Drosophila Caspases Involved in Developmentally Regulated Programmed Cell Death of Peptidergic Neurons during Early Metamorphosis

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1 RESEARCH ARTICLE Drosophila Caspases Involved in Developmentally Regulated Programmed Cell Death of Peptidergic Neurons during Early Metamorphosis Gyunghee Lee, 1 Zixing Wang, 2 Ritika Sehgal, 1 Chun-Hong Chen*, 3 Keiko Kikuno, 1 Bruce Hay, 3 and Jae H. Park 1,2 * 1 Department of Biochemistry and Cellular and Molecular Biology, University of Tennessee, Knoxville, Tennessee Graduate Program of Genome Science and Technology, University of Tennessee, Knoxville, Tennessee Department of Biology, California Institute of Technology, Pasadena, California ABSTRACT A great number of obsolete larval neurons in the Drosophila central nervous system are eliminated by developmentally programmed cell death (PCD) during early metamorphosis. To elucidate the mechanisms of neuronal PCD occurring during this period, we undertook genetic dissection of seven currently known Drosophila caspases in the PCD of a group of interneurons (vcrz) that produce corazonin (Crz) neuropeptide in the ventral nerve cord. The molecular death program in the vcrz neurons initiates within 1 hour after pupariation, as demonstrated by the cytological signs of cell death and caspase activation. PCD was significantly suppressed in dronc-null mutants, but not in null mutants of either dredd or strica. A double mutation lacking both dronc and strica impaired PCD phenotype more severely than did a dronc mutation alone, but comparably to a triple dredd/strica/dronc mutation, indicating that dronc is a main initiator caspase, while strica plays a minor role that overlaps with dronc s. As for effector caspases, vcrz PCD requires both ice and dcp-1 functions, as they work cooperatively for a timely removal of the vcrz neurons. Interestingly, the activation of the Ice and Dcp-1 is not solely dependent on Dronc and Strica, implying an alternative pathway to activate the effectors. Two remaining effector caspase genes, decay and damm, found no apparent functions in the neuronal PCD, at least during early metamorphosis. Overall, our work revealed that vcrz PCD utilizes dronc, strica, dcp- 1, and ice wherein the activation of Ice and Dcp-1 requires a novel pathway in addition to the initiator caspases. J. Comp. Neurol. 519:34 48, VC 2010 Wiley-Liss, Inc. INDEXING TERMS: corazonin; neuropeptide; peptidergic neurons; Drosophila melanogaster; metamorphosis; apoptosis; caspase Most animal species undergo a juvenile phase prior to reproductively mature adulthood. In particular, holometabolous insects, including fruit flies, exhibit the most dramatic case of metamorphosis during which most larval tissues along with a great number of neurons in the central nervous system (CNS) are removed by programmed cell death (PCD), while adult-specific tissues and neurons are formed de novo from imaginal tissues and postembryonic neuroblasts, respectively (Riddiford, 1993; Truman et al., 1993, 1994). Therefore, timely execution of PCD is one of the most essential cellular processes required for successful metamorphic development. Caspases are the most important executioners for PCD and classified into two subgroups, initiators and effectors, based on their activation mode and prodomain length. VC 2010 Wiley-Liss, Inc. Activation of initiator caspases in response to death signals is a prerequisite for subsequent activation of the effector caspases (Shi, 2002; Hay and Guo, 2006). Currently, there are seven caspases known in Drosophila. Additional supporting information may be found in the online version of this article *Chen-hong Chen present address: Division of Molecular and Genomic Medicine, National Health Research Institutes, 35 Keyan Road, Zhunan Town, Miaoli County 350, Taiwan. Grant sponsor: National Science Foundation; Grant number: IOS and Hunsicker award (to J.H.P.); Grant sponsor: National Institutes of Health; Grant number: GM (to B.H.). *CORRESPONDENCE TO: Jae H. Park, Department of Biochemistry and Cellular and Molecular Biology, University of Tennessee, Knoxville, TN jhpark@utk.edu Received March 9, 2010; Revised May 18, 2010; Accepted August 22, 2010 DOI /cne Published online September 14, 2010 in Wiley Online Library (wileyonlinelibrary.com) 34 The Journal of Comparative Neurology Research in Systems Neuroscience 519:34 48 (2011)

2 Caspases-dependent neuronal cell death Three of them, dronc, dredd, and strica, are considered initiators and the remaining four, dcp-1, ice, damm, and decay, as effectors (for a review, see Kumar et al., 2007). Although most dronc-null mutants die during pupal development, flies lacking both maternal and zygotic contributions of dronc function are embryonic lethal, suggesting that dronc is essential for Drosophila PCD during embryogenesis as well as metamorphosis (Chew et al., 2004; Daish et al., 2004; Waldhuber et al., 2005; Xu et al., 2005). However, lack of dronc and dark function for the removal of the larval midgut implies that this death enzyme works in a tissue-specific manner (Daish et al., 2004; Mills et al., 2006). dronc isalsoknowntofunctioninnonapoptotic processes such as dendritic pruning of the C4da sensory neurons (Huh et al., 2004; Kuo et al., 2006; Williams et al., 2006). Unlike dronc, dredd-null mutants are viable and dredd s primary function is so far limited to an innate immune response (Leulier et al., 2000) and sperm individualization (Huh et al., 2004). Despite its unknown activation mode, strica is considered to play a potential role as an initiator due to its rather long prodomain (Doumanis et al., 2001). Flies bearing a deletion of the strica locus are viable and fertile, and strica s function, cooperatively with dronc, is associated with PCD occurring during oogenesis, suggesting a limited role played by this enzyme for developmental PCD (Baum et al., 2007). Nevertheless, the expression of strica transcripts in the larval salivary glands and midgut indicates PCD-relevant function of this gene in these tissues (Doumanis et al., 2001). Multiple lines of evidence suggest that ice is the most important effector caspase for developmental PCD in Drosophila. ice-null mutants show severe defects in PCD during both embryogenesis and metamorphosis and die mostly at late pupal stages, suggesting that ice-mediated PCD is essential for proper pupal development (Kondo et al., 2006; Muro et al., 2006; Xu et al., 2006). By