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. Author manuscript; available in PMC: 2009 Jan 1.
Published in final edited form as: Mech Dev. 2007 Oct 11;125(1-2):91–105. doi: 10.1016/j.mod.2007.10.002

Krüppel homolog 1 (Kr-h1) mediates juvenile hormone action during metamorphosis of Drosophila melanogaster

Chieka Minakuchi 1,1, Xiaofeng Zhou 1,2, Lynn M Riddiford 1,3,4
PMCID: PMC2276646  NIHMSID: NIHMS37789  PMID: 18036785

Abstract

Juvenile hormone (JH) given at pupariation inhibits bristle formation and causes pupal cuticle formation in the abdomen of Drosophila melanogaster due to its prolongation of expression of the transcription factor Broad (BR). In a microarray analysis of JH-induced gene expression in abdominal integument, we found that Krüppel homolog 1 (Kr-h1) was up-regulated during most of adult development. Quantitative real-time PCR analyses showed that Kr-h1 up-regulation began at 10 h after puparium formation (APF), and Kr-h1 up-regulation occurred in imaginal epidermal cells, persisting larval muscles, and larval oenocytes. Ectopic expression of Kr-h1 in abdominal epidermis using T155-Gal4 to drive UAS-Kr-h1 resulted in missing or short bristles in the dorsal midline. This phenotype was similar to that seen after a low dose of JH or after misexpression of br between 21–30 h APF. Ectopic expression of Kr-h1 prolonged the expression of BR protein in the pleura and the dorsal tergite. No Kr-h1 was seen after misexpression of br. Thus, Kr-h1 mediates some of the JH signaling in the adult abdominal epidermis and is upstream of br in this pathway. We also show for the first time that the JH-mediated maintenance of br expression in this epidermis is patterned and that JH delays the fusion of the imaginal cells and the disappearance of Dpp in the dorsal midline.

Keywords: Krüppel homolog 1, broad, juvenile hormone, ecdysteroid, adult development, metamorphosis, Drosophila melanogaster, histoblasts, Dpp

Introduction

In the Coleoptera and Lepidoptera, the epidermal cells make larval, pupal, and adult cuticles sequentially. By contrast, in higher Diptera such as Drosophila melanogaster, the adult epidermis on the head and the thorax is derived from imaginal discs, and the adult epidermis on the abdomen is formed by imaginal cells derived from the histoblast nests that-make larval cuticle, but do not divide during larval life (Fristrom and Fristrom, 1993; Madhavan and Schneiderman, 1977). After puparium formation, the histoblasts begin to proliferate rapidly, displacing the larval epidermal cells that subsequently die after making the pupal cuticle (Madhavan and Madhavan, 1980; Ninov et al., 2007). This process is complete by 40 h after puparium formation (APF). Juvenile hormone (JH) application to D. melanogaster during the final larval instar or during the prepupal stage has little effect on the adult differentiation of the head and the thoracic epidermis, but it prevents the normal adult differentiation of the abdominal epidermis that is derived from the histoblasts (Ashburner, 1970; Postlethwait, 1974; Riddiford and Ashburner, 1991; Zhou and Riddiford, 2002). After JH treatment, the histoblasts continue to divide to form the imaginal epidermis, but the normal outgrowth of abdominal bristles is prevented, and a second pupal, rather than adult, cuticle is formed (Zhou and Riddiford, 2002).

In insects ecdysteroids trigger molting, while JH determines the nature of the molt (Riddiford, 1994). When JH is present, the ecdysteroid-induced molt is to another like stage; whereas in its absence, metamorphosis ensues. Little is known about how the JH signal is mediated in preventing insect metamorphosis. The broad (br) gene, an ecdysone-induced transcription factor in the Broad-Tramtrack-Bric-a-brac (BTB) family, is the key regulator of the onset of metamorphosis, since amorphic D. melanogaster mutants of br (npr) can develop normally until the final larval instar but fail to begin metamorphosis (Kiss et al., 1976, 1988). In the silkworm Bombyx mori, RNAi knock-down of br in imaginal discs and primordia resulted in their failure to undergo metamorphosis properly (Uhlirova et al., 2003). Zhou and Riddiford (2002) showed that the treatment of either D. melanogaster or M. sexta with a JH mimic (JHM) at the onset of adult development induced the re-expression of the br gene in the abdominal epidermis and that misexpression of br during the adult development of D. melanogaster resulted in the truncation of bristles and the formation of pupal cuticle by the imaginal cells, both of the abdomen and of the disc-derived head and thorax. In hemimetabolous insects such as the milkweed bug Oncopeltus fasciatus, br is expressed during embryonic development and each nymphal molt, then disappears at the molt to the adult (Erezyilmaz et al., 2006). In this animal, JH is necessary to maintain br expression during the nymphal stages. Clearly, br can be regulated by JH. Yet little is known about the genes that are either upstream or downstream of br in the JH signaling pathway.

We therefore performed a genome-wide analysis of JH-regulated genes in the abdominal integument of D. melanogaster to which pyriproxyfen, a JHM, had been applied at the time of pupariation to suppress the adult differentiation of abdominal histoblasts. One of the up-regulated genes was Krüppel-homolog 1 (Kr-h1, CG9167). We show here that the misexpression of Kr-h1 in the epidermal cells resulted in missing or short bristles in the dorsal midline of the adult fly, a phenotype similar to that seen after treating wild-type animals with a low dose of JH and to that seen after br was misexpressed early in adult development. This action of KR-H1 was found to be accompanied by the prolongation of BR expression in the abdominal epidermis, indicating that Kr-h1 is a key regulator functioning upstream of br in the JH signaling pathway. We also found that JHM treatment delayed the development of the abdominal epidermis, thus altering the timing of Dpp expression in the dorsal midline.

Materials and methods

Drosophila stocks and JH application

The wild-type Canton S strain of D. melanogaster was reared at 25° C unless otherwise noted. 100 ng of pyriproxyfen (JHM) (gift of Sumitomo Chemical Co., Osaka, Japan) in 0.2 μl acetone was topically applied to the dorsum at the time of puparium formation. The control received the same volume of acetone. In some cases, the larvae were fed on JH-containing diet (Riddiford and Ashburner, 1991). We added 100 μg JH III (Sigma, St. Louis, MO) in 50 μl 95% ethanol to 6 ml standard cornmeal-molasses-based medium (Sullivan et al., 2000) at 60° C, then placed the JH-containing diet into a 35×10 mm Falcon Petri dish (BD Biosciences, Franklin, NJ). About 30 Canton S eggs were placed in each dish which was put inside a 100×20 mm Petri dish to maintain high humidity. The larvae hatched, fed and pupariated in these dishes. The abdominal bristles were scored in pharate adults 5 days after pupariation.

