Defective proboscis extension response (DPR), a member of the Ig superfamily required for the gustatory response to salt

Abstract

Gustatory stimuli, such as sugar, induce a behavioral response in Drosophila that involves extension of the proboscis and consumption of the sugar-containing solution. Addition of salt to the sugar solution inhibits this behavioral response. However, the mechanisms and gene products involved in the salt aversion response have not been described. Here, we report the identification of a locus, defective proboscis extension response (dpr), that is required for salt aversion. dpr was expressed in a subset of primary neurons in the gustatory organs and encoded a protein with two Ig-like domains, a single putative transmembrane domain, and a short region C terminal to the transmembrane segment. In addition, DPR defines a large previously unknown group of > or =20 highly related Ig-containing proteins.

Fig. 1.

Proboscis extension response assay.A, The fraction of flies extending their proboscis in response to presentation of a solution containing different concentrations of sucrose. The wild-type (Canton S) anddpr¹ responses are indicated by thesquares and circles, respectively. The SEMs, indicated by the error bars, were based on samples of 29–32 flies. B, The fraction of flies exhibiting a PER during exposure to 0.1 m sucrose and varying concentrations of NaCl. Mean results were compiled from analyses of 29–32 flies. C, Proportion of flies extending their proboscis in response to either 0.1 m sucrose (black columns) or 0.1 m sucrose in combination with 1 m NaCl (gray columns). The following stocks were analyzed: wild type (Canton S), dpr¹ homozygotes (dpr¹), dpr^{1 in trans} with a deficiency, Df(2R)AA21, which uncoversdpr¹(dpr¹/Df), and an excision allele ofdpr¹(dpr^1ex128). Twenty to 32 flies were analyzed for each stock.

Fig. 2.

Expression of the dpr lacZreporter. A–D and H–M show staining indpr¹/+ flies. E–G show the results of lacZ staining inpoxn^70–28, dpr¹/poxn^70–28flies. A, Anterior wing margin. Cells that displaylacZ activity are indicated with white arrowheads. The positive cells are situated at the bases of both dorsal (red arrows) and ventral (green arrows) chemosensory bristles.B, Dorsal and medial rows of wing sensory bristles. Chemosensory bristles (red arrows) and mechanosensory bristles (blue arrowheads) are indicated.C, Ventral view of chemosensory sensilla with recurved bristles (indicated by arrow). The recurved bristles are separated by several mechanosensory bristles (blue arrowheads). D, Tip of the labelum showing strong lacZ staining. E, NolacZ staining in the anterior wing margin ofpoxn^70–28, dpr¹/poxn^70–28flies. The transformed bristles are indicated by the red arrows. F, Magnification of the transformed bristles (red arrow) inpoxn^70–28, dpr¹/poxn^70–28flies stained for lacZ activity. G, Tip of the labelum did not show staining inpoxn^70–28, dpr¹/poxn^70–28flies. H, Horizontal section of an adult fly head stained for lacZ activity. br, Brain;la, lamina; me, medulla;re, retina. I, Proximal region of the optic lobe and retina stained with anti-β-galactosidase (green) and anti-ELAV (red) antibodies. In the lamina cell layer, there were typically twodpr-positive cells in a single laminal cartridge (indicated by arrows). In the retina, staining was restricted to the R8 photoreceptor cells (indicated with thesmall arrow). la-c, Lamina cell body region; la-n, lamina neuropil region;me-c, medulla cell body region; me-n, medulla neuropil region; R8, nucleus of an R8 cell.J, Optical horizontal section of the ventral region of the adult thoracic ganglion stained with anti-β-galactosidase (green) and anti-ELAV (red) antibodies showing the first (I), second (II), and third (III) segments. The box indicates a region shown at higher magnification in K–M. K, Anti-ELAV. L, Anti-β-galactosidase.M, Merged images of the anti-ELAV and anti-β-galactosidase staining. Indicated with arrowsare examples of cells showing very intense anti-β-galactosidase staining. Scale bars: A, D,J, 50 μm; H, 100 μm;I, 25 μm; K, 10 μm.

Fig. 3.

Distribution and morphology ofdpr-expressing cells examined using GFP. The GFP was expressed using the GAL4/UAS system and examined by confocal microscopy. A–H, GFP expression indpr⁺/dpr heterozygous flies (dpr-GAL4, UAS-dpr/+). I,J, GFP expression in dpr homozygous flies (dpr-GAL4, UAS-dpr/dpr^1RH). Tissues are oriented with the distal side to the right, except in G and H. Scale bar:A–J, 50 μm. A, Anterior wing margin. Two pairs of cells showing strong GFP staining (indicated bysmall arrows). The arrowheads indicate dendrites extending from the cell bodies to the base of chemosensory bristles. B, Merge of GFP (shown in A) with the Nomarski image. The large arrows indicate the bases of chemosensory bristles. No GFP expression was evident at the bases of the mechanosensory bristles. C, Distal region of the anterior wing margin. GFP staining was observed in pairs of cells (small arrows) and in hair shafts (large arrowheads); the spatial relationship of neuronal cell bodies and their corresponding hair shafts is evident. D, Merge of GFP (shown in C) with the Nomarski image. Thelarge arrows indicate GFP staining in hair shafts.E, Ventral view of the third tarsal leg segment. GFP-positive clusters each contain two neurons (arrows).F, Merge of GFP (shown in E) with the Nomarski image. Each GFP-positive pair of neurons was near the base of a recurved bristle, indicated by a large arrow. G, GFP-positive neurons in the proboscis occur singly or in groups of two (indicated by small arrowheads).H, Merge of GFP (shown in G) with Nomarski image. Chemosensory bristles (arrows) are innervated by GFP-positive neurons. I, Anterior wing margin in a dpr homozygote. J, Distal region of anterior wing margin in a dprhomozygote.

