starvation-induced cell death during oogenesis (Laundrie et al., 2003), they are otherwise quite healthy. This stands in contrast to Nc mutants, which show many defects in developmental cell death (Chew et al., 2004; Daish et al., 2004; Waldhuber et al., 2005; Xu et al., 2005), and have a very low rate of survival to adulthood (Xu et al., 2005). By contrast, several observations suggest that Ice may play an important role as a cell death effector. First, depletion of Ice from S2 cells inhibits apoptotic events in response to a variety of stimuli (Fraser et al., 1997; Muro et al., 2002; Muro et al., 2004). In addition, antibodies that recognize the Nc-cleaved, and therefore activated, version of Ice, label dying cells during development (Yoo et al., 2002; Yu et al., 2002), as well as cells exposed to a variety of apoptotic stimuli (cf. Huh et al., 2004a; Perez-Garijo et al., 2004).

To explore roles of Ice as a cell death effector, we generated a null mutation for the Ice locus, Ice¹. Ice¹ animals show defects in some, but not all apoptotic cell deaths, Ice¹ animals also show defects in a non-apoptotic process: spermatid individualization.

	MATERIALS AND METHODS

TOP SUMMARY INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Isolation of a Ice null allele and generation of a rescue construct
We used transposon mobilization to generate the Ice¹ excision allele from The P transposon line EP^GE28489 (Genexel, Seoul, Korea). EP^GE28489 is inserted at base 1711 with respect to the start of the Ice transcription unit. A genomic rescue fragment containing 2.4 kb of DNA flanking the Ice gene was generated using PCR from genomic DNA with the primers 5' CGC CTC GAG CCT CTT TGA GAG TGT GAC CGT GCA TAA and 5' CGC TCT AGA ACG ATC AGG GTC AGC CAA TGG CTG GAC. Products were digested with XhoI and XbaI and cloned into the P element transformation vector pCasper4 (Thummel and Pirotta, 1992). UAS-ark-RNAi and UAS-Ice-RNAi constructs were made by introducing 21 bp sequences complementary to the ark- or Ice-coding regions into one strand of the stem region of a 70 nucleotide fragment encoding the Drosophila microRNA mir-6.1. These fragments were cloned into the UASt vector (Brand and Perrimon, 1993). Details of these constructs will be provided elsewhere (C.H.C. and B.A.H., unpublished).

Fly stocks and genetics
All crosses and stocks were maintained at 25°C. The following fly stocks were used: GMR-rpr and GMR-hid (Hay et al., 1995), GMR-grim and GMR-Nc (Hawkins et al., 2000), GMR-p35 (Hay et al., 1994), GMR-GAL4-UAS-Th-RNAi (Huh et al., 2004a), en-GAL4, UAS-GFP (Kimura et al., 2004), dcp-1^Prev1 (Laundrie et al., 2003) and P[sli-1.0-lacZ] (Wharton and Crews, 1993).

Fly viability determination
Third instar larvae were collected from each genotype and put into vials with fresh food at 25°C. They were followed for 7 days and viability was determined as the fraction of eclosed adults compared with the total number of third instar larvae. At least 200 third instar larvae were scored for each genotype.

Western blotting, Immunohistochemistry and TEM
For Western blotting, adult flies were lysed and processed as described previously (Huh et al., 2004b). Blots were probed with rabbit anti-full-length Ice sera (1:1000) and anti-tubulin (Sigma) at a dilution of 1:500. Embryos and wing discs were fixed and processed for anti-caspase and TUNEL staining as described (Yoo et al., 2002). Anti-active Ice (Yoo et al., 2002) and CM1 (Cell Signaling #9661) were used at 1:50, and the secondary anti-rabbit antibody (Molecular Probes) at 1:500. Similar conditions were used for adult testis staining. Third instar larvae were exposed to 2000 rads of X-irradiation using a Torex 120D X-ray inspection system (Astrophysics Research, Long Beach, CA). They were processed 4 hours after irradiation. Pupal eyes were fixed and stained with anti-Dlg (Developmental Studies Hybridoma Bank) at 1:300. The midline glia were visualized by anti-ß-gal (Sigma Chemical Corp.) immunohistochemistry. TEM of adult testis was carried out as described previously (Huh et al., 2004b).

