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Evolution of expression patterns of two odorant-binding protein genes, Obp57d and Obp57e, in Drosophila
Gene 467 (2010) 25–34
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Gene j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / g e n e
Evolution of expression patterns of two odorant-binding protein genes, Obp57d and Obp57e, in Drosophila Jyunichiro Yasukawa, Sachiko Tomioka, Toshiro Aigaki, Takashi Matsuo ⁎ Department of Biological Sciences, Tokyo Metropolitan University, Minami Osawa 1-1, Hachioji, Tokyo 192-0397, Japan
a r t i c l e
i n f o
Article history: Accepted 9 July 2010 Available online 15 July 2010 Received by L. Marino-Ramirez Keywords: Gene duplication Taste perception Cis-regulatory elements
a b s t r a c t Odorant-binding proteins (OBPs) function in the perception of chemical signals together with odorant and taste receptors. Genes encoding OBPs form a large family in insect genomes. In Drosophila, the evolution of OBP gene repertoire has been well studied by comparisons of the whole genome sequences from 12 closely related species. In contrast, their expression patterns are known only in Drosophila melanogaster. Two OBP genes, Obp57d and Obp57e, arose by gene duplication at the early stage of D. melanogaster species group evolution, followed by the divergence of open reading frame (ORF) sequences from each other. While most species in the melanogaster group maintain both Obp57d and Obp57e, some species have lost either gene, suggesting that the birth-and-death process is a dominating pattern of evolution at the Obp57d/e locus. However, it has not been explored whether the expression patterns of these two OBP genes are diverged or conserved among species. Here, we examined the expression patterns of Obp57d and Obp57e in the selected species from the melanogaster group using a combination of reporter analysis, RNA in situ hybridization, and quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) analysis. As previously reported for D. melanogaster, expression in the chemosensilla on the legs was observed in all the species examined. Unlike in D. melnanogaster, however, additional expression in the chemosensilla on the mouthparts was observed in some species including Drosophila pseudoobscura, which maintains an ancestral OBP gene at the Obp57d/e locus. This result shows that, as well as their ORF sequences, the expression patterns of Obp57d and Obp57e have diverged substantially between closely related Drosophila species. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Odorant-binding proteins (OBPs) are expressed in the extracellular lymph of insect chemosensilla, where they function in the perception of chemical signals together with odorant and taste receptors (ORs and GRs) (Tegoni et al, 2004; Pelosi et al., 2006). As genes encoding ORs and GRs, genes encoding OBPs form a large family in insect genomes (Xu et al., 2003; Forêt and Maleszka, 2006). Intensive comparative analyses using the whole genome sequences from 12 closely related Drosophila species revealed that the evolution of OR, GR, and OBP gene repertoires is much slower in the Drosophila species than in mammalian species (Nozawa and Nei, 2007; Guo and Kim, 2007; McBride and Arguello, 2007; Vieira et al., 2007). Some of these studies proposed that the regulatory mechanisms for chemosensory gene expression may account for the observed difference in the “evolvability” of gene repertoires (Nozawa and Nei, 2007; Nei et al., 2008). In mammalian species, expression patterns of OR genes are dynamically determined, ensuring a newly emerged gene to be expressed exclusively in single neurons (Serizawa et al., 2004). In
⁎ Corresponding author. Tel.: +81 42 677 2574; fax: +81 42 677 2559. E-mail address: [email protected] (T. Matsuo). 0378-1119/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.gene.2010.07.006
Drosophila species, in contrast, cis-regulatory elements statically determines the expression patterns of OR genes (Ray et al., 2007; Ray et al., 2008). In such a system, a newly emerged gene would be coexpressed with other genes in single neurons, being prevented from expressing independent phenotype. Thus, it is proposed that the evolution of expression patterns may interact with the evolution of chemosensory gene repertoires in Drosophila species. However, the expression patterns of OR, GR, and OBP genes are known only in Drosophila melanogaster, and no comparative analysis among closely related species has been done (Clyne et al., 1999; Vosshall et al., 1999; Scott et al., 2001; Throne et al., 2004; Galindo and Smith, 2001). Here, for the first time, we compared the expression patterns of two OBP genes, Obp57d and Obp57e, among closely related Drosophila species. Unlike the other OBP genes in D. melanogaster that are expressed in the olfactory sensilla on the antennae, Obp57d and Obp57e are expressed in the taste sensilla on the legs (Galindo and Smith, 2001). These two OBP genes arose by duplication of an ancestral OBP gene at the early stage of D. melanogaster species group evolution (Matsuo, 2008). Results of comparative analyses of the open reading frame (ORF) sequences suggested that Obp57d and Obp57e are functionally diverged from each other (Matsuo, 2008). Behavioral analyses of the D. melanogaster Obp57d and Obp57e mutants revealed that both of the
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two OBPs are involved in the taste detection of toxic fatty acids in the potential host plants, while Obp57d alone is required for presentation of the appropriate wing posture during male courtship toward females, probably by its additional function in pheromone perception (Matsuo et al., 2007; Harada et al., 2008; Koganezawa et al., 2010). Although both Obp57d and Obp57e are maintained in most species of the melanogaster group, either gene is lost in some lineages, reverting to the single OBP gene at the locus (Matsuo, 2008). In this study, we conducted gene expression analyses of Obp57d and Obp57e in the selected species that represent the evolutionary pattern at the Obp57d/e locus in the melanogaster group. In addition to the expression in the legs as observed in D. melanogaster, expression in the mouthparts was observed in some species including Drosophila pseudoobscura, which maintains an ancestral OBP gene at the Obp57d/ e locus. This result shows that, as well as their ORF sequences, the expression patterns of Obp57d and Obp57e have diverged substantially between closely related Drosophila species.
the GFP expression, two or three most extreme lines were used in the scoring of GFP positive cells. However, for all the constructs, differences between lines were not statistically significant by Fisher's exact test at the confidence level of 0.95. Thus, the data were pooled in Table 1. GFP expression was observed on the third day after eclosion at 25 °C.
2.3. Scanning electron microscopy Legs and labella were dissected from the staged adult flies and mounted with double-sided carbon tape. Samples were sputtercoated with gold using Quick Coater SC 701 S (Sanyu Electron, Tokyo, Japan) and viewed with the Carry Scope JCM 5100 (Japan Electron Optics Laboratory, Tokyo, Japan, http://www.jeol.com/).
2.4. RNA in situ hybridization 2. Materials and methods 2.1. Fly stocks The following strains were previously described (Matsuo, 2008); D. melanogaster w1118, Drosophila simulans S357, Drosophila yakuba L42, Drosophila rufa TMU, and Drosophila ananassae 14024-0371.00. Drosophila pseudoobscura Chiricahua (CH) was originally used for population genetics studies of gene arrangement on the third chromosome (Dobzhansky and Quel, 1938). D. melanogaster and D. simulans were reared at 25 °C. D. yakuba, D. rufa, D. ananassae, and D. pseudoobscura were reared at 20 °C. Newly eclosed adults that were staged for 3–4 days at 25 °C were used for all the assays. 2.2. GFP-reporter analysis D. melanogaster transgenic strains that carry the GFP-reporter transgene for D. melanogaster Obp57e (Dmel\Obp57e) or D. simulans Obp57e (Dsim\Obp57e) were generated previously (Matsuo et al., 2007). For other OBP genes, promoter regions were amplified by polymerase chain reaction (PCR) using the following primer pairs: 5'GCGGCCGC-AGCCACAAACTGGAGGACAG-3' and 5'-AAAGGATCCCAAACTAGTTGAAGATATCATAGGAAACT-3' for D. melanogaster and D. simulans Obp57d (Dmel\Obp57d and Dsim\Obp57d, respectively), 5'GCGGCCGC-AGCCACAAACTGGAGGACAG-3' and 5'-AAAGGATCCCAAACTTGTTGAAGATATCATAGGAAACT-3' for D. yakuba Obp57d (Dyak\Obp57d), 5'-GCGGCCGC-GGTGGCACCGAAAATCAATCT-3' and 5'-AAAGGATCC-ACTTACTATATTCCCGGAGAA-3' for D. yakuba Obp57e (Dyak\Obp57e), 5'-GCGGCCGC-AGCCACAAACTGGAGGACAG-3' and 5'AAAGGATCC-TACTAACGTTCTAAAAATGAGTTAT-3'for D. ananassae Obp57d (Dana\Obp57d), 5'-GCGGCCGC-ATGGAATAACAGAATACAACG-3' and 5'-AAAGGATCC-AGTTTCTGTAATGAAGCAAAATCCG-3' for D. rufa Obp57e (Drua\Obp57e), 5'-GCGGCCGC-AGCCACAAACTGGAGGACAG-3' and 5'-GGATCC-GTTTTCTACTCGAAATGTTGTTTG-3' for D. pseudoobscura Obp57d (Dpse\Obp57d). The amplified regions are shown in Fig. 1B. Obtained DNA fragments were digested with NotI and BamHI, and cloned into the multicloning site of pGreenPelican, a vector for the GFP-reporter construction that is equipped with two insulator sequences from gypsy transposon (Fig. 1C) (Barolo et al., 2000). The resulting vector DNA was injected into D. melanogaster w1118 embryo, and transgenic strains carrying a GFP-reporter transgene were established. Each GFP-reporter transgene is designated as (species abbreviation)\(gene name)>GFP; for example, the GFPreporter transgene using the promoter of D. pseudoobscura Obp57d is designated as Dpse\Obp57d>GFP. More than three transgenic lines were established for each transgene construct. All of the lines were preliminarily examined for possible variability in the GFP expression pattern between lines. For the constructs suspected to be variable in
DNA templates for probe synthesis were amplified from genomic DNA by PCR using the following primer pairs: 5'-TATTTAACCCATGTCGAGGGC-3' and 5'-TCATTCCCAAGTGGTCGCTG-3' for Dpse\Obp57d, 5'ATGAGGATCCCTGTGAGAATC-3' and 5'-ATTTCTTCAGACTGGATTCTG-3' for Dana\Obp57d, 5'-CAGATTCCGTCGTCTTCAATC-3' and 5'-CTTGATATTCTCGGCCGCAAG-3' for Drua\Obp57e, and 5'-AGAACTGCCGATTCTAACGA-3' and 5'-TATAGCAAAAATTCGGCTAC-3' for Dmel\Obp57d, and 5'-CTTCAGTATTTAATCCGTGTG-3' and 5'-TTGAAACATACTTCTCGGC-3' for Dmel\Obp57e. The amplified fragments were cloned into the TOPO pCR4 vector (Invitrogen). RNA probes were synthesized by using the T3/T7 DIG RNA labeling kit (Roche diagnostics). Dissected labella were fixed in 4 % formaldehyde for 30 min at room temperature followed by additional fixation in methanol for 5 min. Samples were treated with proteinase K at a concentration of 0.2 μg/ml for 15 min at room temperature before the overnight hybridization at 65 °C. After 5 times of wash at 65 °C, signals were detected by using the DIG nucleic acid detection kit (Roche diagnostics).