comparison, dcp-1, a close relative of ice, seems to have a limited proapoptotic function. Flies devoid of dcp-1 develop normally into fertile APF CNS Crz damm dark dcp-1 decay dredd dronc ice IHC PCD VNC strica vcrz WL3 WT Abbreviations After puparium formation Central nervous system Corazonin Death-associated molecule related to Mch2 Drosophila Apaf-1-related killer (Flybase: Ark) Death caspase-1 Death executioner caspase related to Apopain/Yama Death related ced-3/nedd2-like Drosophila Nedd-2 like caspase Interleukin-1b-converting enzyme Immunohistochemistry Programmed cell death Ventral nerve cord Serine/Threonine Rich Caspase (Flybase: dream) Ventral corazonergic neuronal group Wandering third instar larva Wildtype adults with occasional extra bristles and display some defective PCD phenotypes in the female germline and developing eye imaginal discs (Laundrie et al., 2003; Kondo et al., 2006; Mendes et al., 2006). Nevertheless, a double mutant lacking both dcp-1 and ice dies around mid-pupal stages with more severe PCD defects than a single ice-null mutant does, supporting that dcp-1 has both overlapping and nonoverlapping functions with ice for proper metamorphic development (Xu et al., 2006). Expression of cell death in vitro and its endogenous expression was observed in various larval tissues including midgut and salivary glands, suggesting its potential role in developmentally regulated PCD (Dorstyn et al., 1999). However, recent studies showed that decay is unnecessary for PCD of the larval midgut (Denton et al., 2009) and for the normal development into adult, implying that decay is a nonessential caspase for developmental PCD (Kondo et al., 2006). Like decay, damm is capable of inducing cell death ectopically in vitro and in vivo to some extent (Harvey et al., 2001). However, its intrinsic PCD-associated functions are not well understood as a loss-of-function mutation for this gene is currently unavailable. During metamorphosis of insects, the CNS is subjected to an extensive remodeling process wherein the larval neurons face either PCD or altercations in their neural processes (Truman et al., 1993; Weeks, 2003). While recent studies have added significant molecular bases to the latter event (reviewed in Luo and O Leary, 2005; Saxena and Caroni, 2007), understandings of developmentally regulated neuronal PCD in depth have been staggered largely due to the lack of a neuronal system that is identifiable and malleable to genetic and molecular analyses. Our previous study identified eight pairs of corazonin-expressing peptidergic neurons (vcrz), each pair located from the 2nd thoracic to the 6th abdominal neuromere in the ventral nerve cord (VNC). The vcrz neurons undergo PCD shortly after the onset of metamorphosis and the ecdysone signal plays a role as a pivotal endocrine cue that initiates the death program in them (Choi et al., 2006). In the present study we undertook comprehensive genetic dissection of all seven caspases to elucidate their individual as well as cooperative roles in the PCD of vcrz neurons. MATERIALS AND METHODS Fly strains Flies were raised at 25 C in food vials containing yeast-cornmeal-dextrose-agar medium supplemented with methyl paraben (Tegosept) as a preservative. Canton-S was used as a wildtype, and yellow white (y w)or w 1118 strain as genetic controls. Two dronc-null mutant alleles, dronc I24 and dronc I29, were examined as either in trans with dronc 51 or homozygotes (Chew et al., 2004; Xu The Journal of Comparative Neurology Research in Systems Neuroscience 35

3 Lee et al. et al., 2005). The following null alleles were obtained as described: dredd D55, dredd L23, and dredd B118 (Leulier et al., 2000); dcp-1 Prev1 (Laundrie et al., 2003); strica 4 (Baum et al., 2007), and ice D1 (Muro et al., 2006). For a damm mutation, we characterized damm f02209 fly line carrying an insertion of a piggybac transposable element PBac{WH} within this locus (Bloomington stock no , Bloomington, IN; Thibault et al., 2004). For a putative decay mutation, we examined P{XP}d07129 flies bearing a P-element in upstream region of the decay (Exelixis Collection d07129). For transgenic manipulations, UAS-dronc (Quinn et al., 2000), UAS-CD8-PARP-Venus (Williams et al., 2006), or UAS-p35 (Hay et al., 1994) were crossed to a Crz-gal4 driver (Choi et al., 2008). A double transgenic Crz-gal4 line (2) combining two independent transgenes in the 2nd and 3rd chromosomes was used in some experiments. To induce targeted micro-rna-mediated gene silencing, UAS-mi-ice, UAS-mi-dcp-1, UAS-miiceþdcp-1, UAS-mi-damm, and UAS-mi-decay lines were constructed, as described in Chen et al. (2007). Histology Anti-Crz Polyclonal rabbit anti-crz (aka anti-cap; Choi et al., 2005, 2006) was raised against a synthetic peptide (VDPDPENSAHPRLSN) corresponding to the associated region of the Crz precursor. The immunoreactive patterns were identical to the Crz mrna expression revealed by in situ hybridization and Crz-gal4 activity, thus validating the specificity of this antibody to the Crz (Choi et al., 2005, 2006, 2008; Lee et al., 2008). Anti-Crz was applied at a dilution of 1:1,200 for wholemount immunohistochemistry (IHC). Anti-cPARP To detect endogenous caspase activity within the Crz neurons, progeny derived from a crossing between Crzgal4 and UAS-CD8-PARP-Venus were immunostained with anti-cleaved PARP (cparp) at 1:300 dilution as described (Williams et al., 2006). The CD8-PARP-Venus fusion proteins, when cleaved by caspases, expose a unique N-terminal epitope, which is readily detectable with anti-cparp in Drosophila neurons (Williams et al., 2006). The anticparp was purchased from AbCam (Cambridge, MA; cat. no. Ab2317), which was originally generated by BioVision (Mountain View, CA; cat. no ). This commercial rabbit polyclonal antibody was raised against a short peptide immunogen (GVDEVAKKKS), as provided by BioVision, and was reported to recognize only the cparp ( The antibody did not produce any immunoreactive signals in an entire CNS of wildtype (data not shown). In addition, all Crz neurons expressing the intact CD8-PARP-Venus (Fig. 3Ai,Bi) were not recognized by this antibody in the larval and white prepupal CNS (Fig. 3A,B), verifying no immunoreactivity between this fusion protein and anticparp. Moreover, no cparp-immunoreactivity was observed in the protocerebral DL neurons that persist into an adult brain (Fig. 3A D). We previously have shown the survival of these larval neurons throughout the