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INTRODUCTIONSteroid hormones are responsible for the coordination and regulationof many aspects of development, growth and differentiation ofmulticellular organisms. In insects and other arthropods, a strictregulation of titers of ecdysteroids, especially ecdysone (so called�-ecdysone) and 20-hydroxyecdysone (20E), plays central roles indevelopment, especially in guiding transition from onedevelopmental stage to the next via molting and metamorphosis(Thummel, 2001; Gilbert et al., 2002; Thummel and Chory, 2002).During larval and pupal development of insects, ecdysone issynthesized from dietary cholesterol or phytosterols via a series ofhydroxylation and oxidation steps in the small endocrine organcalled the prothoracic gland (PG) (Gilbert et al., 2002).

Studies using Drosophila have identified several molecules thatare involved in ecdysteroid biosynthesis in the PG. For example,ecdysone synthesis is regulated by itpr, which encodes inositol 1,4,5-trisphosphate receptor (Venkatesh and Hasan, 1997; Venkatesh etal., 2001); dare, which encodes adrenodoxin reductase (Freeman etal., 1999); ecdysoneless (ecd), which encodes an evolutionallyconserved protein with no known motifs (Warren et al., 1996;Gaziova et al., 2004); and without children, which encodes a putativetranscriptional regulator (Wismar et al., 2000; Warren et al., 2001).Ras-dependent signaling cascade and insulin-dependent PG cellgrowth are also essential for the ecdysone production and/or release(Caldwell et al., 2005; Mirth et al., 2005).

Recently, five hydroxylase genes that are essential for ecdysteroidbiosynthesis have been identified in Drosophila. All of thehydroxylase genes, Cyp306a1/phantom (phm), Cyp302a1/disembodied (dib), Cyp315a1/shadow (sad), Cyp314a1/shade (shd)and Cyp307a1/spook (spo), are named the Halloween genes andencode cytochrome P450 mono-oxygenases (Chávez et al., 2000;Warren et al., 2002; Petryk et al., 2003; Niwa et al., 2004; Warren etal., 2004; Namiki et al., 2005). A combination of molecular andbiochemical experiments have shown that Phm, Dib, Sad and Shdplay pivotal roles in the final four steps of ecdysteroidogenesis,namely the conversion of 5�-ketodiol to 20E (Gilbert and Warren,2005). Orthologs of the Halloween P450 genes have also beenidentified in the silkworm Bombyx mori and the tobacco hornmothManduca sexta. The expression patterns of these lepidopteranhydroxylase genes are spatially restricted to the PG and aretemporally correlated with the ecdysteroids titer during larvaldevelopment (Niwa et al., 2004; Warren et al., 2004; Namiki et al.,2005; Niwa et al., 2005; Rewitz et al., 2006). The identification ofthese genes provides the basis for investigating the regulation ofinsect hormone production in more detail. For example, it has beenshown that the expression of phm and dib is regulated by the �FTZ-F1 transcription factor in Drosophila (Parvy et al., 2005). Similarly,molting defective, which encodes a putative transcription factor inDrosophila, influences the expression level of some Halloween P450genes (Neubueser et al., 2005). In Bombyx, the expression of the dibortholog is significantly induced by steroidogenic neuropeptideprothoracicotropic hormone in cultured PGs (Niwa et al., 2005).

Although the enzymes involved in the final biochemical steps ofecdysteroid biosynthesis are relatively well characterized, little isknown about the molecules involved in earlier steps (Gilbert andWarren, 2005). Dietary cholesterol (C) is first converted to 7-dehydrocholesterol (7dC) by 7,8-dehydrogenation in theendoplasmic reticulum. Conversion of the 7dC to the �4-diketolconstitutes the so called ‘black box’. Subsequently, cytosolic 5�-reduction and microsomal 3�-reduction steps convert the �4-diketol

Neverland is an evolutionally conserved Rieske-domainprotein that is essential for ecdysone synthesis and insectgrowthTakuji Yoshiyama1, Toshiki Namiki1, Kazuei Mita2, Hiroshi Kataoka1,† and Ryusuke Niwa1,*,†

Steroid hormones mediate a wide variety of developmental and physiological events in multicellular organisms. During larval andpupal stages of insects, the principal steroid hormone is ecdysone, which is synthesized in the prothoracic gland (PG) and plays acentral role in the control of development. Although many studies have revealed the biochemical features of ecdysone synthesis inthe PG, many aspects of this pathway have remained unclear at the molecular level. We describe the neverland (nvd) gene, whichencodes an oxygenase-like protein with a Rieske electron carrier domain, from the silkworm Bombyx mori and the fruitflyDrosophila melanogaster. nvd is expressed specifically in tissues that synthesize ecdysone, such as the PG. We also show that loss ofnvd function in the PG causes arrest of both molting and growth during Drosophila development. Furthermore, the phenotype isrescued by application of 20-hydroxyecdysone or the precursor 7-dehydrocholesterol. Given that the nvd family is evolutionallyconserved, these results suggest that Nvd is an essential regulator of cholesterol metabolism or trafficking in steroid synthesis acrossanimal phyla.

KEY WORDS: Bombyx mori, Cholesterol, Drosophila melanogaster, Ecdysone, Growth, Molting, Prothoracic gland, Rieske, Ring gland,Steroidogenesis

Development 133, 2565-2574 (2006) doi:10.1242/dev.02428

1Department of Integrated Biosciences, Rm201, Graduate School of FrontierSciences, The University of Tokyo, 5-1-5 Kashiwanoha, Kashiwa, Chiba 277-8562,Japan. 2Laboratory of Insect Genome, National Institute of Agrobiological Sciences,Owashi 1-2, Tsukuba, Ibaraki 305-8643, Japan.

*Present address: Department of Molecular, Cellular and Developmental Biology,Yale University, KBT 938, P.O. Box 208103, 266 Whitney Ave., New Haven, CT06520, USA†Authors for correspondence (e-mail: ryusuke-niwa@umin.ac.jp; kataoka@k.u-tokyo.ac.jp)

Accepted 5 May 2006

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to the 5�-ketodiol. Further identification and characterization ofthese ‘early’ ecdysteroidogenic genes are important forunderstanding the mechanisms by which ecdysteroidogenesis anddevelopmental timing are precisely controlled in arthropods.

