Duplication and differentiation of common carp (Cyprinus carpio) myoglobin genes revealed by BAC analysis

Gene 548 (2014) 210–216

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Duplication and differentiation of common carp (Cyprinus carpio) myoglobin genes revealed by BAC analysis Zi-Xia Zhao a, Peng Xu a, Ding-Chen Cao b, You-Yi Kuang b, Hai-Xia Deng a, Yan Zhang a, Li-Ming Xu a, Jiong-Tang Li a, Jian Xu a, Xiao-Wen Sun a,b,⁎ a b

The Center for Applied Aquatic Genomics, Chinese Academy of Fishery Sciences, Beijing 100141, PR China Heilongjiang River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Harbin 150070, PR China

a r t i c l e

i n f o

Article history: Received 24 July 2013 Received in revised form 9 May 2014 Accepted 11 July 2014 Available online 12 July 2014 Keywords: Common carp Myoglobin Duplication Differentiation Hypoxia

a b s t r a c t Two distinct myoglobin (mb) transcripts have been reported in common carp, Cyprinus carpio, which is a hypoxia-tolerant fish living in habitats with greatly fluctuant dissolved oxygen levels. Recombinant protein analysis has shown functional specialization of the two mb transcripts. In this work, analysis for mb-containing bacterial artificial chromosome (BAC) clones indicated different genome loci for common carp myoglobin-1 (mb-1) and myoglobin-2 (mb-2) genes. Fluorescence in situ hybridization (FISH) revealed that mb-1 and mb-2 are located on separate chromosomes. In both of the mb-1 and mb-2 containing BAC clones, gene synteny was well conserved with the homologous region on zebrafish chromosome 1, supporting that the common carp specific mb-2 gene originated from the recent whole genome duplication event in cyprinid lineage. Transcription factor binding sites search indicated that both common carp mb genes lacked specificity Protein 1 (Sp1) and myocyte enhancer factor-2 (MEF2) binding sites, which mediated muscle-specific and calcium-dependent expression in the wellstudied mb promoters. Potential hypoxia response elements (HREs) were predicted in the regulatory region of common carp mb genes. These characteristics of common carp mb gene regulatory region well interpreted the hypoxia-inducible, non-muscle expression pattern of mb-1. In the case of mb-2, a 10 bp insertion to the binding site of upstream stimulatory factor (USF), which was a co-factor of hypoxia inducible factor (HIF), might cause the non-response to hypoxia treatment of mb-2. The case of common carp mb gene duplication and subsequent differentiation in expression pattern and protein function provided an example for adaptive evolution toward aquatic hypoxia tolerance. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Myoglobin (mb) gene encodes an oxygen-binding hemoprotein, which mainly serves oxygen supply function by facilitating the delivery of oxygen from the plasma membrane to the energy-producing mitochondria. There used to be a consensus that mb gene was expressed exclusively in muscle tissues, such as oxidative skeletal and cardiac myocytes, where it could be up-regulated via a calcium-dependent Abbreviations: ASR, aquatic surface respiration; ARP, acidic ribosomal phosphoprotein P0; BAC, bacterial artificial chromosome; CDS, coding sequences; DO, dissolved oxygen; FISH, fluorescence in situ hybridization; HIF, hypoxia inducible factor; HIF1, hypoxia inducible factor-1; hpf, hours post fertilization; HRE, hypoxia response element; mb, myoglobin; mb-1, myoglobin-1; mb-2, myoglobin-2; MEF2, myocyte enhancer factor-2; MYA, million years ago; NFAT, nuclear factor of activated T-cell; Ngb, neuroglobin; NRE, NFAT response elements; qRT-PCR, quantitative real time PCR; ROS, reactive oxygen species; Sp1, specificity Protein 1; TSS, transcription start sites; USF, upstream stimulatory factor; WGD, whole genome duplication; zv9, zebrafish whole genome assembly version 9; 5′ UTRs, 5′ untranslated regions. ⁎ Corresponding author at: The Center for Applied Aquatic Genomics, Chinese Academy of Fishery Sciences, Beijing 100141, PR China. E-mail address: [email protected] (X.-W. Sun).

