1117RESEARCH ARTICLE

INTRODUCTIONInsect molting and metamorphosis are governed primarily byecdysone (used in the generic sense) and juvenile hormone (JH),with ecdysone causing molting and JH preventingmetamorphosis. Juvenile hormone has a classic ‘status quo’ actionin preventing the program-switching action of ecdysone duringlarval molts (Riddiford, 1994; Riddiford et al., 2001) and inmaintaining the developmental arrest of imaginal primordiaduring the intermolt periods (Truman et al., 2006). Its effects atthe outset of metamorphosis, though, are more complex. Studiesmainly on Lepidoptera show that for selected tissues JH needs tobe present to allow them to undergo pupal differentiation, ratherthan undertaking a precocious adult differentiation (Williams,1961; Kiguchi and Riddiford, 1978; Champlin and Truman,1998a; Champlin et al., 1999).

The mechanism through which JH maintains the status quo anddirects early development at metamorphosis is still poorlyunderstood. Whether JH has one or multiple receptors (Wheelerand Nijhout, 2003; Riddiford, 2008), and the nature of thesereceptors, is still controversial. The best candidate for a receptoris the product of the Methoprene-tolerant (Met) gene, a PASdomain protein that was originally isolated in Drosophilamelanogaster (Ashok et al., 1998). In vitro transcribed andtranslated Met protein has been shown to bind JH with high

affinity (Miura et al., 2005), and RNAi knock-down experimentsin Tribolium castaneum show that Met is essential for mediatingthe status quo action of JH in this beetle (Konopka and Jindra,2007; Parthasarathy et al., 2008).

In D. melanogaster, JH is thought to have no role in the onsetof metamorphosis, as exogenous JH only delays but does notprevent pupariation (Riddiford and Ashburner, 1991; Riddiford etal., 2003). Although it has no apparent effect on the developmentof the imaginal discs, JH prevents normal adult development ofthe abdominal integument when given at pupariation (for areview, see Riddiford, 1993) (see also Zhou and Riddiford, 2002).Internally, JH at this time affects normal reorganization of thecentral nervous system and development of the thoracicmusculature (Restifo and Wilson, 1998). These effects of JH onmetamorphosis do not occur in Met mutants, unless at least 100times the dose is given (Wilson and Fabian, 1986; Restifo andWilson, 1998). The Met27-null mutants proceed through larvaldevelopment and metamorphosis apparently normally (Wilsonand Ashok, 1998). However, if in addition, RNAi is used tosuppress expression of Germ-Cell Expressed (Gce), a relatedbHLH protein with a high similarity to Met that heterodimerizeswith it (Godlewski et al., 2006), Met-null mutants die as pharateadults (Liu et al., 2009). In the Met-deficient mutant, the adult eyeshows a few (<12) defective ommatidia in the posterior region(Wilson et al., 2006). Also, the females mature fewer eggs at aslower rate than do wild-type females (Wilson and Ashok, 1998),indicating that Met is also important for JH effects in eggmaturation (Soller et al., 1999). While this paper was underreview, Liu et al. (Liu et al., 2009) showed that the fat bodyunderwent precocious cell death beginning in the third larvalinstar in larvae that were allatectomized using the same protocolas we describe below.

Development 137, 1117-1126 (2010) doi:10.1242/dev.037218© 2010. Published by The Company of Biologists Ltd

Janelia Farm Research Campus, Howard Hughes Medical Institute, 19700 HelixDrive, Ashburn, VA 20147, USA.

†Present address: Department of Cell and Developmental Biology, University ofMichigan, Ann Arbor, MI 48109-2200, USA*Author for correspondence (riddifordl@janelia.hhmi.org)

Accepted 26 January 2010

SUMMARYTo elucidate the role of juvenile hormone (JH) in metamorphosis of Drosophila melanogaster, the corpora allata cells, whichproduce JH, were killed using the cell death gene grim. These allatectomized (CAX) larvae were smaller at pupariation and died athead eversion. They showed premature ecdysone receptor B1 (EcR-B1) in the photoreceptors and in the optic lobe,downregulation of proliferation in the optic lobe, and separation of R7 from R8 in the medulla during the prepupal period. All ofthese effects of allatectomy were reversed by feeding third instar larvae on a diet containing the JH mimic (JHM) pyriproxifen orby application of JH III or JHM at the onset of wandering. Eye and optic lobe development in the Methoprene-tolerant (Met)-nullmutant mimicked that of CAX prepupae, but the mutant formed viable adults, which had marked abnormalities in theorganization of their optic lobe neuropils. Feeding Met27 larvae on the JHM diet did not rescue the premature EcR-B1 expressionor the downregulation of proliferation but did partially rescue the premature separation of R7, suggesting that other pathwaysbesides Met might be involved in mediating the response to JH. Selective expression of Met RNAi in the photoreceptors causedtheir premature expression of EcR-B1 and the separation of R7 and R8, but driving Met RNAi in lamina neurons led only to theprecocious appearance of EcR-B1 in the lamina. Thus, the lack of JH and its receptor Met causes a heterochronic shift in thedevelopment of the visual system that is likely to result from some cells ‘misinterpreting’ the ecdysteroid peaks that drivemetamorphosis.

KEY WORDS: Juvenile hormone, Optic lobe, Methoprene-tolerant, Ecdysone receptor-B1

A role for juvenile hormone in the prepupal development ofDrosophila melanogasterLynn M. Riddiford*, James W. Truman, Christen K. Mirth and Yu-chi Shen†

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Here, we have genetically allatectomized Drosophila larvae bytargeting expression of a cell death gene to the corpora allata (CA),the gland that produces JH. These larvae formed smaller puparia andshowed precocious maturation of the visual system, but died aroundhead eversion.

