Proc. Natl. Acad. Sci. USA Vol. 92, pp. 5441-5445, June 1995 Developmental Biology Drosophila 5-HT2 serotonin receptor: Coexpression with fushi-tarazu during segmentation (in situ hybridization/G protein-coupled receptors/pair-rule gene) JEAN-FRAN;OIS CoLAs*, JEAN-MARIE LAUNAYt, ODILE KELLERMANNt, PHILIPPE ROSAY*, AND Luc MAROTEAUX*§ *Institut de Genetique et de Biologie Mol6culaire et Cellulaire, Centre National de la Recherche Scientifique, Institut National de la Sante et de la Recherche Medicale, Universite de Strasbourg, BP 163, 67404 Illkirch Cedex, France; tH6pital Lariboisiere, Service de Biochimie, 2 rue Ambroise Pare, 75475 Paris Cedex 10, France; and tDepartement de Biologie Moleculaire, Institut Pasteur, 25 rue du Dr Roux, 75724 Paris Cedex 15, France Communicated by Walter J. Gehring, University ofBasel, Basel, Switzerland, March 6, 1995 (received for review December 5, 1994) ABSTRACT Serotonin, first described as a neurotrans- ergic neurons (6), and to incomplete sclerotization of the mitter in invertebrates, has been investigated mostly for its cuticule (3). functions in the mature central nervous system of higher We report the characterization of a Drosophila serotonin vertebrates. Serotonin receptor diversity has been described receptor that displays a typical 5-HT2 receptor sequence, gene in the mammalian brain and in insects. We report the isolation organization, and pharmacology.5 It is expressed in the central of a cDNA coding for a Drosophila melanogaster serotonin nervous system (CNS) during larval and adult stages. More receptor that displays a sequence, a gene organization, and surprisingly, this receptor is expressed at the blastoderm stage pharmacological properties typical of the mammalian 5-HT2 of embryogenesis in a pattern similar to that of the pair-rule serotonin receptor subtype. Its mRNA can be detected in the gene fushi-tarazu (ftz). adult fly; moreover, a high level of expression occurs at 3 hr ofDrosophila embryogenesis. This early embryonic expression is surprisingly organized in a seven-stripe pattern that ap- MATERIALS AND METHODS pears at the cellular blastoderm stage. In addition, this pat- Fly Stocks. From the stocks described in ref. 7, we used the tern is in phase with that of the even-parasegment-expressed following, hypomorph or null, alleles: hairy (h), [25]5H07; runt pair-rule gene fushi-tarazu and is similarly modified by (run), [28]YE96; even-skipped (eve), [3]R13; fushi-tarazu (ftz), mutations affecting segmentation genes. Simultaneously with [13]9H34; Kruppel (Kr), [2]; hunchback (hb), Df(3R)p25; this pair-rule expression, the complete machinery ofserotonin knirps (kni), [11]IIE72; giant (gt), YA82; tailless (tll), [1]L10; synthesis is present and leads to a peak of ligand concomitant engrailed (en), [54]IIB86; odd-skipped (odd), [5]IIID36; and with a peak of 5-HT2-specific receptor sites in blastoderm snail (sna), IIGO5. embryos. Drugs and Chemicals. Ketanserin, ritanserin, and setoper- one were kindly provided by Janssen, and ICS 205-930 and In vertebrates, the biogenic amine serotonin (5-hydroxy- MDL 72 222 by Sandoz Pharmaceutical and Merell-Dow tryptamine, 5-HT) affects a wide variety of behavioral and (Strasbourg, France), respectively. Other neurochemicals were physiological functions that are mediated by numerous recep- from Research Biochemicals (Natick, MA) or Sigma. (±)-1- tor subtypes. These receptors can be classified according to the (2,5-Dimethoxy-4-[125I]iodophenyl)-2-aminopropane ([125I]- transduction mechanisms: the 5-HT1 subtype as an adenylyl DOI) (2200 Ci/mmol; 1 Ci = 37 GBq) was from New England cyclase inhibitor, 5-HT2 as a phospholipase C stimulator, and Nuclear. 5-HT4, 5-HT6, and 5-HT7 as adenylyl cyclase activators; these PCR Experiments. For receptor cloning, degenerate oligo- are all G protein-coupled receptors. The 5-HT3 subtype is a nucleotides coding for conserved sequences oftransmembrane ligand-gated channel (1). domains VI and VII and were used for PCR In insects, biogenic amines are similarly implicated as neu- (a) (b c) amp- romodulators and neurotransmitters (2) in addition to their lification as described (8): a, 5'-TACCTCGAGGTCGACGGTI- function of crosslinking proteins and chitin during sclerotiza- ATGTGGTGYCCITTYTTYAT-3'; b, 5'-AGAACTAGTGG- tion of the cuticle (3). In Drosophila melanogaster, the sero- TACCCRTIGTRTADATIAYIGGRTT-3'; and c, 5'-AGAAC- toninergic neurons have been localized (4) and implicated in TAGTGGTACCCSWRCAIACRTAICCDATCCA-3'. salivary gland secretion, heart and oviduct contractions, cir- In Situ Hybridization. Digoxigenin-labeled NP81 cDNA cadian rhythms, and learning and memory (2). 