http://dx.doi.org/10.1016/j.gene.2014.07.034 0378-1119/© 2014 Elsevier B.V. All rights reserved.

signaling pathway, according to intensive in vivo and in vitro studies on mammals (Berenbrink, 2010; Mirceta et al., 2013; Riggs and Gorr, 2006). In 2006, Fraser et al. discovered simultaneous expression of two distinct mb transcripts in common carp (Cyprinus carpio) (Fraser et al., 2006). Myoglobin-1 (mb-1) was expressed in the muscle and some other metabolically active tissues, including the liver, kidney, and gills. Myoglobin-2 (mb-2), a unique novel transcript, was expressed only in the brain, co-existing with neuroglobin (Ngb). Known as a hypoxiatolerant teleost, common carp was often found living in eutrophic habitats with greatly fluctuant dissolved oxygen (DO) levels (Stecyk and Farrell, 2002). In summer, diurnal oxygen tension might range from 30.66 kPa in the afternoon to 6.66 kPa during the pre-dawn hours. In winter, common carp survived in the ice-covered ponds with constantly dropping oxygen supply. Under laboratory observation, common carp was able to endure extreme hypoxia treatment of 0.5 mg/L DO exposure for 42 days without inducing apoptosis (Poon et al., 2007). The unprecedented expression pattern of common carp mb genes, which was inferred related to hypoxia adaption, immediately aroused wide refocus on the function and regulation of globins (Riggs and Gorr, 2006).

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mb's non-muscle expression was soon confirmed in other teleosts, including crucian carp (Roesner et al., 2008), zebrafish (Cossins et al., 2009), and medaka (Wawrowski et al., 2011). However, the unique mb-2, was only found in common carp and its closely related species, crucian carp, which was an extreme hypoxia-tolerant fish (Wittenberg and Wittenberg, 2012). Recent work by Helbo et al. indicated functional specialization between MB-1 and MB-2 for tissue-specific protect roles, since the MB-1 protein displayed higher oxygen affinity and faster catalytic velocity for nitric oxide, while the mb-2 encoded protein proved to be significantly faster at eliminating hydrogen peroxide (Helbo et al., 2012). Gene duplication and alternative splicing isoforms are the two major evolutionary mechanisms that increase gene diversification (Jin et al., 2008; Su and Gu, 2012). In human genome, all the three known transcript variants of mb shared the same gene locus. In this work, genomic sequences and genome location of common carp mb genes were investigated, suggesting that the two different mb transcripts originated from recent whole genome duplication (WGD) event, rather than alternative splicing isoforms. 2. Materials and methods 2.1. Screening of mb containing BAC clones A three-dimension bacterial artificial chromosome (BAC) pool of common carp (Li et al., 2011), containing 18,432 individual clones (1.5 × haploid genome coverage), was screened by PCR amplification for mb-1 and mb-2. Distinctive primers were obtained from earlier work of Fraser et al. (mb-1F: ATGGCCGATCACGAACTGGTT; mb-1R: GCCGTGGGCCTTCACCGCTGCGTT; mb-2F: ATGGCTGATTACGAGCGG TTT; mb-2R: ACCGTGGGACGCCACCAACGTGTC) (Fraser et al., 2006). The mb primers were amplified in 88 wells, representing column pool, row pool, and plate pool. Positive wells served as three-dimensional coordinates to locate the original mb containing clones. Candidate clones were picked up from the BAC library, and then validated by another round of PCR. 2.2. Sequence analysis of mb containing BAC clones Isolated BAC clones, CYC041G12 containing mb-1, CYC001O02 and CYC002I05 containing mb-2, were separately sequenced on 314 chips of Ion Torrent PGM (Life technologies). More than 200× coverage sequencing data was obtained for each BAC clone, at quality cutoff Q20. Read assembly was performed with Torrent Suite and Newbler. Potential mb promoter regions were checked by Sanger sequencing. Gene annotation was performed via BLASTx against Ensembl peptide database of zebrafish whole genome assembly version 9 (zv9). 5′ untranslated regions (5′ UTRs) were identified by BLASTn searching in sequencing data of common carp full-length cDNA library. Schematic diagrams of gene structure and gene synteny were drawn with the online tool fancyGENE v1.4 (Rambaldi and Ciccarelli, 2009). Transcription factor binding sites were checked using the online TFSEARCH program (Akiyama, 1998) against TRANSFAC databases (Heinemeyer et al., 1998), combined with online search by JASPAR (Bryne et al., 2008), PROMO (Farre et al., 2003; Messeguer et al., 2002), and local search by CLC Genomics Workbench.