MATERIALS AND METHODSAnimalsAug21-GAL4 [a stock obtained by T. Siegmund and G. Korge but notreported in the screen for neurosecretory neurons (Siegmund and Korge,2001)], UAS-grim (Wing et al., 1998), and the Met27-null mutant (Wilsonand Ashok, 1998) stocks were used. The GAL4 lines R15E10, R17A12,R23C06, R25B08 and R27G05 were constructed as described by Pfeifferet al. (Pfeiffer et al., 2008). The UAS-EcR-B1 (Lee et al., 2000) and UAS-MetRNAi (45852 and 45854 from the Vienna Drosophila RNAi Center)lines were used. The Aug21-GAL4/CyO, actin-GFP, the R27G05; UAS-mCD8::GFP and the Met27; Aug21-GAL4, UAS-GFP/CyO stock wereconstructed at the Janelia Farm Fly Core facility.

The Met27 fly stocks and the GAL4>UAS-EcR-B1 and the GAL4>UAS-MetRNAi crosses were maintained at 25°C. The Aug21-GAL4/CyO, actin-GFP�UAS-grim crosses were maintained at 29°C until the late third instar,then transferred to 25°C. The Aug21>grim larvae were separated from theCyO, UAS-grim larvae by the expression of a GFP transgene on the CyOchromosome.

Juvenile hormone treatmentsLarvae or white puparia were given the JH mimic (JHM) pyriproxifen(Sumitomo Chemical Company, Osaka, Japan) or 7R-JH III (Toong et al.,1988) (gift of Dr Y. Toong) topically or in the diet. For topical application,0.2 ml cyclohexane containing a specified amount of JH III or JHM wasapplied to wandering larvae under CO2 anesthesia or to white puparia. Forthe feeding experiments, 5 mg JHM in 50-100 ml ethanol was mixed with5 ml of standard diet to give a diet containing 1 part per million (ppm)JHM.

Immunocytochemistry and imagingBrains and other tissues were dissected and fixed in 3.9% formaldehyde(Fisher Scientific, Fairlawn, NJ, USA) in phosphate-buffered saline(PBS; Mediatech, Manassas, VA, USA) for 30 minutes, then rinsed andincubated in PBS-1% Triton X-100 (TX) with 2% normal donkey serumfor 30 minutes. They were then incubated in PBS-1% TX with primaryantibody for 1-2 days at 4°C, rinsed, then incubated in secondary antibodyovernight at 4°C. Primary antibodies used were: ecdysone receptor, B1isoform (EcR-B1; mouse ascites fluid AD 4.4; 1:10,000; from C. S.Thummel, University of Utah, Salt Lake City, UT, USA); Fasciclin II[mouse monoclonal antibody (MAb) 1D4; 1:50; from C. Goodman,University of California, Berkeley, Berkeley, CA, USA]; Deadpan (ratMAb; 1:1; from C. Q. Doe; Boone and Doe, 2008); phosphohistone H3(PH3; 1:1000; Upstate, Millipore, Billerica, MD, USA); and chaoptin(mouse MAb 24B10; 1:50), N-cadherin (rat MAb MNCD2; 1:50) andneuroglian (mouse MAb BP104; 1:40; all from the DevelopmentalStudies Hybridoma Bank, Iowa City, IA, USA). Secondary antibodieswere fluorescein, Texas Red or Cy5 conjugated and were used at 1:500(Jackson ImmunoResearch Laboratories, West Grove, PA, USA). OregonGreen 488 phalloidin (Molecular Probes, Invitrogen, Carlsbad, CA, USA)was sometimes used as a counterstain.

Immunostained preparations were typically dehydrated through a gradedethanol series, cleared in xylene and mounted in DPX (Fluka BioChemika,Sigma-Aldrich, St Louis, MO, USA). Eye discs that were counterstainedwith phalloidin were mounted directly into Vectashield (Vector Laboratories,Burlingame, CA, USA) to preserve the phallodin immunofluorescence. The preparations were imaged on a Zeiss 510 confocal microsocope and processed with Image J. Counts of the number of dividing cells, as indicated by PH3 labeling, were performed using V3D software developed by H. Peng (Janelia Farm Research Campus, HHMI,Ashlam, VA, USA; http://penglab.janelia.org/proj/v3d/v3d2.html).

RESULTSEffects of genetic allatectomy on larvaldevelopment and metamorphosisWe confirmed that Aug21-GAL4 drove UAS-GFP expression in theCA (T. Siegmund and G. Korge, personal communication) andfound that it was also expressed strongly in the salivary glands.When Aug21-GAL4 drove the cell death gene grim (Wing et al.,1998) in these tissues, both sets of glands were either missing or ina state of advanced degeneration by the onset of the third instar (datanot shown), and all remnants of the CA had disappeared by the onsetof wandering (Fig. 1A). Therefore, the larvae had beenallatectomized (CAX).

The Aug21>grim (CAX) larvae developed slightly slower thandid their CyO, UAS-grim counterparts, and pupariated about 8-12hours later. They were also about 75% of the weight of their CyO,UAS-grim siblings (Fig. 1B,C). Survival to pupariation was similarfor both the CAX and the control (CyO, UAS-grim) larvae; of a totalof 1300 puparia scored, 51% were from CAX larvae and 49% fromCyO, UAS-grim larvae. When fed a diet containing 1 ppm JHMduring the third instar, the CAX larvae grew to the normal size (Fig.1B,C), showing that the effects on growth were due to the lack of theCA and not to the degeneration of the salivary glands. Both thecontrol CyO, UAS-grim and the CAX larvae on the JHM dietdelayed pupariation by 1-2 days.