2.5. Quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) Total RNA was extracted from the legs, mouthparts (labella), wings, or antennae of 20 staged females with the QIAshredder and RNeasy Micro kit (QIAGEN, Valencia, CA, http://www1.qiagen.com), and cDNA was synthesized using the SuperScript III first strand synthesis system (Invitrogen, Carlsbad, CA, http:www.invitrogen. com) with the oligo(dT)20 primer. Quantitative PCR was carried out with the Chromo 4 realtime PCR analysis system (BioRad, http:// www.bio-rad. com) using SYBR Premix ExTaq (Takara, http://www. takara-bio.com) with the following primer pairs: 5'-TTATTTTGGAAATTCAATTTAGAACTGCCG-3' and 5'-TGATTCGGCTATATCTTCGTCTATTCCTTG-3' for Dmel\Obp57d, 5'-TGCGCAAATGTTCTCGCTAACACTT3' and 5'-ATTCTCCATCACTTGGTGGGCTTCATA-3' for Dmel\Obp57e, 5'GCAGATTCCGTCGTCTTCAATC-3' and 5'-ATTATGATTGTCCGGCCAG-3' for Drua\Obp57e, 5'-CATTTGTGGGATTATTCTT GTTAATTC-3' and 5'GTAAGTTTGCTGGCCAATTTTC-3' for Dana\Obp57d, and 5'-CTTTTATATGGTTATGCCCACAGTA-3' and 5'-CTCTGCCTTCCAAGTTCTTTAG-3' for Dpse\Obp57d. As the internal reference, transcripts from the Ribosomal protein L32 (RpL32) gene were quantified using the following primer pairs: 5'-GCTAAGCTGTCGCACAAATG-3' and 5'TGTGCACCAGGAACTTCTTG-3' for D. melanogaster and D. rufa, 5'CGAAGTTGTCGCACAAATG-3' and 5'-TGTGCACCAGGAACTTCTTG-3' for D. ananassae, and 5'-AAGTTGTCGCACAAATGGC-3' and 5'-TGTGCACCAGGAATTTCTTG-3' for D. pseudoobscura. Either of the primer pair was designed to be on an exon boundary to ensure PCR amplification only from the matured transcripts.
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Fig. 1. Schematic representation of genomic structure at the Obp57d/e locus and the GFP-reporter transgene. (A) Evolution at the Obp57d/e locus in the D. melanogaster species group (Matsuo, 2008). The phylogenetic relationship between species is based on Da Lage et al. (2007). Obp57d (cyan) and Obp57e (magenta) arose by duplication of an ancestral OBP gene (yellow) at the early stage of the melanogaster group evolution. Either of the two OBP genes was lost in some lineages. (B) Genomic structure at the Obp57d/e locus in the species that were selected for the analysis. Arrows indicate translation initiation sites (ATG), not transcription start sites except for Cpr57A and CG30148 in D. melanogaster. Solid lines under the genomic structure indicate promoter regions used for the GFP-reporter analysis. Numbers in parentheses indicate the distance in base pairs from the translation initiation site. The genomic sequence of D. rufa around the Obp57d/e locus was determined previously (Matsuo, 2008; DDBJ accession #AB370285). (C) Structure of the GFP-reporter transgene. Each promoter fragment was inserted into the pGreenPelican vector (Barolo et al., 2000), which is equipped with the gypsy transposon-derived insulators to minimize the interference on expression with surrounding genomic sequences.
3. Results
3.2. GFP reporter analysis of Obp57d and Obp57e expression patterns
3.1. Rapid evolution of Obp57d and Obp57e
In D. melanogaster, Obp57e was reported to be expressed in four cells on each leg (Matsuo et al., 2007; Galindo and Smith, 2001). By detailed examination of the GFP-reporter-carrying transgenic fly that was used in our previous study, however, we found that Dmel\ Obp57e>GFP is expressed in more than four cells in the prothoracic legs (Fig. 2A–C, Supplementary Figs. S1, S2). All GFP-expressing cells were associated with chemosensilla. According to the proposed nomenclature (Meunier et al., 2003), these chemosensilla were identified as pairs of 5 b, 5 s, and 4 s sensilla, and an outside-specific 4c sensillum. The 4c sensillum was reported to be male-specific (Meunier et al., 2003), but we found that the 4c also exists in females and Dmel\Obp57e>GFP was expressed in 4c of both sexes. GFP expression in 5 b, 4 s, and 4 c sensilla was observed in all the individuals, whereas the frequency of GFP-positive individuals was low for 5 s sensilla (Table 1). In mesothoracic and metathoracic legs, GFP expression was observed only in four cells associated with 5 b and 4 s sensilla, as previously reported (Fig. 2D–F, Supplementary Figs. S3, S4).
Matsuo (2008) revealed that Obp57d and Obp57e have undergone rapid evolution in the D. melanogaster species group (Fig. 1A). They arose by duplication of an ancestral OBP gene, which remains single in the D. obscura species group. After the subsequent diversification in the open reading frame (ORF) sequences, Obp57e was lost in the D. ananassae subgroup, while Obp57d was lost in the auraria-rufa lineage of the montium subgroup. The rest of the species in the melanogaster group maintain both genes. The following species were selected for analyses: D. melanogaster, D. simulans, and D. yakuba because they maintain both OBP genes; D. rufa because it has lost Obp57d; D. ananassae because it has lost Obp57e; and D. pseudoobscura because it maintains an ancestral Obp57d. According to the standard nomenclature, each OBP gene is designated as (species abbreviation)\(gene name); for example, the GFP-reporter transgene using the promoter of D. pseudoobscura Obp57d is designated as Dpse\Obp57d>GFP (Fig. 1C).
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Table 1 Frequency of GFP expression in the chemosensilla on the legs. Species
D. melanogaster
Reporter transgene (N of linesa)
Obp57dNGFP (3) Obp57eNGFP (1)
D. simulans
Obp57dNGFP (2) Obp57eNGFP (1)
D. yakuba
Obp57dNGFP (3) Obp57eNGFP (1)
D. rufa
Obp57eNGFP (2)
D. ananassae
Obp57dNGFP (3)
D. pseudoobscura
Obp57dNGFP (3)
Sex
female male female male female male female male female male female male female male female male female male
Mesothoracic leg
Metathoracic leg
5b
Prothoacic leg 5s
5t
4s
4c
4d
3b
2b
2c
5b
5t
4s
5b
5t
4s
0.07 0.12 1 1 0.99 1 0.98 0.93 0.01 0.02 1 0.88 0.94 0.99 0.98 0.98 1 0.98
0.01 0.01 0.05 0.03 0.04 0.08 0.05 0.08 0.08 0.07 0.05 0.05 0.79 0.99 0.01 0.04 0.96 0.93
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0.74 0.59
0.02 0.01 1 1 0.98 1 0.85 0.5 0 0 0.85 0.6 0.98 0.96 0.98 1 1 0.95
0.2 0.2 1 1 1 1 1 0.85 0.6 0.6 0.95 0.7 0.9 1 0 0 0.8 0.93
0 0 0 0 0 0 0 0 0 0 0 0 0 0.34 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0 0 0.81 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0 0 0.53 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0 0 0.45 0 0 0 0
0.02 0.03 0.98 1 1 1 1 0.9 0 0.03 0.95 0.6 0.96 0.99 0.98 1 0.99 0.99
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0.79 0.85
0 0 1 1 0.99 0.98 0.95 0.75 0 0 0.83 0.5 0.99 0.95 1 0.99 0.98 0.97
0 0 0.93 0.95 0.91 0.91 0.95 0.75 0 0 0.58 0.5 0.94 1 0.81 0.79 1 1
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0.71 0.77
0 0 0.83 0.9 0.8 0.85 0.78 0.5 0 0 0.28 0.18 0.91 1 0.76 0.75 1 0.97
Legs of ten males and ten females were examined for each line. a Number of examined transgenic lines.