To facilitate identification and characterization of componentsresponsible for ecdysteroid biosynthesis, we have used Bombyx toidentify genes predominantly expressed in the PG (Niwa et al., 2004;Namiki et al., 2005; Niwa et al., 2005; Yamanaka et al., 2005). Basedon gene expression analysis using Bombyx cDNA microarrays that wehave previously performed (Niwa et al., 2004), we now describe anovel gene named neverland (nvd), the expression of which isspecifically enriched in ecdysone-synthesizing tissues, including thePG. We show that loss of nvd function in the PG causes growth arrestat the larval stages, and this phenotype is rescued by application of20E or 7dC. Our results suggest that Nvd plays a pivotal role in themetabolism of cholesterol and steroid intermediates duringecdysteroidogenesis. Considering that the nvd gene family isevolutionally conserved, we propose that the nvd family of proteins isan essential regulator of steroid biosynthesis in various animal phyla.

MATERIALS AND METHODSAnimal strains and cultureCulture and staging of silkworms, B. mori (KINSHU � SHOWA F1 hybrid),have been described previously (Niwa et al., 2004). All D. melanogaster flieswere reared on a standard agar-cornmeal medium at 25°C under a 12-hourlight/12-hour dark photoperiod unless otherwise specified. 2-286-GAL4(Timmons et al., 1997; Andrews et al., 2002), AUG21-GAL4 (Siegmund andKorge, 2001) and AKH-GAL4 (Lee and Park, 2004) were kindly providedfrom C. S. Thummel, G. Korge and J. H. Park, respectively. wocrgl (Wismaret al., 2000) was generous gift from J. T. Warren and L. I. Gilbert. breathless-GAL4 (Shiga et al., 1996), elavc155-GAL4 (Luo et al., 1994) and teashirt-GAL4 (Shiga et al., 1996) were provided from Genetic Strains ResearchCenter in National Institute of Genetics, Japan. scabrous-GAL4 (Klaes et al.,1994) and decapentaplegic-GAL4 (Staehling-Hampton et al., 1994) wereobtained from the Bloomington stock center. Lsp2-GAL4 (Cherbas et al.,2003) and pGawB5015 were provided from the Drosophila Genetic ResourceCenter, Kyoto Institute of Technology. Although a previous study shows thata GAL4 in pGawB5015 is active in lymph gland and hematopoietic cells(Sinenko et al., 2004), the GAL4 was also expressed in the larval ring glandcontaining the PG cells (see Fig. S1 in the supplementary materials; J.-B.Peyre and T. Aigaki, personal communication). All flies were maintained inyw background. Cyo[y+] and TM3[y+] were used as balancers.

Molecular cloningBecause an EST clone prgv0382 from the Bombyx EST project (Mita et al.,2003) lacked the 5� region of full-length cDNA of Bombyx neverland (nvd-Bm), the 5� end of nvd-Bm cDNA was obtained by the 5� RapidAmplification of cDNA ends (5� RACE) method using the GeneRacer Kit(Invitrogen). The first 5� RACE product was amplified with a gene-specificprimer (5�-GGGCAGAAGTAAGGAGCGCCATCTCTGTG-3�) andGeneRacer 5� Primer. Then we performed the nested PCR with anothergene-specific primer (5�-CCGCTGTAAAGAAGCCAATTAAGGTG-GCGC-3�) and GeneRacer 5� nested primer. The nucleotide sequence ofDrosophila neverland (nvd-Dm) was identified from the Drosophila ESTdatabase (GenBank Accession Number BT021261). The cDNA containingthe entire open reading frame (ORF) for nvd-Dm was amplified by PCRfrom wild-type Drosophila ring gland-derived cDNA using the followingprimers: forward, 5�-ATGACGAGCTACAGTTTATTTTGGATGTC-3�;reverse, 5�-CTACCAACCAATATTGGTTGCTTCAG-3�. The DNAsequence data of nvd-Bm and nvd-Dm were deposited in GenBank(AB232986 and AB232987, respectively).

Reverse transcription (RT)-PCRFor analyzing tissue expression pattern of nvd-Bm, total RNA was derivedfrom tissue of second-day wandering (W1) fifth instar larvae of Bombyx asdescribed (Niwa et al., 2004). Specific primers for nvd-Bm (forward, 5�-AGATGGCGCTCCTTACTTCTG-3�; reverse, 5�-TCAGACACTTGGT-

CACTCCATC-3�) were used. The expression level of nvd-Dm wasexamined by RT-PCR using the following specific primers: forward, 5�-CGAGCTACAGTTTATTTTGGATGTCATTGC-3�; reverse, 5�-GGGC-ATATAACACAGTCGTCAGC-3�. Quantitative RT-PCR was performed asdescribed (Niwa et al., 2005). rpL3 and rp49 were used as loading controlsfor nvd-Bm and nvd-Dm, respectively (Foley et al., 1993; Matsuoka andFujiwara, 2000).

Northern and in situ hybridizationDigoxigenin (DIG)-labeled RNA probes were synthesized using the DIGRNA labeling kit (Roche) and T3 or T7 RNA polymerase (Invitrogen). Forthe nvd-Bm and nvd-Dm probes, prgv0382 and a pBluescript containing nvd-Dm (nvd-Dm-pBluescript), respectively, were used as templates. Northernblot and hybridization using a DIG-labeled probe was performed asdescribed (Charles et al., 1999). In situ hybridization was performed asdescribed (Lehmann and Tautz, 1994; Buszczak et al., 1999; Niwa et al.,2004).

UAS vector construction and generation of transgenic strainsOverexpression studies and RNAi experiments using hairpin double-stranded RNAs was performed using GAL4/UAS system (Brand andPerrimon, 1993). The construct for nvd-Dm overexpression was generatedby ligation of a BamHI/XhoI fragment isolated from nvd-Dm-pBluescriptinto the BglII/XhoI site of pUAST vector (Brand and Perrimon, 1993). Tocarry out a transgenic RNAi (Kennerdell and Carthew, 2000), we generatedtwo distinct UAS-nvd-Dm-Inverted Repeat constructs, designated UAS-nvd-Dm-IR-1 and UAS-nvd-Dm-IR-2. UAS-nvd-Dm-IR-1 was a genomic-cDNAfusion construct (Kalidas and Smith, 2002) comprising the 488-1131 bpregion of the nvd-Dm gene. This genomic fragment contained a part of thefourth exon downstream of an intrinsic BspHI site, the entire fourth intronand a NotI site at the 3� end (see Fig. S2 in the supplementary materials).These fragments amplified by PCR were ligated into pUAST. To make UAS-nvd-Dm-IR-2 (a cDNA-cDNA RNAi construct), the 77-757 bp region wasamplified and cloned into pUAST as a tail-to-tail inverted repeat (see Fig.S2 in the supplementary material). The Drosophila transformants wereobtained using standard protocols.

