Wing-to-Leg Homeosis by Spineless Causes Apoptosis Regulated

MOLECULAR AND CELLULAR BIOLOGY, Apr. 2005, p. 3140–3150 Vol. 25, No. 80270-7306/05/$08.00�0 doi:10.1128/MCB.25.8.3140–3150.2005Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Wing-to-Leg Homeosis by Spineless Causes Apoptosis Regulated byFish-lips, a Novel Leucine-Rich Repeat Transmembrane Protein†

Takashi Adachi-Yamada,1,2,3*‡ Toshiyuki Harumoto,2‡ Kayoko Sakurai,4‡ Ryu Ueda,5Kaoru Saigo,6 Michael B. O’Connor,7,8 and Hiroshi Nakato4,7*

Department of Earth and Planetary System Sciences, Graduate School of Science and Technology,1 and Department ofSciences for Natural Environment, Faculty of Human Development,2 SORST, and Japan Science and Technology

Agency,3 Kobe University, Kobe, Department of Biology, Faculty of Science, Tokyo Metropolitan University,Hachioji,4 Genetic Strains Research Center, National Institute of Genetics, Mishima,5 and Department

of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, Tokyo,6 Japan,and Department of Genetics, Cell Biology, and Development7 and Howard

Hughes Medical Institute,8 University of Minnesota,Minneapolis, Minnesota

Received 24 May 2004/Returned for modification 6 July 2004/Accepted 21 December 2004

Growth, patterning, and apoptosis are mutually interactive during development. For example, cells thatselect an abnormal fate in a developing field are frequently removed by apoptosis. An important issue in thisprocess that needs to be resolved is the mechanism used by cells to discern their correct fate from an abnormalfate. In order to examine this issue, we developed an animal model that expresses the dioxin receptor homologSpineless (Ss) ectopically in the Drosophila wing. The presence of mosaic clones ectopically expressing ss resultsin a local transformation of organ identity, homeosis, from wing into a leg or antenna. The cells withmisspecified fates subsequently activate c-Jun N-terminal kinase to undergo apoptosis in an autonomous ornonautonomous manner depending on their position within the wing, suggesting that a cell-cell interaction is,at least in some cases, involved in the detection of misspecified cells. Similar position dependence is commonlyobserved when various homeotic genes controlling the body segments are ectopically expressed. The autono-mous and nonautonomous apoptosis caused by ss is regulated by a novel leucine-rich repeat family trans-membrane protein, Fish-lips (Fili) that interacts with surrounding normal cells. These data support a mech-anism in which the lack of some membrane proteins helps to recognize the presence of different cell types anddirect these cells to an apoptotic fate in order to exclude them from the normal developing field.

Signals that regulate cell growth, patterning, and apoptosisare interdependent during development (1, 19, 29, 37). Inparticular, there are likely to be multiple ways to elicit anapoptotic cell fate since apoptosis serves a variety of functionsin multicellular organisms (29). In regard to the cell autonomyof apoptosis, a cell-autonomous apoptosis occurs during cellcompetition, a phenomenon whereby cells that grow slowly dueto the mutation of essential genes such as Minute or ras areremoved later in development (39, 40, 49, 56). In contrast, ithas been reported that nonautonomous cell death is oftenassociated with cell fate changes by altering the morphogenactivities (2, 3) of Decapentaplegic (Dpp) (32, 41) or Wingless(Wg) (42, 60). In these cases, apoptotic cell death occurs bothwithin and outside of the abnormal cell population and isreferred to as morphogenetic apoptosis. Similarly, when spalt(sal), a target gene of the Dpp signal, is ectopically expressed

apoptosis is induced through interaction with the surroundingnormal cells (38). Thus, the removal of physiologically abnor-mal cells by apoptosis can occur by more than one mechanism,although the details of the various processes remain to beelucidated. In order to search for and analyze the ways toremove other types of abnormal cell, we have ectopically ex-pressed various master genes, including homeotic genes, tocreate cell populations with developmentally abnormal fates.In most cases, cells with abnormal fate induced severe apopto-sis in a cell autonomous or nonautonomous manner. Amongthese, we focus on the master gene ss, of which overexpressionin the wing changes the organ identity into a ventral appendagesuch as a leg or antenna.

Based on sequence identity and splice site conservation, Ss isthe closest Drosophila homolog of the mammalian dioxin re-ceptor (arylhydrocarbon receptor [Ahr]) (17, 27). Both mam-malian and Drosophila proteins can also bind to the xenotoxinresponsive element (XRE) and stimulate transcription fromgenes containing this cis-acting element (18). Although Ss hasnot been shown to bind to arylhydrocarbons, it regulates nor-mal morphogenesis of the leg or antenna and bristles, all ofwhich are major Drosophila sensor organs or tissues that re-spond to environmental chemicals.

Our results reveal that ectopic ss provokes a wing-to-legand/or antenna homeosis that subsequently elicits apoptosis inan autonomous or nonautonomous manner. This apoptoticresponse is regulated by a novel transmembrane leucine-rich

* Corresponding authors. Mailing address for T. Adachi-Yamada:Department of Earth and Planetary System Sciences, Graduate Schoolof Science and Technology, Kobe University, Kobe 657-8501, Japan.Phone: 81-78-803-7743. Fax: 81-78-803-7743. E-mail: [email protected]. Mailing address for H. Nakato: Department of Genet-ics, Cell Biology, and Development, University of Minnesota, Minne-apolis, MN 55455. Phone: (612) 625-1727. Fax: (612) 626-5652. E-mail:[email protected].

† Supplemental material for this article may be found at http://mcb.asm.org/.

‡ T.A.-Y., T.H., and K.S. contributed equally to this study.

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repeat (LRR) protein, Fili, and may be a common processinduced by ectopic expression of various homeotic genes.These results indicate that homeosis elicits a complex set ofsignals that influence the survival of the transformed cells andtheir surrounding cells. In addition, these results suggest ahypothesis that different LRR family transmembrane proteinsfunction in different subdomains of a developing field to rec-ognize developmentally misspecified cells and to regulate cellsurvival.