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mb-1 containing chromosome region was stained with green fluorescence, through successive reactions with biotin labeled BAC plasmid probe, avidin-FITC (Sigma-Aldrich), biotinylated anti-avidin D (Vector), and avidin-FITC. Meanwhile, the mb-2 containing chromosome region was stained with red fluorescence, through successive reactions with digoxigenin labeled BAC plasmid probe, mouse anti-digoxin antibody, anti-mouse IgG-TRITC antibody produced in rabbit, and anti-rabbit IgG-TRITC antibody produced in goat (Sigma-Aldrich). After counterstained by DAPI (Sigma-Aldrich), chromosome images were observed and captured on a fluorescence microscopy Axio Imager M2 (Zeiss), with AxioVision software system. More than 30 successfully stained metaphase spreads from 3 individuals were analyzed. 2.4. Sampling Full siblings of common carp were obtained from Hulan hatchery at Harbin, China. Healthy individuals, three-month old, with body weight around 150 g were selected and kept in 25 °C for 14 days before hypoxia treatment. Stop feeding for 24 h, and then stop aeration to make DO value drop naturally. Tissue samples were collected and flash frozen in liquid nitrogen. Five experimental groups were set: (1) normoxia controls, collected before stopping aeration, at DO value around 6.0 mg/L; (2) accelerated breathing group, collected 20 min after stopping aeration, at DO value around 3.0 mg/L; (3) aquatic surface respiration (ASR) group, collected 3 h after stopping aeration, at DO value around 1.0 mg/L; (4) coma group, collected about 6 h after stopping aeration, when individuals lost balance and had no response to gentle touch, at DO value around 0.3 mg/L; and (5) recovery group, move the fishes into fully oxygenated water 10 min after they fell into coma, samples were collected when individuals recovered to normal swimming, at DO value around 6.0 mg/L. Embryos were collected after timed intervals: 0, 3, 6, 12, 18, 24, 36, 48, 72, 96, 120, 144, 192, and 240 hours postfertilization (hpf) at 20 °C and flash frozen in liquid nitrogen. 2.5. Expression analysis of mb-1 and mb-2 Total RNA was extracted using TRIzol reagent (Invitrogen), according to the manufacturer's instruction. DNase I (Sigma-Aldrich) digestion was performed to avoid contamination by genomic DNA, before reverse transcription of mRNA using the First Strand cDNA Synthesis Kit ReverTra Ace-α-RT-PCR (TOYOBO). Quantitative real time PCR (qRT-PCR) was practiced on Applied Biosystems 7500 Real-Time PCR System (Life Technologies), using SYBR Green Realtime PCR Master Mix (TOYOBO). mb-1 and mb-2 were amplified with distinctive primers mentioned in Section 2.1. ARP (acidic ribosomal phosphoprotein P0, GenBank accession

2.3. Chromosome localization of mb containing BAC clones Twelve-month common carps were obtained from Songpu hatchery at Harbin, China. Head kidney was used for mitosis metaphase chromosome preparation, and fluorescence in situ hybridization (FISH) was performed with BAC plasmid probe, by methods described previously (Deng et al., 2013). Using a nick translation kit (Roche), BAC clone CYC041G12 was labeled by biotin-16-dUTP (Fermentas), while BAC clone CYC002I05 was labeled by digoxigenin-16-dUTP (Roche). The

Fig. 1. Results of PCR amplification for mb-1 (left) and mb-2 (right) in BAC clone (A) CYC001O02, (B) CYC002I05, and (C) CYC041G12.