Phenotypic characterization of nvd-Dm RNAi animalsTo assess the phenotype of nvd-Dm RNAi individuals, yw; UAS-nvd-Dm-IRmales of each RNAi strain were crossed with yw; 2-286-GAL4/TM3[y+]females and 0-12 hours after egg laying (AEL) eggs were collected. Theanimals were reared on agar-apple juice plates with yeast paste. nvd-DmRNAi progeny carrying both UAS and GAL4 transgenes (yw; UAS-nvd-Dm-IR/+; 2-286-GAL4/+) were distinguished from the others not carrying eitherUAS or GAL4 by the yellowish color of their mouth hooks and denticle belts,because all of UAS and GAL4 strains were balanced by balancers with y+

marker. Progeny carrying either UAS or GAL4 transgene alone were used asa control. Control and nvd-Dm RNAi larvae were transferred to fresh plates36±6 hours AEL. After 48 hours AEL, we checked the lethality andphenotype of larvae every 24 hours. Larvae were staged by the morphologyof the mouth hook as described (Roberts and Standen, 1998).

Ecdysteroid titer measurementsControl and nvd-Dm RNAi larvae were collected 30-42 hours AEL andstored in 1.5 ml tubes that were weighed before and after the addition oflarvae in order to determine the weight of the larvae. Sample preparation forecdysteroid titer measurement was done as described (Bialecki et al., 2002).The amounts of ecdysone were measured using the ecdysteroidradioimmunoassay (RIA) as described (Mizoguchi et al., 2001). As 20-hydroxyecdysone (20E, Sigma) was used as the standard, the ecdysteroidamount was expressed in 20E equivalents.

Ecdysteroid feeding experimentsControl and nvd RNAi larvae were collected 30±6 hours AEL and placed onagar-apple juice plates. These larvae were fed on yeast paste with a finalconcentration of 1 mg/ml or zero 20E in 3.3% ethanol 30-42, 54-66 and 72hours AEL in order to stimulate the hormone pulse that triggers the molt(Bialecki et al., 2002; Warren et al., 2006). Lethality was scored at 12-hourintervals. Cholesterol (C) and 7-dehydrocholesterol (7dC) were generous

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gifts from Y. Fujimoto. Control and nvd-Dm RNAi larvae were also fed onyeast paste containing 0.5% wet weight of C and 7dC (50 mg dry yeast, 95�l water, 5 �l of 100% ethanol and 0.75 mg of each ecdysteroidintermediate) (Warren et al., 2001) from 30 hours AEL until all animals hademerged or died. The experiment using 7dC was carried out under constantdark conditions because 7dC is unstable in light.

RESULTSBombyx neverland is specifically expressed in theprothoracic glandTo identify components for ecdysteroidogenesis, we have used aBombyx cDNA microarray (Niwa et al., 2004). We have comparedgene expression profiles of the PGs from first day of fifth instar (V0)larvae, in which ecdysone is not produced, with that of second-dayof wandering (W1) larvae, in which ecdysone is actively synthesized.The microarray data have yielded 1883 out of 5760 nonredundantcDNA clones showing at least a twofold increased expression (Niwaet al., 2004). Of these clones, prgv0382 (hereafter referred to as nvd-Bm) showed a 4.7-fold higher expression in the PGs of second-daywandering (W1) larvae than those of first-day fifth instar (V0) larvae.Consistent with the microarray data, Northern blot analysis revealedthat a change in mRNA expression level of nvd-Bm was positivelycorrelated with the changes of hemolymph ecdysteroid titers duringBombyx development (Fig. 1A) (Satake et al., 1998; Mizoguchi et al.,2001). Furthermore, expression of nvd-Bm in various tissues of theW1 Bombyx larvae was examined by RT-PCR. nvd-Bm wasexpressed predominantly in the PG, while weak nvd-Bm was alsodetected in brain and malpighian tubules (Fig. 1B). These resultssuggest that nvd-Bm is involved in the temporal regulation ofecdysteroid biosynthesis in the PG.

The neverland family of proteins is conservedamong animal phylaThe full-length nucleotide sequence of nvd-Bm was obtained bymodified 5� RACE. The predicted ORF for the full-length clone ofnvd-Bm encodes a protein of 453 amino acids in length. A BLASTsearch using the deduced Nvd-Bm protein sequence revealed thatputative nvd-Bm orthologs are present in several animal species (Fig.2A). These Nvd proteins all contain a Rieske [2Fe-2S] center

binding motif (C-X-H-X16-17-C-X2-H) that is known to function asan electron acceptor and is involved in electron transfer to otherproteins (Link, 1999). The putative nvd family of proteins also havea highly conserved domain in the C-terminal region that contains amononuclear, non-heme iron-binding motif (E/D-X3-D-X2-H-X4-H), which is thought to be involved in oxygen binding (Mason andCammack, 1992). The primary structure of the Nvd proteins, whichhas both the Rieske domain and the non-heme iron-binding motif,is typically conserved in class IA terminal oxygenases fromprokaryotes (Jiang et al., 1996), such as KshA (3-ketosteroid 9�-hydroxylase) of Rhodococcus erythropolis (van der Geize et al.,2002) and PrnD (aminopyrrolnitrin dioxygenases) of Pseudomonasfluorescens (Kirner et al., 1998). In the case of Nvd proteins fromBombyx, Drosophila, C. elegans and zebrafish, putativetransmembrane regions are predicted in their N-terminal regions(Fig. 2A). Only one homolog existed in the genome of each of theseeukaryotic species. We could not find nvd orthologs frommammalian or plant genomes using a standard BLAST search.

