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INTRODUCTIONDuring animal development, dynamic cell behaviors are preciselyorchestrated to accurately complete morphogenesis. However, themechanisms that determine precisely how cell behaviors regulatemorphogenesis according to the developmental timetable are stilluncharacterized. Programmed cell death or apoptosis not onlyfunctions in sculpting and deleting structures in developinganimals, but also it plays dynamic roles in coordinating organmorphogenesis (Stenn and Paus, 2001; Toyama et al., 2008). TheDrosophila male terminalia is an asymmetric looping organ; theinternal genitalia (spermiduct) loops dextrally around the hindgut.During the maturation of the internal genitalia, the male terminaliarotates 360° clockwise (Gleichauf, 1936). The orientation defect ofadult male terminalia is thought to occur when this rotation isincomplete (Adam et al., 2003). Apoptosis is thought to contributeto the completion of genitalia rotation, because an orientationdefect of the adult male terminalia is observed in mutants ofapoptotic pathway components, including: hid (head involution

defective; W – FlyBase) (Abbott and Lengyel, 1991), a pro-apoptotic gene; drICE (Muro et al., 2006) (Ice – FlyBase), theDrosophila ortholog of caspase 3; and dronc (Krieser et al., 2007)(Nc – FlyBase), the Drosophila ortholog of caspase 9. Caspasefamily proteases are the central executioners for the geneticallyencoded apoptosis in animals (Degterev et al., 2003). However, thephysiological roles of apoptosis in completing the morphogenesisof male terminalia remain to be elucidated.

We herein used live in vivo imaging to determine the dynamicsof the looping morphogenesis and spatiotemporal apoptosis duringmale genitalia development. Our observations suggest thatapoptosis drives the acceleration of rotation, enabling the completegenitalia morphogenesis to occur within the developmentaltimetable.

MATERIALS AND METHODSFly stocks and temporal gene expressionFlies were raised on standard Drosophila medium at 25°C. The following flystrains were used in this study: en-GAL4, UAS-mCD8-EGFP, UAS-lacZ,Histone2Av (His2Av)-mRFP, tub-GAL80ts (Bloomington Drosophila StockCenter); AbdB-GAL4LDN (de Navas et al., 2006); UAS-p35 (Zhou et al.,1997); UAS-PVR DN (Duchek et al., 2001); UAS-JNK DN (Adachi-Yamadaet al., 1999): UAS-SCAT3 (Takemoto et al., 2007; Kanuka et al., 2005;Kuranaga et al., 2006); and UAS-Histone2B (H2B)-ECFP, UAS-nls-SCAT3(Koto et al., 2009).

Using the TARGET system, we bred flies at the permissivetemperature (18°C) of GAL80ts until the time when the head of the pupaehad just everted, to suppress the activity of GAL4. After head eversion,the flies were moved to the restrictive temperature (29°C) of GAL80ts

(McGuire et al., 2003) for 12 hours. Time-lapse imaging using astereomicroscope (M205FA, Leica) was performed at 22°C after the heatshock.

Development 138, 1493-1499 (2011) doi:10.1242/dev.058958© 2011. Published by The Company of Biologists Ltd

1Department of Genetics, Graduate School of Pharmaceutical Sciences, TheUniversity of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. 2CREST, JST,7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. 3Laboratory for HistogeneticDynamics, RIKEN Center for Developmental Biology, Kobe 650-0047, Japan.4National Research Center for Protozoan Diseases, Obihiro University of Agricultureand Veterinary Medicine, Inada-cho, Obihiro, Hokkaido 080-8555, Japan.5Department of Physiology, Yokohama City University Graduate School of Medicine,Fuku-ura 3-9, Kanazawa-ku, Yokohama, Kanagawa 236-0004, Japan. 6JST, PRESTO,Saitama 332-0012, Japan. 7Laboratory of Biology, Sapporo Campus, HokkaidoUniversity of Education, 3-1 Ainosato 5jo, Kita-ku, Sapporo, Hokkaido 002-8502,Japan.

