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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Fly (Austin). Author manuscript; available in PMC Apr 13, 2009.
Published in final edited form as:
Published online Nov 5, 2008.
PMCID: PMC2668719
NIHMSID: NIHMS104187

The role of the RING-finger protein Elfless in Drosophila spermatogenesis and apoptosis

Abstract

elfless (CG15150, FBgn0032660) maps to polytene region 36DE 5′ (left) of reduced ocelli/Pray for Elves (PFE) on chromosome 2L and is predicted to encode a 187 amino acid RING finger E3 ubiquitin ligase that is putatively involved in programmed cell death (PCD, e.g., apoptosis). Several experimental approaches were used to characterize CG15150/elfless and test whether defects in this gene underlie the male sterile phenotype associated with overlapping chromosomal deficiencies of region 36DE. elfless expression is greatly enhanced in the testes and the expression pattern of UAS-elfless-EGFP driven by elfless-Gal4 is restricted to the tail cyst cell nuclei of the testes. Despite this, elfless transgenes failed to rescue the male sterile phenotype in Df/Df flies. Furthermore, null alleles of elfless, generated either by imprecise excision of an upstream P-element or by FLP-FRT deletion between two flanking piggyBac elements, are fertile. In a gain-of-function setting in the eye, we found that elfless genetically interacts with key members of the apoptotic pathway including the initiator caspase Dronc and the ubiquitin conjugating enzyme UbcD1. DIAP1, but not UbcD1, protein levels are increased in heads of flies expressing Elfless-EGFP in the eye, and in testes of flies expressing elfless-Gal4 driven Elfless-EGFP. Based on these findings, we speculate that Elfless may regulate tail cyst cell degradation to provide an advantageous, though not essential, function in the testis.

Keywords: Drosophila, RING finger, apoptosis, spermatogenesis, male sterile, pPTGAL, piggyBac, dronc, ubcD1

Introduction

Spermatogenesis1-4 is a continuous process that begins at the most immature end of the testis, the gonial region, distal from the seminal vesicle. The stereotypic developmental process of spermatogenesis begins with a single germline cell produced at the gonial tip that travels along the length of the testis, dividing, differentiating and maturing, before ultimately giving rise to fully formed sperm. At the onset of the proliferation phase, occurring at the gonial tip, an unequal cell division from a precursor germline stem cell, tethered to a hub cell, gives rise to a primary spermatogonial cell. In synchrony with the germline stem cell division, two somatic cyst progenitor cells undergo unequal division to form a head cyst cell and a tail cyst cell, which together surround the spermatogonial cell as it undergoes its several divisions. The cyst cells do not divide during the spermatogenesis program. Within the cyst envelope, the primary spermatogonial cell undergoes four rounds of mitotic division to give rise to 16 spermatogonial cells. Cytokinesis is incomplete during these mitotic divisions, leaving cytoplasmic bridges known as ring canals connecting the cytoplasm of the diploid spermatogonial cells.5 A specialized cytoskeleton organelle found in the germ line, called the fusome, runs between all of these ring canals and originates from a spectrin-rich structure, called the spectrosome, found in the spermatogonial cells.6,7 These bridges persist throughout the remaining stages of spermatogenesis8 and are only degraded later when the mature sperm are individualized.

The 16 germline cells in each cyst then enter a prolonged growth phase with marked increase in transcription levels and increase their cell volume by nearly 25-fold before initiating a meiotic program to give rise to a 64 haploid spermatid cyst. As these haploid cells mature, their mitochondria fuse to form a specialized mitochondrial derivative called a nebenkern. At this stage, the post-meiotic spermatocyst has traveled about two-thirds the length of the testes and, during the cytodifferentiation phase of spermatogenesis, the mature nebenkern begins to elongate with the growing axonemal tail extending from the spermatid. In concert, the two cyst cells undergo distinct morphological changes where the head cyst cell surrounds the spermatid nuclei and the tail cyst cell elongates along with the growing flagellar tail assembly.2 The tails of the 64 spermatids reach about 2 mm in length while the spermatid DNA condenses and the unit completes cytodifferentiation (Fig. 1).

Figure 1
Spermatid individualization. (A) Following elongation of the axoneme, mitochondria and the tail cyst cell, the head cyst cell-associated spermatid nuclei condense into needle-like morphologies. For clarity, only three of the 64 spermatid units are shown. ...

Sperm individualization is the final stage of spermiogenesis and this process has recently been demonstrated to be dependent on apoptotic machinery.9-13 Here complexes of actin-rich proteins, the individualization complexes (i.e., the investment cones), travel the length of the spermatids to remove syncytial cytoplasm (Fig. 1B) which eventually collects at the base of the spermatids to form a so-called waste bag; each spermatid is now ensheathed in its own plasma membrane. When each spermatid is fully individualized, the cysts and spermatids coil and the spermatozoa are transferred to the seminal vesicles for storage and, later, fertilization.14

Flies trans-heterozygous for polytene region 36DE deficiencies Df(2L)TW119 and Df(2L)TW201 (reviewed in ref. 15) exhibit male sterility, deafness and reduced ocelli (Fig. 2B). The latter two phenotypes are due to disruptions in btv/DHC36D (FBgn0023096)16 and Pray for Elves/rdo (FBgn0243486),17 respectively. In Df(2L) TW119/Df(2L)TW201 overlapping deficiency males (henceforth Df/Df), the mature sperm are fully formed, yet are amotile (Fig. 2A). The two cyst cells surrounding the mature 64-unit sperm bundle fail to be degraded and the spermatids that constitute this bundle are not individualized (Fig. 2A). Overlapping deficiency males court and mount females, however their initiation and duration of courtship is generally longer and their successful attempted copulation rate is lower due to altered song production and coordination defects caused by btv-associated deafness.18,19 Furthermore, despite the copulation efforts of Df/Df males, the sperm are amotile and therefore fail to enter the seminal vesicle, preventing sperm transfer and fertilization.

Figure 2
The btv-male sterile-rdo overlapping deficiency region. (A) Representative squash of Df/Df testes showing amotile sperm that remain encysted. (B) The genetic map of the btv-male sterile-rdo region derived from overlapping deficiencies TW119 and TW201. ...