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Fig. 2. Chromosome localization of common carp mb-1 (green) and mb-2 (red) genes by fluorescence in situ hybridization (FISH) of BAC clones.

number: KF572122) was utilized as the internal control gene for hypoxia treatment analysis (Roesner et al., 2006; Simpson et al., 2000), while betaactin was utilized as the internal control gene for embryonic development and tissue distribution analysis (Casadei et al., 2011), with primers ARP-F: GGCTTTGGGAATCACCACCA; ARP-R: GCTGTAGACGCTGCCGTTAT; actin-F: TGCAAAGCCGGATTCGCTGG; and actin-R: AGTTGGTGACAATACCGTGC. For each sample, 5 biological replicates with 3 experimental replicates were examined. The comparative CT (2−ΔΔCT) method (Schmittgen and Livak, 2008) was applied to analyze the expression of mb genes. 3. Results

column 5 all yielded positive signals. A second round of PCR in 8 candidate BAC clones verified that clone CYC001O02 and CYC002I05 contained mb-2 gene. As shown in Fig. 1, mb-1 and mb-2 existed in different BAC clones. 3.2. Genome localization of mb-1 and mb-2 By metaphase chromosome FISH of BAC clones, mb-1 and mb-2 genes were localized on separate chromosomes (Fig. 2). Among the 100 chromosomes of common carp, a pair of submetacentric chromosomes was found bearing mb-1 gene (green), while mb-2 gene (red) was located on short arms of a pair of acrocentric chromosomes.

3.1. Screening of mb containing BAC clones BLAST search against EST data from a 2.0 × 105 CFU full-length cDNA library and the transcriptome data of four common carp stains (Xu et al., 2012) was performed to search potential unknown mb transcripts. mb-1 and mb-2 were the only transcripts found. Different PCR results for mb-1 and mb-2 specific primers were found in the BAC screening pool, suggesting different genome loci for the two transcripts. Positive amplification for mb-1 was found in plate 41, row 7, and column 12, indicating that the BAC clone CYC041G12 might contain mb-1 gene. For mb-2, PCR amplification in plate 1, plate 2, row 9, row 15, column 2, and

3.3. Conserved synteny of flanked genes near mb loci BAC clones containing mb-1 and mb-2 were sequenced and assembled, separately. Over 100 kb high-quality genomic sequences for each clone were obtained. BAC clone CYC001O02 and CYC002I05, which both contained mb-2 gene, were found overlapped. Fig. 3 indicated the gene synteny with homologous region on zebrafish chromosome 1, which was the sole locus of mb in zebrafish genome. The available 7 genes adjacent to mb kept in the same order with zebrafish in both of the common carp BAC clones. The coding sequences (CDS)

Fig. 3. Gene synteny of mb containing genomic regions of zebrafish and common carp.

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were conserved, with nucleotide similarity of 86%. Meanwhile, sizes of introns and intergenic regions varied. Most of the introns and intergenic regions of common carp were shorter than their counterparts of zebrafish.

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was detected in the skin, muscle, gill, liver, kidney, and slightly in the brain, while mb-2 was uniquely expressed in the brain.

3.6. Hypoxia response of mb-1 and mb-2 3.4. Comparison of mb gene structures Structure of mb genes of common carp, zebrafish, and human was compared in Fig. 4. Exactly the same genome locus, identical CDS and 3′ UTR were shared by the three human mb transcript variants (mb-1: NM_005368.2, mb-2: NM_203377.1, mb-3: NM_203378.1), which differed in the 5′ UTR due to alternative transcription initiation. No transcript variant of zebrafish mb was reported up to now. Common carp mb-1 (KC342292.1), mb-2 (DQ338464.1), and zebrafish mb (NM_ 200586.1) had similar gene structure to a recently reported mb transcript, human mb-2, which was discovered in breast cancer cells with hypoxia-inducible non-muscle expression (Kristiansen et al., 2011). Compared with the well-known human mb-1, the three teleost mb genes possessed an additional intron in 5′ UTR.

