Open Access
Research article
Expression and water calcium dependence of calcium transporter
isoforms in zebrafish gill mitochondrion-rich cells
Bo-Kai Liao
1,2, Ang-Ni Deng
1,2, Shyh-Chi Chen
1, Ming-Yi Chou
2and
Pung-Pung Hwang*
1,2Address: 1Institute of Cellular and Organismic Biology, Academia Sinica, Taipei, Taiwan, ROC and 2Institute of Fisheries Science, National Taiwan
University, Taipei, Taiwan, ROC
Email: Bo-Kai Liao - lbk@ecophysiology.org.tw; Ang-Ni Deng - andeng@ym.edu.tw; Shyh-Chi Chen - shyhchi@gate.sinica.edu.tw; Ming-Yi Chou - eleber@gate.sinica.edu.tw; Pung-Pung Hwang* - pphwang@gate.sinica.edu.tw
* Corresponding author
Abstract
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.
Published: 4 October 2007
BMC Genomics 2007, 8:354 doi:10.1186/1471-2164-8-354
Received: 13 August 2007 Accepted: 4 October 2007 This article is available from: http://www.biomedcentral.com/1471-2164/8/354
© 2007 Liao et al.; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
Ca2+ is an essential element for almost all organisms and plays comprehensive regulatory roles in eukaryotic cells; therefore, Ca2+ must be maintained within a narrow con-centration range in organisms (cellular free [Ca2+]: 100 nM; plasma total [Ca2+]: 2~3 mM). In terrestrial verte-brates, the whole-body Ca2+ balance is mainly achieved by intestinal absorption and kidney reabsorption. Ca2+ 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 Ca2+ uptake mechanisms [1]. In fish, gills are the main site (> 97% of the whole body) of Ca2+ uptake from the aquatic environment to maintain the Ca2+ bal-ance [2], and skin takes the place of gills during early developmental stages when the gills are not yet developed and functioning [3-5]. Compared with terrestrial verte-brates, Ca2+ regulation mechanisms in aquatic animals are probably more complicated and challenging because of the dramatic fluctuations in ambient Ca2+ concentrations which can occur (seawater, around 10 mM; fresh water, 0.01~3 mM).
According to the current model in mammals, active and transcellular Ca2+ transport is carried out as a 3-step proc-ess [1]. Following entry of Ca2+ through apical epithelial Ca2+ channels (ECaC, TRPV5, and/or TRPV6), Ca2+ is bound to calbindins that facilitate diffusion to the basola-teral membrane, and then it is extruded via the plasma membrane Ca2+-ATPase (PMCA) and/or Na+/Ca2+ exchanger (NCX). In this way, net transepithelial Ca2+ absorption from the luminal compartment (or environ-ment) to the plasma is accomplished. A model of the capability of physiological regulation has also been simi-larly 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 Ca2+ uptake. The ecac gene has recently been cloned and sequenced in fugu, zebrafish, and trout [4,7,8]. In zebrafish embryos, low-Ca2+ fresh water causes upregula-tion of the whole-body Ca2+ influx and zECaC expression in mitochondrion-rich (MR) cells of both gills and skin, providing molecular evidence for the role of ECaC in fish Ca2+ uptake [4]. On the other hand, some biochemical and physiological studies have investigated the possibility of PMCA and NCX's involvement in fish gill Ca2+ uptake [9,10]. Nevertheless, no convincing evidence has been presented demonstrating the existence and involvement of PMCA and NCX in fish Ca2+ 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 Ca2+ from cells for maintaining a gradient across the
plasma membrane [11]. PMCA belongs to the P-type pri-mary ion transport ATPase superfamily with 10 trans-membrane 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 Ca2+ 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 stud-ies have indicated that vitamin D3 modulates Ca2+ 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 non-mammals as well [17,18]. However, there is no molecular physiological evidence to support the involvement of NCX in fish Ca2+ uptake mechanisms.
Because of a theoretical process of whole-genome duplica-tion (WGD) from ancestor vertebrates [19,20], the num-bers of gene isoforms found in teleosts are usually more than those found in mammals and tetrapods. While dif-ferent isoforms coded from distinct genomic loci tend to have tissue-specific expressions, there are some cases of isoforms being differentially regulated upon facing envi-ronmental 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 physiolog-ical needs and environmental changes, have been pro-posed as being associated with transepithelial Ca2+ transport [9,23]. However, nothing is known about which specific isoforms are responsible for Ca2+ uptake mecha-nisms 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 iso-forms 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 identi-fied. 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-Ca2+ environments were also examined by quantita-tive real-time PCR analysis. The present study provides molecular evidence to support the current Ca2+ uptake model in fish gill cells [24].