MATERIALS AND METHODS

Fly strains. We used two different GAL4 enhancer trap lines of scalloped (sd)in the present study. The (s) and (w) lines induce a strong and weak expressionof UAS-driven genes, respectively. The plasmid construct for tgo-RNAi was madeby inserting an inverted tgo cDNA into the 5� region of the standard UAS-tgoconstruct. When tgo-RNAi was induced singly in the wing, the fly wing showed nophenotype (data not shown).

The name fish-lips was derived from the observation that the expression pat-tern of fili in the wing disc made a fish lips-like shape. fili is identical to thetemporarily assigned hypothetical gene CG4054 in the Berkeley Drosophila Ge-nome Project. The fili-lacZ fly was identified during a screen of lacZ enhancertrap strains with P-lacW, a derivative of the transposon P-element (6). The filimutant allele �102 was isolated by an imprecise excision of P-lacW in fili-lacZaccording to the standard procedure. The UAS-fili fly was made by inserting thefili cDNA (2,297 bp, including full-length open reading frame, 18 bp of the 5�untranslated region and 62 bp of the 3� untranslated region) into the pUASTvector (7) with a white� marker and transforming the white� fly according tostandard procedures.

The other lacZ enhancer trap lines we used are sdETX4, Dll01092, hth05745,hid05014, ombP1, fz3SW076-A7.3XW, trnS064117, and pucE69. hid05014 was also used asa mutant (see Fig. 4E and G). At the late-third-instar larval stage, this hid-lacZis expressed irrespective of apoptosis around the dorsal-ventral boundary and atthe two spots in the dorsal and ventral hinge region (data not shown). However,at the mid-third-instar larval stage, these expressions do not begin, and theexpression is associated with apoptosis. Therefore, we observed the hepCA- orss-induced hid-lacZ expression by using the mid-third-instar larvae.

Detection of apoptosis. Caspase-3 plays a central role in many types of apo-ptosis, whereas c-Jun N-terminal kinase (JNK) activation elicits a limited groupof apoptotic events (14). In the Drosophila larval wing disc, activation of JNKalways leads to the activation of caspase-3 (2). Puckered (Puc) is a proteinphosphatase specifically inactivating JNK, and its transcription is induced by JNKsignal, thereby making a negative-regulatory circuit (36). Most of the endoge-nous and ectopic puc-lacZ expression observed in the present study was elimi-nated in a mutant background of the hep gene (20), which encodes the homologof mitogen-activated protein kinase kinase-7 that activates JNK. Immunofluo-rescence was carried out according to the standard procedure. The puc-lacZexpression is detected by measuring mouse anti-�-galactosidase antibody (1:200dilution; Promega Z378) and Cy3-labeled anti-mouse immunoglobulin (1:200dilution; Jackson Immunoresearch). The rabbit anti-active caspase-3 antibody(CM1) was a gift from Idun Pharmaceuticals. It specifically recognizes thecleaved and activated form of mammalian caspase-3 and has been shown to alsorecognize the cleaved form of the Drosophila caspase-3 homolog, Drice (59).Thus, active caspase-3/Drice was detected by CM1 (1:4,000 dilution) and Cy5-labeled anti-rabbit immunoglobulin (1:200 dilution; Jackson Immunoresearch).

Antibodies. The other antibodies we used were rat anti-Al (from G. Campbell[1:1,000 dilution]), rat anti-Sal (from R. Barrio [1:400 dilution]), mouse anti-Dac(MABDAC2-3 from the Developmental Studies Hybridoma Bank [1:10 dilutionof supernatant]), rabbit anti-Vg (from S. Carroll [1:200 dilution]), rabbit anti-p-Mad (PS1 from P. ten Dijke [1:200 dilution]), mouse anti-Arm (N2 7A1 from theDevelopmental Studies Hybridoma Bank [1:10 dilution of supernatant]), mouseanti-Dll (MAb DMDll.1 from D. Duncan [1:500 dilution]), and rabbit anti-Caps(from A. Nose [1:200 dilution]).

Mosaic overexpression analysis. Discs were prepared from larvae carrying theAyGAL4 (actin promoter-FRT-yellow�, terminator-FRT-GAL4 [28]), hs-FLP(yeast FLP recombinase gene driven by heat shock promoter), UAS-ss, andUAS-GFP transgenes. The GAL4-expressing clones were induced by heat treat-ment at 37°C for 20 min at 48 to 72 h after egg-laying and observed 48 to 72 hafter heat treatment. In each experiment, we observed more than 10 wing discsto confirm the results.

RESULTS

Overexpression of ss leads to a disappearance of the wing,which is dependent on tgo. Using the GAL4/UAS system (7),we overexpressed ss in the wing blade region by scalloped (sd)(s)-GAL4 (Fig. 1A). As a result, no adults developed wings(Fig. 1C). When we expressed ss in a more restricted regionusing the weaker allele, sd(w)-GAL4 (Fig. 1B), the wing mar-gin structure disappeared (Fig. 1D and E). These results sug-gest that ectopic expression of ss in the wing leads to massiveapoptosis.

Mammalian Ahr is known to function together with a struc-turally similar protein, Arylhydrocarbon receptor nucleartranslocator (Arnt), by forming a heterodimeric transcriptionfactor (27). To test whether the wing apoptosis phenotype by ssoverexpression is mediated by Tango (Tgo) (43), the Drosoph-ila homolog of Arnt, we examined flies in which we simulta-neously induced both ss expression and tgo-RNAi. Prior to thisexperiment, we examined whether tgo-RNAi can remove en-dogenous tgo function. When tgo-RNAi was induced in theantenna by using Distal-less (Dll)-GAL4, the antenna trans-formed to a leg-like organ as seen in the mutant of tgo (18)

FIG. 1. Activation of ss leads to loss of wing. (A and B) sd-strong(s)and sd-weak(w) expression patterns (green) in the wing imaginal discs.Anterior is to the left, and dorsal is at the top. (C) Wild-type fly (left)and a fly without wing by overexpression of ss under the control of thesd(s)-GAL4 driver (right). (D) Wild-type wing. (E) A wing overex-pressing ss under the control of the sd(w)-GAL4 driver shows anincision of the wing margin. (F) Front view of the normal fly head withwhite� compound eyes for easier recognition of antenna. (G) Frontview of the fly head in which tgo is repressed by UAS-tgo-RNAi withDll-GAL4. The antenna transforms to a leg-like appendage, which issimilar to the antenna mutant for ss or tgo (17, 18). (H) A wingoverexpressing ss but repressing tgo under the control of the sd(w)-GAL4 driver shows a reversion of the wing margin incision found inthe ss overexpression alone.

