A Drosophila Protein Family Implicated in Pheromone Perception Is Related to Tay-Sachs GM2-Activator Protein
2008; Elsevier BV; Volume: 284; Issue: 1 Linguagem: Inglês
10.1074/jbc.m806474200
ISSN1083-351X
AutoresЕ. Г. Старостина, Aiguo Xu, Heping Lin, Claudio W. Pikielny,
Tópico(s)Animal Behavior and Reproduction
ResumoLow volatility, lipid-like cuticular hydrocarbon pheromones produced by Drosophila melanogaster females play an essential role in triggering and modulating mating behavior, but the chemosensory mechanisms involved remain poorly understood. Recently, we showed that the CheB42a protein, which is expressed in only 10 pheromone-sensing taste hairs on the front legs of males, modulates progression to late stages of male courtship behavior in response to female-specific cuticular hydrocarbons. Here we report that expression of all 12 genes in the CheB gene family is predominantly or exclusively gustatory-specific, and occurs in many different, often non-overlapping patterns. Only the Gr family of gustatory receptor genes displays a comparable variety of gustatory-specific expression patterns. Unlike Grs, however, expression of all but one CheB gene is sexually dimorphic. Like CheB42a, other CheBs may therefore function specifically in gustatory perception of pheromones. We also show that CheBs belong to the ML superfamily of lipid-binding proteins, and are most similar to human GM2-activator protein (GM2-AP). In particular, GM2-AP residues involved in ligand binding are conserved in CheBs but not in other ML proteins. Finally, CheB42a is specifically secreted into the inner lumen of pheromone-sensing taste hairs, where pheromones interact with membrane-bound receptors. We propose that CheB proteins interact directly with lipid-like Drosophila pheromones and modulate their detection by the gustatory signal transduction machinery. Furthermore, as loss of GM2-AP in Tay-Sachs disease prevents degradation of GM2 gangliosides and results in neurodegeneration, the function of CheBs in pheromone response may involve biochemical mechanisms critical for lipid metabolism in human neurons. Low volatility, lipid-like cuticular hydrocarbon pheromones produced by Drosophila melanogaster females play an essential role in triggering and modulating mating behavior, but the chemosensory mechanisms involved remain poorly understood. Recently, we showed that the CheB42a protein, which is expressed in only 10 pheromone-sensing taste hairs on the front legs of males, modulates progression to late stages of male courtship behavior in response to female-specific cuticular hydrocarbons. Here we report that expression of all 12 genes in the CheB gene family is predominantly or exclusively gustatory-specific, and occurs in many different, often non-overlapping patterns. Only the Gr family of gustatory receptor genes displays a comparable variety of gustatory-specific expression patterns. Unlike Grs, however, expression of all but one CheB gene is sexually dimorphic. Like CheB42a, other CheBs may therefore function specifically in gustatory perception of pheromones. We also show that CheBs belong to the ML superfamily of lipid-binding proteins, and are most similar to human GM2-activator protein (GM2-AP). In particular, GM2-AP residues involved in ligand binding are conserved in CheBs but not in other ML proteins. Finally, CheB42a is specifically secreted into the inner lumen of pheromone-sensing taste hairs, where pheromones interact with membrane-bound receptors. We propose that CheB proteins interact directly with lipid-like Drosophila pheromones and modulate their detection by the gustatory signal transduction machinery. Furthermore, as loss of GM2-AP in Tay-Sachs disease prevents degradation of GM2 gangliosides and results in neurodegeneration, the function of CheBs in pheromone response may involve biochemical mechanisms critical for lipid metabolism in human neurons. Detection of pheromones in animals involves specialized chemosensory organs of two distinct types. Volatile pheromones are detected, often at great distances from the source, by highly sensitive olfactory organs (1Wyatt T.D. Pheromones and Animal Behaviour: Communication by Smell and Taste.in: Oxford University Press, Oxford2003Crossref Google Scholar). However, many pheromones that trigger specific sexual behaviors are poorly volatile and act through direct contact with chemosensory organs (2Wicker-Thomas C. J. Insect Physiol. 