Previous studies in flies indicate that ultrastuctural or physiological defects associated with disruption of the ciliated neurons of the Drosophila hearing organ are often likewise exhibited in the sperm tails; i.e., deafness and male sterility are intimately linked through dysfunction of common microtubule arrangements.16,20-23 Indeed, sperm flagella and the ciliated chordotonal sensory neurons in the antenna are composed of the typical 9 × 2 arrangement of microtubule doublets (+0 in chordotonals, +2 in sperm flagella). Courtship, coordination and deafness defects are also apparent in two extant alleles of beethoven, btv5P1 and btvk07109; however, males are fertile and their sperm are motile, although their successful copulation rate is reduced because of the coordination defects.24 Based on the evidence that the two extant btv alleles and several P-element derived alleles of DHC36D16 only exhibit deafness, it was likely that the male sterile phenotype associated with deletion of region 36DE was due to disruption of a distinct locus.

Initially, there were several uncharacterized candidates within this large deficiency overlap including several small predicted genes: CG5674 (with exons nested in the introns of btv), CG5693, CG15149, CG31740 and CG15150 (Fig. 2C). CG15150, which we here name elfless, (Fig. 2C and D) was further investigated as this gene had been previously isolated from testis cDNA pools25 and was therefore considered a promising candidate for the fertility defects seen in Df/Df flies. In this paper we describe experiments showing that elfless encodes a RING-finger protein, is expressed specifically in the tail cyst cells, but does not account for the male sterility in this interval. Mis-expressing elfless in most tissues is lethal, but we examine its possible biological role by mis-expressing it in the eye, a tissue dispensable for viability.

Results

elfless encodes a predicted nuclear RING-finger protein

FlyBase33 predicts the genomic region of elfless to be 1300 basepairs, comprising a 15 basepair 5′UTR, a 587 basepair 3′UTR, 3 exons and 2 short introns (Fig. 2D). The full-length cDNA of elfless is 564 basepairs. The Berkeley Drosophila Genome Project (BDGP) isolated thousands of cDNA clones in large-scale screens of libraries from different Drosophila tissues. One such clone, AT24563 (a cDNA from a Drosophila Adult Testes Library), contains a 1,194 basepair full-length elfless cDNA, with 3′ and 5′ UTR ends (Fig. 3); this cDNA clone was used for further analyses, as discussed below.

Figure 3
The elfless nucleotide and amino acid sequences. The elfless 5′UTR is shown in blue, exons are in black (ATG start codon in bold), introns in grey and the 3′UTR in red with putative polyadenylation site (AATAAA) in bold. The elfless protein ...

elfless is predicted to encode a 187 amino acid zinc finger Cys3-His-Cys4 RING protein33 (C3HC4 RING; Fig. 2E); amino acids 135–174 align with the reported RING consensus sequence34 [C-X(2)-C-X(9-39)-C-X(1-3)-H-X(2-3)-C-X(2)-C-X(4-48)-C-X(2)-C] (where X is any amino acid, C is Cys and H is His; Fig. 3). RING domains are involved in protein-protein interactions, including binding to E2 ubiquitin conjugating enzymes and have recently been shown to function as E3 ubiquitin ligases and essential regulators of apoptosis.35-37 Indeed, the RING finger protein Elfless is predicted to encode an E3 ubiquitin ligase.33 Furthermore, in a yeast two-hybrid assay, Elfless was shown to interact with the E2 ubiquitin conjugating enzyme UbcD1 (FlyGrid; http://biodata.mshri.on.ca/fly_grid/).

BLASTp analysis with the Elfless protein sequence revealed no strong matches (>50% identity), but, in general, it was found that Elfless shares some similarity with C3HC4-type RING-finger proteins in D. melanogaster and other organisms, almost exclusively in the RING domain. Similarly, PSORT (http://psort.hgc.jp) analysis also identified a C3HC4 RING domain. Additionally, PSORT identified a sequence (PPNKRIK; Fig. 3) with 65.2% probability to be a nuclear localization signal.

elfless is expressed in the tail cyst cells in testes

Based on the EST data we wanted to determine the elfless expression pattern. Initially, our focus was primarily on adult tissues for two reasons: (1) we were investigating elfless as a candidate for male sterility and (2) data collected from the BDGP in situ screen indicated that elfless has no detectable embryonic expression pattern. Real time PCR analysis indicates that elfless expression is strongly enhanced in the testes and whole males, while it is virtually undetectable in males with testes removed (carcasses) and whole females (Fig. 4; see Fig. for complete statistical analysis).

Figure 4
elfless transcripts are greatly enhanced in testes. Levels of elfless transcripts, isolated from various tissues, relative to Gapdh2 control transcripts. elfless is most abundant in testes (~1 copy of elfless for every 3 copies of Gapdh2), and ...

To understand more completely the spatial and temporal expression of elfless, we employed the pPTGAL vector32 to generate elfless-Gal4. In this construct, elfless regulatory sequences were cloned upstream of Gal4 and therefore Gal4 expression follows an elfless-specific pattern. A 2.27 kilobase fragment that includes the elfless TATA box and presumptive 5′ regulatory elements was cloned into pPTGAL, and transgenic flies with the resulting plasmid were used as elfless-Gal4.

In testes, elfless-Gal4 driven GFP expression was prominent in the cytoplasm of cells whose position and morphology are consistent with tail cyst cells (Fig. 5D–F). Outside of testes, no reproducible GFP expression was detected. Expression was assayed in all embryonic stages, in L1 and L3 larvae, in early- and mid-pupal stages and in adults (data not shown). Sporadic GFP expression was seen in a very small number of cells in the L3 salivary glands and Malpighian tubules—the position of these cells was not reproducible, suggesting that this expression represents noise in the Gal4 system rather than real elfless expression.

Figure 5
elfless is expressed in testes. All UAS-constructs are driven by elfless-Gal4 and the expression pattern is restricted to the testes. ((A–C), UAS-elfless-EGFP; (D–F) UAS-cytoplasmic-GFP) (A) UAS-elfless-EGFP expression is limited to the ...

