Structural and Functional Evolution of Three Cardiac Natriuretic Peptides
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分子生物学进展 2005年第12期
* Ocean Research Institute, The University of Tokyo, Minamidai, Nakano, Tokyo, Japan; Department of Aquatic Biosciences, Tokyo University of Marine Science and Technology, Minato, Tokyo, Japan; Fukui Prefectural Inlandwater General Center, Fukui, Japan; Division of Biological Sciences, Graduate School of Science, Hokkaido University, Sapporo, Japan; || Division of Biological Sciences, Graduate School of Science, Nagoya University, Nagoya, Japan; and ? Departimento di Biologia Cellulare, Università della Calabria, Arcavacata Di Rende (CS), Italy
E-mail: inouek@ori.u-tokyo.ac.jp.
Abstract
Natriuretic peptides (NPs) are a group of hormones playing important roles in cardiovascular and osmoregulatory systems in vertebrates. Among the NP subtypes, atrial NP (ANP), B-type NP (BNP), and ventricular NP (VNP) are circulating hormones expressed exclusively in the heart (cardiac NPs). The constitution of cardiac NPs is variable among species of vertebrates. In order to understand the evolutionary and functional significance of such variation, we performed a systematic survey of cardiac NP cDNAs in nine taxonomically diverse teleosts inhabiting environments of varying salinity. The discovery of the coexistence of the ANP, BNP, and VNP genes in the eel and rainbow trout suggested that the ancestral teleost had all three cardiac NPs. As the VNP cDNA was undetectable in ayu and six species of Neoteleostei, it is possible that VNP was lost before the divergence of Osmeroidei. The ANP gene was also undetectable in the medaka. Thus, only the BNP gene is universal in species examined in the present study. Synthetic medaka BNP preferentially activated two medaka GC-A–type receptors, suggesting that the three cardiac NPs share the same receptor. However, the regulation of BNP expression may be the most strict because ATTTA repeats in the 3'-untranslated region and the dibasic motif in the ring are conserved among teleosts and tetrapods. Linkage analyses in the rainbow trout located ANP, BNP, and VNP genes on the same chromosome, which suggested the generation of the VNP gene by tandem duplication as observed with ANP and BNP genes. If the duplication occurred before the divergence of tetrapods and teleosts, VNP may exist in the tetrapod lineage.
Key Words: natriuretic peptides ? guanylyl cyclase ? evolution ? tandem duplication ? linkage mapping
Introduction
Natriuretic peptides (NPs) are a group of hormones characterized by the conserved ring structure consisting of 17 amino acids with N-terminal and C-terminal extensions, which are called "head" and "tail" segments, respectively. NPs play important roles in the regulation of circulatory and fluid homeostasis in vertebrates (Loretz and Pollina 2000; Takei 2000; Toop and Donald 2004). Four major members, atrial NP (ANP), B-type NP (BNP), ventricular NP (VNP), and C-type NP (CNP), have been identified thus far in vertebrates. ANP, BNP, and VNP are circulating hormones expressed mainly in the heart (cardiac NPs), whereas CNP is a paracrine/autocrine factor mainly expressed in the brain. CNP, characterized by the absence of the tail segment, has been found in all vertebrates previously examined except in the hagfish, which has a unique NP, hfNP (Kawakoshi et al. 2003). Although hfNP has a tail segment, it is thought to be a derivative of CNP (Kawakoshi, A., S. Hyodo, and Y. Takei, unpublished data). Thus, CNP has been suggested to be the ancestral molecule of the NP family (K. Inoue et al. 2003a). The constitution of the cardiac NPs is variable among different species of vertebrates. In tetrapods, ANP and BNP have been found in mammals and amphibians, but ANP has not been found in birds (Takei 2000). In teleosts, the ANP and BNP genes are also isolated from the tilapia and pufferfish. In the eel and trout, however, BNP has not been found but, instead, VNP was isolated (Takei et al. 1991; Takei, Ueki, and Nishizawa 1994). In addition, we recently found that the sturgeon Acipenser transmontanus, which is a species of Chondrostei, has both the BNP and VNP genes in addition to the ANP and CNP genes (Kawakoshi et al. 2004). However, information on the significance of such variations in the cardiac NPs is insufficient, and thus, systematic surveys of NPs are necessary to understand the complex NP system of vertebrates totally.
In the present study, we isolated cDNAs encoding precursors of cardiac NPs from taxonomically diverse teleosts, including seawater (SW), freshwater, and migratory species, and compared constituents of cardiac NP genes and their structure. We subsequently identified the positions of the ANP, BNP, and VNP genes on the chromosomes of the rainbow trout by linkage analyses. We also prepared synthetic BNP of medaka, in which NP receptors have been extensively studied (Kusakabe and Suzuki 2000), and examined its activity using the homologous NP receptors expressed in cultured cells. Based on the results of such analyses, we discuss the structural and functional evolution of the three cardiac NP genes in the lineage of ray-finned fish.
Materials and Methods
Fish
The four-spine sculpin, Cottus kazika, and ayu, Plecoglossus altivelis, were obtained from Fukui Prefectural Inlandwater General Center, Fukui, Japan. The icefish Chionodraco hamatus was sampled at Terranova Bay, Ross Sea, Antarctica. The killifish Fundulus heteroclitus was obtained from Fumi Katoh of Ocean Research Institute, The University of Tokyo. The medaka, Oryzias latipes (outbred, orange-red type), pufferfish, Takifugu rubripes, rainbow trout, Oncorhynchus mykiss, Japanese eel, Anguilla japonica, and tilapia, Oreochromis mossambica, were obtained as previously described (Takei et al. 2001; K. Inoue et al. 2003a, 2003b; Kawakoshi et al. 2004). The taxonomic relationships according to phylogenetic trees based on the whole mitochondrial genome sequences (J. G. Inoue et al. 2003; Miya et al. 2003) and salinity conditions of habitats of species examined in this study are indicated in figure 1.