Although the CAX larvae showed normal viability up topupariation, they typically died around the time of head eversion.Video imaging showed that they initiated head eversion at thenormal time but were unable to eject the mouth hooks completelyand evert the pupal head (six out of seven animals). One of these didcomplete head eversion. Such ‘escapers’ were not found at 29°C, butoccurred 20% of the time when the white puparia were placed at

RESEARCH ARTICLE Development 137 (7)

Fig. 1. Genetic allatectomy and its effect on puparial size. (A) Thering gland of control CyO, UAS-grim (above) and allatectomized (CAX)Aug21>grim (below) wandering larvae showing the loss of the corporaallata (CA) in the latter. a, aorta; PG, prothoracic gland. Scale bar: 25mm. (B,C) Sample puparia (B) and the pupal weights (C) of CyO, UAS-grim (control) and Aug21>grim (allatectomized) larvae raised on normaldiet containing 1% ethanol or 1 ppm pyriproxyfen (JHM) in 1%ethanol. Bars represent the average ± s.d. for 50-60 puparia (one dayafter wandering). Different letters denote average weights that aresignificantly different (P≤0.0001, using one-way ANOVA and Tukey-Kramer HSD comparison of means).

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18°C (n=20). When fed a 1 ppm JHM diet during the third instar,89% (n=64) continued beyond head eversion and arrested as pharateadults just before eclosion (Table 1). These showed truncated andmissing abdominal bristles, the typical effects of JH application atpupariation (Postlethwait, 1974; Riddiford and Ashburner, 1991).When 10 ng JHM was applied to early wandering larvae, 37%(n=54) formed pharate adults, but only one pharate adult was formedafter the same treatment of white puparia (Table 2). A similar rescuewas obtained after treatment with 500 ng JH III during earlywandering (Table 2).

Effect of allatectomy on development of thevisual systemIn the tobacco hornworm, Manduca sexta, lack of JH during theprepupal molt leads to premature adult differentiation of the eye(Kiguchi and Riddiford, 1978; Champlin and Truman, 1998a). Todetermine whether JH has a similar effect in Drosophila, we lookedat the early development of the visual system in the CAX animals.We used the appearance of the B1 isoform of the Ecdysone Receptor(EcR-B1) as a molecular marker for developmental timing in theretina and the optic lobes. In controls EcR-B1 is absent from the eyedisc at pupariation (Fig. 2A). It becomes evident in the crystallinecone cells and underlying photoreceptors by 12 hours after pupariumformation (APF). Its expression then transiently fades in these cellsbut returns at 18 to 22 hours APF. Although expression is prominentin the cones, it is absent from most of the surrounding pigment cells.In the optic lobes, the only neurons to express EcR-B1 at pupariationare the three larval visual interneurons (Truman et al., 1994). Thefirst traces of EcR-B1 expression appear in neurons in the innerproliferation zone at about 6 hours APF (data not shown). By 8hours, EcR-B1 expression is evident in the lamina, medulla and

lobula; it is missing from the inner and outer proliferation zone stemcells, which are Deadpan positive (Egger et al., 2007), and thestrongest EcR-B1 expression is in the neurons that are the farthestfrom the proliferation zones, suggesting that neurons need to be acertain time after their birth before they are competent to expressEcR-B1 (Fig. 3A,D). EcR-B1 expression is maintained in optic lobeneurons through about 50 hours APF (Truman et al., 1994) (see alsoFig. 6C).

The expression of EcR-B1 was significantly advanced in theCAX prepupae, such that it was evident in both the eye disc and theoptic lobes at the time of pupariation. In the eye disc, it wasexpressed in the cone cells and photoreceptors in the posterior regionof the disc (Fig. 2B). It was absent in cells anterior to the furrow(data not shown) and in the youngest ommatidia that wereorganizing just posterior to the furrow (Fig. 2B). In the optic lobes,EcR-B1 expression was seen in neurons of the medulla and the‘central plug’ (Fig. 3B), but not in the neuroblasts of the inner orouter proliferation zones (Fig. 3B). When third instar CAX larvaewere fed on a diet containing 1 ppm JHM, no EcR-B1 wasdetectable throughout the prepupal period (Fig. 3C; data not shown).Therefore, the early appearance of EcR-B1 in the photoreceptors andoptic lobe neurons in CAX larvae is due to the lack of JH during thelate third larval instar.

We assessed the cellular effects of the lack of JH by examiningthe patterns of proliferation in the visual system and also themorphogenesis of the growth cones of the photoreceptors thatproject into the medulla. Proliferation was monitored byimmunostaining for PH3 to detect mitotic cells. We saw no obviouseffect of removal of JH on the proliferation pattern in the imaginaldiscs (data not shown). We did, however, see a strong effect onproliferation in the optic lobes. At pupariation both intact and CAXanimals had comparable amounts of proliferation in both the innerand outer proliferation zones (Fig. 4A,B), although the brains of theCAX larvae were somewhat smaller because of the overall reductionin body size. In control animals, proliferation continued at a highlevel in the outer proliferation zone through the next 8 hours (Fig.4C), but had markedly declined by 12 hours APF (data not shown).In the CAX prepupae, by contrast, proliferation in the outer zone haddropped down to about 15% of control values by 8 hours APF (Fig.4D,F). When the CAX larvae were fed on a JHM-containing diet,the number of mitotic cells in the outer proliferation zone waselevated to levels seen in intact prepupae (Fig. 4E,F). Proliferation

1119RESEARCH ARTICLEJH and prepupal optic lobe development

Table 1. Fate of allatectomized (CAX) larvae fed on a 1 ppmpyriproxifen (JHM) dietGenotype, stage Number puparia Percentage head eversion