The Drosophila homolog of neverland (nvd-Dm) was notoriginally annotated in the Drosophila euchromatin genomedatabase. Our BLAST search revealed that nvd-Dm is composed ofsix exons distributed over 76 kb and located on the thirdchromosome heterochromatin scaffold (Hoskins et al., 2002). The5� region of nvd-Dm overlapped with a predicted gene designatedCG40050 that might have been mis-annnotated in FlyBase (see Fig.S3 in the supplementary materials).

Drosophila neverland is expressed in tissues thatsynthesize steroid hormonesTo analyze the expression pattern of nvd-Dm during Drosophiladevelopment, in situ RNA hybridizations were carried out onembryos, larvae and adult ovaries. We detected no nvd-Dm mRNAin unfertilized eggs, suggesting that there is no maternal contributionof nvd-Dm (Fig. 3A), as was previously noted for phm, dib and sad(Chávez et al., 2000; Warren et al., 2002; Niwa et al., 2004; Warrenet al., 2004). Moreover, no nvd-Dm expression was detected at theblastoderm, gastrulation, germ band elongation and retraction stages(Fig. 3A,B; see Fig. S4 in the supplementary materials). nvd-Dmexpression was first seen at stage 14 in the primordia of the ringgland, which contains Drosophila PG cells (see Fig. S4 in thesupplementary material). nvd-Dm expression in the ring glandbecame higher at stage 15, and continued through the remainder ofembryogenesis (Fig. 3C). nvd-Dm mRNA was also expressedspecifically in the ring gland at the larval stage (Fig. 3D,E). In thering gland, this expression was exclusively observed in the PG, butnot in the corpus allatum or corpus cardiacum cells (Fig. 3D,D�).nvd-Dm expression in the PG was downregulated after ecdysis fromthe second instar to the third instar, and then was significantlyupregulated at the late third instar larval stage (Fig. 3F). Theseresults suggest that the transcriptional activity of nvd correlates withecdysone titer changes during the larval molting cycle, similar to thatof phm, dib, sad and Start1 (Drosophila homolog of StAR) (Roth etal., 2004; Gilbert and Warren, 2005).

In adult females, nvd-Dm mRNA was expressed in the nurse cellsof developing egg chambers (Fig. 3G-I). The nurse cells areconsidered to be the source of ecdysteroids in adult females(Riddiford, 1993; Gilbert et al., 2002) and a number ofecdysteroidogenic genes, such as dare, Start1 and ecdysoneless, areknown to be expressed in the nurse cells (Freeman et al., 1999;Gaziova et al., 2004; Roth et al., 2004). These results indicate thatthe expression of nvd-Dm is enriched in tissues that synthesizesteroid hormones.

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Fig. 1. Spatiotemporal expression pattern of nvd-Bm. (A) Northernblot analysis showing temporal expression profiles of nvd-Bm. Thelength of the nvd-Bm cDNA (3747 bp) is similar to the 3.7 kb banddetected by northern hybridization. No other bands were detected (datanot shown). (B) Tissue expression profiles of nvd-Bm and a control generpL3 in the W1 fifth instar larvae by reverse transcription-PCR.

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RNAi of Drosophila neverland in the PG causeslarval arrestTo assess the importance of nvd-Dm during development, weexamined phenotypes of loss- or gain-of-nvd-Dm function indeveloping flies. As nvd-Dm is located in heterochromatin region(see Fig. S3 in the supplementary materials), it is difficult to isolateor create genetic mutations within the nvd-Dm locus by geneticmutant screens or homologous recombination techniques. Wetherefore examined the effects of overexpression or knock down ofnvd-Dm in developing flies using the GAL4/UAS system (Brand andPerrimon, 1993). In a wild-type background, overexpression of nvd-Dm using any of GAL4 drivers in Table 1 had no visible effect ondevelopment (data not shown). To knock down nvd-Dm indeveloping flies, we used transgenic RNA interference (RNAi),known to be an effective method of degrading endogenous targetmRNAs in Drosophila (Kennerdell and Carthew, 2000; Kalidas andSmith, 2002). We established transgenic lines in which double-stranded RNA molecules corresponding to nvd-Dm mRNA weregenerated using an inverted repeat construct under the control of theUAS promoter (UAS-nvd-Dm-IR; see Fig. S2 in the supplementarymaterials). We found that all of the RNAi animals failed to developinto adults when the UAS lines were crossed with two GAL4 lines,2-286-GAL4 and pGawB5015, in which GAL4 transgenes are activein the PG cells (Table 1). By contrast, the progeny resulting fromcrosses between UAS-nvd-Dm-IR flies and GAL4 lines, in which theGAL4 transgenes are not active in the PG, were fully viable (Table1), consistent with the expression pattern of nvd-Dm in wild-type

animals. This lethal phenotype by PG-GAL4 strains was observedusing two distinct nvd-Dm RNAi constructs (UAS-nvd-Dm-IR-1 andUAS-nvd-Dm-IR-2), which target different regions of nvd-DmmRNA (Table 1; see Fig. S2 in the supplementary material). Thereduction of nvd-Dm mRNA level in the RNAi larvae was confirmedby RT-PCR (Fig. 4A). These results suggest that nvd-Dm plays anessential role in the PG during fly development.

The lethal phase for animals carrying UAS-nvd-Dm-IR-1 and 2-286-GAL4 was examined in more detail. For simplicity, we refer tothese animals as ‘nvd-Dm RNAi animals’. nvd-Dm RNAi animalscompleted embryogenesis and hatched normally. Followinghatching, the first instar larvae of nvd-Dm RNAi animals showed noapparent morphological or behavioral defects (Fig. 4C-E; data notshown). However, ~48 hours after egg laying (AEL), nvd-Dm RNAianimals showed apparent growth arrest in body size compared withcontrol animals (Fig. 4E-G). To examine whether larval moltingoccurs in nvd-Dm RNAi animals, the larval stages were determinedby stage-specific size and morphology of mouth hook (Roberts andStanden, 1998). By 48 hours AEL, both the control and RNAianimals had the small, one-toothed mouth hook that is characteristicof the first instar larvae (Fig. 4H,I,M,N). Forty-eight hours AEL, thecontrol animals underwent the first molt and showed the secondinstar larva-type mouth hook with two to five teeth (Fig. 4O,P). After72 hours AEL, the control animals molted to the third instar larvaeand possessed larger mouth hooks with numerous small teeth (Fig.4Q). By contrast, nvd-Dm RNAi animals still retained the mouthhook of the first instar larva-type, even 96 hours AEL (Fig. 4J-L). At

