EcR expression in the prothoracicotropic hormone- producing neurosecretory cells of the Bombyx mori brain An indication of the master cells of insect metamorphosis Monwar Hossain 1 , Sakiko Shimizu 2 , Haruhiko Fujiwara 3 , Sho Sakurai 1,2 and Masafumi Iwami 1,2 1 Division of Life Sciences, Graduate School of Natural Science and Technology, Kanazawa University, Japan 2 Division of Biological Science, Graduate School of Natural Science and Technology, Kanazawa University, Japan 3 Department of Integrated Biosciences, Graduate School of Frontier Sciences, The University of Tokyo, Kashiwa, Japan The insect brain is the center of developmental control. In the brain of the silkworm Bombyx mori, two pairs of lateral neurosecretory cells (LNCs) produce the prothoracicotropic hormone (PTTH) [1,2]. The peptide PTTH stimulates the prothoracic glands to synthesize and release ecdysone [3]. The active form of ecdysone, 20-hydroxyecdysone (20E), controls many physiologi- cal and developmental processes of insect molting and metamorphosis [4]. Beside PTTH, the brain produces many neurosecretory hormones that orchestrate devel- opmental processes. It is also the target organ of 20E, effecting dynamic morphological changes and rear- rangement of the neural network during processes such as the formation of the optic lobe and mushroom body differentiation during metamorphosis [5,6]. Hence, the analysis of 20E-induced gene expression in the brain is of critical importance in understanding insect develop- ment. The 20E signal is mediated intrinsically through binding with its heterodimeric nuclear receptor consist- ing of the ecdysone receptor (EcR) and ultraspiracle (USP) [7,8]. The 20E–receptor complex directly induces several early genes and the products of these genes activate late effector genes that control stage- and tissue-specific developmental responses to 20E [9]. Two EcR isoforms, EcR-A and EcR-B1, and two USP iso- forms, usp-1 and usp-2, which have been identified in Bombyx [10–13], are involved in specific responses to Keywords Bombyx mori; brain; ecdysone receptor; metamorphosis; prothoracicotropic hormone-producing neurosecretory cell Correspondence M. Iwami, Division of Life Sciences, Graduate School of Natural Science and Technology, Kanazawa University, Kanazawa 920-1192, Japan Fax: +81 76 2646251 Tel: +81 76 2646255 E-mail: masafumi@kenroku.kanazawa-u.ac.jp (Received 18 May 2006, revised 22 June 2006, accepted 26 June 2006) doi:10.1111/j.1742-4658.2006.05398.x The steroid hormone 20-hydroxyecdysone (20E) initiates insect molting and metamorphosis through binding with a heterodimer of two nuclear recep- tors, the ecdysone receptor (EcR) and ultraspiracle (USP). Expression of the specific isoforms EcR-A and EcR-B1 governs steroid-induced responses in the developing cells of the silkworm Bombyx mori. Here, analysis of EcR-A and EcR-B1 expression during larval-pupal development showed that both genes were up-regulated by 20E in the B. mori brain. Whole- mount in situ hybridization and immunohistochemistry revealed that EcR-A and EcR-B1 mRNAs and proteins were exclusively located in two pairs of lateral neurosecretory cells in the larval brain known as the pro- thoracicotropic hormone (PTTH)- producing cells (PTPCs). In the pupal brain, EcR-A and EcR-B1 expression was detected in tritocerebral cells and optic lobe cells in addition to PTPCs. As PTTH controls ecdysone secre- tion by the prothoracic gland, these results indicate that 20E-responsive PTPCs are the master cells of insect metamorphosis. Abbreviations 20E, 20-hydroxyecdysone; EcR, ecdysone receptor; LNC, lateral neurosecretory cell; MEF2, myocyte enhancer factor 2; OLC, optic lobe cell; NaCl ⁄ P i ⁄ Tween, phosphate