Zebrafish β-adrenergic receptor mRNA expression and control of pigmentation

Gene 446 (2009) 18–27

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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

Zebrafish β-adrenergic receptor mRNA expression and control of pigmentation Zhipeng Wang a, Yuhei Nishimura a,b,c, Yasuhito Shimada a,b,c, Noriko Umemoto a, Minoru Hirano a, Liqing Zang a, Takehiko Oka a, Chikara Sakamoto a, Junya Kuroyanagi a, Toshio Tanaka a,b,c,⁎ a b c

Department of Molecular and Cellular Pharmacology, Pharmacogenomics and Pharmacoinformatics, Mie University Graduate School of Medicine, Tsu, Mie, Japan Department of Bioinformatics, Mie University Life Science Research Center, Tsu, Mie, Japan Department of Medical Chemogenomics, Mie University Venture Business Laboratory, Tsu, Mie, Japan

a r t i c l e

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Article history: Received 29 January 2009 Received in revised form 12 June 2009 Accepted 15 June 2009 Available online 18 June 2009 Received by M. Schartl Keywords: Zebrafish beta-Adrenergic receptors Pigmentation F1F0-ATPase complex

a b s t r a c t Beta adrenergic receptors (β-ARs) are members of the G-protein-coupled receptor superfamily and mediate various physiological processes in many species. The expression patterns and functions of β-ARs in zebrafish are, however, largely unknown. We have identified zebrafish β-AR orthologs, which we have designated as adrb1, adrb2a, adrb2b, adrb3a and adrb3b. adrb1 was found to be expressed in the heart and brain. Expression of adrb2a predominated in the brain and skin, whereas adrb2b was found to be highly expressed in muscle, pancreas and liver. Both adrb3a and adrb3b were exclusively expressed in blood. Knock-down of these β-ARs by morpholino oligonucleotides revealed a functional importance of adrb2a in pigmentation. Expression of atp5a1 and atp5b, genes that encode subunits of F1F0-ATPase, which is known to be involved in pigmentation, was significantly increased by knock-down of adrb2a. Our data suggest that adrb2a may regulate pigmentation, partly by modulating F1F0-ATPase. © 2009 Elsevier B.V. All rights reserved.

1. Introduction Beta adrenergic receptors (β-ARs) are members of the G-proteincoupled receptor superfamily. β-ARs mediate various physiological processes and are involved in many diseases, including hypertension, cardiovascular diseases, nocturnal asthma, type II diabetes mellitus, obesity, and hyperpigmentation (Dulloo, 2002; Xiang and Kobilka, 2003; Grando et al., 2006; Sivamani et al., 2007; Taylor, 2007). Animal models with genetic manipulations of β-ARs are important to understand the signaling pathways involved in these diseases (Rohrer et al., 1996; Chruscinski et al., 1999; Rohrer et al., 1999; Bachman et al., 2002). Although these rodent models have contributed to our understanding of the functions of β-ARs in various pathophysiologies, several aspects of rodent biology limit the use of rodent models in large-scale genetic and therapeutic screening (Lieschke and Currie, 2007). Being vertebrates, zebrafish have been used to model various human diseases (Lieschke and Currie, 2007; Tanaka et al., 2008);

Abbreviations: β-AR, beta adrenergic receptor; MC, melanocyte; hpf, hours postfertilization; dpf, days post-fertilization; mpf, months post-fertilization; EST, expressed sequence tag; Ct, threshold cycle; TM, transmembrane domain; Mbp, megabase pairs; mM, millimole; μg, microgram; WT, wild-type; MO, morpholino oligonucleotide; 5-mis MO, 5-base mismatch morpholino oligonucleotide; qPCR, quantitative real-time polymerase chain reaction; FSGD, fish-specific genome duplication; FFA, free fatty acid; cAMP, cyclic adenosine monophosphate; CREB, cAMP response element binding protein; MITF, microopthalmia-associated transcription factor; TYR, tyrosinase. ⁎ Corresponding author. 2-174 Edobashi, Tsu, Mie 514-8507, Japan. Tel.: +81 59 231 5411; fax: +81 59 232 1765. E-mail address: [email protected] (T. Tanaka). 0378-1119/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.gene.2009.06.005

indeed, their organs are functionally and morphologically similar to those of humans (Shin and Fishman, 2002). Therefore, it is important to identify β-ARs in zebrafish and characterize their functions. The objectives of this study were i) to identify zebrafish genes that encode β-ARs, and ii) to characterize the functions of the zebrafish βARs. We identified five zebrafish β-AR genes, which we have designated as adrb1, adrb2a, adrb2b, adrb3a, and adrb3b, and our analyses suggest that three subtypes of β-AR arose before the divergence of tetrapods and ray-finned fish. The expression profiles of the five β-AR genes revealed that the mRNA tissue distributions of adrb1, adrb2a, and adrb2b were comparable with those in humans. Expression of adrb3a and adrb3b was restricted to blood, consistent with a previous report showing high expression of rainbow trout adrb3 in erythrocytes (Nickerson et al., 2003). To characterize the functions of these β-ARs in zebrafish, we knocked down each β-AR using morpholino antisense oligonucleotides and analyzed the phenotypic abnormalities. The analysis revealed that knock-down of adrb2a induced significant hypopigmentation, consistent with previous reports showing important functions of β2-AR in pigmentation (Fujii, 2000; Schallreuter et al., 2008). It has been shown that human melanocytes (MCs) express ADRB2 and that stimulation of ADRB2 with salbutamol promotes pigmentation in human MCs (Gillbro et al., 2004). Also, ADRB2 expression was induced in response to ultraviolet radiation in human MCs (Yang et al., 2006). Furthermore, ADRB2 has been identified as a melanoma-specific dysregulated gene by at least three microarray studies (Györffy and Lage, 2007). Research on pigmentation using zebrafish is a rapidly evolving field (Pickart et al., 2004), and both forward and reverse