We also reexamined the previously generated transgenic strain for Dsim\Obp57e>GFP (Matsuo et al., 2007), and found that the expression pattern was exactly the same as that of Dmel\Obp57e>GFP in the following terms (Table 1, Supplementary Figs. S5, S6): (1) In the prothoracic legs, GFP expression was observed in 5b, 4 s, and 4c sensilla at high frequency, and in 5 s at low frequency. (2) In the mesothoracic and metathoracic legs, GFP expression was observed only in 5 b and 4 s sensilla. (3) No difference was observed between sexes. Galindo and Smith (2001) reported that Obp57d and Obp57e are expressed in the same cells associated with 5 b and 4 s chemosensilla (Galindo and Smith, 2001). Following the identification of Obp57e expression in more chemosensilla, however, it should be reexamined whether Obp57d is also expressed in these newly found chemosensilla. To this end, we generated Dmel\Obp57d>GFP and Dsim\Obp57d> GFP transgenic strains. The frequency of GFP-positive cells was quite low for Dmel\Obp57d>GFP, whereas that for Dsim\Obp57d>GFP was as high as that for Obp57e>GFP (Table 1, Supplementary Figs. S7–S9). For Dmel\Obp57d>GFP, GFP expression was not observed in 4 s sensilla on mesothoracic legs, and in 5b and 4 s on metathoracic legs. Except for this difference, the Obp57d expressing sensilla were essentially identical to that of Obp57e in both species. We then generated GFP reporter using the promoter sequences of Obp57d and Obp57e from other species. D. yakuba belongs to the melanogaster subgroup and maintains both Obp57d and Obp57e. The expression patterns were essentially the same as those of D. melanogaster, except for 4 s sensilla on prothoracic legs and 5b on mesothoracic legs where Dyak\Obp57d>GFP expression was not observed (Table 1, Supplementary Figs. S10–S12). D. rufa belongs to the montium subgroup and maintains only Obp57e. GFP expression from the Drua\Obp57e>GFP transgene was observed in more sensilla than in the case of D. melanogaster. In addition to 5b, 5s, 4s, and 4c sensilla, GFP expression was also observed in 4d, 3b, 2b, and 2c sensilla on the prothoracic leg (Table 1, Fig. 2G–L, Supplementary Figs. S13–S16). Interestingly, these expressions in the additional sensilla were observed only in males. D. ananassae belongs to the ananassae subgroup and maintains only Obp57d. GFP expression was observed in a similar set of sensilla as those observed for Dsim\Obp57d>GFP, except for 4c on the prothoracic leg where Dana\Obp57d>GFP was not expressed (Table 1, Supplementary Figs. S17, S18). D. pseudoobscura belongs to the obscura group, an outgroup of the melanogaster group. D. pseudoobscura has Obp57d, which is an ancient form of both Obp57d and Obp57e, and thus distinct from Obp57d in the
melanogaster group (Matsuo, 2008). GFP expression from Dpse \Obp57d>GFP was observed in 5t sensilla in addition to 5b, 5s, 4s, and 4c sensilla (Table 1, Fig. 2M–O, Supplementary Figs. S19, S20). The results of GFP reporter analysis in the legs are summarized as follows: expression in 5b and 4s sensilla of all legs and additional expression in 5s and 4c in the prothoracic legs were observed for all GFP-reporter transgenes. The conserved expression pattern among the GFP-reporter transgenes with promoter sequences from various species indicates that the cis-regulatory function of the promoter region is essentially conserved among these species. Nevertheless, the proportion of GFP-positive sensilla was different among the GFPreporter transgenes, suggesting that the activity level of cis-regulatory elements is different between promoter sequences from different species. In addition, D. rufa and D. pseudoobscura promoter sequences resulted in the expression in the extra sensilla, suggesting that the cisregulatory elements in these species have a slightly diverged function from those of other species. 3.3. Morphological identification of gustatory sensilla on the legs in each species Because GFP reporter expression was observed in the D. melanogaster background, it was necessary to ensure that the GFP-positive sensilla actually exist in the original species. Morphological examination of the prothoracic legs revealed that there is a significant difference in the number and position of chemosensilla between sexes and species (Fig. 3). It was confirmed, however, that GFP-positive 5b, 5s, 4c, and 4s sensilla are well conserved in terms of their number and position among species and sexes, suggesting that the OBP expression itself is also conserved in these sensilla. In contrast, OBP expression in the sensilla that exist in the original species but not in D. melanogaster may have been overlooked in our GFP reporter analysis. 3.4. GFP reporter expression in the labellum For promoters from species that belong to the melanogaster subgroup (D. melanogaster, D. simulans, and D. yakuba), the expression of the GFP reporter was restricted to the legs. To our surprise, however, the promoters from D. rufa, D. ananassae, and D. pseudoobscura resulted in the GFP expression not only in the legs but also in the labellum, the most distal structure of the fly's mouthparts (Fig. 4). Like in the legs, GFP-expressing cells in the labellum were associated with the gustatory sensilla, which are classified into three types on the
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Fig. 2. GFP-reporter expression in the legs. GFP expression from each reporter transgene was observed in the D. melanogaster background. Arrows indicate sockets of sensilla. Bar = 50 μm. (A–C) Dmel\Obp57e N GFP expression in the female prothoracic leg. Lateral (outside) view of the right leg is shown. (D–F) Dmel\Obp57e N GFP expression in the female mesothoracic leg. Lateral view of the right leg is shown. (G–L) Drua\Obp57e N GFP expression in the male prothoracic leg. (G–I) Lateral view of the right leg is shown. (J–L) Dorsal view of the fourth tarsal segment is shown. (M–O) Dpse\Obp57d expression in the male metathoracic leg. Medial view of the left leg is shown. The 5t sensillum was not identified in the previous study (Meunier et al., 2003). Although it was not physiologically confirmed as chemosensilla, we named it expediently to score the GFP expression in this sensillum.
basis of their morphology (Hiroi et al., 2002). GFP expression was most frequently observed in the L-type sensilla in all the three species. The frequencies of GFP-expressing sensilla were low for I-type and Stype sensilla for Drua\Obp57e>GFP and Dana\Obp57d>GFP, while it was high for Dpse\Obp57d>GFP (Fig. 4G–I). Morphological examination of chemosensilla on the labellum confirmed that the number and position of L-type sensilla are well conserved among species (Fig. 5), while those of I-type and S-type sensilla were variable. 3.5. Direct observation of OBP expression in the original species In our GFP reporter analysis, the function of promoters from the other species was examined in the D. melanogaster background. Thus, it was possible that trans-regulatory factors in D. melanogaster functioned less properly on cis-regulatory elements of the more distantly related species, resulting in wider expression patterns in the D. melanogaster background than in the original species. To exclude this possibility, OBP expression in the labellum was directly examined by RNA in situ hybridization. In consistent with the result of GFP reporter analysis, OBP transcripts were detected in the labellum of D. pseudoobscura, D. ananassae, D. rufa, but not in that of D. melanogaster (Fig. 6). The stained cells were associated to L type sensilla when it was able to be identified.
The Obp57d and Obp57e transcript levels in the other tissues were determined by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) analysis (Fig. 7). OBP transcripts were detected in the legs from all the tested species. The transcript levels were higher in D. rufa and D. pseudoobscura than in the other species, in consistent with the result of the GFP reporter analysis showing that more sensilla express GFP in these species (Table 1). OBP transcripts were also detected in the labellum of all the tested species, but at an extremely low level in D. melanogaster, in consistent with the results of GFP reporter analysis and RNA in situ hybridization in which OBP expression was not observed in the labellum of D. melanogaster. The transcript levels were low in the wings and antennae, again in consistent with the result of the GFP reporter analysis in which expression of GFP was not observed in these tissues. 4. Discussion 4.1. GFP-reporter expression reproduced the intrinsic OBP expression patterns In the GFP-reporter analysis, we used 5' region of the OBP genes. Thus, our results do not reflect the effect of cis-regulatory elements in the 3' and intronic regions, if there are any of such elements.
30 J. Yasukawa et al. / Gene 467 (2010) 25–34 Fig. 3. Morphological identification of chemosensilla on the legs. Number and position of chemosensilla in each species were examined by scanning electron microscopy (SEM). Chemosensilla and other bristles were distinguished on the basis of their morphology (shape of shaft and socket), not by physiological analyses. (A–H) Schematics of chemosensilla positions on the legs. Name of each sensillum was assigned by relative position to other sensilla (Meunier et al., 2003). Dorsal view of the right prothoracic leg is shown (medial side is upward). Circles indicate position of chemosensilla. Each pair of symmetrical chemosensilla is connected by a line. Filled circles indicate previously unidentified sensilla that show chemosensilla-like morphology. Because they are not physiologically confirmed as chemosensilla, however, they are unnamed except for 5t sensilla, in which the GFP expression was observed for Dpse\Obp57d N GFP. (I–P) SEM images of dorsal view of the right prothoracic legs. Arrows indicate sockets of chemosensilla. Arrowheads indicate sockets of previously unidentified chemosensilla (corresponding to the filled circles in A–H). (Q–X) Lateral (outside) view of the prothoracic legs. (Y-e) Basolateral view of the fifth tarsal segment. Bar = 50 μm.
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Fig. 4. GFP-reporter expression in the labellum. (A-C) GFP expression from the reporter transgenes in D. melanogaster background. Lateral view of the labellum, the most distal structure of the fly's mouthparts, is shown. Anterior is to the right. Bar = 30 μm. (D–F) Schematics of chemosensilla positions on the labellum. The number and position of L-type sensilla were highly stable. The other sensilla were identified on the basis of their relative position to L-type sensilla. The S0 sensillum was previously unidentified (Hiroi et al., 2002), and S9 sensilla was not found in our observation. The fill colors of symbols indicate the frequency of GFP expression at each sensillum: black for more than 0.1, gray for less than 0.1, and white for no GFP expression. (G–I) Frequency of GFP expression in each chemosensillum. Twenty individuals (10 females and 10 males) were examined. No difference was observed between sexes, and the data were pooled. Counts for S0 and S1 were pooled.
