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The Dh gene of Drosophila melanogaster encodes a diuretic peptide that acts through cyclic AMP

Pablo Cabrero¹, Jonathan C. Radford¹, Kate E. Broderick¹, Laurence Costes², Jan A. Veenstra², Eric P. Spana³, Shireen A. Davies¹ and Julian A. T. Dow^1,*

¹ IBLS Division of Molecular Genetics, University of Glasgow, Glasgow G11 6NU, UK
² Laboratoire de Neuroendocrinologie des Insectes, Université Bordeaux I, Avenue des Facultés, 33405 Talence Cedex, France
³ Syngenta Biotechnology, Inc., Research Triangle Park, NC 27709, USA

View larger version (54K): [in a new window]   Fig. 1. The Drosophila melanogaster CRF-like gene. (A) cDNA and deduced peptide sequence of Dh. Putative dibasic convertase cleavage sites are underlined. The C-terminal amidation signal glycine residue is in italic. The positions of introns in the genomic sequence are marked with ><. The predicted peptide is indicated in blue. The encoded peptide has a signal peptide, with a predicted cleavage site between amino acid residues 24 and 25, marked with ^, as predicted by PSORTII Prediction program (http://psort.nibb.ac.jp/form2.html). The polyadenylation signal is indicated in yellow. Differences between the genomic sequence and the cDNA are marked in red. (B) Genomic context of Dh. View of region 85E of chromosome 3R. The neighbours of Dh are, on the negative strand, Scm, an RNA polymerase II transcription factor implicated in gene silencing, and CG9492, a dynein ATPase implicated in microtubule-based movement. Within an intron of Dh, on the opposite strand, is frost (fst), a gene implicated in response to cold. Dh (old) denotes the original, incomplete, Gadfly annotation of the Dh gene.

View larger version (17K): [in a new window]   Fig. 2. Alignment of insect corticotropin-releasing factor-related peptides. The sequences producing significant alignments against Drome-DH44 peptide after a protein Blast search are shown below in red. Mac Vector clustal analysis was performed to align the sequences. Residues highlighted in yellow have 50% identity. Swissprot accession numbers for each peptide are as follows: Musca domestica (house fly), DIUH_MUSDO; Periplaneta americana (American cockroach), DIUH_PERAM; Zootermopsis nevadensis (termite), DIUH_ZOONE; Diploptera punctata (Pacific beetle cockroach) class I, DIUH_DIPPU; Locusta migratoria (migratory locust), DIUH_LOCMI; Tenebrio molitor (yellow mealworn), DIU2_TENMO; Acheta domesticus (house cricket), DIUH_ACHDO; Manduca sexta (tobacco hornworm) precursor 1, DIU1_MANSE; DromeDH, dh gene product (present study); Culex salinarus, CCRF-DP.

View larger version (15K): [in a new window]   Fig. 3. Drome DH44 increases fluid secretion rates. (A) Typical experiment, showing DromeDH44-stimulated fluid secretion at 10-7 moll-1. The peptide was added at 30 min (arrow), and secretion rates measured as described in Materials and methods. Values are mean fluid secretion rates (nl min-1) ± S.E.M. (N=6-9). Rates of DromeDH44-stimulated secretion at 40-60 min differ significantly from control (asterisks; P<0.05). (B) Dose—response plot for Drome DH44 stimulation of fluid secretion. Stimulated secretion rates were obtained from the typical assays shown in A. To normalise and compare data between different experiments, the results are expressed as the percentage increase of basal rate on peptide stimulation (maximal stimulated rate—mean basal rate)/basal ratex100) ± S.E.M. (N=7-9). Stimulation is significant (P<0.05) at concentrations above 10-8 moll-1.

View larger version (28K): [in a new window]   Fig. 4. Effect of Drome-DH44 on cyclic nucleotide levels. (A) Drome-DH44 elevates intracellular cAMP levels. Intracellular cAMP levels in wild-type (Oregon R) tubules were measured under control and Drome-DH44 stimulated conditions by radioimmunoassay. The results are expressed as percentage increase of basal rate on peptide stimulation (stimulated rate—mean basal rate)x100/mean basal rate). Values are means ± S.E.M. (N=5-9;): stimulation of cAMP is significant (P<0.05) for concentrations above 10-8 moll-1. The mean basal cAMP level was 383±43 fmol/20 tubules. (B) cGMP levels are not affected by Drome-DH44. Intracellular levels of cyclic GMP did not show a significant difference between control and Drome-DH44 (10-7 moll-1) stimulated conditions by RIA. The results are expressed as means ± S.E.M., N=4 independent samples, each pooled from 20 tubules. (C,D) Drome-DH44-induced increase in cAMP is confined to principal cells. Immunocytochemistry of whole-mount tubules in control cells (C) and 10 min after application of 10-7 moll-1 Drome-DH44 (D). Images were recorded under identical filter and exposure settings, using a matched-pair protocol. A typical stellate cell is outlined in each photograph.

View larger version (16K): [in a new window]   Fig. 5. Drome-DH44 elevates cAMP-, but not cGMP-, phosphodiesterase. Values are means ± S.E.M., N=4 determinations. Where error bars are not shown, they are too small to be visible. *, P<0.05.

View larger version (10K): [in a new window]   Fig. 6. Drome-DH44 has no effect on intracellular calcium levels. [Ca2+]i levels in real time were measured after stimulation with mock-injection (Control), CAP2b, DLK and Drome-DH44 at 10-7 mol l-1. DLK and CAP2b are known to stimulate [Ca2+]i levels in stellate and principal cells of the main segment, respectively, and were used as a positive control. To enable comparison between experiments, [Ca2+]i values are expressed as mean percentage increases ± S.E.M. (N=4-8). Significant differences, relative to controls, are marked with an asterisk (P<0.05).

View larger version (53K): [in a new window]   Fig. 7. CRF is expressed within a small set of neuroendocrine cells in the CNS. (a) Immunocytochemical localization of Drome-DH44 in the larval nervous system. There are three cells (arrows) in each cerebral lobe with axons running to the corpus cardiacum (CC). (b) In the adult these six neuroendocrine cells are located in the pars intermedia. (c) In situ hybridization with antisense RNA identifies the same cells in the larval nervous system.

View larger version (38K): [in a new window]   Fig. 8. Cells expressing Dh also express the leucokinin receptor. Larval brain, double stained for LKR, the Drosophila leucokinin receptor (Radford et al., 2002) and DromeDH44. (a) Immunostaining for LKR (green), showing widespread expression; (b) immunostaining for DromeDH44 (red), showing expression in cell triplets, as in Fig. 7 (not all visible in this plane of focus), and in their axons in the retrocerebral complex of the corpus cardiacum (CC); (c) merged image showing colocalisation.