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(Fig. 1F and G). Thus, the tgo-RNAi was confirmed to repressthe endogenous tgo. When ss was induced together with thistgo-RNAi, the wing phenotype was completely suppressed (Fig.1H), suggesting that the loss of the wing due to ectopic expres-sion of ss requires tgo activity and is not simply due to a stresscaused by ectopic expression of ss.

Overexpression of ss leads to a homeotic transformationfrom wing to ventral appendage before disappearance of thecells. ss is known to specify the identities of tarsus (distal leg)and arista (distal antenna) during normal development (17,18). Thus, we thought that the ss overexpression in the wingmay have removed the organ identity as a wing and underwenta homeotic change to a leg, antenna, or other ventral append-age that does not actually exist in the normal fly prior todisappearance of the wing. To investigate this possibility, wenext examined the expression of marker genes specific to thewing or haltere or to leg or antenna. The sd gene, which isnormally expressed exclusively in the wing and haltere (10)(Fig. 2A), was found to be less strongly expressed in ss-over-expressing clones (Fig. 2C and C�). This result suggests that thess-overexpressing clones had lost, at least partially, their wing

identity. In contrast, Dll, a master gene that directs the identityof the distal leg or whole antenna (13, 45) (Fig. 2A�), wasectopically and cell autonomously induced in the ss-overex-pressing clones (Fig. 2D and D�). During normal wing devel-opment, Dll expression is induced in response to Wg signaling.However, Dll induction in the ss-overexpressing clones ap-peared to be independent of the Wg signal since Dfrizzled-3(fz3) induction and Armadillo (Arm) accumulation did notcoincide with the ss-overexpressing region, as demonstratedbelow. Therefore, we hypothesized that this Dll inductionmight provoke a different Dll function to redirect the identityof the wing to that of the distal leg or whole antenna. In fact,high-level induction of Dll in the wing has been reported tolead to its transformation into a distal leg (21). Although nor-mal expression of ss in the leg is known to be downstream ofDll (17), Dll expression is regulated by ectopic ss in the wing,indicating the presence of a positive feedback loop between Dlland ectopic ss.

Next, we examined the expression of various genes that aremarkers for different subdomains of the leg or antenna (Fig.2E to H) (8, 9, 15, 16, 35, 45; Flybase [http://flybase.bio.indiana

FIG. 2. Homeotic transformation of organ identity from that of a wing to that of a leg or antenna by overexpression of ss. (A and A�) Expressionof sd and Dll in the wild-type imaginal discs. Haltere (halt.), leg, wing, antenna (ant.), and eye discs are shown. (B) Expression of various genesas markers for the leg or antenna segments in the ss-overexpressing clones. A schematic representation at the bottom of the panel shows normalexpression in the antenna (left) and leg (right). The open bar in the sal expression in the antenna indicates the broad expression (in the a2 anda3 segments) found only in the early stage. This expression is restricted to a narrower region (only a2) later in development. A table indicatingincreases (�) or decreases (�) in gene expression in the ss-overexpressing clones in the wing blade and hinge regions is shown at the top. NE, noexpression was seen. (C to H and C� to H�) Wing discs showing an increase (white arrows) or decrease (gray arrows) of gene expression (magenta)in the ss-overexpressing clones (green). ss overexpression was induced, together with DIAP1 (IAP), to delay apoptosis except for panel H. Asterisksin panel G� indicate normal expression of Dac in the peripodial membrane. (I) An ectopic tarsus-like structure generated in the wing bycoexpression of ss and DIAP1 (IAP) with sd(s)-GAL4 driver. (I�) t1 segment of the distal mid-leg in wild type. (J and J�) Normal expression of ptc(green) and Wg (magenta) in the leg and wing. An asterisk indicates a ventral stripe of Wg expression in the leg. (K and K�) Expression of ptc(green) and Wg (magenta) in the wing in which ss is overexpressed by ptc-GAL4 driver. Asterisk indicates an ectopic stripe of Wg expression inthe ventral wing. The gray arrow indicates the reduced Wg expression that is not found in the normal wing.

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.edu/]). In summary, ss-overexpressing clones in the blade re-gion of the wing have most likely transformed their identity tothat of a distal leg (t1 area) rather than that of an antenna (Fig.2B). In the hinge region of the wing, Dll expression was notinduced by ss (Fig. 2D). Instead, the expression of hth, a mastergene that directs the identity of the proximal leg or wholeantenna (12, 44) was enhanced in the ss-overexpressing clones(Fig. 2H and H�). However, in the normal leg or antenna therewas no segment that expresses both ss and hth but not Dll (Fig.2B). Thus, the fate of the ss-overexpressing clones in the hingeregion of the wing should have transformed to that of a ventralappendage, although we have not identified which normal ap-pendage segment that the ss-overexpressing clones resemble.In comparison, a leg-antenna intermediate and a hypotheticalancestor appendage have been observed in Dll and ss mutants,respectively (17, 18).

