Hormonal regulation and developmental role of Krüppel homolog 1,a repressor of metamorphosis, in the silkworm Bombyx mori

Takumi Kayukawa a, Mika Murata a, Isao Kobayashi a, Daisuke Muramatsu b, Chieko Okada a,Keiro Uchino a, Hideki Sezutsu a, Makoto Kiuchi a, Toshiki Tamura a, Kiyoshi Hiruma b,Yukio Ishikawa c, Tetsuro Shinoda a,n

a National Institute of Agrobiological Sciences, Ohwashi 1-2, Tsukuba, Ibaraki 305-8634, Japanb Faculty of Agriculture and Life Sciences, Hirosaki University, Hirosaki 036-8561, Japanc Laboratory of Applied Entomology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo 113-8657, Japan

a r t i c l e i n f o

Article history:Received 3 September 2013Received in revised form17 January 2014Accepted 26 January 2014Available online 4 February 2014

Keywords:Krüppel homolog 1Juvenile hormoneEcdysteroidMoltMetamorphosisEcdysone-inducible gene

a b s t r a c t

Juvenile hormone (JH) has an ability to repress the precocious metamorphosis of insects during their larvaldevelopment. Krüppel homolog 1 (Kr-h1) is an early JH-inducible gene that mediates this action of JH; however,the fine hormonal regulation of Kr-h1 and the molecular mechanism underlying its antimetamorphic effect arelittle understood. In this study, we attempted to elucidate the hormonal regulation and developmental role ofKr-h1. We found that the expression of Kr-h1 in the epidermis of penultimate-instar larvae of the silkwormBombyx mori was induced by JH secreted by the corpora allata (CA), whereas the CA were not involved in thetransient induction of Kr-h1 at the prepupal stage. Tissue culture experiments suggested that the transient peakof Kr-h1 at the prepupal stage is likely to be induced cooperatively by JH derived from gland(s) other than theCA and the prepupal surge of ecdysteroid, although involvement of unknown factor(s) could not be ruledout. To elucidate the developmental role of Kr-h1, we generated transgenic silkworms overexpressing Kr-h1.The transgenic silkworms grew normally until the spinning stage, but their development was arrested at theprepupal stage. The transgenic silkworms from which the CA were removed in the penultimate instar did notundergo precocious pupation or larval–larval molt but fell into prepupal arrest. This result demonstrated thatKr-h1 is indeed involved in the repression of metamorphosis but that Kr-h1 alone is incapable of implementingnormal larval molt. Moreover, the expression profiles and hormonal responses of early ecdysone-induciblegenes (E74, E75, and Broad) in transgenic silkworms suggested that Kr-h1 is not involved in the JH-dependentmodulation of these genes, which is associated with the control of metamorphosis.

& 2014 Elsevier Inc. All rights reserved.

Introduction

Insect metamorphosis is coordinated by the actions of ecdysteroidsand juvenile hormone (JH), which are secreted by the prothoracicgland and corpora allata (CA), respectively. In holometabolous insects,20-hydroxyecdysone (20E), the active metabolite of ecdysteroids,induces the larval–larval molt in the presence of JH, whereas inthe absence of JH, it induces larval–pupal and pupal–adult molts(Riddiford, 1994). In many insects, the topical application of JHanalogs during larval development inhibits normal metamorphosis,leading to supernumerary larval molt or prepupal arrest (Retnakaranet al., 1985; Kamimura and Kiuchi, 2002). Conversely, the deprivationof JH by the removal of CA (allatectomy) from the penultimate larvaeinduces precocious metamorphosis (Dominick and Truman, 1985).

Thus, the major function of JH is to prevent larvae from precociouslyturning into adults (status quo action) (Riddiford, 1994).

The molecular actions of 20E in larval–pupal metamorphosishave been elucidated in detail: 20E binds to a complex of theecdysone receptor (EcR) and ultraspiracle (USP), both of whichbelong to the nuclear receptor superfamily. The liganded EcR/USPcomplex directly activates the transcription of early ecdysone-inducible genes such as E74, E75, and the Broad complex (Broad),all of which are transcriptional regulatory factors. The earlyecdysone-inducible genes subsequently regulate large groups oflate ecdysone-inducible genes, some of which are involved in theformation of pupal traits (Dubrovsky, 2005; Nakagawa andHenrich, 2009). JH has been reported to modify the expressionof several isoforms of the early ecdysone-inducible genes inManduca sexta (Stilwell et al., 2003; Keshan et al., 2006; Zhouet al., 1998), but the molecular mechanism underlying thismodulation remains largely unknown.