Flies carrying broad transgenes under the control of the heat shock protein (hsp) 70 promoter were: Z1/Z1; Z1/Z1 [w1118; 708-14; 708-1], Z2/Z2; Z2/Z2 [w1118; cD5-4c; cD5-1], Z3/Z3; Z3/Z3 [w1118; 797-3; 797-E8] and Z4/Z4; Z4/Z4 [w1118; Z4-13; Z4-11] (Bayer et al., 1997; Crossgrove et al., 1996) (gift from Dr. Cynthia A. Bayer). Enhancer-trap lines cn1 P{ry+t7.2=PZ}Kr-h110642/CyO; ry506 and y1w1118; P{w+mC=dpp-lacZ.Exel.1}2 were obtained from Bloomington Stock Center. P{EP}Kr-h1EP2289 (Rørth, 1996) with the UAS sequence inserted in the upstream of Kr-h1α was provided by Szeged Stock Center. The En-Gal4 line (from the Bloomington Stock Center) was used to drive UAS-mCD8-GFP in the posterior part of each segment. An enhancer-trap line T155 that is ubiquitously expressed in abdominal imaginal cells (Harrison et al., 1995; Kopp et al., 1999) was kindly provided by Dr. Carl S. Thummel. The mutant linesKr-h1(1)/CyO, P{w+mC=GAL4-Hsp70.PB}TR1, P{w+mC=UAS-GFP.Y}TR1 and Kr-h1(1)/CyO, P {w+mC=GAL4-Hsp70.PB}TR1, P{w+mC=UAS-GFP.Y}TR1; hs-Kr-h1(1)α were provided by Dr. Geoffrey Richards.

Genome-wide microarray analysis

The abdominal integuments (cuticle, epidermis and muscle mostly cleaned of fat body and gut) of JHM- or acetone-treated Canton S prepupae/pupae were dissected every 6 h from 6–36 h APF in phosphate-buffered saline (PBS) (10 mM sodium phosphate, pH 7.4, 150 mM NaCl) for RNA extraction. Three replicate RNA samples of JHM-treated and of acetone-treated animals were isolated for each time point. Total RNA extractions were made with Trizol (Gibco BRL, Invitrogen Corp., Carlsbad, CA) from at least 30 prepupae/pupae at each time point. The isolated total RNA was then purified using the RNeasy kit (Qiagen Inc., Valencia, CA). The samples were tested for their integrity by Agilent 2100 Bioanalyser (Agilent Technologies Inc, Palo Alto, CA). Affymetrix eukaryotic target preparation protocol was followed to synthesize double stranded cDNA and then biotin-labeled cRNA. Fragmented cRNA was hybridized on an Affymetrix DrosGenome1 oligonucleotide array (Affymetrix Inc, Santa Clara, CA) that contains 13,966 oligonucleotide sequences representing the genome of D. melanogaster. The hybridized GeneChip was then washed and scanned in the Center for Array Technologies at the University of Washington.

The expression values for each gene were calculated by using Affymetrix Microarray Suite (MAS) 5.0 software. To allow inter-array comparisons, all probe sets were scaled from each array to a target intensity value of 100. The data were log (2-based) transformed and normalized using the RMA algorithm (Irizarry et al, 2003). The ANOVA method was used to compare gene expression in JHM-treated and control flies.

Quantitative real-time PCR and RT-PCR analysis

The abdominal integument of Canton S pupae to which 100 ng JHM or acetone had been applied at pupariation was dissected as described above at several time points from 0 h to 96 h APF. At least 35 pupae were dissected and mixed for RNA isolation at each time point. Total RNA was extracted using Trizol reagent (Gibco BRL). To reduce the contamination of genomic DNA and proteins, the extracted RNA was further purified by repeating the extraction procedure with Trizol. cDNA was synthesized using Oligo (dT)12–18 primers (Invitrogen) and M-MLV reverse transcriptase (Invitrogen). Quantitative real-time PCR with Brilliant SYBR Green QPCR Master Mix (Stratagene, La Jolla, CA) was performed using Chromo 4 real-time PCR instrument (MJ Research, Bio-Rad Laboratories, Hercules, CA). Primers detecting both α and γ isoforms of Kr-h1 were TCA CAC ATC AAG AAG CCA ACT and GCT GGT TGG CGG AAT AGT AA. Primers for Kr-h1β isoform were TAT CCA AGA GTC CCG CAA AG and GGT CGT CGC TGT TAG TGG AG. Primers for rp49 were CCA GTC GGA TCG ATA TGC TAA and GTT CGA TCC GTA ACC GAT GT. The signal intensity of Kr-h1 was normalized to that of rp49. Analysis was performed three times on each sample, and the average and standard deviation were calculated.

cDNA was synthesized from the whole body of pupae between 0 h and 18 h after overexpression of BR-Z2 or BR-Z4 at 27 h APF by a half hour heat-shock (37° C). Expression of Kr-h1 mRNA was detected using RT-PCR with rp49 as the internal standard, and visualized with ethidium bromide staining. Primers for rp49 used here were AGC TTC AAG ATG ACC ATC CG and TCC GAC CAC GTT ACA AGA AC. Primers for common region of Kr-h1 isoforms were TCG CAC TCG AAG AAC CAA GA and AAC AGC TTG TGG CAG AAC TC.

Ectopic expression of Kr-h1α and broad

T155-GAL4 (Harrison et al., 1995) was crossed with P{EP}Kr-h1EP2289 (Rørth, 1996) in order to express the Kr-h1α isoform ectopically in the histoblast nests. They were reared at 22–25° C until puparium formation, and then transferred to 29° C to enhance the expression of T155-GAL4.

Broad isoforms were misexpressed at particular times during adult development using the four transgenic lines described above by exposure to 37° C for 30 min (Bayer et al., 1997). In our experience br mRNA is present from the end of the heat shock to about 6 h after the heat shock, and the Broad protein is present from 2–12 h (Zhou and Riddiford, 2002).