buffered saline containing Tween; PTPC, PTTH-producing neurosecretory cell; PTTH, prothoracicotropic hormone; TCC, tritocerebral cell; USP, ultraspiracle. FEBS Journal 273 (2006) 3861–3868 ª 2006 The Authors Journal compilation ª 2006 FEBS 3861 20E. EcR-A expression regulates neuronal maturation, while EcR-B1 expression controls neuronal regression both in Drosophila melanogaster [14,15] and Manduca sexta [16]. Mutational analysis of EcR isoforms in Drosophila identified several different lethal phases, including developmental arrest late in embryogenesis, failure of pupariation [17,18], and disruption of neur- onal remodeling [18]. Although the effect of ecdysone on neurons of the central nervous system has been extensively studied in Drosophila and Manduca, little information is available on the role of EcR expression in the brain. In the present study, we examined EcR-A and EcR-B1 expression in the Bombyx brain and demonstrate stage- and cell-specific expression of EcR isoforms, which is especially prominent in PTTH- producing cells (PTPCs). These results provide an insight into ecdysone receptor function in the brain during larval-pupal-adult development. Results Expression of EcR-A and EcR-B1 in the Bombyx brain Day-2 fifth instar B. mori larvae were injected with 1 lg 20E (+20E) or Ringer’s solution ()20E), and RNA was extracted from the brains 2 h after the injec- tions. RT-PCR revealed that both EcR-A and EcR-B1 were weakly expressed in the absence of 20E, and that 20E up-regulated the expression of both genes (Fig. 1A). EcR-A was shown to be expressed at a slightly higher level than EcR-B1 following induction by 20E. No transcript was amplified after 25 cycles of RT-PCR for both +20E and )20E samples. Expres- sion of the control housekeeping gene RpL32 showed no difference between +20E and )20E samples after 25 and 35 cycles of RT-PCR. To examine spatial expression of EcR isoforms, we carried out RT-PCR on RNA purified from the anterior and the posterior sections of B. mori brains (Fig. 1B). Intense EcR-A expression was observed exclusively in the anterior part while no expression was detected in the posterior part after 35 cycles of RT-PCR (Fig. 1C). Strong EcR-B1 expression was also detected in the anterior part of the brain and weaker expression was detected in the posterior section, indi- cating that both genes exhibit a spatial-specific pattern of expression. RpL32 expression did not differ between the two parts of the brain. Cell-specific expression of EcR isoforms as revealed by in situ hybridization The spatial specificity of EcR-A and EcR-B1 expres- sion was also determined by whole-mount in situ hybridization of larval and pupal brains using EcR iso- form-specific probes. EcR-A expression was detected exclusively in two pairs of lateral neurosecretory cells (LNCs) in day-2 (Fig. 2A) and day-7 (Fig. 2C) fifth instar larval brains. The location of the EcR-A-positive cells was assumed to be the same as that of PTTH- producing cells (PTPCs), as shown in Fig. 2E. To con- firm this, we performed in situ hybridization using a mixture of EcR-A and PTTH probes. The hybridiza- tion signal was again detected exclusively in the two pairs of LNCs (Fig. 2G), indicating that EcR-A and PTTH mRNAs were colocalized in PTPCs of the lar- val brain. EcR-B1 expression was also detected exclu- sively in the same LNCs in day-2 (Fig. 2B) and day-7 (Fig. 2D) fifth instar larval brains, while EcR-B1 and PTTH expression was shown to colocalize in PTPCs of the larval brain (Fig. 2H). In the pupal brain, EcR-A expression was observed in PTPCs (Fig. 2I,L) and in several tritocerebral cells (TCCs) (Fig. 2K) and optic lobe cells (OLCs) (Fig. 2J). EcR-B1 expression was