2007; 53: 1089-1100Crossref PubMed Scopus (87) Google Scholar, 3Zufall F. Leinders-Zufall T. Curr. Opin. Neurobiol. 2007; 17: 483-489Crossref PubMed Scopus (77) Google Scholar). In Drosophila melanogaster, pheromones modulate multiple aspects of mating behavior (4Ferveur J.F. Behav. Genet. 2005; 35: 279-295Crossref PubMed Scopus (385) Google Scholar, 5Vosshall L.B. Neuron. 2008; 59: 685-689Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). The past few years have brought remarkable progress in our understanding of the mechanisms underlying pheromone perception by the olfactory system. In particular, cis-vaccenyl acetate (cVA), 4The abbreviations used are: cVA, cis-vaccenyl acetate; CheB, chemosensory B; CheBr, CheB-related; AP, activator protein; Gr, gustatory receptors; ML, MD-like; MD, myeloid differentiation protein; NPC2, Niemann-Pick gene 2 protein; PBP, pheromone-binding protein; RT, reverse transcriptase; GFP, green fluorescent protein; PBS, phosphate-buffered saline. a volatile pheromone produced in the male ejaculatory bulb, is detected by one or perhaps two olfactory receptor proteins expressed in specific subsets of olfactory hairs on the antennae of both sexes (6Ha T.S. Smith D.P. J. Neurosci. 2006; 26: 8727-8733Crossref PubMed Scopus (202) Google Scholar, 7Kurtovic A. Widmer A. Dickson B.J. Nature. 2007; 446: 542-546Crossref PubMed Scopus (513) Google Scholar, 8van der Goes van Naters W. Carlson J.R. Curr. Biol. 2007; 17: 606-612Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar). Despite the apparently identical responses it engenders in the peripheral olfactory organs of males and females (8van der Goes van Naters W. Carlson J.R. Curr. Biol. 2007; 17: 606-612Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar), cVA inhibits male courtship behavior, but accelerates female mating (7Kurtovic A. Widmer A. Dickson B.J. Nature. 2007; 446: 542-546Crossref PubMed Scopus (513) Google Scholar). Another olfactory receptor is tuned to male-specific odors distinct from cVA, and two others respond indistinguishably to male and female odors (8van der Goes van Naters W. Carlson J.R. Curr. Biol. 2007; 17: 606-612Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar). Less is known about the molecular mechanisms involved in detecting a number of pheromones that have very low volatility, which also play critical roles in modulating Drosophila mating behavior (2Wicker-Thomas C. J. Insect Physiol. 2007; 53: 1089-1100Crossref PubMed Scopus (87) Google Scholar, 4Ferveur J.F. Behav. Genet. 2005; 35: 279-295Crossref PubMed Scopus (385) Google Scholar). These long-chain hydrocarbons (C23-C27) are produced by specialized epidermal cells in the abdomen. In particular, female-specific cuticular hydrocarbons are required for normal stimulation of male courtship behavior (9Marcillac F. Ferveur J.F. J. Exp. Biol. 2004; 207: 3927-3933Crossref PubMed Scopus (47) Google Scholar, 10Savarit F. Sureau G. Cobb M. Ferveur J.-F. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 9015-9020Crossref PubMed Scopus (127) Google Scholar), and even trigger homosexual male courtship when ectopically produced by males (11Ferveur J. Savarit F. O'Kane C. Sureau G. Greenspan R. Jallon J. Science. 1997; 276: 1555-1558Crossref PubMed Scopus (183) Google Scholar). Remarkably, whereas these compounds have very low volatility and are only effective over a radius of less than 1 cm (12Gailey D.A. Lacaillade R.C. Hall J.C. Behav. Genet. 1986; 16: 375-405Crossref PubMed Scopus (90) Google Scholar), their detection may involve both olfactory (13Stockinger P. Kvitsiani D. Rotkopf S. Tirian L. Dickson B.J. Cell. 2005; 121: 795-807Abstract Full Text Full Text PDF PubMed Scopus (426) Google Scholar, 14Ejima A. Smith B.P. Lucas C. Levine J.D. Griffith L.C. Curr. Biol. 2005; 15: 194-206Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar) and gustatory organs (15Boll W. Noll M. Development. 2002; 129: 5667-5681Crossref PubMed Scopus (111) Google Scholar, 16Bray S. Amrein H. Neuron. 