Staining procedures

For immunocytochemistry, the abdominal integument was dissected from pupae and fixed with 3.7% formaldehyde in PBS for 30 min as previously described (Zhou and Riddiford, 2002). It was then incubated with a monoclonal antibody against the Drosophila BR core region (1:250) (Emery et al., 1994) followed by 1:500 FITC-conjugated donkey anti-mouse secondary antibody (Jackson ImmunoResearch, West Grove, PA). Texas-Red phalloidin (1:50) (Molecular Probes, Carlsbad, CA) was used as an F-actin counterstain (data not shown). The tissue was mounted in a drop of Fluoromount-G (Southern Biotech, Birmingham, AL). Fluorescent visualization was performed with a BioRad MRC-600 confocal laser scanning microscope, and images were processed with Scion Image and Adobe Photoshop software.

For the detection of Kr-h1-lacZ or dpp-lacZ expression, the abdominal integument was dissected from the pupae of the enhancer-trap lines cn1 P{ry+t7.2=PZ}Kr-h110642/CyO; ry506 (Russell et al., 1998) and y1w1118; P{w+mC=dpp-lacZ.Exel.1}2 respectively and fixed with 2% formaldehyde in PBS for 30 min. For pharate adults at 93 h APF, animals were cut in the ventral midline and fixed. Signal was detected by staining with 0.2% X-gal solution as described (Hama et al., 1990). Otherwise, signal was detected by incubating the tissue with 1:2000 rabbit anti-β-galactosidase antibody (Cappel, MP Biomedicals, Irvine, CA) followed by 1:500 FITC-conjugated donkey anti-rabbit secondary antibody. Texas-Red-conjugated phalloidin (1:50) was used as an F-actin counterstain.

When the cleaned abdominal integument was stained with phalloidin only, it was fixed with 2% formaldehyde in PBS for 5 min, washed three times in PBS-0.1% Triton X-100 (TX), blocked in 1% bovine serum albumin (BSA) and PBS-0.1% TX for 5 min, and then stained with phalloidin conjugated to Texas Red (1:50) at room temperature for 30 min in the dark.

Results

JH up-regulates Kr-h1 in the abdominal integument

To identify the genes whose expression is regulated by JH during metamorphosis, we compared genome-wide gene expression levels in JHM-treated and control prepupae/pupae at designated time points using Affymetrix DrosGenome1 oligonucleotide arrays. We found that 217 genes were significantly up-regulated and 114 genes were significantly down-regulated at least two-fold (P ≤ 0.01) at one or more time points. One of the JHM-up-regulated genes was Kr-h1. Our microarray data showed that in the Canton S line, the signal intensity of Kr-h1 transcripts was very low and not distinguishable from background noise from 12 h to 36 h APF (Fig. 1A). By contrast, Kr-h1 was significantly up-regulated by JHM treatment after 12 h APF throughout the rest of the test period except at 18 h and 24 h APF, though the signal intensities were low.

Figure 1.

Figure 1

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Expression pattern of Kr-h1 mRNA. Solid circles, acetone-treated control animals. Open circles, JHM-treated animals. (A) Signal intensity of Kr-h1 mRNA from 6 h to 36 h after acetone- or JHM-treatment was analyzed with the Affymetrix Drosophila GeneChip. Each circle represents a pool of at least 30 abdomens. (B) Relative signal intensity of Kr-h1α and Kr-h1γ mRNA after acetone- or JHM-treatment was analyzed by quantitative RT-PCR. Signal for Kr-h1α and Kr-h1γ was normalized with that of rp49. The signal intensity at puparium formation (0 h APF) was considered as 100. Analysis was performed three times for each sample, and the average and standard deviation are shown. (C) Relative signal intensity of Kr-h1β mRNA after acetone- or JHM-treatment was analyzed by quantitative RT-PCR. Signal for Kr-h1β was normalized with that of rp49. The signal intensity in embryonic stage from 0 h to 12 h after egg laying (AEL) was determined and considered as 100. Analysis was performed three times for each sample, and the average and standard deviation are shown.

To verify this microarray result, we performed quantitative real-time PCR analysis on cDNA of abdominal integuments. In acetone-treated animals, the levels of Kr-h1α and Kr-h1γ [indistinguishable with the probe used and from here forward referred to as Kr-h1α since Kr-h1γ is 10-fold less abundant (Pecasse et al., 2000)] mRNAs were high at puparium formation and 2 h APF (Fig. 1B). Kr-h1α mRNA then decreased to undetectable levels by 8 h APF and was absent up to 84 h APF. In JHM-treated animals, the Kr-h1α mRNA also decreased between 2 h and 8 h APF, but reappeared at 10 h APF, and remained high until 36 h APF followed by a slight decrease but still significantly higher than controls until 84 h APF. This result was basically consistent with the microarray data.

We also analyzed the expression of the Kr-h1β isoform that is dominant during the embryonic stage in the nervous system (Beck et al., 2004; Pecasse et al., 2000) (Fig. 1C). The signal intensity in embryos 0 h to 12 h after egg laying (AEL) was high (considered as 100 in Fig. 1C). In the abdominal integuments of acetone-treated controls, Kr-h1β was expressed weakly at pupariation, but was absent between 12 h and 84 h APF. By contrast, after JHM treatment, Kr-h1β was significantly up-regulated from 12 h to 84 h APF. The signal intensity for the β isoform was much lower than that for the α isoform during pupal and adult development.

Up-regulation of KR-H1 in persisting larval muscles, imaginal epidermis, and oenocytes of the abdomen by JHM treatment

To determine the tissues in which KR-H1 protein was up-regulated by JHM treatment, we used a Kr-h1-lacZ reporter line, and the signal was detected either by X-gal staining or by a specific antibody against β-galactosidase since a reliable KR-H1 antibody was not available. A potential drawback of this method is that the β-galactosidase reporter is known to have a long half life so does not accurately reflect the time of inactivation of the gene. As shown in Figs. 2C and 2E, lacZ was expressed in the larval epidermal cells and persisting larval muscles for both acetone-treated and JHM-treated pupae at 28 h APF, with no significant difference between the two evident. This finding likely reflects the persistence of the β-galactosidase reporter since in the controls Kr-h1 mRNA was not detected after 8 hr APF (Fig. 1B, C). In acetone-treated pupae at 45 h APF, little lacZ signal was detected in the abdominal epidermis, and a very weak signal was detected in persisting larval muscles (Fig. 2D). By contrast, in JHM-treated pupae at 45 h APF, strong lacZ signal was found in the persisting larval muscles, and a weak signal in imaginal epidermal cells (Fig. 2F). The signal was also detected in persisting larval oenocytes in JHM-treated pupae at 27 h, 51 h, 72 h (data not shown) and 93 h APF (Fig. 3E). In acetone-treated pupae, no signal was detected in oenocytes by 51 h (data not shown).