To determine the subcellular localization of Elfless, a UAS construct containing elfless cDNA C-terminally fused with EGFP (UAS-elfless-EGFP) was generated. elfless-EGFP expression driven by elfless-Gal4 is restricted to a subset of nuclei whose location is again consistent with the tail cyst cell nuclei. Expression is apparent neither in the 64-unit sperm bundle nuclei nor in the head cyst cell nuclei that are proximal to the spermatid nuclei (Fig. 5A). Nuclear localization is consistent with the PSORT prediction of a nuclear localization signal. To confirm nuclear localization, a genetic approach to counter-staining nuclei was undertaken with the reporter construct, UAS-RedStinger-nls, that expresses a nuclear localized RFP (nucDsRed), cloned from the coral Discosoma.29 In w; UAS-RedStinger-nls/+; UAS-elfless-EGFP/elfless-Gal4 testes, elfless-EGFP expression co-localizes with nucDsRed, confirming that Elfless-EGFP is indeed a nuclear localized fusion protein (Fig. 5B and C). When testes from the parental UAS-RedStinger-nls flies are observed in the absence of a Gal4 driver, significant, spurious expression of nucDsRed in the spermatid bundle and head and tail cyst cell nuclei is evident (data not shown); this accounts for the incomplete co-localization in Figure 5C. Since GFP alone localizes to the cytoplasm (Fig. 5D–F), the nuclear localization of Elfless-EGFP most likely relies on the predicted nuclear localization signal in the Elfless protein; formal confirmation would require mutating this predicted signal.

To confirm the identity of expressing cells as tail cyst cells, we introduced the tail cyst cell lacZ enhancer trap, P{lacW}498 (reviewed in ref. 2), into the elfless-Gal4, UAS-elfless-EGFP genotype. X-gal staining coincides to a large extent with elfless-EGFP expression (Fig. 6). In these flies we found the number of X-gal positive cells to vary considerably from fly to fly; Figure 6 is an example with a larger fraction of the tail cyst cells, but not all of them, labelled (reviewed in ref. 2).

Figure 6
Elfless-EGFP is co-expressed with a tail cyst cell marker. A testis from a male with elfless-Gal4 driving UAS-elfless-EGFP in the presence of the tail cyst cell enhancer trap P{lacW}498 was processed for X-gal staining to reveal the tail cyst cell nuclei ...

elfless fails to rescue male sterility in Df/Df

Because of the specific elfless expression in testes, we wanted to test whether elfless is responsible for the male sterility in Df/Df males. To this end, a UAS rescue construct, UAS-elfless, and a genomic rescue construct, Casper-elfless, were generated. The genomic elfless construct contains the entire elfless coding region and the presumptive upstream regulatory elements (discussed above). Two independent genomic construct insertions, one each on chromosomes 2 and 3, failed to rescue male sterility when Df/Df males were crossed to w1118 virgin females (N = 10; no offspring produced). In testis squashes, the sperm were amotile and remained encysted like those of the Df/Df testes shown in Figure 2A (data not shown).

UAS-elfless was generated for expression with the Gal4/UAS system.31 The UAS-elfless construct was generated from the full-length elfless cDNA, AT24563, by subcloning into the pUAST vector.31 As with the genomic elfless, UAS-elfless driven by elfless-Gal4 fails to rescue male sterility in Df/Df (N = 10; no offspring produced). In testis squashes the sperm were amotile and remained encysted like those of Df/Df (data not shown).

elfless null alleles do not disrupt fertility

To rule out the possibility that failure of elfless transgenes to rescue male sterility in a Df/Df background was due to a problem with the transgenes, or the alternative possibility that Df/Df uncovered more than one fertility locus, we wanted to examine elfless-specific loss of function. Therefore, elfless null alleles were generated using two genetic techniques: P-element excision and piggyBac-mediated FLP-FRT deletion. The P-element, KG02815,38 inserted just downstream of the 5′UTR of PFE (rdo+) and 3′ of elfless (Fig. 2C), was used in a P-element mobilization screen for imprecise excisions that disrupt elfless. 146 total putative excision lines were PCR screened and in two lines, elflessKG02815.8b and elflessKG02815.15a, the entire elfless presumptive upstream regulatory region and coding region were deleted while the neighboring predicted genes, CG31740, CG15149 and CG5693, remained intact. These lines are also rdo likely due to a flanking deletion into PFE; the exact breakpoints of these deletions were not determined. At the same time as the P-element mobilization screen, the more precise heat shock FLP-FRT technique for generating deletions through recombination between nearby transposon insertions was also performed.30,39 Two piggyBac insertions, WHf06603 (rdo+) and WHf06680 (rdo), are inserted on either side of elfless (Fig. 2C) and FLP-mediated recombination was used to delete the intervening region that includes elfless, CG31740, CG15149 and CG5693. Heat shock and crosses were performed as described30 and two deficiency strains Df(2L)JCC.YA2 and Df(2L)JCC.PE2 were isolated.

We wanted to determine if deletion elfless blocked or reduced fertility. Fertility of P-element derived deletion lines, elflessKG02815.8b and elflessKG02815.15a was not significantly reduced compared to wild type controls, and FLP-FRT-derived deletion lines, Df(2L)JCC.YA2 and Df(2L)JCC.PE2 showed modest, but significantly reduced fertility that was later determined to be due to the reduced fecundity of the original WHf06680 piggyBac strain (data not shown). Altogether, these data indicate that elfless is not essential for fertility. Furthermore, since the FLP-mediated deletions also remove three additional predicted genes, these four genes neither solely nor together are responsible for the male sterility in Df/Df.

A gain-of-function role for elfless in apoptosis

Since many developmentally restricted genes have no phenotype when expressed ubiquitously, we were surprised to find that UAS-elfless mis-expression is embryonic or first instar larval lethal with most Gal4 drivers including: (1) a ubiquitous Gal4 driver spaghetti-squash-Gal4 (reviewed in ref. 40), (2) nervous system drivers elav-Gal4 (FBst0008760)41 and NP6215-Gal4 (FBst0304010),42 and (3) salivary gland driver sgs3-Gal4 (FBst0006870).43 In contrast, a photoreceptor driver GMR-Gal4 (FBal0052387)44 and the cyst cell driver elfless-Gal4 that we describe here, both allow adult survival. Expression with GMR-Gal4 resulted in eye defects, as described below. This, together with the lethality associated with mis-expression of elfless in other tissues, shows that this molecule is detrimental in many tissues. This conclusion introduced the possibility that this molecule might inappropriately regulate the apoptosis pathway in these various tissues, as other E3 ubiquitin ligases can induce or inhibit apoptosis.13,35,37,45

To determine whether ectopic elfless could be interfering with the apoptosis pathway, we further explored the gain-of-function eye phenotype with GMR-Gal4. We found that w; GMR-Gal4; UAS-elfless flies exhibit morphological defects including missing or extra inter-ommatidial bristles, rather than the stereotypical single inter-ommatidial bristle seen in wild type eyes (Fig. 7A). Furthermore, the eye itself is prone to deformation (Fig. 7B), due to a presumptive loss of ommatidial ultrastructural integrity, and the individual ommatidia also exhibit pigment loss in random patterns over the surface of the eye (Fig. 7A)—pigment loss and structural defects become more advanced as the fly ages (data not shown). Notably, the size of the eye, despite the roughness and pigment loss, is largely unchanged in this gain-of-function line (Fig. 7A and B).