Results
Molecular cloning and bioinformatics analysis of pmca and ncx
In this whole-genome survey, 6 distinct PMCAs (Figure 1) and 7 distinct NCXs (Figure 2) were found in zebrafish. According to the phylogenetic analysis, these transporters were annotated as zPMCA1a, zPMCA1b, zPMCA2, zPMCA3a, zPMCA3b, zPMCA4 (Figure 3a, and in stand-ard nomenclature ATP2B1a, ATP2B1b, ATP2B2, ATP2B3a, ATP2B3b, and ATP2B4, respectively) and zNCX1a, zNCX1b, zNCX2a, zNCX2b, zNCX3, zNCX4a, and zNCX4b (Figure 3b, in standard nomenclature SLC8a1a, SLC8a1b, SLC8a2a, SLC8a2b, SLC8a3, SLC8a4a, and SLC8a4b, respectively). zNCX1a and zNCX1b were previously described and named zNCX1h and zNCX1n, respectively [25], and zNCX2a, zNCX3, zNCX4a, and zNCX4b have also been published [17,18]. However, zNCX2b and the other zPMCAs are first described in this study.
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 anal-ysis, 10 putative transmembrane domains were predicted for zPMCA (Figures 1, 4a), whereas NCX has 11 hydro-phobic segments and 9 putative transmembrane domains (Figures 2, 4b) due to an intracellular helix front trans-membrane domain 6 and a P loop-like structure of hydro-phobic segment 8, which contained the amino acid sequence "GIG" [26]. PMCA and sarco-endoplasmic retic-ulum calcium ATPase (SERCA), the closest kin to PMCA, share a similar topological structure: hydrophobic seg-ments span the membrane 10 times, and 3 intracellular loops consist of Ca2+ binding and transporting, and cal-modulin-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-termi-nal (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 iso-forms 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 analy-sis, 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 karyo-type, zPMCAs and zNCXs are located on different chro-mosomes (see additional file 1), suggesting that these paralogous groups may have originated from genome duplication but not from tandem duplication. Relation-ships among these pairs of duplicates were evident not only in the phylogenetic tree, but also in their gene struc-tures (Figure 4). All teleost PMCAs and NCXs examined in this study showed an outgroup topology to their ortho-logues of mammals (Figure 3a,b), and the paralogous tel-eosts 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 Ca2+ 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), suggest-ing that these genes may regulate intracellular Ca2+ home-ostasis at earlier stages. zpmca1a, zpmca2, zpmca4, zncx1, and zncx4a were expressed in gills (Figure 5).
Effects of environmental Ca2+ levels on ecac, pmca, and
ncx mRNA expressions
According to the quantitative real time-PCR analysis, low-Ca2+ FW-acclimated zebrafish expressed significantly higher levels of zecac in gills than did high-Ca2+ FW-accli-mated fish (Figure 6). However, no significant difference was found between the 2 groups in the expressions of any of the zpmcas and zncxs (Figure 6). Consistent with the results of the tissue scans shown in Figure 5, zpmca4,
Alignment of the deduced amino acid sequences of zebrafish plasma membrane Ca2+-ATPases (PMCAs)
Figure 1
Alignment of the deduced amino acid sequences of zebrafish plasma membrane Ca2+-ATPases (PMCAs). Residues in black and gray regions indicate identical and similar residues between isoforms, respectively. TM1-10, the postulated transmembrane domains. TM1 TM2 TM4 TM5 TM6 TM7 TM8 TM9 TM10 TM3
Alignment of the deduced amino acid sequences of the zebrafish Na+/Ca2+ exchangers (NCXs)
Figure 2
Alignment of the deduced amino acid sequences of the zebrafish Na+/Ca2+ exchangers (NCXs). Residues in black and gray regions indicate identical and similar residues between isoforms, respectively. The intracellular helixes (Helix, gray box) and the "GIG" P loops (P loop, open box) of NCX are also indicated. TM1-9, the postulated transmembrane domains.