Nevertheless, the GFP expression patterns in labella were confirmed by direct observation of transcripts using RNA in situ hybridization. In addition, the GFP expression patterns matched with the transcript levels in various tissues determined by quantitative RT-PCR analysis. Furthermore, in D. melanogaster, the higher frequency of GFP-positive
cells for Obp57e>GFP than Obp57d>GFP was also consistent with the higher transcript level for Obp57e than Obp57d. All of these results support that the 5' region used in our analysis is sufficient to reproduce the intrinsic expression patterns for Obp57d and Obp57e. However, we should note that in the case of Dsim\Obp57d, the high
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Fig. 5. Morphological identification of chemosensilla on the labellum. Number and position of chemosensilla in each species were examined by scanning electron microscopy. Three types (L, I, and S) were distinguished on the basis of size (Hiroi et al., 2002). Sensilla of each type were numbered from the apical to the basal side; thus, the sensilla of different species with the same name do not necessarily correspond to each other.
frequency of GFP-positive cells in the reporter analysis was not supported by the other methods. In our previous study (Matsuo et al., 2007), the amount of Dsim\Obp57d transcripts was below the detection level in quantitative RT-PCR analysis. Interestingly, when the D. simulans genomic fragment including the Obp57d/e region was introduced into D. melanogaster background by P element-based transformation, now Dsim\Obp57d transcripts were detected at a level slightly higher than that of Dsim\Obp57e. Although it should be examined further, the observed alteration of the expression level is supposed to be caused by the difference in trans-acting factors between species. 4.2. Variation in the promoter sequence controlling the expression of Obp57d and Obp57e In the GFP reporter analysis, the promoters of D. rufa, D. ananassae, and D. pseudoobscura resulted in different expression patterns from
those of D. melanogaster promoters, even though the GFP expression was controlled by a same set of trans-acting factors in the D. melanogaster background. Thus, the observed variation in the GFP expression patterns should be caused by the evolution in cisregulatory elements that reside within the relatively short promoter regions used in our reporter analysis. Comparative analyses of the promoter sequences from the species used in this study, as well as of those from other closely related species, by using OLIGO-ANALYSIS and DYAD-ANALYSIS (van Helden et al., 1998; van Helden et al., 2000), however, failed to find any conserved motifs (data not shown). In our previous study, a 4 bp insertion was found in the D. sechellia Obp57e promoter region, and the removal of the insertion restored the promoter activity in the D. melanogaster background, suggesting that the sequence around this insertion site is important for the Obp57e expression (Matsuo et al., 2007). However, the promoter sequence of D. yakuba Obp57e does not have significant homology in this region, while it resulted in the similar expression pattern as those of D.
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Fig. 6. Detection of OBP transcripts by RNA in situ hybridization in the labellum of original species. (A) Dpse\Obp57d anti-sense probe. (B) Dana\Obp57d anti-sense probe. (C) Drua\Obp57e anti-sense probe. (D) Dpse\Obp57d sense probe (negative control). (E) Dmel\Obp57d anti-sense probe. (F) Dmel\Obp57e anti-sense probe.
melanogaster and D. simulans Obp57e. Thus, although the function of cis-regulatory elements seems to be conserved, their positions would be variable among species. An experimental approach, such as the GFP reporter analysis with shorter promoter sequences, may be required to identify the position of the cis-regulatory elements. 4.3. Expression patterns and the evolutionary fate of duplicated genes There are two alternative evolutionary consequences of gene duplication: degeneration of either copy or maintenance of both
(Lynch and Conery, 2000). Functional divergence between the duplicates prevents them from degeneration, because both genes independently contribute to the fitness and both are necessary for survival of the organism. Functional divergence occurs not only in the structure of the encoded gene products but also in the expression patterns; if the expression patterns of the duplicated genes have diverged from each other, loss of either gene may not be complemented by the other, even though their structures are similar. Obp57d and Obp57e arose by gene duplication, and the comparisons of their ORF sequences showed that their structures have diverged from each
Fig. 7. Quantitative analysis of the transcript level in various tissues. Levels of Obp57d and Obp57e transcripts in the tissues carrying chemosensilla were determined by quantitative RT-PCR. Primers were independently designed for each species. Transcript levels are shown as relative to that of the Ribosomal protein L32 gene. Each bar represents the mean of three to twelve independent replicates. Error bars indicate standard error.
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other, suggesting that Obp57d and Obp57e have acquired different functions (Matsuo, 2008). In contrast, as shown in this study, their expression patterns overlap each other, suggesting that the expression patterns might not be involved in the functional divergence between Obp57d and Obp57e. Nevertheless, the observed expression frequency was low for Dmel\Obp57d>GFP and Dyak\Obp57d>GFP. Further analysis is required to examine a possible effect of the diverged expression levels on preservation of the both OBP genes. Although the difference in the expression pattern was not observed between the duplicated genes, another type of association between the expression pattern and the gene duplication was observed in this study. In the species that maintain both Obp57d and Obp57e, the expression was restricted to the chemosensilla on the legs, while in the species that lost either gene, as well as in D. pseudoobscura that maintains an ancestral Obp57d gene, the additional expression in the chemosensilla on the labellum was observed. In D. melanogaster, biological roles of Obp57d and Obp57e have been shown by the behavioral analyses of the knock-out mutants of these OBP genes. Obp57d and Obp57e are involved in the egg-laying site determination by their function in the taste detection of toxic octanoic acid in the medium, while they had no effect on the feeding response to octanoic acid (Harada et al., 2008). It may be interesting to see if the ectopic expression of Obp57d and Obp57e in the labellum changes the feeding response of D. melanogaster. Further functional analyses of Obp57d and Obp57e in the evolutionary context may also elucidate a possible link between the evolution of expression pattern and gene duplication. 5. Conclusions We have analyzed the spatial expression patterns of Obp57d and Obp57e in the closely related Drosophila species. In addition to the expression in the legs as previously observed in D. melanogaster, expression in the labellum was observed in some species including D. pseudoobscura, which maintains an ancestral OBP gene at the Obp57d/e locus, showing that the expression patterns of Obp57d and Obp57e have diverged substantially between closely related Drosophila species. Acknowledgments This work was supported by a grant-in-aid for Young Scientists (B) 19770210 from the Ministry of Education, Culture, Sports, Science and Technology, Japan to TM. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.gene.2010.07.006. References Barolo, S., Carver, L.A., Posakony, J.W., 2000. GFP and β-galactosidase transformation vectors for promoter/enhancer analysis in Drosophila. Biotechniques 29, 726–732.
Clyne, P., Warr, C., Freeman, M., Lessing, D., Kim, J., Carlson, J., 1999. A novel family of divergent seven-transmembrane proteins: candidate odorant receptors in Drosophila. Neuron 22, 327–338. Da Lage, J.L., Kergoat, G.J., Maczkowiak, F., Silvain, J.F., Cariou, M.L., Lachaise, D., 2007. A phylogeny of Drosophilidae using the Amyrel gene: questioning the Drosophila melanogaster species group boundaries. J. Zool. Syst. Evol. Res 45, 47–63. Dobzhansky, T., Quel, M.L., 1938. Genetics of natural populations. II. Genetic variation in population of Drosophila pseudoobscura inhabiting isolated mountain ranges. Genetics 23, 463–484. Forêt, S., Maleszka, R., 2006. Function and evolution of a gene family encoding odorant binding-like proteins in a social insect, the honey bee (Apis mellifera). Genome Res. 16, 1385–1394. Galindo, K., Smith, D.P., 2001. A large family of divergent Drosophila odorant-binding proteins expressed in gustatory and olfactory sensilla. Genetics 159, 1059–1072. Guo, S., Kim, J., 2007. Molecular evolution of Drosophila odorant receptor genes. Mol. Biol. Evol. 24, 1198–1207. Harada, E., Haba, D., Aigaki, T., Matsuo, T., 2008. Behavioral analyses of mutants for two odorant-binding protein genes, Obp57d and Obp57e, in Drosophila melanogaster. Genes Genet. Syst. 83, 257–264. Hiroi, M., Marion-Poll, F., Tanimura, T., 2002. Differentiated response to sugars among labellar chemosensilla in Drosophila. Zool. Sci. 19, 1009–1018. Koganezawa, M., Haba, D., Matsuo, T., Yamamoto, D., 2010. The shaping of male courtship posture by lateralized gustatory inputs to male-specific interneurons. Curr. Biol. 20, 1–8. Lynch, M., Conery, J.S., 2000. The evolutionary fate and consequences of duplicate genes. Science 290, 1151–1155. Matsuo, T., Sugaya, S., Yasukawa, J., Aigaki, T., Fuyama, Y., 2007. Odorant-binding proteins OBP57d and OBP57e affect taste perception and host-plant preference in Drosophila sechellia. PLoS Biol. 5, e118. Matsuo, T., 2008. Rapid evolution of two odorant-binding protein genes, Obp57d and Obp57e, in the Drosophila melanogaster species group. Genetics 178, 1061–1072. McBride, C.S., Arguello, J.R., 2007. Five Drosophila genomes reveal nonneutral evolution and the signature of host specialization in the chemoreceptor superfamily. Genetics 177, 1395–1416. Meunier, N., Marion-Poll, F., Rospars, J.P., Tanimura, T., 2003. Peripheral coding of bitter taste in Drosophila. J. Neurobiol. 56, 139–152. Nei, M., Niimura, Y., Nozawa, M., 2008. The evolution of animal chemosensory receptor gene repertoires: roles of chance and necessity. Nat. Rev. Genet. 12, 951–963. Nozawa, M., Nei, M., 2007. Evolutionary dynamics of olfactory receptor genes in Drosophila species. Proc. Natl. Acad. Sci. USA 104, 7122–7127. Pelosi, P., Zhou, J.J., Ban, L.P., Calvello, M., 2006. Soluble proteins in insect chemical communication. Cell. Mol. Life Sci. 63, 1658–1676. Ray, A., van der Goes van Naters, W., Shiraiwa, T., Carlson, J.R., 2007. Mechanisms of odor receptor gene choice in Drosophila. Neuron 53, 353–369. Ray, A., van der Goes van Naters, W., Carlson, J.R., 2008. A regulatory code for neuronspecific odor receptor expression. PLoS Biol. 6, 1069–1083 (e125). Scott, K., Brady, R., Cravchik, A., Morozov, P., Rzhetsky, A., Zuker, C., et al., 2001. A chemosensory gene family encoding candidate gustatory and olfactory receptors in Drosophila. Cell 104, 661–673. Serizawa, S., Miyamichi, K., Sakano, H., 2004. One neuron-one receptor rule in the mouse olfactory system. Trends Genet. 20, 648–653. Tegoni, M., Campanacci, V., Cambillau, C., 2004. Structural aspects of sexual attraction and chemical communication in insects. Trends Biochem. Sci. 29, 257–264. Throne, N., Chromey, C., Bray, S., Amrein, H., 2004. Taste perception and coding in Drosophila. Curr. Biol. 14, 1065–1079. van Helden, J., Andre, B., Collado-Vides, J., 1998. Extracting regulatory sites from the upstream regions of yeast genes by computational analysis of oligonucleotide frequencies. J. Mol. Biol. 281, 827–842. van Helden, J., Rios, A., Collado-Vides, J., 2000. Discovering regulatory elements in noncoding sequences by analysis of spaced dyads. Nucleic Acids Res. 28, 1808–1818. Vieira, F.G., Sanchez-Gracia, A., Rozas, J., 2007. Comparative genomic analysis of the odorant-binding protein family in 12 Drosophila genomes: purifying selection and birth-and-death evolution. Genome Biol. 8, R235. Vosshall, L., Amrein, H., Morozov, P., Rzhetsky, A., Axel, R., 1999. A spatial map of the olfactory receptor expression in the Drosophila antenna. Cell 96, 725–736. Xu, P.X., Zwiebel, L.J., Smith, D.P., 2003. Identification of a distinct family of genes encoding atypical odorant-binding proteins in the malaria vector mosquito, Anopheles gambiae. Insect Mol. Biol. 12, 549–560.
