Expression and water calcium dependence of calcium transporter isoforms in zebrafish gill mitochondrion-rich cells

Background Freshwater fish absorb Ca2+ predominantly from ambient water, and more than 97% of Ca2+ uptake is achieved by active transport through gill mitochondrion-rich (MR) cells. In the current model for Ca2+ uptake in gill MR cells, Ca2+ passively enters the cytosol via the epithelium Ca2+ channel (ECaC), and then is extruded into the plasma through the basolateral Na+/Ca2+ exchanger (NCX) and plasma membrane Ca2+-ATPase (PMCA). However, no convincing molecular or cellular evidence has been available to support the role of specific PMCA and/or NCX isoforms in this model. Zebrafish (Danio rerio) is a good model for analyzing isoforms of a gene because of the plentiful genomic databases and expression sequence tag (EST) data. Results Using a strategy of BLAST from the zebrafish genome database (Sanger Institute), 6 isoforms of PMCAs (PMCA1a, PMCA1b, PMCA2, PMCA3a, PMCA3b, and PMCA4) and 7 isoforms of NCXs (NCX1a, NCX1b, NCX2a, NCX2b, NCX3, NCX4a, and NCX4b) were identified. In the reverse-transcriptase polymerase chain reaction (RT-PCR) analysis, 5 PMCAs and 2 NCXs were ubiquitously expressed in various tissues including gills. Triple fluorescence in situ hybridization and immunocytochemistry showed the colocalization of zecac, zpmca2, and zncx1b mRNAs in a portion of gill MR cells (using Na+-K+-ATPase as the marker), implying a subset of ionocytes specifically responsible for the transepithelial Ca2+ uptake in zebrafish gills. The gene expressions in gills of high- or low-Ca2+-acclimated zebrafish by quantitative real-time PCR analysis showed that zecac was the only gene regulated in response to environmental Ca2+ levels, while zpmcas and zncxs remained steady. Conclusion The present study provides molecular evidence for the specific isoforms of Ca2+ transporters, zECaC, zPMCA2, and zNCX1b, supporting the current Ca2+ uptake model, in which ECaC may play a role as the major regulatory target for this mechanism during environmental challenge.


Background
Ca 2+ is an essential element for almost all organisms and plays comprehensive regulatory roles in eukaryotic cells; therefore, Ca 2+ must be maintained within a narrow concentration range in organisms (cellular free [Ca 2+ ]: 100 nM; plasma total [Ca 2+ ]: 2~3 mM). In terrestrial vertebrates, the whole-body Ca 2+ balance is mainly achieved by intestinal absorption and kidney reabsorption. Ca 2+ uptake mechanisms in the mammalian kidney have been most extensively studied, while fish gills, a specialized organ in non-mammalian vertebrates, is another model for studying Ca 2+ uptake mechanisms [1]. In fish, gills are the main site (> 97% of the whole body) of Ca 2+ uptake from the aquatic environment to maintain the Ca 2+ balance [2], and skin takes the place of gills during early developmental stages when the gills are not yet developed and functioning [3][4][5]. Compared with terrestrial vertebrates, Ca 2+ regulation mechanisms in aquatic animals are probably more complicated and challenging because of the dramatic fluctuations in ambient Ca 2+ concentrations which can occur (seawater, around 10 mM; fresh water, 0.01~3 mM).
According to the current model in mammals, active and transcellular Ca 2+ transport is carried out as a 3-step process [1]. Following entry of Ca 2+ through apical epithelial Ca 2+ channels (ECaC, TRPV5, and/or TRPV6), Ca 2+ is bound to calbindins that facilitate diffusion to the basolateral membrane, and then it is extruded via the plasma membrane Ca 2+ -ATPase (PMCA) and/or Na + /Ca 2+ exchanger (NCX). In this way, net transepithelial Ca 2+ absorption from the luminal compartment (or environment) to the plasma is accomplished. A model of the capability of physiological regulation has also been similarly proposed in fish gills [6]. However, only very little molecular evidence is currently available to support this model in fish gills, which is a specialized organ for Ca 2+ uptake. The ecac gene has recently been cloned and sequenced in fugu, zebrafish, and trout [4,7,8]. In zebrafish embryos, low-Ca 2+ fresh water causes upregulation of the whole-body Ca 2+ influx and zECaC expression in mitochondrion-rich (MR) cells of both gills and skin, providing molecular evidence for the role of ECaC in fish Ca 2+ uptake [4]. On the other hand, some biochemical and physiological studies have investigated the possibility of PMCA and NCX's involvement in fish gill Ca 2+ uptake [9,10]. Nevertheless, no convincing evidence has been presented demonstrating the existence and involvement of PMCA and NCX in fish Ca 2+ uptake mechanisms.