Aug21>grim (CAX)

Second, early third 64 8924-hour third 13 100

CyO, UAS-grim (Control)

Second, early third 69 91

Table 2. Rescue of head eversion defect of allatectomized (CAX) larvae by topical application of JH III or its mimic pyriproxifenCyO,UAS-grim (Control) Aug21>grim (CAX)

Hormone, dose, stage Number puparia Percentage head eversion* Number puparia Percentage head eversion*

10 ng pyriproxifen

Early wander 25 88 54 37Mid-wander ND ND 4 100Pupariation ND ND 31 3

100 ng JH III

Early wander 8 88 8 12Mid-wander 7 100 4 0Pupariation 9 100 12 0

500 ng JH III

Early wander 21 95 15 53Pupariation 10 100 8 12†

*These pupae went on to form pharate adults showing typical JH effects when treated with pyriproxifen. The controls when treated with either 100 or 500 ng JH III emergedas apparently normal adults, whereas only three out of the eight pharate adults formed after 500 ng JH III treatment of CAX larvae eclosed and were apparently normal.†This pupa died soon after head eversion.ND, not determined. D

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in the inner proliferation zone in the CAX prepupae showed only aslight, but significant (P=0.003), reduction, which was also restoredby feeding on a JHM-containing diet (Fig. 4C-F).

The adult optic lobe depends on incoming photoreceptors fromthe eye to direct its development (Meinertzhagen and Hanson, 1993;Ting and Lee, 2007). At mid-third instar, the morphogenetic furrowbegins to move across the eye disc, behind which the photoreceptorsin each ommatidium begin to differentiate (Wolff and Ready, 1993).This process continues through the prepupal period until headeversion, when all the ommatidia have formed. We examined theeffects of JH on neuronal morphogenesis by focusing on the

outgrowth of photoreceptors R7 and R8, which extend axons intothe medulla. For a given ommatidium, the R8 growth cone is the firstto arrive, followed many hours later by the R7 growth cone, whichextends slightly beyond it (e.g. Fig. 5, Control). The growth conesstart to separate from each other at 18 hours APF and are clearlyseparated by 24 hours (Ting et al., 2005). Ingrowing laminainterneurons fill much of the space in between. In the CAXprepupae, we already see the distinct separation between R7 and R8soon after pupariation (Fig. 5). When CAX larvae were fed on a 1ppm JHM-containing diet in the third instar, the prematureseparation did not occur (Fig. 5).

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Fig. 2. Developmental appearance of theecdysone receptor EcR-B1 in thephotoreceptors in CyO, UAS-grim(control), Aug21>grim (allatectomized,CAX), and Met27 prepupae and pupae.(A) Confocal sections of developingommatidia at the levels of the crystallinecone cells (top) and the photoreceptors(bottom) showing the time course ofappearance of EcR-B1 (red) in wild-typeprepupae and pupae. Cellular outlines aredelineated by phalloidin staining (green).Times shown are hours after pupariumformation (APF). Note that EcR-B1 is firstevident at 12 hours APF. Scale bar: 5mm. (B) Confocal sections showing theprecocious presence of EcR-B1 in thephotoreceptors of allatectomized (CAX) andMet27 eye discs at the time of pupariation.N-cadherin (green) stains developingphotoreceptors. Scale bar: 20mm. (C) Ommatidia from the eye disc of a Met27

prepupa at 2 hours APF showing theprecocious appearance of EcR-B1. In A andC, the asterisks indicate an example of acone cell (top) or a photoreceptor (bottom).

Fig. 3. Precocious appearance of the ecdysone receptor EcR-B1 in the optic lobes (OL) of Aug21>grim allatectomized (CAX) prepupaeand its suppression by feeding larvae on a diet containing 1 ppm pyriproxyfen (JHM). (A-E) OL confocal sections at different times (Xhours; hr) APF of (A,D) CyO, UAS-grim (control) prepupae, (B) CAX prepupae after feeding on normal diet, (C) CAX prepupa after feeding on theJHM diet, and (E) Met27 prepupae. Arrows indicate EcR-B1-positive neurons. Insets in A-C show endogenous EcR-B1 in the mushroom bodyneurons. (D,E) Deadpan-positive neuroblasts (green); EcR-B1 neurons (red). cp, ‘central plug’ neurons produced by the neuroblasts of the innerproliferation zone (ipz); la, lamina; m, medulla; opz, outer proliferation zone. Scale bar: 25mm. D

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Processes that occur prematurely in CAX animals are delayed orsuppressed in wild-type larvae that are treated with JHM. As seen inFig. 6A, puparia treated with JHM show only a partial separation ofR7 from R8 at 24 hours, although separation looks normal by 30

hours APF. Also, in the JHM-treated animals, proliferation wasextended by about 12 hours (Fig. 6B) and EcR-B1 expression in theoptic lobe was severely suppressed (Fig. 6C).

The role of the Methoprene-tolerant (Met) gene inJH regulation of optic lobe developmentThe Met gene encodes a likely JH receptor. The Met27 null mutantsdid not show the small size or prepupal lethality characteristic of theCAX larvae, but they did show similar effects on the developmentof the optic lobe. As in the CAX prepupae, EcR-B1 appearedprecociously in the photoreceptors (Fig. 2B,C) and in the optic lobe(Fig. 3E, Fig. 7A) of Met27 prepupae. Also, proliferation in both theouter and the inner proliferation zones showed an early suppression(Fig. 7B,G), and R7 showed precocious separation from R8 (Fig.7C).