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Gene j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / g e n e
Evolution of expression patterns of two odorant-binding protein genes, Obp57d and Obp57e, in Drosophila Jyunichiro Yasukawa, Sachiko Tomioka, Toshiro Aigaki, Takashi Matsuo ⁎ Department of Biological Sciences, Tokyo Metropolitan University, Minami Osawa 1-1, Hachioji, Tokyo 192-0397, Japan
a r t i c l e
i n f o
Article history: Accepted 9 July 2010 Available online 15 July 2010 Received by L. Marino-Ramirez Keywords: Gene duplication Taste perception Cis-regulatory elements
a b s t r a c t Odorant-binding proteins (OBPs) function in the perception of chemical signals together with odorant and taste receptors. Genes encoding OBPs form a large family in insect genomes. In Drosophila, the evolution of OBP gene repertoire has been well studied by comparisons of the whole genome sequences from 12 closely related species. In contrast, their expression patterns are known only in Drosophila melanogaster. Two OBP genes, Obp57d and Obp57e, arose by gene duplication at the early stage of D. melanogaster species group evolution, followed by the divergence of open reading frame (ORF) sequences from each other. While most species in the melanogaster group maintain both Obp57d and Obp57e, some species have lost either gene, suggesting that the birth-and-death process is a dominating pattern of evolution at the Obp57d/e locus. However, it has not been explored whether the expression patterns of these two OBP genes are diverged or conserved among species. Here, we examined the expression patterns of Obp57d and Obp57e in the selected species from the melanogaster group using a combination of reporter analysis, RNA in situ hybridization, and quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) analysis. As previously reported for D. melanogaster, expression in the chemosensilla on the legs was observed in all the species examined. Unlike in D. melnanogaster, however, additional expression in the chemosensilla on the mouthparts was observed in some species including Drosophila pseudoobscura, which maintains an ancestral OBP gene at the Obp57d/e locus. This result shows that, as well as their ORF sequences, the expression patterns of Obp57d and Obp57e have diverged substantially between closely related Drosophila species. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Odorant-binding proteins (OBPs) are expressed in the extracellular lymph of insect chemosensilla, where they function in the perception of chemical signals together with odorant and taste receptors (ORs and GRs) (Tegoni et al, 2004; Pelosi et al., 2006). As genes encoding ORs and GRs, genes encoding OBPs form a large family in insect genomes (Xu et al., 2003; Forêt and Maleszka, 2006). Intensive comparative analyses using the whole genome sequences from 12 closely related Drosophila species revealed that the evolution of OR, GR, and OBP gene repertoires is much slower in the Drosophila species than in mammalian species (Nozawa and Nei, 2007; Guo and Kim, 2007; McBride and Arguello, 2007; Vieira et al., 2007). Some of these studies proposed that the regulatory mechanisms for chemosensory gene expression may account for the observed difference in the “evolvability” of gene repertoires (Nozawa and Nei, 2007; Nei et al., 2008). In mammalian species, expression patterns of OR genes are dynamically determined, ensuring a newly emerged gene to be expressed exclusively in single neurons (Serizawa et al., 2004). In
⁎ Corresponding author. Tel.: +81 42 677 2574; fax: +81 42 677 2559. E-mail address: [email protected] (T. Matsuo). 0378-1119/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.gene.2010.07.006
Drosophila species, in contrast, cis-regulatory elements statically determines the expression patterns of OR genes (Ray et al., 2007; Ray et al., 2008). In such a system, a newly emerged gene would be coexpressed with other genes in single neurons, being prevented from expressing independent phenotype. Thus, it is proposed that the evolution of expression patterns may interact with the evolution of chemosensory gene repertoires in Drosophila species. However, the expression patterns of OR, GR, and OBP genes are known only in Drosophila melanogaster, and no comparative analysis among closely related species has been done (Clyne et al., 1999; Vosshall et al., 1999; Scott et al., 2001; Throne et al., 2004; Galindo and Smith, 2001). Here, for the first time, we compared the expression patterns of two OBP genes, Obp57d and Obp57e, among closely related Drosophila species. Unlike the other OBP genes in D. melanogaster that are expressed in the olfactory sensilla on the antennae, Obp57d and Obp57e are expressed in the taste sensilla on the legs (Galindo and Smith, 2001). These two OBP genes arose by duplication of an ancestral OBP gene at the early stage of D. melanogaster species group evolution (Matsuo, 2008). Results of comparative analyses of the open reading frame (ORF) sequences suggested that Obp57d and Obp57e are functionally diverged from each other (Matsuo, 2008). Behavioral analyses of the D. melanogaster Obp57d and Obp57e mutants revealed that both of the
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two OBPs are involved in the taste detection of toxic fatty acids in the potential host plants, while Obp57d alone is required for presentation of the appropriate wing posture during male courtship toward females, probably by its additional function in pheromone perception (Matsuo et al., 2007; Harada et al., 2008; Koganezawa et al., 2010). Although both Obp57d and Obp57e are maintained in most species of the melanogaster group, either gene is lost in some lineages, reverting to the single OBP gene at the locus (Matsuo, 2008). In this study, we conducted gene expression analyses of Obp57d and Obp57e in the selected species that represent the evolutionary pattern at the Obp57d/e locus in the melanogaster group. In addition to the expression in the legs as observed in D. melanogaster, expression in the mouthparts was observed in some species including Drosophila pseudoobscura, which maintains an ancestral OBP gene at the Obp57d/ e locus. This result shows that, as well as their ORF sequences, the expression patterns of Obp57d and Obp57e have diverged substantially between closely related Drosophila species.
the GFP expression, two or three most extreme lines were used in the scoring of GFP positive cells. However, for all the constructs, differences between lines were not statistically significant by Fisher's exact test at the confidence level of 0.95. Thus, the data were pooled in Table 1. GFP expression was observed on the third day after eclosion at 25 °C.
2.3. Scanning electron microscopy Legs and labella were dissected from the staged adult flies and mounted with double-sided carbon tape. Samples were sputtercoated with gold using Quick Coater SC 701 S (Sanyu Electron, Tokyo, Japan) and viewed with the Carry Scope JCM 5100 (Japan Electron Optics Laboratory, Tokyo, Japan, http://www.jeol.com/).