similarly detected in PTPCs (Fig. 2F), TCCs, and OLCs (Fig. 3E) but at a lower level than EcR-A. In addition to the brain, faint EcR-A expression was detected in the subesophagal ganglion (Fig. 2K). Both EcR isoforms are therefore A B C Fig. 1. EcR-A and EcR-B1 are induced by 20E and are predominantly expressed in the anterior of day-2 fifth instar larval brains. (A) RT-PCR analysis of EcR-A and EcR-B1 expression following injection of 20E (+20E) or Ringer’s solution ()20E). The number of PCR cycles is indica- ted above the panel. The housekeeping gene RpL32 was used as a control. (B) Total RNA was extracted either from the anterior (A) or pos- terior (P) part of the brains and analyzed by RT-PCR to assess the spatial distribution of gene expression. (C) RT-PCR analysis of EcR expression using RNA from the anterior (A) and posterior (P) part of the brains. The number of PCR cycles is indicated above the panel. EcR expression in PTTH cells of Bombyx mori brain M. Hossain et al. 3862 FEBS Journal 273 (2006) 3861–3868 ª 2006 The Authors Journal compilation ª 2006 FEBS expressed exclusively in PTPCs at the larval stage and in PTPCs, TCCs, and OLCs in the pupal stage of B. mori development. Immunohistochemical confirmation of EcR-A and EcR-B1 cell-specific expression To confirm the in situ hybridization results, we performed double-labeled immunohistochemistry with anti-EcR-A and anti-EcR-B1 sera, and anti-PTTH IgG on the brains of day-2 and day-7 fifth instar larvae and day-2 pupae. EcR-A and PTTH expression was detected exclusively in two pairs of LNCs in day-2 fifth instar larvae (Fig. 3A, left). The merged image shows that EcR-A and PTTH expression colocalizes in PTPCs. This colocalization was also observed in day-7 larval and day-2 pupal brains (Fig. 3B,C, left). The same results were obtained for EcR-B1 (Fig. 3A–C, right), confirming that both isoforms are colocalized in PTPCs in the brain. In the pupal brain, EcR-A and EcR-B1 expression was observed in TCCs and weakly in OLCs in addi- tion to PTPCs (Fig. 3D,E, also Fig. 2I,J). It is note- worthy that EcR-B1 fluorescence was strongest just beneath the cell membrane of TCCs (Fig. 3F) and was weaker in the axons, while EcR-A fluorescence was more diffuse throughout the cytoplasm (Fig. 3D, also Fig. 3A), indicating that the distribution of the two isoforms differed slightly. EcR-A and EcR-B1 expression was also detected in the subesophagal gan- glion (Fig. 3D,E). Discussion In the present study, we have shown for the first time that EcR-A and EcR-B1 are up-regulated by 20E AB DC EF HG I J K L Fig. 2. Localization of EcR-A, EcR-B1 and PTTH mRNAs by whole-mount in situ hybridization. EcR-A and EcR-B1 mRNA was detected in day-2 (A,B) and day-7 (C,D) fifth instar larval brains and day-2 pupal brains (F,I). For fifth instar day-2 specimens, brains were dissected 2 h after 20E injection. PTTH mRNA was detected in day-2 fifth instar larval brains (E). (G) EcR-A and PTTH as well as (H) EcR-B1and PTTH probes were used simultaneously for hybridization of day-2 larval brains. (A–E,G,H) show the anterior portion of the larval brain. EcR-A expression in the pupal brain (I) is magnified in panels J–L, where OLCs (J, white arrows), TCCs (K, white arrows) and PTPCs (L, white arrows) are shown in green, blue, and black boxes, respectively. The blue arrows in K indicate faint signals in the sub- esophagal ganglion. Scale bar ¼ 100 lm. M. Hossain et al. EcR expression in PTTH cells of Bombyx mori brain FEBS Journal 273 (2006) 3861–3868 ª 2006 The Authors Journal compilation ª 2006 FEBS 3863 exclusively in PTPCs in the B. mori larval brain. The spatial specificity of EcR-A and EcR-B1 expression was determined by in situ hybridization