5-HT receptors (see Results) was synthesized and was detected with alkaline have also been detected as a heterogeneous population by phosphatase, using 5-bromo-4-chloro-3-indolyl phosphate as [3H]5-HT binding experiments or by second-messenger cou- substrate, on whole-mount embryos (8). For double-staining pling to adenylyl cyclase or to phospholipase C (2). So far, only experiments, after in situ hybridization, embryos were washed receptors coupled to adenylyl cyclase and close to the mam- and incubated with a mouse anti-en monoclonal antibody malian 5-HT1 receptor subfamily have been described in (provided by M. Haenlin, Institut de Genetique et de Biologie Drosophila (5). Moleculaire et Cellulaire) or with a rabbit anti-ftz antiserum The pathway leading to 5-HT uses tryptophan as a substrate, (1:1000; provided by W. J. Gehring, University of Basel, which is converted to 5-hydroxytryptophan by tryptophan Basel). The signal was detected by a peroxidase-coupled rabbit 5-hydroxylase (TPH; EC 1.14.16.4, the rate-limiting enzyme in anti-mouse or donkey anti-rabbit secondary antibody (1:350; 5-HT synthesis). 5-Hydroxytryptophan is converted to 5-HT by Cappel) with diaminobenzidine as substrate. dopa decarboxylase (EC 4.1.1.28). Depletion of 5-HT and dopamine in Drosophila mutants lacking dopa decarboxylase Abbreviations: AEL, after egg laying; CNS, central nervous system; leads to learning deficits, to an aberrant pattern of serotonin- DOI, (±)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane; 5-HT, 5-hydroxytryptamine (serotonin); TPH, tryptophan 5-hydroxylase. §To whom reprint requests should be addressed. The publication costs of this article were defrayed in part by page charge TThe sequences reported in this paper have been deposited in the payment. This article must therefore be hereby marked "advertisement" in GenBank data base (accession nos. X81835 and X85407 for the accordance with 18 U.S.C. §1734 solely to indicate this fact. cDNA and genomic sequences, respectively). 5441 Downloaded by guest on September 29, 2021 5442 Developmental Biology: Colas et at Proc. NatL Acad Sck USA 92 (1995) DNQ8Y8QRG PRLZZAD WOPRQPFXD CZG8NI!B YYPSELPTSX 80 FIG. 1. Deduced amino acid se- ULIXZGIITF QCARQZDXM ZIFY8CD0 ZPLB8TL RLKARLLRP YZAIXIRLA IXNA8LSDA Da.BDSWSCD 160 quence of the NP81 receptor. Roman numerals above boxes identify the trans- ZGUIVLQCKL GQANZLLLCN LQQLURZPLZ WG8AZ3BFBPD8LLQA?QZ ZLGA W 87 I P ZII240 membrane domains. The intron/exon VI!SLDWL a TABTADUAZ ?AVTK8TLLIDY UP F TVFXAG LGAULVCLLV A Nw 320 boundary locations are indicated by the XII 12 inI exon numbers (Arabic numerals above IG 0 IVD3VLFI V4 the sequence). Putative N-glycosylation LTOIRIAIW VMAIPVs8BsI TVi 8=. IKpzpNICV I LVATIpIUm mvTTJLTxP L*x A 480 sites are circled and the consensus phos- ZI1P tRi LGJRQ HBO NOZGQ GVZPL tI!A 560 phorylation sites by protein kinase A or 4 5 C are circled and labeled with left infe- S tRNVNG'P AGS0RR!OT A 8O8QFL OXILRIIIP AUM i RIERWIO GP 640 rior "A" or right inferior "C," respec- INLELBLVPP P YPTNVOOUG RTMTQZVGL OGGE T NELGO!L MI ATIQOPI 720 tively. The hydrophobic region near the RVXTQIPQLG 56 N terminus and the serine/threonine- region in the third intracellular loop UIQ8QAAGI0 XVXPTA LEZPQZE VARPWAFRVATPTh LNN4&3f--- 8AMVA!ZQK AN 800 rich FWVLCWSPI ILEZIF ZCQVP CLWLGTVRS TN LLKNVKR* 869 are underlined. Binding Assay. [15I]DOI (9) was used as the radioligand on (Fig. 1). The genomic sequence of the NP81 gene, investigated membrane fractions isolated from embryos or from NP81- by screening a Drosophila genomic library in A phage EMBL4, transfected COS-1 cells as described (10). spans 10 kb and consists of six exons interspersed with five 5-HT Dosage. Extracts from staged embryos were used for introns. binding experiments or for 5-HT quantification by enzymatic- In situ hybridization on salivary gland polytene chromo- isotopic microassay using partially purified N-acetyltransferase somes (data not shown) located the NP81 receptor gene on the (11) or for determination of TPH activity by a radioenzymatic right arm of the third chromosome in the region 82C-82F assay (12). The resulting global concentration was deduced uncovered by the deficiency Df(3R)110, which contains the from the calculated maximum number of binding sites (Bmax) pair-rule gene odd-paired (opa) located in 82E (14). However, by assuming the Drosophila embryo volume as 15 nl and a the NP81 gene is still present in embryos homozygous for the uniform distribution over the embryo. RESULTS NP81 cDNA Encodes a 5-HT2-Like Receptor. By PCR amplification, we cloned aDrosophila genomic DNA fragment, NP81, that we used to screen a library of Drosophila head cDNA. The complete cDNA sequence (3889 bp) encodes a A C protein of 868 aa (Fig. 1) which displays eight hydrophobic regions, seven of which can be identified as transmembrane domains (Fig. 1). The NP81 protein shares homologies with the G protein-coupled receptor family, including (i) potential N-glycosylation sites in its amino terminus, (ii) consensus sequences for phosphorylation by different protein kinases in B D the cytoplasmic regions, and (iii) an identical location for conserved amino acids within the transmembrane regions (13) I NP81 RP49 FIG. 3. NP81 mRNA is expressed in seven stripes. Wild-type embryos were probed by in situ hybridization with digoxigenin-labeled NP81 cDNA. Embryos are shown anterior to the left, ventral side for the Oh 6h 13 Ad down. (A-D) Reaction products are seen as blue/violet staining I 112 ihl Ipupl I NP81 mRNA.