2003; 39: 1019-1029Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar, 17Park S.K. Mann K.J. Lin H. Starostina E. Kolski-Andreaco A. Pikielny C.W. Curr. Biol. 2006; 16: 1154-1159Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). Although there have been recent breakthroughs in the characterization of the olfactory perception of these cuticular hydrocarbon pheromones (8van der Goes van Naters W. Carlson J.R. Curr. Biol. 2007; 17: 606-612Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar, 18Ejima A. Smith B.P. Lucas C. van der Goes van Naters W. Miller C.J. Carlson J.R. Levine J.D. Griffith L.C. Curr. Biol. 2007; 17: 599-605Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar), more is known about the gustatory organs and molecules involved. Differences in the number and innervation patterns of taste sensilla between the sexes (19Nayak S.V. Singh R.N. Int. J. Insect Morphol. Embryol. 1983; 12: 273-291Crossref Scopus (148) Google Scholar, 20Possidente D.R. Murphey R.K. Dev. Biol. 1989; 132: 448-457Crossref PubMed Scopus (66) Google Scholar), as well as amputation and masking experiments (21Robertson H.M. Experientia (Basel). 1983; 39: 333-335Crossref Scopus (32) Google Scholar, 22Venard R. Anthony C. Jallon J.-M. Neurobiology of Sensory Systems.in: Singh R.N. Strausfeld N.J. Plenum Press, New York1989: 377-385Crossref Google Scholar), provided early evidence that taste sensilla on male forelegs are involved in detection of pheromones. We and others therefore identified genes that are specifically expressed in subsets of gustatory sensilla on male front legs, at least two of which are required for normal male courtship response to female-specific cuticular hydrocarbons (16Bray S. Amrein H. Neuron. 2003; 39: 1019-1029Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar, 17Park S.K. Mann K.J. Lin H. Starostina E. Kolski-Andreaco A. Pikielny C.W. Curr. Biol. 2006; 16: 1154-1159Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 23Xu A. Park S.K. D'Mello S. Kim E. Wang Q. Pikielny C.W. Cell Tissue Res. 2002; 307: 381-392Crossref PubMed Scopus (45) Google Scholar). CheA29a and CheB42a, two genes expressed specifically in subsets of gustatory sensilla on male front legs, defined two novel and unrelated families of small secreted proteins (23Xu A. Park S.K. D'Mello S. Kim E. Wang Q. Pikielny C.W. Cell Tissue Res. 2002; 307: 381-392Crossref PubMed Scopus (45) Google Scholar). More recently, we have shown that CheB42a is expressed in the same taste hairs as Gr68a (17Park S.K. Mann K.J. Lin H. Starostina E. Kolski-Andreaco A. Pikielny C.W. Curr. Biol. 2006; 16: 1154-1159Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar), a putative gustatory pheromone receptor (16Bray S. Amrein H. Neuron. 2003; 39: 1019-1029Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar). Furthermore, loss of CheB42a results in a remarkably specific effect on the elaborate courtship behavior that males direct at females (17Park S.K. Mann K.J. Lin H. Starostina E. Kolski-Andreaco A. Pikielny C.W. Curr. Biol. 2006; 16: 1154-1159Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). Although unaffected in several other behaviors, including the early steps in the courtship sequence, CheB42a mutant males attempt to copulate earlier and more frequently than controls with other individuals of the same species, whether male or female, as long as they express female-specific hydrocarbons. These results indicate that CheB42a functions in gustatory perception of female cuticular hydrocarbon pheromones that modulate male courtship. By what mechanism does CheB42a modulate pheromone perception? What is the function of the other 11 CheBs encoded by the D. melanogaster genome? Based on the evidence in this report, we propose that CheB42a and other CheBs are gustatory-specific pheromone-binding proteins that modulate detection of specific contact pheromones. Analysis of the Expression of Drosophila CheB Genes—RNA preparation, Northern blots, semi-quantitative PCR, and in situ hybridization were described previously (23Xu A. Park S.K. D'Mello S. Kim E. Wang Q. Pikielny C.W. Cell Tissue Res. 2002; 307: 381-392Crossref PubMed Scopus (45) Google Scholar). For Northern blots, RNA was extracted from appendages (antennae, legs, and wings), or heads (without antennae) separated in bulk by sieving (24Pikielny C.W. Hasan G. Rouyer F. Rosbash M. Neuron. 