FIG. 1.— The phylogenetic and taxonomic status of the teleosts that were examined in this study, and the distribution of cardiac NPs identified by cDNA cloning. The sturgeon was also included for comparison. Phylogenetic relations are indicated according to the phylogenetic trees proposed by J. G. Inoue et al. (2003) and Miya et al. (2003) based on whole mitochondrial genome sequences. The possible timing of the loss of the VNP and ANP genes are indicated by a and b, respectively. The asterisk indicates that the sturgeon belongs to Chondrostei, whereas others to Teleostei.
cDNA Cloning
After anesthesia with 0.05% 2-phenoxyethanol, the heart of each species was dissected free. The total RNA was isolated using Isogen (Nippon Gene, Toyama, Japan), and the cDNA pool was synthesized using the SMART cDNA Library Construction Kit (Clontech Laboratories, Palo Alto, Calif.), as described by Inoue, Iwatani, and Takei (2003). Partial NP cDNAs were isolated by 3'-rapid amplification of cDNA ends (3'-RACE) using the primers shown in table 1: primers fishANP-S1, fishBNP-S1, and fishVNP-S1 were designed based on specific sequences of ANP, BNP, and VNP of teleosts, respectively. Partial NP cDNAs were amplified by "touchdown polymerase chain reaction (PCR)" in the condition as follows: for fishANP-S1, eight cycles of amplifications using the thermal steps consisting of denaturation at 94°C for 30 s, annealing at 72–64°C for 30 s, and extension at 72°C for 105 s, decreasing annealing temperature 1°C after each cycle finished. Then 35 cycles of amplification were performed using the fixed thermal steps of denaturation at 94°C for 30 s, annealing at 64°C for 30 s, and extension at 72°C for 105 s. Annealing temperature was 4°C and 6°C decreased for fishBNP-S1 and fishVNP-S1, respectively. Because partial sequences of ANP and VNP cDNAs of the eels and those of ANP and BNP cDNAs of the pufferfish and tilapia obtained by the first screening by 3'-RACE agreed completely with sequences previously reported (Takei, Ueki, and Nishizawa 1994; Takei et al. 1997; Kawakoshi et al. 2004), further analyses were not performed for these cDNAs. For other cDNAs, upstream regions were obtained by 5'-RACE using specific primers designed from the sequences obtained by 3'-RACE (table 1). Finally, the whole cDNA sequences were obtained by 3'-RACE using specific primers designed based on 5'-untranslated regions (UTRs) (table 1). PCR conditions for 5'-RACE and final 3'-RACE were described by J. G. Inoue et al. (2003). All DNA sequences were determined using ABI 3100 sequencer and BigDye cycle sequencing kit ver. 3.1 (Applied Biosystems, Foster City, Calif.).
Table 1 Primers Used for Cloning and RT-PCR Analyses
Screening of Medaka Bacterial Artificial Chromosome Library
The medaka bacterial artificial chromosome (BAC) library (Matsuda et al. 2001) was screened using BNP cDNA labeled by Alkphos Direct Labelling Reagents (Amersham Biosciences, Buckinghamshire, United Kingdom) as a probe, and positive clones mHDR188J4 and mHDR164P14 were obtained. EcoRI fragments bearing BNP and CNP-3 genes were isolated from the former clone and inserted into pBluescriptII (Stratagene, La Jolla, Calif.). After sequencing of cloned fragments, sequences of peripheral region from 23.6-kb upstream to 14.3-kb downstream of the BNP gene were determined by primer walking.
Deposition of Nucleotide Sequences
The sequences obtained in this paper have been deposited in the DNA Data Bank of Japan/GenBank/European Molecular Biology Laboratory Database with accession numbers AB162775, AB162776, AB162777, AB162778, AB162779, AB162780, AB087286, AB179821, AB076603, AB162781, AB076604, and AB179820 for cDNAs of ayu ANP, ayu BNP, four-spine sculpin ANP, four-spine sculpin BNP, icefish ANP, icefish BNP, killifish ANP, killifish BNP, rainbow trout ANP, rainbow trout BNP, rainbow trout VNP, eel BNP and accession numbers AB204712, AB204714, and AB207138 for the second intron of rainbow trout VNP gene, the second intron of the rainbow trout BNP gene, and the medaka genomic fragment containing CNP-3 and BNP genes, respectively.
Analyses of Genomic Databases
Genomic information of the pufferfish and medaka was obtained from the Web sites http://fugu.biology.qmul.ac.uk/ and http://dolphin.lab.nig.ac.jp/medaka/, respectively.
Receptor Activation Assays
Predicted medaka BNP was synthesized at Peptide Institute (Minoo, Osaka, Japan). Activities of the synthetic BNP on the three endogenous NP receptors OlGC1 (GC-B homolog) (Takeda and Suzuki 1999), OlGC2, and OlGC7 (GC-A homologs) (Yamagami, K. Suzuki, and N. Suzuki 2001) expressed in COS-7 cells were performed according to K. Inoue et al. (2003a). CNP-4, CNP-3, and CNP-1 were also examined for comparison. Experiments are performed in quadruplicate or triplicate and repeated twice.
Reverse Transcription–Polymerase Chain Reaction
The tissue distribution of transcripts of the ANP, BNP, and VNP genes in rainbow trout was examined by reverse transcription (RT)–PCR as described by K. Inoue et al. (2003b). Primer sets ANP-S11/ANP-A5, TroutBNP-S1/TroutBNP-A1, VNP-S4/VNP-A3, and GAPDH-S2/GAPDH-A2 (table 1) were used for the detection of ANP, BNP, VNP, and glyceraldehyde-3-phosphate dehydrogenase (internal control) transcripts, respectively.
Linkage Mapping
To use as genotyping markers, nucleotide polymorphisms in rainbow trout ANP, BNP, and VNP genes were searched by PCR followed by direct sequencing. Genomic DNA (0.1 μg) of the male and female parent fish of the linkage panel established by Sakamoto et al. (2000) was used as the template. Primers rtANP-S2/rtANP-A2, rtVNP-S2/rtVNP-A2, and rtBNP-S1/rtBNP-A1 were used for the amplification of ANP, BNP, and VNP genes, respectively, and amplified fragments were sequenced using the same primers from both ends. Linkage mapping was performed according to Sakamoto et al. (2000). The polymorphism in the length of cytosine repeat starting from the 65th base of the 3'-UTR of ANP gene was detected by PCR using primers rtANP-S7 and rtANP-A7 followed by 5% polyacrylamide gel electrophoresis. The single-nucleotide polymorphisms (SNPs) in the VNP gene, at 250 and 20 bases upstream from the end of the second intron, were detected by PCR using primer sets rtVNP-S4/rtVNP-A4 and rtVNP-S5/rtVNP-A5. The SNPs in the BNP gene were detected using a PRISM SNaPshot multiplex kit (Applied Biosystems). Primers TroutBNP-S2 and TroutBNP-A3 were used for the detection of the SNP at the 62nd base of the coding region in the gene of the father fish and that at 97 bases upstream from the end of the second intron of the gene of the mother, respectively.