and confirmed by immunohistochemistry using EcR isoform-specific antibodies. Expression of EcR-A and EcR-B1 in PTPCs has not been reported previously. Recently, Vafopoulou et al. [19] used immunohistochemistry to demonstrate EcR expression in the medial neurosecretory cells of Rhodn- ius prolixus. The postembryonic development of Rhodnius and Bombyx is strikingly different. Unfed Rhodnius larvae exist in an arrested developmental state caused by an ecdysteroid deficiency [20], which is immediately met by the consumption of a blood meal, thus initiating development. No such phenomenon exists in Bombyx larvae, and it is possible that the difference in 20E-responsive cells between the two insects reflects the different developmental systems. Our study indicates that the expression level of EcR- A is slightly higher than that of EcR-B1, as previously observed during the neuronal maturation of Drosophila and Manduca [16]. The developmental function of EcR-A is distinct from those mediated by the EcR-B1 and EcR-B2 isoforms in Drosophila.AnEcR-A mutant is arrested during early to mid-pupal development, indicating that EcR-A is required for the formation of the basic pupal body plan prior to the differentiation of most adult structures [15]. By contrast, an EcR-B1 mutant is lethal at the first and second larval molts [18] and fails to undergo pupariation [17]. PTPCs are the only cells to express EcR isoforms in the Bombyx larval brain, and these cells also express A B C D F E Fig. 3. Immunohistochemical localization of EcR-A, EcR-B1, and PTTH. (A) Day-2 larval, (B) day-7 larval and (C) day-2 pupal brains. For fifth instar day-2 specimens, brains were dissected 6 h after 20E injection. Anti-EcR-A, anti-EcR-B1, and anti-PTTH were used as primary antibodies. A FITC-conjugated anti- rabbit secondary antibody was used to detect EcR-A or EcR-B1, and a Texas-red conjugated anti-mouse secondary antibody was used to detect PTTH. The middle panel (A–C) shows a merged (yellow) image of the upper (green, EcR-A or EcR-B1) and lower (red, PTTH) panels. (D) EcR-A and (E) EcR-B1 expression in day-2 pupal brains. White arrows in (D) and (E) indicate the EcR-A- and EcR-B1-producing cells in the tritocerebral region of the pupal brain, respectively. Red arrows indicate the EcR-A- or EcR-B1-producing cells in the subesopha- gal ganglion, respectively. A magnified image of the red box in (E) is shown in (F), where EcR-B1 expression is indicated by white arrows. Scale bar ¼ 100 lm. EcR expression in PTTH cells of Bombyx mori brain M. Hossain et al. 3864 FEBS Journal 273 (2006) 3861–3868 ª 2006 The Authors Journal compilation ª 2006 FEBS usp isoforms (MDM Hossain & M Iwami, unpub- lished data). As the 20E signal is transduced via the EcR–USP complex, it can be concluded that PTPCs are the only cells to respond to 20E at the transcrip- tional level, and these cells therefore play important roles in 20E-dependent larval-pupal metamorphosis. PTTH controls the hemolymph 20E level by stimula- ting ecdysone synthesis and coordinating its release from the prothoracic glands. EcR-A and EcR-B1 expression in the PTPCs suggests that 20E modulates PTTH expression through a feedback loop. Beside its prothoracicotropic effect, PTTH may act as a growth factor as it shares a common ancestor with the verteb- rate growth factor superfamily peptides such as nerve growth factor, transforming growth factor, and plate- let-derived growth factor [21]. It also enhances the syn- thesis of several short-lived proteins that mediate a variety of extracellular signals [22,23]. Despite the first demonstration of PTTH almost three decades ago in LNCs in Manduca [24] and later in Bombyx [1,2], the molecular mechanisms of PTTH