1994; 12: 35-49Abstract Full Text PDF PubMed Scopus (306) Google Scholar). To allow for a rough comparison of the expression levels from different genes, all probes were of ∼500 nucleotides, labeled at similar specific activities with [32P]dCTP, and hybridized to identical filters that were exposed for the same amount of time (except for the exposure of the rp49 filter for 1/10th the time) to x-ray film. For semi-quantitative RT-PCR analysis, body parts of each type were hand-dissected from male or females, total nucleic acids were extracted, and cDNA generated using oligo(dT) primers. Amplification primers were designed to flank an intron so that the ratio of the short product resulting from amplification of the cDNA, to the long product resulting from amplification of genomic DNA, provides a relative measure of the mRNA levels in different samples (25Diviacco S. Norio P. Zentilin L. Menzo S. Clementi M. Biamonti G. Riva S. Falaschi A. Giacca M. Gene (Amst.). 1992; 122: 313-320Crossref PubMed Scopus (249) Google Scholar). Expression of CheB38c was further analyzed by generating a CheB38c-Gal4 fusion, in which 5.2 kb of genomic DNA upstream of the CheB38c initiation codon were amplified by PCR from genomic DNA and inserted 5′ of the hsp70 TATA box in the pGATB vector (26Brand A.H. Perrimon N. Development. 1993; 118: 401-415Crossref PubMed Google Scholar). Transgenic flies were generated using standard methods (27Rubin G.M. Spradling A.C. Science. 1982; 218: 348-353Crossref PubMed Scopus (2336) Google Scholar) and GFP expression was analyzed for several independent insertions in crosses to UAS-GFP lines obtained from the Drosophila stock center (Bloomington, IN). For quantitative real-time PCR, RNA was extracted from whole flies with TRIzol (Invitrogen), treated with RNase-free DNase (Qiagen), and further purified on RNeasy/QIAamp columns (Qiagen). First-strand cDNA was synthesized using oligo(dT)20 and SuperScript III reverse transcriptase (Invitrogen). Real time PCR were performed in 96-well thin-wall plates (Applied Biosystems) using an Applied Biosystems 7300/7500 Real Time PCR System according to the manufacturer’s suggested procedure, with the following modifications. One primer in each pair was designed to span an exon-exon junction, resulting in specific amplification of cDNA as confirmed in pilot experiments (data not shown). For every sample, specificity of amplifications was further confirmed by analysis of the dissociation curve. In every experiment, the relative concentration of each mRNA was obtained by comparison to a standard curve generated by amplification of serial dilutions of the corresponding cDNA product. To account for possible differences in total RNA concentration between samples, all values were normalized to the relative concentration of a ubiquitously expressed ribosomal protein mRNA (rp49). Independent replications of these measurements were performed at least once for seven of the 12 CheB genes, in four cases with different primers, with similar results (not shown). Sequence Analysis—PSI-BLAST searches (28Altschul S.F. Madden T.L. Schaffer A.A. Zhang J. Zhang Z. Miller W. Lipman D.J. Nucleic Acids Res. 1997; 25: 3389-3402Crossref PubMed Scopus (59933) Google Scholar, 29Schaffer A.A. Aravind L. Madden T.L. Shavirin S. Spouge J.L. Wolf Y.I. Koonin E.V. Altschul S.F. Nucleic Acids Res. 2001; 29: 2994-3005Crossref PubMed Scopus (1122) Google Scholar) were performed for multiple iterations until no new significant similarities were uncovered, on the National Center for Biotechnology Information web server (www.ncbi.nlm.nih.gov). The sequences of over 100 ML domain proteins were obtained from the SMART server (30Schultz J. Milpetz F. Bork P. Ponting C.P. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5857-5864Crossref PubMed Scopus (3020) Google Scholar). Sequences were aligned using ClustalW (31Thompson J.D. Higgins D.G. Gibson T.J. Nucleic Acids Res. 1994; 22: 4673-4680Crossref PubMed Scopus (55767) Google Scholar) at the European Bioinformatics Institute server, and alignments edited and displayed using BOXSHADE. PSI-BLAST significance scores depend on a number of parameters, including the number of sequences in the data base at the time of the search, but in all cases discussed here, p < 1 × 10-10. Several PSI-BLAST searches using one of the CheBs as query indicate significant similarities with CheBrs and vice versa, and searches with either type of insect protein indicate significant similarities with human GM2-AP and related proteins from other organisms. In the same searches, similarities with NPC2, Der f2, and many other ML proteins are either not detected, or associated with much poorer significance scores than GM2-APs. In addition, PSI-BLAST searches using human GM2-AP as query yield significant scores with CheBrs but not with NPC2, Der f2, or many other MLs. An identical search restricted to arthropod sequences also identifies significant similarities with D. melanogaster CheBs but not to Drosophila NPC2 (32Huang X. Warren J.T. Buchanan J. Gilbert L.I. Scott M.P. Development. 2007; 134: 3733-3742Crossref PubMed Scopus (109) Google Scholar). Immunochemistry—Our immunohistochemistry protocol was modified from Ref. 33Pitts R.J. Fox A.N. Zwiebel L.J. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 5058-5063Crossref PubMed Scopus (172) Google Scholar. Briefly, front legs of Canton S males were dissected by hand and fixed overnight at 4 °C in phosphate-buffered saline (PBS) containing 4% paraformaldehyde. After three rinses in PBS solution, the legs were dehydrated in a series of ethanol solutions (25, 50, 75, and 90% and three times at 100%), and embedded in Paraplast Plus (Fisher Scientific) tissue embedding media at 60 °C. Samples were sectioned on a microtome (Olympus Cut 4060), collected on Superfrost plus microslides (Fisher Scientific), and dried at 39 °C overnight. The slides were then dewaxed in xylenes, rehydrated in 100, 75, 50, and 25% ethanol, rinsed in PBS, and blocked in PBT (PBS with 1% bovine serum albumin and 0.1% Triton X-100) containing 5% normal goat serum (Roche) for 1 h at room temperature. Slides were incubated for 4 h at room temperature with guinea pig anti-CheB42a antibody (23Xu A. Park S.K. D'Mello S. Kim E. Wang Q. Pikielny C.W. Cell Tissue Res. 2002; 307: 381-392Crossref PubMed Scopus (45) Google Scholar) at a 1:200 dilution, and when applicable, rat anti-PBPRP2 (34Park S.K. Shanbhag S.R. Wang Q. Hasan G. Steinbrecht R.A. Pikielny C.W. Cell Tissue Res. 2000; 300: 181-192Crossref PubMed Scopus (51) Google Scholar) at a 1:300 dilution for 4 h at room temperature, followed by three washes in PBT. The sections were then incubated for 2 h at room temperature with goat anti-guinea pig antibodies conjugated to Alexa Fluor 488 (Invitrogen), and when appropriate, with goat anti-rat antibodies conjugated to Alexa Fluor 568 (Molecular Probes), at dilutions of 1:200. After three washes in PBT, the stained sections were mounted in Vectashield and analyzed under a Leica TCS confocal microscope. Genes of the Drosophila CheB Family Are Expressed in a Variety of Sexually Dimorphic, Gustatory-specific Patterns in Adult Flies—We have previously shown that, in addition to CheB42a, the mRNAs for three other D. melanogaster CheB genes are enriched and present at sexually dimorphic levels in a preparation of mixed fly appendages with chemosensory function (legs, wings, and third antennal segments) (23Xu A. Park S.K. D'Mello S. Kim E. Wang Q. Pikielny C.W. Cell Tissue Res. 2002; 307: 381-392Crossref PubMed Scopus (45) Google Scholar). The same mRNAs are not detected in heads lacking the olfactory third antennal segment. We have now confirmed and extended this observation using Northern blots to analyze the distribution of all 12 CheB mRNAs (Fig. 1A). The mRNAs for 8 CheB genes (CheB38a, -38b,-42a,-53a,-53b,-74a,-93a, and -93b) are detected only in male appendages, whereas CheB98a mRNA is found at higher levels in the appendages of males than those of females. In the above experiments, RNA was extracted from a pool of appendages with different chemosensory functions: legs and wings carry large numbers of gustatory sensilla, whereas the third antennal segment is the main olfactory organ of the fly (35Vosshall L.B. Stocker R.F. Annu. Rev. Neurosci. 2007; 30: 505-533Crossref PubMed Scopus (640) Google Scholar). We therefore further analyzed expression of all 12 CheB genes in specific appendages (Fig. 1B) using a semi-quantitative RT-PCR method that allows comparison of relative concentrations of an mRNA among small samples of different tissues (23Xu A. Park S.K. D'Mello S. Kim E. Wang Q. Pikielny C.W. Cell Tissue Res. 2002; 307: 381-392Crossref PubMed Scopus (45) Google Scholar, 25Diviacco S. Norio P. Zentilin L. Menzo S. Clementi M. Biamonti G. Riva S. Falaschi A. Giacca M. Gene (Amst.). 