As shown in Fig. 6, mb-1 expression in the muscle and gill significantly fluctuated under severe hypoxia, but mb-2 seemed unaffected by various hypoxia conditions. mb-1 expression was not affected under temporary slight hypoxia, as shown in the accelerated breathing group. While common carp suffered from severe hypoxia, the fish moved to water surface to increase oxygen supply, and mb-1 was two-fold up-regulated. Then DO level continued dropping, the fish fell into a coma status with minimized vital movements, accompanied with decreasing mb-1 expression. Unlike many hypoxia-sensitive teleosts, common carp was able to recover from hypoxia induced coma, once re-oxygenated. The fish quickly returned to normal behavior before the hypoxia conditions in internal tissues were relieved, suggested by higher muscle mb-1 expression in the recovery group.

3.5. Expression divergence of mb-1 and mb-2

3.7. Analysis of transcription factor binding sites in mb-1 and mb-2

QRT-PCR analysis indicated that mb-1 and mb-2 had different expression patterns, as shown in Fig. 5(a) for representative stages during embryonic development, and Fig. 5(b) for tissue distribution in adult common carp (3 month old). No significative expression of both mb genes could be observed during the first 12 hours post fertilization (hpf), corresponding to the end of gastrulation by embryos at tail bud stage. Until 18 hpf, significant expression was detected for both mb-1 and mb-2. The expression of mb-1 gradually increased during segmentation (12–48 hpf), pharyngula (48–72 hpf), and hatching (72–96 hpf), then experienced a sharp increase in the larval period after hatching, observed from 96 to 240 hpf. Meanwhile, mb-2 exhibited significantly lower expression level all along the examined periods, with no more than 1/10 of mb-1 mRNA expression quantity at 240 hpf. And no great fluctuation of mb-2 expression was observed during the developmental periods from 18 to 240 hpf. In adult common carp, expression of mb-1

Potential transcription factor binding sites were searched in 1 kb upstream regions of mb-1 and mb-2 transcription start sites (TSS). Classic mb promoter consisted of an A/T motif for myocyte enhancer factor-2 (MEF2) binding, a CCAC box for Specificity Protein 1 (Sp1) binding, and 2 NFAT response elements (NRE) for nuclear factor of activated T-cell (NFAT) binding (Kanatous and Mammen, 2010; Kanatous et al., 2009). Neither Sp1 nor MEF2 binding site was found in common carp mb-1 or mb-2, while no less than 3 NREs appeared in both mb genes. Hypoxia response elements (HREs), which were commonly considered not existing in mb gene, were found in both mb-1 and mb-2. Although mb-2 was expressed exclusively in the brain, no neuron-specific transcription factor binding sites was found. Near TATA box, there was an upstream stimulatory factor (USF) binding site in mb-1, while the homologous site in mb-2 was interrupted by a 10 bp insertion. Comparison for mb-1 and mb-2 regulatory regions was shown in Fig. 7. In the

Fig. 4. Comparison of mb gene structures for human (mb-1: NM_005368.2, mb-2: NM_203377.1, mb-3: NM_203378.1), zebrafish (NM_200586.1), and common carp (mb-1: KC342292.1, mb-2: DQ338464.1).

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selected 599 bp region, only 75% sequence similarity was observed, which was much lower than that of the CDS region (86%).

4. Discussion 4.1. Origin of common carp specific mb-2 gene Unlike human mb transcript variants, the common carp mb-2 gene appeared to originate from gene duplication, rather than alternative transcription or splicing, since BAC library screening (Fig. 1) and chromosome localization (Fig. 2) revealed distinct genome loci for mb-1 and mb-2. Compared with zebrafish (2n = 50), a typical member and model organism in cyprinid lineage, the chromosome number of common carp (2n = 100) doubled. It's estimated that 5.6 to 11.3 million years ago (MYA), whole genome duplication (WGD) occurred to the ancient cyprinid(s), resulting in the current common carp speciation (Wang et al., 2012). Crucian carp (2n = 100 and 3n = 150) might experienced the same duplication event, which also possessed the mb-2 gene copy. Identical syntenic flank genes with the mb region in zebrafish genome (Fig. 3) confirmed that the specific mb-2 gene copy was derived from large scaled genome duplication, instead of random translocation.