Results
Cloning of ANP, BNP, and VNP cDNAs
cDNAs encoding ANP and VNP were isolated from the heart of the eel and rainbow trout. BNP cDNAs were also isolated from these two species, in which the BNP had not been isolated. Thus, it was found that ANP, BNP, and VNP genes are present in the eel and rainbow trout, as they are in the sturgeon (fig. 1). Although cDNAs encoding ANP and BNP were obtained from the heart of the ayu, four-spine sculpin, pufferfish, icefish, tilapia, and killifish, extensive search for VNP cDNA was not successful in these species. We also screened the pufferfish genome database but failed to detect the VNP gene. From the heart of medaka, BNP cDNA was isolated but neither ANP nor VNP cDNAs were found in spite of repeated attempts using various primers. Blast searches of the medaka whole-genome shotgun database also failed to detect ANP- and VNP-like sequences, although multiple clones encoding precursors of BNP and CNPs have been identified. Moreover, we isolated genomic BAC clones of the medaka containing BNP and CNP-3 genes and determined the sequence neighboring the two genes. However, neither ANP nor VNP genes were found (fig. 2). In the downstream region of the BNP gene, where the ANP gene is present in mammals and Tetraodon, we found a sequence similar to a region of the retrotransposon sushi-ichi of the pufferfish (Poulter and Butler 1998). This sequence was followed by a repetitive region containing tandem repeats of an approximately 3-kb sequence. Similar repeats frequently appear in many scaffolds of the medaka genome. Thus, the ANP gene was not found in the vicinity of the BNP and CNP-3 genes. This result suggests that the ANP gene is absent in the medaka.
FIG. 2.— Comparison of the arrangement of genes around the BNP gene in humans, Tetraodon, and medaka. Shaded arrows indicate position and direction of genes. Exons and introns were not distinguished in this figure. The Tetraodon gene was predicted using the Takifugu cDNA sequences. Hatched arrow indicates the region containing a tandem repeat of a 3-kb sequence.
Predicted Structure of ANP, BNP, and VNP
Mature ANP, BNP, and VNP sequences predicted from cDNAs isolated in this study are shown in figure 3, in comparison with sturgeon NP sequences previously reported (Kawakoshi et al. 2004). As for ANP precursors, the amino acid just before the termination codon was glycine in all species examined, except rainbow trout. Thus, it was suggested that teleost ANPs have an amidated tail, as reported in the eel ANP (Takei 2000), except those of salmonid fishes.
FIG. 3.— Structure of teleost ANP, BNP, and VNP predicted from cDNAs isolated in this study. The sequences of the sturgeon reported by Kawakoshi et al. (2004) are also indicated for comparison. The carboxyl terminus of ANP was indicated as an amidated form when the terminal amino acid was glycine. Amino acid residues conserved completely in each subtype were reversed, and those conserved in more than half the species are shaded. BNP-specific dibasic motif is indicated by asterisks.
BNP sequences were highly conserved among teleosts. Basic amino acids at fourth and fifth residues of the predicted ring domain, which are characteristic of most tetrapod BNPs, were conserved (fig. 3). Repeated ATTTA sequences, which are reported to decrease the stability of mRNA (Wilson and Treisman 1988), are also observed in the 3'-UTR of BNP cDNAs of all species examined in this study (fig. 4).
FIG. 4.— Distribution of the ATTTA motif in the 3'-UTR of the teleost BNP cDNAs isolated in this study. The sturgeon BNP cDNA is also shown for comparison. Positions of the first base of the ATTTA motif are indicated by arrowheads. Dotted boxes and lines represent the end of the coding region and 3'-UTR, respectively.
The mature trout VNP sequence predicted from the cDNA was identical to that obtained previously by peptide isolation (Takei et al. 1994). It was found that VNP sequences were conserved among the eel, rainbow trout, and sturgeon (fig. 3).
Activity of Medaka BNP on Three Medaka NP Receptors
Synthetic medaka BNP, at the concentration of 10–8 M or higher, induced cyclic guanosine monophosphate (cGMP) accumulation in COS cells expressing two medaka NP receptors, OlGC2 and OlGC7, both of which are homologs of GC-A of other vertebrates. However, medaka BNP barely increased cGMP in OlGC1, a GC-B homolog (fig. 5). Thus, it was found that BNP preferentially activates the two GC-A–type receptors.
FIG. 5.— Dose-dependent activation of the medaka NP receptors OlGC1 (a GC-B homolog), OlGC2, and OlGC7 (GC-A homologs) by the synthetic medaka BNP ligand. Receptors were expressed in COS-7 cells, and cGMP concentration was measured by enzyme-linked immunosorbent assay. The activities of CNP-4, CNP-3, and CNP-1, which exhibited highest activity on OlGC1, OlGC2, and OlGC7, respectively, in a previous study (K. Inoue et al. 2003a), were also indicated for comparison. Open circles and solid lines indicate BNP. Filled squares, triangles, and circles with hatched lines indicate CNP-1, CNP-3, and CNP-4, respectively. Analyses were performed in quadruplicate or triplicate, and values are shown as means ± standard error of mean.
Tissue Specificity of Expression of ANP, BNP, and VNP Genes
Among tissues of the rainbow trout examined, transcripts of ANP, BNP, and VNP genes were detected exclusively in the atrium and ventricle of the heart (fig. 6). This pattern occurred even after 40 cycles of amplifications in RT-PCR.
FIG. 6.— Expression of ANP, BNP, and VNP genes in various tissues of the rainbow trout. Transcripts were amplified by RT-PCR and electrophoresed onto 1.2% agarose gel containing ethidium bromide. Glyceraldehyde-3-phosphate dehydrogenase expression was also shown as an internal control.
Linkage Mapping
In the linkage panel of the rainbow trout, nucleotide polymorphisms between two haploid genomes were successfully found in ANP and BNP genes of both parents. In the VNP gene, however, only one SNP was found between the haploid sets of the father and none was found in those of the mother. As a result of linkage mapping using such polymorphisms as markers, all three genes were mapped on the same region of a linkage group (LG. C) in the male linkage map (fig. 7). ANP and BNP genes are also mapped to the same linkage group in the female map.
FIG. 7.— Positions of ANP, BNP, and VNP genes determined by linkage mapping in the rainbow trout. Microsatellite markers closely linked to the three genes are also shown. Maps constructed according to inheritance of markers and genes from male and female parents are indicated separately (see Sakamoto et al. 2000). LG implies linkage group. The position of the VNP gene was not determined in the female map because no sequence polymorphism was found in the VNP gene of the mother fish of the mapping panel.