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genetic analyses have identified many genes that influence the development and migration of melanophores (Pickart et al., 2004). Chemical screens using zebrafish have also been performed and have identified novel pigmentation modulators (Jung et al., 2005; NiKomatsu and Orlow, 2007). However, to date no molecular evidence has been presented showing that adrb2 has a role in fish pigmentation. Therefore, we screened genes potentially involved in the pigmentation regulated by adrb2a and revealed that expression of atp5a1 and atp5b, genes that encode subunits of F1F0-ATPase, were induced by knockdown of adrb2a in zebrafish. It has been shown that F1F0-ATPase modulates pigmentation negatively in both zebrafish and cultured mammalian melanocytes (Jung et al., 2005; Ni-Komatsu and Orlow, 2007). These data suggest that adrb2a may regulate pigmentation, partly by modulating F1F0-ATPase. 2. Materials and methods 2.1. Fish strains The zebrafish used were the AB strain supplied by the Zebrafish International Resource Center (University of Oregon, Eugene, OR, USA). Fish and embryos were maintained in our own facility according to standard procedures (Westerfield, 2000). Embryos were staged based on equivalent hours post-fertilization (hpf) at 28.5 °C (Kimmel et al., 1995). 2.2. Cloning and sequencing of zebrafish β-AR genes Potential zebrafish β-AR genes were identified by TBLASTN searches of the zebrafish genome assembly with the human β-AR amino acid sequences (ADRB1, NP_000675; ADRB2, NP_000015; ADRB3, NP_000016). The top ten ranked hits were then used in a reciprocal search against the human assembly. The zebrafish hits that identified the original human sequence as the best hit were then designated as orthologs. We identified five different β-AR genes in the zebrafish genome. The gene structures of these β-ARs were subsequently predicted using Genscan (Burge and Karlin, 1997), and were then further refined by comparing these predictions with EST evidence. Primers were designed to amplify the putative open reading frames of these β-ARs (Supplementary Table 1). To clone zebrafish β-AR genes, total RNA from 1-month postfertilization (mpf) zebrafish was prepared using Isogen reagent (Nippon Gene, Tokyo, Japan) and then treated with RNase-Free DNase (QIAGEN, Venlo, The Netherlands). Reverse transcription was performed using Superscript II Reverse Transcriptase (Invitrogen, Carlsbad, USA). PCR reactions were performed using LA Taq (TaKaRa Bio, Shiga, Japan). Non-reverse transcribed total RNA was used as a negative control. The PCR reactions were analyzed on 1.0% agarose gels and single bands were identified, excised, and purified using the Wizard SV Gel and PCR Clean-Up System (Promega, Madison, USA). The fragments were A/T-ligated using a DNA Ligation kit Ver2.1 (TaKaRa Bio, Shiga, Japan), and introduced into XL-1 Blue supercompetent cells (Stratagene, San Diego, USA). Plasmid DNA was isolated using the Wizard Plus SV Minipreps DNA Purification System (Promega). Sequencing was performed using a CEQ 2000 sequencer (Beckman Coulter, Fullerton, USA). Raw sequence assembly and more detailed sequence analyses were performed using the Sequencher software package (Gene Codes, Ann Arbor, USA). 2.3. Genomic and phylogenetic analysis Zebrafish β-AR sequences were used as queries to search for potential homologs in other teleost genomes using the TBLASTN program (http://www.ensembl.org). The following teleost genome assemblies were included in this study: zebrafish (Zv7: http://www.

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sanger.ac.uk/Projects/D_rerio/Zv7_assembly_information.shtml), medaka (version 1.0: http://dolphin.lab.nig.ac.jp/medaka/), takifugu (version 4.0: http://genome.jgi-psf.org/Takru4/Takru4.home.html), stickleback (gasAcu1.0: http://www.broad.mit.edu/models/stickleback) and tetraodon (v7: http://www.genoscope.cns.fr/externe/ tetranew/). β-AR homologs were predicted based on similarity to zebrafish β-ARs. Phylogenetic analysis was performed based on these homologs and the β-AR sequences of human, mouse, chicken and frog, which were available from the public genome databases. Phylogenetic reconstruction was performed with ClustalX 2.0.10 (http://www.clustal.org/) using the neighbor-joining method. The protein weight matrix setting used was “Gonnet” and default settings of other parameters were used. Bootstrap sampling was reiterated 1000 times. Trees were drawn using TreeView (Page, 1996). Maximum-likelihood phylogenetic analysis was performed using TREE-PUZZLE 5.2 (Schmidt et al., 2002). The following program settings were used: quartet puzzling tree search, approximate quartet likelihood, 10,000 puzzling steps, outgroup = the amphioxus dopamine D1/β-AR sequence, no clocklike branch lengths, and the VT model of substitution (Müller and Vingron, 2000). Amino acid frequencies were estimated from the data set, and the model of rate heterogeneity was 1 invariable + 8 gamma rates. Sequence analyses were performed on database entries with the following accession numbers: ADRB1: human (NP_000675), mouse (NP_031445), frog (NP_001084152), chicken (ENSGALP00000014538), Oryzias latipes (medaka) (ENSORLP00000006043), Gasterosteus aculeatus (stickleback) (ENSGACP00000008698), Takifugu rubripes (Fugu) (ENSTRUP00000031392), Tetraodon nigroviridis (tetraodon) (ENSTNIP00000022083), zebrafish (AB294717). ADRB2: human (NP_000015), mouse (NP_031446), rainbow trout (NP_001117912), chicken (XP_425195). ADRB2A: medaka (ENSORLP00000009383), stickleback (ENSGACP00000024398), Fugu (ENSTRUP00000036148), tetraodon (ENSTNIP00000020193), zebrafish (AB294718). ADRB2B: medaka (ENSORLP00000002752), stickleback (ENSGACP00000027046), Fugu (ENSTRUP00000022798), zebrafish (AB469813). ADRB3: human (NP_000016), mouse (NP_038490), chicken (XP_428541), medaka (ENSORLP00000014229), stickleback (ENSGACP00000014582), Fugu (ENSTRUP00000020757). ADRB3A: zebrafish (AB469812), rainbow trout (NP_001118100). ADRB3B: zebrafish (AB294719), rainbow trout (NP_001117924). Dopamine D1/β-AR receptor: Branchiostoma floridae (amphioxus) (AAQ91625). 2.4. Analysis of conserved synteny The genes neighboring those encoding human and zebrafish β-ARs were identified using the NCBI Sequence Viewer (http://www.ncbi. nlm.nih.gov/projects/sviewer/). Pairs of predicted orthologous Entrez Gene IDs were identified using the HomoloGene (http://www.ncbi. nlm.nih.gov/homologene/) and UC Santa Cruz assembly (http:// genome.ucsc.edu/) databases. 2.5. qPCR analysis Total RNA was extracted from the tissues and organs of 6 mpf zebrafish to determine their tissue expression profiles, from whole bodies of zebrafish at different time points to determine their developmental profiles, and from whole bodies of 26 and 36 hpf adrb2a morphants to investigate the expression of pigmentationrelated genes. The RNA was then treated with RNase-Free DNase (QIAGEN). cDNA was synthesized from total RNA using random primers and SuperScript II Reverse Transcriptase (Invitrogen). Quantitative real-time polymerase chain reaction (qPCR) was performed on triplicate samples of cDNA using Power SYBR Green PCR Master Mix (Applied Biosystems, Foster City, USA). Gene-specific primers for qPCR were designed using Perlprimer (Marshall, 2004) (Supplementary Table 1). The fluorescence signal intensities were recorded using an