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Fig. 2. neverland is conserved amongseveral animal species. (A) Predictedpolypeptides that are produced from nvd-Bm (Bombyx mori) (GenBank AccessionNumber AB232986) and its closestrelatives from Drosophila melanogaster(AB232987), Anopheles gambiae(XP_309236.1), Caenorhabditis elegans(NP_505629), Ciona intestinalis (ESTclones cicl031b24 and cicl077j15 fromthe Ciona cDNA database;http://ghost.zool.kyoto-u.ac.jp/cgi-bin/getblast1.cgi), Danio rerio(NP_001002612), Gallus gallus(XP_425346), Rhodococcus erythropolis(kshA gene, AAL96829) andPseudomonas fluorescens (prnD gene,U74493). These polypeptides share aRieske domain (dark gray) and a highlyconserved domain in the C-terminalregion (light gray). The bars above eachdomain indicate the positions of the [2Fe-2S] binding motif (C-X-H-X16-17-C-X2-H)and the non-heme iron-binding motif[Fe(II); E-X3-D-X2-H-X4-H]. Percentagesrepresent amino acid identities betweeneach domain of nvd-Bm and that ofothers. Also indicated are the totalnumbers of residues for individualproteins. The Anopheles and Cionaorthologs are incompletely predicted onboth ends. A black box indicates thetransmembrane domain predicted byTMHMM software (http://www.cbs.dtu.dk/services/TMHMM/). (B) Sequence alignment of the Rieske motif and the non-heme iron-binding motif ofNvd-Bm with its closest relatives. Identical residues in all nine species are white on black, while identical residues in five or more species are boxed.The evolutionally conserved amino acids of the Rieske domain and non-heme iron binding motif are indicated above the alignment.

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34% 22%3981Rhodococcus

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C H C HBombyx 166 YCPHLGANLAVGGTV-RGSCIECPFHK 191Drosophila 137 YCPHLGANLGIGGSV-ADDCVICPFHQ 162Anopheles 93 YCPHLGANLGVGGIV-RGDCIECPFHH 118Caenorhabditis 167 YCPHIGANFNIGGRVVRDNCIQCPFHG 193Ciona 107 YCPHMGGNLAVGGIV-KNDCLECPFHG 132Danio 163 YCPHLGANLAVGGRVV-GGCIECPFHG 188Gallus 56 YCPHLGADLAAGGRVV-GSCIECPFHG 81Rhodococcus 79 YCRHMGGNLAHG-TVKGDS-IACPFHD 103Pseudomonas 68 HCSHLGANLADG-R-IKDGCIQCPFHH 92

E D H H269 PENGADVPHLNAVHS 283243 PENGADIAHFNAIHK 257201 PENGADVAHLSAVHG 215275 PENGADIAHLNYLHK 289212 PENGSDLAHLSHLHV 226269 PENAADIAHLAHLHT 283162 PENAADTAHLAFLHG 176186 VDNVVDMAHFFYVHY 200178 VENFYDAQHATPVHA 192

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around 72 hours AEL, most of the nvd-Dm RNAi animals graduallyceased feeding and became stationary. Ninety-eight percent (54/55)of these animals died before 108 hours AEL as first instar larva, asjudged from the mouth-hook morphology (Fig. 4B, Fig. 6I). Wenoted that all of the nvd-Dm RNAi animals failed to show thedouble-mouth-hook phenotype that frequently occurs in mutantswith defects in ecdysone signaling (Li and Bender, 2000; Gaziovaet al., 2004), implying that loss of nvd-Dm function prevents larvalgrowth prior to the initiation of the molting process. These resultssuggest that nvd-Dm plays a pivotal role in larval development inDrosophila. On this basis of the prolonged first instar larvalphenotype, we named this gene ‘neverland’, which is the fictionalisland featured in J. M. Barrie’s play Peter Pan, where children ceaseto age.

Larval arrest of nvd-Dm RNAi animals is due to areduction of ecdysteroid titerThe PG-specific expression of nvd-Dm suggests that nvd-Dm couldfunction in ecdysteroidogenesis in the PG and that nvd-Dm RNAianimals might have a reduced ecdysteroid titer during development.As a direct test of this hypothesis, we measured the ecdysteroid titerin these animals using a radioimmunoassay. nvd-Dm RNAi andcontrol animals were collected 36±6 hours AEL, at which time wild-type first instar larvae show a peak of 20-hydroxyecdysone (20E)

(Kraminsky et al., 1980; Sullivan and Thummel, 2003; Warren et al.,2006). Organic extracts prepared from nvd-Dm RNAi animals had asignificantly reduced level of ecdysteroid compared with extractsfrom control animals (Fig. 5), indicating that nvd-Dm activity isrequired for ecdysteroid biosynthesis in PG.

To address whether the larval arrest of nvd-Dm RNAi animals isdue to reduced ecdysteroid titers, we attempted to rescue the larvalarrest phenotype by feeding ecdysteroids to nvd-Dm RNAi animals.When newly-hatched nvd-Dm RNAi animals were fed yeast pastesupplemented with active ecdyseroid (20E) 30-42 hours AEL in thefirst instar period, the arrest phenotype was rescued and the animalsgrew to the second instar larvae. However, the animals later died asprolonged second instar larvae (Fig. 6A,B,I). Similarly, when therescued second instar larvae were fed the 20E-containing foodduring 54-66 hours AEL in the second instar period, they molted intothe third instar larvae but failed to become pupae (Fig. 6C,D,I).Furthermore, when the rescued third instar animals received foodwith 20E after 78 hours AEL, these RNAi animals were partiallyrescued and developed into pupae. A number of the pupaecompleted metamorphosis and emerged as fertile adults (Fig. 6E,I),although almost all died within one week of eclosion. Like the nvd-Dm animals without 20E, second and third instar larvae fed with 20Efor 12 hours during each instar also showed the prolonged larvalphenotype, surviving for 3-4 days after each molting. In spite of the