2.4. RNA in situ hybridization 2. Materials and methods 2.1. Fly stocks The following strains were previously described (Matsuo, 2008); D. melanogaster w1118, Drosophila simulans S357, Drosophila yakuba L42, Drosophila rufa TMU, and Drosophila ananassae 14024-0371.00. Drosophila pseudoobscura Chiricahua (CH) was originally used for population genetics studies of gene arrangement on the third chromosome (Dobzhansky and Quel, 1938). D. melanogaster and D. simulans were reared at 25 °C. D. yakuba, D. rufa, D. ananassae, and D. pseudoobscura were reared at 20 °C. Newly eclosed adults that were staged for 3–4 days at 25 °C were used for all the assays. 2.2. GFP-reporter analysis D. melanogaster transgenic strains that carry the GFP-reporter transgene for D. melanogaster Obp57e (Dmel\Obp57e) or D. simulans Obp57e (Dsim\Obp57e) were generated previously (Matsuo et al., 2007). For other OBP genes, promoter regions were amplified by polymerase chain reaction (PCR) using the following primer pairs: 5'GCGGCCGC-AGCCACAAACTGGAGGACAG-3' and 5'-AAAGGATCCCAAACTAGTTGAAGATATCATAGGAAACT-3' for D. melanogaster and D. simulans Obp57d (Dmel\Obp57d and Dsim\Obp57d, respectively), 5'GCGGCCGC-AGCCACAAACTGGAGGACAG-3' and 5'-AAAGGATCCCAAACTTGTTGAAGATATCATAGGAAACT-3' for D. yakuba Obp57d (Dyak\Obp57d), 5'-GCGGCCGC-GGTGGCACCGAAAATCAATCT-3' and 5'-AAAGGATCC-ACTTACTATATTCCCGGAGAA-3' for D. yakuba Obp57e (Dyak\Obp57e), 5'-GCGGCCGC-AGCCACAAACTGGAGGACAG-3' and 5'AAAGGATCC-TACTAACGTTCTAAAAATGAGTTAT-3'for D. ananassae Obp57d (Dana\Obp57d), 5'-GCGGCCGC-ATGGAATAACAGAATACAACG-3' and 5'-AAAGGATCC-AGTTTCTGTAATGAAGCAAAATCCG-3' for D. rufa Obp57e (Drua\Obp57e), 5'-GCGGCCGC-AGCCACAAACTGGAGGACAG-3' and 5'-GGATCC-GTTTTCTACTCGAAATGTTGTTTG-3' for D. pseudoobscura Obp57d (Dpse\Obp57d). The amplified regions are shown in Fig. 1B. Obtained DNA fragments were digested with NotI and BamHI, and cloned into the multicloning site of pGreenPelican, a vector for the GFP-reporter construction that is equipped with two insulator sequences from gypsy transposon (Fig. 1C) (Barolo et al., 2000). The resulting vector DNA was injected into D. melanogaster w1118 embryo, and transgenic strains carrying a GFP-reporter transgene were established. Each GFP-reporter transgene is designated as (species abbreviation)\(gene name)>GFP; for example, the GFPreporter transgene using the promoter of D. pseudoobscura Obp57d is designated as Dpse\Obp57d>GFP. More than three transgenic lines were established for each transgene construct. All of the lines were preliminarily examined for possible variability in the GFP expression pattern between lines. For the constructs suspected to be variable in
DNA templates for probe synthesis were amplified from genomic DNA by PCR using the following primer pairs: 5'-TATTTAACCCATGTCGAGGGC-3' and 5'-TCATTCCCAAGTGGTCGCTG-3' for Dpse\Obp57d, 5'ATGAGGATCCCTGTGAGAATC-3' and 5'-ATTTCTTCAGACTGGATTCTG-3' for Dana\Obp57d, 5'-CAGATTCCGTCGTCTTCAATC-3' and 5'-CTTGATATTCTCGGCCGCAAG-3' for Drua\Obp57e, and 5'-AGAACTGCCGATTCTAACGA-3' and 5'-TATAGCAAAAATTCGGCTAC-3' for Dmel\Obp57d, and 5'-CTTCAGTATTTAATCCGTGTG-3' and 5'-TTGAAACATACTTCTCGGC-3' for Dmel\Obp57e. The amplified fragments were cloned into the TOPO pCR4 vector (Invitrogen). RNA probes were synthesized by using the T3/T7 DIG RNA labeling kit (Roche diagnostics). Dissected labella were fixed in 4 % formaldehyde for 30 min at room temperature followed by additional fixation in methanol for 5 min. Samples were treated with proteinase K at a concentration of 0.2 μg/ml for 15 min at room temperature before the overnight hybridization at 65 °C. After 5 times of wash at 65 °C, signals were detected by using the DIG nucleic acid detection kit (Roche diagnostics).
2.5. Quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) Total RNA was extracted from the legs, mouthparts (labella), wings, or antennae of 20 staged females with the QIAshredder and RNeasy Micro kit (QIAGEN, Valencia, CA, http://www1.qiagen.com), and cDNA was synthesized using the SuperScript III first strand synthesis system (Invitrogen, Carlsbad, CA, http:www.invitrogen. com) with the oligo(dT)20 primer. Quantitative PCR was carried out with the Chromo 4 realtime PCR analysis system (BioRad, http:// www.bio-rad. com) using SYBR Premix ExTaq (Takara, http://www. takara-bio.com) with the following primer pairs: 5'-TTATTTTGGAAATTCAATTTAGAACTGCCG-3' and 5'-TGATTCGGCTATATCTTCGTCTATTCCTTG-3' for Dmel\Obp57d, 5'-TGCGCAAATGTTCTCGCTAACACTT3' and 5'-ATTCTCCATCACTTGGTGGGCTTCATA-3' for Dmel\Obp57e, 5'GCAGATTCCGTCGTCTTCAATC-3' and 5'-ATTATGATTGTCCGGCCAG-3' for Drua\Obp57e, 5'-CATTTGTGGGATTATTCTT GTTAATTC-3' and 5'GTAAGTTTGCTGGCCAATTTTC-3' for Dana\Obp57d, and 5'-CTTTTATATGGTTATGCCCACAGTA-3' and 5'-CTCTGCCTTCCAAGTTCTTTAG-3' for Dpse\Obp57d. As the internal reference, transcripts from the Ribosomal protein L32 (RpL32) gene were quantified using the following primer pairs: 5'-GCTAAGCTGTCGCACAAATG-3' and 5'TGTGCACCAGGAACTTCTTG-3' for D. melanogaster and D. rufa, 5'CGAAGTTGTCGCACAAATG-3' and 5'-TGTGCACCAGGAACTTCTTG-3' for D. ananassae, and 5'-AAGTTGTCGCACAAATGGC-3' and 5'-TGTGCACCAGGAATTTCTTG-3' for D. pseudoobscura. Either of the primer pair was designed to be on an exon boundary to ensure PCR amplification only from the matured transcripts.
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Fig. 1. Schematic representation of genomic structure at the Obp57d/e locus and the GFP-reporter transgene. (A) Evolution at the Obp57d/e locus in the D. melanogaster species group (Matsuo, 2008). The phylogenetic relationship between species is based on Da Lage et al. (2007). Obp57d (cyan) and Obp57e (magenta) arose by duplication of an ancestral OBP gene (yellow) at the early stage of the melanogaster group evolution. Either of the two OBP genes was lost in some lineages. (B) Genomic structure at the Obp57d/e locus in the species that were selected for the analysis. Arrows indicate translation initiation sites (ATG), not transcription start sites except for Cpr57A and CG30148 in D. melanogaster. Solid lines under the genomic structure indicate promoter regions used for the GFP-reporter analysis. Numbers in parentheses indicate the distance in base pairs from the translation initiation site. The genomic sequence of D. rufa around the Obp57d/e locus was determined previously (Matsuo, 2008; DDBJ accession #AB370285). (C) Structure of the GFP-reporter transgene. Each promoter fragment was inserted into the pGreenPelican vector (Barolo et al., 2000), which is equipped with the gypsy transposon-derived insulators to minimize the interference on expression with surrounding genomic sequences.
3. Results
3.2. GFP reporter analysis of Obp57d and Obp57e expression patterns
3.1. Rapid evolution of Obp57d and Obp57e
In D. melanogaster, Obp57e was reported to be expressed in four cells on each leg (Matsuo et al., 2007; Galindo and Smith, 2001). By detailed examination of the GFP-reporter-carrying transgenic fly that was used in our previous study, however, we found that Dmel\ Obp57e>GFP is expressed in more than four cells in the prothoracic legs (Fig. 2A–C, Supplementary Figs. S1, S2). All GFP-expressing cells were associated with chemosensilla. According to the proposed nomenclature (Meunier et al., 2003), these chemosensilla were identified as pairs of 5 b, 5 s, and 4 s sensilla, and an outside-specific 4c sensillum. The 4c sensillum was reported to be male-specific (Meunier et al., 2003), but we found that the 4c also exists in females and Dmel\Obp57e>GFP was expressed in 4c of both sexes. GFP expression in 5 b, 4 s, and 4 c sensilla was observed in all the individuals, whereas the frequency of GFP-positive individuals was low for 5 s sensilla (Table 1). In mesothoracic and metathoracic legs, GFP expression was observed only in four cells associated with 5 b and 4 s sensilla, as previously reported (Fig. 2D–F, Supplementary Figs. S3, S4).
Matsuo (2008) revealed that Obp57d and Obp57e have undergone rapid evolution in the D. melanogaster species group (Fig. 1A). They arose by duplication of an ancestral OBP gene, which remains single in the D. obscura species group. After the subsequent diversification in the open reading frame (ORF) sequences, Obp57e was lost in the D. ananassae subgroup, while Obp57d was lost in the auraria-rufa lineage of the montium subgroup. The rest of the species in the melanogaster group maintain both genes. The following species were selected for analyses: D. melanogaster, D. simulans, and D. yakuba because they maintain both OBP genes; D. rufa because it has lost Obp57d; D. ananassae because it has lost Obp57e; and D. pseudoobscura because it maintains an ancestral Obp57d. According to the standard nomenclature, each OBP gene is designated as (species abbreviation)\(gene name); for example, the GFP-reporter transgene using the promoter of D. pseudoobscura Obp57d is designated as Dpse\Obp57d>GFP (Fig. 1C).