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ABI PRISM® 7700 Sequence Detection System (Applied Biosystems). Typically, cycling parameters were 95 °C for 10 min to activate DNA polymerase, then 40 cycles of 95 °C for 15 s, 55 °C for 30 s and 72 °C for 30 s. Data were analyzed using SDS 1.6.3 (Applied Biosystems). The baseline and threshold were set manually according to the manufacturer's instructions. The relative amount of cDNA in a sample was calculated by comparing the threshold cycle (Ct) in the sample against a standard curve generated from a series of 1:4 diluted cDNA prepared from a concentrated pool of zebrafish cDNA. To ensure that SYBR Green was not being incorporated into primer dimers or non-specific amplicons during qPCR, PCR products were analyzed by agarose gel electrophoresis in initial experiments; single bands of the expected size were obtained in all instances. Furthermore, the construction of SYBR Green dissociation curves after the completion of 40 cycles revealed the presence of single amplicons for each primer pair. The relative expression of mRNA levels was determined using β-actin as an endogenous standard. The 15 genes related to pigmentation were selected based on the following criteria: i) genes with a central role in melanogenesis, (for example, tyr); ii) pigmentary genes validated in zebrafish (for example, kitlga, slc24a5 and atp7a); and iii) genes suggested by a literature review to possibly be responsible for the hypopigmentation induced by adrb2a knock-down. PathwayStudio (Ariadne Genomics, Rockville, USA) was used to identify genes satisfying the third criterion. 2.6. Microinjection of MOs Morpholino oligonucleotides (MOs) (Gene Tools LLC, Oregon, USA) were designed to inhibit the predicted translational start sites of zebrafish β-ARs (Supplementary Table 2). MO microinjection was performed according to common practice (Nasevicius and Ekker, 2000) with the following modifications: approximately 1 nl of solution containing morpholino and 0.1% Phenol Red was injected into 1–4-cell stage embryos. MOs were used at concentrations of 0.1, 0.2, 0.4 and 0.8 mM (about 0.8, 1.7, 3.4 and 6.7 ng/embryo) and suitable doses were determined based on the phenotypes of each morphant. We decided to inject morpholino as follows: 1.7 ng/ embryo for adrb2a, adrb2b and adrb3b, 6.7 ng/embryo for adrb3a and two doses (1.7 ng and 6.7 ng/embryo) for adrb1. 2.7. Melanin contents and image analysis Melanin contents were spectrometrically determined as described previously (Choi et al., 2007) except for the following modifications: five zebrafish larvae, at 72 hpf, were used per measurement, and pellets were dissolved in 0.2 ml of 1 N NaOH. Measurements were made in polystyrene 96-well flat-bottomed plates (Greiner Bio One, Frickenhausen, Germany) using a Varioskan Flash® spectral scanning multimode reader (Thermo Electron, Waltham, USA). Zebrafish larvae were partially immobilized in 3% methylcellulose and photographs were taken using a DS-2Mv digital camera mounted on an SMZ800 stereo-microscope (Nikon, Tokyo, Japan). Larval yolk width was measured using NIS Elements D2.20 image analysis software (Nikon). 2.8. Heart rate measurement Among the five zebrafish β-AR genes we identified, adrb1 and adrb2a were highly expressed in heart, so we examined the effects of knock-down of adrb1 and adrb2a on heart rate. Embryos injected with 1.7 ng of MO and wild-type embryos born in the same lot to the same parent fishes were subjected to heart rate measurements. Twelve randomly selected pairs of fish were included. Heart rate was measured using the following method at 2, 3, 4 and 5 dpf. Data were statistically analyzed using paired t-tests.