Discussion
Original Set of Cardiac NPs in Teleosts Is ANP, BNP, and VNP
VNP was found in the eel and trout in the early 1990s, but BNP was not found in these species at that time. Thus, VNP was thought to be a teleost homolog of BNP. The recent finding of the coexistence of BNP and VNP in the sturgeon suggested that the BNP and VNP genes are not orthologous (Kawakoshi et al. 2004). The BNP gene was subsequently isolated from pufferfish and tilapia (Kawakoshi et al. 2004), but VNP was not found in these species. Thus, information about the NPs in teleosts has been insufficient to explain the constitution of cardiac NPs in extant species. For example, it has been difficult to explain how species retaining ANP and BNP (tilapia and pufferfish) and those retaining ANP and VNP (eel and rainbow trout) have diverged. In the first part of this study, we performed a systematic survey of the cardiac NP cDNAs in teleosts. The discovery of BNP cDNA in the rainbow trout and eel provides a new insight into the evolutionary history of the cardiac NPs. Because the sturgeon, eel, and trout, which have diverged in the early evolution of ray-finned fish, still retain ANP, BNP, and VNP genes (fig. 1), it was suggested that the ancestral ray-finned fish had these three genes. In addition, the coexistence of BNP and VNP genes in the eel, known as diploid species, clearly shows that BNP and VNP are not generated by polyploidization.
BNP Is Conserved Throughout Teleosts and Tetrapods
A BNP cDNA was detected in all teleost species examined in this study. Considering the taxonomic positions of the species examined in this study, it is probable that BNP is distributed throughout the teleost lineage. As BNP has been found also in amphibians, birds, and mammals (Takei 2000), BNP may be universal throughout tetrapod and teleost lineages. The structural characteristics of tetrapod BNPs, for example, two consecutive basic amino acids at the fourth and fifth residues of the ring (fig. 2) and ATTTA repeats in 3'-UTR (fig. 3), were also conserved in the species examined in this study. These characteristics may be indispensable for the specific function of BNP. It is known that ANP and BNP share the same receptor GC-A in mammals (Suga et al. 1992) and ANP and VNP also share the GC-A in the eel (Kashiwagi et al. 1999). We also found that medaka BNP activated the two GC-A homologs of medaka (fig. 4). Thus, it seems that ANP, BNP, and VNP share the same GC-A receptor. In addition, we found that the sites of expression are also common among the three NP genes in the rainbow trout (fig. 5); thus, the three cardiac NPs seem functionally equivalent. It has been shown that the ATTTA sequence decreases the stability of mRNA and contributes to the rapid degradation of the massage (Wilson and Treisman 1988). In addition, it is generally believed that the BNP gene is an immediate early gene that participates in immediate adjustments of cardiovascular and body fluid regulation (Gardner 2003). Thus, it is possible that precise temporal regulation of BNP mRNA expression is a special characteristic of BNP, and rapid clearance of the message may be indispensable for this. It is also possible that the other conserved characteristic, the dibasic motif in the ring, is related to the clearance of BNP at the peptide level.
Absence of VNP and ANP in Some Teleost Species
We could not find the VNP cDNA in the ayu (Osmeroidei), tilapia, pufferfish, icefish, medaka, killifish, and pufferfish (Neoteleostei). The absence of the VNP gene was also confirmed by the analysis of the genomic databases of the pufferfish and medaka. Thus, it is supposed that the VNP gene, which existed in the ancestoral ray-finned fish, was lost or deformed during the course of evolution in the actinopterygian lineage. According to the new phylogenetic tree of teleosts that was proposed based on the whole mitochondrial genome (J. G. Inoue et al. 2003; Miya et al. 2003), it is possible that the loss of the VNP gene occurred before the divergence of Osmeroidei (fig. 1). In medaka, we failed to even find ANP cDNA in spite of extensive cDNA screening using various primers. In addition, we could not find the ANP gene in the whole-genome shotgun database of medaka. Moreover, we screened a genomic BAC library of medaka (Matsuda et al. 2001) using the trout ANP cDNA as a probe, but we failed to detect a clone containing the ANP gene (data not shown). Furthermore, we isolated the genomic BAC clones bearing the BNP and CNP-3 genes and determined the sequences around the two genes (fig. 2) because the synteny of CNP-3, BNP, and ANP genes is conserved in all tetrapods and teleosts in which whole genomic sequences are available. However, the ANP gene was not found, and therefore, the ANP gene seems absent in this species. Because the ANP cDNA was found in the killifish, the loss of the ANP gene seems to have occurred in Beloniformes (fig. 1). It has been suggested that, in eel, ANP plays an important role in the first phase of SW adaptation (Takei 2000; Takei and Hirose 2002). In addition, we showed that medaka is a euryhaline fish but lacks the ability for immediate adaptation to high salinity (Inoue and Takei 2002, 2003). It is an interesting question whether the absence of ANP is related to the lack of the ability for immediate osmoregulation.
Generation of the VNP Gene by Tandem Duplication
In a previous study, we presumed the evolutionary history of NPs based on linkage mapping in the medaka and comparative genomics in the pufferfish, medaka, and humans (K. Inoue et al. 2003a). As a result, it was suggested that ANP and BNP genes were generated by the tandem duplications of one of the four CNP genes, CNP-3. However, the origin of VNP still remains unknown because, in both species, the VNP gene is not detectable by screening of cDNA or by database searches. In the present study, we found all three cardiac NP genes in the rainbow trout, in which panels for linkage mapping have been already established (Sakamoto et al. 2000). By linkage mapping, the ANP, BNP, and VNP genes were located on the same position of the same linkage group. Namely, the three genes localize tandemly on the same chromosome. This result suggests that the VNP gene has also been generated by tandem duplication of one of the ANP, BNP, and CNP-3 genes. The exact timing of the generation of the VNP gene is still unknown at present, but if it occurs before the divergence of the tetrapod and teleost lineages, the VNP gene may exist in the tetrapods.