Figure 2.

Figure 2

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(A, B) Schematic representation of larval epidermal cells (gray) and imaginal epidermal cells (blue) in an abdominal segment at approximately 28 h APF (A) and 45 h APF (B). Abbreviations in these figures: DM, dorsal midline; VM, ventral midline; Sp, spiracle; A, anterior; P, posterior. The area observed in Figs. 2C–F is boxed in red. (C-F) Expression pattern of β-galactosidase reporter of Kr-h1 activity in the abdominal integument at 28 h APF (C, E) and 45 h APF (D, F). Abdominal integuments from the pupae of the enhancer-trap line cn1P{ry+t7.2=PZ}Kr-h110642/CyO; ry506 were stained with rabbit anti-β-galactosidase antibody followed by FITC-conjugated donkey anti-rabbit secondary antibody (shown in green). The F-actin counterstain with Texas-Red-phalloidin is shown in red. Animals were treated with either acetone (C, D) or JHM (E, F) at puparium formation. Persisting larval muscles are indicated by white arrows.

Figure 3.

Figure 3

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Expression pattern of the β-galactosidase reporter of Kr-h1 activity in the enhancer-trap line cn1P{ry+t7.2=PZ}Kr-h110642/CyO; ry506 after acetone (A, B) or JHM (C, D, E) treatment at puparium formation. Signal was detected by staining with 0.2% X-gal solution. (A, C) Legs (indicated by arrows) and wings at 72 h APF. (B, D) Ventral view of the head at 93 h APF. (E) Abdominal epidermis of a JHM-treated animal at 93 h APF. Persisting larval oenocytes are indicated by arrows.

After JH treatment at pupariation, the animal develops to a pharate adult with an apparently normal adult head and thorax in terms of epidermal development (Ashburner, 1970; Postlethwait, 1974), but internally the development of the flight muscles and the central nervous system is disrupted (Restifo and Wilson, 1998). Therefore, it was a surprise to find Kr-h1-lacZ reporter staining in the developing legs and wings in JHM-treated animals at 51 h (data not shown) and 72 h APF (Fig. 3C). No signal was seen in these tissues of acetone-treated pupae at these stages (Fig. 3A). At 93 h APF, lacZ expression was observed in the legs and eyes, but again there was no signal in control animals (Figs. 3B and D).

These findings suggest that JH treatment at pupariation allows the re-expression of Kr-h1 in the abdominal epidermis, the persisting larval muscles of the abdomen, and the persisting oenocytes during adult development. In addition, it appears to be also up-regulated in parts of the adult structures developing from imaginal discs that show no external morphological effects.

Misexpression of either Kr-h1α or br mimicked JH effects on bristle formation in the dorsal midline

In order to express Kr-h1α ectopically in the histoblasts, P{EP}Kr-h1EP2289 (Rørth, 1996; Abdelilah-Seyfried et al., 2000) was crossed with the T155-Gal4-driver line. This driver causes expression primarily in the histoblasts and the derived imaginal epidermis throughout adult development (Harrison et al., 1995; X. Zhou, personal observation). The larvae were reared at 22–25°C until pupariation, then placed at 29° C during adult development. In the F1 animals, the patterning of the adult bristle formation in the abdominal dorsal midline was disrupted. As shown in Figures 4C and D, some bristles along the dorsal midline were short or missing, and the bristle orientation around the dorsal midline was irregular as compared to the control animals (Figs. 4A and B). Their phenotype was similar to that of pharate adults to which an intermediate dose of JHM had been applied at puparium formation (Riddiford and Ashburner, 1991). At low doses eclosion was inhibited, but the pharate adult had normal bristles; with much higher doses, most of the bristles on the abdominal tergite were missing or very short (Fig. 4E, left). At intermediate doses only the dorsal midline lacked bristles (Fig. 4E, right). After the ectopic expression of Kr-h1, 13 animals died as pharate adults and 7 animals eclosed successfully (N = 20); all of them had irregular or missing bristles in the dorsal midline. In the eclosed adults, the pigmentation pattern of the abdominal tergite along the dorsal midline was also abnormal (Fig. 4D).

Figure 4.

Figure 4

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Effect of either ectopic expression of Kr-h1αor JHM application at the time of pupariation on the abdominal epidermis. The dorsal views of pharate adults that had been taken out from their pupal case (A, C, E) and the dorsal view of the male adult abdomen (B, D) are shown. The dorsal midline with missing bristles was indicated by red arrows. (A, B) Control animals. (C, D) Animals that were formed after ectopic expression of Kr-h1α. (E) Wild-type (Canton S) pharate adults with severe (left) and mild (right) effects after JHM application (100 ng pyriproxyfen; PPX) at puparium formation (left) or feeding on a JH III diet containing 16.7 μg/ml JH III (right). (F-H) Confocal images of abdominal dorsal epidermis with arrows pointing to the dorsal midline. (F) w1118 at 48 h APF at 25°C. (G) Abdominal epidermis misexpressing Kr-h1α at 40 h APF at 29°C. The arrowheads point to the bristles. (G’) A higher magnified view of the rectangle in (G). (H) JHM-treated Canton S (100 ng pyriproxyfen) at 48 h APF at 25°C. Note the absence of the adult hairs and bristles in the midline of the experimental animals in G, G’, and H (arrow). Scale bar, 20 μm.

The larval epidermal cells (LECs) along the dorsal midline are the last cells to be replaced by the imaginal cells (Madhavan and Madhavan, 1980), and the failure to replace these LECs leads to the absence of bristles in the dorsal midline (Ninov et al., 2007). We examined whether the lack of bristles in this area after misexpression of Kr-h1α was due to the incomplete replacement of LECs. By staining with Texas Red-conjugated phalloidin, we found that the dorsal fusion of the imaginal cells was complete and no LEC remained (Fig. 4G). Thus, the lack of bristles may due to the failure of outgrowth of the bristles rather than the incomplete fusion of imaginal cells. Interestingly, many of the imaginal cells, which likely expressed BR (see below), had no hairs and appeared larger (Fig. 4G′). Some patches of cells were able to form hairs, but with abnormal polarity, indicating that the planar polarity information had been altered. Similarly, the JHM-treated developing adults were also able to complete the fusion of imaginal cells at the dorsal midline (Fig. 4H). Most of the imaginal cells in these JH-treated animals do not have hairs, except the ones in the a2 regions where br seems not to be re-expressed after JHM-treatment (see below).