Zebrafish larvae were partially immobilized in 3% methylcellulose and transferred onto a Thermo plate MATS-55AXKAF20 (Tokai Hit, Shizuoka, Japan) at least 1 min before measurement. The temperature was set to the incubation temperature of the larvae (28.5 °C). Timelapse images of heart beating were captured using an AXIOCAM MRC digital camera (Carl Zeiss, Oberkochen, Germany) mounted on an inverted microscope (Zeiss Axiovert 200M) (Carl Zeiss). Image capture was controlled by the AxioVision Rel. 4.7 package (Carl Zeiss). Photographs were continuously captured over a 15-second period for each measurement, typically producing 344 frames per measurement. Heart rates were determined by reviewing the movies. 3. Results 3.1. cDNA cloning of zebrafish β-ARs The protein sequences of human ADRB1, ADRB2 and ADRB3 were used as queries for BLAST searches of the translated zebrafish genomic DNA. These searches revealed potential orthologs of β-ARs in the zebrafish genome; one for ADRB1, two for ADRB2, and two for ADRB3. RT-PCR analysis revealed that the five predicted β-AR genes were expressed in zebrafish. Sequence analysis of the PCR products confirmed that they were zebrafish orthologs of β-ARs (Supplementary Fig. S1). We named the five β-AR genes: adrb1, adrb2a, adrb2b, adrb3a and adrb3b (DDBJ accession numbers AB294717, AB294718, AB469813, AB469812 and AB294719, respectively). The overall percentage amino acid sequence identities between zebrafish and human sequences were 50.7% (adrb1 and ADRB1), 54.5% (adrb2a and ADRB2), 52.9% (adrb2b and ADRB2), 42.8% (adrb3a and ADRB3), and 38.8% (adrb3b and ADRB3). The percentage amino acid sequence identities in the seven transmembrane domains (TM) between these five zebrafish genes and their human orthologs were 77.3%, 76.0%, 73.4%, 64.8%, and 62.3%, respectively. β-ARs in fish have previously been reported to be encoded by adrb2 (Nickerson et al., 2001), adrb3a and adrb3b (Nickerson et al., 2003) in Oncorhynchus mykiss (rainbow trout), and adrb1, adrb2 and adrb3 in Ameiurus melas (black bullhead) (Dugan et al., 2008). The amino acid sequences of rainbow trout and black bullhead adrb2 are more similar to zebrafish adrb2b than adrb2a (Supplementary Table 3), suggesting that rainbow trout and black bullhead adrb2 may be orthologs of zebrafish adrb2b. The amino acid sequences of rainbow trout adrb3a and adrb3b and black bullhead adrb3 are more similar to zebrafish adrb3a than adrb3b (Supplementary Table 3), suggesting that the two rainbow trout adrb3 genes and the black bullhead adrb3 gene may be orthologs of zebrafish adrb3a. The two orthologs of rainbow trout β3-AR are reported to have 84% overall amino acid sequence identity with each other (Nickerson et al., 2003), whereas the two orthologs of zebrafish β3-AR were found to have 47.4% overall amino acid sequence identities with each other. 3.2. Genomic and phylogenetic analyses To investigate if the β-ARs identified in zebrafish reflected a common characteristic of teleost fish, we searched for orthologs of βARs in other fish species. Using the BLAST algorithm, we searched the public genome databases for Fugu, medaka, stickleback and tetraodon. The analysis predicted one ortholog of adrb1 and two orthologs of adrb2 in these four fish species and one ortholog of adrb3 in Fugu, medaka and stickleback. Based on the sequence similarity to zebrafish adrb2, we designated the predicted two orthologs of adrb2 in these fish as adrb2a and adrb2b. To obtain a more complete picture of β-AR evolution we performed phylogenetic analysis of β-ARs using the neighbor-joining method (Fig. 1) and maximum-likelihood algorithm (Supplementary Fig. S2). These analyses demonstrated that β1, β2 and β3-ARs were clearly distinguished. For each β-AR subtype, fish orthologs showed higher

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Fig. 1. Unrooted phylogenetic trees of β-ARs constructed using the neighbor-joining algorithm. Analysis was based on an alignment of amino acids from TM 1 to TM 7. Main branches corresponding to β1-AR, β2-AR and β3-AR are indicated. Zebrafish β-ARs are boxed. Percentage frequencies that were replicated after 1000 iterations are indicated. Scale bar indicates 10% sequence dissimilarity. Abbreviations: Dre (Danio rerio), Gac (Gasterosteus aculeatus), Gga (Gallus gallus), Hsa (Homo sapiens), Mmu (Mus musculus), Ola (Oryzias latipes), Omy (Oncorhynchus mykiss), Tru (Takifugu rubripes), Tni (Tetraodon nigroviridis), Xtr (Xenopus tropicalis).

similarity to each other than to those of mammals (Fig. 1 and Supplementary Fig. S2).

3.3. Syntenic analysis Because synteny between zebrafish and human genomes is relatively well conserved (Postlethwait et al., 2000; Woods et al., 2000), we compared the syntenic relationships of every probable or possible orthologous pair to provide a more definitive assessment of orthology. We found that synteny was conserved between three zebrafish β-AR genes, adrb1, adrb2a, and adrb2b, and their human counterparts (Fig. 2). Zebrafish adrb1 was located in a well conserved syntenic group, including orthologs of human DCLRE1A, NHLRC2, ADRB1, C10orf118, TDRD1, VWA2, AFAPIL2 and ABLIM1 (Fig. 2A). Portions of the chromosomes that harbor zebrafish adrb2a and adrb2b showed conserved syntenies with the region of human chromosome 5 that contains ADRB2. Numerous other orthologous genes can be found in these regions of conserved synteny; however, a considerable amount of reorganization is evident (Fig. 2B). Conservation of synteny was less strong between zebrafish regions containing adrb3a and adrb3b and their human counterparts (Fig. 2C). However, the orthologous genes zgc:152691 and GOT1L1 are linked to adrb3a and human ADRB3, respectively; likewise, zgc:100856 and BRF2 are linked to adrb3b and human ADRB3, while NPM2, the human homolog of the immediate neighbor of adrb3a, LOC797865, was within 16.0 megabasepairs (Mbp) of ADRB3, supporting the argument for their orthology.