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Fig. 3. Expression pattern of nvd-Dm. (A-C) In situ embryonicexpression (see also Fig. S4 in thesupplementary material). nvd-DmmRNA was not detected during earlystages of embryogenesis, such aspreblastoderm stage (A) or stage 8(B). At stage 17 (C), nvd-Dmexpression was detected specificallyin the ring gland (arrowhead).(D) Brain-ventral nerve cord-ringgland complex of wandering stage ofthird instar larva. (D��) Highermagnification of D. Expression wasdetected only in the region of PGcells, but not in either the corpusallatum (CA) or the corpus cardiacum(CC). (E) Quantitative RT-PCR analysisof nvd-Dm transcript in several larvaltissues from wandering third instar larvae. nvd-Dm/rp49 indicates the levels of nvd-Dm mRNA normalized to the levels of internal rp49 mRNA. Thenormalized nvd-Dm mRNA level in the ring gland is set as 1. (F) Cyclic expression of nvd-Dm in the ring glands during the second and third larvalstages shown by the quantitative RT-PCR. The normalized nvd-Dm mRNA level from the wandering third instar larvae is set as 1 (±s.e.m.; n=3). (G-I)Ovarian expression. nvd-Dm mRNA was detected in the nurse cells (G,H; arrow), whereas oocytes was devoid of nvd-Dm expression (H; arrowhead).No staining was obtained for the sense RNA probes in ovaries (I), embryos and larvae (data not shown).

Table 1. Targeted expression of UAS-nvd-Dm-IR transgenes using several GAL4 driversParental genotype Expression domain Lethality

UAS-nvd-Dm-IR-1 � 2-286-GAL4 PG (ring gland), salivary glands, some neurons Larval lethalUAS-nvd-Dm-IR-1 � pGawB5015 PG (ring gland), lymph gland, haemolymph Larval lethalUAS-nvd-Dm-IR-1 � AUG21-GAL4 Corpus allatum Fully viableUAS-nvd-Dm-IR-1 � AKH-GAL4 Corpus cardiacum Fully viableUAS-nvd-Dm-IR-1 � bth-GAL4 Tracheae Fully viableUAS-nvd-Dm-IR-1 � scabrous-GAL4 Neuronal cells, epithelial cells Fully viableUAS-nvd-Dm-IR-1 � elavC155-GAL4 Post-mitotic neurons Fully viableUAS-nvd-Dm-IR-1 � Lsp2-GAL4 Fat body Fully viableUAS-nvd-Dm-IR-1 � dpp-GAL4 Numerous (not in PG) Fully viableUAS-nvd-Dm-IR-1 � tsh-GAL4 Numerous (not in PG) Fully viableUAS-nvd-Dm-IR-2 � 2-286-GAL4 PG (ring gland), salivary glands, some neurons Larval lethal

Transgenic male adults bearing UAS-nvd-Dm-IR were crossed to females of several GAL4 drivers.

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prolonged phenotype, both the rescued second and third instar larvaedid not overgrow, and their body sizes were almost the same asmature second and third instar wild-type larvae, respectively (Fig.6A,C). We confirmed that PG cells looked normal in the prolongednvd-Dm RNAi larvae (see Fig. S5 in the supplementary materials),indicating that the nvd-Dm RNAi phenotype is not due to loss of PGcells. Taken together, these results suggest that nvd-Dm is essential

for insect molting, metamorphosis and body growth throughoutdevelopment via the regulation of ecdysteroid biosynthesis in thePG.

The lethality of nvd-Dm RNAi animals iscompletely rescued by 7-dehydrocholesterolTo examine which ecdysteroid conversion step is affected by loss-of-function of nvd-Dm, we performed a feeding experiment withprecursors of ecdysone biosynthesis. If Neverland functions in aspecific conversion step of the ecdysteroid biosynthesis pathway, wewould expect that an exogenously applied intermediate, which isdownstream of the conversion step by Nvd, would overcome thedevelopmental arrest phenotype observed in the nvd-Dm RNAianimals. We performed the feeding experiment with two precursorsof ecdysteroid biosynthesis, cholesterol (C) and 7-dehydrocholesterol (7dC) (Gilbert et al., 2002). C and 7dC areinvolved in the first step of ecdysteroidogenesis in the PG; 7dC issynthesized from C by 7,8-dehydrogenation by an uncharacterizedenzyme (Grieneisen et al., 1993; Gilbert et al., 2002; Gilbert andWarren, 2005). When 7dC was added to the food, all nvd-Dm RNAianimals were completely rescued and developed into adults (Fig.6F,I). The food with 7dC also restored the ecdysone titer in nvd-DmRNAi animals (Fig. 5), suggesting that the nvd-Dm mutants do nothave a defect in the conversion steps from 7dC to ecdysone in its PG.However, nvd-Dm RNAi animals raised on food containingexcessive C were partially rescued; about 47% (18/38) of theseanimals molted to the second instar larvae (Fig. 6G,H,I), but no nvd-Dm RNAi animal was observed to molt to the third instar larvae. Itshould be noted that although the nvd-Dm RNAi larvae rescued byC arrested in the second instar larvae, they survived for about 3weeks and their body size reached almost the same size as wild-typethird instar larvae (Fig. 6G). These results strongly suggest that theNvd proteins act upstream of 7dC production in the ecdysteroidsynthesis pathway in the PG.

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Fig. 4. Developmental arrest of nvd-DmRNAi animals. (A) RT-PCR analysis of nvd-Dmand control gene rp49 in RNAi and controlanimals 48 hours AEL. (B) Comparison of thesurvival rate and developmental progression ofnvd-Dm RNAi animals versus control animals.nvd-Dm RNAi animals did not become pupaeand died as larvae. (C-G) Comparison of bodysize between nvd-Dm RNAi and controlanimals. Anterior is leftwards. RNAi animals(upper) grew at the same rate as controlanimals (lower) until about 48 hours after egglaying (AEL) (C-E). Forty-eight hours AEL, RNAianimals failed to grow in size (F,G).(H-Q) Dissected mouth hooks of nvd-Dm RNAianimals (H-L) and control animals (M-Q) atdifferent developmental stages. Redarrowheads indicate teeth of the mouth hooks.Control first instar larvae had one-tooth mouthhooks (M,N), control second instar larvae hadtwo to five teeth (O,P), and control third instarlarvae had numerous small teeth (Q). Bycontrast, mouth hooks of all nvd-Dm RNAianimals remained small and showed thecharacteristic morphology of first instar larvae,even 96 hours AEL (H-L). Scale bar: 1.2 mm forC-G; 10 �m for H-Q.