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Developmental Biology

http://dx.doi.org/10.1016/j.ydbio.2014.01.0220012-1606 & 2014 Elsevier Inc. All rights reserved.

n Corresponding author. Fax: þ81 29 838 6075.E-mail address: shinoda@affrc.go.jp (T. Shinoda).

Developmental Biology 388 (2014) 48–56

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Minakuchi et al. (2008) revealed for the first time that theKrüppel homolog 1 (Kr-h1) gene, a C2H2 zinc-finger type tran-scription factor, is a JH-early-inducible gene and represses themetamorphic differentiation of adult abdominal epidermis inDrosophila melanogaster. This gene plays a key role in the repres-sion of larval–pupal and nymphal–adult transitions in holometa-bolous and hemimetabolous insects, respectively (Minakuchi et al.,2009; Lozano and Belles, 2011; Konopova et al., 2011). Recentstudies clarified the mechanism underlying JH-mediated inductionof Kr-h1 in insects: JH binds to its receptor, methoprene tolerant(Met) (Wilson and Fabian, 1986; Ashok et al., 1998; Miura et al.,2005; Charles et al., 2011); then, JH-liganded Met interacts withsteroid receptor coactivator (SRC) (Li et al., 2011; Zhang et al.,2011; Kayukawa et al., 2012), and the JH/Met/SRC complexactivates Kr-h1 by interacting with JH response element (kJHRE)in the Kr-h1 gene (Kayukawa et al., 2012, 2013).

Although Kr-h1 plays a crucial role in the repression of insectmetamorphosis, only a few studies have attempted to elucidate itsmolecular mechanisms. In D. melanogaster, mutations in Kr-h1cause changes in the expression patterns of ecdysone-induciblegenes such as E74A, E75B, E93, HR3, and βFTZ-F1 during metamor-phosis (Pecasse et al., 2000). Recently, Kr-h1 was found to berequired for the suppression of Broad in the fat body of youngD. melanogaster larvae (Huang et al., 2011). These studies suggestthat Kr-h1 may repress metamorphosis via modification of theexpression of early-ecdysone inducible genes. However, no furtherstudies have been conducted to elucidate the molecular mechanismsof the repression of metamorphosis by Kr-h1.

In the present study, we investigated the hormonal regulationof Kr-h1 across the postembryonic development of B. mori, and weanalyzed the functions of Kr-h1 in the larval–larval molt andlarval–pupal transition by using transgenic silkworms overexpres-sing Kr-h1. Based on our findings, we reviewed the JH signalingcascade from the viewpoint of the mode of action of Kr-h1 in therepression of metamorphosis.

Material and methods

Experimental animals and dissection

B. mori (Kinsyu� Showa strain) larvae were reared on anartificial diet (Nosan Corp, Japan) at 25 1C under a 12-h light/darkphotoperiod. The epidermis was dissected from the insects atvarious stages in phosphate-buffered saline (137 mM NaCl, 8 mMNa2HPO4, 2.7 mM KCl, and 1.5 mM KH2PO4, pH 7.4).

piggyBac vectors and transgenic silkworms

The open reading frame (ORF) of BmKr-h1α anchored with theXbaI site at both ends was amplified from a full-length cDNA(Kayukawa et al., 2012) by using the BmKrh1α_XbaI_FW1 andBmKrh1α_XbaI_RV1 primers (Table S1). The amplified ORF frag-ment of BmKr-h1αwas digested with XbaI and inserted into the BlnIsite of the plasmid pBacMCS[UAS-3xP3-EGFP] (Sakudoh et al., 2007)to generate the piggyBac vector pBac[UAS-BmKr-h1α, 3xP3-EGFP].The piggyBac vector and the helper plasmid pHA3PIG were dis-solved in the injection buffer (5 mM KCl, 0.5 mM sodium phos-phate, pH 7), each at a concentration of 0.2 mg/mL. To generate atransgenic line harboring UAS-BmKr-h1α, the piggyBac plasmidwas coinjected with the helper plasmid into preblastodermembryos of the w-1 pnd strain of B. mori (Tamura et al., 2000).G1 embryos were screened under a fluorescence stereomicroscopeequipped with an EGFP filter to obtain the UAS-BmKr-h1α line.By crossing the UAS-BmKr-h1α line with the A3-Gal4 line, whichhas a promoter derived from actin A3 of B. mori (Imamura et al.,