Pupal histology
Flies were maintained at 25°C and aged to 24 hours after puparium formation. For histology, whole pupae were fixed and processed as described previously (Martin and Baehrecke, 2004) for paraffin sectioning and light microscopy. The number of abnormal masses in the head and in the wing and leg discs was counted from every fifth histological section of the pupa. Sections were counted throughout the entire pupa and, owing to the size of the pupa, on average eight sections were counted for each pupa. The average number of masses per section was determined for each pupa and at least five different pupae were counted per genotype to determine the average number of masses per section for each genotype.

Ex vivo hemocyte analyses
Hemocyte analysis was performed essentially as previously described (Chew et al., 2004). Hemocytes were plated in Schneider's media with 10% FBS. Hemocytes were seeded for 1 hour and then washed with media and treated with 20 µg/ml of cycloheximide. Four hours after treatment, cells were visualized for cell membrane blebbing. The fraction of blebbing cells was used as a measure of apoptosis.

	RESULTS

TOP SUMMARY INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Generation of Ice¹, a Ice null allele
To generate a Ice mutant, we mobilized the P transposon EP^GE28489, which is located within the 3' UTR of the Ice transcription unit. One excision line, designated Ice¹, lacks the entire Ice-coding region, but has no effect on the structure of the surrounding genes (Fig. 1A). We generated Ice¹ flies that also carried a 2.4 kb fragment (Ice^+2.4) encompassing the Ice transcription unit (Ice^+2.4; Ice¹ flies). As expected, Ice¹ animals lacked detectable Ice protein, while Ice^+2.4; Ice¹ flies expressed Ice at wild-type levels, as did flies that lacked dcp-1 (Fig. 1B). In addition, all the phenotypes detailed below for Ice¹ mutants were suppressed in the presence of Ice^+2.4, demonstrating that the Ice locus, and only the Ice locus, is altered in the Ice¹ flies.

Ice¹ flies have decreased viability and pupae contain abnormal masses
Ice¹ embryos and larvae showed normal levels of survival. However most animals (80%) died following puparium formation (Fig. 2A). Ice¹ pupal lethality is due to loss of Ice as it was suppressed in the presence of the Ice^+2.4 rescue DNA. Survival during embryonic and larval stages, and pupal lethality, are not due to perdurance of maternally deposited protein, as a similar survival profile was observed for Ice¹ animals derived from homozygous Ice¹ mothers (data not shown). Flies lacking dcp-1 (dcp-1^Prev1) (Laundrie et al., 2003) showed a modest decrease in survival compared with wild-type animals, whereas animals lacking Ice that were heterozygous for dcp-1^Prev1 eclosed only rarely (1%) (data not shown). Animals lacking Ice and dcp-1 (dcp-1^Prev1; Ice¹) all died prior to, or during, early pupal stages. Together, these observations suggest that Ice and dcp-1 both contribute to pupal development, perhaps playing partially redundant roles.

View larger version (19K): [in this window] [in a new window]   Fig. 1. Ice1 is a null allele of Ice. (A) Genomic organization of the Ice region. The P transposon element EPGE28489 is inserted at base 1711 in the Ice transcription unit. The Ice1 excision line removes the entire Ice-coding region, fusing small remaining fragments of the Ice 5'- and 3'-UTR sequences. A 1.5 kb fragment of EPGE28489 also remains. The gray region indicates the Ice-coding region; the black boxes indicate the UTR sequences of Ice and of two neighboring genes. Locations of the EPGE28489 element, the Ice1 deletion and the Ice genomic rescue construct (Ice+2.4) are indicated to scale. (B) Western blot of adults of various genotypes probed with anti-Ice antibodies. The genotypes are wild type, Ice1, Ice1; Ice+2.4 and dcp-1Prev1 (Dcp-1), a null allele of dcp-1.