Three NCX genes (SLC8A1, SLC8A2, and SLC8A3) and 4 PMCA genes (ATP2B1, ATP2B2, ATP2B3, and ATP2B4) have been identified in mammals so far. The fundamental work of PMCA is the highly regulated active extrusion of Ca 2+ from cells for maintaining a gradient across the plasma membrane [11]. PMCA belongs to the P-type primary ion transport ATPase superfamily with 10 transmembrane domains and 1 calmodulin-binding site. At least 20 alternative splicing variants of PMCA have been found in mammals [12]. In mammals, PMCA1 and PMCA4 are ubiquitously expressed, whereas PMCA2 and PMCA3 are more tissue specific; it has been suggested that PMCA1 and PMCA4 are housekeeping isoforms involved in the maintenance of cellular Ca 2+ homeostasis [13]. However, no molecular data about PMCAs are available in fish. On the other hand, at least 32 alternatively spliced isoforms of NCX1 gene products have been identified [14]. Knock-out of NCX1 in mice is lethal due to cardiac defects before birth [15], while many physiological studies have indicated that vitamin D 3 modulates Ca 2+ balance by upregulating NCX1 expression [16]. Recently, NCX1-3 and NCX4, another putative prototype of NCX, have been reported in fish, indicating the existence of NCXs in nonmammals as well [17,18]. However, there is no molecular physiological evidence to support the involvement of NCX in fish Ca 2+ uptake mechanisms.
Because of a theoretical process of whole-genome duplication (WGD) from ancestor vertebrates [19,20], the numbers of gene isoforms found in teleosts are usually more than those found in mammals and tetrapods. While different isoforms coded from distinct genomic loci tend to have tissue-specific expressions, there are some cases of isoforms being differentially regulated upon facing environmental changes (reviewed by Schulte [21]. Isoforms also serve compensatory functions; upregulation of TRPV6 was found in the kidneys of TRPV5-deficient mice [22]. In biochemical studies on fish, the gill activities of PMCA and NCX, which are altered based upon physiological needs and environmental changes, have been proposed as being associated with transepithelial Ca 2+ transport [9,23]. However, nothing is known about which specific isoforms are responsible for Ca 2+ uptake mechanisms in teleost gills. Therefore, using a functional genomic approach to investigate all isoforms of PMCA and NCX in a species may provide both physiological and genomic insights into this issue. In the present study, a strategy of a whole-genome survey was used to uncover any unidentified PMCA and NCX isoforms in zebrafish, and then these candidates were refined with the EST database and cloned. Six isoforms of PMCAs (PMCA1a, PMCA1b, PMCA2, PMCA3a, PMCA3b, and PMCA4) and 7 isoforms of NCXs (NCX1a, NCX1b, NCX2a, NCX2b, NCX3, NCX4a, and NCX4b) were identified. Moreover, we report that some duplication of PMCA and NCX occurred, and the tissue distributions and expression patterns of these isoforms were analyzed by reverse-transcriptase polymerase chain reaction (RT-PCR) and triple fluorescence labeling, respectively. The gene expressions in gills of zebrafish acclimated to high-or low-Ca 2+ environments were also examined by quantitative real-time PCR analysis. The present study provides molecular evidence to support the current Ca 2+ uptake model in fish gill cells [24].