4.2. Ancestral form of mb gene transcripts The CCAC box in well studied human mb-1 promoter, related to muscle-specific expression (Kanatous and Mammen, 2010), was absent in common carp mb genes. So did the A/T rich motif, responsive to the calcineurin/MEF2 signaling pathway (Kanatous and Mammen, 2010). Existence of HREs and NREs suggested that common carp mb might be regulated by hypoxia inducible factor-1 (HIF1) and calcineurin/NFAT signaling pathway. This HIF-inducible, non-muscle expression pattern was also observed in human mb-2, which had a similar three-intron gene structure to the teleost mb genes. Thus, human mb-2, although recently reported (Kristiansen et al., 2011) and not universally expressed, might be close to the primitive transcript of mb gene in the aquatic ancient ancestors of mammals and modern teleosts. After landing on earth, steady oxygen level in the atmosphere relieved the stress of oxygen supply, and then the new mb transcripts evolved and became dominant in terrestrial animals, such as human mb-1, which was not directly regulated by HIF, and solely expressed in metabolically active muscles. On the other hand, modern teleosts conserved the ancestral form of mb gene transcript, but with shortened introns during additional rounds of WGD.

4.3. Differentiation of common carp mb-1 and mb-2 As previously reported (Helbo et al., 2012; Wittenberg and Wittenberg, 2012), MB-1 and MB-2 shared 73% similarity in amino acid sequences, and MB-2 protein was evolutionarily optimized for new function or new environment, since it exhibited higher efficiency in eliminating reactive oxygen species (ROS).

Fig. 5. Expression analysis of common carp mb-1 and mb-2 genes during early developmental stages (a) and in adult tissues (b), revealed by qRT-PCR. Error bars represented standard error of the mean (n = 5).

Fig. 6. Hypoxia expression of common carp mb-1 (a) and mb-2 (b) genes, determined by qRT-PCR. Brain, gill, and muscle tissue samples were examined for (1) normoxia controls, (2) slight hypoxia with accelerated breathing, (3) severe hypoxia with aquatic surface respiration (ASR), (4) hypoxic coma, and (5) recovery groups. Error bars represented standard error of the mean (n = 5). Statistical differences relative to normoxia controls were calculated by using Student's t test (*, P b 0.01).

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Fig. 7. Alignment of common carp mb-1 and mb-2 gene upstream sequences. Hypoxia response elements (HRE), NFAT response elements (NRE), upstream stimulatory factor (USF) binding site, and TATA boxes were indicated.