We also obtained other evidence demonstrating that thecells with ss overexpression display characters of the ventralappendages. First, an ectopic appendage-like structure wasoften generated (Fig. 2I) when ss-induced apoptosis was par-tially inhibited by coexpression of Drosophila inhibitor of ap-optosis protein 1 (DIAP1 [26]). The structure most similar tothis appendage that is seen in external morphology of normalfly is the tarsus of leg (Fig. 2I�). Second, during normal devel-opment expression of patched (ptc) was dependent on Hedge-hog signaling, which induces a band-like expression of dpp inthe wing and the dorsal leg region and that of wg in the ventralleg region (5) (Fig. 2J and J�). When ss expression was induced

by ptc-GAL4, Wg was ectopically induced only in the ventralhalf of the ptc-expressing region (Fig. 2K and K�), as found inthe normal leg or antenna. However, the level of wg inductionin the ptc-expressing region was weaker than that found in thenormal leg. Therefore, the ss-expressing wing appears to havepartially transformed its identity to that of a leg or antenna.This mixed or conflicted identity may also be responsible forinduction of apoptosis. Although it has been shown previouslythat the ectopic expression of ss by ptc-GAL4 leads to a dele-tion of the central wing (17), the molecular mechanism was notidentified. Our results suggest that the deletion is triggeredprimarily by homeotic transformation.

Overexpression of ss leads to apoptosis in which cell auton-omy is position dependent. To study the detailed mechanismleading to apoptosis by ectopic expression of ss, we next exam-ined the cell autonomy of apoptosis in ss-overexpressingclones. We monitored caspase-3 and JNK activation by usingan antibody that recognizes active caspase-3 and the expressionof a reporter gene, puckered (puc)-lacZ, respectively. After48 h of mosaic induction, we observed two types of apoptosis(Fig. 3A). The ss-overexpressing clones inside the wing bladeprimordium showed strong activation of caspase-3 and a weakactivation of JNK (Fig. 3A� and A�). Activation of caspase-3 inthe vicinity of the dorsoventral compartment boundary (redline in Fig. 3A) was relatively weak and delayed. We canrecognize the delay of the cell removal around the dorsoven-tral boundary more readily at later stage after the clone induc-tion (data not shown). All of these responses were always cell

FIG. 3. Overexpression of ss leads to autonomous and nonautonomous apoptosis in a Dll-dependent manner. (A) A wing imaginal disc withmosaic clones overexpressing ss (green). JNK activation visualized by puc-lacZ expression (magenta) and caspase-3 activation (cyan) are shown.Red line denotes the D/V boundary. (A�-A��) Higher magnification of each clone in panel A. The total number of clones examined is 229. Amongthese, the number of clones exhibiting autonomous puc-lacZ induction is 30, whereas the number of those exhibiting nonautonomous puc-lacZinduction is 99. (B to B�) Wing imaginal disc with ss overexpression driven by the ptc-GAL4 (green). Either cell autonomous or nonautonomousapoptosis was observed symmetrically as a function of the distance of the cells from the dorsoventral boundary. Arrowheads and arrows denotea nonautonomous activation of JNK (magenta) and autonomous activation of caspase-3 (cyan), respectively. (C) Wing subdomains based on theresponse to ss. The Dll region is white, the vg region is red, and the hth region is blue. (D) Wing imaginal disc with mosaic clones overexpressingss (green) in the Dll5/Dll9 mutant background. JNK activation visualized by puc-lacZ expression (magenta) and caspase-3 activation (cyan) aregreatly reduced. (E) A wing with ss overexpression by sd(w)-GAL4 in the Dll5/� mutant background shows a strongly suppressed phenotype. (Fand F�) Wing imaginal disc with mosaic clones overexpressing Dll (green). DIAP1 (IAP) is coexpressed to delay apoptosis. puc-lacZ expression(magenta). Caspase-3 activation is indicated in cyan. Arrows and arrowheads denote an autonomous and nonautonomous activation of JNK,respectively. (F�) High magnification of the boxed area in panel C. (G) A wing with Dll overexpression by sd(w)-GAL4 shows an incision of thewing margin.



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autonomous. In contrast, the ss-overexpressing clones outsideof the wing blade region (that is the hinge region) showed weakactivation of caspase-3 and strong activation of JNK in cells oneither side of the clone boundary (Fig. 3A��). In this region,the outline of the clones which were probably induced at anearlier stage, are severely disrupted with strong activation ofboth caspase-3 and JNK (Fig. 3A��).

The presence of the two types of apoptosis can clearly berecognized in ss overexpression by ptc-GAL4, which is ex-pressed in a band intersecting the dorsoventral boundary (Fig.3B to B�). Strong, autonomous caspase-3 activation was ob-served in the blade region, whereas strong, nonautonomousJNK activation was observed around the hinge region. Theregion around the dorsoventral boundary did not show bothJNK and caspase-3 activation. Thus, the wing disc can bedivided into three subdomains based on the nature of theapoptotic response to ss overexpression in these regions (Fig.3C). Interestingly, these subdomains correlate with the expres-sion domains of several key transcriptional regulators. TheDll-expressing subdomain showed an autonomous and delayedresponse. The vestigial (vg)-expressing subdomain showed anautonomous and acute response. The homothorax (hth)-ex-pressing subdomain showed a nonautonomous response.These subdomains can be recognized by the foldings of the disccell layer or by distance between the nuclei, through counterstaining with DAPI (4�,6�-diamidino-2-phenylindole). Thevariance of apoptotic responses are summarized in Table 1.Similar traits in position-dependent cell autonomy of apoptosishave also been found in the clones mutant for the Dpp recep-tor Thick veins (Tkv) (3).