Figure 7
Ectopic mis-expression of elfless induces apoptosis. (A, C and E) Light microscopy (B, D and F) SEM with insets. (A and B) w; GMR-Gal4/+; UAS-elfless/+ flies exhibit pigment loss and other defects such as loss or duplication of the interommatidial bristles ...

The ommatidial bristle loss and gain and the pigment loss phenotypes are similar to those exhibited by several members of the apoptosis pathway including w; GMR-Gal4; UAS-Dronc.46 In this strain, like in w; GMR-Gal4; UAS-elfless, the size of the eye is largely unaffected whereas in other apoptosis pathway lines, like GMR-hid and GMR-rpr (where hid or rpr are expressed by direct GMR promoter fusion), the size of the eye is greatly reduced.47-49 These observations suggested that elfless is not a pro-apoptotic gene like rpr or hid, but may be acting further downstream in the apoptosis pathway.

A key regulator of apoptosis is DIAP1, the Drosophila Inhibitor of Apoptosis 1.27,37,46,50-53 DIAP1 directly inhibits downstream caspases, Dronc46,54,55 and Drice56,57 and prevents cell death. The regulation of Dronc is accomplished through ubiquitination of Dronc via the C-terminal RING domain of DIAP1 in a UbcD1-dependent manner.37 Contrarily, when the cell receives an intrinsic or extrinsic signal to die, the N-Terminal RHG domains of proapoptotic factors Rpr, Hid and Grim bind to the C-terminal BIR (baculovirus IAP repeat) domain of DIAP1,58 and thereby antagonize DIAP1/UbcD1-mediated inhibition on the zymogen form of the caspase Dronc. Furthermore, binding of Grim, Rpr and Hid to DIAP1 promotes auto-ubiquitination of DIAP1, again in a UbcD1-dependent fashion.27,51-53,59 This second level of downregulation of DIAP1 ensures liberation of the initiator caspase, Dronc, which is then able to associate with the adaptor molecule, Dark,60 and this active complex then processes and activates downstream effector caspases, Drice and Dcp-1,46,61 that initiate PCD.

Elfless has been experimentally demonstrated to interact with UbcD1 by yeast two-hybrid and, as mentioned above, UbcD1 has been implicated as a key regulator of the apoptosis pathway at the intersection between DIAP1 and the caspases. When w; GMR-Gal4; UAS-elfless was crossed into a ubcD1 heterozygous mutant background, the eye phenotype became significantly more severe, indicating increased apoptosis, and the vast majority of these flies die as pharate adults (Fig. 7C and D). Conversely, reducing the level of the downstream caspase, Dronc, suppressed the eye phenotype, as the eyes of w; GMR-Gal4; UAS-elfless flies in a Dronc heterozygous mutant background show improvement in both the extent of pigmentation loss and overall eye morphology/deformation (Fig. 7E and F). Together these data show that elfless genetically interacts with key members of the apoptosis pathway that are downstream of the pro-apoptotic genes (Fig. 9).

Figure 9
Model for elfless in apoptosis. In these simplified models of apoptosis, the strength of inhibition (t-bars), activation (arrowheads) or ubiquitination (inverted arrowheads) is represented by line weight. (A) In a wild type non-apoptotic cell, the key ...

To determine whether protein levels of DIAP1 or UbcD1 are affected upon Elfless-EGFP mis-expression in the eye, or overexpression in the tail cyst cells, we isolated protein from heads or testes of flies carrying the appropriate Gal4 driver, with or without the UAS-elfless-EGFP construct, and probed Western blots with either DIAP1 or UbcD1 antibodies. We found that in both tissues, DIAP1 protein levels were increased, while UbcD1 protein levels remained unchanged (Fig. 8). These results, together with the genetic interactions, indicate that the apoptosis pathway is affected by these genetic manipulations.

Figure 8
DIAP1, but not UbcD1, is increased upon Elfless expression. Western blot of protein isolated from GMR-Gal4 control heads (H), or Elfless-Gal4 control testes (T), was probed with antibodies against DIAP1, UbcD1 or the loading control, Hsp60. Compared to ...

Discussion

Emerging data are now elucidating the roles of key members of the apoptosome, including the caspases Dronc and Drice, in spermatid individualization in Drosophila.9,13 The molecules are thought to be inhibited by dBruce, a ubiquitin-conjugating enzyme with a BIR domain.9,10 In effect, dBruce may be acting in the spermatid cysts in a manner similar to another documented BIR-domain protein, DIAP1. In this study, we examined the role of the E3 ubiquitin ligase, Elfless, and show that, consistent with its prediction as a RING finger protein, Elfless interacts with key members of the apoptotic pathway.

Based on our results and the following published observations we propose that Elfless acts to directly or indirectly regulate UbcD1 activity in the apoptotic pathway (Fig. 9). w; GMR-Gal4; UAS-elfless in an otherwise wild type background produces pigment cell defects reminiscent of those of w; GMR-Gal4; UAS-Dronc (Fig. 7A and B). As previously shown, w; GMR-Gal4; UAS-Dronc in a ubcD1 heterozygous mutant background produces an eye phenotype slightly worse than w; GMR-Gal4; UAS-Dronc alone37 which suggests that Dronc may also be a target of UbcD1. Finally, inhibition of apoptosis through Dronc is effectively lost in w; GMR-Gal4; UAS-Dronc in a diap1/+ background; the eye defects exhibited in these lines are severe and flies die as pharate adults.55 w; GMR-Gal4; UAS-elfless flies in a ubcD1/+ mutant background similarly die as pharate adults (sublethal in males and females; analyzed by χ2: p < 0.05, N = 25 vials/allele; data not shown) and the eye phenotype is significantly worse than that of w; GMR-Gal4; UAS-elfless alone (Fig. 7C and D); pigmentation is absent in these flies and the size of the eye is reduced due to decreased UbcD1-mediated inhibition of Dronc and DIAP1. These data suggested that Elfless may be regulating UbcD1 activity.