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Table 1 Frequency of GFP expression in the chemosensilla on the legs. Species
D. melanogaster
Reporter transgene (N of linesa)
Obp57dNGFP (3) Obp57eNGFP (1)
D. simulans
Obp57dNGFP (2) Obp57eNGFP (1)
D. yakuba
Obp57dNGFP (3) Obp57eNGFP (1)
D. rufa
Obp57eNGFP (2)
D. ananassae
Obp57dNGFP (3)
D. pseudoobscura
Obp57dNGFP (3)
Sex
female male female male female male female male female male female male female male female male female male
Mesothoracic leg
Metathoracic leg
5b
Prothoacic leg 5s
5t
4s
4c
4d
3b
2b
2c
5b
5t
4s
5b
5t
4s
0.07 0.12 1 1 0.99 1 0.98 0.93 0.01 0.02 1 0.88 0.94 0.99 0.98 0.98 1 0.98
0.01 0.01 0.05 0.03 0.04 0.08 0.05 0.08 0.08 0.07 0.05 0.05 0.79 0.99 0.01 0.04 0.96 0.93
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0.74 0.59
0.02 0.01 1 1 0.98 1 0.85 0.5 0 0 0.85 0.6 0.98 0.96 0.98 1 1 0.95
0.2 0.2 1 1 1 1 1 0.85 0.6 0.6 0.95 0.7 0.9 1 0 0 0.8 0.93
0 0 0 0 0 0 0 0 0 0 0 0 0 0.34 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0 0 0.81 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0 0 0.53 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0 0 0.45 0 0 0 0
0.02 0.03 0.98 1 1 1 1 0.9 0 0.03 0.95 0.6 0.96 0.99 0.98 1 0.99 0.99
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0.79 0.85
0 0 1 1 0.99 0.98 0.95 0.75 0 0 0.83 0.5 0.99 0.95 1 0.99 0.98 0.97
0 0 0.93 0.95 0.91 0.91 0.95 0.75 0 0 0.58 0.5 0.94 1 0.81 0.79 1 1
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0.71 0.77
0 0 0.83 0.9 0.8 0.85 0.78 0.5 0 0 0.28 0.18 0.91 1 0.76 0.75 1 0.97
Legs of ten males and ten females were examined for each line. a Number of examined transgenic lines.
We also reexamined the previously generated transgenic strain for Dsim\Obp57e>GFP (Matsuo et al., 2007), and found that the expression pattern was exactly the same as that of Dmel\Obp57e>GFP in the following terms (Table 1, Supplementary Figs. S5, S6): (1) In the prothoracic legs, GFP expression was observed in 5b, 4 s, and 4c sensilla at high frequency, and in 5 s at low frequency. (2) In the mesothoracic and metathoracic legs, GFP expression was observed only in 5 b and 4 s sensilla. (3) No difference was observed between sexes. Galindo and Smith (2001) reported that Obp57d and Obp57e are expressed in the same cells associated with 5 b and 4 s chemosensilla (Galindo and Smith, 2001). Following the identification of Obp57e expression in more chemosensilla, however, it should be reexamined whether Obp57d is also expressed in these newly found chemosensilla. To this end, we generated Dmel\Obp57d>GFP and Dsim\Obp57d> GFP transgenic strains. The frequency of GFP-positive cells was quite low for Dmel\Obp57d>GFP, whereas that for Dsim\Obp57d>GFP was as high as that for Obp57e>GFP (Table 1, Supplementary Figs. S7–S9). For Dmel\Obp57d>GFP, GFP expression was not observed in 4 s sensilla on mesothoracic legs, and in 5b and 4 s on metathoracic legs. Except for this difference, the Obp57d expressing sensilla were essentially identical to that of Obp57e in both species. We then generated GFP reporter using the promoter sequences of Obp57d and Obp57e from other species. D. yakuba belongs to the melanogaster subgroup and maintains both Obp57d and Obp57e. The expression patterns were essentially the same as those of D. melanogaster, except for 4 s sensilla on prothoracic legs and 5b on mesothoracic legs where Dyak\Obp57d>GFP expression was not observed (Table 1, Supplementary Figs. S10–S12). D. rufa belongs to the montium subgroup and maintains only Obp57e. GFP expression from the Drua\Obp57e>GFP transgene was observed in more sensilla than in the case of D. melanogaster. In addition to 5b, 5s, 4s, and 4c sensilla, GFP expression was also observed in 4d, 3b, 2b, and 2c sensilla on the prothoracic leg (Table 1, Fig. 2G–L, Supplementary Figs. S13–S16). Interestingly, these expressions in the additional sensilla were observed only in males. D. ananassae belongs to the ananassae subgroup and maintains only Obp57d. GFP expression was observed in a similar set of sensilla as those observed for Dsim\Obp57d>GFP, except for 4c on the prothoracic leg where Dana\Obp57d>GFP was not expressed (Table 1, Supplementary Figs. S17, S18). D. pseudoobscura belongs to the obscura group, an outgroup of the melanogaster group. D. pseudoobscura has Obp57d, which is an ancient form of both Obp57d and Obp57e, and thus distinct from Obp57d in the
melanogaster group (Matsuo, 2008). GFP expression from Dpse \Obp57d>GFP was observed in 5t sensilla in addition to 5b, 5s, 4s, and 4c sensilla (Table 1, Fig. 2M–O, Supplementary Figs. S19, S20). The results of GFP reporter analysis in the legs are summarized as follows: expression in 5b and 4s sensilla of all legs and additional expression in 5s and 4c in the prothoracic legs were observed for all GFP-reporter transgenes. The conserved expression pattern among the GFP-reporter transgenes with promoter sequences from various species indicates that the cis-regulatory function of the promoter region is essentially conserved among these species. Nevertheless, the proportion of GFP-positive sensilla was different among the GFPreporter transgenes, suggesting that the activity level of cis-regulatory elements is different between promoter sequences from different species. In addition, D. rufa and D. pseudoobscura promoter sequences resulted in the expression in the extra sensilla, suggesting that the cisregulatory elements in these species have a slightly diverged function from those of other species. 3.3. Morphological identification of gustatory sensilla on the legs in each species Because GFP reporter expression was observed in the D. melanogaster background, it was necessary to ensure that the GFP-positive sensilla actually exist in the original species. Morphological examination of the prothoracic legs revealed that there is a significant difference in the number and position of chemosensilla between sexes and species (Fig. 3). It was confirmed, however, that GFP-positive 5b, 5s, 4c, and 4s sensilla are well conserved in terms of their number and position among species and sexes, suggesting that the OBP expression itself is also conserved in these sensilla. In contrast, OBP expression in the sensilla that exist in the original species but not in D. melanogaster may have been overlooked in our GFP reporter analysis. 3.4. GFP reporter expression in the labellum For promoters from species that belong to the melanogaster subgroup (D. melanogaster, D. simulans, and D. yakuba), the expression of the GFP reporter was restricted to the legs. To our surprise, however, the promoters from D. rufa, D. ananassae, and D. pseudoobscura resulted in the GFP expression not only in the legs but also in the labellum, the most distal structure of the fly's mouthparts (Fig. 4). Like in the legs, GFP-expressing cells in the labellum were associated with the gustatory sensilla, which are classified into three types on the
J. Yasukawa et al. / Gene 467 (2010) 25–34
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Fig. 2. GFP-reporter expression in the legs. GFP expression from each reporter transgene was observed in the D. melanogaster background. Arrows indicate sockets of sensilla. Bar = 50 μm. (A–C) Dmel\Obp57e N GFP expression in the female prothoracic leg. Lateral (outside) view of the right leg is shown. (D–F) Dmel\Obp57e N GFP expression in the female mesothoracic leg. Lateral view of the right leg is shown. (G–L) Drua\Obp57e N GFP expression in the male prothoracic leg. (G–I) Lateral view of the right leg is shown. (J–L) Dorsal view of the fourth tarsal segment is shown. (M–O) Dpse\Obp57d expression in the male metathoracic leg. Medial view of the left leg is shown. The 5t sensillum was not identified in the previous study (Meunier et al., 2003). Although it was not physiologically confirmed as chemosensilla, we named it expediently to score the GFP expression in this sensillum.
basis of their morphology (Hiroi et al., 2002). GFP expression was most frequently observed in the L-type sensilla in all the three species. The frequencies of GFP-expressing sensilla were low for I-type and Stype sensilla for Drua\Obp57e>GFP and Dana\Obp57d>GFP, while it was high for Dpse\Obp57d>GFP (Fig. 4G–I). Morphological examination of chemosensilla on the labellum confirmed that the number and position of L-type sensilla are well conserved among species (Fig. 5), while those of I-type and S-type sensilla were variable. 3.5. Direct observation of OBP expression in the original species In our GFP reporter analysis, the function of promoters from the other species was examined in the D. melanogaster background. Thus, it was possible that trans-regulatory factors in D. melanogaster functioned less properly on cis-regulatory elements of the more distantly related species, resulting in wider expression patterns in the D. melanogaster background than in the original species. To exclude this possibility, OBP expression in the labellum was directly examined by RNA in situ hybridization. In consistent with the result of GFP reporter analysis, OBP transcripts were detected in the labellum of D. pseudoobscura, D. ananassae, D. rufa, but not in that of D. melanogaster (Fig. 6). The stained cells were associated to L type sensilla when it was able to be identified.