Fig. 4.

Molecular analysis of the dpr gene.A, Genomic region flanking the P-element insertion site. The positions of EcoRI (E) sites are shown. The P-element is represented by the large inverted triangle. The site of insertion of the P-element is indicated by the vertical line connecting the inverted triangle to the genomic DNA. The orientation of the 5′ and 3′ ends of the P-element is indicated. The dpr exons, deduced by comparison of the cDNA and genomic sequences, are indicated by the black boxes below the genomic map.B, Expression of dpr RNA in wild type anddpr¹. Ten micrograms of wild-type (w.t.) and dpr¹polyadenylated RNA were fractionated on 3% formaldehyde–0.8% agarose gels, transferred to membranes, and probed with the c-6705–6 cDNA labeled with ³²P. Filters were reprobed withrp49 to ascertain whether the RNAs were comparably loaded in each lane. C, Developmental expression ofdpr mRNA. Polyadenylated RNA was prepared after collecting embryos for 4 hr from the Canton S (wild-type) strain and incubating at 25°C for either 0–16 hr (0–20 hr embryos) or for 1, 2, 4, 6, 8, and 9–10 d (adults). The 1, 2, and 4 d collections coincided approximately with the first, second, and third instar larval stages, and the 6 and 8 d collections corresponded approximately with the early and late pupal stages. Lanes were loaded as follows:lane 1, embryo RNA; lanes 2–6, contained RNA from samples prepared after 1, 2, 4, 6, and 8 d of development, respectively; lane 7, adult RNA. The size of the 3.8 kb mRNA was estimated based on the migration of RNA size markers. D, Hydrophobicity analysis of DPR (Kyte and Doolittle, 1982). The putative signal sequence (SS) (Nielsen et al., 1997) and the transmembrane domain (TMD) (Kyte and Doolittle, 1982) are indicated.E, Schematic of the domain organization of DPR.SS, N-terminal signal sequence; Ig-I andIg-II, the two Ig domains; TMD, transmembrane domain.

Fig. 5.

Expression of the DPR protein. A, Western blot of the DPR protein. Extracts from wild type,dpr¹, anddpr^1e5 were fractionated by SDS-PAGE, transferred to a membrane, and probed with the polyclonal anti-DPR antibody. The positions of protein size markers and the 40 kDa DPR band is indicated to the left and right, respectively. B, Spatial distribution of DPR in an optical horizontal section of a dpr⁺thoracic ganglion. Anti-DPR staining is shown in green, and neuronal nuclei are labeled with anti-ELAV antibody (red). The first (I), second (II), and third (III) segments of the thoracic ganglion are indicated. C, Enlarged portion of B, indicated by the white box. DPR expression is detected in a subset of neural cell bodies, some of which are indicated witharrows. A bundle of the wing nerve (wn) also shows a relatively high level of anti-DPR staining (indicated with the arrowhead). D, Horizontal section of adult head (dpr¹/+) double stained with anti-DPR antibodies (green) and anti-β-galactosidase antibodies (red). The proximal region of the laminal cell layer showed a high level of anti-DPR staining (indicated with an arrow). la-c, Lamina distal region (containing cell bodies); la-n, lamina proximal region (neuropil); me-c, medulla distal region (containing cell bodies); me-n, medulla proximal region (neuropil); re, retina. Scale bars:B–D, 50 μm. It was not possible to determine the distribution of the DPR protein in the appendages because the cuticle prevented penetration of the antibodies.

Fig. 6.

The DIG group of proteins. Alignment of the deduced amino acid sequences of DPR (DPR1) and DPR2–20. The DPR1-related proteins were assigned numbers in descending order of the E values generated from the BLAST search (for E values and BDGP numbers, see Materials and Methods). The sequences of some of the putative DPR-related proteins, corresponding to portions of DPR1 residues 55–84, were not available and are indicated. Other sequences unavailable for DPR7, DPR11, DPR13, DPR14, and DPR19 are indicated byX symbols; however, the exact number of missing residues is not known. Gaps in some DPR proteins, relative to others, are represented by dashes. The double asterisks in the DPR15 and DPR20 represent possible insertions of 160 and 10 residues, respectively, not included in this alignment. The predicted cleavage site (after residue 32 in DPR) after the putative signal sequence (Nielsen et al., 1997) is indicated by thetriangle. The predicted transmembrane domain (TMD) (amino acids 276–293) is boxed. The numbers above the sequences show the running tally of amino acids in DPR1. The GenBank accession number for the DPR1 sequence is AF489698.

Fig. 7.

Correlation between relatedness of DIG proteins and chromosomal positions of corresponding genes. A, Dendrogram showing the relatedness of DIG proteins generated using MacVector 6.5.3 (Accelrys, Burlington, MA). The chromosome encoding each of the corresponding genes is shown to theright. The three pairs of tightly clustered genes areboxed. The bracket indicates a group of four dpr genes clustered on chromosome III.B, Chromosomal map positions of dprgenes. Those genes that are tightly clustered areboxed.