Ice¹ adults display a number of defects
Homozygous Ice¹ adults showed several phenotypes suggestive of decreased cell death during development. The arista is a feather-like structure derived from the third antennal segment. It consists of a central core of epidermal cells and a series of lateral branches. The number of lateral branches is regulated by apoptotic cell death (Cullen and McCall, 2004; He and Adler, 2001). Thus, adults homozygous for the tissue-specific Th loss-of-function allele th¹ lack essentially all lateral branches, a phenotype that is suppressed by mutations in ark (Cullen and McCall, 2004). By contrast, flies mutant for hid, an inhibitor of Th, have extra lateral branches (Cullen and McCall, 2004). As shown in Fig. 3, the arista of Ice¹ flies (Fig. 3B) has a much thicker central shaft and many more lateral branches than does the arista from wild-type flies (Fig. 3A). In addition, removal of Ice suppressed the loss of lateral shafts seen in th¹ flies (Fig. 3C,D). Finally, although the arista appeared wild type in dcp-1^Prev1 flies (data not shown), adult Ice¹ flies heterozygous for dcp-1^Prev1 had an increased number of lateral branches when compared with Ice¹ alone (Fig. 3E).

The adult male terminalia, which derives from the genital imaginal disc, undergoes a 360° clockwise rotation during development (Gleichauf, 1936; Adam et al., 2003). Mutations in hid (Abbott and Lengyel, 1991), as well as overexpression of the baculovirus caspase inhibitor p35 (Macias et al., 2004), give rise to adults in which complete rotation fails to occur. This results in adults in which the genitalia and analia are mislocalized with respect to the abdomen. Approximately 50% of Ice¹ males showed a similar phenotype (Fig. 3F,G), as did all Ice¹ males heterozygous for dcp-1^Prev1 (n=30). Finally, we noted that more than 50% of Ice¹ adults had an open `scar' of varying severity along the dorsal midline (Fig. 3I; compare with wild type, Fig. 3H). The tissue appeared thin, white and fragile, with some flies possessing holes or tears in this area that were associated with leakage of hemolymph. The integument of the adult abdomen is derived from four groups of abdominal histoblast nest cells that are present at characteristic positions in each segment of the larval abdomen. Following pupariation, histoblasts replace larval epidermal cells, which undergo apoptosis and are phagocytosed by hemocytes (Madhavan and Madhavan, 1980). We speculate that loss of Ice compromises some aspect of the process of cell replacement, histoblast nest fusion or cell differentiation.

In addition to the above-noted defects, Ice¹ flies also displayed several phenotypes, in the wing and eye, which could be directly attributed to defects in cell death. Epidermal cells that make up the adult Drosophila wing undergo death within the first hour after eclosion (Kimura et al., 2004). When this death fails to occur, the wing appears opaque when compared with wild type, and sometimes contains trapped fluid. An opaque wing phenotype has also been reported for hid (Abbott and Lengyel, 1991), ark (Rodriguez et al., 1999) and Nc (Xu et al., 2005) mutants. Cell death in the adult wing can be visualized in living animals that express a nuclear-localized green fluorescent protein. Live cells show GFP fluorescence, while dead cells do not. Importantly, these deaths are inhibited - and thus GFP fluorescence retained -in cells that express baculovirus p35 (Kimura et al., 2004), or that are mutant for Nc (Xu et al., 2005). All Ice¹ flies have opaque wings, which sometimes contain trapped fluid or never fully extend (data not shown). We used en-GAL4 and UAS-GFP (en::GFP) to visualize cell death in wild-type and Ice¹ backgrounds. In wild-type adults less than 1 hour after eclosion, GFP was seen throughout the posterior compartment, the region in which en::GFP is expressed (Fig. 4A). By 2 hours post-eclosion GFP fluorescence was largely absent from posterior compartment (Fig. 4B). By contrast, in Ice¹ animals, GFP fluorescence could be observed in the posterior compartment for greater than 24 hours (Fig. 4C).