Previously we reported that the misexpression of isoforms of br resulted in the phenotype of short or missing bristles on the entire abdomen (Zhou and Riddiford, 2002). Here we show that heat shock-induced expression of BR-Z4 between 21 h and 30 h APF specifically caused the absence of bristles in the dorsal midline (Fig. 5A), but did not prevent dorsal midline fusion (Fig. 5C). In some cases, misexpression of BR-Z4 caused the loss of the cellular hairs in the posterior dorsal midline (Fig. 5C right). Unlike the bristles adjacent to the dorsal midline in the control animals that point towards the posterior and the dorsal midline (Fig. 4F), the bristles in animals in which BR-Z4 had been misexpressed were directed away from the dorsal midline (Fig. 5C). BR-Z4 misexpression at 18 h APF had no effect on the differentiation of any bristles (data not shown). Misexpression of BR-Z4 at 33 h or 36 h APF resulted in shorter bristles on the entire abdomen, whereas misexpression at 27 h or 30 h caused truncation of the thoracic bristles. Misexpression of other isoforms of br caused similar phenotypes with BR-Z2 and BR-Z4 generally having stronger effects than did the BR-Z1 and BR-Z3 isoforms (Fig. 5D). After the misexpression of BR-Z4 at 27 h APF, 39 animals died as pharate adults, and 9 animals eclosed (N = 48). In the eclosed adults, the dorsal midline lacked the normal pigmentation (Fig. 5B, right). This phenotype was not as severe as that seen after the ectopic expression of Kr-h1.

Figure 5.

Figure 5

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Effect of misexpression of broad (br) on adult development. In (A, C, D), all developed to the pharate adult stage at which time they were removed from their puparia for scoring and photography, while in (B) the phenotype was observed in eclosed adults. (A) Misexpression of BR-Z4 in the line w1118; Z4-13; Z4-11 by a 30 min heat shock (hs) at 37°C at 21 h, 24 h, 27 h, 30 h, 33 h or 36 h APF. (B) Normal male abdomen (left) and abdomen of male given BR-Z4 (same line as in A) at 27 h APF (right). (C) Misexpression of BR-Z4 at 30 h APF, then the abdomen was dissected and stained with phalloidin at 48 h APF. An arrow points to the dorsal midline. Right, a higher magnified view of the rectangle in the left. Scale bar, 20 μm. (D) Misexpression of BR-Z1 [w1118; 708-14; 708-1], BR-Z2 [w1118; cD5-4c; cD5-1] or BR-Z3 [w1118; 797-3; 797-E8] at 30 h APF.

Interaction between Kr-h1 and br

As discussed above, the misexpression of Kr-h1α resulted in a similar phenotype as that seen after the misexpression of br between 21 and 30 h APF, and both showed similarities to the effect of a low dose of JH given at puparium formation. These results raised the possibility that Kr-h1α and br might be working in the same pathway of JH signaling. To examine the interaction between Kr-h1 and br, we first asked whether Kr-h1 expression is induced after the misexpression of br. No significant induction of Kr-h1 mRNA was seen from 0 h to 18 h after the misexpression of either BR-Z2 or BR-Z4 by heat shock at 27 h APF (data not shown). Since BR protein is stable up to 12 h after the misexpression, then disappears (Zhou et al., 2004), we conclude that Kr-h1 is not up-regulated by br.

To determine if Kr-h1 is upstream of br, we used a specific antibody against BR protein (that detects all isoforms) after the misexpression of Kr-h1α in the epidermal cells using the T155-Gal4-driver line. These animals were reared at 29° C from puparium formation to enhance the expression of T155-Gal4 in the histoblasts and the derivative imaginal epidermis, and were dissected at several time points between 24 h and 70 h APF at 29° C. In our hands, wild-type animals finished adult differentiation by 70–75 h at 29° C, and eclosed around 80 h APF, although the timing of the eclosion itself is quite variable due to photoperiodic gating of eclosion (Truman, 2005). As shown in Figure 6A, in the control line where Kr-h1α is not misexpressed, BR is present in the larval epidermal cells as well as in the imaginal cells at 24 h APF at 29° C, and then disappears by 27 h APF. This observation is basically consistent with that in the wild-type animals reared at 25° C where BR disappears by about 36 h with the disappearance of the last larval cells (Zhou and Riddiford, 2002; XZ, unpublished observations). Thus, in both cases BR protein disappears just at the time when the imaginal epidermal cells have completely replaced the larval epidermal cells by fusing together in the dorsal midline. By contrast, when Kr-h1α was expressed ectopically, the expression of BR protein in the imaginal epidermal cells was prolonged, especially in the pleura and in the dorsal part of the tergite (Figs. 6A, B, C). Also, the larval epidermal cells were still prominent at 27 h APF (Fig. 6A). After ectopic expression of Kr-h1α, BR remained high until 38 h APF, then decreased gradually, disappearing from the lateral areas first. At 70 h APF, a weak BR signal was still detected in some imaginal cells in the dorsal midline (Fig. 6B). These results suggested that Kr-h1α is involved in the regulation of br expression.

Figure 6.

Figure 6

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Immunostaining of Broad protein in the abdominal epidermis during adult development. Schematic representations of larval epidermal cells (light gray) and imaginal epidermal cells (dark gray) are shown, and the observed area is boxed. Abbreviations in these figures: DM, dorsal midline; LM, lateral midline; VM, ventral midline; Sp, spiracle; A, anterior; P, posterior. Animals were reared at 29°C after puparium formation. Phalloidin staining showed integrity of the epidermis (data not shown). (A) BR immunostaining after the ectopic expression of Kr-h1α (T155>Kr-h1α) is compared with that of control (T155>+) at 24 h, 27 h and 30 h APF. (B) BR immunostaining after the ectopic expression of Kr-h1α (T155>Kr-h1α) during adult development from 38 h to 70 h APF. At 60 hr APF, the bristles can be faintly seen due to cuticular autofluoresence. The pictures shown in (A) and (B) of the epidermis were from different cohorts of pupae. (C) BR immunostaining after the ectopic expression of Kr-h1α (T155>Kr-h1α) at 30 h APF.