TM2 TM1 TM3 TM4 TM5 TM6 TM7 P loop TM8 TM9 Helix
Phylogenetic analysis of plasma membrane Ca2+-ATPase (PMCA) and Na+/Ca2+ exchanger (NCX) amino acid sequences
Figure 3
Phylogenetic analysis of plasma membrane Ca2+-ATPase (PMCA) and Na+/Ca2+ exchanger (NCX) amino acid sequences. Con-sensus trees were generated using a Neighbor-joining (NJ) method with 1000-time bootstraps. The GenBank accession num-bers of the sequences used are as follows: human PMCA 1 (hPMCA1), P20020; human PMCA 2 (hPMCA2), Q01814; human PMCA 3 (hPMCA3), Q16720; human PMCA 4 (hPMCA4), P23634; Norway rat PMCA 1 (ratPMCA1), P11505; Norway rat PMCA 2 (ratPMCA2), P11506; Norway rat PMCA 3 (ratPMCA3), Q64568; Norway rat PMCA 4 (ratPMCA4), Q64542; mouse PMCA 2 (mPMCA2), NP_033853; mouse PMCA 3 (mPMCA3), NP_796210; mouse PMCA 4.b (mPMCA4.b), AY560895; dog PMCA 1 (dogPMCA1), NP_777121; pig PMCA 1 (pigPMCA1), P23220; bullfrog PMCA 1bx (bullfrogPMCA1bx), AAK11272; bullfrog PMCA 2av (bullfrogPMCA2av), AAK11273; Caenorhabditis elegans PMCA (CelegansPMCA), CAA09303;Oreochromis
mossambicus PMCA 2 (tilapiaPMCA2), AAK15034; Tetraodon nigroviridis unnamed protein product (tetradonPMCA1a),
CAF95990; T. nigroviridis unnamed protein product (tetradonPMCA1b), CAG08760; T. nigroviridis unnamed protein product (tetradonPMCA4), CAF90203; human NCX 1 (hNCX1), P32418; human NCX 2 (hNCX2), Q9UPR5; human NCX 3 (hNCX3), NP_150287; mouse NCX 1 (mouseNCX1), P70414; mouse NCX 2 (mouseNCX2), NP_683748; mouse NCX 3 (mouseNCX3), NP_536688; Norway rat NCX 1 (ratNCX1), Q01728; Norway rat NCX 2 (ratNCX2), NP_511174; Norway rat NCX 3 (ratNCX3), NP_511175; T. nigroviridis unnamed protein product (tetraodonNCX1), CAG00274; T. nigroviridis unnamed protein product (tetraodonNCX2a), CAF95011; T. nigroviridis unnamed protein product (tetraodonNCX2b), CAG06357; T. nigroviridis unnamed protein product (tetraodonNCX3), CAG13245; T. nigroviridis unnamed protein product (tetraodonNCX4a), CAG05743; T. nigroviridis unnamed protein product (tetraodonNCX4b), CAG01582; Ore. mossambicus NCX1a (tilapiaNCX1a), AAP37041; Oncorhynchus mykiss NCX 1a (troutNCX1a) AAF06363; Rhodopirellula baltica NCX (Pirel-lulaNCX), NP_868143; C. elegans NCX (CelegansNCX), CAA04574; Loligo opalescens NCX (squidNCX), AAB52920; and
Dro-sophila melanogaster NCX (flyNCX), NP_524423. The un-annotated proteins of Takifugu rubripes obtained from Ensembl
browser with the Ensembl gene ID are as follows: Takifugu PMCA 3b (fuguPMCA3b), NEWSINFRUG00000123527; Takifugu PMCA 2 (fuguPMCA2), NEWSINFRUG00000164849; Takifugu PMCA 1a (fuguPMCA1a), NEWSINFRUG00000133763;
Tak-ifugu PMCA 4 (fuguPMCA4), NEWSINFRUG00000161238; TakTak-ifugu PMCA 1b (fuguPMCA1b), NEWSINFRUG00000135851; Takifugu NCX 4b (fuguNCX4b), NEWSINFRUG00000165032; Takifugu NCX 4a (fuguNCX4a), NEWSINFRUG00000131148; Takifugu NCX 1a (fuguNCX1a), NEWSINFRUG00000136894; Takifugu NCX 1b (fuguNCX1b), NEWSINFRUG00000145936; Takifugu NCX 3 (fuguNCX3), NEWSINFRUG00000135815; Takifugu NCX 2b (fuguNCX2b), NEWSINFRUG00000136441;
and Takifugu NCX 2a (fuguNCX2a), NEWSINFRUG00000160221. An asterisk (*) indicates a hypothetical protein predicted from other studies.