Acknowledgements
We express thanks to John A. Donald of Deakin University for critical reading of the manuscript, to Masato Kinoshita of Kyoto University and Atsushi Fujiwara of National Research Institute of Aquaculture for cooperation in screening of NP genes and chromosomal analyses, respectively, and to Fumi Katoh for the gift of the killifish. We also thank Susumu Hyodo and Sanae Hasegawa for valuable comments and discussion. This work was supported in part by grants-in aid for Creative Basic Research (12NP0201) from the Ministry of Education, Culture, Sports, Science and Technology of Japan and for Scientific Research (16207004, 15580157) from the Japan Society for Promotion of Science to Y.T. and K.I., respectively.
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E-mail: inouek@ori.u-tokyo.ac.jp.
Abstract
Natriuretic peptides (NPs) are a group of hormones playing important roles in cardiovascular and osmoregulatory systems in vertebrates. Among the NP subtypes, atrial NP (ANP), B-type NP (BNP), and ventricular NP (VNP) are circulating hormones expressed exclusively in the heart (cardiac NPs). The constitution of cardiac NPs is variable among species of vertebrates. In order to understand the evolutionary and functional significance of such variation, we performed a systematic survey of cardiac NP cDNAs in nine taxonomically diverse teleosts inhabiting environments of varying salinity. The discovery of the coexistence of the ANP, BNP, and VNP genes in the eel and rainbow trout suggested that the ancestral teleost had all three cardiac NPs. As the VNP cDNA was undetectable in ayu and six species of Neoteleostei, it is possible that VNP was lost before the divergence of Osmeroidei. The ANP gene was also undetectable in the medaka. Thus, only the BNP gene is universal in species examined in the present study. Synthetic medaka BNP preferentially activated two medaka GC-A–type receptors, suggesting that the three cardiac NPs share the same receptor. However, the regulation of BNP expression may be the most strict because ATTTA repeats in the 3'-untranslated region and the dibasic motif in the ring are conserved among teleosts and tetrapods. Linkage analyses in the rainbow trout located ANP, BNP, and VNP genes on the same chromosome, which suggested the generation of the VNP gene by tandem duplication as observed with ANP and BNP genes. If the duplication occurred before the divergence of tetrapods and teleosts, VNP may exist in the tetrapod lineage.
Key Words: natriuretic peptides ? guanylyl cyclase ? evolution ? tandem duplication ? linkage mapping
Introduction
Natriuretic peptides (NPs) are a group of hormones characterized by the conserved ring structure consisting of 17 amino acids with N-terminal and C-terminal extensions, which are called "head" and "tail" segments, respectively. NPs play important roles in the regulation of circulatory and fluid homeostasis in vertebrates (Loretz and Pollina 2000; Takei 2000; Toop and Donald 2004). Four major members, atrial NP (ANP), B-type NP (BNP), ventricular NP (VNP), and C-type NP (CNP), have been identified thus far in vertebrates. ANP, BNP, and VNP are circulating hormones expressed mainly in the heart (cardiac NPs), whereas CNP is a paracrine/autocrine factor mainly expressed in the brain. CNP, characterized by the absence of the tail segment, has been found in all vertebrates previously examined except in the hagfish, which has a unique NP, hfNP (Kawakoshi et al. 2003). Although hfNP has a tail segment, it is thought to be a derivative of CNP (Kawakoshi, A., S. Hyodo, and Y. Takei, unpublished data). Thus, CNP has been suggested to be the ancestral molecule of the NP family (K. Inoue et al. 2003a). The constitution of the cardiac NPs is variable among different species of vertebrates. In tetrapods, ANP and BNP have been found in mammals and amphibians, but ANP has not been found in birds (Takei 2000). In teleosts, the ANP and BNP genes are also isolated from the tilapia and pufferfish. In the eel and trout, however, BNP has not been found but, instead, VNP was isolated (Takei et al. 1991; Takei, Ueki, and Nishizawa 1994). In addition, we recently found that the sturgeon Acipenser transmontanus, which is a species of Chondrostei, has both the BNP and VNP genes in addition to the ANP and CNP genes (Kawakoshi et al. 2004). However, information on the significance of such variations in the cardiac NPs is insufficient, and thus, systematic surveys of NPs are necessary to understand the complex NP system of vertebrates totally.
In the present study, we isolated cDNAs encoding precursors of cardiac NPs from taxonomically diverse teleosts, including seawater (SW), freshwater, and migratory species, and compared constituents of cardiac NP genes and their structure. We subsequently identified the positions of the ANP, BNP, and VNP genes on the chromosomes of the rainbow trout by linkage analyses. We also prepared synthetic BNP of medaka, in which NP receptors have been extensively studied (Kusakabe and Suzuki 2000), and examined its activity using the homologous NP receptors expressed in cultured cells. Based on the results of such analyses, we discuss the structural and functional evolution of the three cardiac NP genes in the lineage of ray-finned fish.
Materials and Methods
Fish
The four-spine sculpin, Cottus kazika, and ayu, Plecoglossus altivelis, were obtained from Fukui Prefectural Inlandwater General Center, Fukui, Japan. The icefish Chionodraco hamatus was sampled at Terranova Bay, Ross Sea, Antarctica. The killifish Fundulus heteroclitus was obtained from Fumi Katoh of Ocean Research Institute, The University of Tokyo. The medaka, Oryzias latipes (outbred, orange-red type), pufferfish, Takifugu rubripes, rainbow trout, Oncorhynchus mykiss, Japanese eel, Anguilla japonica, and tilapia, Oreochromis mossambica, were obtained as previously described (Takei et al. 2001; K. Inoue et al. 2003a, 2003b; Kawakoshi et al. 2004). The taxonomic relationships according to phylogenetic trees based on the whole mitochondrial genome sequences (J. G. Inoue et al. 2003; Miya et al. 2003) and salinity conditions of habitats of species examined in this study are indicated in figure 1.