In light of these results, we have examined in further detail the pattern of BR expression in the JHM-treated Canton S flies at 25° C. To describe the observed pattern, we have used the nomenclature of the abdominal segmental subregions as defined by Struhl et al. (1997b) (Fig. 7, top left). engrailed (en)-driven green fluorescent protein (GFP), which marks the posterior compartment of the abdominal segment, was used to facilitate definition of the spatial pattern of br expression. Figure 7 (bottom) represents typical BR immunocytochemical staining in the JHM-treated wild type abdominal epidermis at 42 h APF at 25° C, when the histoblast-derived cells have fused at the dorsal midline. BR protein was detected in about a 10 cell-width stripe in the dorsal midline. Away from this region, BR staining was lost in the cells in the a1 and a2 domains, but was present in most of the epidermal cells in the a3 to a5 regions. The cells at either edge of the en-GFP-expressing “belt” contained BR, but those in the middle of the “belt”, which may correspond to the p2 region, had lost BR staining. The trichogen cells all contained significant amounts of BR. Thus, BR is not re-expressed in all the abdominal imaginal cells in JH-treated animals, but in a manner presumably reflecting underlying patterning elements.

Figure 7.

Figure 7

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Patterning of Broad (BR) in the dorsal abdominal epidermis of an animal treated with JHM at the time of pupariation. Top Left. The designation of the regions of the abdominal segmental tergite as regions defined by Struhl et al. (1997b) (reproduced with permission of the Company of Biologists). Top Right. A diagram indicating the region of the segment (red box) that was imaged in the bottom series. Bottom. BR antibody staining (red) and en>GFP (green) imaging in dorsal abdominal epidermis of a developing Canton S adult at 42 h APF after treatment with 100 ng pyriproxifen at pupariation. An arrow points to the dorsal midline. Scale bar: 50 μm.

JHM treatment delayed the development of the abdominal epidermis and altered the timing of Dpp expression in the dorsal midline

Both JHM- and Kr-h1-induced BR expression patterns showed that the cells at the dorsal midline and near the boundary of the anterior and posterior compartments tended to re-express BR. This pattern was reminiscent of the expression of the morphogen Decapentaplegic (Dpp) in the abdominal segments (Kopp et al., 1999). To determine whether dpp signaling in this pathway was affected by the JHM treatment, we examined the expression of presumed Dpp protein using an enhancer-trap line y1w1118; P{w+mC=dpp-lacZ.Exel.1}2 in the entire tergite (Fig. 8A, B). In acetone-treated control animals at 29 h APF (25° C), lacZ staining was detected in both larval and imaginal epidermal cells in the posterior edge of the anterior compartment (Fig. 8A, B). Dpp expression was especially strong in the larval epidermal cells between the contralateral dorsal histoblast nests and in the imaginal cells derived from the ventral histoblast nests. The imaginal cells continued to proliferate and migrate, until they replaced larval epidermal cells completely and merged at the dorsal midline between 33 h and 37 h APF. During this time the lacZ staining decreased except in the dorsal midline and in the spiracular area. At 37 h and 40 h APF, lacZ staining was only found at the posterior edge of the anterior compartment as well as in a wedge-shaped stripe in the dorsal midline. The large stained cells seen in the midline at 37 hr in Figure 8B are pericardial cells (which are known to be scavenger cells) (Crossley, 1985; Locke and Russell 1998; Shanmugavelu et al., 2000), not larval epidermal cells. These observations were consistent with the findings of Kopp et al. (1999). In JHM-treated animals at 29 h APF, lacZ staining was similar to the control (Fig. 8B, right). But this staining remained high between 33 and 40 h APF, as many larval epidermal cells remained in the dorsal tergite. By 45 h APF, the staining pattern (the typical staining pattern is shown in Fig. 8C) was similar to that seen in the control at 37 h APF (Fig. 8B), reflecting the delay in fusion of the imaginal cells at the dorsal midline until between 40 h and 45 h APF in the JHM-treated pupae. No large pericardial cells are seen at the dorsal midline in the JHM-treated animals due to their removal during dissection. These results indicated that JHM treatment somehow delayed either differentiative cell division or migration of the imaginal abdominal cells resulting in a delay of fusion of the imaginal cells in the dorsal midline. Concomitant with this delay was a prolongation of the normal pattern of Dpp expression in the abdominal epidermis.

Figure 8.

Figure 8

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(A) Schematic representation of larval epidermal cells (light gray) and imaginal epidermal cells (dark gray) in an abdominal segment of acetone-treated (control, left) and JHM-treated (right) animals at approximately 29 h APF at 25° C were shown. The area observed in Fig. 8B is dashed. The boundary between the anterior and posterior compartments of a segment is outlined by a dotted line. Abbreviations in these figures: DM, dorsal midline; VM, ventral midline; Sp, spiracle; A, anterior; P, posterior. (B) Differentiation of adult abdominal epidermis in control animals and JHM-treated animals. Abdominal integument of either control or JHM-treated animals was stained to detect dpp-lacZ at 29 h, 33 h, 37 h, 40 h and 45 h APF. At 29 h APF, the boundary between the anterior and posterior compartments of a segment is outlined by a broken line, and the boundary between polyploid larval epidermal cells and diploid imaginal cells is outlined by a solid line. (C) A wedge-shaped Dpp staining in the dorsal midline of JHM-treated animals at 45 h APF. In the schematic representation of an abdominal segment, the area observed in the pictures is dashed, and the boundary between the anterior and posterior compartments of a segment is outlined by a dotted line.

Discussion

In this study we have identified Kr-h1 as one of the genes up-regulated by JHM treatment of Drosophila at pupariation that then persists during the entire pupal-adult developmental period. Moreover, our studies indicate that the presence of KR-H1 during early adult development can induce the abnormal re-expression of br in the abdomen that results in the formation of a second pupal cuticle (Zhou and Riddiford, 2002).

KR-H1 is a zinc-finger type transcription factor with three putative isoforms with different N-terminal sequences (Pecasse et al., 2000). There are two main isoforms with the βisoform being expressed mainly during nervous system development in the embryo (Beck et al., 2004). Normally Kr-h1α is expressed at low levels in midembryogensis, at high levels during larval life, then declines rapidly after pupariation (Pecasse et al., 2000) and is not expressed again until just before adult eclosion (Y. Beck and G. Richards, personal communication). KR-H1α appears to be necessary for metamorphosis since most of the mutants lacking Kr-h1α function die at the time of head eversion to the pupa or shortly thereafter (Pecasse et al., 2000).