metamorphosis using in situ hybridization and immunohistochemistry (Choi et al., 2005; Lee et al., 2008). These data together suggest that the cparp-immunoreactivity is specific to the vcrz neurons that are undergoing caspase-dependent apoptosis. A Western blot experiment using the same transgenic UAS-CD8-PARP-Venus and anti-cparp also showed that the antibody detects specifically cleaved PARP-Venus in the Drosophila retina, only when the death of this tissue was induced (Mendes et al., 2009). This result further supports the relevance of this system as a readout of caspase activity in vivo. Whole-mount CNS IHC CNSs were dissected in phosphate-buffered saline (PBS) and fixed in Zamboni s fixative containing 4% paraformaldehyde and 7.5% picric acid in 0.1 M sodium phosphate buffer (SPB, ph7.4) at 4 C overnight (Helfrich-Förster et al., 2000). Fixed samples were rinsed three times in SPB and subsequently three times in TNT buffer (0.1 M Tris, ph7.4, 0.3M NaCl, 0.5% Triton X-100) for 15 minutes each. After rinsing, tissues were preincubated in blocking buffer containing 4% normal donkey serum and 0.02% NaN 3 in TNT at room temperature for hours. Incubation with primary antibody continued overnight at 4 C, followed by washing in TNT (6 10 minutes) at room temperature. Primary antibodies were detected by incubating tissues in a solution containing rabbit anti-igg secondary antibody conjugated with tetramethyl rhodamine (TRITC) (Jackson ImmunoResearch, West Grove, PA) at 1:200 dilution in TNT including 2% normal donkey serum for 1 2 hours at room temperature in dark. The tissues were rinsed in TNT (3 10 minutes) and subsequently in SPB (3 10 minutes) to remove unbound secondary antibodies. The samples were mounted in medium containing 80% glycerol, SPB, and 2% n-propyl gallate and viewed with an Olympus BX61 epifluorescence microscope equipped with a CC12 camera. Images were taken at serial optical sections (thickness of 2 3 lm); these images were further processed to generate a composite image with Olympus Microsuite software, analysis 3.1 version (Soft Imaging System, Lakewood, CO). The composite images were edited for size, brightness, and contrast using Adobe Photoshop (San Jose, CA). Observation of male reproductive organs Male reproductive organs were dissected in PBS, fixed in 4% paraformaldehyde for 1 2 hours with gentle shaking at 36 The Journal of Comparative Neurology Research in Systems Neuroscience

4 Caspases-dependent neuronal cell death room temperature, washed in PBS (3 5minutes),and mounted with 100% glycerol. Snapshots were taken with a brightfield setting of an Olympus BX61 microscope. TUNEL (terminal deoxynucleotide transferase dutp nick end labeling) assay This assay was performed with a commercial kit (Deadend Fluorometric TUNEL system, Promega, Madison, WI) to detect fragmented DNA in dying neurons following the manufacturer s recommendation. To collect precisely aged prepupae, newly formed white prepupae were taken every 30 minutes and aged on a wet filter paper at 25 C. CNSs dissected from the prepupae were fixed in PBS containing 4% paraformaldehyde overnight at 4 C, and then washed three times with PBS containing 0.1% Triton X- 100 at room temperature. Tissues were transferred into a 0.25-mL conical cup (Fisher Scientific, Pittsburgh, PA) and incubated in equilibration buffer (50 ll) for 10 minutes. Afterward, samples were incubated with a reaction mixture of the equilibrium buffer (45 ll), fluorescein- 12-dUTP (5 ll), and terminal deoxynucleotidyl transferase (1 ll) at 37 C for 1 hour in a dark chamber. The reaction was terminated by rinsing tissues with 2 SSC once for 15 minutes at room temperature and then with PBS (5 5 minutes). Samples were mounted and fluorescent signals were acquired as described above. RT-PCR To detect decay or damm transcripts, total RNA was isolated from a group of 40 adult flies homozygous for w 1118 (control), damm f02209,ordecay d07129 using TRIZOL reagent (Invitrogen, La Jolla, CA). Total RNA was also extracted from 50 dissected adult male reproductive organs including the testes, anterior ejaculatory ducts, testicular ducts, and accessory glands, or from 50 CNSs obtained from both wandering 3rd instar larvae and white prepupae. Total RNA (200 ng, unless indicated otherwise) was added to a one-step reverse-transcription polymerase chain reaction RT-PCR kit (SuperScript: Invitrogen). In each PCR reaction, one of the following sets of primers was included: damm-f (5 0 -CTAACCCAGCGTT GCCGGACGTT) and damm-r (5 0 -TCGCGTCAGCTCTCAA CGGACCC); decay-f (5 0 -TCTCCGAGATCAACGACACGCTC) and decay-r (5 0 -GACGCCCTCCGGCGTGGCAGCT); b-tubulin-f (5 0 -GCAACAACTGGGCCAAGGGTCATTAC) and b-tubulin-r (5 0 -CTTGGCATCGAACATCTGCTGGGTCAG). RESULTS Temporal regulation of the PCD of vcrz neurons In response to a surge of ecdysone hormone toward the end of larval period, a wandering 3rd instar larva (WL3) ceases its movement and initiates puparium formation. The larval cuticle hardens and tans to form the prepupal cuticle, which then separates from the underlying epidermis (apolysis) around 4 6 hours after puparium formation (APF) at 25 C (Fig. 1A) (Yin and Thummel, 2005). At 12 hours APF, a small ecdysone pulse triggers head eversion, a hallmark of pupation. During this early metamorphic period, most larval tissues along with a large number of neurons are removed by PCD (Truman et al., 1993, 1994). To construct a developmental profile of dying neurons in the prepupal CNS, we performed a time-lapse TUNEL analysis. The TUNEL signals in the VNC became apparent around 2 hours APF, peaked between 3 and 8 hours APF, and gradually diminished afterward (Fig. 1B I). Since the signals reflect an accumulation of chromosomal breakdown, a hallmark of apoptotic cell death, peak TUNEL signals are likely to precede a complete removal of these cells. In this aspect, a progressive elimination of the vcrz neurons between 3 and 7 hours APF is noteworthy (Fig. 1J R; see also Choi et al., 2006). Despite unknown identities of most of the dying cells in the VNC, we speculate that underlying PCD mechanism ongoing in the vcrz neurons is likely shared by other doomed neurons during a similar developmental period. Previously, we used lacz reporter as a means to mark doomed vcrz neurons (Choi et al., 2006). These neurons were stained by using X-gal histochemistry; however, this method, albeit rather convenient, proved unable to reveal subtle morphological changes associated with cell death due to its