3.4. mRNA expression of adrb family in zebrafish Our results showed that all five adrb are expressed at appreciable levels during embryonic and larval development and that levels of expression increased gradually with time. adrb1 reached maximal expression around 10 dpf, adrb2a expression peaked in adulthood (6 mpf), while expression of adrb2b, adrb3a and adrb3b peaked at 1 mpf (Fig. 3, A–E). To examine the tissue distribution of adrb family in zebrafish, we performed a qPCR analysis using adult zebrafish tissues (Fig. 3, F–K). Zebrafish adrb1 was highly expressed in brain, heart and eye. adrb2a was highly expressed in brain and skin, whereas adrb2b was highly expressed in skeletal muscle, liver and pancreas. The expression of both of adrb3a and adrb3b was restricted to the blood. 3.5. Phenotypes induced by knock-down of the adrb family in zebrafish To analyze the functions of zebrafish β-ARs, we used MO technology, which has been widely used to knock down the expression of proteins in zebrafish (Eisen and Smith, 2008). We designed MOs to knock down the expression of zebrafish β-ARs (Supplementary Table 2) and injected these MOs into zebrafish embryos. As shown in Fig. 4A and B, zebrafish treated with MOs against adrb2a had significant hypopigmentation of skin melanophores. The difference in pigmentation between adrb2a morphants and wild-type fish became recognizable at 36 hpf. Melanosomes of larvae treated with MOs against adrb2a were fewer in number, smaller, less pigmented, and irregular compared with wild-type fish,

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Fig. 2. Syntenic relationships between regions containing zebrafish β-AR genes and their putative human orthologs. A. Syntenic relationships between regions containing zebrafish adrb1 and human ADRB1. Approximately 0.5 Mbp of zebrafish chromosome 12, and 1.0 Mbp of human chromosome 10, are shown. B. Syntenic relationships between regions containing zebrafish adrb2a and adrb2b and human ADRB2. Approximately 1.0 Mbp of zebrafish chromosome 14, 0.4 Mbp of zebrafish chromosome 17 and 1.6 Mbp of human chromosome 5 are shown. Human genes located outside the 1.6-Mbp interval, and their approximate distances from human ADRB2, are WDR55 (8.2 Mbp), CDC23 (10.6 Mbp), WNT8A (10.7 Mbp), EBF1 (10.1 Mbp), RNF145 (10.4 Mbp), UBLCP1 (10.4 Mbp), IL12B (10.5 Mbp), RARS (19.7 Mbp), FOXI1 (21.3 Mbp), LCP2 (21.4 Mbp) and UBTD2 (23.4 Mbp). Zebrafish genes located outside the 1.0-Mbp interval on chromosome 14, and their approximate distances from zebrafish adrb2a, are LOC556843 (17.2 Mbp), pde6a (17.4 Mbp), ppargc1b (17.5 Mbp), LOC793180 (17.6 Mbp), csnk1a1 (4.8 Mbp) and LOC799091 (5.1 Mbp). C. Syntenic relationships between regions containing zebrafish adrb3a and adrb3b and human ADRB3. Approximately 0.4 Mbp of zebrafish chromosome 8 and chromosome 3 and 0.5 Mbp of human chromosome 8, are shown. A human gene located outside the 0.5-Mbp interval, and its approximate distance from human ADRB3, is NPM2 (16.0 Mbp). Zebrafish β-AR genes and their putative human orthologs are indicated in bold.

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at 72 hpf (Fig. 4A and Supplementary Fig. S3). The difference in skin pigmentation remained distinguishable until at least 10 dpf (Supplementary Fig. S3). To ascertain the specificity of the observed effects of the MO against adrb2a, we performed experiments using 5-base mismatch MOs for adrb2a (5-mis MOs). As shown in Fig. 4A, the 5-mis MOs did not produce any observable effects on zebrafish embryonic development. Melanin levels were significantly decreased in the adrb2a morphants (Fig. 4B). Low pigmentation was not the only characteristic of larvae microinjected with adrb2a MOs. As shown in Fig. 4C, the yolks of adrb2a morphants were significantly wider than those of wild-type fish, suggesting slower consumption of yolk by adrb2a morphants. Preliminary data suggest that knock-down of adrb2a did not reduce heart rate significantly (data not shown); however, further study is needed to ascertain the possible role of adrb2a in zebrafish cardiac function. Zebrafish treated with the MO against adrb1 at high dose showed curved bodies, larger yolks and early larval lethality (Supplementary Fig. S4A), while those treated with a low dose showed a reduction in heart rate (Supplementary Fig. S5). Zebrafish treated with MOs against adrb2b had a phenotype characterized by abnormal body shape and small head size (Supplementary Fig. S4B), followed by highly edematous swelling at 120 hpf (Supplementary Fig. S4C). Zebrafish treated with adrb3b MOs showed malformations of the cervical region and early larval lethality (Supplementary Fig. S4E and F). The adrb3a MO produced no detectable change (Supplementary Fig. S4D). 3.6. qPCR analysis of genes related to pigmentation To explore the mechanism underlying the hypopigmentation induced by knock-down of adrb2a, qPCR analysis was performed. We selected genes known to be involved in pigmentation and designed primers to analyze the expression of these genes (Table 1). Using total RNA extracted from adrb2a morphants and wildtype embryos, we tried to find genes whose expression was dysregulated by knock-down of adrb2a. To detect the genes responsible for the hypopigmentation in adrb2a morphants, we analyzed embryos at 26 hpf and 36 hpf. Among the 15 genes tested (Table 1), the expression levels of atp5a1 and atp5b were significantly different between adrb2a morphants and wild-type embryos (Table 1, Fig. 5A and B). The expression levels of atp5a1 and atp5b were also elevated in zebrafish 26 hpf after treatment with adrb2a MOs (Fig. 5A and B). The expression level of tyrosinase (tyr), the rate-limiting enzyme in melanogenesis (Mishima, 1994), was not significantly different between adrb2a morphants and wild-type fish (Fig. 5C). 4. Discussion 4.1. Comparison of zebrafish β-ARs with those of other species In this study, we demonstrate that zebrafish possess five β-AR orthologs, adrb1, adrb2a, adrb2b, adrb3a and adrb3b. Like zebrafish, medaka, Fugu, tetraodon and stickleback also have one ortholog for β1-AR and two orthologs for β2-AR. In contrast to zebrafish, these species have only one ortholog for β3-AR. These results suggest that the three subtypes of β-AR arose before the divergence of rayfinned fish and tetrapods. Ray-finned fish are believed to have undergone additional genome duplication (third round genome duplication or fish-specific genome duplication (FSGD) (Meyer and Van de Peer, 2005), from which the two copies of β1, β2 and β3-AR in ray-finned fish probably originated. Meanwhile one copy of β1AR and one copy of β3-AR were lost in most teleost species, except rainbow trout (Nickerson et al., 2003) and zebrafish. These examples of duplication have also been shown for many other