2003), silkworms harboring two transgenes, UAS-BmKr-h1α andA3-Gal4, were generated.

Allatectomy and JHA treatment

The CA were removed from B. mori larvae on day 0 or day 3 ofthe 4th instar under a stereomicroscope by using fine forceps, andthe larvae were allowed to recover for 3 h after the operation. Thelarvae allatectomized on day 0 underwent precocious metamor-phosis, whereas those allatectomized on day 3, which had alreadycommitted to undergo larval–larval molt, normally molted to the5th instar and developed into normal adults. To examine the effectof exogenous JH on gene expression in the allatectomized larvae,2 μL of an acetone solution of methoprene (juvenile hormoneanalog [JHA], 0.5 μg/μL, SDS Biotech) or 2 μL of acetone (control)was topically applied to the dorsal abdomen of the allatectomizedlarvae. The epidermis was dissected from each larva at 24 h postapplication, and the levels of gene expression in the tissue weredetermined by quantitative real time PCR (qPCR). The effect ofexogenous JH on gene expression in adults derived from allatec-tomized larvae (referred to as “allatectomized adults”) was alsoexamined: 4 μL of an acetone solution of JHA (0.5 μg/μL) or 4 μL ofacetone (control) was topically applied to the dorsal abdomen ofthe adults at 4 h after eclosion, and the epidermis of each adultwas dissected at 6 h post application.

Tissue culture

Dorsal abdominal integuments of larvae were cultured inGrace's insect medium (Gibco, Invitrogen) at 25 1C according tothe method reported by Muramatsu et al. (2008). The integumentswere precultured with hormone-free medium for 3 h and subse-quently incubated in medium containing either JH I (SciTech) or20-hydroxyecdysone (20E, Sigma-Aldrich), or both, for 3 h.

qPCR

Total RNA was extracted from the epidermis by using theRNeasy Plus mini kit (Qiagen) and was used to synthesize thecDNAwith the PrimeScript RT reagent kit (Takara Bio). The primersdesigned to quantify the transcripts of BmKr-h1 (all isoforms),BmKr-h1α, BmKr-h1β, BmBRC (all isoforms), BmE74A, BmE74B,BmE75A, BmE75B, BmE75C, BmE75D, and BmRp49 are shown inTable S1. BmRp49 was used as the internal reference. The reactionwas carried out in a 10-μL reaction volume containing thetemplate cDNA derived from 1 ng of total RNA, 5 μL of SYBRPremix Ex Taq (Takara Bio), and 0.2 μM of each primer by usingthe LightCycler 480 real-time thermal cycler (Roche). The PCRconditions were 95 1C for 5 min, followed by 55 cycles each at95 1C for 5 s and 60 1C for 20 s. The relative molarities of the genetranscripts were obtained with crossing point analysis involvingstandard curves generated using plasmids containing a fragmentof each gene. The expression levels of the target genes werenormalized against that of BmRp49.

Results

Developmental expression profile

Developmental changes in the expression of BmKr-h1α andBmKr-h1β, two isoforms of Kr-h1, in the epidermis of B. mori weredetermined by qPCR (Fig. 1). Although the levels of BmKr-h1αwereapproximately tenfold higher than those of BmKr-h1β (see theinset of Fig. 1), the overall expression patterns of the two isoformswere very similar. The transcripts of BmKr-h1α/β were