When third instar Met27 larvae were fed a diet containing 1 ppmJHM, the hormone mimic was unable to block most of theprecocious changes. EcR-B1 still appeared precociously in theprepupal optic lobe (Fig. 7D) and proliferation in the outerproliferation zone was reduced (Fig. 7E,G), just as in prepupae fromMet27 larvae fed on a normal diet (control; Fig. 7A,B). By contrast,the proliferation in the inner proliferation zone was significantlyincreased (P=0.003), although not back to the levels seen in the wild-type prepupae (Fig. 7G). Furthermore, the R7-R8 separation was notas pronounced (Fig. 7F) as in the Met27 prepupae (Fig. 7C), but wasmore extended than in control prepupae (Fig. 5). Therefore, JHapparently is acting via the Met pathway in controlling EcR-B1expression and proliferation in the outer proliferation zone, but onlypartially so to prevent premature shutdown of proliferation in theinner proliferation zone and growth cone separation.

In the Met27 mutant, the separation of the R7 and R8 terminals isevident by 6 hours APF, about 12 hours earlier than normal (Ting etal., 2005) (Fig. 7). To determine if Met was causing an advancementof the development of the entire visual system, relative topupariation, we used a GAL4 driver line from the Rubin collectionof enhancer tester lines (Pfeiffer et al., 2008). R27G05-GAL4 drivesexpression in lamina neurons that project into the medulla. Using amouse CD8 (mCD8)::GFP reporter, we counted the number of rowsof lamina axon terminals that had grown into the medulla at varioustimes after pupariation. Over the 6 to 9 hours of the prepupal period,both wild-type and Met27 prepupae added approximately one row ofterminals per hour, but the Met27 individuals in the early stages weretwo rows ahead of the controls (Fig. 8A,B). Hence, the loss of Metresults in a two-hour advancement in the ingrowth of lamina

1121RESEARCH ARTICLEJH and prepupal optic lobe development

Fig. 4. Precocious decline in proliferation in the OL inAug21>grim allatectomized (CAX) prepupae and its preventionby feeding larvae on diet containing 1 ppm pyriproxyfen (JHM).(A-E) The OL at different times (hours) APF of (A,C) CyO, UAS-grim(control) prepupae, (B,D) CAX prepupae after feeding on normal diet,and (E) a CAX prepupa after feeding on the JHM diet. (C-E) Frontal z-projection of the optic lobe (left) and a 90° projection along the x-axis(right), showing an end-on view to discriminate the inner (ipz) andouter (opz) proliferation zones; (A,B) x-axis projections. Aqua representsPH3 staining of mitotic cells; red is chaoptin staining of the incomingphotoreceptors, except in E where it was omitted. Scale bar: 50mm. (F) Mean number (±s.d.) of proliferating cells in the opz (black bars) andthe ipz (white bars) of 3-5 animals at 8 hours APF.

Fig. 5. Dorsal views of the medullashowing the effects of JH manipulationson the timing of separation of the R7and R8 growth cones. (Left) Controlprepupae showing that growth conesbecome completely separated by 18 to 24hours (h) APF. (Middle) Allatectomized (CAX)prepupae show complete separation by 6hours APF. (Right) Feeding CAX larvae on aJHM diet blocks the precocious separation.Scale bar: 10mm.

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neurons, relative to the time of puparium formation. However, thisdifference in the timing of ingrowth is much less than the 12-houradvancement seen for the separation of R7 from R8.

As the Met27 mutants became viable adults, we were able to seethe final outcome of the lack of JH signaling on the development ofthe optic lobes. The gross anatomy of the lamina and medullaappeared normal and we did not see a mistargeting of photoreceptoraxons to inappropriate layers (Fig. 8D). The lobula, however,showed gross abnormalities with irregular lobes that projectedtowards, or sometimes penetrated, the medulla neuropil (Fig. 8C).These projections severely disrupted the layering of the lobula andthe lobula plate, such that neurons that normally had their dendritesconfined to a single layer, now projected in a disorganized fashionthroughout the lobula (Fig. 8D). The beginnings of these irregularprojections were evident at 12 hours APF, the same time that cellularchanges were occurring in the more superficial neuropils (Fig. 8E).

Cell-cell interactions and the action of JHPossible interactions between different neuronal populations ingenerating some of the Met27 mutant phenotypes were tested usingR27G05-GAL4 (Fig. 9A), which expresses in the lamina, andR25B08-GAL4, which expresses strongly in photoreceptors R1through R7 (Fig. 9C). We used UAS-MetRNAi constructs to reduceMet levels in the photoreceptors or the lamina neurons. We lacked areliable antibody to measure the level of Met reduction in responseto the RNAi, but immunostaining for EcR-B1 at 3 hours APFshowed that driving UAS-MetRNAi by R25B08-GAL4 or byR27G05-GAL4 resulted in the precocious appearance of EcR-B1 inthe photoreceptors or the lamina neurons, respectively (Fig. 9B,D).Similar results were seen when UAS-MetRNAi was driven in the

photoreceptors by two other GAL4 lines (data not shown). Thelevels of EcR-B1 expression in these cells was similar to that seenin comparably aged, Met27 null mutants, and both RNAi constructsgave similar results. Therefore, the RNAi constructs give sufficientMet reduction to mimic at least one aspect of the Met nullphenotype.