ss-induced apoptosis is dependent on Dll that is responsiblefor homeotic transformation from wing to leg or antenna. Toelucidate the role of Dll in ss-induced apoptosis, we placed thess-overexpressing clones in a strong Dll mutant Dll5/Dll9 back-ground. As shown in Fig. 3D, the activation of both JNK andcaspase-3 was strongly suppressed by this Dll mutation in thewing blade region. Activation was also suppressed in the hingeregion, although ss did not induce Dll expression in this region(Fig. 2D). A possible explanation is that broader expression ofDll in the hinge region at an earlier stage of development (datanot shown) is required for transformation by ss in later stages.The Dll mutation also suppressed the ss-induced adult wingphenotype (Fig. 3E). To test whether Dll overexpression is alsoable to lead to apoptosis, we next generated Dll-overexpressingclones in the wing. As shown in Fig. 3F, JNK and caspase-3activation are observed cell autonomously in the Dll-overex-pressing clones in the wing blade region. Other areas shownonautonomy. The wing notching phenotype induced by Dlloverexpression is found to be similar to that induced by ss

overexpression (Fig. 3G). Thus, ss-induced apoptosis is likelymediated by a pathway that is dependent upon Dll. A discrep-ancy existed in that the normal expression of Dll in the wing didnot induce leg identity or apoptosis. However, the elevatedexpression of Dll in the wing can induce some leg structure(21). Thus, the level of Dll expression, rather than its timing orplace, may be crucial for determining the fate as wing or leg.

Coincidence of round shape and apoptotic response. Apo-ptosis in each of these regions was dependent on Hemipterous(Hep, an activator of JNK [20]), DIAP1, Head involution de-fective (Hid; a proapoptotic protein [23]) and Tgo (Fig. 4B toF). DIAP1 is known to degrade caspase through its ubiquitin-protein ligase activity. Hid can bind to DIAP1 to stimulateautoubiquitination of DIAP1, which prevents the degradationof caspase (58). Interestingly, the ss-overexpressing clones inhep or hid mutant or in DIAP1 overproducer backgroundsretained a round shape, as seen with ss-overexpressing clonesin the wild-type background (Fig. 4A to E). In contrast, thess-overexpressing clones in the tgo-RNAi background lost theirround shape and invaded the surrounding normal cells (Fig.4F). These results indicate that hep and hid only affect theapoptosis pathway, whereas tgo mediated events more up-stream, such as the cell fate change induced by ss. Ectopicexpression of ss may make a difference in cell affinity betweenthe clones and surrounding normal cells, which leads to around shape of the clones and subsequent apoptotic response.

Consistently, activation of JNK pathway by constitutivelyactive Hep leads to hid expression (Fig. 4G). Besides, thess-overexpressing clone induces hid expression autonomouslyin the wing blade region but nonautonomously in the winghinge region (Fig. 4H).

Effect of ss overexpression on the intensity of Dpp and Wgsignaling. The JNK-dependent cell death on either side of theclone boundary is quite similar to what has been referred to as“morphogenetic apoptosis,” through which discontinuities inthe Dpp and Wg morphogen activity gradients are corrected(2). We next examined the activities of certain morphogensignals in the vicinity of the ss-overexpressing clones. Withregard to Dpp signaling, we can find a slight reduction in theexpression of the lacZ enhancer trap of optomotor-blind (omb),a target gene of the Dpp signal (24) (see Fig. S1A� and B� inthe supplemental material). However, the reduction is alwaysfound around the center of the clones and does not coincidewith the outline of the clones. We also see a similarly moderatealteration of the level of phospho-Mad, an active form of theDpp signal transducer “Mothers against dpp” (Mad) (48, 52,57) (see Fig. S1A� and B� in the supplemental material). In thecase of Wg signaling, we examined expression of the fz3-lacZenhancer trap (51, 55), a target gene of the Wg signal, and thelevel of Arm (46, 54), a Wg signal transducer that is known toaccumulate in response to the Wg signal (25, 47). We observeda reduction of fz3-lacZ expression interior to the clone bound-ary of the ss-overexpressing clones (see Fig. S1C, C�, and D toD� in the supplemental material). However, in the most centralcells, cytoplasmic accumulation of Arm was conversely ob-served (see Fig. S1C, C�, D, and D� in the supplemental ma-terial). These results suggested that the level of Wg signal isnot uniformly received throughout the ss-overexpressingclones and is not directly regulated by Ss. Similar responseswere observed in clones both in the wing blade and in the hinge

TABLE 1. Position-dependent variance of apoptotic responses to ss

Subdomaina JNKvariance

Caspase-3variance Cell autonomy Response

Around DVboundary (Dll)

Weak Weak Autonomous Delayed

Rest of blade(vg)

Weak Strong Autonomous Acute

Hinge (hth) Strong Weak Nonautonomous Variable

a Representative genes expressed in each subdomain are indicated in paren-theses.



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regions, as seen in Fig. S1C in the supplemental material.Taken together, these results indicate that discontinuities inthe strength of Dpp and Wg signal reception does not preciselycoincide with the boundary of the ss-overexpressing clones,suggesting that other factors contribute to the nonautonomousJNK activation in this case.

Position-dependent variance in the autonomy of apoptosis isfound also in clones ectopically expressing homeotic genes. Wenext examined whether the position-dependent variance in cellautonomy of apoptosis is specific for ss-induced homeosis or ageneral trait exhibited by ectopic expression of diverse ho-meotic genes. The identity of each body segment is defined bya series of homeotic genes belonging to ANTP-C and BX-C.However, the Drosophila wing requires no input from ANTP-Cand BX-C during its development (11). Instead, these ho-meotic genes repress wing development in all segments, exceptfor the mid-thorax, where the wing normally develops. In orderto produce a misspecified fate in wing cells, we expressedvarious homeotic genes in the wing discs. When labial (lab),proboscipedia (pb), Antennapedia (Antp), Ultrabithorax (Ubx),abdominal-A (abd-A), or Abdominal-B (Abd-B) were expressedby using sd-GAL4 driver, the adult wings showed abnormalvein patterns and incision of the wing margin (Fig. 5A to F). Inthe cases of Ubx or abd-A expression, wing incision was lessfrequently observed. In these cases, the wing has been shownto transform to an organ similar to a haltere (22). When mo-saic clones expressing these homeotic genes are generated,JNK activation was found autonomously in the blade regionbut nonautonomously in the hinge region. All of these re-sponses are quite similar to the results of ss overexpression, ashas been described above. Thus, the position-dependent vari-ance in cell autonomy of apoptosis may be a general phenom-enon accompanying homeotic transformation.