View larger version (94K): [in this window] [in a new window]   Fig. 2. Ice is required for normal pupal development. (A) The percent viability to adulthood for wandering third instar larvae of several different genotypes is shown: wild type, Ice1, Ice+2.4; Ice1, dcp-1Prev1 and dcp-1Prev1; Ice1. (B-G) Paraffin sections of pupae aged 24 hours after puparium formation (apf). (B) In control pupae, salivary glands and larval muscle have both disappeared and the larval mid-gut has degenerated, as previously described (Lee and Baehrecke, 2001). (C) Seventy-three percent of Ice1 pupae develop normally. By 24 hours apf, only a few remaining small degraded fragments of salivary glands and larval muscles are observed and the mid-gut is normal. (D) Twenty-seven percent of Ice1 pupae were arrested in development. Of those that arrested, one-third did not undergo head eversion and arrested prior to the commencement of salivary gland cell death. The remaining pupae appear to have arrested 1 or 2 hours after head eversion, and thus the salivary glands and larval muscle are not degraded, and the larval gut has failed to condense properly. Ice1 pupae have abnormal masses in the head (C,E), wing and leg discs (C,F), and abdomen (G). Arrows indicate abnormal masses; arrowheads indicate muscle; red circles are placed around salivary glands, salivary gland fragments and regions where degraded salivary glands would be if they had failed to die. Animals homozygous for Ice1 lack zygotic but not maternal Ice. Scale bars: 200 µm in B-D; 40 µm in E-G.

Ice is important for cell death during embryogenesis
Cell death is extensive during embryogenesis and is regulated by the RHG family of proteins (White et al., 1994; Grether et al., 1995; Zhou et al., 1995; Huh and Hay, 2002), Nc (Quinn et al., 2000; Chew et al., 2004; Daish et al., 2004; Xu et al., 2005) and Th (Goyal et al., 2000; Lisi et al., 2000; Wang et al., 1999). These deaths are also sensitive to the expression of baculovirus p35, suggesting important roles for one or more effector caspases (Hay et al., 1994; Zhou et al., 1997). To explore roles for Ice in normally occurring cell death during embryogenesis, we characterized wild-type embryos and embryos lacking both maternal and zygotic Ice using the TUNEL assay, which labels fragmented DNA within dying cells. Dying cells were present throughout the wild-type stage 14 embryo, and were particularly prevalent in the head region (Fig. 5A). Ice¹ embryos showed a modest reduction in TUNEL labeling (Fig. 5B) that was restored to wild-type levels in the presence of the genomic rescue construct (Fig. 5C).

View larger version (81K): [in this window] [in a new window]   Fig. 3. Adult Ice1 show multiple defects suggestive of defects in developmental cell death. (A-E) SEM images of arista of different genotypes. (A) Wild type, (B) Ice1, (C) th1, (D) th1, Ice1 and (E) dcp-1Prev1/+; Ice1. (F) Ventral view of the abdomen of a wild-type male, The genitalia (G) and analia (A) are marked. (G) Similar view of the abdomen of a Ice1 adult. Genitalia are markedly rotated. (H) Dorsal view of the abdomen of a wild-type adult male. (I) Similar view of the abdomen of a Ice1 adult male, showing a pale line that runs along the dorsal abdominal midline. Animals homozygous for Ice1 lack zygotic but not maternal Ice.