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genes, including HOX cluster genes (Amores et al., 1998). Mammals have four HOX clusters, HOXA, HOXB, HOXC and HOXD, whereas zebrafish have seven hox clusters, hoxaa, hoxab, hoxba, hoxbb, hoxca, hoxcb and hoxda. It has been presumed that all hox clusters were duplicated during FSGD, with the subsequent loss of one hoxd cluster (Amores et al., 1998). Generally, gene duplications have three outcomes: i) one copy may become silenced by a degenerative mutation; ii) one copy may acquire a novel function, with the other copy retaining original functions; iii) both copies may become partially compromised by mutations to the point at which their total capacity is reduced to the level of a single copy (Lynch and Conery, 2000). It seems that zebrafish β1-AR corresponds to the first outcome and β2-AR corresponds to the third outcome (described below). We showed that zebrafish adrb1 is expressed abundantly in the brain, heart and eye. An avian adrb1 ortholog was first isolated from chicken erythrocytes (Chen et al., 1994) and is expressed in multiple tissues, including heart (Chen et al., 1994) and brain (Chen et al., 1994). Mammalian β1-AR was reported to be detected in different kinds of tissues (Machida et al., 1990; Kawarai and Koss, 1999). The most eminent effector tissue is the heart (Rockman et al., 2002), while its presence in the central nervous system indicates a probable supraneural function (Guo and Li, 2007). These findings indicate that the expression and functions of β1-AR are well conserved among mammals, birds and zebrafish. Indeed, it was shown that application of isoproterenol and propranolol to zebrafish increased and decreased heart rate, respectively (Schwerte et al., 2006). Consistent with these reports, adrb1 morphants showed slight but significant reductions in heart rate, suggesting a functional conservation of zebrafish adrb1 in heart. We demonstrated that zebrafish adrb2b is highly expressed in skeletal muscle, pancreas and liver. An ortholog of adrb2 in rainbow trout is highly expressed in skeletal muscle and liver (Nickerson et al., 2001) and probably involved in anabolism in muscle (Salem et al., 2006) and metabolism of glucose and free fatty acids (FFA) in liver (Van Heeswijk et al., 2006). Phylogenetic analysis also indicated that the rainbow trout adrb2 is an ortholog of zebrafish adrb2b. These results suggest that zebrafish adrb2b may have functions similar to those of adrb2 in rainbow trout. In contrast to adrb2b, zebrafish adrb2a is highly expressed in brain and skin. Among the five zebrafish adrb, adrb2a is the only one expressed predominantly in skin, which is consistent with the fact that knock-down of adrb2a, but not knockdown of other β-AR orthologs, induces hypopigmentation in zebrafish (described later). Mammals have only one copy of β2-AR gene, which is expressed in many tissues, including brain, skin, skeletal muscle, and liver, and which possesses multiple functions including regulating liver metabolism (Erraji-Benchekroun et al., 2005) and skin pigmentation (Gillbro et al., 2004). These findings suggest that zebrafish adrb2a and adrb2b share the functions of a common β2-AR ancestor. We identified two orthologs of adrb3, adrb3a and adrb3b, which are expressed exclusively in blood. This is consistent with a previous report showing that an ortholog of adrb3 in rainbow trout was exclusively expressed in erythrocytes (Nickerson et al., 2003). In rainbow trout, adrb3 regulates the activity of the Na+/H+ exchanger in erythrocytes (Nickerson et al., 2003), suggesting that its counterparts in zebrafish may have similar functions. The two rainbow trout adrb3 share 84% similarity to each other and both of them showed higher similarity to zebrafish adrb3a than to adrb3b, suggesting that the two copies of adrb3 in rainbow trout are orthologs of zebrafish adrb3a and probably originated from a more recent duplication than the FSGD. Genomic analysis of Cyprinus carpio (common carp), a close relative of zebrafish, will be helpful to determine the conservation of the two zebrafish adrb3 orthologs with respect to the minnow family. Further studies are needed to analyze the functions of zebrafish adrb3a and adrb3b.