diffusive nature. Thus, as an alternative tool, we employed Crz-IHC to analyze cytological events occurring in the vcrz neurons prior to their death. Consistent with the X-gal histochemistry, Crz-immunoreactive signals progressively disappeared during a period of 3 7 hours APF (Fig. 1M R). Although the disappearance of vcrz neurons was becoming evident at 3 4 hours APF, close inspection of the immunolabeled vcrz neurons revealed cytological signs of PCD much earlier than their actual removal time. In WL3 and white prepupae (0 hours APF), contralaterally projecting axons showed a smooth surface (Fig. 2A,B). Around 1 hour APF, however, the axons began to develop small dense spots, giving rise to a beads-on-string appearance (Fig. 2C), which is a structural hallmark of axonal degeneration (Nikolaev et al., 2009). These spots became more prominent and increased in number by 2 hours APF (Fig. 2D). The somata also displayed an uneven cell surface and budding structures during the same period (Fig. 2E,F). These changes in the vcrz neurons match distinct morphological characteristics of apoptosis (Kerr, 2002; reviewed in Elmore, 2007). Taken together with previous detection of TUNEL signals within vcrz neurons (Choi The Journal of Comparative Neurology Research in Systems Neuroscience 37

5 Lee et al. Figure 1. Time-course events of vcrz neuronal PCD. A: A schematic diagram of major events occurring during early stages of metamorphosis in D. melanogaster. B I: Time-course TUNEL signals in the VNC during early metamorphosis. Horizontal dotted lines indicate an approximate boundary between the thoracic (Th) and abdominal (Ab) ganglia. J R: Progressive elimination of the vcrz neurons indicated by brackets was observed by Crz-immunofluorescence. Overt signs of dying vcrz neurons, such as fragmented neural projections (arrow) and disappearance of vcrz somata (arrowheads), begin to be revealed around 3 hours APF and complete removal of them by 7 hours APF. Numbers indicate hours after puparium formation. Scale bars ¼ 100 lm. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.] et al., 2006), we conclude that the death of vcrz neurons is indeed apoptotic. Our data also suggest that degeneration occurs simultaneously in both soma and axon. This is in contrast to the pruning events of persisting larval neurons in which the degeneration is limited to axons and/or dendrites (Schubiger et al., 1998; Lee et al., 2000; Kuo et al., 2005; Williams et al., 2006). To gain insight into the roles of caspases in the apoptotic events of vcrz neurons, we first analyzed the activity of endogenous caspases by expressing CD8-PARP-Venus fusion protein directed by a Crz-gal4. PARP (poly-adpribose polymerase) is a well-characterized substrate for effector caspases and the cleaved PARP (cparp) is readily detectable with a specific antibody (Williams et al., 2006). While expression of the CD8-PARP-Venus in all Crz neurons was clearly visualized by the Venus (YFP) signals (Fig. 3Ai Di), none of these neurons were detected by 38 anti-cparp in larval and white prepupal CNS (Fig. 3A,B). cparp-immunoreactivity was first noticeable in a few vcrz neurons and their projections at 1 hour APF (Fig. 3C), which is coincident with early visible signs of vcrz neuronal degeneration (Fig. 2C). At 2 hours APF, cparp signals were more significant in most vcrz neurons and their projections (Fig. 3D). Importantly, the cparp-immunoreactivity was not observed in a group of the Crzexpressing protocerebral DL neurons (Fig. 3A D), which survive throughout metamorphosis and persist into adulthood (Choi et al., 2005; Lee et al., 2008). Furthermore, coexpression of p35, a versatile caspase inhibitor, completely eliminated cparp-immunoreactivity in the doomed vcrz neurons (data not shown, n ¼ 6). These results together support that endogenous caspases become active within the vcrz neurons shortly after the onset of puparium formation. Simultaneous detection of The Journal of Comparative Neurology Research in Systems Neuroscience

6 Caspases-dependent neuronal cell death Figure 2. Early apoptotic signs observed in the vcrz neurons and their axonal projections. A D: Progressive axonal degeneration during early prepupal development. The axonal projections look sleek and evenly stained prior to the onset of metamorphosis (A,B). Shortly after entry into metamorphic development, they begin to show a discontinuous look and distinct bulges (asterisks) along the axonal tract (C,D). E,F: Somata of the vcrz neurons also lose their integrity and the cell surface takes an increasingly irregular shape and forms apoptotic bodies, as indicated by arrowheads. WL3, wandering third instar larva. Scale bar ¼ 25 lm. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.] the cparp in the somata and their axonal projections was also consistent with our earlier description of apoptotic signs, suggesting that caspases act in both subcellular structures to induce synchronous degeneration of an entire neuron. Role of initiator caspases in the PCD of vcrz neurons Given the importance of caspase activities in the timely removal of vcrz neurons, we conducted a comprehensive genetic analysis to define the roles of each caspase. Previously we have shown that dronc plays an important role in the normal course of vcrz death (Choi et al., 2006). Here we further investigated whether dronc functions alone or with other putative initiator caspases. Consistent with our previous data, prepupae homozygous for dronc I24, dronc I29, or hetero-allelic combinations (dronc I24/51 and dronc I29/51 ), all of which are known to be null (Chew et al., 2004; Xu et al., 2005), displayed surviving vcrz neurons at 7 hours APF (Table 1; Fig. 4A). However, these vcrz neurons gradually succumbed to death by 48 hours APF (Table 1, Fig. 4B,C), thus showing that death of the vcrz neurons was markedly delayed, but not prevented in the absence of dronc function. Nonetheless, a noteworthy fact that 25% of vcrz neurons in droncnull mutants undergo normal death by 7 hours APF suggests a role for other initiator caspase(s), besides dronc, for the vcrz PCD. To address this issue, we extended our studies to analyze a possible involvement of other initiator caspases, dredd and strica. Neither dredd-null alleles (dredd D55, dredd L23, and dredd B118 ), nor strica-null (strica 4 ) were able to delay or block the vcrz PCD single-handedly, as no viable vcrz neurons detected at 7 hours APF (Table 2; Fig. 4D). Despite these results, we