Based on the topological structures of PMCA and NCX summarized in mammals [12,26], zebrafish PMCAs and NCXs share similar patterns as mammals at the deduced amino acid sequences. According to the hydropathy analysis, 10 putative transmembrane domains were predicted for zPMCA (Figures 1, 4a), whereas NCX has 11 hydrophobic segments and 9 putative transmembrane domains (Figures 2, 4b) due to an intracellular helix front transmembrane domain 6 and a P loop-like structure of hydrophobic segment 8, which contained the amino acid sequence "GIG" [26]. PMCA and sarco-endoplasmic reticulum calcium ATPase (SERCA), the closest kin to PMCA, share a similar topological structure: hydrophobic segments span the membrane 10 times, and 3 intracellular loops consist of Ca2+ binding and transporting, and calmodulin-regulating functions [12,27]; the zPMCAs were also found to have 3 cytoplasmic domains 1 each between TM2 and TM3, TM4 and TM5, and TM10 and the C-terminal ( Figure 1). As for NCX, a large intracellular loop for ion translocation [28,29] was located between TM5 and TM6 ( Figure 2). There were 20 alternative splicing isoforms of zPMCAs found when cloning was conducted, while 9 alternative splicing isoforms were found in zNCXs (data not shown). The hot spots of alternative splicing regions in the zPMCAs are at the 5' untranslated regions (UTRs), the fragments around the 300th amino acid including from 1 to 3 small exons (less than 100 bp each in those cases), and 3' amino acid tails that usually stretch to the 3' UTR and alter the location of stop codons. On the other hand, a region of alternative splicing sites was found in zNCX1b and zNCX3 around the 610th amino acid, which is a part of the intracellular loop.
zPMCAs share 63%~86% identities with each other, while zNCXs exhibit higher variety among isoforms (i.e., 36%~76% identity). According to the phylogenetic analysis, zebrafish and 2 pufferfish species so far all have 6 PMCA and 7 NCX isoforms (Figure 3a,b). Compared with the isoforms annotated in tetrapods, duplication from the ancestor ray-finned fish occurred in PMCA1, PMCA3, NCX1, NCX2, and NCX4, according to the latest version of the genomic database utilized. Moreover, in the karyotype, zPMCAs and zNCXs are located on different chromosomes (see additional file 1), suggesting that these paralogous groups may have originated from genome duplication but not from tandem duplication. Relationships among these pairs of duplicates were evident not only in the phylogenetic tree, but also in their gene structures ( Figure 4). All teleost PMCAs and NCXs examined in this study showed an outgroup topology to their orthologues of mammals (Figure 3a,b), and the paralogous teleosts formed an inner-group topology. The 2 prototypes of NCXs, NCX4a and NCX4b, found in teleosts were most closely related to NCX1 and NCX3 in both phylogenetic trees ( Figure 3) and gene structures (Figure 4b). In gene structures, all of them had a relatively large first exon, even though some small introns were found in the split exons in zNCX4s (Figure 4b).

mRNA expressions of pmca and ncx in various tissues
RT-PCR analysis revealed that zpmca2 and zpmca4 were ubiquitously expressed in various tissues compared with zpmca1a, zpmca1b, zpmca3a, and zpmca3b ( Figure 5). All zpmcas were abundant in the brain and eye except zpmca1b, which was mainly expressed in the eye. These findings agree with the Ca 2+ homeostasis function of PMCAs found in the mammalian neuron system [12]. zncx1b and zncx4a were also ubiquitously expressed, while zncx2b and zncx3 were only detected in the brain and eyes. zpmca1a, zpmca1b, zpmca2, zpmca3a, and all the zncxs were expressed in ovary and 1-cell embryo ( Figure 5), suggesting that these genes may regulate intracellular Ca 2+ homeostasis at earlier stages. zpmca1a, zpmca2, zpmca4, zncx1, and zncx4a were expressed in gills ( Figure 5).

Colocalization of ecac, pmca, and ncx mRNAs and Na + -K + -ATPase
Fluorescence in situ hybridization and immunocytochemistry were used to determine the isoforms of zpmca and zncx that were specifically expressed in gill ionocytes. Specific mRNA probes of all of the zpmca and zncx isoforms were used to conduct the in situ hybridizations for wholemount embryos and adult gills. Among the isoforms, only zpmca2 and zncx1b mRNA signals were found in specific groups of cells in embryonic skin and adult gills (data not shown).