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Table 1 Change in expression of 15 pigmentation-related genes, induced by knock-down of adrb2a.a,b Gene

Ratioc

P valued

Reference

atp5a1 atp5b kitlga tyrp1 tyr tgfb1 gnb2l1 tnfa ddc mitfa slc24a5 dct prkcb1 atp7a tnfb

1.248 1.472 0.776 0.794 1.150 1.206 1.153 1.903 1.939 0.840 1.111 1.061 1.036 1.010 1.699

0.007 0.005 0.142 0.184 0.380 0.498 0.796 0.618 0.222 0.156 0.711 0.764 0.793 0.908 0.203

Jung et al., 2005 Jung et al., 2005 Hultman et al., 2007 del Marmol and Beermann, 1996; Jimbow et al., 1997 Braasch et al., 2007 Martínez-Esparza et al., 1999; Kim et al., 2004 Park et al., 2004 Martínez-Esparza et al., 1998; Nakamura et al., 2003 Koch et al., 1998; Gillbro et al., 2004 Lister et al., 1999 Lamason et al., 2005 Braasch et al., 2007 Park et al., 1999 Madsen et al., 2008 Krasagakis et al., 1995

a

Primers used are shown in Supplementary Table 1. Total RNA used was from 8 independent experiments of the adrb2a morphant and wild-type groups at 36 hpf. c Ratio is the expression of adrb2a morphant/wild-type. d P is the probability calculated by the Student's t-test, after being normalized to the expression of β-actin. b

Fig. 4. Zebrafish adrb2a loss of function experiments. adrb2a MOs were injected into 1–4-cell stage embryos. (A) Representative images of the dorsal view of wild-type (upper), 5-base mismatch control MO (middle) and adrb2a MO-injected larvae (lower) at 72 hpf. Scale bar indicates 500 μm. (B) Effect of adrb2a MOs on melanin. Five larvae were collected at 72 hpf and dissolved in lysis buffer. After centrifugation, melanin pigment was redissolved in 1 N NaOH, and then total melanin content was quantified by spectrophotometry. Data are presented relative to the wild-type value and are the mean values ± SEM of 12 independent experiments. (C) Width of yolk was measured using NIS Elements D2.20 image analysis software (Nikon). Data are presented as the means ± SEM of 20 larvae at 96 hpf. ⁎P b 0.05; ⁎⁎P b 0.01; ⁎⁎⁎P b 0.001.

4.2. Regulation of pigmentation by β2-AR and the possible role of F1F0ATPase We demonstrated that zebrafish treated with MOs for adrb2a showed significant hypopigmentation. The involvement of β2-AR in pigmentation has been previously reported (Fujii, 2000; Gillbro et al., 2004; Yang et al., 2006). Human keratinocytes, the cells that make up the majority of the epidermis, express only ADRB2 (Orenberg et al., 1983; Steinkraus et al., 1996). MCs also expressed ADRB2, but not

ADRB1 (Grando et al., 2006). Human MCs treated with epinephrine and the selective β2-AR agonist salbutamol showed upregulated melanin production, accompanied by a cyclic adenosine monophosphate (cAMP) response (Gillbro et al., 2004). It was presumed that this cAMP activated protein kinase A and the transcription factor cAMP response element binding protein (CREB), leading to the upregulation of the expression of microphthalmia-associated transcription factor (MITF) (Buscà and Ballotti, 2000). MITF binds to the TYR promoter, thereby increasing TYR expression, which results in increased melanin synthesis (Buscà and Ballotti, 2000). However, direct evidence of the interplay between β2-AR stimulation and expression of TYR is not available. We could not detect a significant difference in tyr expression between adrb2 morphants and controls. Given the redundancy of cAMP signaling in pigmentation (Schallreuter et al., 2008), it seems surprising that knock-down of adrb2a had such effects on pigmentation in zebrafish. However, it is reported that ADRB2 signaling is very effective in promoting pigmentation in human epidermal melanocytes (Gillbro et al., 2004). Biosynthesis and release of epinephrine by surrounding keratinocytes activates ADRB2 in melanocytes, which in turn increases the intracellular cAMP levels ([cAMP]i) (Gillbro et al., 2004). ADRB2 has a CRE binding domain in its promoter that induces transcription of itself in response to an increase in [cAMP]i (Collins et al., 1990). The zebrafish adrb2 gene also has a putative CRE binding domain, TGATGTCA (Smith et al., 2007) (chr14:39,485,691–39,485,698, zebrafish Zv7 assembly). These reports suggest that there might be a positive autoregulation of β2-AR signaling in melanogenesis, which might account for the significant hypopigmentation observed in adrb2a morphants. In fact, zebrafish treated with ICI-118551, a selective antagonist of β2-AR, showed hypopigmentation, albeit to a lesser extent than that of adrb2a morphants (data not shown). By screening 15 genes that are known to be involved in pigmentation, only atp5a1 and atp5b were found to be significantly upregulated by knock-down of adrb2a. In humans, ATP5A1 and ATP5B are α and β subunits of F1F0-ATPase. F1F0-ATPase has been identified to negatively modulate pigmentation (Jung et al., 2005; Ni-Komatsu and Orlow, 2007). It is an evolutionarily conserved enzyme that catalyzes ATP synthesis, using an electrochemical gradient of protons

Fig. 3. mRNA expression of zebrafish adrb family in developmental stages and adult tissues. Developmental profiles of gene expression are shown on the right (A–E); relative mRNA expression levels in tissues of zebrafish at 6 mpf are shown on the left (F–J). Six individual zebrafish at 6 mpf were used for organ-specific RNA isolation. SYBR Green-based quantitative real-time PCR was used to quantify the expression levels of adrb family. Relative transcript ratios were determined by normalizing threshold cycles (Ct) values to β-actin expression. Data are presented relative to the value in the most highly expressing time point or tissue.