While Elfless and UbcD1 had been shown to interact by yeast-two hybrid, we have not been able to confirm a direct association between Elfless and either DIAP1 or UbcD1 by co-immunoprecipitation experiments (data not shown). Nevertheless, it is clear from Western blot analysis that mis-expression of Elfless-EGFP in the eye or testes does not significantly change the level of UbcD1 protein but does increase DIAP1 protein levels (Fig. 8). Thus, if Elfless is downregulating UbcD1 activity, it seems to be doing so without changing UbcD1 levels. Since DIAP1 auto-ubiquitination is UbcD1-dependent, Elfless-mediated downregulation of UbcD1 activity is consistent with reduced DIAP1 auto-ubiquitination and degradation, resulting in higher DIAP1. While on the one hand this would increase the anti-apoptotic activity of DIAP1, UbcD1 downregulation on the other hand would also increase the pro-apoptotic activity of Dronc, effectively circumventing DIAP1, and producing the somewhat mild eye phenotype evident in w; GMR-Gal4; UAS-elfless in an otherwise wild type background. Consistent with this model, in ubcD1 heterozygotes the eye phenotype is severely worsened and lethality is evident, while in Dronc heterozygotes, the eye phenotype is improved.

Although we clearly demonstrate a genetic interaction of elfless with Dronc and ubcD1 in PCD and we propose that mis-expressed Elfless in the eye negatively regulates UbcD1 activity, we were unable to ascribe an essential role in fertility to this locus, despite the promising role of this molecule in sperm development based on expression profiling and the Df/Df male sterility phenotype. Furthermore, an elfless transgene is not sufficient to rescue the male sterility associated with these deficiencies in polytene region 36DE. Despite the lack of a sterility phenotype when the RING finger protein encoded by elfless is deleted from the genome, the fact that elfless is functionally retained in the genome by selection indicates that elfless performs an important and advantageous, albeit redundant, function in the testes. Thus, while in laboratory vials, males are able to produce normal numbers of offspring, subtle functional differences can be quite significant in wild populations. What advantage could Elfess provide in the tail cyst cell? One possibility is that mobilization of Elfless in the nuclei of these cells facilitates tail cyst cell degeneration for more efficient resorption; sperm release from the cyst can still take place, but there may be a physiological burden on the testis during the life-time of the fly. The fact that Elfless is in the nucleus may suggest that it targets gene expression at this transitional stage of tail cyst cells. Full activation of apoptosis by Elfless in tail cyst cells is unlikely, as Elfless is nuclear, and no TUNEL staining in wildtype testes appears in the late stage cysts (data not shown). Nevertheless, apoptosis signaling can be quite different in varying developmental contexts (reviewed in refs. 10 and 13), and there is precedent for enlisting branches of the apoptotic pathway in different developmental processes. For example, Caspase-3 is activated in the cystic bulge of the developing spermatocyst,9 while the correct balance of DIAP1 levels is important for cellular mobilization of border cells and other cells during oogenesis.62,63 More incisive future approaches will be needed to discern the roles that Elfless and other subtle modulators play in mating and evolution.

Materials and Methods

Animals

Control strains used for fertility tests were of the genotypes w1118 (Exelixis isogenic, henceforth isow; FBst0006326) and y w. Df(2L)TW119, cn bw/CyO (FBab0001642) and Df(2L) TW201, b/CyO (FBab0001650)15 lines were used for deficiency mapping. We will refer to flies carrying these two overlapping deficiencies as Df/Df. Genetic interaction studies were performed with the following stocks: w;; DroncI24 FRT80/TM3, Sb (reviewed in ref. 26), Sco/Cyo; GMR-rpr (reviewed in ref. 27), w1118; ubcD1XS347/TM3, Sb GMR-sina (reviewed in ref. 28) and w1118; ubcD1598/TM3, Sb GMR-sina (reviewed in ref. 27). Co-localization with UAS-elfless-EGFP was analyzed with w1118; P{UAS-RedStinger}4/CyO (FBst0008546)29 and with the y w; P{lacW, w+}498 tail cyst cell enhancer trap line.2 See references in text for genotypes of Gal4 drivers used in these analyses. Transposable element strains are described below.

Fertility assays

Single newly emerged males were isolated and were allowed to mate with five isow or y w virgin females, as appropriate, for 2 days. Prior to crossing, isolated males and females were stored separately for 1 day to allow cuticles to sufficiently harden. After 2 days of mating, females were then moved into separate vials and the original vial was likewise retained. Male fertility for each genotype was scored as the average of the total number of offspring that emerged from all vials in a 5-day period (N = 10 males for all genotypes tested).

P-element excisions and piggyBac deletions

The y1 w67c23; P{SUPor-P}KG02815 (FBst0012989) P-element was mobilized by crossing to transgenic flies carrying a source of P-transposase, wgSp-1/CyO; ry506 Sb1 P{Δ2-3}99B/TM6,UbxP15 (FBst0002535). y w derivatives were screened and independent lines established. Heat shocking larvae for generating FLP-FRT deletions with Exelixis piggyBac insertion lines isow; Pbac{WH}f06603 (FBst1020693) and isow; Pbac{WH}f06680 (FBst1020745) was performed as described.30 Likewise, putative deletion lines were isolated and stable stocks generated as described using the Exelixis isogenized w strains, P{hsFLP}1, w1118; Adv1/CyO (FBst0000006) and w1118; P{hs-hid}2, wgSp-1/CyO (FBst0007757). Primers used to screen for P-element and piggyBac deletions in the full-length coding regions of elfless and the neighboring predicted genes are as follows (Table 1): elfless-FullF and elfless-FullR, CG31740Fwd and CG31740Rev, CG15149Fwd and CG15149Rev and CG5693Fwd and CG5693Rev and the PCR program [{95°C/30 s, 55.9°C/30 s, 72°C/1 min} × 30; {72°C/5 min} × 1].

Table 1
Primers used in this study

Cloning

The UAS-elfless construct was generated by subcloning the full length elfless cDNA from the AT24563 EST clone (Accession #AY113298) into pUAST;31 this UAS construct was injected into w1118 embryos using standard techniques. UAS-elfless-EGFP was generated by amplifying the elfless cDNA from the AT24563 vector template with primers elf-GWF and elf-GWRv2 (Table 1) and the PCR program [{95°C/30 s, 55.0°C/30 s, 72°C/30 s} × 30; {72°C/5 min} × 1]. The elf-GWF primer has an engineered CACC sequence before the ATG start, to ensure directional cloning into the pENTr entry of the Drosophila Gateway vector system (Invitrogen). The amplified elfless cDNA was cloned in the pENTr/D-TOPO vector, sequenced to verify and subcloned into the Drosophila Gateway pTWG destination vector by the LR recombination reaction as described (Invitrogen and Murphy Lab http://www.ciwemb.edu/labs/murphy/Gateway vectors.html). The final construct was injected at the Model System Genomics Facility at Duke University (http://www.biology.duke.edu/model-system/).