Characterization of the novel gene fili that encodes a LRRfamily transmembrane protein. Recently, clones with sal-in-duced misspecified fate were reported to be recognized by thesurrounding normal cells through the LRR family transmem-brane proteins, Capricious (Caps) and Tartan (Trn), to preventapoptosis (38). The expression pattern of both caps and trn arecomplementary to the pattern of sal expression in the late thirdinstar wing (38) (Fig. 6C). Thus, we investigated the role of anewly identified LRR family protein, Fili, which was expressedin the wing in a nearly complementary pattern as that of Dll(Fig. 6A and B), in regulating apoptosis in ss-overexpressingclones.

The fly strain fili-lacZ was isolated by a screen of enhancertrap lines that displayed a previously unknown expression pat-tern. The transposon P-element carrying the lacZ gene wasinserted at �56 relative to the presumptive transcription startsite, TTAGTT (Fig. 7A). The deduced protein, Fili, was com-posed of 738 amino acid residues and had a single transmem-brane domain and 14 LRR domains (Fig. 7B). These featuresare common to Caps and Trn, which share 55 and 56% ho-mologies with Fili in the LRR domains, respectively (Fig. 7Cand D). The homologies also extend to the amino- and car-boxyl-terminus domains (Fig. 7E and F), suggesting that Filiplays a role that is especially related to Caps and Trn amongnumerous members of LRR family with a variety of functions.A mutant, �102, was isolated by imprecise excision of theP-element in fili-lacZ. It lacks 534 bp, including a part of the

FIG. 4. Involvement of clone shape and hid expression. (A) Clonesexpressing green fluorescent protein alone. All of the clones show anirregular shape. (B) ss and DIAP1 (IAP)-overexpressing clones. Mostof the clones are rounded. (C) Higher magnification of a clone boxedin panel B. JNK activation is found nonautonomously but highly ac-cumulated inside the clone, whereas caspase-3 activation is found onlyoutside of the clone, which may be due to the ability of DIAP1 tobreakdown caspase-3. (D) ss-overexpressing clones in a hep mutantbackground. Most of the clones are rounded. (E) ss-overexpressingclones in a hid mutant background. Most of the clones are rounded.(F) ss-overexpressing clones under the control of tgoRNAi. Most of theclones show an irregular shape and do not induce activation of JNKand caspase-3 significantly. (G and G�) Expression of constitutivelyactive Hep (green) induces hid-lacZ expression (magenta). To createan intact shape of the clone by repressing apoptosis, the enhancer trapmutation hid-lacZ (hid05014) is homozygous in this experiment. (H andH�) ss overexpression (green) induces hid-lacZ expression (magenta)autonomously in the wing blade region (arrow) and nonautonomouslyin the wing hinge region (arrowheads).



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first exon, and is lethal at late embryonic stages. The maincause of this lethality is unknown. In the wing disc, fili exhibiteda pattern of gene expression that was nearly complementary tothat of Dll (Fig. 6A and B). In addition, fili expression wascomplementary to Dll expression in antenna discs (Fig. 6A�and B�). In contrast, in the leg disc, fili seemed to partiallyoverlap with Dll (Fig. 6A� and B�). However, we found thatmost of the cells expressing the respective genes were in dif-ferent layers of the leg disc folding.

ss-induced apoptosis can be controlled by Fili. If fili is in-volved in the control of apoptosis in ss-overexpressing clone, itsexpression should be downregulated by ss. As expected, filiexpression is repressed in the ss-overexpressing clones (Fig.8A�). Interestingly, in contrast, expression of caps (Fig. 8A�)and trn (data not shown) is enhanced by ss. Similar responsesin fili and caps can also be observed in Dll-overexpressingclones (Fig. 8B to B�).

When ss was induced together with fili, JNK and caspase-3activation was greatly reduced (Fig. 3A and 9A to A�). Resid-ual JNK and caspase-3 activation was observed only in themost peripheral hinge region, where fili is not expressed in thenormal wing. Similarly, in the central wing region, where fili isnot expressed in the normal wing, caspase-3 activation was notsuppressed by fili coexpression. As a control experiment, weobserved that coexpression of both caps and ss did not suppressthe ss-induced apoptotic response (Fig. 9B and B�). Consis-tently, ss overexpression does not repress but rather elevatesthe caps expression (Fig. 8A�). Thus, we conclude that filiexpression is sufficient for survival of ss-overexpressing cells inthe wing region where endogenous fili is expressed, probablythrough recognition by surrounding normal cells transmitting asurvival signal, as shown for Caps and Trn (38).

We further carried out a control experiment to test the effectof fili on sal-induced apoptosis because sal-induced apoptosis

FIG. 5. Ectopic expression of various homeotic genes commonlyinduces autonomous death in the wing blade region and nonautono-mous death in the wing hinge region. (A to F) Wings were generatedwith ectopic expression of the indicated homeotic genes by using sd-GAL4(s) or sd-GAL4(w) drivers. All of the wings show abnormal veinpatterns and incision of the wing margin at various frequencies. (A� toF� and A� to F�) Wing imaginal discs with mosaic clones ectopicallyexpressing the indicated homeotic genes (green). puc-lacZ expressionis indicated in magenta, and caspase-3 activation is indicated in cyan.Arrows and arrowheads denote an autonomous and nonautonomousactivation of JNK, respectively. The total numbers of clones examinedwere 30 (lab), 22 (pb) 44 (Antp), 16 (Ubx), 48 (abd-A), and 33 (Abd-B).Among these, the numbers of clones exhibiting autonomous puc-lacZinduction are 15 (lab), 6 (pb) 6 (Antp), 4 (Ubx), 2 (abd-A), and 9(Abd-B), whereas the numbers of those exhibiting nonautonomouspuc-lacZ induction are 10 (lab), 4 (pb) 14 (Antp), 4 (Ubx), 4 (abd-A),and 16 (Abd-B).