Subsequently, double-fluorescence in situ hybridizations were used for zecac and zpmca2 ( Figure 7A-C), and zecac and zncx1b ( Figure 7D-F), respectively. More gill cells expressed zpmca2 and zncx1b mRNAs than zecac mRNA ( Figure 7A-F). Notably, zpmca2 and zncx1b were also abundantly expressed in gill lamellae, where fewer MR cells are usually found [24]. zecac and zpmca2 mRNAs were colocalized only in a portion of gill cells, but were not colocalized all of the time. Around half of the zecacpositive cells co-expressed zpmca2 signals (51.7 +/-3.50%, Quantitative real-time PCR analysis of plasma membrane Ca 2+ -ATPases (PMCAs) and Na + /Ca 2+ exchangers (NCXs) Figure 6 Quantitative real-time PCR analysis of plasma membrane Ca 2+ -ATPases (PMCAs) and Na + /Ca 2+ exchangers (NCXs). HCa (filled bar), cDNA from the gill of high-Ca 2+ -acclimated zebrafish; LCa (opened bar), cDNA from the gill of low-Ca 2+acclimated zebrafish. An asterisk (*) indicates a significant difference between the 2 groups (Student's t-test, p < 0.05).  Expression patterns of zebrafish plasma membrane Ca 2+ -ATPases (PMCAs) and Na + /Ca 2+ exchangers (NCXs) in vari-ous tissues by RT-PCR analysis Figure 5 Expression patterns of zebrafish plasma membrane Ca 2+ -ATPases (PMCAs) and Na + /Ca 2+ exchangers (NCXs) in various tissues by RT-PCR analysis. The zebrafish beta-actin was used as internal control to evaluate the relative amounts of cDNAs. The expected amplicon sizes are shown on the right. 1Cell, cDNA from the 1-cell stage of zebrafish embryos.  Figures 7A-C, 8). Similar results were found in the case of double labeling of zecac and zncx1b ( Figures 7D-F, 8). The proportion of zncx1bpositive cells that co-expressed zecac was 77.1 +/-1.35% (n = 3), while zncx1b signals could be detected in all zecacpositive cells (Figures 7D-F, 8).

Discussion
So far, 4 PMCA genes have been identified in mammals, and PMCA1 and PMCA4 were suggested to be housekeeping isoforms. Mammalian PMCA2 was found to be expressed in the hair bundle of hair cells of the inner ear [30], suggesting that it is responsible for endolymph Ca 2+ homeostasis [11]. A null mutation of PMCA2 caused balance and hearing defects in mice [31]. On the other hand, mammalian PMCA1 was deemed a major Ca 2+ extrusion transporter for Ca 2+ absorption in intestine and kidney [32][33][34]. Our data demonstrated opposite phenomena in fish PMCAs. zpmca2 was expressed with a housekeeping pattern ( Figure 5), while zpmca1a showed a hair cell expression pattern during embryonic stages (data not shown). This functional substitution of paralogues found in Ca 2+ transporters is not a unique example. In zebrafish, the expression profiles suggest the hypothesis that zebrafish hoxB1a and hoxB1b are functional equivalents of mouse Hoxb1 and Hoxa1, respectively. On the contrary, the zebrafish orthologue of mouse Hoxa1, zebrafish hoxA1a, is not expressed in a similar pattern [20]. Further examination of the promoter regions of these genes may provide insights into this phenomenon. As far as our data, zPMCA2 was proposed to participate in the transcellular Ca 2+ uptake pathway in fish gills. NCX1a and NCX1b from teleosts including zebrafish and pufferfish were not clustered within an inner group on the phylogenetic tree ( Figure 3). Further collection is necessary to enlarge the sequence pool, which may allow us to reconstruct the phylogenetic tree or to rename members of the gene family. In the present study, zNCX1b was demonstrated to be responsible for the Ca 2+ extrusion mechanism in fish gill cells, while the paralogue, zNCX1a, was proven to be a heart-specific isoform that causes cardiac fibrillations in zNCX1a mutants [25]. The combined functions from both zNCX1s are equivalent to those of mammalian NCX1; however, investigations of the direct function of NCX1 in Ca 2+ absorption were not possible due to fatal heart failure in knockout mice. Therefore, teleosts may serve as a good model to examine the role of NCX in Ca 2+ uptake without resulting heart failure.