Fig. 5. nvd-Dm RNAi animals show reduced ecdysteroid titer.Larvae were collected 30-42 hours after egg laying (AEL),corresponding to the first instar larval stages. Ecdysteroid titers ofcontrol (black box) and the nvd-Dm RNAi animals (shaded box) on foodwith or without 7-dehydrocholesterol (7dC) were determined byradioimmunoassay. The results are depicted as picograms (pg) of 20-hydroxyecdysone equivalents/mg initial body weight on the vertical axis.Each bar represents the mean±s.e.m. from multiple independentsamples. The following number of independent samples was examined:four (control), five (RNAi) and six (control + 7dC and RNAi + 7dC).*P<0.01 by Student’s t-test.

control RNAi

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DISCUSSIONThe neverland genes play an essential role inecdysteroid biosynthesis in insectsIn this study, we identified the neverland genes from Bombyx (nvd-Bm) and Drosophila (nvd-Dm), which belong to an evolutionallyconserved family of Rieske-domain proteins and are expressedspecifically in tissues that synthesize ecdysone, including the PG.We also showed through molecular genetics and feeding assays inDrosophila that loss of nvd function in the PG causes growth arrestduring larval stages that is due to a reduced ecdysteroid titer. All ofour results are consistent with the hypothesis that the nvd family ofproteins plays a pivotal role in steroidogenesis. The primary proteinstructure of Nvd is completely different from that of proteinsencoded by other known genes expressed predominantly in the PG,i.e. Halloween P450s (Gilbert and Warren, 2005), Dare (Freeman etal., 1999) and Start1 (Roth et al., 2004).

Spatiotemporal expression pattern of nvdThe spatial and temporal expression pattern of nvd during lateembryogenesis and larval development is similar to that ofHalloween P450 genes, i.e., phm, dib and sad, in both Bombyx andDrosophila. The expression of nvd-Bm is detected specifically inthe PG, and nvd-Dm mRNA is enriched in both the PG and ovary,tissues that synthesize ecdysteroids. Temporal changes inexpression are also observed in both nvd-Bm and nvd-Dm, and arecorrelated with changes in ecdysone titers during larvaldevelopment. However, it should be emphasized that the earlyembryonic expression pattern of nvd-Dm is completely differentfrom the patterns of the Halloween P450 genes. During Drosophilaembryogenesis, ecdysteroid levels begin to rise around the onset ofgastrulation (stages 6-7) and peak at stages 11-12 during germ bandretraction (Kraminsky et al., 1980; Moróy et al., 1988; Sullivan andThummel, 2003). The expression levels of the Halloween genes are

strongly correlated with embryonic ecdysteroid titers, andmutations in any of these genes cause ecdysteroid deficiency in theembryo (Gilbert and Warren, 2005; Namiki et al., 2005). Bycontrast, nvd-Dm expression is not detected at this early embryonicstage, suggesting that nvd-Dm is not necessary to regulate ecdysoneproduction during embryogenesis. Although it is known thatmaternally synthesized ecdysteroids are provided in Drosophilaeggs (Bownes et al., 1988), it has been unclear what type ofecdysteroid precursor is required for ecdysone production duringembryogenesis (Chávez et al., 2000). Identifying the exact steps inwhich Nvd is involved, as discussed below, would shed light onhow ecdysone synthesis varies across different developmentalstages.

Molecular functions of the neverland family ofproteinsThe Nvd proteins have strong similarities to class IA oxygenases,which have both a consensus [2Fe-2S] Rieske-type domain and adomain that is highly similar to the proposed mononuclear non-heme Fe (II) binding motif (Mason and Cammack, 1992). Amongthe enzymes in this class, the protein encoded by Rhodococcus kshAfunctions in steroid hydroxylation (van der Geize et al., 2002),raising the possibility that the Nvd proteins could catalyze oxidationor hydroxylation reactions in the ecdysteroid biosynthesis pathway.

In which step of ecdysteroid biosynthesis does Nvd function?Considering the results of our rescue experiments with cholesterol(C) and 7-dehydrocholesterol (7dC), it is likely that the Nvd proteinmight play a role in transport and/or metabolism of cholesteroland/or its derivatives. This idea is also supported by the recentlyreported phenotype of Drosophila NPC1 mutants (Fluegel et al.,2005; Huang et al., 2005). Drosophila NPC1 is an ortholog ofmammalian Niemann-Pick type C disease genes that are well knownto encode cholesterol-binding proteins (Chang et al., 2005). NPC1

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Fig. 6. Developmentalprogression of nvd-Dm RNAianimals by feeding 20-hydroxyecdysone, cholesteroland 7-dehydrocholesterol. nvd-Dm RNAi (black arrowheads) andcontrol larvae were fed yeast pastesupplemented with either 3.3%ethanol, 1 mg/ml 20-hydroxyecdysone (20E) or 0.5% wetweight of cholesterol (C) or 7-dehydrocholesterol (7dC). All controlanimals grew to the third instarlarvae 96 hours AEL. Red arrowheadsindicate teeth of the mouth hooks.(A,B) nvd-Dm RNAi animals fed 20E-containing food during only 30-42hours after egg laying (AEL) moltedto the second instar larvae. However,96 hours AEL they showed smallbody size (A) and still possessed thesecond instar-type mouth hook (B).(C,D) nvd-Dm RNAi animals with 20E30-42 hours and 54-66 hours AELmolted to the third instar larvae. (E) nvd-Dm RNAi animals fed 20E 30-42 hours, 54-66 hours and 78 hours AEL. After becoming third instar larvae,45% and 5% of the nvd-Dm animals pupariated and eclosed (E), respectively. (F) nvd-Dm RNAi animals with 7dC. All the animals grew to adults.(G,H) nvd-Dm RNAi animals with excessive C had the second instar-type mouth hook 96 hours AEL (H). Their body size nearly reached the size ofcontrol animals (G). Scale bar: 1.0 mm for A,C,E,F,G; 10 �m for B,D,H. (I) Lethality of animals in the feeding experiments. The stage of developmentat which an animal died is depicted as a percentage of animals that died at that stage. ‘30-42’, ‘54-66’ and ‘78-‘ in brackets refer to the periods(AEL) at which animals were fed 20E-treated yeast pastes. ’n’ refers to the total number of animals.