FIG. 6. Expression patterns of LRR protein genes. (A to A�) Ex-pression of fili-lacZ (magenta) and Dll (green) in the wing, leg, andantenna discs; (B to B�) in situ hybridization showing fili mRNA; (C)expression of Caps (blue), trn-lacZ (red), and Sal (green) in the wingdisc.



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was reported to be suppressed by coexpression of caps or trn.At first, we observed that the sal-overexpressing clones dis-played a nonautonomous activation of JNK in the wing regionwhere sal is not normally expressed (Fig. 9C to C�). Thisnonautonomy was quite similar to the morphogenetic apopto-sis and ss-induced apoptosis, suggesting that the regulation ofall of these apoptotic responses may share similar molecularmechanism(s). When sal was induced together with fili, JNK,and caspase-3 activation was not affected significantly (Fig. 9Dto D�). Thus, these results indicate that fili does not suppressall cases of nonautonomously induced apoptosis.

Shape and apoptosis of clones mutant for or overexpressingfili. Next, we examined the relationship of the clone shape andapoptosis in a simple fili mutant or in its overexpression. Thefili mutant clones showed an irregular shape irrespective oftheir position in the wing disc (Fig. 10A) and did not displayany apoptotic signs. These results are not always discrepantfrom the other results and are similar to observations in mu-tants for caps or trn alone (38). The other LRR family proteinsmight function with Fili in a redundant manner in the gener-ation of affinity with surrounding normal cells. In contrast tothe fili mutant, the fili-overexpressing clones showed a smoothoutline (Fig. 10B). The smooth outlines were found evenwhere endogenous fili was expressed, which may be due to theimmense amount of its expression by using UAS/GAL4 system.However, the clone in the central region where endogenous filiwas not expressed exhibited the most rounded shape (Fig.10B�). Interestingly, unlike the case of caps and trn, the over-expression of fili alone induced apoptosis in a nonautonomousmanner. When UAS-fili was induced by ptc-GAL4 (Fig. 10D),nonautonomous activation of JNK was apparent in the cellsaround the posterior edge of ptc expression but not in the cellsaround the anterior edge (Fig. 10D to D�). The posterior edgeis known to create a sharp discontinuity of ptc expressionlevels, whereas the anterior edge does not (5). More interest-

ingly, the area where JNK was activated did not overlap thearea where endogenous fili was expressed. As a result, theareas with JNK activation were divided into three regions(arrowheads in Fig. 10D�). Also, forced expression of fili in thecentral wing blade in the adult wing generated a severelynotched phenotype by apoptosis (Fig. 10C). These datastrongly suggest that the occurrence of a large discontinuity inthe fili expression level activates JNK nonautonomously.

DISCUSSION

Ectopic ss induces a homeosis-dependent apoptosis thatmay cause a rare frequency of wing-to-leg transdetermination.Homeosis is a naturally occurring rare phenomenon with for-mation of a local body part having characteristics that arenormally found in a related part at another location in thebody. This phenomenon had been studied exclusively in Dro-sophila melanogaster but can also be found in a variety ofinsects and other organisms (53). Although homeosis is rarelyfound in wild-type flies, transdetermination, a similar phenom-enon, can be easily induced by in vivo culture and subsequenttransplantation of imaginal discs between different individualsor by directly manipulating functions of various master and/orhomeotic genes. Although such developmentally abnormalcells occurring in homeosis might a priori be expected to beremoved during development to guarantee normal morpho-genesis, apoptosis has not been previously observed to accom-pany homeosis. Our results suggest that the cells undergoinghomeosis do in fact induce apoptotic responses to help preventthem from developing abnormal structures. The rare fre-quency in wing-to-leg transdetermination, which was observedabout 30 years ago (30), may be derived partly from the highfrequency of apoptosis associated with this particular transfor-mation.

FIG. 7. Structure of fili gene and Fili protein. (A) The horizontal line represents a segment of the genomic DNA around fili gene. The five exonsin fili gene are indicated below the DNA. The P-element inserted in the fili-lacZ fly genome is indicated by the downward stippled arrowhead atthe position �56 relative to the potential transcription start site. The deleted region in fili�102 mutant is indicated around �56. “cen” and “telo”denote the direction toward centromere and telomere, respectively. (B) Domain organization of Fili protein. The deduced Fili protein possessestwo hydrophobic stretches (orange), signal peptide (SP), and transmembrane domain (TM). In the extracellular domain, 14 LRRs are found(green). NF and CF refer to the amino-flanking and carboxy-flanking motifs, respectively. (C) Comparison of 14 LRRs in Fili. The identical aminoacid residues are masked with orange. (D) Comparison of LRR consensus among five LRR family members. Variable amino acid residues areshown with hyphens. (E and F) Comparison of NF and CF among Fili, Trn, and Caps. Identical and similar amino acid residues are shown inorange and cyan, respectively.



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ss-induced autonomous apoptosis is related to cell compe-tition. Cell competition is a phenomenon whereby cell cloneswith a slow growth rate are eliminated during development.The autonomous cell death in cell competition has been dem-onstrated to be caused by an impaired reception of extracel-lular survival factors. As representative examples, clones withcells heterozygous for the M(2)60E mutation show an auton-omous death as a result of reduced Dpp reception (40). Thus,the autonomous death in ss-overexpressing cells in the wingblade might be a type of cell competition response. In this case,however, the extracellular factor that the ss-overexpressingcells fail to receive is not likely to be Dpp, since we observedthat the Dpp signaling level is not strongly affected in thess-overexpressing cells (see Fig. S1A and B in the supplementalmaterial). Also, ss-overexpressing cells can be removed fromthe lateral region of the wing blade where the level of Dppsignaling is normally low. In contrast, cells heterozygous forM(2)60E can survive in this area due to the loss of competitionfor Dpp (40). Therefore, Wg or other extracellular survivalfactors might be affecting the survival of ss-overexpressingcells, as discussed below.