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mRNA-protecting protein HuR (Klöss et al., 2004). HuR was reported to bind to the AU-rich element in the 3′-UTR of ATP5B and to stabilize the mRNA (Izquierdo, 2006). A consensus motif of the HuR binding site, WAUUUAWW (Barreau et al., 2005), is also located in the 3′-UTR of zebrafish atp5b and atp5a1. Knock-down of adrb2a in zebrafish may stabilize atp5a1 and atp5b mRNA through HuR. Application of carteolol, a non-selective β-AR blocker, resulted in a significant increase in F1F0-ATPase activity in the hearts of turkeys with dilated cardiomyopathy (Gwathmey et al., 1999). The mechanisms regulating expression of F1F0-ATPase by β2-AR remain to be studied. It is interesting to note that treatment of differentiated adipocytes with small molecule inhibitors of F1F0-ATPase or antibodies against its α and β subunits leads to a decrease in cytosolic lipid droplet accumulation (Arakaki et al., 2007), consistent with our observation that the adrb2a morphant exhibited slow yolk consumption. Another possibility is that knock-down of adrb2a may reduce metabolism in yolk. It has been shown that adrb2 expressed in rainbow trout hepatocytes regulates glucose and FFA metabolism (Van Heeswijk et al., 2006; Dugan et al., 2008). Knock-down of adrb2a may reduce glucose and FFA metabolism in both liver and yolk. We also cannot exclude the possibility that adrb2a morphants are developmentally delayed. Although further research is needed to confirm this, the induction of atp5a1 and atp5b may be involved in lipid metabolism in yolk. In summary, we cloned and characterized five zebrafish orthologs of β-AR genes representing homologs of three β-AR subtypes. Loss-offunction analysis revealed an important role for zebrafish adrb2a in the regulation of pigmentation, consistent with mammalian β2-AR. qPCR analysis suggested the involvement of F1F0-ATPase in the dysregulation of pigmentation following knock-down of adrb2a. Further studies should be performed to clarify the functional relationships between adrb2a and F1F0-ATPase in the regulation of pigmentation. Acknowledgments

Fig. 5. Effects of adrb2a knock-down on atp5a1, atp5b, and tyr mRNA expression. MOs were injected into 1–4-cell stage embryos. The mRNA expression in wild-type embryos and in embryos injected with adrb2a MOs was quantified by qPCR at 26 and 36 hpf as the hypopigmentation phenotype induced by adrb2a MOs became identifiable at 36 hpf. (A) atp5a1 mRNA expression. (B) atp5b mRNA expression. (C) tyr mRNA expression. Data are presented as the means ± SEM of 12 independent experiments, after normalization to the expression of β-actin. ⁎P b 0.05; ⁎⁎P b 0.01; ⁎⁎⁎P b 0.001.

across the mitochondrial inner membrane during oxidative phosphorylation (Saraste, 1999). It was proposed that inhibition of F1F0ATPase-mediated transport of H+ ions resulted in the alkalinization of the cytosol (Jung et al., 2005; Ni-Komatsu and Orlow, 2007). Alkalinization of the cytosol facilitates the proper folding and release of Tyr from endoplasmic reticulum (ER)–Golgi intermediate compartments, the early Golgi compartments, and subsequent trafficking of Tyr to the melanosomes (Ni-Komatsu and Orlow, 2007). Chemical genetic screenings using melan-p cells (cultured melanocytes from p gene null mouse) and zebrafish have identified compounds that inhibit F1F0-ATPase as pigmentation enhancers (Jung et al., 2005; NiKomatsu and Orlow, 2007). Compounds that increase the pH of the ER–Golgi intermediate (for example, vacuolar ATPase inhibitor bafilomycin A1, the ionophore monensin, ammonium chloride) have also been identified for correction of albinism in melan-p cells (Chen et al., 2004). The expression levels of zebrafish atp5a1 and atp5b were significantly increased by knock-down of adrb2a. It has been reported that increased intracellular cAMP levels induce downregulation of the

This work was supported in part by the New Energy and Industrial Technology Development Organization, the Ministry of Education, Culture, Sports, Science and Technology-Japan, the Mitsubishi Pharma Research Foundation, the Japan Atherosclerosis Research Foundation, the Suzuken Memorial Foundation, the Nakatomi Foundation, Toyama Hospital and the Japan Chemical Industry Association. We also thank T. Murata, Y. Yoshikawa, K. Nishiguchi, A. Kamakura and C. Hirota for their experimental assistance and R. Ikeyama, Y. Yoshida and K. Ito for secretarial assistance. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.gene.2009.06.005. References Amores, A., et al., 1998. Zebrafish hox clusters and vertebrate genome evolution. Science 282, 1711–1714. Arakaki, N., Kita, T., Shibata, H., Higuti, T, 2007. Cell-surface H+-ATP synthase as a potential molecular target for anti-obesity drugs. FEBS Lett. 581, 3405–3409. Bachman, E., et al., 2002. betaAR signaling required for diet-induced thermogenesis and obesity resistance. Science 297, 843–845. Barreau, C., Paillard, L., Osborne, H, 2005. AU-rich elements and associated factors: are there unifying principles? Nucleic Acids Res. 33, 7138–7150. Braasch, I., Schartl, M., Volff, J, 2007. Evolution of pigment synthesis pathways by gene and genome duplication in fish. BMC Evol. Biol. 7, 74. Burge, C., Karlin, S, 1997. Prediction of complete gene structures in human genomic DNA. J. Mol. Biol. 268, 78–94. Buscà, R., Ballotti, R, 2000. Cyclic AMP a key messenger in the regulation of skin pigmentation. Pigment Cell Res. 13, 60–69. Chen, X., Harden, T., Nicholas, R, 1994. Molecular cloning and characterization of a novel beta-adrenergic receptor. J. Biol. Chem. 269, 24810–24819.

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