FIG. 1.— The phylogenetic and taxonomic status of the teleosts that were examined in this study, and the distribution of cardiac NPs identified by cDNA cloning. The sturgeon was also included for comparison. Phylogenetic relations are indicated according to the phylogenetic trees proposed by J. G. Inoue et al. (2003) and Miya et al. (2003) based on whole mitochondrial genome sequences. The possible timing of the loss of the VNP and ANP genes are indicated by a and b, respectively. The asterisk indicates that the sturgeon belongs to Chondrostei, whereas others to Teleostei.
cDNA Cloning
After anesthesia with 0.05% 2-phenoxyethanol, the heart of each species was dissected free. The total RNA was isolated using Isogen (Nippon Gene, Toyama, Japan), and the cDNA pool was synthesized using the SMART cDNA Library Construction Kit (Clontech Laboratories, Palo Alto, Calif.), as described by Inoue, Iwatani, and Takei (2003). Partial NP cDNAs were isolated by 3'-rapid amplification of cDNA ends (3'-RACE) using the primers shown in table 1: primers fishANP-S1, fishBNP-S1, and fishVNP-S1 were designed based on specific sequences of ANP, BNP, and VNP of teleosts, respectively. Partial NP cDNAs were amplified by "touchdown polymerase chain reaction (PCR)" in the condition as follows: for fishANP-S1, eight cycles of amplifications using the thermal steps consisting of denaturation at 94°C for 30 s, annealing at 72–64°C for 30 s, and extension at 72°C for 105 s, decreasing annealing temperature 1°C after each cycle finished. Then 35 cycles of amplification were performed using the fixed thermal steps of denaturation at 94°C for 30 s, annealing at 64°C for 30 s, and extension at 72°C for 105 s. Annealing temperature was 4°C and 6°C decreased for fishBNP-S1 and fishVNP-S1, respectively. Because partial sequences of ANP and VNP cDNAs of the eels and those of ANP and BNP cDNAs of the pufferfish and tilapia obtained by the first screening by 3'-RACE agreed completely with sequences previously reported (Takei, Ueki, and Nishizawa 1994; Takei et al. 1997; Kawakoshi et al. 2004), further analyses were not performed for these cDNAs. For other cDNAs, upstream regions were obtained by 5'-RACE using specific primers designed from the sequences obtained by 3'-RACE (table 1). Finally, the whole cDNA sequences were obtained by 3'-RACE using specific primers designed based on 5'-untranslated regions (UTRs) (table 1). PCR conditions for 5'-RACE and final 3'-RACE were described by J. G. Inoue et al. (2003). All DNA sequences were determined using ABI 3100 sequencer and BigDye cycle sequencing kit ver. 3.1 (Applied Biosystems, Foster City, Calif.).
Table 1 Primers Used for Cloning and RT-PCR Analyses
Screening of Medaka Bacterial Artificial Chromosome Library
The medaka bacterial artificial chromosome (BAC) library (Matsuda et al. 2001) was screened using BNP cDNA labeled by Alkphos Direct Labelling Reagents (Amersham Biosciences, Buckinghamshire, United Kingdom) as a probe, and positive clones mHDR188J4 and mHDR164P14 were obtained. EcoRI fragments bearing BNP and CNP-3 genes were isolated from the former clone and inserted into pBluescriptII (Stratagene, La Jolla, Calif.). After sequencing of cloned fragments, sequences of peripheral region from 23.6-kb upstream to 14.3-kb downstream of the BNP gene were determined by primer walking.
Deposition of Nucleotide Sequences
The sequences obtained in this paper have been deposited in the DNA Data Bank of Japan/GenBank/European Molecular Biology Laboratory Database with accession numbers AB162775, AB162776, AB162777, AB162778, AB162779, AB162780, AB087286, AB179821, AB076603, AB162781, AB076604, and AB179820 for cDNAs of ayu ANP, ayu BNP, four-spine sculpin ANP, four-spine sculpin BNP, icefish ANP, icefish BNP, killifish ANP, killifish BNP, rainbow trout ANP, rainbow trout BNP, rainbow trout VNP, eel BNP and accession numbers AB204712, AB204714, and AB207138 for the second intron of rainbow trout VNP gene, the second intron of the rainbow trout BNP gene, and the medaka genomic fragment containing CNP-3 and BNP genes, respectively.
Analyses of Genomic Databases
Genomic information of the pufferfish and medaka was obtained from the Web sites http://fugu.biology.qmul.ac.uk/ and http://dolphin.lab.nig.ac.jp/medaka/, respectively.
Receptor Activation Assays
Predicted medaka BNP was synthesized at Peptide Institute (Minoo, Osaka, Japan). Activities of the synthetic BNP on the three endogenous NP receptors OlGC1 (GC-B homolog) (Takeda and Suzuki 1999), OlGC2, and OlGC7 (GC-A homologs) (Yamagami, K. Suzuki, and N. Suzuki 2001) expressed in COS-7 cells were performed according to K. Inoue et al. (2003a). CNP-4, CNP-3, and CNP-1 were also examined for comparison. Experiments are performed in quadruplicate or triplicate and repeated twice.
Reverse Transcription–Polymerase Chain Reaction
The tissue distribution of transcripts of the ANP, BNP, and VNP genes in rainbow trout was examined by reverse transcription (RT)–PCR as described by K. Inoue et al. (2003b). Primer sets ANP-S11/ANP-A5, TroutBNP-S1/TroutBNP-A1, VNP-S4/VNP-A3, and GAPDH-S2/GAPDH-A2 (table 1) were used for the detection of ANP, BNP, VNP, and glyceraldehyde-3-phosphate dehydrogenase (internal control) transcripts, respectively.
Linkage Mapping
To use as genotyping markers, nucleotide polymorphisms in rainbow trout ANP, BNP, and VNP genes were searched by PCR followed by direct sequencing. Genomic DNA (0.1 μg) of the male and female parent fish of the linkage panel established by Sakamoto et al. (2000) was used as the template. Primers rtANP-S2/rtANP-A2, rtVNP-S2/rtVNP-A2, and rtBNP-S1/rtBNP-A1 were used for the amplification of ANP, BNP, and VNP genes, respectively, and amplified fragments were sequenced using the same primers from both ends. Linkage mapping was performed according to Sakamoto et al. (2000). The polymorphism in the length of cytosine repeat starting from the 65th base of the 3'-UTR of ANP gene was detected by PCR using primers rtANP-S7 and rtANP-A7 followed by 5% polyacrylamide gel electrophoresis. The single-nucleotide polymorphisms (SNPs) in the VNP gene, at 250 and 20 bases upstream from the end of the second intron, were detected by PCR using primer sets rtVNP-S4/rtVNP-A4 and rtVNP-S5/rtVNP-A5. The SNPs in the BNP gene were detected using a PRISM SNaPshot multiplex kit (Applied Biosystems). Primers TroutBNP-S2 and TroutBNP-A3 were used for the detection of the SNP at the 62nd base of the coding region in the gene of the father fish and that at 97 bases upstream from the end of the second intron of the gene of the mother, respectively.