Position-dependent variance in cell autonomy is found inthe apoptotic response to ss overexpression. We have not yetaddressed the question of why the variance in cell autonomy ofss-induced apoptosis is related to dorsoventral patterning orblade/hinge subdomains. However, the variance suggests aninteresting model in which signals that normally regulate dor-

soventral appendage patterning or blade/hinge subdomainsalso directly affect the apoptotic response or specification oforgan identity as cryptic mechanisms. Interestingly, ectopicexpression of most of the segment-specifying homeotic genescommonly shows a similar position-dependent variance in cellautonomy (Fig. 5). In this view, Wg, the morphogen controllingthe dorsoventral patterning (42, 60), or Vg, the selector of wingsubdomain (31, 34), may be involved in the regulation of po-sition-dependent variance in the cell autonomy of apoptosis.However, definitively demonstrating the involvement of Wg orVg is difficult because changing Wg signal or Vg activity alonealso induces a severe unrelated apoptosis (2). Thus, the simul-taneous manipulation of ss and Wg/Vg activities may lead to acomplicated result that is difficult to interpret.

Implication of various LRR family transmembrane proteinsin the regulation of apoptosis to remove misspecified cells. Arecent study concerning the relation of apoptosis in sal-over-expressing cells and LRR family proteins Caps/Trn has pro-vided a new insights into how cell survival of misspecified cellsis controlled (38). Cells that overexpress sal lack caps/trn ex-pression and are removed from the area where sal is notnormally expressed. However, cells expressing both sal andcaps can survive. In order to control cell survival in the wing,Caps and Trn presumably provide position-dependent recog-nition cues along the anteroposterior axis, whereas Fili appears

FIG. 8. Ss and Dll regulate the expression of fili and Caps. (A toA�) fili-lacZ expression (red) is repressed, whereas Caps expression(blue) is induced in the ss-overexpressing clones (green). (B to B�)fili-lacZ expression (red) is repressed, whereas Caps expression (blue)is induced in the Dll-overexpressing clones (green). Areas of repres-sion (gray arrows) and induction (white arrows) are indicated.

FIG. 9. Fili regulates ss-induced apoptosis. Areas of puc-lacZ ex-pression (red), caspase-3 activation (cyan), and Caps expression (blue)are indicated. (A to A�) Wing imaginal disc with mosaic clones over-expressing ss and fili (green). JNK activation and caspase-3 activationare suppressed. (B to B�) Wing imaginal disc with mosaic clonesoverexpressing ss and caps (green). JNK activation is not suppressed byCaps. (C to C�) Wing imaginal disc with mosaic clones overexpressingsal (green). Nonautonomous activation of JNK is found at the regionwhere endogenous sal is not expressed normally. (D to D�) Wingimaginal disc with mosaic clones overexpressing sal and fili (green).JNK activation is not suppressed.



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to provide such cues to neighboring cells along the dorsoven-tral axis. These results raise the possibility that cells can rec-ognize the type of neighboring cells using distinct LRR familymembers, depending on the position of the cells in the primor-dial tissues. The Drosophila genome contains at least 10 mem-bers of LRR family, most of which have not been examined forfunction (Flybase).

Functional relationship between Drosophila Ss and mamma-lian Ahr. In the present study, several cellular responses to ssoverexpression have been described. Clonal overexpression ofss induces a sequential response composed of an induction ofleg- or antenna-specific genes, autonomous homeosis, round-ing, and autonomous or nonautonomous apoptosis. The acti-

vation of mammalian Ahr is known to affect apoptosis, cellproliferation, differentiation, and morphogenesis in various tis-sues (50). Polycyclic aromatic hydrocarbon, a ligand for Ahr,induced apoptosis accompanied by the activation of JNK (33).It should also be noted that dioxin-induced responses such asP450 expression in the liver are not observed in all cells ex-posed to dioxin but are observed in a subset of centrilobularcells with the highest sensitivity to dioxin (4). Thus, the mosaicactivation of Ss and subsequent JNK activation in surroundingcells in Drosophila induce circumstances similar to that of livercells exposed to dioxin. With regard to morphogenesis, thetype of morphogenetic signals that are modified or distortedduring dioxin action is unknown, despite many examples ofdioxin-induced mismorphogenesis that have been reported.Therefore, our studies on Ss in Drosophila may provide at leastin part, a common molecular and cellular basis for understand-ing Ahr-induced apoptosis and mismorphogenesis. It couldinvolve homeotic transformation of organ identity, recognitionby surrounding cells through LRR protein, and apoptosis-me-diated large deletion of tissues that can cause a malformationduring organogenesis. Interestingly, all of the genes involved inthis sequential process are evolutionarily conserved in mam-mals. Similar and detailed analysis in mammalian system willresolve these issues.

ACKNOWLEDGMENTS

We thank R. Barrio, S. Campbell, S. T. Crews, A. Garcıa-Bellido,B. A. Hay, Y. Hiromi, Y. H. Inoue, K. Irvine, W. Janning, E. Martın-Blanco, A. Martinez-Arias, A. Nose, S. Noselli, C. Rauskolb, M.Shinza-Kameda, G. Struhl, K. Takahashi, and D. Yamamoto, Bloom-ington Stock Center, and GETDB (GAL4-Enhancer Trap Data Base)in the National Institute of Genetics in Japan, for the fly strains; R.Barrio, G. Campbell, S. B. Carroll, D. Duncan, A. Nose, M. Shinza-Kameda, and P. ten Dijke, Developmental Studies Hybridoma Bank,Iowa University, and Idun Pharmaceutical, Inc., for antibodies; J. Kimand Y. Lee for technical advice; and Y. Aoki and O. Habara fortechnical assistance.

This study was supported by grants from the Japan Science andTechnology Agency; the Ministry of Education, Science, Sports, andCulture in Japan (to T.A.-Y.); and NIH and the Human FrontierScience Program (to H.N.). M.B.O. is an Investigator of the HowardHughes Medical Institute.

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Wing-to-Leg Homeosis by Spineless Causes Apoptosis Regulated