The gene encoding capaR, CG14575, is highly expressed in Drosophila tubules; microarray analysis of CG14575 gene expression in the tubule versus whole fly [14] demonstrates that CG14575 is almost uniquely expressed in both of the adult and larval tubule. Given the importance of the tubule as a key tissue for homeostasis, it is possible that capaR has a significant role in organismal survival. A capaR promoter-specific GAL4 line allows expression mapping of endogenous capaR to tubule principal cells. Targeted expression of capaR RNAi in these cells decreases [Ca2+]I under stimulation of capa-1 and abolishes capa-1 induced fluid transport. Finally, targeting of capaR RNAi to only tubule principal cells increases organismal survival to desiccation or water stress, demonstrating that capaR signaling in the tubule impacts on fluid homeostasis and on organismal survival. Materials and Methods Drosophila stocks and generation of transformants All lines were maintained on a standard Drosophila diet at 22uC, 55% humidity on a 12:12 h light:dark photoperiod. Wild-type flies were obtained from a Canton-S (CS) stock. In order to drive cell- and tissue-specific gene expression of gene(s) of choice in vivo, the GAL4/ UAS system was used, in which cell- or tissue-specific GAL4 ‘drivers’ enable binary expression of genes cloned downstream of the GAL4-binding Upstream Activating Sequence [15]. Thus, for intact tubules, principal cell-specific expression can be driven using either c42-GAL4 [16] or Urate-Oxidase-GAL4 [17] drivers in an otherwise normal fly. To assess in vivo calcium signals, doubly homozygous c42-GAL4.UAS-apoaequorincyto (c42aeq) flies were used, which specifically express the apoaequorin luminescent calcium reporter in the cytosol of the principal cells of the tubule main segment (upon which the diuretic neuropeptide capa-1 acts) [18]. The ubiquitous actin-GAL4 and the UAS-GFP lines were obtained from the Bloomington Stock Center (Bloomington, IN). To assess the impact of capaR and capaR RNAi on calcium signaling in vivo, lines were crossed to the doubly homozygous c42aeq flies. The ORF of the capaR (CG14575) was amplified from whole fly cDNA as template using the primers 59-CGCG- GCCGCATGAATTCATCGACCG-39 and 59-GCGGTACCT- TAAATACAAGTCTC-39 and cloned into the pUAST vector. To generate construct for heritable RNA interference (RNAi) of the capaR gene, an inverted repeat of a 615 base pair fragment was generated by PCR using the primers 59-GCACTCTAGAA- CAAGGCAGTTTTGATAAC-39 and 59-GCACTCTAGAGTT- CGAGATCGAATCTTGGC-3, and cloned as a tail-tail inverted repeat flanking the white intron into the P-element vector pWIZ [19]. Validation of the capaR RNAi line was confirmed by quantitative Q-RT-PCR using the primers 59-GCTCTCCT- TTGTGCGGGGGCACAT-39 and 59-GCACGTCAGAGCC- AGCCAGCATCC-39 (Fig. S1). To generate the capaR-GAL4 driver, the putative promotor sequence of the capaR gene was amplified by PCR using wild-type genomic DNA as a template with the primers 59-CAGTCGACACCGGCAACCAC-39and 59-TT- TAGCCCAGAGCTGAATGT-39. The resulting amplicon (corre- sponding to bases 21 to 21501 from the transcriptional start site of the capaR coding region) was digested with KpnI and subcloned into pinGAL4 vector (gift of Dr. Jean-Christophe Billeter), which had previously digested with KpnI and CIP-treated. The ORF of the human neuromedin U receptor 2 (also referred to as FM4) was amplified from full-length cDNA clone (MHS1010-98075312, Thermo scientific) using specific primers: 59-CACCATGTCAGG- GATGGAAAAACTTC-39 and 59-TCAGGTTTTGTTAAAGT- GGAAGCTTTG-39. The ORF of the NMUR 2 gene was cloned