Examination of the terminal projections of R7 and R8 at 6 hoursAPF showed that UAS-MetRNAi driven in the photoreceptors causedmany of the R7 photoreceptors to separate from the R8photoreceptors (Fig. 9I), just as in the Met27 mutant (Fig. 9F). Bycontrast, when R27G05-GAL4 was used to knock down Met levelsin the lamina neurons, the photoreceptor terminals stayed in theirpre-18 hour APF positions (Fig. 9H). To see if the precociousexpression of EcR-B1 in the photoreceptors could also cause earlyseparation of R7 and R8, we used R25B08-GAL4 to drive UAS-EcR-B1 in the photoreceptors. As seen in Fig. 9G, the two receptorterminals remained associated despite the expression of EcR-B1 inR7. Therefore, the loss of Met function must be producing othereffects that are needed for the precocious separation of the growthcones.

DISCUSSIONAlthough a number of studies have reported the effects of applyingexogenous JH or JH mimics to Drosophila (for a review, seeRiddiford, 1993; Riddiford and Ashburner, 1991; Restifo andWilson, 1998), there are only two very recent studies of the effectsof manipulating endogenous JH on larval growth andmetamorphosis, both of which appeared while this paper was underreview. JH is normally present in the early larval instars, declinessubstantially during the last (third) larval stage and then returns

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Fig. 6. The effect of 100 ngpyriproxifen (JHM) applied atthe time of pupariation on OLdevelopment. (A) Dorsal view ofthe medulla showing that JHMtreatment retards the separationof the R7 and R8 growth cones.(B) Frontal projections of theoptic lobe showing theprolongation of proliferation inthe JHM-treated animals: red,chaoptin; aqua, phosphohistoneH3. (C) JHM treatmentsuppresses the appearance ofEcR-B1. h, hours APF; MB,mushroom body. Scale bars: 10mm in A; 25mm in B,C.

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transiently around the time of pupariation (Bownes and Rembold,1987; Sliter et al., 1987). The CAX larvae undergo the expected twolarval molts, but because we sometimes see remains of degeneratingCA cells at the start of the last larval stage, we cannot concludeanything about the requirements of JH for these larval molts.Recently, Jones et al., using 3-hydroxy-3-methylglutaryl CoA

reductase (HMGCR) RNAi to depress the level of JH and itsfarnesoid precursors in early larvae, showed that the larvae mainlydie during the molt to the third instar (Jones et al., 2010), indicatingthat JH may be required for that molt.

The destruction of the CA by the third instar allowed us to examinethe role of JH during the last instar and early metamorphosis. Ourfinding that these larvae were smaller than their CyO, UAS-grimsiblings at pupariation could be explained by either the loss of JH orby the loss of the salivary glands, as these glands are also destroyed.Because dietary JH in the final instar rescued these larvae to normalsize, the lack of the CA, rather than the lack of the salivary glands, isthe cause of their reduced growth. Preliminary studies show that CAXlarvae grow more slowly in the third instar (C.K.M. and L.M.R.,unpublished), but the underlying basis for this retardation is not yetunderstood. Similarly, allatectomized third instar larvae displayedpremature apoptosis of the fat body and downregulation of severalenzymes involved in energy metabolism at the onset of wandering(Liu et al., 2009). These fat body effects could underlie the reducedlarval growth seen in CAX larvae.

A major effect of the removal of JH was on the timing of eventsduring the prepupal period. Studies on the wild silkmothHyalophora cecropia first showed that removal of the CA in the lastlarval stage resulted in the formation of a pupa with adultcharacteristics (Williams, 1961). Other moths, like Manduca sexta,showed more subtle responses to allatectomy, with premature adultdifferentiation most evident in the patterned region of the compoundeye, posterior to the morphogenetic furrow (Kiguchi and Riddiford,1978; Champlin and Truman, 1998a). Subsequent studies on avariety of tissues in Manduca showed that the eye, the optic lobe andthe ventral diaphragm each had a prolonged period of proliferationthat extended from the prepupal period through early adultdifferentiation (Champlin and Truman, 1998a; Champlin andTruman, 1998b; Champlin et al., 1999). This proliferation wasmaintained by a-ecdysone or low levels of 20-hydroxyecdysone(20E), but was terminated by high levels of 20E, which induceddifferentiation. These tissues are exposed to differentiation-inducingtiters of 20E that occur during the larval-pupal transition early intheir growth, but studies on the ventral diaphragm showed that JH‘protects’ them from these high 20E levels, allowing them tocontinue proliferating (Champlin et al., 1999). Removal of JHresulted in these tissues undergoing premature termination of tissuegrowth and precocious adult differentiation.

The response of Drosophila larvae to the loss of JH is in linewith the effects seen in Manduca, and is also most evident in thedeveloping visual system. In normal individuals, the appearanceof EcR-B1 in the optic lobe (Truman et al., 1994) and thetermination of proliferation in the outer proliferation zone (T. A.Awad, PhD thesis, University of Washington, 1995; Fig. 6B)coincide with the ecdysteroid peak at head eversion (Handler,1982) and become more pronounced at 18 hours APF with the riseof ecdysteroid for adult differentiation (Handler, 1982). Theseparation of the R7 and R8 growth cones also begins about thislatter time (Ting et al., 2005) (Fig. 5). The only one of thesetissues that has been directly tested in vitro for 20E sensitivity isthe optic lobe (T. A. Awad, PhD thesis, University of Washington,1995) and in this case high levels of 20E do indeed suppressproliferation. We assume that the other processes also respond tothe changing ecdysteroid levels. The lack of JH results in aheterochronic advance of these events by 10 to 12 hours,consistent with the tissues now responding to the earlierecdysteroid peak that causes pupariation. Although the removalof JH advances these processes, we find that the application of JH