The full-length genomic region of elfless was PCR-cloned from Canton S DNA (4040 basepairs into TOPO-XL) with the primers SKF-5′F and SKF-5′R (Table 1) and the PCR program [{95°C/30 s, 54.0°C/30 s, 72°C/5 min} × 30; {72°C/10 min} × 1] and sequenced to verify clone accuracy against the published sequence. This 4040-basepair clone was used to generate an elfless genomic rescue clone by restriction enzyme digest subcloning into pCaSpeR4. To generate an elfless-Gal4 driver, a 2.27 kilobase fragment that includes the elfless TATA box and presumptive regulatory elements was subcloned from the genomic clone in TOPO-XL into the pPTGAL vector.32 Both resulting plasmids were injected by the Model System Genomics Facility at Duke University.

Real time PCR

Total RNA was isolated from tissues indicated in text using the RNeasy mini kit (Qiagen) with DNaseI treatment (Qiagen) per kit instructions. For real time PCR, first strand synthesis was performed on isolated total RNA using the Superscript III First Strand Synthesis System for RT-PCR (Invitrogen) and oligo(dT)20 primer per kit instructions. Real time PCR was carried out with isolated cDNAs and the SYBR Green PCR Master Mix (Applied Biosystems) with Gapdh2 control primers Gapdh2-f and Gapdh2-r and elfless primers elfless-realtimeF and elfless-realtimeR (Table 1). To control for pipetting errors, cDNAs from a biological sample were amplified in triplicate for both control and elfless primers; three biological samples per strain per genotype were analyzed. Real time PCR was performed using the following cycling conditions: [{95°C/10 min} × 1; {95°C/15 s, 60°C/1 min} × 40] and a single dissociation protocol {60°C + 35°C/20 min}. In the dissociation step, a single peak was obtained for Gapdh2 and elfless primers indicating only a single, specific product was amplified.

Scanning electron microscopy

Heads were isolated and fixed in 2.5% glutaraldehyde for 24 hours at 4°C, postfixed in 1% OsO4/Phosphate Buffer for 1 hour, dehydrated in an ethanol series, critical-point dried and finally sputter coated with a layer of Gold/Palladium. Images were taken on a Hitachi S-4000.

Western analysis

Fifty pairs of testes or 20 heads from control and Elfless-EGFP-expressing animals were homogenized in buffer (150 mM NaCl, 0.05% Nonidet p40, 50 mM Tris-HCl, pH 8.0 and protease inhibitors (Roche)). After 3 h of incubation at 4°C followed by 15 min centrifugation at 4000 g, crude extract was twofold diluted with lysis buffer (50 mm Tris/HCl, pH 8.0, 150 mm NaCl, 0.05% NP40, 5 mm EDTA, 0.2% BSA, 0.02% NaN3 and protease inhibitors). Lysate (20 μg/lane) from each preparation was run out on a 4–12% polyacrylamide gel. Blots were probed with antibodies against DIAP1 (gift of Hermann Steller), UbcD1 (gift of Hyung Don Ryoo) and Hsp60 (Sigma).

Statistical analysis

All data were analyzed using SAS version 6.12 (Sas Institute Inc., Cary, NC, USA). Real time PCR and fertility data were analyzed by ANOVA with pair-wise Tukey and counts for the w; GMR-Gal4; UAS-elfless with ubcD1 and Dronc alleles were analyzed by χ-square with an expected 1:1:1 Mendelian ratio.

Acknowledgements

We would like to thank Grace Boekhoff-Falk for critical reading of the manuscript. Steve DiNardo, Andreas Bergmann, Yehuda Ben-Shahar, Toshi Kitamoto, Wayne Johnson, Barbara Sisson and the Bloomington Drosophila stock center for providing fly stocks, the Duke University Model Systems Genomics Facility for injection of the elfless constructs, and the University of Iowa Central Microscopy Facility for SEM use. This work was supported by NIH grant DC04848 to Daniel F. Eberl.

Abbreviations

RING
really interesting new gene
GMR
glass multimer reporter
PCD
programmed cell death