1992; 122: 313-320Crossref PubMed Scopus (249) Google Scholar). RT-PCR was performed on total nucleic acids extracted from distinct body parts dissected from either males or females: front legs, second and third pairs of legs (pooled), wings, third antennal segment, heads (without third antennal segment, but retaining many taste hairs on the labelum as well as inside the pharynx), and bodies (decapitated and with no legs or wings). As primers were designed to hybridize to either side of an intron, the relative concentrations of the short product resulting from amplification of the cDNA, and the long product, resulting from amplification of genomic DNA, provides a measure of the relative levels of mRNA in different samples (Fig. 1B). This analysis confirms that expression of all 12 CheB genes is specific to appendages and undetectable in heads and bodies. Furthermore, expression of all 12 CheB genes is found almost exclusively in the legs and wings, the two gustatory appendages. Only CheB42b and CheB93a are expressed detectably, albeit at levels lower than in wings and legs, in an olfactory appendage: the third antennal segment. The coincidence of the expression of CheB family genes with gustatory function is further reinforced by their preferential expression in the front legs relative to the other two pairs of legs, mirroring the higher concentrations of taste sensilla found on the front legs (19Nayak S.V. Singh R.N. Int. J. Insect Morphol. Embryol. 1983; 12: 273-291Crossref Scopus (148) Google Scholar, 20Possidente D.R. Murphey R.K. Dev. Biol. 1989; 132: 448-457Crossref PubMed Scopus (66) Google Scholar). The only exceptions to this rule are CheB98a, which is not detectably expressed in the legs of animals of either gender, and the remarkable absence of CheB38c expression in the front legs of males, see below. Although expression of the 12 CheBs is almost exclusively gustatory-specific, it occurs in a variety of patterns. CheBs can be assigned to one of two groups based on their expression (Table 1). CheB42a and seven other CheBs in Group I are expressed exclusively or at highest levels in the front legs of males. As expression of CheB42a is restricted to a subset of hairs on the front legs of males (17Park S.K. Mann K.J. Lin H. Starostina E. Kolski-Andreaco A. Pikielny C.W. Curr. Biol. 2006; 16: 1154-1159Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 23Xu A. Park S.K. D'Mello S. Kim E. Wang Q. Pikielny C.W. Cell Tissue Res. 2002; 307: 381-392Crossref PubMed Scopus (45) Google Scholar), we compared it to that of CheB93a, another Group I gene, by double label in situ hybridization (Fig. 2). As previously reported for CheB42a (17Park S.K. Mann K.J. Lin H. Starostina E. Kolski-Andreaco A. Pikielny C.W. Curr. Biol. 2006; 16: 1154-1159Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 23Xu A. Park S.K. D'Mello S. Kim E. Wang Q. Pikielny C.W. Cell Tissue Res. 2002; 307: 381-392Crossref PubMed Scopus (45) Google Scholar), both genes are expressed in punctate patterns that correspond to cells or groups of cells associated with gustatory sensilla. However, the mRNAs for CheB93a and CheB42a are never found in the same cell, or even in association with the same sensillum. Therefore, expression of CheB42a and CheB93a is associated with two distinct subsets of taste hairs on the front legs of males.TABLE 1D. melanogaster CheBs can be classified into two groups based on their expression patternsGroup I: specific expression in male front legsGroup II: specific or preferential expression on wings of either sexCheB38a, CheB38b, CheB42a, CheB53a, CheB53b, CheB74a, and CheB93b are only expressed in male front legsCheB38c is also expressed in legs, except male front legsCheB93a is expressed in a distinct subset of hairs from CheB42a and, at lower levels, in the third antennal segmentCheB42b is also expressed in the third antennal segment of males, and front legs of both males and femalesCheB42c is also expressed in the front legs of both males and femalesCheB98a is wing-specific, higher in males Open table in a new tab In contrast to Group I genes, all four CheB genes in Group II are expressed in both males and females. Furthermore, whereas three Group II genes are detectably expressed in legs, all are also expressed at high levels in the wings, another appendage with large numbers of gustatory sensilla. We therefore investigated in more detail the expression pattern of the Group II gene CheB38c by generating a transgenic construct in which 5.2 kb of sequences upstream of the CheB38c gene are fused to the yeast transcriptional activator Gal4 (26Brand A.H. Perrimon N. Development. 