could not rule out the possibility that strica or dredd functions redundantly with dronc. To determine whether this is the case, we examined double homozygous mutants, strica 4 ; dronc I24 and dredd L23 ; dronc I24/51. Interestingly, while vcrz PCD phenotype in dredd; dronc double mutants is indistinguishable from that in dronc mutants, nearly all vcrz neurons survived in the CNS of strica; dronc double mutant at 7 hours APF (Table 2; Fig. 4E). Such abnormal PCD phenotype is significantly more severe than the one observed in either dronc I24 or strica 4 mutation alone (Tables 1, 2). When strica 4 ; dronc I24 /TM6B was examined, no surviving vcrz neurons were found at 7 hours APF, suggesting that a heterozygosity of dronc is sufficient to compensate the lack of strica function. We also examined triple null mutants lacking all three initiator caspases, dredd B118 ; strica 4 ; dronc I24. This mutant displayed results comparable to that of strica 4 ; dronc I24 double mutant (Table 2; Fig. 4F). From these data together, we conclude that strica plays a minor role that overlaps with dronc s, while dredd has no function in the vcrz neuronal death. Dronc is insensitive to inhibition by p35 in vcrz neurons Baculoviral p35, a broad-spectrum caspase inhibitor, is cleaved by caspases at a specific site, and the cleaved product makes a stable association with a caspase, thereby suppressing its proteolytic activity (reviewed in Clem, 2001). According to a biochemical study, p35 is not recognized by Dronc, thus not cleaved by it. Consistently, cell death induced by dronc overexpression in the retina was shown to be insuppressible by the coexpression of p35 (Hawkins et al., 2000; Meier et al., 2000). These results suggest that inhibitory action of the p35 is mainly on the effector caspases. However, contradictory data were also presented (Quinn et al., 2000). Expression of p35 blocks developmental PCD of vcrz neurons in which Dronc plays a major role (Table 1; see The Journal of Comparative Neurology Research in Systems Neuroscience 39

7 Lee et al. Figure 3. in vivo detection of caspase activity using cleaved PARP (cparp) as an indicator of caspase action. A D: Progressive activation of caspases in the vcrz neurons. Shortly after the onset of metamorphosis, cparp-immunoreactivity is evident in the somata and projections (C,D). Note that no cparp was detected in a brain Crz neuronal group (DL) that persists into adulthood. Ai Di: Venus (YFP) expression in the samples corresponding to A D. A magenta-green copy of this figure is available as a supporting figure. Scale bar ¼ 100 lm. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.] TABLE 1. Time-course vcrz PCD in dronc Mutants Genotype 7 h APF 16 h APF 24 h APF 48 h APF Wildtype 0 (20) 0 (20) 0 (16) nd Crz-gal4 x UAS-p35 16 (16) 16 (8) 16 (5) 16 (5) dronc I (9) (3) (3) 0 (1) dronc I (3) (4) (3) (6) dronc I24/ (8) (13) (7) (3) dronc I29/ (10) (6) nd (3) Values indicate mean number of vcrz neurons 6 SEM. (n) number of specimens; nd, not determined. also Choi et al., 2006). To test if p35 is able to suppress Dronc-induced death of vcrz neurons in the larval CNS in which the endogenous PCD program is not yet operative, the expression of dronc either alone or with p35 was directed by the Crz-gal4 and then Crz-immunoreactivity was performed. As expected, dronc overexpression killed all vcrz neurons but, unexpectedly, none of the protocerebral corazonergic neurons (Fig. 5A vs. B). Coexpression of the p35 did not prevent such dronc-induced killing of vcrz neurons (Fig. 5C), thus supporting that Droncinduced ectopic killing is insensitive to p35 inhibition. Our foregoing data suggest that the mechanisms of induced death by overexpressed dronc differ from those of normal death by endogenous dronc. In the latter case, main function of the Dronc, as an initiator caspase, is to activate effector caspases, which are effectively blocked by ectopic p35 expression. The former case, however, unlikely involves activation of effector caspases, because Diap1, an inhibitor of apoptosis protein, is likely to form an inhibitory complex with effector caspases in the larval tissues as the death program has not yet been initiated at this stage (Zachariou et al., 2003; Yan et al., 2004). Rather, overproduced Dronc may act like an effector caspase. In line with this notion, a mammalian Dronc homolog, caspase 9, was shown to directly degrade vimentin, a type of intermediate filament (Nakanishi et al., 2001). These results raise the possibility that overproduced Dronc acts like effector caspases to process cytoplasmic 40 The Journal of Comparative Neurology Research in Systems Neuroscience

8 Caspases-dependent neuronal cell death TABLE 2. vcrz PCD in the Mutants of Initiator Caspases Genotype 7 h APF 16 h APF dredd L23 0 (5) nd dredd B118 0 (3) nd dredd D55 0 (3) nd dredd L23 ; dronc I24/ (8) (5) strica 4 0 (4) nd strica 4 ; dronc I24 /TM6C 0 (3) 0 (2) strica 4 ; dronc I (6) (3) dredd B118 ; strica 4 ; dronc I (16) (5) Values indicate mean number of vcrz neurons 6 SEM. (n) number of specimens; nd, not determined. Figure 4. Roles of initiator caspases for the vcrz PCD. A C: Delayed vcrz neuronal PCD in dronc I24 null mutant CNS. D: Normal PCD in a strica-null mutant at 7 hours APF. E,F: Strong but incomplete suppression of vcrz PCD was observed at 7 hours APF in strica 4 ; dronc I24 double mutant (E), and dredd B118 ; strica 4 ; dronc I24 triple mutant (F). See also Tables 1 and 2. Scale bar ¼ 50 lm. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.] proteins, leading to the cell death. Since Dronc is insensitive to p35, ectopic expression of p35 is unable to block Dronc-induced cell death. Role of effector caspases Since Ice and Dcp-1 are the best-characterized effector caspases for their proapoptotic functions in Drosophila and they are sensitive to inhibition by p35, it is likely that these two are the primary executioners for the vcrz PCD. Thus, we determined vcrz PCD in the mutant lacking dcp-1 or ice function. In a dcp-1-null mutant (dcp-1 Prev1 ), an average of (mean 6 SEM, n ¼ 19) vcrz somata remained detectable at 7 hours APF (Fig. 6A), but none did at 16 hours APF (n ¼ 17, Fig. 6B). Moreover, the general morphology of the vcrz neurons at 7 hours APF was comparable to those of wildtype at 4 5 hours APF (Fig. 1C), indicating that the PCD has been slowed down in the absence of dcp-1 function. These results suggest that Dcp-1 is necessary but not a sole effector caspase for