References

1. Castrillon DH, Gönczy P, Alexander S, Rawson R, Eberhart CG, Viswanathan S, DiNardo S, Wasserman SA. Toward a molecular genetic analysis of spermatogenesis in Drosophila melanogaster: characterization of male-sterile mutants generated by single P element mutagenesis. Genetics. 1993;135:489–505. [PMC free article] [PubMed]
2. Gönczy P, Viswanathan S, DiNardo S. Probing spermatogenesis in Drosophila with P-element enhancer detectors. Development. 1992;114:89–98. [PubMed]
3. Gönczy P, DiNardo S. The germ line regulates somatic cyst cell proliferation and fate during Drosophila spermatogenesis. Development. 1996;122:2437–47. [PubMed]
4. Fuller MT. Spermatogenesis. In: Bate M, Martinez Arias A, editors. The Development of Drosophila. Cold Spring Harbor Press; Cold Spring Harbor: 1993. pp. 71–147.
5. Williamson A, Lehmann R. Germ cell development in Drosophila. Annu Rev Cell Dev Biol. 1996;12:365–91. [PubMed]
6. Gönczy P, Matunis E, DiNardo S. bag-of-marbles and benign gonial cell neoplasm act in the germline to restrict proliferation during Drosophila spermatogenesis. Development. 1997;124:4361–71. [PubMed]
7. Hime GR, Brill JA, Fuller MT. Assembly of ring canals in the germ line from structural components of the contractile ring. J Cell Sci. 1996;109:2779–88. [PubMed]
8. de Cuevas M, Lilly MA, Spradling AC. Germline cyst formation in Drosophila. Annu Rev Genet. 1997;31:405–28. [PubMed]
9. Arama E, Agapite J, Steller H. Caspase activity and a specific cytochrome c are required for sperm differentiation in Drosophila. Dev Cell. 2003;4:687–97. [PubMed]
10. Cagan RL. Spermatogenesis: borrowing the apoptotic machinery. Curr Biol. 2003;13:600–2. [PubMed]
11. Fabrizio JJ, Hime G, Lemmon SK, Bazinet C. Genetic dissection of sperm individualization in Drosophila melanogaster. Development. 1998;125:1833–43. [PubMed]
12. Noguchi T, Miller KG. A role for actin dynamics in individualization during spermatogenesis in Drosophila melanogaster. Development. 2003;130:1805–16. [PubMed]
13. Steller H. Regulation of apoptosis in Drosophila. Cell Death Differ. 2008;15:1132–8. [PubMed]
14. Tokuyasu KT, Peacock WJ, Hardy RW. Dynamics of spermiogenesis in Drosophila melanogaster. Z Zellforsch mikros Anat. 1972;127:492–525. [PubMed]
15. Wright TRF, Hodgetts RB, Sherald AF. The genetics of dopa decarboxylase in Drosophila melanogaster I. Isolation and characterization of deficiencies that delete the dopa-decarboxylase-dosage-sensitive region and the α-methyl-dopa-hypersensitive locus. Genetics. 1976;84:267–85. [PMC free article] [PubMed]
16. Sharma Y. Biological Sciences. University of Iowa; Iowa City: 2004. The Drosophila deafness gene beethoven encodes the Dynein Heavy Chain 1b isoform required for intraflagellar transport.
17. Caldwell JC, Fineberg SK, Eberl DF. reduced ocelli encodes the leucine rich repeat protein Pray For Elves in Drosophila melanogaster. Fly. 2007;1:146–52. [PMC free article] [PubMed]
18. Tauber E, Eberl DF. Song production in auditory mutants of Drosophila: the role of sensory feedback. J Comp Physiol A. 2001;187:341–8. [PubMed]
19. Tauber E, Eberl DF. Acoustic communication in Drosophila. Behav Proc. 2003;64:197–210.
20. Baker JD, Adhikarakunnathu S, Kernan MJ. Mechanosensory-defective, male-sterile unc mutants identify a novel basal body protein required for ciliogenesis in Drosophila. Development. 2004;131:3411–22. [PubMed]
21. Caldwell JC, Eberl DF. Towards a molecular understanding of Drosophila hearing. J Neurobiol. 2002;53:172–89. [PMC free article] [PubMed]
22. Eberl DF, Hardy RW, Kernan M. Genetically similar transduction mechanisms for touch and hearing in Drosophila. J Neurosci. 2000;20:5981–8. [PubMed]
23. Kernan M, Cowan D, Zuker C. Genetic dissection of mechanosensory transduction: mechanoreception-defective mutations of Drosophila. Neuron. 1994;12:1195–206. [PubMed]
24. Eberl DF, Duyk GM, Perrimon N. A genetic screen for mutations that disrupt an auditory response in Drosophila melanogaster. Proc Natl Acad Sci (USA) 1997;94:14837–42. [PMC free article] [PubMed]
25. Stapleton M, Carlson J, Brokstein P, Yu C, Champe M, George R, Guarin H, Kronmiller B, Pacleb J, Park S, Wan K, Rubin GM, Celniker SE. A Drosophila full-length cDNA resource. Genome Biol. 2002;3:1–8. [PMC free article] [PubMed]
26. Xu D, Li Y, Arcaro M, Lackey M, Bergmann A. The CARD-carrying caspase Dronc is essential for most, but not all, developmental cell death in Drosophila. Development. 2005;132:2125–34. [PMC free article] [PubMed]
27. Ryoo HD, Bergmann A, Gonen H, Ciechanover A, Steller H. Regulation of Drosophila IAP1 degradation and apoptosis by reaper and ubcD1. Nature Cell Biol. 2002;4:432–8. [PubMed]
28. Cadavid ALM, Ginzel A, Fischer JA. The function of the Drosophila Fat facets deubiquitinating enzyme in limiting photoreceptor cell number is intimately associated with endocytosis. Development. 2000;127:1727–36. [PubMed]
29. Barolo S, Castro B, Posakony JW. New Drosophila transgenic reporters: insulated P-element vectors expressing fast-maturing RFP. Biotechniques. 2004;36:436–42. [PubMed]
30. Parks AL, Cook KR, Belvin M, Dompe NA, Fawcett R, Huppert K, Tan LR, Winter CG, Bogart KP, Deal JE, Deal-Herr ME, Grant D, Marcinko M, Miyazaki WY, Robertson S, Shaw KJ, Tabios M, Vysotskaia V, Zhao L, Andrade RS, Edgar KA, Howie E, Killpack K, Milash B, Norton A, Thao D, Whittaker K, Winner MA, Friedman L, Margolis J, Singer MA, Kopczynski C, Curtis D, Kaufman TC, Plowman GD, Duyk G, Francis-Lang HL. Systematic generation of high-resolution deletion coverage of the Drosophila melanogaster genome. Nature Genet. 2004;36:288–92. [PubMed]
31. Brand AH, Perrimon N. Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development. 1993;118:401–15. [PubMed]
32. Sharma Y, Cheung U, Larsen EW, Eberl DF. pPTGAL, a convenient Gal4 P-element vector for testing expression of enhancer fragments in Drosophila. Genesis. 2002;34:115–8. [PMC free article] [PubMed]
33. Wilson RL, Goodman JL, Strelets VB. The Flybase Consortium. Flybase: integration and improvements to query tools. Nucl Acids Res. 2008;36:588–93. [PMC free article] [PubMed]