1123RESEARCH ARTICLEJH and prepupal optic lobe development

Fig. 7. The Met27 mutant shows precocious OL development andreduced sensitivity to a JHM (1 ppm pyriproxifen) diet.(A,D) Frontal sections through the brain and OL showing precociousEcR-B1 immunostaining in the OL of both Met27 prepupae (A) andthose fed a JHM diet (D). (B,E) An x-projection of the OL showing thatactivity in the outer proliferation zone (OPZ) is also precociously reducedin both groups. red, chaoptin; aqua, phosphohistone H3; IPZ, innerproliferation zone. (C,F) Dorsal view of the medulla showing that theprominent separation of the R7 and R8 growth cones is partiallysuppressed by feeding the mutant JHM. In the above series, the Met27

examples were at 6 hours APF and the Met27 fed JHM were at 8 hoursAPF. (G) Mean number (±s.d.) of proliferating cells in the OPZ (blackbars) and IPZ (white bars) of 4-5 animals at 8 hours APF. The reducedproliferation in the OPZ is not rescued in the mutants that were fed onthe JHM diet, whereas the number of dividing cells in the IPZ is partiallyrestored. Scale bars: in A, 25mm for A,B,D,E; in C, 10mm for C,F.

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mimics delays them (Fig. 6). Consequently, in selective tissues inDrosophila, JH acts to direct the nature of tissue responses toecdysone.

The removal of JH or of one its receptors, Met, has a mixedeffect on the developing visual system. We saw no effect onproliferation or inductive events in the eye disc itself, in that inCAX animals the morphogenetic furrow continues to move andsimilar rows of ommatidia have sent R8 axons into the medullaby 6 hours APF, as compared with controls (Fig. 5). Likewise, inMet27 individuals there is only a slight advance (about 2 hours) inthe schedule of lamina interneuron ingrowth into the medulla(Fig. 8). However, for some of the cellular and molecular events,like the appearance of EcR-B1 and the separation of R7 from R8,there is a 10- to 18-hour advance in their occurrence. Hence, thelack of JH or of its receptor Met causes a heterochronic shiftwithin the developing visual system with some differentiationresponses being advanced relative to the normal schedule ofneuronal birth and axon ingrowth. At least in the case of thephotoreceptors, the effect of Met removal is largely cellautonomous, with the reduction of Met function in just those cellsbeing sufficient to cause the precocious appearance of EcR-B1and the early separation of R7 from R8 (Fig. 9D). By contrast, the

reduction of Met in lamina interneurons allowed these cells toprecociously express EcR-B1 but did not affect the behavior ofthe R7 and R8 growth cones. This suggests that the separation ofR7 and R8 is an active response of the photoreceptors, which islikely to be caused by the rising ecdysteroid titer driving adultdifferentiation. Although the lack of JH or Met function at theoutset of metamorphosis results in the cell-autonomousexpression of EcR-B1 in the photoreceptors, misexpressionexperiments (Fig. 9F) show that the appearance of this receptoralone is not sufficient to bring about the early separation of R7and R8. Therefore, although the upregulation of EcR-B1 is aprominent response to rising ecdysteroid titers, it is not the keychange responsible for the repositioning of the receptor terminals.

As it is viable, the Met mutant allowed us to see the final resultsof the mistiming of development in the optic lobes. We could notdetect any permanent effect of the early separation of the R7 and R8growth cones on the final anatomy of these projections in themedulla, or on the structure of the later neuropil. However, thelobula was grossly distorted and the normal layering of dendriticarbors disrupted (Fig. 8C-E). This aberrant morphogenesis alsostarts early, being already evident by 12 hours APF. The cellularbasis for the lobula distortion, however, is not yet known.

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Fig. 8. Comparison of OL development in wild-type and Met27 mutants. (A,B) R27G05-GAL4 driving mCD8::GFP revealed lamina axoningrowth into the medulla in wild-type and Met27 backgrounds. (A) The mean number (±s.d.) of rows of lamina neuron terminals seen in themedulla of wild-type (black bars) and Met27 (white) individuals at various times after pupariation; 3-5 samples per bar. (B) Confocal projections ofrows of lamina neuron terminals in the medulla at various times APF. Scale bar: 10mm. (C,D) Frontal sections of the adult OL. (C) The lobula (lo) ofthe Met27 mutant has abnormal lobes (arrows) that project towards the medulla (m); (D) expression of GFP in the R23C06 line reveals lobulainterneurons whose dendrites are normally confined to defined layers of the lobula, but in the mutant they extend throughout this neuropil: red,chaoptin; blue, N-cadherin. Insets show a z-projections of the entire dendritic tree. Below are horizontal sections at the level of the arrow; lp, lobulaplate. (E) Sections through the developing optic lobe of Met27 mutants show that lobula irregularities are evident as early as 12 hours APF. Stainingin C and E: N-cadherin, aqua; neuroglian, red. Scale bars: 20mm in B; 50mm in C-E.

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Heterochronic shifts in the timing of development that extendbeyond the visual system are likely to be the cause of the lethalityseen in the CAX puparia. Puparia appear normal through the first 6to 7 hours after pupariation but then abruptly undergo tissuecollapse. In normal flies, the early part of metamorphosis isaccomplished by a complicated replacement of histolyzing larvaltissues by the growing adult tissues. Diverse tissues showindividualized times of histolysis that are tied to the ecdysteroid titer.For instance, the larval midgut cells degenerate in response to thepupariation peak of ecdysone, whereas the larval salivary glanddegeneration is triggered by the small rise of ecdysteroid at the endof the prepupal period (Jiang et al., 1997). We suspect that withoutJH, some of the histolysis events are mistimed, leading to the rapiddeath of the prepupa. Liu et al. have recently shown in CAX larvaethat the fat body undergoes precocious programmed cell deathbeginning in the third larval instar (Liu et al., 2009). Interestingly,this lethal effect was not seen in animals in which Aug21-GAL4drove RNAi for JH acid O-methyltransferase, the enzyme thatconverts JH acid to JH, in the CA (Niwa et al., 2008). Whether this

indicates that JH acid plays a role in prepupal development ormerely reflects the incomplete loss of JH in these animals isunknown.