A
PMCA4 PMCA3 PMCA2 PMCA1 hPMCA1 pigPMCA1 dogPMCA1 ratPMCA1 bullfrogPMCA1bx zPMCA1a fuguPMCA1a* tetradonPMCA1a* zPMCA1b fuguPMCA1b* tetradonPMCA1b* hPMCA2 mPMCA2 ratPMCA2 bullfrogPMCA2av zPMCA2 fuguPMCA2* tilapiaPMCA2 hPMCA3 mPMCA3 ratPMCA3 zPMCA3a zPMCA3b fuguPMCA3b* hPMCA4 mPMCA4.b ratPMCA4 zPMCA4 fuguPMCA4* tetradonPMCA4* CelegansPMCA 99 100 99 100 96 100 100 100 64 100 64 72 100 100 97 100 100 99 87 70 99 99 85 99 70 99 91 64 0.05B
NCX4 NCX2 NCX3 NCX1 hNCX1 ratNCX1 mNCX1 zNCX1b fuguNCX1b* tetradonNCX1b* zNCX1a troutNCX1a tilapiaNCX1a fuguNCX1a* tetradonNCX1a* hNCX3 mNCX3 ratNCX3 zNCX3 fuguNCX3* tetradonNCX3* zNCX4a fuguNCX4a* tetradonNCX4a* zNCX4b fuguNCX4b* tetradonNCX4b* hNCX2 ratNCX2 mNCX2 zNCX2a fuguNCX2a* tetradonNCX2a* zNCX2b fuguNCX2b* tetradonNCX2b* squidNCX flyNCX CelegansNCX PirellulaNCX 100 100 100 100 100 100 100 97 100 100 99 99 100 99 100 100 100 75 100 98 86 57 90 98 92 49 89 89 85 86 89 73 51 0.2zpmca1a, and zpmca2 exhibited more than 20-fold higher
expression levels than did the other zpmca isoforms in gills, whereas zncx1b was found to have an approximately 5-times stronger signal compared with the other zncxs (Figure 6).
Colocalization of ecac, pmca, and ncx mRNAs and Na+ -K+-ATPase
Fluorescence in situ hybridization and immunocytochem-istry were used to determine the isoforms of zpmca and
zncx that were specifically expressed in gill ionocytes.
Spe-cific mRNA probes of all of the zpmca and zncx isoforms were used to conduct the in situ hybridizations for whole-mount 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 zecac-positive cells co-expressed zpmca2 signals (51.7 +/- 3.50%,
Quantitative real-time PCR analysis of plasma membrane
Ca2+-ATPases (PMCAs) and Na+/Ca2+ exchangers (NCXs)
Figure 6
Quantitative real-time PCR analysis of plasma membrane Ca2+-ATPases (PMCAs) and Na+/Ca2+ exchangers (NCXs). HCa (filled bar), cDNA from the gill of high-Ca2+-acclimated zebrafish; LCa (opened bar), cDNA from the gill of low-Ca2+ -acclimated zebrafish. An asterisk (*) indicates a significant dif-ference between the 2 groups (Student's t-test, p < 0.05).
Normal
ized quantiti
es
* High Ca2+
Low Ca2+
Gene structures of plasma membrane Ca2+-ATPases
(PMCAs) and Na+/Ca2+ exchangers (NCXs)
Figure 4
Gene structures of plasma membrane Ca2+-ATPases (PMCAs) and Na+/Ca2+ exchangers (NCXs). Exons are showed in gradient bars separated by introns that are drawn in lines. The scale bar for the length of exon is shown, and introns are shown as 1/10 the size of exons. Red-filled region, the predicted transmembrane domain; blue-filled region, the intracellular helixes of NCX; orange-filled exon region, the GIG P loops of NCX.