View larger version (145K): [in this window] [in a new window]   Fig. 4. Ice is required for normally occurring cell death in the adult wing and pupal retina. (A) en::GFP expression in the wing of a wild-type adult (eclosed for less than 1 hour). (B) en::GFP expression in the wing of a wild-type adult (eclosed for 2 hours). (C) en::GFP expression in the wing of a Ice1 adult (eclosed for 24 hours). (D,E) Forty-hour pupal eyes stained with anti-Dlg. (D) Wild type; (E) Ice1. The area in which the number of interommatidial cells was counted for each genotype is indicated in D by the broken line. Several regions with extra cells are indicated with arrows in the mutant (E). Animals homozygous for Ice1 lack zygotic but not maternal Ice.

View larger version (56K): [in this window] [in a new window]   Fig. 5. Ice is required for some normally occurring cell death during embryogenesis. (A-C) TUNEL stains of stage 14 embryos of different genotypes. (A) Wild type; (B) Ice1; (C) Ice+2.4; Ice1. (D-F) Stage 17 embryos of different genotypes, each of which carries the P[sli-1.0-lacZ] enhancer trap line, which is expressed in midline glia. (D) Wild type, (E) Ice1 and (F) Ice+2.4; Ice1. Embryos homozygous for Ice1 lack maternal and zygotic Ice.

View larger version (61K): [in this window] [in a new window]   Fig. 6. Ice is required for cell death induced by X-irradiation and inhibition of protein synthesis. (A-F) TUNEL-stained third instar wing discs of various genotypes. (A) Untreated wild type; (B) X-irradiated wild type; (C) untreated Ice1; (D) X-irradiated Ice1; (E) X-irradiated Ice+2.4; Ice1; (F) X-irradiated dcp-1Prev1. (G) Wild-type hemocytes exposed to cyclohexamide died within 4 hours. Removal of zygotic Ice (Ice1)prevented almost all this death, which was restored in the presence of a wild-type Ice rescue transgene (Ice1,Ice+2.4). Animals homozygous for Ice1 lack zygotic but not maternal Ice. Animals homozygous for dcp-1Prev1 lack both maternal and zygotic dcp-1.

View larger version (82K): [in this window] [in a new window]   Fig. 7. Ice is important for cell death induced by rpr, hid, grim and loss of Th, but not Nc overexpression. (A-J) Adult eyes of various genotypes: (A) GMR-rpr; (B) GMR-hid; (C) GMR-grim; (D) GMR-Th-RNAi; (E) GMR-Nc; (F) GMR-rpr; Ice1; (G) GMR-hid;Ice1; (H) GMR-grim; Ice1; (I) GMR-Th-RNAi; Ice1; (J) GMR-Nc; Ice1. (K-M) Tangential sections through adult eyes of various genotypes: (K) wild type; (L) GMR-Nc; (M) GMR-Nc, Ice1. Pigment cells (red) and photoreceptors (blue) form a regular array in the wild-type adult eye (K). These cells are largely absent in the eyes of flies expressing Nc, in the presence (L) or absence (M) of Ice. Animals homozygous for Ice1 lack zygotic but not maternal Ice.

Cleavage-specific anti-Ice antibodies function as accurate reporters of Ice activation in wing disc cells induced to die in response to X-irradiation
We previously generated a polyclonal rabbit antiserum containing antibodies directed against the C terminus of the Ice p20 fragment (QRSQTETD) that is generated following cleavage by Nc (Yoo et al., 2002). These antibodies (anti-active-Ice) recognize versions of Ice that have been cleaved, and label dying cells in several different contexts (Yoo et al., 2002). Cleaved versions of Ice, but not Dcp-1, and dying cells in Drosophila are also recognized by antibodies present in a polyclonal rabbit antiserum (CM1) raised against a related peptide, that corresponds to the C-terminus of the mammalian caspase 3-cleaved p20 fragment (Yu et al., 2002).