*Author for correspondence (kuranaga@cdb.riken.jp)

Accepted 2 February 2011

SUMMARYIn metazoan development, the precise mechanisms that regulate the completion of morphogenesis according to a developmentaltimetable remain elusive. The Drosophila male terminalia is an asymmetric looping organ; the internal genitalia (spermiduct)loops dextrally around the hindgut. Mutants for apoptotic signaling have an orientation defect of their male terminalia,indicating that apoptosis contributes to the looping morphogenesis. However, the physiological roles of apoptosis in the loopingmorphogenesis of male terminalia have been unclear. Here, we show the role of apoptosis in the organogenesis of maleterminalia using time-lapse imaging. In normal flies, genitalia rotation accelerated as development proceeded, and completed afull 360° rotation. This acceleration was impaired when the activity of caspases or JNK or PVF/PVR signaling was reduced.Acceleration was induced by two distinct subcompartments of the A8 segment that formed a ring shape and surrounded themale genitalia: the inner ring rotated with the genitalia and the outer ring rotated later, functioning as a ‘moving walkway’ toaccelerate the inner ring rotation. A quantitative analysis combining the use of a FRET-based indicator for caspase activation withsingle-cell tracking showed that the timing and degree of apoptosis correlated with the movement of the outer ring, andupregulation of the apoptotic signal increased the speed of genital rotation. Therefore, apoptosis coordinates the outer ringmovement that drives the acceleration of genitalia rotation, thereby enabling the complete morphogenesis of male genitaliawithin a limited developmental time frame.

KEY WORDS: Caspase, Apoptosis, In vivo imaging, Drosophila

Apoptosis controls the speed of looping morphogenesis inDrosophila male terminaliaErina Kuranaga1,2,3,*, Takayuki Matsunuma1, Hirotaka Kanuka4, Kiwamu Takemoto5,6, Akiko Koto1,Ken-ichi Kimura7 and Masayuki Miura1,2

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Fig. 1. In vivo imaging and quantitative analysis of genitalia rotation. (A)An image (left) and schematic drawing (right) of the malegenitalia of His2Av-mRFP flies at 24 hours APF. Each segment is highlighted in a different color: A8 (green), A9 (orange) and A10 (yellow).(B,D,E) Time-lapse series of genitalia rotation in (B) His2Av-mRFP/+, (D) en-GAL4 UAS-H2B-ECFP/+ and (E) en-GAL4 UAS-H2B-ECFP/UAS-p35flies are shown. Ventral is towards the top in all figures. (C)Image (left) and schematic drawing (right) of genitalia in en-GAL4 UAS-H2B-ECFP/+ at 24 hours APF. The posterior region of the A8 segment is highlighted in green. (F,G)Scanning electron micrograph of the adult malegenitalia of en-GAL4/UAS-lacZ (F) and en-GAL4/UAS-p35 (G). (H)The genitalia angle () in control (black) and p35-expressing flies (red) wasmeasured every 30 minutes, and the mean angle is shown. Error bars indicate s.d. (control, n10; p35, n8). (I)Velocity (Vd/dt) and(J) acceleration rate (adV/dt) were quantified by measuring and V as a function of time t in control (black) and p35-expressing flies (red).The initiation of genitalia rotation (>1 hour) in p35-expressing flies was similar to control flies (indicated by the gray area). Genotypes of thecontrol flies were as follows: en-GAL4 UAS-mCD8-GFP/+, en-GAL4 UAS-H2B-ECFP/+ and en-GAL4 UAS-nls-ECFP-venus/+. (K)The meangenitalia angle () in flies expressing dominant-negative JNK (JNK-DN; blue) and dominant-negative PVR (PVR-DN; green) were plotted every30 minutes. Error bars indicate the s.d. (JNK-DN, n12; PVR-DN, n11). control and p35 in Fig. 1H are represented for reference as black andred lines, respectively. (L)The average velocity (Vd/dt) was quantified by measuring as a function of time t in control (black), p35 (red),JNK-DN (blue) and PVR-DN (green) flies (mean±s.d.) (**P<0.01, *P<0.05). D

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Sample preparation for time-lapse imaging and scanning electronmicroscopyStaged pupae (24 hours APF) were washed in water and mounted on aglass slide using double-sided tape. The pupal case covering the caudal partof the abdomen was removed. A very wet filter paper was placed aroundthe pupae to keep them hydrated. The pupae were covered with acoverglass in a small drop of water to avoid desiccation. Silicon (Shinetsu)was used to seal the chamber. In most cases, the animal survived the dataacquisition and developed into an adult. Time-lapse images were capturedusing an SP5 confocal microscope (Leica) or an inverted microscope(Olympus) with a spinning disc-type confocal unit (CSU10, Yokogawa,Tokyo) equipped with the Aquacosmos/Ashura system (HamamatsuPhotonics) (Kuranaga et al., 2006). The FRET analysis was performedusing the Aquacosmos (Hamamatsu Photonics) and MetaMorph software(Molecular Devices) programs. For the scanning electron microscopy, weused the VE-8800 microscope system (Keyence).