T. Kayukawa et al. / Developmental Biology 388 (2014) 48–56 49

continuously detected during the 3rd and 4th instar stages, withfluctuations (Fig. 1). The BmKr-h1α/β transcripts disappearedcompletely by day 1 of the 5th instar, reappeared when the larvaebegan spinning (day 6), peaked during the prepupal stage (days 7–8), and disappeared again at 1 d before pupation (day 9) (Fig. 1). Inthe pupal stage, the expression of BmKr-h1α/β remained unde-tectable for the first half of the stage, but gradually increased fromday 6 toward the end of the stage, and the high level wasmaintained during the adult stage (Fig. 1). Little difference wasobserved between the expression levels of BmKr-h1α/β in femalesand males. The expression patterns of BmKr-h1α/β were closelycorrelated with the titer of ecdysteroids in the hemolymph of B.mori. Notably, the peaks of the transcripts on day 3 of the 4th-instar larvae and at the prepupal stage corresponded well withthose of ecdysteroids (Fig. 1).

Hormonal regulation of BmKr-h1 expression at the prepupal andadult stages

In the epidermis of the 4th-instar larvae, the transcription ofBmKr-h1 is positively regulated by JH (Kayukawa et al., 2012). Toexamine whether BmKr-h1 is regulated by JH also in other stages,we removed the CA from 4th-instar larvae on day 3. Because theallatectomized larvae had already committed to larval–larval molt,they normally molted to the 5th instar and became normal pupaeand normal adults. The BmKr-h1 transcripts in the allatectomizedadults were drastically decreased irrespective of the sex (Fig. 2A).The transcript levels in the allatectomized adults treated with JHAremained high in both sexes (Fig. 2A), suggesting that the

expression of BmKr-h1 in the adult stage is also induced by JHsecreted by the CA. In contrast, the BmKr-h1 transcript level in theprepupal stage was barely affected by the allatectomy (Fig. 2B).Given the occurrence of a peak ecdysteroid titer at the prepupalstage (Fig. 1), this finding led us to consider the possibility that theexpression of BmKr-h1 in the allatectomized larvae was induced bythe ecdysteroids (Fig. 1). We therefore examined the effect of 20Eand JH on the cultured epidermis of day-5 5th-instar larvae, inwhich the expression of Kr-h1 was undetectable (Fig. 1). Contraryto our expectations, 20E alone did not induce the expression of Kr-h1 (Fig. 2C). However, JHA significantly induced the Kr-h1 expres-sion and 20E enhanced the induction by 2.3-fold (Fig. 2C).

Phenotypes of B. mori overexpressing BmKr-h1α

To investigate the function of Kr-h1 in vivo, we generatedtransgenic silkworms overexpressing BmKr-h1α (A3-GAL4/þ ,UAS-BmKr-h1α/þ; referred to as BmKr-h1αO/E silkworms forbrevity). Because the expression level of BmKr-h1β was by farlower than that of BmKr-h1α throughout the development (Fig. 1)(Kayukawa et al., 2012), we focused on the function of BmKr-h1αin this study.

The level of the BmKr-h1 transcripts in the epidermis of the4th-instar BmKr-h1αO/E silkworms was approximately fourfoldhigher than that of the control (þ /þ , UAS-BmKr-h1α/þ) (Fig. 3Aand B, IV0). The allatectomy of the control 4th-instar larvae on day0 resulted in the depletion of the BmKr-h1 transcripts, and thelevel of transcripts was restored by the topical application of JHA(Fig. 3A). In contrast, allatectomy and JHA barely affected the

Fig. 1. Developmental expression profiles of BmKr-h1 in the epidermis of Bombyx mori. The expression levels of BmKr-h1α and BmKr-h1β were determined by qPCR. The datarepresent the mean 7SD (3 replicates) values. The numerals under the horizontal axis indicate the days in the respective stage. HCS, head capsule slippage; SP, spinning.Blue and red triangles indicate male and female, respectively. The sex of the 3rd and 4th instar larvae was not discriminated (closed circle). (Inset) The scale of the BmKr-h1βtranscript is fitted to that of BmKr-h1α. The changes in the ecdysteroid titer are drawn based on the data from Koyama et al. (2004), Sakurai et al. (1998), and Kaneko et al.(2006). The changes in the juvenile hormone (JH) titer are adopted from Niimi and Sakurai (1997).