Regulation of Kr-h1 expression by ecdysteroid and JH

In insects ecdysteroids cause the molt and JH is present during larval life to ensure that the molt is to another larval stage by preventing the developmental program-switching action of ecdysteroids necessary for metamorphosis (Riddiford, 1994, 1996). In most holometabolous insects where the epidermal cells are polymorphic so that they produce sequentially larval, then pupal, then adult cuticles, this switching occurs in the final larval instar when the JH titer declines and ecdysone appears in the absence of JH. By contrast, in the highly derived Drosophila, the onset of metamorphosis triggered by ecdysone in the absence of JH results in the death of most of the larval tissues and the development of the pupa and subsequent adult from the imaginal discs. One exception is the larval abdominal epidermis which switches from production of larval cuticle to that of pupal cuticle. The subsequent adult cuticle is then made by imaginal cells derived from the abdominal histoblasts that begin proliferation shortly after pupariation (Fristrom and Fristrom, 1993). Importantly, in Drosophila JH cannot prevent the metamorphosis of the imaginal discs or the proliferation of the histoblasts but can delay the onset of metamorphosis; it also causes the formation of a pupal rather than an adult cuticle by the new imaginal cells of the abdomen (Riddiford, 1993; Zhou and Riddiford, 2002; Riddiford et al., 2003).

Kr-h1α is regulated at least in part by 20-hydroxyecdysone (20E) and in turn regulates the ecdysone-regulated processes (Pecasse et al., 2000; Beckstead et al., 2005). It shows a dynamic pattern of binding to certain ecdysteroid-regulated chromosomal sites during the 20E-induced cell death of the salivary glands at metamorphosis (Beck et al., 2005). In mutants that lack Kr-h1α function, the normal ecdysteroid cacade of transcription factors at pupariation is disrupted with some appearing precociously and others being delayed or reduced in amount (Pecasse et al., 2000). The result of this misregulation is retention of the salivary glands and death around the time of head eversion that signals completion of pupal development. Interestingly, although overexpression of Kr-h1 suppressed the initial morphogenesis of mushroom body neurons, its loss caused no detectable defects in neuronal morphogenesis, but rather affected the patterning of EcR-B1 expression in the central nervous system at the onset of metamorphosis (Shi et al., 2007). Thus, KR-H1 is clearly necessary for the proper coordination of the ecdysone response.

Interestingly, the few escapers among the Kr-h1α mutants formed cryptocephalic pupae that developed into adults with pigmented eyes and wings but no adult abdominal differentiation beyond the proximal segments (Pecasse et al., 2000). Thus, they resemble pharate adults formed after treatment with JH at the time of pupariation (Ashburner, 1970; Postlethwait, 1974; Zhou and Riddiford, 2002). In these JH-treated animals, we find in this study that Kr-h1 was up-regulated in imaginal abdominal epidermal cells, derivatives of imaginal discs (wing, leg, and eye), persisting larval muscles necessary for eclosion and wing-spreading behavior (Kimura and Truman, 1990), and in larval oenocytes during the ecdysteroid rises for pupal head eversion and adult development. Yet the adult head and thorax appeared grossly normal after JH treatment. Likewise, there was no significant difference in the number of persisting larval muscles in JH-treated pupae, but the normal differentiation and attachment of adult muscles and the outgrowth of abdominal bristles were either inhibited or delayed at 45 h APF (see Figure 2) as had previously been seen for the thoracic muscles (Sandstrom et al., 1997; Sandstrom and Restifo, 1999) and the abdominal bristles (Zhou and Riddiford, 2002). The larval oenocytes are involved in lipid metabolism during growth and larval development, then persist through much of adult development where they appear critical for normal utilization of the stored lipid (Gutierrez et al., 2007). In other insects such as Tenebrio, the oenocytes have been shown to produce ecdysteroids (Delbecque et al., 1990). Whether the presence of KR-H1 in these oenocytes during this latter period is responsible for any of the defects seen in JH-treated animals is unknown.

The role of JH in the normal developmental expression of Kr-h1α in the Drosophila larva is not known. In the red flour beetle, Tribolium castaneum, the transcript level of Kr-h1 is high during larval life, decreases at the end of the final larval stage and disappears just before pupation, then remains very low during the pupal stage and the ensuing adult development (Minakuchi and Shinoda, in preparation) just as in Drosophila (Pecasse et al., 2000). Importantly, RNAi-mediated knockdown of Kr-h1 in young (pre-final instar) Tribolium larvae resulted in precocious metamorphosis, indicating that Kr-h1 is necessary for mediating JH signals in normal larvae (Minakuchi and Shinoda, in preparation). Our finding that Kr-h1α reappears abnormally in the abdomen of pupae that were treated with JH at pupariation suggests that its appearance is the normal larval response to ecdysone in the presence of JH. Clearly further work is necessary to work out the details of the normal hormonal regulation of Kr-h1 in the Drosophila larva.

In the brain of the honeybee, Apis mellifera, a homolog of Kr-h1 was identified as one of the genes down-regulated by queen mandibular pheromone (Grozinger et al., 2003) and up-regulated during the transition to foraging behavior of the adult, which is initiated by JH (Grozinger and Robinson, 2006). Whether JH directly or indirectly controls the transcription of this gene has not been determined.

Interaction of Kr-h1 and br during development

In Drosophila at the onset of metamorphosis, 20E induces a cascade of transcription factors including the different isoforms of br, E74, and E75 that serve to regulate tissue-specific genes involved in metamorphosis (Thummel, 1995, 2001; Zhou and Riddiford, 2002). For most tissues, this is the first appearance of Broad (BR), a BTB-domain containing transcription factor, which is necessary for metamorphosis (Bayer et al., 1996). At this time, br is apparently regulated by KR-H1 since this protein has been localized to the 2B5 br gene site on the salivary gland chromosomes (Beck et al., 2005). In the abdominal epidermis, br specifies pupal cuticle formation, whether the cells are initially larval or whether the cells are the imaginal cells derived from the histoblasts (Zhou and Riddiford, 2002). We found that misexpression of Kr-h1α during adult development caused the re-expression of br in the imaginal epidermis of both the pleura and the dorsal abdominal tergites, but that misexpression of br during normal adult development did not lead to Kr-h1 misexpression. These data strongly suggest that the JH-induced KR-H1α is acting somehow to prevent the permanent cessation of br expression in at least some of the imaginal abdominal epidermal cells during the onset of adult development. The nature of this action is not understood including whether or not it acts directly or indirectly on br transcription.

Importantly, this interaction of Kr-h1α and br is not essential for the normal expression of BR during the late third larval instar since in Kr-h1α mutants, BR is present at the time of wandering as in wild type larva (data not shown). Whether the normal time course of br activation and expression occurs in these mutants has yet to be studied.