B 1 kbp A zPMCA1a zPMCA1b zPMCA2 zPMCA3a zPMCA3b zPMCA4 zNCX1b zNCX2a zNCX2b zNCX3 zNCX4a zNCX4b
Expression patterns of zebrafish plasma membrane Ca2+
-ATPases (PMCAs) and Na+/Ca2+ exchangers (NCXs) in
vari-ous tissues by RT-PCR analysis
Figure 5
Expression patterns of zebrafish plasma membrane Ca2+ -ATPases (PMCAs) and Na+/Ca2+ exchangers (NCXs) in vari-ous 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.
zpmca1a zpmca1b zpmca2 zpmca3a zpmca3b zpmca4 zncx1b zncx2a zncx2b zncx3 zncx4a zncx4b beta-actin 428bp 556bp 444bp 457bp 614bp 388bp 358bp 276bp 477bp 244bp 426bp 350bp 514bp GillInte stin e Kid ney Liv er Sp lee n He art Blo od Bra in EyeSkinFin Mu scle Te stis Ova ry 1C ell GillInte stin e Kid ne y Liv er Sp lee n He art Blo od Bra in EyeSkinFin Mu scle Te stis Ova ry 1C ell
n = 3), while only 40.3 +/- 1.35% (n = 3) of
zpmca2-posi-tive cells showed zecac signals (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 zncx1b-positive cells that co-expressed zecac was 77.1 +/- 1.35% (n = 3), while zncx1b signals could be detected in all zecac-positive cells (Figures 7D–F, 8).
The subsequent triple-labeling experiments further dem-onstrated the colocalization of zecac mRNA/zpmca2 mRNA/Na+-K+-ATPase (Figure 8A–E), and zecac mRNA/
zncx1b mRNA/Na+-K+-ATPase (Figure 8F–J), respectively, in specific groups of gill ionocytes. Taken all together, only a portion of gill ionocytes co-expressed the mRNA of
zecac, zpmca2, and zncx1b, and Na+-K+-ATPase.
Discussion
So far, 4 PMCA genes have been identified in mammals, and PMCA1 and PMCA4 were suggested to be housekeep-ing 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 Ca2+
homeostasis [11]. A null mutation of PMCA2 caused bal-ance and hearing defects in mice [31]. On the other hand, mammalian PMCA1 was deemed a major Ca2+ extrusion transporter for Ca2+ absorption in intestine and kidney [32-34]. 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 Ca2+ 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 con-trary, 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 Ca2+ 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 fam-ily. In the present study, zNCX1b was demonstrated to be responsible for the Ca2+ 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 Ca2+ 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 Ca2+ uptake without resulting heart failure.
As described above, Flik et al. [6] proposed a model of a fish gill Ca2+uptake mechanism, which is similar to that for Ca2+ reabsorption in mammalian kidneys [1]. There has been no convincing or comprehensive molecular evi-dence 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 Ca2+ 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 cur-rent view that MR cells are the predominant site for branchial Ca2+ uptake [36], and the authors also reported that hypercapnia, implantation with cortisol, and infu-sion with CaCl2 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 Double fluorescence in situ hybridizations of zecac/zpmca2
(A-C) and zecac/zncx1b (D-F) in frozen sections of zebrafish gills
Figure 7
Double fluorescence in situ hybridizations of zecac/zpmca2 (A-C) and zecac/zncx1b (D-F) in frozen sections of zebrafish gills. A and D, zecac mRNA; B, zpmca2 mRNA; C, merged image of A and B; E, zncx1b mRNA; F, merged image of D and E. Arrow indicates the colocalization of zecac/zpmca2 (A-C) and zecac/zncx1 (D-F), respectively. An asterisk (*) indicates the zpmca2 signal without zecac colocalization (A and B) and the zncx1b signal without zecac colocalization (D and E). Scale bar = 20 µm.
A
ecac
B
pmca2
D
ecac
E
ncx1b
F
merge
*
merge
*
*
*
C
45Ca2+ 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 Ca2+ 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 immunocytochem-ical experiments demonstrate the co-expressions of zecac,
zpmca2, and zncx1b in a specific group of gill MR cells,
pro-viding comprehensive molecular evidence for the involve-ment of these transporters in a fish gill Ca2+ 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 hybridiza-tion. 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 Ca2+ uptake in zebrafish gills. It was noted that some gill cells express only 1 or 2 (never all) of the 3 major Ca2+ transporters. These cells may be in the process of terminal differentia-tion as Hsiao et al. [38] reported in skin ionocytes in zebrafish embryos. Alternatively, the Ca2+ 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 sec-tion, 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.