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expression of BmKr-h1 in the BmKr-h1αO/E silkworm (Fig. 3B),indicating that the high level of BmKr-h1 expression is mostlyattributable to the transgene. The BmKr-h1αO/E silkwormsshowed normal development until the 5th instar and startedspinning at the same time as the control insects (day 5). However,they spun less silk than the control insects and produced anunusually thin-skinned cocoon (Fig. 4A and B). The growth ofBmKr-h1αO/E silkworms was eventually arrested at the prepupalstage (prepupal arrest) (Table 1 and Fig. 4B). At this stage, theformation of a new pupal cuticle was observable in the dorsalabdomen under the apolysed old cuticle (Fig. 4B' and B″ arrow-heads). Although no difference was found between the bodyweights of BmKr-h1αO/E and control silkworms until the spinningstage, the BmKr-h1αO/E larvae thereafter tended to be heavierthan the controls (Fig. S1). The silk glands in BmKr-h1αO/Esilkworms on day 3 in the spinning stage were substantially largerthan those of the control larvae (Fig. 4E), suggesting that thedifference in the larval weight is mainly attributable to theremaining unspun silk protein. Thus, the overexpression ofBmKr-h1α appeared to have no significant effect on the larvaldevelopment but did affect the metamorphic processes.

To further analyze the function of BmKr-h1 as a repressor ofmetamorphosis, we allatectomized the 4th-instar larvae of BmKr-h1αO/E on day 0 and observed their phenotypes. The allatecto-mized control larvae (þ /þ , UAS-BmKr-h1α/þ) underwent preco-cious metamorphosis at the end of the 4th instar. They formedsmall thick cocoons and became miniature pupae (Table 1, Fig. 4Cand C'). The allatectomized BmKr-h1αO/E larvae formed thincocoons and entered precocious prepupal arrest, without moltinginto the 5th instar (Table 1 and Fig. 4B, B', D, and D'). When JHAwas topically applied to the allatectomized BmKr-h1αO/E larvae,they molted into the normal 5th instar and eventually enteredprepupal arrest, as observed in the intact BmKr-h1αO/E larvae(Table 2).

Overexpression of BmKr-h1α does not affect early ecdysone-induciblegenes

To address the molecular actions of BmKr-h1 in the repressionof the metamorphosis, we investigated the expression of earlyecdysone-inducible genes in BmKr-h1αO/E silkworms. Althoughthe expression level of BmKr-h1 in normal 5th-instar larvaedecreased as they aged, that in the transgenic silkworms was highthroughout the feeding stage, including the time critical for pupalcommitment (Muramatsu et al., 2008) (Fig. 5A).

BmBRC is a transcription factor found in the epidermis and iscrucial for pupal commitment (Muramatsu et al., 2008); however,the ectopic expression of BmKr-h1α did not induce any change inthe expression of BmBRC, compared with that for the controls(Fig. 5B). The expression of other transcription factors examined inthis study (i.e., BmE74A, BmE74B, BmE75A, BmE75B, BmE75C, andBmE75D) was also barely affected by the overexpression of BmKr-h1α (Fig. 5), indicating that BmKr-h1 does not modulate theexpression of the early ecdysone-inducible genes in the epidermisduring the larval period investigated in this study, which includesthe time critical for larval–pupal commitment.

We further analyzed whether the ectopic expression of BmKr-h1 altered the responsiveness of the early ecdysone-induciblegenes to JH and/or ecdysone by tissue culture experiments. Whenthe epidermis of day-2 5th-instar larvae of normal and transgenicB. mori were cultured with the hormones for 24 h, the expressionof BmBRC was induced by 20E regardless of the overexpression ofBmKr-h1α (Fig. 6A and B). The induction of BmBRC by 20E wassuppressed by JH I also, regardless of overexpression (Fig. 6).Moreover, there was no or little difference between the controland BmKr-h1αO/E silkworms with respect to the responsiveness of

Fig. 2. Effects of allatectomy (CAX) and hormones on BmKr-h1 expression. Fourth-instar larvae were allatectomized or sham-operated on day 3, and the expressionlevel of BmKr-h1 in the epidermis was determined by qPCR. (A) Day 0 in the adultstage. Acetone (control) or 2 μg of methoprene was topically applied to adults at4 h after eclosion, and the expression of BmKr-h1 in the epidermis was determinedby qPCR 6 h later. (B) Day 1 in the spinning stage. (C) Pieces of the integument ofday-5 5th-instar larvae were cultured with combinations of 1 μM 20E and 10 μMJHA for 3 h after being precultured with hormone-free medium for 3 h, and theexpression of BmKr-h1 in the epidermis was quantified. Mean 7SD (3–4 replicates)values have been provided.