RESULTS AND DISCUSSIONTo visualize the genitalia rotation in living animals, we first usedthe His2Av-mRFP Drosophila line whose nuclei are ubiquitouslymarked by a fluorescent protein (Pandey et al., 2005). The genitaldisc is a compound disc comprised of cells from three differentembryonic segments: A8 (male eighth tergite), A9 (maleprimordium) and A10 (anal). During metamorphosis, the genitaldisc is partially everted, exposing its apical surface, and adopts acircular shape (Fig. 1A) (Keisman et al., 2001). Our resultscaptured the male genitalia undergoing a 360° clockwise rotation(Fig. 1B; see Movie 1 in the supplementary material). Inhibitingapoptosis by expressing the baculovirus pan-caspase inhibitor p35driven by engrailed-GAL4 (en-GAL4), which is expressed in theposterior compartment of each segment, results in genital mis-orientation at the adult stage (Macias et al., 2004).

In flies expressing nuclear fluorescent protein driven by en-GAL4, we observed that the posterior part of the A8 segment (A8p)formed a ring of cells surrounding the A9-A10 part of the disc (Fig.1C). First, we recorded the images at a low resolution (10�objective lens) to measure the rotation speed accurately in controland p35-expressing flies, because long-term time-lapse imaging ata high resolution can cause photodamage, and thus alter pupaldevelopment. Most of the cells in the A8p that seem to disappearat the end of Movies 2 and 3 in the supplementary material actuallymoved out of the plane of focus. In our imaging results, the rotationstarted around 24 hours APF (after puparium formation) andstopped about 12 hours later (12 hours 5 minutes±58 minutes;n10) (Fig. 1D; see Movie 2 in the supplementary material). Toconfirm whether the mis-oriented genital phenotype in the caspase-inhibited flies was caused by incomplete rotation, we observed therotation in flies expressing p35 under the en-GAL4 driver. In thep35-expressing flies, the rotation began, but it stopped before it wascomplete, after about 12 hours (12 hours 8 minutes±1 hour 27minutes; n8), i.e. with the same timing as in control flies (Fig. 1E;see Movie 3 in the supplementary material). This suggested that thereduced caspase activation in A8p prevented the genitalia fromcompleting the rotation, resulting in mis-oriented adult genitalia(Fig. 1F,G).

To compare complete rotation with incomplete rotation, wecalculated the rotation speed by measuring the angle (control andp35) of the A9 genitalia every 30 minutes on time-lapse images.The normal rotation was composed of at least four steps: initiation,acceleration, deceleration and stopping (Fig. 1H). We calculatedthe velocity of rotation Vd/dt by measuring as a function oftime t. At first, the genitalia rotated at an average velocity (Vcontrol)of 7.67±3.72°/hour by 1 hour after initiation, then the rotation

1495RESEARCH REPORTApoptosis controls speed of morphogenesis

Fig. 2. Cellular behaviors and apoptosis in the A8p region. (A)A time-lapse series of genitalia rotation in en-GAL4 UAS-nls-ECFP-venus/+. Theposterior compartment is visible in this fly. Cells represented by magenta rotated with A9-A10, and cells colored green rotated later. (B)Image (left)and schematic drawing (right) of genitalia in en-GAL4 UAS-nls-ECFP-venus/+ at 24 hours APF. A8p was divided into two parts, A8pa and A8pp.Each part is highlighted in a different color: A8pa (green) and A8pp (magenta). (C)Caspase activity was examined by the imaging of a FRET-basedprobe, nls-ECFP-venus (nls-SCAT3), and is shown in pseudo-color. White circles indicate the cell that underwent apoptosis, pseudo-color graduallychanged from red to blue. (D)Result of cell tracing in the A8p region. Cells that underwent apoptosis are marked by yellow dots. Magenta dotsshow cells located in A8pp, which moved with A9, and green dots represent cells in A8pa, which rotated later. Genotype was en-GAL4 UAS-nls-SCAT3/+; His2Av-mRFP/+. Three flies were examined and a typical example is shown.