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mutants show a larval arrest phenotype similar to that of the nvd-DmRNAi animals discussed above, and this phenotype is rescued tovarying degrees by feeding with 20E, C or 7dC.

Our feeding experiments reveal that food containing 7dCcompletely rescues the nvd-Dm RNAi phenotype, indicating thatNvd acts upstream of 7dC synthesis. Given the fact that suchcomplete rescue activity is not observed when food supplementedwith excessive C was used, it is possible that Nvd might function inthe conversion of C to 7dC. This hypothesis is consistent with thefollowing points.

(1) nvd-Dm is not expressed during mid-embryogenesis.Although ecdysteroidogenesis takes place at this stage, embryos donot consume food containing dietary C. By contrast, the larvalexpression of nvd-Dm coincides with the uptake of C, suggesting arole for nvd in C metabolism.

(2) The nvd ortholog is conserved in the nematode C. elegans, inwhich the C to 7dC conversion takes place during steroidbiosynthesis even though C. elegans does not have ecdysteroids(Chitwood, 1999).

(3) A mutant phenotype of C. elegans ortholog of nvd, asdiscussed below, is rescued by feeding with 7dC, but not with C(Rottiers et al., 2006).

It is possible, however, that Nvd itself does not directly catalyzeC to 7dC. Previous biochemical studies have shown that theconversion of C to 7dC is mediated by an enzyme with cytochromeP450 characteristics under the control of the Zn-finger proteinWithout Children (Woc) (Grieneisen et al., 1993; Warren et al.,1995; Warren et al., 2001). Our RT-PCR analysis did not detect asignificant difference of nvd-Dm expression level between wocmutant and wild type (see Fig. S6 in the supplementary materials).We also tested whether the Nvd proteins can catalyze cholesterol andcholesterol derivatives (22-hydroxycholesterol, 25-hydroxycholesterol and 7dC) using a S2 cell system previouslydescribed in biochemical studies of Halloween P450s (Warren et al.,2002; Niwa et al., 2004). However, no metabolites have yet beendetected (data not shown).

Although the proposed function for Nvd on the conversion fromC to 7dC is most likely, we should point out that food containingexcessive C partially rescues the defect of nvd-Dm RNAi animals.This raises the possibility that the partial rescue activity by C mightbe due to a hypomorphic nature of the nvd-Dm RNAi. In nvd-DmRNAi animals, we detect a significantly reduced but specific level ofnvd-Dm mRNA (Fig. 4A) and substantial 20E (Fig. 5), suggestingthat the nvd-Dm RNAi animals might still possess low levels of Nvdactivity and thus these animals may be able to produce smallamounts of 7dC from C. An alternative explanation is that Nvdwould function in the ‘black box’ in which 7dC is converted to the�4-diketol by an uncharacterized mechanism, because 7dC is aslightly unstable chemical and it is possible that the rescuing activityof 7dC might be caused by a contaminant oxidated in the food. Inorder to determine the function of Nvd in more detail, isolation ofgenetic null mutants of nvd and the biochemical properties of Nvdproteins will need to be investigated.

The functional role of the Neverland proteins inother organismsOur current studies do not address the functional role of the Nvdproteins in organisms other than insects. Notably, a nvd ortholog isfound in the C. elegans genome. According to recent advances intaxonomy, both insects and nematodes belong to Ecdysozoa, whichare characterized by molting (ecdysis) behavior (Aguinaldo et al.,1997). In C. elegans, it has been suggested that uncharacterized

steroid hormones play a role in both molting and dauer formation(Thummel, 2001; Entchev and Kurzchalia, 2005). Therefore, wepostulate that Nvd may be important in controlling proper larvaldevelopment in nematode species. Indeed, during the revision of thispaper, it has been demonstrated that the daf-36 gene, which encodesthe C. elegans homolog of nvd, is required to bypass the C. elegansdauer diapause (Rottiers et al., 2006). It is thought that daf-36mutants cannot produce a steroid-like hormone that prevents dauerformation, suggesting that nvd/daf-36 is also essential for a steroid-like synthesis pathway in nematodes. daf-36 mutants are also shownto displays adult aging phenotypes (Rottiers et al., 2006). As reduced20E in Drosophila is associated with expanded lifespan (Tu et al.,2002; Simon et al., 2003; Colombani et al., 2005), it would beintriguing to explore the role of nvd on insect aging.

Interestingly, no ortholog of nvd has been found in genomes ofmammalian species, while nvd orthologs are conserved in otherchordates, such as ascidian, zebrafish and chicken. This suggests thatnvd orthologs might have been lost during mammalian evolution.Currently, we do not know the exact differences of steroidbiosynthesis pathways between mammals and other animals. Futurestudies on the nvd family could provide new insight into thesimilarities and differences between steroid hormone biosynthesisamong animal clades.

We are grateful to Toshiro Aigaki, Adam Antebi and Veerle Rottiers forinforming us their unpublished data. We also thank Yoshinori Fujimoto,Lawrence I. Gilbert, Günter Korge, Joe H. Park, Carl S. Thummel, TadashiUemura, James T. Warren, the Bloomington stock center, National Institute ofGenetics of Japan and Drosophila Genetic Resource Center of Kyoto Instituteof Technology for stocks and reagents; and Frank J. Slack, Diya Banerjee,Katherine Olsson Carter, Aurora Esquela-Kerscher and Helge Großhans forcritical reading of the manuscript. R.N. was a recipient of SPD researchfellowship of the Japan Society for the Promotion of Science (JSPS). This workwas supported by grants to K.M. from the Program for Promotion of BasicResearch Activities for Innovative Biosciences and the Insect Technology Projectof the Ministry of Agriculture, Forestry and Fisheries of Japan; and to H.K. fromResearch for the Future Program of the JSPS.

Supplementary materialSupplementary material for this article is available athttp://dev.biologists.org/cgi/content/full/132/13/2565/DC1

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RESEARCH ARTICLE Development 133 (13)