To explore the origins of anti-active Ice staining in dying cells, we exposed wing discs from larvae of several different genetic backgrounds to X-irradiation and stained for TUNEL, anti-active Ice and CM1. Un-irradiated wild-type wing discs showed only occasional TUNEL, anti-active Ice or CM1-positive cells (data not shown). By contrast, wild-type discs exposed to X-irradiation showed high levels of TUNEL staining (Fig. 9A,C,D,F), and of active-Ice (Fig. 9B,C) and CM1 (Fig. 9E,F) staining. Wing discs from irradiated Ice¹ animals showed essentially no TUNEL (Fig. 9G,I,J,L), anti-active Ice (Fig. 9H,I) or CM1 (Fig. 9K,L) staining. As an internal control, and to demonstrate that the loss of Ice was responsible for the lack of TUNEL and anti-active Ice and CM1 staining, we also examined irradiated wing discs from animals expressing a microRNA targeting Ice (en-Ice-RNAi) in the posterior wing compartment. Consistent with the results obtained with the Ice¹ mutant, TUNEL (Fig. 9M,O,P,R), anti-active Ice (Fig. 9N,O) and CM1 (Fig. 9Q,R) staining were each largely absent, specifically in the posterior wing compartment. Together, these results argue that the anti-active Ice and CM1 staining observed in wing disc cells stimulated to die is Ice dependent. Although we cannot rule out the possibility that the epitope recognized in dying cells is a product of Ice-dependent cleavage of substrates, the most parsimonious explanation is that this staining reflects recognition of cleaved Ice itself.

View larger version (171K): [in this window] [in a new window]   Fig. 8. Ice is important for spermatid individualization. (A-C) EM sections of elongated cysts of different genotypes: (A) wild type; (B) Ice1; (C) Ice1. Scale bars: 1 µm in A,B; 2.4 µm in C. Arrows in C indicate some regions within cysts in which individualization has failed. Animals homozygous for Ice1 lack maternal and zygotic Ice.

Elongated wild-type spermatids undergoing individualization also stain intensely with anti-active Ice and CM1 (Arama et al., 2003; Huh et al., 2004b) (Fig. 9W,Y). The observations presented in Fig. 8 demonstrate that Ice participates in normal spermatogenesis. To test the hypothesis that this staining represents the presence of activated Ice, we stained testes from Ice¹ with anti-active Ice and CM1 sera. Both antisera (Ice, Fig. 9X; CM1, Fig. 9Z) labeled individualizing spermatids from mutant testis to the same extent as in wild type (Fig. 9W,Y), demonstrating that although these antibodies recognize dying cells very specifically in some contexts, they cannot be (primarily) labeling cleaved Ice in individualizing spermatids. Instead, this intense and specific staining must reflect the presence of an unknown individualization-specific epitope.

	DISCUSSION

TOP SUMMARY INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
In mammals the effector caspases, caspase 3 and caspase 7, which are most closely related to Ice and Dcp-1, show a high level of functional redundancy or compensation. Thus, single mutants for either caspase are viable and healthy, while double mutants die before birth with many defects in apoptosis (Lakhani et al., 2006). By contrast, while loss of dcp-1 has little effect on viability or apoptosis in most Drosophila tissues, loss of Ice results in many phenotypes attributable to, or suggestive of, defects in developmental or stress-induced apoptosis, indicating a nonredundant role for this caspase. Related phenotypes have recently been reported for flies carrying a missense mutation in Ice (Xu et al., 2006). All of the deaths we identify as being Ice dependent are also Nc dependent. However, our observations do not directly address the issue of whether the requirement we observe for Ice is achieved only through activation by Nc, or whether it also involves the activity of other proteases, such as Damm, Decay and Strica.