the degeneration of the vcrz neurons. Next, we determined the death of vcrz neurons in an ice-null allele, ice D1. Remarkably, we found all 16 vcrz neurons remained at 7 hours APF (n ¼ 16, Fig. 6C); however, the surviving neurons showed budding structures (Fig. 6D) that were somewhat similar to what we described earlier for dying wildtype vcrz neurons at 1 2 hours APF (Fig. 2E,F). Such morphological similarities led us to wonder whether PCD of vcrz neurons is delayed in the absence of ice function. Indeed, the number of vcrz neurons was progressively reduced to at hours APF (n ¼ 4, Fig. 6E) and at 24 hours APF (n ¼ 6, Fig. 6F). Delayed death in the absence of either dcp-1 or ice implies that these two effector caspases act cooperatively to draw a full destructive power, ensuring the death of all vcrz neurons in a timely manner. To test this hypothesis, we performed Crz-IHC in a double null mutant, dcp- 1 Prev1 ; ice D1. Intriguingly, all 16 vcrz neurons remained detectable at 7 hours APF (n ¼ 5, Fig. 6G) and even at 16 hours APF (n ¼ 2, Fig. 6H). Although developmental arrest and lethality of the double mutant did not permit us to examine surviving vcrz neurons beyond this stage, we can clearly infer from our results that both dcp-1 and ice are necessary and sufficient for the vcrz death. In addition, comparisons in the number of surviving vcrz neurons and their structural altercations from different developmental stages between dcp-1 Prev1 and ice D1 mutants suggest that ice is a major player in the execution of vcrz death, while dcp-1 plays a lesser but nonetheless essential role. Autonomous effects of dcp-1 and ice Since dcp-1 Prev1 ; ice D1 double mutants display developmental arrest around the mid-pupal stage, we wanted to confirm that the suppression of vcrz PCD in the mutant CNS does not result from the systemic developmental arrest. To investigate an autonomous role of dcp-1 and ice, we employed a targeted knockdown of these caspases using a micro-rna-based silencing tool (mi-rna) (Chen et al., 2006, 2007). For this, UAS-mi- The Journal of Comparative Neurology Research in Systems Neuroscience 41

9 Lee et al. Figure 5. p35-insensitive Dronc function. A: Wildtype (WT) Crz-immunoreactivity in the larval CNS. Dorsal lateral (DL) neurons in the brain are indicated by arrowheads. B: Expression of dronc by Crz-gal4 caused premature death of the vcrz neurons, while DL neurons remained alive (arrowheads). Genotype: UAS-dronc/þ; Crz-gal4/þ. C: Coexpression of p35 did not prevent dronc-induced death of vcrz neurons. Genotype: UAS-dronc/UAS-p35; Crz-gal4/þ. Scale bar ¼ 100 lm. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.] Figure 6. Effect of a dcp-1 or ice null mutation on vcrz PCD. A,B: Surviving vcrz neurons in dcp-1 Prev1 at 7 hours APF (A), and at 16 hours APF (B). C F: Surviving vcrz neurons and their projections in ice D1 mutant at indicated timepoints. The boxed area in (C) was recaptured with a higher magnification (D). Blebbings in the somata of the surviving vcrz neurons are indicated by arrowheads. G,H: Complete rescue of doomed vcrz neurons in dcp-1 Prev1 ; ice D1 double mutant CNS. Severe tissue deformity seen at 16 hours APF (H) is due to the systemic developmental arrest of the double mutant. Scale bars ¼ 50 lm ina;25lm in D. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.] 42 The Journal of Comparative Neurology Research in Systems Neuroscience

10 Caspases-dependent neuronal cell death TABLE 3. Autonomous Apoptotic Roles of dcp-1 and ice Crz-gal4 1X 2X mi-rna 7 h APF 7 h APF 16 h APF mi-ice (7) (5) nd mi-dcp (6) (7) nd mi-iceþdcp (9) (4) (2) Values indicate mean number of vcrz neurons 6 SEM. (n) number of specimens; nd, not determined. RNA lines for dcp-1 and ice, which are referred to as UAS-mi-dcp-1 and UAS-mi-ice, respectively, were crossed to a single Crz-gal4 (1) or a double Crz-gal4 line (2) to alter the dosage of mi-rna expression. Remarkably, the expression of mi-dcp-1 (Fig. 7A,B) and mi-ice (Fig. 7C) blocked vcrz PCD to the extent comparable to those obtained from each corresponding null mutation. Expression of mi-iceþdcp-1 also showed a similar level of cell survival to that observed with the dcp-1/ice double mutation (Fig. 7D F; Table 3), providing clear evidence for the cell-autonomous roles of dcp- 1 and ice. Figure 7. Cell autonomous function of dcp-1 and ice. A,B: The death of vcrz neurons is rescued partially by mi-dcp-1 expression driven by 1 Crz-gal4 (A) and 2 Crz-gal4 (B) at 7 hours APF. C: Complete rescue of vcrz cell death by expressing mi-ice by 1 Crz-gal4 at 7 hours APF. D F: Coexpression of mi-dcp-1 and mi-ice (mi-dcp-1þice). The death of vcrz neurons is completely blocked even at 16 hours APF (F). G,H: Complete suppression of the vcrz PCD in dcp-1 Prev1 ; dronc I29 mutant. See also tables for the quantitative data. Scale bars ¼ 100 lm ina;50lm inf. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.] Dronc downstream caspases It is currently believed that initiator caspases, such as dronc, play a proapoptic role through the activation of effector caspases, thus placing both ice and dcp-1 in the downstream of dronc (Hawkins et al., 2000). Our foregoing data, however, do not robustly support this canonical model, as the rescue of the vcrz PCD is greater in icenull mutant than in either dronc or dronc/strica-double mutation. To test further if dronc is the activator of dcp-1, we analyzed a mutant null for both dcp-1 and dronc (dcp-1 Prev1 ; dronc I24 and dcp-1 Prev1 ; dronc I29 ). If dcp-1 is an obligatory downstream of dronc, one can expect the defective PCD phenotype of the double mutants to be similar to those of dronc single mutants. Surprisingly, we found a full rescue of vcrz PCD by dcp-1/dronc double mutation at both 7 hours (n ¼ 6, Fig. 7G) and 16 hours APF (n ¼ 2, Fig. 7H), compared to a partial rescue by dronc single mutations (Fig. 4A,B). Based on these results, we propose that an activation of Ice and Dcp-1 requires a novel pathway in addition to the Dronc and Strica. No roles found for damm and decay in vcrz death Our sequence analysis of the damm f transgenic line revealed that a piggybac transposon is inserted into the 3rd intron and carries a deletion of 4 bp (TCAA) in The Journal of Comparative Neurology Research in Systems Neuroscience 43