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accelerated, with Vcontrol gradually increasing to 53.83±7.11°/hourby 7 hours after initiation (Fig. 1I). Interestingly, in the p35-expressing flies, the rotation normally started at 24 hours APF, andthe average velocity (Vp35) from the initial rotation to 1 hour laterwas 7.45± 2.98°/hour, which was not significantly different fromthe normal rotation. However, the acceleration of the rotation in thep35-expressing flies was lower than normal, with Vp35 graduallyincreasing to 21.35±7.45°/hour at 5.5 hours after initiation (Fig. 1I).As shown in Fig. 1J, the first peak of the acceleration rate, whichwas defined as the initiation of rotation, was observed in the p35-expressing flies (ap35) and was the same as in the control flies(acontrol). However, the duration of the acceleration period wasshorter in the p35-expressing flies (Fig. 1J). These data suggest arelationship between apoptosis and the acceleration of genitaliarotation.

Next, we examined the signaling mechanism(s) involved in theacceleration of genitalia rotation. The inhibition of JNK (c-Jun N-terminal kinase) and PVF (platelet vascular factor) signaling inmale flies has been shown to result in mis-oriented adult maleterminalia, and it has been hypothesized that the PVF/PVR (PVFreceptor) may affect the genitalia rotation via JNK-mediatedapoptosis (Macias et al., 2004; Benitez et al., 2010). Consistentwith previous reports, the acceleration of genitalia rotation wassignificantly impaired in flies expressing dominant-negative forms

of JNK (JNK-DN) and PVR (PVR-DN) (Fig. 1K,L). These dataimplied that caspase activation and JNK signaling contribute todriving the acceleration of genitalia rotation.

To analyze how the genitalia accelerate their rotation, we tracedthe movement of A8p at the single-cell level. For this experiment,we performed live imaging at a high resolution (20� objectivelens), which enabled the cells in A8p to be tracked at single-cellresolution. As shown in Fig. 2A, cells (magenta) that wereneighbors of A9 rotated with A9, whereas cells (green) located inthe anterior half of A8p rotated later than A9. Based on ourimaging, we divided A8p into two sheets, named A8pa (anterior ofA8p) and A8pp (posterior of A8p), as shown in Fig. 2B. We foundthat a part of the cells in A8p underwent apoptosis.

To observe caspase activation in living animals, we generated aFRET (fluorescence resonance energy transfer)-based indicator,SCAT3 (sensor for activated caspases based on FRET) (Takemotoet al., 2003; Takemoto et al., 2007). To precisely evaluateapoptosis, we used a nuclear localization signal-tagged SCAT3(nls-SCAT3; UAS-nls-ECFP-venus) (Koto et al., 2009). The nls-SCAT3 signal was clearly observed in A8p (Fig. 2C). Cellsexhibiting high caspase activity were extruded into the body cavityand disappeared, consistent with their apoptotic death andengulfment by circulating hemocytes. We tracked each cell in theA8p region during the first half of the rotation and found that at

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Fig. 3. Two distinct rotations occur in the genitalia rotation, and the outer ring rotation is impaired by caspase inhibition. (A)Time-lapseseries of genitalia rotation in UAS-nls-ECFP-venus/+; AbdB-GAL4LDN/+. Representative paths of cells are shown. Ventral side is towards the top in allpanels. Different colored dots and lines indicate the tracks of three different cells. (B)Schematic drawing of the AbdB-expression region in the malegenitalia based on the image in A. The AbdB-expressing region is highlighted in green (outer ring; A8a and A8pa). AbdB was not expressed inA8pp (inner ring). (C)Mean of the turning angle of cells in the AbdB-expressing region (AbdB) from the initial point of rotation. Error bars indicates.d. (n4 flies). control in Fig. 1H is represented for reference as a gray line. (D)Time-lapse series of genitalia rotation in en-GAL4 UAS-nls-ECFP-venus/UAS-p35; His2Av-mRFP/+. Cells in the inner ring (magenta) rotated only 180° and the rotation of cells in the outer ring (green) was impaired.(E)Means of the turning angle of cells in the outer ring (p35_ outer) and the inner ring (p35_ inner) from the initial point of rotation are shown. Errorbars indicate s.d.

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least three types of cellular behavior were observed, as shown inFig. 2D: cells located in A8pp (magenta) moved with A9, cellsunderwent apoptosis (yellow) and cells located in A8pa (green)rotated later (Fig. 2D).