As described above, Flik et al. [6] proposed a model of a fish gill Ca 2+ uptake mechanism, which is similar to that for Ca 2+ reabsorption in mammalian kidneys [1]. There has been no convincing or comprehensive molecular evidence to support the existence of the 3 major transporters, ECaC, PMCA, and NCX, in fish gill ionocytes until the present study. Pan et al. [4] for the first time demonstrated the expression of zecac in a subset of MR cells in zebrafish skin/gills and a correlation between zecac expression and in vivo Ca 2+ uptake function. Subsequently in rainbow trout, Perry and colleagues found the expression of ECaC mRNA and/or protein in both pavement and MR cells in rainbow trout [8,35], which is inconsistent with the current view that MR cells are the predominant site for branchial Ca 2+ uptake [36], and the authors also reported that hypercapnia, implantation with cortisol, and infusion with CaCl 2 affected ECaC expression in MR cells and/ or pavement cells [35]. In a recent study on isolated trout gill cells, PNA + MR cells showed an over 3-fold higher  45 Ca 2+ uptake capacity compared to either PNA -MR or pavement cells [37]. The subset of gill MR cells and/or pavement cells that expressed ECaC is not necessarily involved in transepithelial Ca 2+ uptake as Hwang and Lee [24] claimed, because those previous studies lacked molecular and physiological data to demonstrate the transport pathway across the basolateral membrane. The present triple in situ hybridization and immunocytochemical experiments demonstrate the co-expressions of zecac, zpmca2, and zncx1b in a specific group of gill MR cells, providing comprehensive molecular evidence for the involvement of these transporters in a fish gill Ca 2+ uptake mechanism.
Double in situ hybridization and immunocytochemistry experiments indicated that about 90% of gill MR cells expressed zecac mRNA, although this may have been underestimated because the antigenicity of Na + -K + -ATPase (the marker for MR cells) is attenuated during the high-temperature treatments used for in situ hybridization. All zecac-expressing cells co-expressed zncx1b, and about half of the zecac-expressing cells co-expressed zpmca2, indicating that about 50% of zecac-expressing cells co-expressed both zncx1b and zpmca2. Taking all these findings into account, only about 45% of the entire population of MR cells co-express zecac, zpmca2, and zncx1b, and this subset of ionocytes may specifically carry out the function of transepithelial Ca 2+ uptake in zebrafish gills. It was noted that some gill cells express only 1 or 2 (never all) of the 3 major Ca 2+ transporters. These cells may be in the process of terminal differentiation as Hsiao et al. [38] reported in skin ionocytes in zebrafish embryos. Alternatively, the Ca 2+ transporter(s) expressed in those cells may be involved in intracellular Triple fluorescence in situ hybridizations (ISH) and immunocytochemistry (ICC) for zecac mRNA/zpmca2 mRNA/Na + -K + -ATPase (A-E) and zecac mRNA/zncx1b mRNA/Na + -K + -ATPase (F-J) in frozen sections of zebrafish gills Figure 8 Triple fluorescence in situ hybridizations (ISH) and immunocytochemistry (ICC) for zecac mRNA/zpmca2 mRNA/Na + -K + -ATPase (A-E) and zecac mRNA/zncx1b mRNA/Na + -K + -ATPase (F-J) in frozen sections of zebrafish gills. A-E are the same section, and F-J are another section. A, zpmca2 mRNA; B and G, zecac mRNA; C and H, Na + -K + -ATPase (NKA); D and I, bright field (BF); E, merged image of A-D; J, merged image of F-I. Colocalization of the 3 transporter mRNAs and protein was found respectively in E and J. Scale bar = 20 µm. Ca 2+ homeostasis or other cellular events [4,39,40]. This remains to be confirmed in future studies.