11 Lee et al. Figure 8. Characterization of damm PB and d07129 mutants. A: The sequence shows partial third intron (lowercase letters) and fourth exon (capitals) of damm. A triangle indicates the damm PB insertion in a 3 0!5 0 direction. Bold letters (tcaa) designate a 4-bp deletion. B: damm expression assessed by RT-PCR. damm expression was not detected in damm PB (boxed). Primers for the RT-PCR were derived from the 3rd and 4th exons of the damm. The same primers produced slightly larger size of genomic product due to the presence of the 50-bp 3rd intron. b-tubulin was used as a control. C: Lack of expression of both damm and decay in the CNS of wildtype (w 1118 ) wandering 3rd instar larvae and white prepupae. D,E: PCD of vcrz neurons is unaffected by a damm PB insertion (D) or by mi-decay expression driven by 2 Crz-gal4 (E) at 7 hours APF. F: Expression of decay assessed by RT-PCR. Decay RT-PCR product was detected in the male body (B) of w 1118, but not in the reproductive organs (R). However, decay expression was detected in both body as well as reproductive organs of d07129 homozygous males (arrowhead). Scale bar ¼ 100 lm in D (applies to E). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.] which the last A is part of the splicing acceptor site (AG) (Fig. 8A). As a result, we presume that the damm f allele suffers a splicing error. Consistent with this prediction, RT-PCR using a set of primers specific to the 3rd and 4th exons failed to produce PCR products from the homozygous mutant (for short, damm PB in Fig. 8B), while the same primers produced damm-specific 252-bp products from control w 1118 adults. This result indicates that damm f is likely a null or at least a strong hypomorphic allele. Nevertheless, damm f homozygous adult flies appear normal, suggesting that damm is not vital for fly development. RT-PCR was unable to detect damm transcripts in the CNS of both late 3rd instar larvae and white prepupae of w 1118 (Fig. 8C) and vcrz neurons underwent normal PCD in the homozygous damm f mutants (n ¼ 7, Fig. 8D). The expression of mi-damm by Crz-gal4 1 (n ¼ 5) or 2 (n ¼ 12) also did not alter the PCD of vcrz neurons (data not shown). These data support a lack of damm function in the early metamorphosing CNS including the death of vcrz neurons. Like damm, decay-null mutant was reported to be healthy and fertile (Kondo et al., 2006). Due to a patent issue, we were unable to test this mutant; instead, we employed mi-rna-mediated knockdown of decay. Expression of mi-decay by Crz-gal4 2 did not affect the normal course of vcrz PCD (n ¼ 13, Fig. 8E). Moreover, decay expression, as determined by RT-PCR, was clearly detectable in the body of w 1118 adults (Fig. 8F), but not in the CNS of WL3 and white prepupae (Fig. 8C), leading us to speculate that decay has no particular role in the CNS. As a putative decay mutant allele, we examined P{XP}d07129 (for short, d07129) homozygous flies, which, as we confirmed, contain a P-element in 398 bp upstream from the transcription start site of decay. Although the homozygous d07129 animals develop normally and PCD of vcrz neurons was unaffected by this allele (n ¼ 7, data not shown), males were reproductively abnormal, ranging from sterile to weakly fertile. RT-PCR result showed no detectable expression of decay in the wildtype male reproductive organs, implying the lack of endogenous decay function in this tissue. Interestingly, however, decay expression was clearly observed in the same tissues of homozygous d07129 males(arrowheadinfig.8f).suchmisexpression of decay is likely due to the insertion of a P{XP} element containing two UAS sequences that were intended to induce ectopic expression of a nearby gene in a Gal4-dependent manner (Thibault et al., 2004). It is not uncommon that UAS activity, depending on the genomic location, is not silent even in the absence of Gal4 (Markstein et al., 2008). Thus, we speculate that decay s upstream regulatory region is affected by the UAS sequence, resulting in the misexpression of decay in the male reproductive organs without thepresenceofgal4. To explore whether male sterility of the d07129 is due to abnormal reproductive activity, we examined the 44 The Journal of Comparative Neurology Research in Systems Neuroscience

12 Caspases-dependent neuronal cell death Figure 9. Defective reproductive organs in d07129 homozygous males. Arrowheads indicate joints of the seminal vesicles (SV) with testes (T) or ejaculatory duct (ED). A: Distended SV reflects massive amounts of mature sperms stored in this organ in wildtype (n ¼ 4). B: An area indicated by a box in (A) is magnified. C: Reproductive organs of the decay d07129 mutant. D: A magnified image of the dotted box in (C). The SV of the d07129 male is slender and transparent due to no or very little amounts of sperm stored (n ¼ 4). The magnified images are rotated to show better anatomical comparison. AG, Accessory glands. Scale bars ¼ 100 lm. morphology of the male reproductive organs. The testes of d07129 males looked relatively normal but the accessory glands and anterior part of the ejaculatory duct of the mutant males were substantially smaller than the wildtype counterparts (Fig. 9A vs. C). In addition, the seminal vesicles that store mature sperm were slender and transparent, a likely result from the near-absence of stored sperms (Fig. 9B vs. D). Thus, it is likely that decay misexpression in the d07129 male reproductive organs is causally associated with an abnormal sperm production, perhaps as a result of an inappropriate male germ cell death. DISCUSSION Major changes in the mode of locomotion from larval (peristaltic crawling) to adult (walking and flying) stages in holometabolous insects requires the degeneration of obsolete larval motor neurons followed by de novo generation of adult-specific motor neurons. Notably, a majority of the motor neurons innervating larval abdominal muscles from the abdominal ganglia are eliminated during early metamorphosis in a moth (reviewed in Weeks, 2003). Similar removal of larval motor neurons is expected in Drosophila. Consistently, our results revealed progressively increasing TUNEL signals, particularly in the VNC shortly after the entry into metamorphosis in Drosophila, indicating that neuronal PCD plays a significant role in the remodeling of this part of the CNS. In addition to the motor neurons, certain larva-specific interneurons including vcrz neurons are also targets for PCD during this early phase of metamorphosis (Weeks, 2003; Choi et al., 2006; Lee et al., 2008). Despite these studies, it has been poorly understood how such neuronal PCD is executed. Using vcrz neurons as a model system and various neurogenetic tools, we revealed intricate actions of initiator and effector caspases in the doomed vcrz neurons. Roles of dronc and strica for the vcrz neuronal death Among three putative initiator caspases, we found dronc as a major one, while strica plays a minor role that overlaps with dronc. None of our evidence is in favor of the proapoptotic roles of dredd in the death of vcrz neurons. According to a conventional model, the initiators activate downstream effectors, which then carry out the cellular degeneration process. However, normal PCD of a few vcrz neurons still occurs even in the absence of all three initiators, raising the possibility that there is either a yet-unidentified initiator The Journal of Comparative Neurology Research in Systems Neuroscience 45