The phenotype of ectopic expression of Kr-h1α in the abdominal epidermis

As stated above, the ectopic expression of Kr-h1α in the abdominal epidermis resulted in missing or short bristles in the dorsal midline. We also observed a similar phenotype after low JHM was applied at pupariation (Fig. 4E), or after misexpression of br during early adult development between 21 h to 30 h APF (Fig. 5). The abdominal epidermis around the dorsal midline is the most sensitive to JHM treatment (Riddiford and Ashburner, 1991; LMR, unpublished observation). As the dose of JHM is increased, more bristles on the tergite are affected. In wild-type Canton S, 100 ng of pyriproxyfen prevents the outgrowth of abdominal bristles, resulting in a bald abdomen with few or no bristles (Zhou and Riddiford, 2002) as shown in Figure 4E. When JHM was applied to JH-resistant lines such as the methoprene-tolerant mutant, the outgrowth of the majority of abdominal bristles was not completely blocked except for the bristles in the dorsal midline (Ashok et al., 1998; Wilson and Ashok, 1998; Wilson and Fabian, 1986).

By misexpressing Kr-h1 with the Gal4 driver T155 that expresses in the abdominal histoblasts and the derivative imaginal epidermal cells, we were unable to mimic the complete loss of bristles as seen with the higher dose of JHM. Whether this is due to the strength of the T155 driver or whether this indicates the involvement of other pathways in the action of JH is unknown. The fact that BR persists longer in the dorsal midline cells of the tergite under control of KR-H1 driven by T155 than it does in the dorsolateral cells suggests that patterning elements may also be affected by JH. Zhou and Riddiford (2002) showed that misexpression of the various isoforms of br between 30 and 39 h caused a truncated bristle phenotype, at early times on the head and thorax and at later times on the abdomen. Misexpression of BR-Z1 between 44 and 60 h had little effect on bristle formation but caused the formation of pupal cuticle. In the present study, we have shown that the misexpression of br at even earlier times in developing adults (between 21 h and 30 h APF) caused the loss of bristles and hairs in the dorsal midline of the tergite. This effect was mimicked by the ectopic expression of Kr-h1α in the larval and imaginal epidermis that in turn caused the prolongation of BR protein in the dorsal midline until at least 70 h (at 29°C). Presumably this BR misexpression prevented hair formation, bristle outgrowth and normal adult cuticle formation. Whether this effect of Kr-h1α on br expression is constrained to the dorsal midline by Dpp signaling or is modulated by other patterning genes is unclear.

Interestingly, the pattern of BR expression in Kr-h1α-containing cells is different from the JH-induced BR pattern. In the Kr-h1α-directed BR expression, there appear to be more BR-expressing cells near the mid-dorsal line and fewer such cells laterally. Also, in the a5 region, more of the Kr-h1α-directed cells express BR than in the JH-treated animals, whereas fewer of those in a3 and a4 express BR. Thus, the KR-H1α-directed BR pattern looks like a triangle with most of the cells at the dorsal midline and very few cells in the lateral area expressing BR. Possibly this patchy BR staining in the anterior tergite directed by Kr-h1α is dependent on other local patterning elements that are not disturbed by JH. Further study of this difference is warranted.

JH delayed the adult development of the abdominal epidermis

The histoblast nests start rapid mitosis after puparium formation, and the cell division continues until the imaginal cells from several nests migrate and fuse (Madhavan and Madhavan, 1980; Ninov et al., 2007). After the imaginal cells replace the larval epidermal cells, they start the formation of abdominal bristles followed by the secretion of adult cuticle (Fristrom and Fristrom, 1993; Madhavan and Madhavan, 1980). Using the enhancer trap line (dpp-lacZ), Kopp et al. (1999) showed that Dpp expression is confined to the pleura and the dorsal midline in the posterior edge of the anterior compartment at 45 h APF. As shown in Fig. 8, we also observed a similar pattern of Dpp expression. Importantly, the spatial patterns of Dpp expression were very similar between control and JHM-treated animals, but the expression of Dpp and the differentiation of the abdominal epidermis were delayed in JHM-treated animals. Zhou and Riddiford (2002) previously reported that the proliferation of imaginal epidermal cells on the abdomen occurred normally after JHM treatment at puparium formation such that there was no apparent difference in the size of the imaginal nest between JHM-treated and control animals at 18 h and 30 h APF. In this study we investigated the migration of imaginal cells between 29 h and 45 h APF in detail, and found that JHM treatment at puparium formation delayed the migration of imaginal epidermal cells and/or the death of the larval epidermal cells during this period. Since death of the larval cells is tightly linked to the migration of the adult cells (Ninov et al., 2007), one may not be able to separate the effects on the two. Whether JH delays the earlier rapid proliferative events needs to be restudied using the techniques of Ninov et al. (2007). These results indicate that JHM treatment disrupts the coordination of events in development of the adult abdominal epidermis, including the timing of Dpp expression, which is likely a consequence of the delayed development. Whether the JH-induced re-expression of br in the abdominal epidermis mediates this effect on dpp expression or whether there is a direct effect of JH on dpp expression is unknown.

These studies have shown for the first time that the JH-mediated maintenance of br expression is patterned in the developing adult dorsal abdominal epidermis (the ventral epidermal cells were not investigated). The cells in the a1 region, the anterior-most of the segment (Struhl et al., 1997b) (Fig. 7), do not express BR in response to JHM. These cells, under normal conditions, show high levels of hedgehog (hh), and do not receive wingless (wg) and optomotor-blind (omb) signals (Kopp et al., 1997; Lawrence et al., 2002; Struhl et al., 1997a, b). The lack of BR expression in these cells may be due to the lack of Wg signal. Wg signaling has been shown to be required for BR expression in the follicle cells of the dorsal appendage primordia of egg chambers (Jordan et al., 2005; Ward et al., 2006). However, the loss of br expression in the cells in the p2 region is not likely to be caused by lack of Wg signal, because Wg is present at higher levels in the p2 region than in the p1 region where BR is expressed (Lawrence et al., 2002). Thus, various patterning elements apparently are also involved in the regulation of the JHM-induced br re-expression during adult development.

Acknowledgments

We thank Thanh Nguyen for technical assistance with the microarray analysis, Dr. Cynthia Bayer for the hs-BR transgenic Drosophila lines, Dr. Geoffrey Richards for Kr-h1 mutant lines and many helpful discussions, Dr. Carl S. Thummel for the enhancer-trap line T155, and the Comparative Genomics Center in the Department of Biology, University of Washington, for the use of the quantitative PCR equipment. This study was supported by NIH grant GM60122 to L. M. R. and a fellowship to C. M. from the Japan Society for the Promotion of Science.

Footnotes

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