ECaC is a vitamin D-sensitive transporter in mammals [22], and the expression of PMCA1 was also found to be stimulated by 1,25(OH) 2 D 3 in mammals and chicken [34,41,42]. 1,25(OH) 2 -vitamin D 3 was shown to have a hypercalcemic effect on both freshwater and saltwater teleosts [6,43], whereas stanniocalcin, a predominant hypocalcemic hormone produced by Stannius bodies, was found to show opposite effect on fish [44]. Stanniocalcin did not show a significant effect on basolateral Ca 2+ transport [45], but quickly reduced the permeability of Ca 2+ at the apical membrane [6,46]. It has been proposed that ECaC is the rate-limiting step and the gatekeeper channel for active Ca 2+ transport [47]. The present study provides further molecular physiological evidence to support this notion. The present study for the first time demonstrates that acclimation to a low-Ca 2+ environment, which would stimulate Ca 2+ uptake capacity in fish [3,48,49], caused upregulation of mRNA expression of zECaC but not zPMCA2 or zNCX1b, implying that the steady-state expressions of zPMCA2 and zNCX1b may fulfill the requirement for the transepithelial transport machinery under all situations. Moreover, this study provides molecular evidence to support a previous biochemical study by Flik et al. [50], who found that the basolateral Ca 2+ extrusion mechanisms by PMCA and NCX is far below their maximum capacity in fish gills based on the kinetic properties of the 2 enzymes.

Conclusion
The major findings of the present study were that (1) 6 PMCAs and 7 NCXs were identified from zebrafish tissues; (2) differential expressions of these genes were found in various tissues including gills; (3) acclimation to low-Ca 2+ environment stimulated the mRNA expression of ecac in gills, but not those of pmcas or ncxs; and (4) only a portion of gill MR cells co-expressed zecac, zpmca2, and zncx1b mRNAs.

Molecular cloning and sequences analysis
The peptide sequences from other species (teleost had a higher priority) were used to BLAST the genome databases (of NCBI and Ensembl) for zebrafish, pufferfish (Fugu rubripes), and tetradon (Tetraodon nigroviridis). The putative full-length or partial open reading frames of PMCAs and NCXs obtained were confirmed using the EST database, and/or were used to design primers for cloning and the RT-PCR analysis. PCR products thus obtained were subcloned into a pGEM-T Easy vector (Promega, Madison, WI, USA), and the nucleotide sequences were determined with an ABI 377 sequencer (Applied Biosystems, Warrington, UK). Sequence analysis was conducted with a BLASTx program (NCBI). The specific primers of 5' and 3'rapid amplification of cDNA ends (RACE) were designed from the partial sequences obtained from the PCR with degenerate primers. The RACE PCR program followed a commercial protocol (Clontech, Mountain View, CA, USA), and the RACE PCR products were also subcloned into pGEM-T Easy vectors and sequenced. The full-length deduced amino-acid sequences were aligned with Clus-talX and then phylogenetically analyzed with Mega3.1 [51]. For the proteomic structure analysis, the hydropathy plot and transmembranes were predicted with EMBOSS (the European Molecular Biology Open Software Suite, version 2.8.0 [52] algorithm according to Persson and Argos [53].

RT-PCR analysis
An appropriate amount of zebrafish adult tissues was collected for the total RNA preparation with TRIZol reagent (Invitrogen, Carlsbad, CA, USA). The amount and quality of total RNA were determined by measuring the absorbances at 260 and 280 nm with a spectrophotometer (Hitachi U-2000) and RNA denatured gels. For cDNA synthesis, 5 µg of total RNA was reverse-transcribed in a final volume of 20 µL containing 0.5 mM dNTPs, 2.5 µM oligo (dT) 18  A 514-bp fragment of zebrafish beta-actin was used as an internal control to evaluate the relative amounts of complementary DNAs (cDNAs), and the design of the primer pairs followed Hsiao et al. [54]. The amplicons were all sequenced to make sure that the PCR products were the desired gene fragments.