All these effects of allatectomy can be rescued by JH either fedduring the third instar or applied at the time of early wandering, butnot at pupariation. Both Bownes and Rembold (Bownes andRembold, 1987) and Sliter et al. (Sliter et al., 1987) found a declineof JH III in the third instar; this was followed by a peak of JH duringlate wandering (Sliter et al., 1987). When JH begins to rise isunknown, as their measurements were made every 24 hours.Presumably it is the lack of this JH during wandering when theecdysteroid titer is rising and peaking that leads to the optic lobeanomalies and the premature histolysis.

The finding that the Met27 null mutant has the same defects inoptic lobe development as are found in CAX prepupae stronglysuggests that JH is acting via the Met pathway in controlling thetiming of some events in the optic lobe. Accordingly, JHM treatmentcannot suppress most of the premature development seen inprepupae lacking Met (Fig. 7). However, a major difference betweenthe CAX animals and the Met27 mutants is that the CAX prepupaedied before head eversion, whereas the Met27 animals were viable.This difference is also seen in the precocious cell death of the fatbody caused by allatectomy, which does not occur in the Met nullmutant even in the presence of gce RNAi (Liu et al., 2009). Insteadprecocious cell death of the fat body was seen when Met wasoverexpressed in that tissue and the death could be suppressed byexogenous methoprene (a JH mimic). This latter finding suggeststhat JH would act in this case to suppress Met-mediated cell death.We tested this idea by seeing whether the removal of Met wouldprotect the prepupa from the death caused by early allatectomy.When Met27; Aug21-GAL4>UAS-GFP/CyO females were crossedwith UAS-grim males, 44% (n=88; 18 males and 22 females)eclosed, all showing the CyO phenotype. The remainder died at headeversion, and should have been half CAX, Met-heterozygousfemales and half CAX, Met-null males. We then separated anothergroup by sex prior to pupariation. Forty-nine percent of the females(n=57) and 48% of the males (n=52) died at head eversion. All of theadults that emerged were CyO, showing that all the CAX prepupaedied regardless of whether or not they were lacking Met function.

These results together with our findings that JHM treatment of theMet27 mutant gave a partial rescue of the premature separation of R7and R8, and of the decreased proliferation in the inner proliferationzone (Fig. 7), indicate that there may be more than one receptor forJH. Thus, we support the idea summarized by Wheeler and Nijhout(Wheeler and Nijhout, 2003) that JH might act through multiplepathways. A major pathway involves Met, but Gce or some othermediator may serve as an alternate pathway in some tissues. Asimilar protective role of JH at pupation mediated by Met is foundin Tribolium, as injection of Met RNAi into either fourth instarlarvae (Konopova and Jindra, 2007) or final instar larvae(Parthasarathy et al., 2008) caused the precocious appearance ofadult eyes, adult antennae and other features in the resulting pupae.

These studies show that JH has an endogenous function inregulating Drosophila metamorphosis, a specific example being inorchestrating the timing of differentiation events in the developingvisual system. These effects of JH are primarily mediated throughthe Met pathway. JH also is necessary for normal larval growth andhas another, as yet undefined, crucial role in prepupal developmentthat prevents death at head eversion. The latter effect is not mediatedthrough Met, indicating that JH might act through multiplepathways.

1125RESEARCH ARTICLEJH and prepupal optic lobe development

Fig. 9. Effects of targeted expression of genes involved in theresponse to JH. (A,C) Images of wandering larval brains showing that(A) R27G05 drives expression in lamina neurons (la), and (C) R25B08drives expression in the photoreceptors, but not in any intrinsic neuronsin the lamina or medulla. (B,D) Confocal projection showing EcR-B1expression in the brain hemisphere and attached eye imaginal disc (ED)at 3 hours APF. Expression of Met RNAi under the control of R27G05and R25B08 results in precocious appearance of EcR-B1 in laminaneurons (la) and photoreceptors, respectively. Mushroom bodies (MB)show strong endogenous expression. (E-I) Transverse slices through themedulla at 6 hours APF, showing the relative position of the terminalsof photoreceptors R7 and R8. (E) Control showing the overlap of R7and R8. F) Met27 mutant in which R7 appears to have a distinct stalk(arrowhead) that separates it from R8. (G) Expression of EcR-B1 in thephotoreceptors does not produce an early separation of the twoterminals. (H) Knockdown of Met in lamina neurons by RNAi does notproduce a separation. (I) Knockdown of Met in the photoreceptorsresults in early, stalked (arrowheads) terminals for R7. Scale bars: in D,100mm for A-D; in I, 10mm for E-I.

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AcknowledgementsWe thank Drs G. Korge, J. R. Nambu, T. G. Wilson and A. Shingleton for giftsof the Aug21-GAL4, UAS-grim, Met27 and UAS-MetRNAi stocks, respectively;Dr G. M. Rubin for the R15E10, R17A12, R23C06, R25B08,and R27G05 lines;Drs C. S. Thummel, C. Goodman and C. Q. Doe for the EcR-B1, Fasciclin II andDeadpan antibodies, respectively; and Karen Hibbard and James McMahon ofthe Janelia Farm Fly Core facility for stock construction. The study wassupported by the Howard Hughes Medical Institute. Early preliminaryexperiments were supported by NSF IBN0344933 to L.M.R. Deposited in PMCfor release after 6 months.

Competing interests statementThe authors declare no competing financial interests.

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