Results
Cloning of ANP, BNP, and VNP cDNAs
cDNAs encoding ANP and VNP were isolated from the heart of the eel and rainbow trout. BNP cDNAs were also isolated from these two species, in which the BNP had not been isolated. Thus, it was found that ANP, BNP, and VNP genes are present in the eel and rainbow trout, as they are in the sturgeon (fig. 1). Although cDNAs encoding ANP and BNP were obtained from the heart of the ayu, four-spine sculpin, pufferfish, icefish, tilapia, and killifish, extensive search for VNP cDNA was not successful in these species. We also screened the pufferfish genome database but failed to detect the VNP gene. From the heart of medaka, BNP cDNA was isolated but neither ANP nor VNP cDNAs were found in spite of repeated attempts using various primers. Blast searches of the medaka whole-genome shotgun database also failed to detect ANP- and VNP-like sequences, although multiple clones encoding precursors of BNP and CNPs have been identified. Moreover, we isolated genomic BAC clones of the medaka containing BNP and CNP-3 genes and determined the sequence neighboring the two genes. However, neither ANP nor VNP genes were found (fig. 2). In the downstream region of the BNP gene, where the ANP gene is present in mammals and Tetraodon, we found a sequence similar to a region of the retrotransposon sushi-ichi of the pufferfish (Poulter and Butler 1998). This sequence was followed by a repetitive region containing tandem repeats of an approximately 3-kb sequence. Similar repeats frequently appear in many scaffolds of the medaka genome. Thus, the ANP gene was not found in the vicinity of the BNP and CNP-3 genes. This result suggests that the ANP gene is absent in the medaka.
FIG. 2.— Comparison of the arrangement of genes around the BNP gene in humans, Tetraodon, and medaka. Shaded arrows indicate position and direction of genes. Exons and introns were not distinguished in this figure. The Tetraodon gene was predicted using the Takifugu cDNA sequences. Hatched arrow indicates the region containing a tandem repeat of a 3-kb sequence.
Predicted Structure of ANP, BNP, and VNP
Mature ANP, BNP, and VNP sequences predicted from cDNAs isolated in this study are shown in figure 3, in comparison with sturgeon NP sequences previously reported (Kawakoshi et al. 2004). As for ANP precursors, the amino acid just before the termination codon was glycine in all species examined, except rainbow trout. Thus, it was suggested that teleost ANPs have an amidated tail, as reported in the eel ANP (Takei 2000), except those of salmonid fishes.
FIG. 3.— Structure of teleost ANP, BNP, and VNP predicted from cDNAs isolated in this study. The sequences of the sturgeon reported by Kawakoshi et al. (2004) are also indicated for comparison. The carboxyl terminus of ANP was indicated as an amidated form when the terminal amino acid was glycine. Amino acid residues conserved completely in each subtype were reversed, and those conserved in more than half the species are shaded. BNP-specific dibasic motif is indicated by asterisks.
BNP sequences were highly conserved among teleosts. Basic amino acids at fourth and fifth residues of the predicted ring domain, which are characteristic of most tetrapod BNPs, were conserved (fig. 3). Repeated ATTTA sequences, which are reported to decrease the stability of mRNA (Wilson and Treisman 1988), are also observed in the 3'-UTR of BNP cDNAs of all species examined in this study (fig. 4).
FIG. 4.— Distribution of the ATTTA motif in the 3'-UTR of the teleost BNP cDNAs isolated in this study. The sturgeon BNP cDNA is also shown for comparison. Positions of the first base of the ATTTA motif are indicated by arrowheads. Dotted boxes and lines represent the end of the coding region and 3'-UTR, respectively.
The mature trout VNP sequence predicted from the cDNA was identical to that obtained previously by peptide isolation (Takei et al. 1994). It was found that VNP sequences were conserved among the eel, rainbow trout, and sturgeon (fig. 3).
Activity of Medaka BNP on Three Medaka NP Receptors
Synthetic medaka BNP, at the concentration of 10–8 M or higher, induced cyclic guanosine monophosphate (cGMP) accumulation in COS cells expressing two medaka NP receptors, OlGC2 and OlGC7, both of which are homologs of GC-A of other vertebrates. However, medaka BNP barely increased cGMP in OlGC1, a GC-B homolog (fig. 5). Thus, it was found that BNP preferentially activates the two GC-A–type receptors.
FIG. 5.— Dose-dependent activation of the medaka NP receptors OlGC1 (a GC-B homolog), OlGC2, and OlGC7 (GC-A homologs) by the synthetic medaka BNP ligand. Receptors were expressed in COS-7 cells, and cGMP concentration was measured by enzyme-linked immunosorbent assay. The activities of CNP-4, CNP-3, and CNP-1, which exhibited highest activity on OlGC1, OlGC2, and OlGC7, respectively, in a previous study (K. Inoue et al. 2003a), were also indicated for comparison. Open circles and solid lines indicate BNP. Filled squares, triangles, and circles with hatched lines indicate CNP-1, CNP-3, and CNP-4, respectively. Analyses were performed in quadruplicate or triplicate, and values are shown as means ± standard error of mean.
Tissue Specificity of Expression of ANP, BNP, and VNP Genes
Among tissues of the rainbow trout examined, transcripts of ANP, BNP, and VNP genes were detected exclusively in the atrium and ventricle of the heart (fig. 6). This pattern occurred even after 40 cycles of amplifications in RT-PCR.
FIG. 6.— Expression of ANP, BNP, and VNP genes in various tissues of the rainbow trout. Transcripts were amplified by RT-PCR and electrophoresed onto 1.2% agarose gel containing ethidium bromide. Glyceraldehyde-3-phosphate dehydrogenase expression was also shown as an internal control.
Linkage Mapping
In the linkage panel of the rainbow trout, nucleotide polymorphisms between two haploid genomes were successfully found in ANP and BNP genes of both parents. In the VNP gene, however, only one SNP was found between the haploid sets of the father and none was found in those of the mother. As a result of linkage mapping using such polymorphisms as markers, all three genes were mapped on the same region of a linkage group (LG. C) in the male linkage map (fig. 7). ANP and BNP genes are also mapped to the same linkage group in the female map.
FIG. 7.— Positions of ANP, BNP, and VNP genes determined by linkage mapping in the rainbow trout. Microsatellite markers closely linked to the three genes are also shown. Maps constructed according to inheritance of markers and genes from male and female parents are indicated separately (see Sakamoto et al. 2000). LG implies linkage group. The position of the VNP gene was not determined in the female map because no sequence polymorphism was found in the VNP gene of the mother fish of the mapping panel.