Only 20% of Ice¹ third instar larvae survived to adulthood, with the rest dying during pupal development. Cell death plays a major role in pupal development, removing many larval structures, including gut, salivary glands, epidermis, muscle and neurons. Ice cannot be absolutely required for these deaths as a significant fraction of Ice¹ animals survive to adulthood. However, defects suggestive of compromised cell death, such as persistent larval muscle and immature gut were often observed in Ice¹ pupae. These were never seen in Ice¹/+ pupae. We also observed large numbers of abnormal masses in Ice¹ pupae. Similar masses were seen only very rarely in Ice¹/+ animals. Interestingly, the frequency of these masses was increased in Ice¹ animals that had been exposed to X-irradiation as first instar larvae. Although the origin of these structures is unknown, we speculate that they may arise as a consequence of defective cell death signaling. For example, in the larval wing disc, cells that are stressed, but prevented from dying, send signals to neighbors that promote their proliferation (Huh et al., 2004a; Perez-Garijo et al., 2004; Ryoo et al., 2004). Perhaps the masses represent cells or populations of cells that have failed to die and/or that have responded in some way to the prolonged presence of signals generated by undead or slowly dying cells that lacked Ice. If such signals exist in undead pupal cells, this may drive increased proliferation or other cell fate changes in surrounding cells that manifest themselves by the presence of these masses. A characterization of the origins and cell types that make up the masses will be required to address this hypothesis.

Although Ice has non-redundant roles as a death effector, several observations suggest that effector caspase redundancy and/or compensation does play important roles in Drosophila. As noted above, whereas most animals lacking Ice die during pupal stages, about 20% survive as fertile adults. This, in conjunction with the recent observation that ark mutants are completely pupal lethal (Akdemir et al., 2006; Mills et al., 2006; Srivastava et al., 2006), suggests that other (presumably caspase-dependent) pathways are important for bringing about cell death and phagocytosis of corpses. The fact that heterozygosity for dcp-1^Prev1 resulted in a further reduction in the survival of Ice¹ pupae suggests that dcp-1 might be a component of such a pathway. A similar conclusion is suggested by several observations of embryos and the larval salivary gland. We observed some decrease in cell death in embryos that lacked maternal and zygotic Ice, but many TUNEL-positive cells were still present. By contrast, embryos that lacked maternal and zygotic Nc showed very few TUNEL-positive cells (Xu et al., 2005). The stronger phenotype observed in embryos that lack Nc may reflect the fact that Nc itself cleaves targets that cooperate with Ice activity. Alternatively, and/or in addition, other Nc-dependent effector caspases such as Dcp-1 may be important. The fact that p35 is a potent inhibitor of cell death in the embryo is consistent with this latter possibility (Hay et al., 1994; Zhou et al., 1997). So, also, is our observation that antiactive Ice and CM1 recognize many cells in embryos lacking Ice (Fig. 7), but not embryos lacking rpr, hid and grim, or Nc (Yoo et al., 2002; Xu et al., 2005). The basis for the residual anti-active Ice and CM1 staining in Ice¹ embryos is presently unknown. However, the fact that it requires upstream death activators suggests it may represent, in part, recognition of one or more cleaved caspase substrates with a sequence similar to that of cleaved Ice. Again, one likely candidate is Dcp-1. Embryos lacking dcp-1 contain many antiactive Ice- and CM1-positive cells (data not shown). However, this is not unexpected as cleaved Ice also contributes to this staining. Mutants that remove both maternal and zygotic Ice and dcp-1 will be needed to address the nature of this epitope. The above hypothesis is not inconsistent with our observation that anti-active Ice and CM1 act as accurate reporters of Ice activation in response to X-irradiation in wing discs. The irradiated wing disc may simply lack the unknown epitope-carrying protein(s) present in the embryo. Alternatively, X-irradiation may lead to the activation of a caspase cascade that does not promote the cleavage of the relevant protein(s), an issue that requires further exploration.

View larger version (100K): [in this window] [in a new window]   Fig. 9. Antibodies that recognize cleaved versions of Ice are accurate reporters of Ice activation in irradiated wing discs, but not in embryos and spermatids. (A-R) X-irradiated wing discs of various genotypes stained for TUNEL (A,C,D,F,G,I,J,L,M,O,P,R), anti-active Ice (B,C,H,I,N,O) or CM1 (E,F,K,L,Q,R). Genotypes are as indicated in the figure. Wild-type (S,U) or Ice1 (T,V) embryos stained with anti-active Ice (S,T) or CM1 (U,V). (W-Z) Wild-type (W,Y) or Ice1 testis stained with anti-active Ice (W,X) or CM1 (Y,Z).