1993; 118: 401-415Crossref PubMed Google Scholar) (Fig. 3). In the presence of a UAS-GFP reporter construct, this transgene results in specific, punctate production of GFP on the legs and wings of males and females but, conspicuously, not on the front legs of males (Fig. 3A). This pattern is consistent with the distribution of CheB38c mRNA (Fig. 1B), suggesting that it faithfully replicates expression of the endogenous CheB38c gene. Higher resolution imaging shows that all GFP-expressing cells or groups of cells on the wings and legs are associated specifically with gustatory sensilla (Fig. 3B and data not shown). Therefore, the three CheB genes whose expression we have analyzed at the cellular level, CheB42a, CheB93a, and CheB38c, are expressed specifically in three non-overlapping subsets of taste hairs. Intriguingly, the combined expression of the 12 CheB genes is also restricted to a subset of all taste hairs of Drosophila, and is almost completely excluded from sensilla involved in detection of food stimuli. In particular, we have not detected any CheB expression in the many gustatory hairs of the head. Those include a high concentration of taste hairs on the surface of the labelum, many of which have been shown to function in gustatory detection of food components, as well as a number of taste hairs located inside the pharynx where they may allow the last sampling of any food before it is transferred to the digestive organs (35Vosshall L.B. Stocker R.F. Annu. Rev. Neurosci. 2007; 30: 505-533Crossref PubMed Scopus (640) Google Scholar). Furthermore, whereas almost all sensilla on the front legs of males and females express CheB42a, CheB93a, or CheB38c, none of these genes is expressed in the two terminal sensilla that respond to sugars and salts (36Meunier N. Ferveur J.F. Marion-Poll F. Curr. Biol. 2000; 10: 1583-1586Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar) (Fig. 3C and data not shown). In addition, expression of CheB42a, CheB93a, or CheB38c is also undetectable in the four shorter sensilla on segments 4 and 5 that respond to bitter compounds (37Meunier N. Marion-Poll F. Rospars J.P. Tanimura T. J. Neurobiol. 2003; 56: 139-152Crossref PubMed Scopus (177) Google Scholar). The only food-tasting sensilla in which we have found CheB expression are four sugar-sensing sensilla on the second and third tarsal segments of female legs (36Meunier N. Ferveur J.F. Marion-Poll F. Curr. Biol. 2000; 10: 1583-1586Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar) (Fig. 3C). Gustatory-specific expression of the 12 CheB genes therefore occurs largely in taste hairs that are unresponsive to food components such as sugars, salts, and bitter compounds. In addition to displaying a variety of gustatory-specific patterns of expression, at least 11 of the 12 CheB genes are expressed in a sexually dimorphic manner. Expression of the 8 genes in Group I is detected only in males (Table 1). Although all four genes in Group II are expressed in both males and females, expression in the two sexes is either qualitatively or quantitatively different in at least three cases. In a pattern strikingly complementary to that of Group I genes, CheB38c is expressed in all legs and wings of both sexes, with the exception of the front legs of males (Figs. 1 and 3). CheB42b mRNA, whereas present at comparable levels in the legs and wings of both sexes, is expressed in the third antennal segments of males but not females (Fig. 1). Finally, whereas CheB98a expression is only detected in the wings, it is higher in males than females (Fig. 1). CheB42c is the only member of t
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