production and release are still at an early level of understanding, although these are of critical importance in insect developmental control [25]. Since the work of Truman [26], it has been believed that PTTH release is controlled by a circadian clock in the brain [27–29]. The close association between clock cells and PTPCs in the brain protocerebral region is seen in the three divergent genera Rhodnius, Dro- sophila, and Bombyx, suggesting that there are routes of communication between these two cell populations [30,31]. The association with clock cells and the responsiveness of PTPCs for ecdysteroidogenesis sug- gests that 20E influences PTTH release from PTPCs through neurons that provide input to clock cells [31,32]. The PTTH titer in Bombyx larval hemolymph is, however, not exclusively controlled by photoperiod and ⁄ or circadian clock mechanisms [27,28], as protho- racicostatic hormones have been shown to influence ecdysteroidogenesis and ecdysteroid release from the PTTH-stimulated prothoracic gland [33,34]. At the transcriptional level, Bombyx PTTH is regulated by trans-regulatory factors such as myocyte enhancer fac- tor 2 (MEF2) [32]. In the brain, MEF2 is expressed at an elevated level in PTPCs and corazonin-like immu- noreactive-LNCs. Over-expression of MEF2 increases PTTH expression in Bombyx brain [32], while up-regu- lation of MEF2 by 20E was reported in adult Dro- sophila myoblasts [35]. An emerging hypothesis is that MEF2 is regulated by 20E, which in turn modulates PTTH expression. In addition to PTPCs, EcR-A and EcR-B1 expres- sion was detected in the tritocerebrum and the optic lobe of day-2 pupae: a crucial stage for the morpholo- gical and neurological reorganization of the brain. 20E induces differentiation of the optic lobe and adult eye in Manduca [5,36], while EcR is expressed during the puparium stage in the optic lobe of Drosophila [16]. In the present study, EcR expression in B. mori OLCs is consistent with these findings. In the tritocerebrum, the number and position of EcR-A-producing cells differed from EcR-B1-producing cells. The intracellular local- ization of EcR-A was also distinct from that of EcR-B1: EcR-A was localized in the cytoplasm while EcR-B1 was beneath or close to the cell membrane of PTPCs and TCCs. The intracellular distribution of EcR-B1 could indicate a nongenomic role for 20E in these cells [37]. The present results clearly show that EcR isoform expression in the brain is exclusive to the PTPCs until pupation. This indicates that PTPCs are the master cells during larval-pupal metamorphosis and that they control ecdysteroidgenesis. At the pupal stage, the number of cells expressing EcR isoforms increases to include TCCs and OLCs. This unique expression pro- file indicates the importance of EcR isoform expression in TCCs and OLCs during larval-pupal metamorpho- sis, although the roles of the individual isoforms in these cells remain to be elucidated. Experimental procedures Animals and hormones B. mori eggs of a racial hybrid, Kinshu · Showa were obtained from Ueda Sanshu (Ueda, Japan), and larvae were reared on an artificial diet (Silkmate II, Nihon Nou- san Kogyo, Yokohama, Japan) under a 12 h light ⁄ 12 h dark photoperiod at 25 ± 1 °C [38]. Ages were counted in days, consisting of a photophase followed by a scotophase. Fifth instar day-2 and day-7 larvae and day-2 pupae were studied. 