13 Lee et al. caspase in the Drosophila genome or a novel pathway capable of activating effector caspases without involving these three initiator caspases. This notion was also supported by our genetic data that an ice mutation alone shows a greater level of suppression of the vcrz PCD than either a dronc alone or a dronc/strica double mutation. Likewise, a dcp-1/dronc double mutation displayed a greater inhibition of vcrz PCD than did a dronc or a dronc/ strica double mutation. Results similar to ours have been reported for different tissues. For instance, a triple dredd/ strica/dronc mutation did not completely stop oogenesisassociated PCD (Baum et al., 2007). Studies on the larval midgut also indicated initiator-independent activation of effector caspases, although the caspases do not seem to cause the PCD of this tissue (Denton et al., 2009). Dronc-independent cell death was also observed in some embryonic cells (Xu et al., 2005). These independent lines of evidence found in various tissue types strongly support the idea that Drosophila has an unconventional activation mode of effector caspases. This possibility warrants further investigation. Roles of effector caspases Thus far, Ice and Dcp-1, two closely related effector caspases, are known to play major functions in the PCD of diverse tissues in Drosophila. However, their roles seem to vary in a tissue-specific manner. For instance, PCD of nurse cells during late oogenesis is slightly reduced in the absence of either ice or dcp-1 function alone, while the PCD is additively suppressed by the lack of both, implicating their cooperative or independent actions in this tissue type (Baum et al., 2007). In developing embryos, global embryonic cell death is nearly unaffected by a dcp-1 mutation, but substantially reduced in ice mutants (Kondo et al., 2006; Xu et al., 2006). The extent of PCD is similar to or slightly lower in dcp-1/ice double mutant embryos, suggesting a predominant role for ice with a minor redundant dcp-1 function during the embryonic PCD. In the imaginal discs of the developing eye, a great reduction in the cell death is observed in either ice or dcp-1 mutants, while the PCD is completely absent in dcp-1/ice mutants (Kondo et al., 2006). These data imply largely overlapping, but cooperative roles played by these two death enzymes for eye development. By comparison, the PCD of the larval salivary glands is surprisingly normal in an ice mutant (Muro et al., 2006), although this event is significantly impaired in a dronc-null mutant (Daish et al., 2004). Thus, it remains to be seen whether dronc s sole benefactor is dcp-1 for the salivary gland cell death. On a different occasion, ice alone is likely to be sufficient for the irradiation-induced cell death (Kondo et al., 2006; Muro et al., 2006). These studies together demonstrate that dcp-1 and ice are essential effector caspases for the PCD of various types of tissues, but required differently in a tissue type-dependent manner. At first glance, ice seemed to be sufficient for vcrz neuronal PCD, as all vcrz neurons remained at 7 hours APF in ice-null mutants. However, the surviving neurons were progressively eliminated as pupal development proceeded, presumably via the action of dcp-1. This is supported by a marginal suppression of vcrz PCD in the absence of dcp-1 function, and a complete block of death in the tissue lacking both dcp-1 and ice functions. Therefore, PCD of vcrz neurons requires the cooperative action of Ice and Dcp-1. Such a joint force seems to be essential for the timely elimination of neurons in the rapidly developing CNS during early metamorphosis, otherwise delayed removal of juvenile neurons could interfere with the proper establishment and elaboration of the adult neural circuitries. Tight temporal as well as spatial regulation of the levels of active caspases in work is critical for proper postembryonic CNS development. For the latter type of regulation, localization of active caspases in neurites is important for the pruning of neuronal processes, leaving the soma intact. Such limited degeneration of persisting larval neurons is required for their respecification in order to make new synaptic contacts during metamorphic development (reviewed in Luo and O Leary, 2005; Saxena and Caroni, 2007). This is in contrast to global activation of caspases in the doomed neurons (this study). Therefore, it appears that neurons are preprogrammed to provide a minimal optimum of total caspase activity to bring about the self-degeneration of a whole cell or specific subcellular structures within a narrow developmental window. ACKNOWLEDGMENTS We thank K. McCall for the kind supply of dcp-1 and double and triple caspase mutant stocks. We thank I. Muro for ice null mutant, D.W. Williams for a UAS-CD8- PARP-Venus, B. Lemaitre for dredd mutants, A. Bergmann for dronc mutant stocks, H. Richardson for a UAS-dronc, and P. Valvo for a kind gift of a fly food ingredient. We also thank B.D. McKee, J. Hall, and M. Labrador for helpful discussion and comments on the article. LITERATURE CITED Baum JS, Arama E, Steller H, McCall K The Drosophila caspases Strica and Dronc function redundantly in programmed cell death during oogenesis. Cell Death Differ 14: Chen C-H, Guo M, Hay BA Identifying microrna regulators of cell death in Drosophila. Methods Mol Biol 342: Chen C-H, Huang H, Ward CM, Su JT, Schaffer LV, Guo M, Hay BA A synthetic maternal-effect selfish genetic element drives population replacement in Drosophila. Science 316: Chew S-K, Akdemir F, Chen P, Lu W-J, Mills K, Daish T, Kumar S, Rodriguez A, Abrams JM The apical caspase 46 The Journal of Comparative Neurology Research in Systems Neuroscience