T. Kayukawa et al. / Developmental Biology 388 (2014) 48–56 51

other early genes (E74 isoforms and E75 isoforms) to JH and/or 20E(Fig. S2).

Discussion

Kr-h1 has been shown to be induced by JH and to mediate theantimetamorphic action of JH during the larval or nymphal stagein several insect species (Minakuchi et al., 2009; Lozano andBelles, 2011; Konopova et al., 2011). In a previous study, wereported that the expression of BmKr-h1 is also induced by JH inthe penultimate-instar larvae of B. mori (Kayukawa et al., 2012).However, in the present study, we found that the expression ofKr-h1 is not always regulated in the same way across postem-bryonic development.

In the adult stage, Kr-h1 is induced by JH and has beensupposed to contribute to the reproductive maturation in Aedesaegypti and T. castaneum (Zhu et al., 2010; Parthasarathy et al.,2010). Kr-h1 is also induced by JH in the adults of the male mothAgrotis ipsilon and is suggested to be involved in the maturation ofthe behavioral and central nervous responses to female sexpheromone (Duportets et al., 2012). The drastic decrease inBmKr-h1 transcript levels in the allatectomized adults and theirrestoration by JHA suggested that the expression of BmKr-h1 in theadult stage of B. mori is also induced by JH secreted by the CA.However, since these adults showed no abnormality in reproduc-tive maturation and sexual behavior, the function of Kr-h1 in adultB. mori is yet to be clarified.

Allatectomy of B. mori larvae showed that the CA are notinvolved in the induction of BmKr-h1 at the prepupal stage. InD. melanogaster, the expression of Kr-h1 is reported to be upregu-lated by 20E, and Kr-h1 is involved in the ecdysone signalingpathway (Pecasse et al., 2000; Beckstead et al., 2005). Our tissueculture experiments have shown that 20E alone does not induceBmKr-h1 at the prepupal stage, but significantly synergizes theinduction by JHA (Fig. 2C). The prepupal peak of Kr-h1 also occursin T. castaneum (Minakuchi et al., 2009). This peak coincided withthe peak of expression of JHAMT, a key enzyme for JH biosynthesis(Minakuchi et al., 2008), suggesting the involvement of JH in theprepupal induction of Kr-h1. Although the CA did not show any JHsynthetic activity at the prepupal stage (Kinjoh et al., 2007), JH wasindeed detected in the hemolymph of the larvae at this stage(Niimi and Sakurai, 1997; Furuta et al., 2013). Taken together, thetransient peak of BmKr-h1 at the prepupal stage is likely to be

induced cooperatively by JH originating from non-CA tissues andthe prepupal surge of ecdysteroid. Nevertheless, we cannot ruleout the possibility that the induction is due to unknown factor(s) other than JH and ecdysteroid.

In the tobacco hornworm, M. sexta, the CA of the late last instarlarvae do not produce JH; instead, the CA secrete JH acid into thehemolymph, which is supposedly converted to JH by JHAMTpresent in the imaginal discs (Sparagana et al., 1985). Meanwhile,de novo biosynthesis of JH by non-CA tissues (accessory glands)has been found in male mosquitoes of A. aegypti (Borovsky et al.,1994). Since the expression of JHAMT was not detected in the CAbut was clearly detected in the testes and ovaries of the prepupalsilkworm (Shinoda and Itoyama, 2003), these tissues are likelycandidates for JH synthesis during this stage. Whether thesetissues produce JH de novo or by the conversion of JH acid secretedby the CA remains to be determined.