into the pUAST vector using the Gateway system. The entry and Capa GPCR and NMUR in Fluid Homeostasis destination vectors used were obtained from the Drosophila Gateway Vector collection (Invitrogen). All Transgenic lines were generated using standard methods for P-element-mediated germline transfor- mation (BestGene Inc, USA). Plasmid construction for expression in Drosophila S2 cells The ORF of capaR was amplified from whole fly cDNA as template using the primers 59-GCGGTACCATGAATTCATC- GACCG-39 and 59-GCGGTACCTTAAATACAAGTCTC-39 and cloned into pMT/V5-His TOPO vector (Invitrogen). The eYFP ORF was fused in-frame at the C-terminus of the capaR using KpnI and ApaI sites and the tagged construct was cloned into the pMT/V5-His TOPO vector. The construction of capaR-Renilla luciferase was realized by subcloning the full-length cDNA encoding Renilla luciferase (Rluc; 312 amino-acid) into a capaR-pMT/V5-His TOPO vector. The b-arrestin-2-eYFP pCDNA3 construct [20] was digested with KpnI and ApaI and subcloned into the pMT/V5-His TOPO vector. The ORF of the CG8795 was amplified from fly cDNA using the primers 59-ATGGCAGTCAAAATGCTGCCC-39 and 59-AAGGCGGCCCGCTCTTCA-39 and was cloned into pMT/ V5-His TOPO vector for expression in S2 cells. The ORF of the NMUR 2 gene was cloned into pMT/V5-His TOPO vector for expression in S2 cells. Peptide synthesis, peptide antibody production, immunofluoescence The capa peptides Drm-capa-1 (GANMGLYAFPRVamide), Drm- capa-2 (ASGLVAFPRVamide), Drm-PK-1 (TGPSASSGLWFGPR- Lamide), Drm-PK-2 (SVPFKPRLamide) and hugin gamma (hugg, pQLQSNGEPAYRVRTPRL-amide) [3] were synthesised as C-terminally amidated peptides (Biomatik Corporation, Canada). Peptides were dissolved in distilled ACN/H2O to a concentration of 1 mM and then diluted to the required working concentration in Schneider’s medium (Invitrogen Inc.). Human neuromedin U-25 was purchased from Sigma. Rabbit polyclonal antibody to porcine neuromedinU-8 was purchased from Progen biotechnik (Heidelberg, Germany). Rabbit capa precursor peptide used was described in [21] Rabbit anti-peptide antibody was raised against the capaR epitope (CQQGTNNRETRNSQM) by Genosphere Biotechnologies (Paris, France) and purified on a HiTrap NHS-activated HP column (Amersham Pharmacia Bio- tech; Buckinghamshire, UK) according to the manufacturer’s instructions. Immunohistochemistry was carried out as described previously [21]. Mouse anti-GFP primary antibody (Zymed) and the antiserum to neuromedinU-8 were all diluted 1:1000 or, in the case of the pre-immune serum, the affinity-purified capaR antibody and the antiserum to the capa precursor peptide, 1:500. Incubations in the primary antibodies were performed overnight. A FITC-conjugated affinity-purified goat anti-mouse antibody (Jackson Immunologicals) was used in a dilution of 1:1,000 for visualization of the mouse monoclonal anti-GFP. A Texas red-conjugated affinity-purified goat anti-rabbit antibody (Jackson Immunologicals) was used at a dilution of 1:1,000 for visualization of the rabbit capaR antiserum. For double labelling, larval brains were incubated with the antiserum to neuromedinU-8, which was visualised using a fluorescein-labeled F(ab) fragment of goat anti-rabbit IgG (Jackson Immunologicals), and subsequently with tetrarhodamine-labeled purified rabbit anti-capa precursor peptide serum [18]. For tubule immunohistochemistry, the nuclear stain DAPI (1 mg ml21 for 1 min, Sigma) was used. The samples were cleared in a glycerol series (20%, 50%, and 80% glycerol/0.04 M Sodium Carbonate PLoS ONE | www.plosone.org 2 January 2012 | Volume 7 | Issue 1 | e29897