pmca2
A
B
ecac
C
NKA
D
E
ISH
ISH
ICC
BF
Merge
ncx1b
F
G
ecac
H
NKA
I
J
Ca2+ 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)2D3 in mammals and chicken [34,41,42]. 1,25(OH)2-vitamin D3 was shown to have a hypercalcemic effect on both freshwater and saltwater tel-eosts [6,43], whereas stanniocalcin, a predominant hypocalcemic hormone produced by Stannius bodies, was found to show opposite effect on fish [44]. Stannio-calcin did not show a significant effect on basolateral Ca2+ transport [45], but quickly reduced the permeability of Ca2+ at the apical membrane [6,46]. It has been proposed that ECaC is the rate-limiting step and the gatekeeper channel for active Ca2+ transport [47]. The present study provides further molecular physiological evidence to sup-port this notion. The present study for the first time dem-onstrates that acclimation to a low-Ca2+ environment, which would stimulate Ca2+ 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 ful-fill the requirement for the transepithelial transport machinery under all situations. Moreover, this study pro-vides molecular evidence to support a previous biochem-ical study by Flik et al. [50], who found that the basolateral Ca2+ 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-Ca2+ 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.
Methods
Animals and embryos
Zebrafish (Danio rerio) brood stocks at the Institute of Cel-lular and Organismic Biology, Academia Sinica were kept in fresh water (local tap water, FW) at 28.5°C under a 14-h:10-h light: dark photoperiod. Embryos were collected within 30 min after fertilization and incubated in a Petri dish until the desired developmental stages. For whole-mount in situ hybridization, PTU (1-phenyl 2-thiourea) at a final concentration of 0.003% was added to prevent melanogenesis. Fish were anesthetized with buffered MS222 before sampling following the guidelines of the Academia Sinica Institutional Animal Care and Utiliza-tion Committee (approval no.: RFiZOOHP2006086).
Acclimation experiment
Following a previous process in our laboratory [49], high-Ca2+ (2.00 mM, control) and low-Ca2+ (0.02 mM) artifi-cial fresh waters were prepared with double-deionized water (Milli-RO60, Millipore, Billerica, MA, USA) supple-mented with adequate CaSO4·2H2O, MgSO4·7H2O, NaCl, K2HPO4, and KH2PO4. Nominal Ca2+ concentra-tions of the high- and low-Ca2+ media were 2.00 and 0.02 mM, respectively, but the other ion concentrations of the media were the same ([Na+], 0.5 mM; [Mg2+], 0.16 mM; and [K+], 0.3 mM) as those in the local tap water. Varia-tions in the ion concentraVaria-tions were maintained within 10% of the predicted values by examination with an atomic absorption spectrophotometer (Hitachi Z-8000, Tokyo, Japan). Zebrafish were transferred to high- and low-Ca2+ media, respectively, for 2 wk. At the end of accli-mation, fish gills were sampled for quantitative real-time PCR analysis.
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
puta-tive full-length or partial open reading frames of PMCAs and NCXs obtained were confirmed using the EST data-base, 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, War-rington, 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 col-lected for the total RNA preparation with TRIZol reagent (Invitrogen, Carlsbad, CA, USA). The amount and quality of total RNA were determined by measuring the absorb-ances at 260 and 280 nm with a spectrophotometer (Hitachi U-2000) and RNA denatured gels. For cDNA syn-thesis, 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, 5 mM dithiothreitol, and 200 units superscript reverse transcriptase III (Invitrogen) for 1.5 h at 42°C, fol-lowed by a 15-min incubation at 70°C. For the PCR amplification, 1 µL cDNA was used as a template in a 25-µL final reaction volume containing 0.25 mM dNTP, 1.25 units Gen-Taq polymerase (Genemark, Taipei, Taiwan), and 0.2 µM of each primer. The primer sets for the PCR of tissue scans were zPMCA1a (428-bp fragment) forward 5'-AGACAGGGTGGATAGAAGGT-3', reverse 5'-CCCCAAT AATGTGAAGATGA-3'; zPMCA1b (556-bp fragment), for-ward 5'-AGCCCCTTATTTCCCGCAC-3', reverse 5'-GCC CCCTTCTCAGCTCCATT-3'; zPMCA2 (444-bp fragment) forward 5'-GGGATCGGGATGAGATGGTA-3', reverse 5'-G CGTGTGTTTGTCTGTGGGT-3'; zPMCA3a (457-bp frag-ment) forward 