Intriguingly, allatectomy of the final instar larvae of otherlepidopteran species such as Hyalophora cecropia and M. sextacaused partial precocious adult development during larval–pupaltransition (Williams, 1961; Kiguchi and Riddiford, 1978), suggest-ing that JH originating from the CA is necessary for preventingpremature development of imaginal structures at the prepupalstage (Riddiford, 2012). However, as mentioned above, allatecto-mized B. mori developed into normal pupae. We hypothesizedthat the role of Kr-h1 at the prepupal stage in holometabolousinsects is to prevent precocious adult differentiation and that theallatectomized B. mori can pupate normally because the expressionof BmKr-h1 at the prepupal stage is induced in a CA-independentmanner characteristically in this species. In order to test thishypothesis, the prepupal expression of Kr-h1 in allatectomizedlarvae of H. cecropia or M. sexta should be examined in thefuture.

We investigated the functions of BmKr-h1 by using transgenicsilkworms. Silkworms overexpressing BmKr-h1α enter prepupalarrest at the end of the 5th instar (Table 1 and Fig. 4). RNAi-mediated knockdown of Kr-h1 in several insects has shown thatKr-h1 plays a crucial role in the repression of insect metamorpho-sis (Minakuchi et al., 2009; Lozano and Belles, 2011; Konopovaet al., 2011). However, RNAi cannot be used for functional analysisof the BmKr-h1 gene because RNAi does not work efficientlyin B. mori. Using a gain-of-function approach, the present studydirectly proved that BmKr-h1 functions as a repressor of larval–pupalmetamorphosis. Moreover, when the BmKr-h1αO/E silkworms wereallatectomized in the 4th instar, they did not undergo the next larval

Fig. 3. Expression of BmKr-h1 in transgenic silkworms. Acetone (control) or 1 μg methoprene was topically applied to fourth-instar larvae after they were allatectomized at12 h of Day 0, and the expression of BmKr-h1 in the epidermis was determined by qPCR 24 h later. (A) Control larvae (þ /þ , UAS-BmKr-h1α/þ) and (B) larvae overexpressingBmKr-h1α (A3-GAL4/þ , UAS-BmKr-h1α/þ). JHA, JH analog (methoprene); Sham, sham operation; CAX, allatectomy. Mean 7SD (4 replicates) values have been provided.

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molt and eventually entered prepupal arrest, while the topicalapplication of JHA rescued this failure (Table 2). These results suggestthat BmKr-h1 alone is not capable of completely repressing thelarval–pupal transition; that is, an additional JH-mediated factor(s) isrequired to implement larval–larval molt normally.

Several early ecdysone-inducible genes, such as E74, E75, andBroad, are involved in larval–pupal transition (Dubrovsky, 2005;Nakagawa and Henrich, 2009), and their expression has beenreported to be modulated by JH. Experiments using the epidermisof the final instar M. sexta larvae showed that E74B, E75D, and

Fig. 4. Phenotypes of transgenic silkworms overexpressing BmKr-h1α. Phenotypes of cocoons (A, B, C and D) and animals (A', B', B″, C' and D'). A, A', C and C': controlsilkworm (þ /þ , UAS-BmKr-h1α/þ). B, B', B″, D and D': silkworms overexpressing BmKr-h1α (A3-GAL4/þ , UAS-BmKr-h1α/þ). A, A', B, B', and B″ were observed at the end ofthe 5th larval instar. C, C', D and D' were allatectomized on day 0 of the 4th instar and observed at the end of the same instar. (B″) Larval cuticle of B' was removed by forceps.Green arrowheads indicate pupal cuticles. (E) The silk glands were dissected from silkworms overexpressing BmKr-h1α (lower) and control silkworms (upper) on day 3 ofthe spinning stage. Scale bar, 1 cm.

Table 1Effect of allatectomy on the phenotype of B. mori overexpressing BmKr-h1α.

Operationa N 4th 5th

Molting arrest Prepupal arrest Pupation Prepupal arrest Pupation

þ/þ , UAS-BmKr-h1α/þ Sham 15 – – – – 15CAX 19 – – 19 – –

A3-GAL4/þ , UAS-BmKr-h1α/þ Sham 15 1 – – 12 2CAX 14 – 14 – – –

Sham, sham operation; CAX, allatectomy.a The corpora allata (CA) were removed from the larvae on day 0 of the 4th instar.