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The common carp mb genes differentiated not only in protein functions, but also in expression patterns, as shown in Figs. 5 and 6, which might be caused by the apparent differences in the promoter region and other transcription factor binding sites. Expression of mb-1 was up-regulated by hypoxia treatment and greatly fluctuated under different hypoxia conditions, while expression of mb-2 seemed not affected by hypoxia stress. Promoter analysis indicated that the common carp mb genes differed at a USF binding site 20 bp upstream to TATA box, where a 10 bp insertion caused mutation of the binding site in mb-2. USF itself was an oxygen independent transcription factor, however, USF sites were often adjacent to HREs and interacted with hypoxia inducible factor (HIF) binding (Borley and Sidell, 2011; Dimova and Kietzmann, 2006; Hankeln et al., 2005; Hu et al., 2011; Kajimura et al., 2006; Small et al., 1998, 2003). The mutation at the USF binding site might lead to the different hypoxia responses of mb-1 and mb-2. Genome duplication was an important source for biodiversity and environmental adaption, especially for teleosts (Santini et al., 2009; Vandepoele et al., 2004). Duplication of ancestral cyprinid mb gene made the survival stress shared and provided opportunities to evolutionary specialization for individual paralogs. As a result, subsequent differentiation in expression patterns and protein functions of the duplicated mb genes arose, contributing to the common carp hypoxia tolerance. Conflict of interest The authors declare that there are no conflicts of interest. Acknowledgments The financial supports from the National High-tech R&D Program of China (863 Program, 2011AA100401), and the Special Scientific Research Funds for Central Non-profit Institutes, Chinese Academy of Fishery Sciences (2012C014, 2014C010) are well acknowledged. References Akiyama, Y., 1998. TFSEARCH: Searching Transcription Factor Binding Sites. http://www. rwcp.or.jp/lab/pdappl/papia.html. Berenbrink, M., 2010. Myoglobin's old and new clothes: from molecular structure to integrated function and evolution. J. Exp. Biol. 213, 2711–2712. Borley, K.A., Sidell, B.D., 2011. Evolution of the myoglobin gene in Antarctic icefishes (Channichthyidae). Polar Biol. 34, 659–665. Bryne, J.C., Valen, E., Tang, M.H., Marstrand, T., Winther, O., da Piedade, I., Krogh, A., Lenhard, B., Sandelin, A., 2008. JASPAR, the open access database of transcription factor-binding profiles: new content and tools in the 2008 update. Nucleic Acids Res. 36, D102–D106. Casadei, R., Pelleri, M.C., Vitale, L., Facchin, F., Lenzi, L., Canaider, S., Strippoli, P., Frabetti, F., 2011. Identification of housekeeping genes suitable for gene expression analysis in the zebrafish. Gene Expr. Patterns 11, 271–276. Cossins, A.R., Williams, D.R., Foulkes, N.S., Berenbrink, M., Kipar, A., 2009. Diverse cellspecific expression of myoglobin isoforms in brain, kidney, gill and liver of the hypoxia-tolerant carp and zebrafish. J. Exp. Biol. 212, 627–638. Deng, H., Zhao, Z., Xu, P., Li, Y., Wang, S., Sun, X., 2013. Construction of a BAC-FISH experimental system for localizing specific sequences on mirror carp chromosomes. Acta Hydrobiol. Sin. 37, 467–472. Dimova, E.Y., Kietzmann, T., 2006. Cell type-dependent regulation of the hypoxiaresponsive plasminogen activator inhibitor-1 gene by upstream stimulatory factor2. J. Biol. Chem. 281, 2999–3005. Farre, D., Roset, R., Huerta, M., Adsuara, J.E., Rosello, L., Alba, M.M., Messeguer, X., 2003. Identification of patterns in biological sequences at the ALGGEN server: PROMO and MALGEN. Nucleic Acids Res. 31, 3651–3653. Fraser, J., de Mello, L.V., Ward, D., Rees, H.H., Williams, D.R., Fang, Y., Brass, A., Gracey, A.Y., Cossins, A.R., 2006. Hypoxia-inducible myoglobin expression in nonmuscle tissues. Proc. Natl. Acad. Sci. U. S. A. 103, 2977–2981. Hankeln, T., Ebner, B., Fuchs, C., Gerlach, F., Haberkamp, M., Laufs, T.L., Roesner, A., Schmidt, M., Weich, B., Wystub, S., Saaler-Reinhardt, S., Reuss, S., Bolognesi, M., De Sanctis, D., Marden, M.C., Kiger, L., Moens, L., Dewilde, S., Nevo, E., Avivi, A., Weber, R.E., Fago, A., Burmester, T., 2005. Neuroglobin and cytoglobin in search of their role in the vertebrate globin family. J. Inorg. Biochem. 99, 110–119. Heinemeyer, T., Wingender, E., Reuter, I., Hermjakob, H., Kel, A.E., Kel, O.V., Ignatieva, E.V., Ananko, E.A., Podkolodnaya, O.A., Kolpakov, F.A., Podkolodny, N.L., Kolchanov, N.A.,

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