Thus, to observe the behavior of the cells in A8pa, we usedAbdominal B (AbdB) as an A8 marker. AbdB is a homeotic genethat is required for the correct development of the genital disc(Estrada et al., 2003; Gorfinkiel et al., 2003), and AbdB-GAL4LDN

is expressed in the segment A8 (in A8a and A8p) of the genitaldisc during the 3rd instar larval stage (de Navas et al., 2006;Benitez et al., 2010; Rousset et al., 2010). At 24 hours APF, AbdBwas expressed in A8 and formed a ring (Fig. 3A,B). We tooktime-lapse images, and unexpectedly found that most of the cells

in the AbdB-expressing region underwent a 180° clockwisemovement, suggesting that AbdB was not expressed in the A8ppregion that moved 360° with A9 (Fig. 3A; see Movie 4 in thesupplementary material). To determine the speed of the AbdB-expressing cells, we traced three individual cells in each fly(Nfly4), and calculated the value of the turning angle of the cells(AbdB) (Fig. 3C). Our findings confirmed that the AbdB-expressing region moved halfway around. Although cells in theAbdB-expressing region moved only 180°, the A8pp (inner ring),which was encircled by the AbdB-expressing region (outer ring),still moved 360°. Furthermore, our imaging data indicated thatthe movement of the outer ring started 1-2 hours later than thatof the A9 region (Fig. 3C), when the acceleration of the genitalia

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Fig. 4. Initiation of outer ring rotation correlates with apoptosis. (A)Acceleration of the rotation of the AbdB-expressing region wasquantified by measuring AbdB and VAbdB as a function of time t (green line). Histogram showing the frequency of apoptosis every 10 minutes from0 hours, when rotation started, to 8 hours. Rapoptosis was normalized to the total number of apoptotic cells in each individual. Error bars indicate s.d.(n3 flies). (B)Data points represent the relationship between aAbdB and Rapoptosis for 0-3 hours using linear regression (R20.951). (C)The genitaliaangle () in lacZ-expressing flies (black) and Rpr-expressing flies (gray) was measured every 30 minutes and the mean angle is shown. Error barsindicate s.d. (lacZ, n8; Rpr, n8). Genotypes of flies were as follows: en-GAL4 UAS-nls-ECFP-venus/UAS-lacZ; tub-GAL80ts/+ and en-GAL4 UAS-nls-ECFP-venus/UAS-Rpr; tub-GAL80ts/+. (D)The average velocity (Vd/dt) was quantified by measuring as a function of time t in lacZ- (black) andRpr-expressing (gray) flies (mean±s.d.) (**P<0.01, *P<0.05). (E)Model of acceleration of genitalia rotation. The inner ring (magenta) rotates inconcert with A9 genitalia (light gray), then the outer ring (green) that encircles inner ring begins to move, which functions like a ‘moving walkway’to accelerate the speed of the inner ring. The initiation of outer ring movement strongly correlated with apoptosis (yellow); moreover, thismovement was impaired by the inhibition of apoptosis. Therefore, the apoptosis increases the rotation of genitalia faster in the direction it is alreadymoving, enabling the full 360° rotation to occur with the correct timing.

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rotation occurred (Fig. 1H-J). These observations raise thepossibility that the outer ring movement is related to theacceleration of the genitalia rotation.

We therefore considered that the outer ring movement wasrestricted in the p35-expressing flies, resulting in an incompletegenitalia rotation of about 180°, which mimics the movement of onlythe inner ring. To verify this possibility, we examined the movementof the outer ring in the p35-expressing flies (en-GAL4+UAS-p35).Although the inner ring rotated normally, the rotation of the outerring was impaired in the p35-expressing flies (Fig. 3D). Wedetermined the turning angles by tracing cells in the p35-expressingflies and found that p35 _inner increased, while the increase ofp35 _outer was impaired (Fig. 3E). These data suggest that the A8segment was composed of two independently regulated rings, andwhen apoptosis was inhibited, the inner ring could move only 180°with no outer ring movement, resulting in incomplete genitaliarotation.