Discussion
Original Set of Cardiac NPs in Teleosts Is ANP, BNP, and VNP
VNP was found in the eel and trout in the early 1990s, but BNP was not found in these species at that time. Thus, VNP was thought to be a teleost homolog of BNP. The recent finding of the coexistence of BNP and VNP in the sturgeon suggested that the BNP and VNP genes are not orthologous (Kawakoshi et al. 2004). The BNP gene was subsequently isolated from pufferfish and tilapia (Kawakoshi et al. 2004), but VNP was not found in these species. Thus, information about the NPs in teleosts has been insufficient to explain the constitution of cardiac NPs in extant species. For example, it has been difficult to explain how species retaining ANP and BNP (tilapia and pufferfish) and those retaining ANP and VNP (eel and rainbow trout) have diverged. In the first part of this study, we performed a systematic survey of the cardiac NP cDNAs in teleosts. The discovery of BNP cDNA in the rainbow trout and eel provides a new insight into the evolutionary history of the cardiac NPs. Because the sturgeon, eel, and trout, which have diverged in the early evolution of ray-finned fish, still retain ANP, BNP, and VNP genes (fig. 1), it was suggested that the ancestral ray-finned fish had these three genes. In addition, the coexistence of BNP and VNP genes in the eel, known as diploid species, clearly shows that BNP and VNP are not generated by polyploidization.
BNP Is Conserved Throughout Teleosts and Tetrapods
A BNP cDNA was detected in all teleost species examined in this study. Considering the taxonomic positions of the species examined in this study, it is probable that BNP is distributed throughout the teleost lineage. As BNP has been found also in amphibians, birds, and mammals (Takei 2000), BNP may be universal throughout tetrapod and teleost lineages. The structural characteristics of tetrapod BNPs, for example, two consecutive basic amino acids at the fourth and fifth residues of the ring (fig. 2) and ATTTA repeats in 3'-UTR (fig. 3), were also conserved in the species examined in this study. These characteristics may be indispensable for the specific function of BNP. It is known that ANP and BNP share the same receptor GC-A in mammals (Suga et al. 1992) and ANP and VNP also share the GC-A in the eel (Kashiwagi et al. 1999). We also found that medaka BNP activated the two GC-A homologs of medaka (fig. 4). Thus, it seems that ANP, BNP, and VNP share the same GC-A receptor. In addition, we found that the sites of expression are also common among the three NP genes in the rainbow trout (fig. 5); thus, the three cardiac NPs seem functionally equivalent. It has been shown that the ATTTA sequence decreases the stability of mRNA and contributes to the rapid degradation of the massage (Wilson and Treisman 1988). In addition, it is generally believed that the BNP gene is an immediate early gene that participates in immediate adjustments of cardiovascular and body fluid regulation (Gardner 2003). Thus, it is possible that precise temporal regulation of BNP mRNA expression is a special characteristic of BNP, and rapid clearance of the message may be indispensable for this. It is also possible that the other conserved characteristic, the dibasic motif in the ring, is related to the clearance of BNP at the peptide level.
Absence of VNP and ANP in Some Teleost Species
We could not find the VNP cDNA in the ayu (Osmeroidei), tilapia, pufferfish, icefish, medaka, killifish, and pufferfish (Neoteleostei). The absence of the VNP gene was also confirmed by the analysis of the genomic databases of the pufferfish and medaka. Thus, it is supposed that the VNP gene, which existed in the ancestoral ray-finned fish, was lost or deformed during the course of evolution in the actinopterygian lineage. According to the new phylogenetic tree of teleosts that was proposed based on the whole mitochondrial genome (J. G. Inoue et al. 2003; Miya et al. 2003), it is possible that the loss of the VNP gene occurred before the divergence of Osmeroidei (fig. 1). In medaka, we failed to even find ANP cDNA in spite of extensive cDNA screening using various primers. In addition, we could not find the ANP gene in the whole-genome shotgun database of medaka. Moreover, we screened a genomic BAC library of medaka (Matsuda et al. 2001) using the trout ANP cDNA as a probe, but we failed to detect a clone containing the ANP gene (data not shown). Furthermore, we isolated the genomic BAC clones bearing the BNP and CNP-3 genes and determined the sequences around the two genes (fig. 2) because the synteny of CNP-3, BNP, and ANP genes is conserved in all tetrapods and teleosts in which whole genomic sequences are available. However, the ANP gene was not found, and therefore, the ANP gene seems absent in this species. Because the ANP cDNA was found in the killifish, the loss of the ANP gene seems to have occurred in Beloniformes (fig. 1). It has been suggested that, in eel, ANP plays an important role in the first phase of SW adaptation (Takei 2000; Takei and Hirose 2002). In addition, we showed that medaka is a euryhaline fish but lacks the ability for immediate adaptation to high salinity (Inoue and Takei 2002, 2003). It is an interesting question whether the absence of ANP is related to the lack of the ability for immediate osmoregulation.
Generation of the VNP Gene by Tandem Duplication
In a previous study, we presumed the evolutionary history of NPs based on linkage mapping in the medaka and comparative genomics in the pufferfish, medaka, and humans (K. Inoue et al. 2003a). As a result, it was suggested that ANP and BNP genes were generated by the tandem duplications of one of the four CNP genes, CNP-3. However, the origin of VNP still remains unknown because, in both species, the VNP gene is not detectable by screening of cDNA or by database searches. In the present study, we found all three cardiac NP genes in the rainbow trout, in which panels for linkage mapping have been already established (Sakamoto et al. 2000). By linkage mapping, the ANP, BNP, and VNP genes were located on the same position of the same linkage group. Namely, the three genes localize tandemly on the same chromosome. This result suggests that the VNP gene has also been generated by tandem duplication of one of the ANP, BNP, and CNP-3 genes. The exact timing of the generation of the VNP gene is still unknown at present, but if it occurs before the divergence of the tetrapod and teleost lineages, the VNP gene may exist in the tetrapods.
Acknowledgements
We express thanks to John A. Donald of Deakin University for critical reading of the manuscript, to Masato Kinoshita of Kyoto University and Atsushi Fujiwara of National Research Institute of Aquaculture for cooperation in screening of NP genes and chromosomal analyses, respectively, and to Fumi Katoh for the gift of the killifish. We also thank Susumu Hyodo and Sanae Hasegawa for valuable comments and discussion. This work was supported in part by grants-in aid for Creative Basic Research (12NP0201) from the Ministry of Education, Culture, Sports, Science and Technology of Japan and for Scientific Research (16207004, 15580157) from the Japan Society for Promotion of Science to Y.T. and K.I., respectively.
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