T. Kayukawa et al. / Developmental Biology 388 (2014) 48–56 53

Table 2Effect of allatectomy and JHA treatment on the phenotype of B. mori overexpressing BmKr-h1α.

Operationa Treatment N 4th 5th

Molting arrest Prepupal arrest Pupation Molting or molting arrest Prepupal arrest Pupation

þ/þ , UAS-BmKr-h1α/þ Sham Acetone 10 – – – – – 10JHA 10 – – – – 1 9

CAX Acetone 10 – – 9 – – 1JHA 9 – – – – 1 8

A3-GAL4/þ , UAS-BmKr-h1α/þ Sham Acetone 9 – – – – 7 2JHA 10 – – – 6 4 –

CAX Acetone 10 – 9 – – 1 –JHA 10 – – – – 9 1

JHA, juvenile hormone analog (methoprene); Sham, sham operation; CAX, allatectomy.a The CA was removed from the larvae on day 0 of 4th instar.

Fig. 5. Effect of the overexpression of BmKr-h1α on the developmental expression of the early ecdysone-inducible genes. The expression of BmKr-h1 (A), BmBRC (B), BmE74A(C), BmE74B (D), BmE75A (E), BmE75B (F), BmE75C (G), and BmE75D (H) in the epidermis of days 3–7 5th-instar larvae were determined by q-PCR. Mean 7SD (4 replicates)have been provided.

T. Kayukawa et al. / Developmental Biology 388 (2014) 48–5654

Broad (unspecified isoform) were induced by 20E and that theirinduction was suppressed by JH (Stilwell et al., 2003; Keshan et al.,2006; Zhou et al., 1998). The current knowledge on the function ofKr-h1 in the control of metamorphosis is summarized as follows:(1) Kr-h1, which is induced by JH, represses the prematureexpression of Broad during the larval stage. (2) At the onset ofmetamorphosis, when JH is depleted, Broad is induced by a smallsurge of ecdysteroid in the absence of Kr-h1, which leads to larval–pupal transition (Riddiford, 2012; Hiruma and Kaneko, 2013).Indeed, the larval–pupal metamorphosis was interrupted by theoverexpression of BmKr-h1α (Table 1 and Fig. 4). However, theformation of the pupal cuticle and the induction of Broad by 20E inthe epidermis were not suppressed by the overexpression ofBmKr-h1α although they were suppressed by JH until the occur-rence of pupal commitment (Figs. 4 and 7) (Muramatsu et al.,2008). These findings indicate that (a) JH regulates metamorphosisthrough Met and Kr-h1, and (b) a separate JH signaling pathway,which does not involve Kr-h1, plays an essential role in themodulation of ecdysone response (Fig. 7).

In conclusion, the following two important findings on the JHsignal pathways in B. mori were obtained in the present study(Fig. 7): first, ecdysteroid synergizes the induction of Kr-h1 by JH.Second, Kr-h1 regulates metamorphosis but not via modulating

early ecdysone-inducible genes. Identification of the targets ofKr-h1 will contribute to better understanding of the molecularmechanism underlying the control of metamorphosis in insects.

Acknowledgments

We thank Drs. Chieka Minakuchi and Toshiki Namiki fortechnical assistance and Ms. Yumiko Satoh for rearing the silk-worms. This study was supported by the Program for Promotion ofBasic Research Activities for Innovative Biosciences (PROBRAIN),JSPS KAKENHI Grant Number 25850230, and the NIAS StrategicResearch Fund.

Appendix A. Supporting information

Supplementary data associated with this article can be found inthe online version at http://dx.doi.org/10.1016/j.ydbio.2014.01.022.

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Hormonal regulation and developmental role of Krüppel homolog 1, a repressor of metamorphosis, in the silkworm Bombyx moriIntroductionMaterial and methodsExperimental animals and dissectionpiggyBac vectors and transgenic silkwormsAllatectomy and JHA treatmentTissue cultureqPCR

ResultsDevelopmental expression profileHormonal regulation of BmKr-h1 expression at the prepupal and adult stagesPhenotypes of B. mori overexpressing BmKr-h1αOverexpression of BmKr-h1α does not affect early ecdysone-inducible genes

DiscussionAcknowledgmentsSupporting informationReferences