20E (Sigma, St. Louis, MO, USA) was dissolved in ethanol and its concentration was determined spectrophoto- metrically at 243 nm (e EtOH ¼ 12 300). An aliquot of the stock solution was evaporated and dissolved in insect Ring- er’s solution (130 mm NaCl, 4.7 mm KCl, 1.9 mm CaCl 2 ). RNA isolation and semiquantitative RT-PCR Total RNA was isolated from whole brains 2 h after the injection of 20E (1 lg per larva) (+20E) or insect Ringer’s solution ()20E) as a control. To determine the region of expression in the brain, total RNA was isolated from the anterior and posterior sections of brains. RNA was purified by the acid guanidinium-thiocyanate ⁄ phenol ⁄ chloroform method [39] with minor modifications [40]. M. Hossain et al. EcR expression in PTTH cells of Bombyx mori brain FEBS Journal 273 (2006) 3861–3868 ª 2006 The Authors Journal compilation ª 2006 FEBS 3865 One microgram total RNA was reverse-transcribed in a 20 lL reaction using 100 U ReverTra Ace (Toyobo, Osaka, Japan) and 5 pmol oligo(dT) 12)18 at 42 °C for 1 h accord- ing to the manufacturer’s instructions. After reverse tran- scription, the reaction was stopped by heating the solution at 99 °C for 5 min, and the cDNA solution was diluted five-fold with water. PCR primers were designed according to the nucleotide sequences: EcR-A 5¢-TGGAGCTGAAA CACGAGGTGGC-3¢ and 5¢-TCCCATTAGGGCTGTAC GGACC-3¢, EcR-B1 5¢-ATAACGGTGGCTTCCCGCTG CG-3¢ and 5¢-CGGTGTTGTGGGAGGCATTGGTA-3¢, and RpL32 5¢-GAGGACGAAGAGATTTATCAGGCA-3¢ and 5¢-CGAAGAGACACCATGAGCGAT-3¢. PCR was carried out in a 10 lL volume containing 10 mm Tris ⁄ HCl (pH 8.8), 50 mm KCl, 1.5 mm MgCl 2 , 0.08% (v ⁄ v) Noni- det P40, 0.2 mm each dNTPs, 0.5 mm each primer, 0.25 U Taq DNA polymerase (Fermentas, Burlington, Ontario, Canada), and 1 lL synthesized cDNA. The reaction was subjected to 25 or 35 cycles of amplification in a thermo- cycler (GeneAmp PCR System 9700, Applied Biosystems, Foster City, CA, USA) using a thermal cycle (94 °C, 30 s; 60 °C, 30 s; 72 °C, 30 s). PCR products were separated on a 1.5% (w ⁄ v) agarose gel and visualized by ethidium bro- mide staining. There was no amplification without reverse transcriptase even at 35 cycles of PCR (data not shown), indicating the specificity of EcR-A and EcR-B1 mRNA amplification. In situ hybridization Whole-mount in situ hybridization of brains was performed as described previously [41]. After dissecting, brains were washed with 10 mm phosphate buffered saline, pH 7.4 NaCl ⁄ P i ⁄ 0.05% Tween20 and fixed in 85% (v ⁄ v) ethanol, 4% (w ⁄ v) formaldehyde, and 5% (v ⁄ v) acetic acid on ice for 40 min. The brains were then incubated in a solution of NaCl ⁄ P i containing 15% (w ⁄ v) sucrose at 4 °C for 15–20 h. After washing with NaCl ⁄ Pi ⁄ 0.05% Tween20, the brains were treated with proteinase K (0.05 mgÆmL )1 in NaCl ⁄ Pi ⁄ 0.05% Tween20) at 37 °C for 40 min, and fixed again with 3% (w ⁄ v) paraformaldehyde in NaCl ⁄ P i at room tem- perature for 20 min. The brains were washed three times with NaCl ⁄ P i ⁄ 0.05% Tween20 and hybridized with 60 ng EcR-A probe (5¢-TCCCATTAGGGCTGTACGGACC-3¢) or EcR-B1 probe (5¢-CGGTGTTGTGGGAGGCATTG GTA-3¢) and ⁄ or PTTH probe (5¢-GTACACAAACACGCC ACGCTGACG-3¢)3¢-labeled with digoxigenin using a Dig-labeled kit (Roche Diagnostics, Mannheim, Germany). Hybridization was carried out at 37 °C for 20–48 h in a 100 lL hybridization solution [50% (v ⁄ v) formamide, 5· standard sodium citrate (NaCl ⁄ Cit; 0.15 m NaCl, 0.015 m sodium citrate), 5% (w ⁄ v) dextran sulphate]. After sequential washing with 50% (v ⁄ v) formamide, 5 · NaCl ⁄ Cit and NaCl ⁄ P i ⁄ 0.05% Tween20 at room temperature, the brains were treated with 5% (v ⁄ v) sheep serum (Rockland, Gilbertsville, PA, USA) at 4 °C for 15–20 h, followed by a 2 h incubation with a 1 : 500 diluted alkaline phosphatase conjugated anti-digoxigenin IgG (Roche Diagnostics) at room temperature. Color development was performed with 4-nitroblue-tetrazolium chloride (336.4 lgÆmL )1 ) and 5-bromo-4-chloro-3-indolyl phos- phate (175 lgÆmL )1 ) in the presence of 1 mm levamisole, a