Thus, to determine whether apoptosis correlates with the outerring movement, we quantified the apoptosis in A8pa every 10minutes from 0-8 hours after the start of genitalia rotation. Thefrequency of apoptosis (Rapoptosis) was normalized to the totalnumber of apoptotic cells in each individual. Pulsatile increases inRapoptosis were observed, with peaks at 1, 2.5 and 4 hours after thestart of genitalia rotation (Fig. 4A). To verify the participation ofRapoptosis in the initiation of outer ring movement, we calculated theacceleration rate of AbdB (aAbdB) by measuring VAbdB as a functionof time t, and compared Rapoptosis with aAbdB. The starting time ofouter ring movement was characterized by the early peaks of aAbdB

(Fig. 4A). Our analysis suggested that the aAbdB was related to theRapoptosis, because aAbdB showed its first two peaks at about 1 and2.5 hours after genitalia rotation started (Fig. 4A). To quantify theseobservations, we calculated the correlation between Rapoptosis andaAbdB. This analysis confirmed that there was a strong correlationbetween these parameters (R20.951), because the correlationbetween aAbdB and Rapoptosis is approximately linear during this time(Fig. 4B). Therefore, these data implied a possible mechanism ofapoptosis that facilitates the outer ring movement.

To verify this possibility, we examined whether the upregulation ofapoptotic signals induces an increase in genitalia rotation speed.Because the expression of apoptotic genes using an en-GAL4 driver,which is expressed at the embryonic stage, is lethal, we used theTARGET system to control gene expression temporally (McGuire etal., 2003). Flies were allowed to develop at 18°C until the head of thepupae had just everted, to inhibit gene expression. The pupae werethen heat-shocked at 29°C for 12 hours to induce gene expression.Live imaging was performed at 22°C, after the heat shock. At thistemperature, the genitalia rotation in the control flies was slower thanin control flies bred at 25°C, because a low breeding temperatureaffects the rate of fly development, including genitalia rotation.Therefore, it was necessary in this experiment to compare the rotationspeeds at the same temperature. The expression of reaper (rpr), a pro-apoptotic gene, using the TARGET system, showed that theupregulation of apoptotic signaling significantly increased the timingof acceleration and speed of genitalia rotation (Fig. 4C,D). Theseobservations led us to propose that the outer ring functions like a‘moving walkway’ to accelerate the speed of the inner part of thestructure, including the A9 genitalia, enabling genitalia to completerotation within the appropriate developmental time window (Fig. 4E).

According to our observations, we found that apoptosis drivesthe movement of cell sheets during the morphogenesis of maleterminalia. Further questions remain with regard to how apoptosiscontributes to the cell sheet movement. A recent study indicated the

possibility that local apoptosis acts as a brake release to regulategenitalia rotation, coupled with left-right determination (Suzanneet al., 2010). However, it has been reported that the cell shapechange by apoptosis enables not only the extrusion of dying cells,but also the reorganization of the actin cytoskeleton in neighboringcells (Rosenblatt et al., 2001). Therefore, apoptosis could affect thebehavior of neighboring cells, to act as a main driving force of thecell-sheet movement. Taken together, apoptosis may generallyparticipate in the morphogenetic process of cell-sheet movementduring morphogenesis.

AcknowledgementsWe thank E. Sanchez-Herrero, B. Hay, the Bloomington Drosophila ResourceCenter and the Drosophila Genetic Resource Center (Kyoto) for fly strains; A.Tonoki, Y. Fujioka, K. Tomioka, A. Isomura and A. Tsukioka for technicalsupport; all members of the M.M. laboratory for helpful discussions; Y.Takahashi, K. Matsuno, S. Hayashi, A. Bergmann, H. Okano and A. Kakizukafor kind support and encouragement; and M. Sato for helpful discussion andtechnical support. We especially thank S. Kuroda for the generous suggestionfor the quantification analysis. We thank the University of Tokyo and LeicaMicrosystems Imaging Center for imaging. This work was supported by grantsfrom the Japanese Ministry of Education, Science, Sports, Culture, andTechnology (to E.K. and M.M.) and by grants from the Takeda ScienceFoundation (to E.K.), the Naito Foundation (to M.M.), the Cell ScienceResearch Foundation (to M.M.), and a RIKEN Bioarchitect Research Grant (toM.M.).

Competing interests statementThe authors declare no competing financial interests.

Supplementary materialSupplementary material for this article is available athttp://dev.biologists.org/lookup/suppl/doi:10.1242/dev.058958/-/DC1

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1499RESEARCH REPORTApoptosis controls speed of morphogenesis

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