The Obp57d and Obp57e transcript levels in the other tissues were determined by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) analysis (Fig. 7). OBP transcripts were detected in the legs from all the tested species. The transcript levels were higher in D. rufa and D. pseudoobscura than in the other species, in consistent with the result of the GFP reporter analysis showing that more sensilla express GFP in these species (Table 1). OBP transcripts were also detected in the labellum of all the tested species, but at an extremely low level in D. melanogaster, in consistent with the results of GFP reporter analysis and RNA in situ hybridization in which OBP expression was not observed in the labellum of D. melanogaster. The transcript levels were low in the wings and antennae, again in consistent with the result of the GFP reporter analysis in which expression of GFP was not observed in these tissues. 4. Discussion 4.1. GFP-reporter expression reproduced the intrinsic OBP expression patterns In the GFP-reporter analysis, we used 5' region of the OBP genes. Thus, our results do not reflect the effect of cis-regulatory elements in the 3' and intronic regions, if there are any of such elements.
30 J. Yasukawa et al. / Gene 467 (2010) 25–34 Fig. 3. Morphological identification of chemosensilla on the legs. Number and position of chemosensilla in each species were examined by scanning electron microscopy (SEM). Chemosensilla and other bristles were distinguished on the basis of their morphology (shape of shaft and socket), not by physiological analyses. (A–H) Schematics of chemosensilla positions on the legs. Name of each sensillum was assigned by relative position to other sensilla (Meunier et al., 2003). Dorsal view of the right prothoracic leg is shown (medial side is upward). Circles indicate position of chemosensilla. Each pair of symmetrical chemosensilla is connected by a line. Filled circles indicate previously unidentified sensilla that show chemosensilla-like morphology. Because they are not physiologically confirmed as chemosensilla, however, they are unnamed except for 5t sensilla, in which the GFP expression was observed for Dpse\Obp57d N GFP. (I–P) SEM images of dorsal view of the right prothoracic legs. Arrows indicate sockets of chemosensilla. Arrowheads indicate sockets of previously unidentified chemosensilla (corresponding to the filled circles in A–H). (Q–X) Lateral (outside) view of the prothoracic legs. (Y-e) Basolateral view of the fifth tarsal segment. Bar = 50 μm.
J. Yasukawa et al. / Gene 467 (2010) 25–34
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Fig. 4. GFP-reporter expression in the labellum. (A-C) GFP expression from the reporter transgenes in D. melanogaster background. Lateral view of the labellum, the most distal structure of the fly's mouthparts, is shown. Anterior is to the right. Bar = 30 μm. (D–F) Schematics of chemosensilla positions on the labellum. The number and position of L-type sensilla were highly stable. The other sensilla were identified on the basis of their relative position to L-type sensilla. The S0 sensillum was previously unidentified (Hiroi et al., 2002), and S9 sensilla was not found in our observation. The fill colors of symbols indicate the frequency of GFP expression at each sensillum: black for more than 0.1, gray for less than 0.1, and white for no GFP expression. (G–I) Frequency of GFP expression in each chemosensillum. Twenty individuals (10 females and 10 males) were examined. No difference was observed between sexes, and the data were pooled. Counts for S0 and S1 were pooled.
Nevertheless, the GFP expression patterns in labella were confirmed by direct observation of transcripts using RNA in situ hybridization. In addition, the GFP expression patterns matched with the transcript levels in various tissues determined by quantitative RT-PCR analysis. Furthermore, in D. melanogaster, the higher frequency of GFP-positive
cells for Obp57e>GFP than Obp57d>GFP was also consistent with the higher transcript level for Obp57e than Obp57d. All of these results support that the 5' region used in our analysis is sufficient to reproduce the intrinsic expression patterns for Obp57d and Obp57e. However, we should note that in the case of Dsim\Obp57d, the high
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J. Yasukawa et al. / Gene 467 (2010) 25–34
Fig. 5. Morphological identification of chemosensilla on the labellum. Number and position of chemosensilla in each species were examined by scanning electron microscopy. Three types (L, I, and S) were distinguished on the basis of size (Hiroi et al., 2002). Sensilla of each type were numbered from the apical to the basal side; thus, the sensilla of different species with the same name do not necessarily correspond to each other.
frequency of GFP-positive cells in the reporter analysis was not supported by the other methods. In our previous study (Matsuo et al., 2007), the amount of Dsim\Obp57d transcripts was below the detection level in quantitative RT-PCR analysis. Interestingly, when the D. simulans genomic fragment including the Obp57d/e region was introduced into D. melanogaster background by P element-based transformation, now Dsim\Obp57d transcripts were detected at a level slightly higher than that of Dsim\Obp57e. Although it should be examined further, the observed alteration of the expression level is supposed to be caused by the difference in trans-acting factors between species. 4.2. Variation in the promoter sequence controlling the expression of Obp57d and Obp57e In the GFP reporter analysis, the promoters of D. rufa, D. ananassae, and D. pseudoobscura resulted in different expression patterns from
those of D. melanogaster promoters, even though the GFP expression was controlled by a same set of trans-acting factors in the D. melanogaster background. Thus, the observed variation in the GFP expression patterns should be caused by the evolution in cisregulatory elements that reside within the relatively short promoter regions used in our reporter analysis. Comparative analyses of the promoter sequences from the species used in this study, as well as of those from other closely related species, by using OLIGO-ANALYSIS and DYAD-ANALYSIS (van Helden et al., 1998; van Helden et al., 2000), however, failed to find any conserved motifs (data not shown). In our previous study, a 4 bp insertion was found in the D. sechellia Obp57e promoter region, and the removal of the insertion restored the promoter activity in the D. melanogaster background, suggesting that the sequence around this insertion site is important for the Obp57e expression (Matsuo et al., 2007). However, the promoter sequence of D. yakuba Obp57e does not have significant homology in this region, while it resulted in the similar expression pattern as those of D.
J. Yasukawa et al. / Gene 467 (2010) 25–34
33
Fig. 6. Detection of OBP transcripts by RNA in situ hybridization in the labellum of original species. (A) Dpse\Obp57d anti-sense probe. (B) Dana\Obp57d anti-sense probe. (C) Drua\Obp57e anti-sense probe. (D) Dpse\Obp57d sense probe (negative control). (E) Dmel\Obp57d anti-sense probe. (F) Dmel\Obp57e anti-sense probe.
melanogaster and D. simulans Obp57e. Thus, although the function of cis-regulatory elements seems to be conserved, their positions would be variable among species. An experimental approach, such as the GFP reporter analysis with shorter promoter sequences, may be required to identify the position of the cis-regulatory elements. 4.3. Expression patterns and the evolutionary fate of duplicated genes There are two alternative evolutionary consequences of gene duplication: degeneration of either copy or maintenance of both
(Lynch and Conery, 2000). Functional divergence between the duplicates prevents them from degeneration, because both genes independently contribute to the fitness and both are necessary for survival of the organism. Functional divergence occurs not only in the structure of the encoded gene products but also in the expression patterns; if the expression patterns of the duplicated genes have diverged from each other, loss of either gene may not be complemented by the other, even though their structures are similar. Obp57d and Obp57e arose by gene duplication, and the comparisons of their ORF sequences showed that their structures have diverged from each
Fig. 7. Quantitative analysis of the transcript level in various tissues. Levels of Obp57d and Obp57e transcripts in the tissues carrying chemosensilla were determined by quantitative RT-PCR. Primers were independently designed for each species. Transcript levels are shown as relative to that of the Ribosomal protein L32 gene. Each bar represents the mean of three to twelve independent replicates. Error bars indicate standard error.
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other, suggesting that Obp57d and Obp57e have acquired different functions (Matsuo, 2008). In contrast, as shown in this study, their expression patterns overlap each other, suggesting that the expression patterns might not be involved in the functional divergence between Obp57d and Obp57e. Nevertheless, the observed expression frequency was low for Dmel\Obp57d>GFP and Dyak\Obp57d>GFP. Further analysis is required to examine a possible effect of the diverged expression levels on preservation of the both OBP genes. Although the difference in the expression pattern was not observed between the duplicated genes, another type of association between the expression pattern and the gene duplication was observed in this study. In the species that maintain both Obp57d and Obp57e, the expression was restricted to the chemosensilla on the legs, while in the species that lost either gene, as well as in D. pseudoobscura that maintains an ancestral Obp57d gene, the additional expression in the chemosensilla on the labellum was observed. In D. melanogaster, biological roles of Obp57d and Obp57e have been shown by the behavioral analyses of the knock-out mutants of these OBP genes. Obp57d and Obp57e are involved in the egg-laying site determination by their function in the taste detection of toxic octanoic acid in the medium, while they had no effect on the feeding response to octanoic acid (Harada et al., 2008). It may be interesting to see if the ectopic expression of Obp57d and Obp57e in the labellum changes the feeding response of D. melanogaster. Further functional analyses of Obp57d and Obp57e in the evolutionary context may also elucidate a possible link between the evolution of expression pattern and gene duplication. 5. Conclusions We have analyzed the spatial expression patterns of Obp57d and Obp57e in the closely related Drosophila species. In addition to the expression in the legs as previously observed in D. melanogaster, expression in the labellum was observed in some species including D. pseudoobscura, which maintains an ancestral OBP gene at the Obp57d/e locus, showing that the expression patterns of Obp57d and Obp57e have diverged substantially between closely related Drosophila species. Acknowledgments This work was supported by a grant-in-aid for Young Scientists (B) 19770210 from the Ministry of Education, Culture, Sports, Science and Technology, Japan to TM. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.gene.2010.07.006. References Barolo, S., Carver, L.A., Posakony, J.W., 2000. GFP and β-galactosidase transformation vectors for promoter/enhancer analysis in Drosophila. Biotechniques 29, 726–732.
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