potent inhibitor of endogenous alkali phosphatases. After dehydration with ethanol, the brains were clarified with methyl salicylate and observed with a microscope (BX-50F, Olympus, Tokyo, Japan). Negative controls omitted the labeled probes, and no signal was detected (data not shown). Anti-peptide serum for Bombyx EcR isoforms An EcR-A specific peptide [H 2 N-GQVKAEPGVSHN GHP(15–29)C-COOH] [13] (Accession no D87118) and an EcR-B1 specific peptide [H 2 N-CPLPMPPTTPKSENES MSSG(94–112)-COOH] [12] (Accession no D43943) were synthesized, HPLC purified, and used to immunize rabbits (Sawady Technology, Tokyo, Japan). Handling of rabbits was performed according to regulation and guidelines of the local authority. The anti-peptide serum titers for EcR-A and EcR-B1, determined using an enzyme-linked immuno- sorbent assay, were 4700 and 119 700, respectively. Immunohistochemistry Double-labeled fluorescent immunohistochemistry was used to detect EcR-A, EcR-B1, and PTTH expression. Brains were washed with NaCl ⁄ P i , fixed in Bouin’s solution over- night, then washed with 70% (v ⁄ v) ethanol. The brains were then soaked in 0.1% (w ⁄ v) sodium deoxycholate and 2% (v ⁄ v) Tween20 in NaCl ⁄ P i at 4 °C for 4 days to facili- tate the penetration of antibodies through the brain sheath [2]. The pretreated brains were incubated with rabbit anti- Bombyx EcR-A serum (1 : 200) or anti-Bombyx EcR-B1 serum (1 : 300) and mouse anti-Bombyx PTTH monoclonal IgG (1 : 100) [2] overnight. The brains were then incubated simultaneously at 4 °C overnight with the secondary anti- bodies, mouse anti-rabbit IgG conjugated with fluorescein isothiocyanate (Cappel, Aurora, OH, USA) at a dilution of 1 : 400 for EcR-A and EcR-B1, and goat anti-mouse IgG conjugated with Texas-Red (Cappel) at a dilution of 1 : 400 for PTTH. The brains were washed with NaCl ⁄ P i ⁄ 0.05% Tween20 and clarified with methyl salicylate. Fluorescence was detected using a fluorescence microscope (BX-50F, Olympus) using NIBA filters for fluorescein isoth- iocyanate and WIY filters for Texas-Red. Negative controls replaced the primary antibodies with NaCl ⁄ P i ⁄ 0.05% Tween20 (data not shown). The absence of detectable flo- rescence in these controls demonstrated the specificity of the reaction. Images were processed with Adobe Photoshop (Adobe Systems Inc, San Jose, CA, USA). EcR expression in PTTH cells of Bombyx mori brain M. Hossain et al. 3866 FEBS Journal 273 (2006) 3861–3868 ª 2006 The Authors Journal compilation ª 2006 FEBS Acknowledgements We are grateful to Dr A. Mizoguchi for the gift of the anti-PTTH antibody. This work was supported by Grants-in-Aid for Scientific Research (15580039 and 18380040) from the Japan Society for the Promotion of Science. References 1 Kawakami A, Kataoka H, Oka T, Mizoguchi A, Kim- ura-Kawakami M, Adachi T, Iwami M, Nagasawa H, Suzuki A & Ishizaki H (1990) Molecular cloning of the Bombyx mori prothoracicotropic hormone. 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Insect Biochem Mol Biol 31, 321–331. 41 Iwami M, Tanaka A, Hano N & Sakurai S (1996) Bombyxin gene expression in tissues other than brain detected by reverse transcription-polymerase chain reac- tion (RT-PCR) and in situ hybridization. Experientia 52, 882–887. EcR expression in PTTH cells of Bombyx mori brain M. Hossain et al. 3868 FEBS Journal 273 (2006) 3861–3868 ª 2006 The Authors Journal compilation ª 2006 FEBS . EcR expression in the prothoracicotropic hormone- producing neurosecretory cells of the Bombyx mori brain An indication of the master cells of insect. available on the role of EcR expression in the brain. In the present study, we examined EcR- A and EcR- B1 expression in the Bombyx brain and demonstrate stage-