Fluorescence double in situ hybridization
Fragments of the target genes obtained by PCR were inserted into pGEM-T Eeasy vectors. Digoxigenin-(Dig) (Roche, Penzberg, Germany) or dinitrophenol (DNP)labeled (Perkin-Elmer, Boston, MA, USA) RNA probes were synthesized by in vitro transcription with T7 and SP6 RNA polymerase (Takara, Shiga, Japan). The qualities of the probes were examined using RNA gels, and the concentrations were determined by a dot-blot assay with standard DIG-labeled RNA (100 ng/µl) (Roche). Excised gills were fixed with 4% paraformaldehyde overnight at 4°C and then washed several times with PBS. For the cryosections, fixed samples were immersed in PBS containing 30% sucrose overnight, and embedded in Optimal Cutting Temperature (OCT) compound embedding medium (Sakura, Tokyo, Japan) at -20°C, and 10-µm frozen cross-sections were cut with a CM 1900 rapid sectioning cryostat (Leica, Heidelberg, Germany) and attached to poly-L-lysine coated slides (Electron Microscopy Sciences, Ft. Washington, PA, USA). Prepared samples from either cryosections or methanol-dehydrated whole gills were washed several times with phosphate-buffered saline with 0.1% tween-20 (PBST). After a brief washing with PBST, samples were incubated with hybridization buffer (HyB) containing 50% formamide, 5× SSC, and 0.1% Tween-20 for 5 min at 65°C. Prehybridization was performed for 2 h at 65°C with HyB+, which is the hybridization buffer supplemented with 500 ng/mL yeast tRNA and 50 µg/mL heparin. For hybridization, samples were incubated in 100 ng of the RNA probe in 200 µL HyB+ at 65°C overnight. Then, slides were washed at 65°C for 10 min in 75% HyB and 25% 2× SSC, 10 min in 50% HyB and 50% 2× SSC, 10 min in 25% HyB and 75% 2× SSC, 10 min in 2× SSC, and 30 min for 2 times in 0.2× SSC at 70°C. Further washes were performed at room temperature for 5 min in 75% 0.2× SSC and 25% PBST, 5 min in 50% 0.2× SSC and 50% PBST, 5 min in 25% 0.2× SSC and 75% PBST, and 5 min in PBST. Fluorescence staining was conducted with a commercial kit, TSA Plus Fluorescence Systems (Perkin-Elmer). The hybridization signals detected by the DIG-labeled RNA probes were amplified through fluorescein-tyramide signal amplification (TSA), while cyanine 3-TSA was used for the DNP-labeled probes. For triple labeling, samples were first subjected to in situ hybridizations of the 2 transporter genes and then to Na + / K + -ATPase immunocytochemistry (see below).

Immunohistochemistry
Zebrafish embryos and gills were fixed in 4% paraformaldehyde for 10 min at 4°C. After washing in PBS, fixed embryos were treated with 100% ethanol for 10 min at -20°C and subsequently subjected to blocking with 3% BSA at room temperature for 30 min. Embryos were then incubated with 1:200 PBS-diluted mouse anti-chicken Na + /K + -ATPase α subunit (cytosolic epitope for all isoforms) monoclonal immunoglobulin G (IgG) (Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA, USA) at room temperature for 2 h. Samples were washed twice in PBS for 10 min each, and then incubated with 1:200 PBS-diluted goat anti-mouse IgG conjugated with Cy5 (Jackson Immunoresearch Laboratories, West Grove, PA, USA) for 2 h at room temperature. Images were acquired with a confocal laser scanning microscope (TCS-SP5, Leica Lasertechnik, Heidelberg, Germany).

Quantitative real-time PCR
Quantitative real-time PCR (qPCR) was carried out using a SYBR green dye (Applied Biosystems)-based assay with an ABI Prism 7000 Sequence Detection System (Applied Biosystems) according to the manufacturer's instructions. Primers targeting Ca 2+ transporters (see additional file 1) and the endogenous control gene, beta-actin, were designed using the Primer Express 2.0 software (Applied Biosystems). In each assay, 25 ng cDNA was amplified in a 20-µL reaction containing 2x SYBR green master mix, 100 nM of forward and reverse primers, and nuclease-free water. Beta-actin, with a 151-bp amplicon (forward 5'-CACCTTCCAGCAGATGTGGA-3' and reverse 5'-AAAAGCCATGCCAATGTTGTC-3'), was used as an internal control to construct the standard curves.

Statistical analysis
Values are presented as the mean ± SE and were compared by the 2-sample t-test.