5'-TTGCTGGTACAGATGTGGC-3', reverse 5'-GGAGGAGAGTGAAGCGGAG-3'; zPMCA3b (614-bp fragment) forward 5'-GGTCTGCTGGGTTTCCTACT-3', reverse 5'-TCCTGCTCAATCTCTCCTTT-3'; zPMCA4 (388-bp fragment) forward 5'-GTCAAGGCTGTGATGTGGGC-3', reverse 5'-TGGTTGGGAGTGGAGAAGGG-3'; zNCX1b (358-bp fragment) forward 5'-AGAGACGAGGAGAAG-GAGGT-3', reverse 5'-GCACGAAAGCAAAGAGAACT-3'; zNCX2a (276-bp fragment) forward 5'-TGATGAAGAAG-GAGGTGAAC-3', reverse 5'-CTTGCGAATGTGTCTGG-TAT-3'; zNCX2b (477-bp fragment) forward 5'-GCAGCC GCATTTCCTTCG-3', reverse 5'-CGCCAGAGTTTCGGAC-CAC-3'; zNCX3 (244-bp fragment) forward 5'-GAG-GAAGCAAGAAGAATAGC-3', reverse 5'-AGTCAAAACAA GATGGCAGA-3'; zNCX4a (462-bp fragment) forward 5'-GGAAGAGAGCGGAGAAGAGC-3', reverse 5'- ATGGT-GAAGAGGGTGACGGA-3'; and zNCX4b (350-bp frag-ment) forward 5'-ATGCAGCAGCAGAAGAGC-3', reverse 5'-ACATTCTCGCCAGGTTTG-3'. 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 con-centrations 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 cry-osections, fixed samples were immersed in PBS contain-ing 30% sucrose overnight, and embedded in Optimal
Cutting Temperature (OCT) compound embedding medium (Sakura, Tokyo, Japan) at -20°C, and 10-µm fro-zen cross-sections were cut with a CM 1900 rapid section-ing 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 over-night. 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. Fur-ther 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 con-ducted with a commercial kit, TSA Plus Fluorescence Sys-tems (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% paraformal-dehyde 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 iso-forms) monoclonal immunoglobulin G (IgG) (Develop-mental 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 incu-bated with 1:200 PBS-diluted goat anti-mouse IgG conju-gated 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 Ca2+ 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 CACCTTCCAGCAGATGTGGA-3' and reverse 5'-AAAAGCCATGCCAATGTTGTC-3'), was used as an inter-nal control to construct the standard curves.
Statistical analysis
Values are presented as the mean ± SE and were compared by the 2-sample t-test.
Abbreviations
BSA, bovine serum albumin; Dig, digoxigenin; DNP, din-itrophenol; ECaC, epithelium Ca2+ channel; EST, expressed sequence tag; FW, freshwater; HCa, high Ca2+; HyB, hybridization buffer; LCa, low Ca2+; MR, mitochon-drion-rich; NCX, Na+/Ca2+ exchanger; OCT, Optimal Cut-ting Temperature; PBS, phosphate-buffered saline; PBST, phosphate-buffered saline with 0.1% tween-20; PMCA, plasma membrane Ca2+-ATPase; PNA, peanut lectin agglutinin; qPCR, Quantitative real-time PCR; RACE, rapid amplification of cDNA ends; RT-PCR, reverse-tran-scriptase polymerase chain reaction; SE, standard error; SERCA, sarco-endoplasmic reticulum calcium ATPase; SSC, SSC, saline-sodium citrate; TRP, transient receptor potential cation channel; TSA, fluorescein-tyramide signal amplification; UTR, untranslated regions; WGD, whole-genome duplication.
Authors' contributions
PPH, the correspondence author, organized the whole project and manuscript. BKL designed and conducted the experiments and the data analysis; AND performed molecular cloning experiments; SCC, and MYC per-formed other biochemical experiments. All authors have read and approved the final manuscript.
Additional material
Acknowledgements
This study was financially supported by grants to P.P.H. from the National Science Council, and Academia Sinica, Taiwan, R.O.C. We extend our thanks to Ms. Y. C. Tung and Mr. J. Y. Wang for their assistance during the experiments, and to the Core Facility of the Institute of Cellular and Organ-ismic Biology, the Institute of Botany and Microbiology, Academia Sinica, for assistance with array printing and scanning, sequencing and microscopy.
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Additional file 1
Table S1: Primers for quantitative real-time PCR and the chromosome loci of zebrafish plasma membrane Ca2+-ATPase (PMCA) and Na+/Ca2+
exchanger (NCX). The chromosome number and loci were obtained by the SSAHA program from the Ensembl database (zebrafish genome assemble version 6).
Click here for file
[http://www.biomedcentral.com/content/supplementary/1471-2164-8-354-S1.xls]
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