34. Saurin AJ, Borden KLB, Boddy MN, Freemont PS. Does this have a familiar RING? Trends Biochem Sci. 1996;21:208–14. [PubMed]
35. Freemont PS. Ubiquitination: RING for destruction? Curr Biol. 2000;10:84–7. [PubMed]
36. Lorick KL, Jensen JP, Fang S, Ong AM, Hatakeyama S, Weissman AM. RING fingers mediate ubiquitin-conjugating enzyme (E2)-dependent ubiquitination. Proc Natl Acad Sci (USA) 1999;96:11364–9. [PMC free article] [PubMed]
37. Wilson R, Goyal L, Ditzel M, Zachiariou A, Baker DA, Agapite J, Steller H, Meier P. The DIAP1 RING finger mediates ubiquitination of Dronc and is indispensible for regulating apoptosis. Nature Cell Biol. 2002;4:445–50. [PubMed]
38. Roseman RR, Johnson EA, Rodesch CK, Bjerke M, Nagoshi RN, Geyer PK. A P element containing suppressor of Hairy-wing binding regions has novel properties for mutagenesis in Drosophila melanogaster. Genetics. 1995;141:1061–74. [PMC free article] [PubMed]
39. Thibault ST, Singer MA, Miyazaki WY, Milash B, Dompe NA, Singh CM, Buchholz R, Demsky M, Fawcett R, Francis-Lang HL, Ryner L, Cheung LM, Chong A, Erickson C, Fisher WW, Greer K, Hartouni SR, Howie E, Jakkula L, Joo D, Killpack K, Laufer A, Mazzotta J, Smith RD, Stevens LM, Stuber C, Tan LR, Ventura R, Woo A, Zakrajsek I, Zhao L, Chen F, Swimmer C, Kopczynski C, Duyk G, Winberg ML, Margolis J. A complementary transposon tool kit for Drosophila melanogaster using P and piggyBac. Nature Genet. 2004;36:283–7. [PubMed]
40. Kiehart DP, Galbraith CG, Edwards KL, Rickoll WL, Montague RA. Multiple forces contribute to cell sheet morphogenesis for dorsal closure in Drosophila. J Cell Biol. 2000;149:471–90. [PMC free article] [PubMed]
41. Yao KM, White K. Neural specificity of elav expression: defining a Drosophila promotor for directing expression to the nervous system. J Neurochem. 1994;63:41–51. [PubMed]
42. Hayashi S, Ito K, Sado Y, Taniguchi M, Akimoto A, Takeuchi H, Aigaki T, Matsuzaki F, Nakagoshi H, Tanimura T, Ueda R, Uemura T, Yoshihara M, Goto S. GETDB, a database compiling expression patterns and molecular locations of a collection of Gal4 enhancer traps. Genesis. 2002;34:58–61. [PubMed]
43. Cherbas L, Hu X, Zhimulev I, Belyaeva E, Cherbas P. EcR isoforms in Drosophila: testing tissue-specific requirements by targeted blockade and rescue. Development. 2003;130:271–84. [PubMed]
44. Freeman M. Reiterative use of the EGF receptor triggers differentiation of all cell types in the Drosophila eye. Cell. 1996;87:651–60. [PubMed]
45. Nishito Y, Hasegawa M, Inohara N, Núñez G. MEX is a testis-specific E3 ubiquitin ligase that promotes death receptor-induced apoptosis. Biochem J. 2006;396:411–7. [PMC free article] [PubMed]
46. Meier P, Silke J, Leevers SJ, Evan GI. The Drosophila caspase DRONC is regulated by DIAP1. EMBO J. 2000;19:598–611. [PMC free article] [PubMed]
47. Bergmann A, Yang AY-P, Srivastava M. Regulators of IAP function: coming to grips with the grim reaper. Curr Opin Cell Biol. 2003;15:717–24. [PubMed]
48. Grether ME, Abrams JM, Agapite J, White K, Steller H. The head involution defective gene of Drosophila melanogaster functions in programmed cell death. Genes Dev. 1995;9:1694–704. [PubMed]
49. White K, Tahaoglu E, Steller H. Cell killing by the Drosophila gene reaper. Science. 1996;9:805–7. [PubMed]
50. Hay BA, Wassarman DA, Rubin GM. Drosophila homologs of baculovirus inhibitor of apoptosis proteins function to block cell death. Cell. 1995;83:1253–62. [PubMed]
51. Hays R, Wickline L, Cagan R. Morgue mediates apoptosis in the Drosophila melanogaster retina by promoting degradation of DIAP1. Nature Cell Biol. 2002;4:425–31. [PubMed]
52. Holley CL, Olson MR, Colón-Ramos DA, Kornbluth S. Reaper eliminates IAP proteins through stimulated IAP degradation and generalized translational inhibition. Nature Cell Biol. 2002;4:439–44. [PMC free article] [PubMed]
53. Yoo SJ, Huh JR, Muro I, Yu H, Wang L, Wang SL, Feldman RMR, Clem RJ, Müller H-AJ, Hay BA. Hid, Rpr and Grim negatively regulate DIAP1 levels through distinct mechanisms. Nature Cell Biol. 2002;4:416–24. [PubMed]
54. Dorstyn L, Colussi PA, Quinn LM, Richardson H, Kumar S. DRONC, an ecdysoneinducible Drosophila caspase. Proc Natl Acad Sci (USA) 1999;96:4307–12. [PMC free article] [PubMed]
55. Quinn LM, Dorstyn L, Mills K, Colussi PA, Chen P, Coombe M, Abrams J, Kumar S, Richardson H. An essential role for the caspase Dronc in developmentally programmed cell death in Drosophila. J Biol Chem. 2000;275:40416–24. [PubMed]
56. Fraser AG, McCarthy NJ, Evan GI. drICE is an essential caspase required for apoptotic activity in Drosophila cells. EMBO J. 1997;16:6192–9. [PMC free article] [PubMed]
57. Kaiser WJ, Vucic D, Miller LK. The Drosophila inhibitor of apoptosis D-IAP1 suppresses cell death induced by the caspase drICE. FEBS Lett. 1998;440:243–8. [PubMed]
58. Vucic D, Kaiser WJ, Miller LK. Inhibitor of apoptosis proteins physically interact with and block apoptosis induced by Drosophila proteins HID and GRIM. Molec Cell Biol. 1998;18:3300–9. [PMC free article] [PubMed]
59. Wing JP, Schreader BA, Yokokura T, Wang Y, Andrews PS, Huseinovic N, Dong CK, Ogdahl JL, Schwartz LM, White K, Nambu JR. Drosophila Morgue is an F box/ubiquitin conjugase domain protein important for grim-reaper mediated apoptosis. Nature Cell Biol. 2002;4:451–6. [PubMed]
60. Rodrigues A, Oliver H, Zou H, Chen P, Wang X, Abrams JM. Dark is a Drosophila homologue of Apaf-1/CED-4 and functions in an evolutionarily conserved death pathway. Nature Cell Biol. 1999;1:272–9. [PubMed]
61. Song Z, McCall K, Steller H. DCP-1, a Drosophila cell death protease essential for development. Science. 1997;275:536–40. [PubMed]
62. Geisbrecht ER, Montell DJ. A role for Drosophila IAP1-mediated caspase inhibition in Rac-dependent cell migration. Cell. 2004;118:111–25. [PubMed]
63. Zhao M, Szafranski P, Hall CA, Goode S. Basolateral junctions utilize Warts signaling to control epithelial-mesenchyme transition and proliferation crucial for migration and invasion of Drosophila ovarian epithelial cells. Genetics. 2008;178:1947–71. [PMC free article] [PubMed]
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