Finally, we identified a non-apoptotic role for Ice in spermatid individualization. Testes from Ice¹ animals consistently showed defects in spermatid individualization similar to those seen when ark, Nc or hid activity was decreased (Huh et al., 2004b; Arama et al., 2005), with some spermatids failing to undergo individualization. However, other spermatids developed normally, and males lacking Ice are fertile. In the present work, we show that that CM1 and anti-active Ice staining of individualizing spermatids does not reflect the presence of active Ice, as high levels of spermatid-specific staining are observed in the complete absence of this protein. This staining is also not eliminated when Nc is downregulated through expression of a dominant-negative protein, or eliminated through mutation (Huh et al., 2004b; Arama et al., 2005). Wild-type levels of staining are also present in males lacking dcp-1 (data not shown), hid, fadd or dredd (Huh et al., 2004b). Therefore, although we cannot exclude the possibility that spermatid anti-active Ice staining reflects the activity of an uncharacterized caspase, at this point a relationship between this staining and caspase activity remains to be demonstrated. It will be interesting to determine if the cleavage-specific caspase 3 immunostaining observed in mammalian spermatids has a similar unexpected origin (cf. Kissel et al., 2005). That said, the epitope recognized by these sera serves as an excellent marker for the process of individualization, becoming apparent throughout spermatids just as the process initiates. Characterization of loci such as Iceless, mutation of which result in elimination of this staining (Huh et al., 2004b), may provide insight into the identity of the target recognized and its role in individualization.

Supplementary material
Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/133/17/3305/DC1

	ACKNOWLEDGMENTS

 
Financial support was provided by NIH grant GM057422 to B.A.H., by NIH grant GM59136 to E.H.B., and by NIH grants NS042580 and NS048396 to M.G.

	REFERENCES

TOP SUMMARY INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

Abbott, M. K. and Lengyel, J. (1991). Embryonic head involution and rotation of male terminalia require the Drosophila locus head involution defective.Genetics 129,783 -789.[Abstract]

Adam, G., Perrimon, N. and Noselli, S. (2003). The retinoic-like juvenile hormone controls the looping of left-right asymmetric organs in Drosophila. Development 130,2397 -2406.[Abstract/Free Full Text]

Akdemir, F., Farkas, R., Chen, P., Juhasz, G., Medved'ova, L., Sass, M., Wang, L., Wang, X., Chittaranjan, S., Gorski, S. M. et al. (2006). Autophagy occurs upstream or parallel to the apoptosome during histolytic cell death. Development 133,1457 -1465.[Abstract/Free Full Text]

Arama, E., Agapite, J. and Steller, H. (2003). Caspase activity and a specific cytochrome c are required for sperm differentiation in Drosophila. Dev. Cell 4, 687-697.[CrossRef][Medline]

Arama, E., Bader, M., Srivastava, M., Bergmann, A. and Steller, H. (2005). The two Drosophila cytochrome C proteins can function in both respiration and caspase activation. EMBO J. 25,232 -243.[CrossRef][Medline]

Arbeitman, M. N., Furlong, E. E., Imam, F., Johnson, E., Null, B. H., Baker, B. S., Krasnow, M. A., Scott, M. P., Davis, R. W. and White, K. P. (2002). Gene expression during the lifecycle of Drosophila melanogaster. Science 297,2270 -2275.[Abstract/Free Full Text]

Baehrecke, E. H. (2002). How death shapes life during development. Nat. Rev. Mol. Cell Biol. 3, 779-787.[CrossRef][Medline]

Baehrecke, E. H. (2003). Autophagic programmed cell death in Drosophila. Cell Death Differ. 10,940 -945.[CrossRef]