The involvement of SLC26 anion transporters in chloride uptake in zebrafish (Danio rerio) larvae

Fishes inhabiting freshwater (FW) continually lose Cl^– and other ions by diffusion across the gill to the surrounding water and to a much lesser extent by urinary excretion. Although there are some notable exceptions of fish lacking appreciable Cl^– uptake capacity [Lepomis macrochirus (Tomasso and Grosell,2005), Anguilla rostrata or A. anguilla(Goss and Perry, 1994) and Fundulus heteroclitus (Patrick et al., 1997)], in most FW fish species that have been examined,internal Cl^– balance is maintained in the face of these diffusive losses by constant replenishment via the active absorption of Cl^– at the gill. More than 70 years ago it was suggested that Cl^– uptake across the gill occurs by Cl^–/HCO₃^– exchange(Krogh, 1937) yet the specific identity of the exchange protein(s) still remains unknown. Although evidence is sparse, members of two gene families have been implicated in branchial Cl^–/HCO₃^– exchange; the SLC4 bicarbonate transporters (for reviews, see Alper et al., 2002; Romero et al., 2004; Alper, 2006; Pushkin and Kurtz, 2006) and the SLC26 anion transporters (for reviews, see Mount and Romero, 2004; Romero et al., 2006; Soleimani and Xu, 2006; Sindic et al., 2007; Ohana et al., 2009). Members of the SLC4 family specifically translocate HCO₃^–or CO₃^2–(Pushkin and Kurtz, 2006)either in a sodium-independent (AE1–4) or sodium-dependent manner.

The SLC26 proteins can be distinguished from the SLC4 transporters because they are largely sodium-independent and promiscuously transfer a variety of anions including Cl^–, HCO₃^–,OH^–, SO₄^2–, formate, oxalate and iodide. Immunohistochemical evidence for the involvement of SLC4A1 (AE1) in branchial Cl^–/HCO₃^– exchange was provided by Wilson (Wilson et al.,2000) who reported the presence of AE1-like protein on the gills of tilapia (Oreochromis mossambicus) using a heterologous antibody. Similarly, evidence for a role of SLC26A4 was obtained by its immunolocalization in gills of FW Atlantic stingray, Dasyatis sabina(Piermarini et al., 2002).

In recent years, the zebrafish (Danio rerio) has emerged as a powerful model for studying piscine ionic regulation owing to the nearly complete annotation of its genome and the ability to perform gene silencing experiments on larvae (Pan et al.,2005; Lin et al.,2005; Yan et al.,2007). Adult zebrafish have an extremely high affinity and high capacity Cl^– uptake mechanism which is appropriately regulated depending on environmental constraints(Boisen et al., 2003). Thus,zebrafish would seem to offer significant advantages over other species in ongoing attempts to delineate the molecular mechanisms of branchial Cl^–/HCO₃^– exchange. In the present study we have focused on three members of the SLC26 gene family as possible candidates for Cl^– uptake in zebrafish; SLC26A3(also known as down-regulated in adenoma; DRA), SLC26A4 (pendrin) and SLC26A6(also known as proton-coupled amino acid transporter 1; PAT1). These three members were selected because they all are known to exhibit Cl^–/HCO₃^– exchange activity(Mount and Romero, 2004). Using this approach, we were able to develop morpholino antisense oligonucleotides to knockdown (Nasevicius and Ekker, 2000) these three SLC26 genes during development and thereby evaluate their roles in Cl^– uptake and base excretion.

Adult zebrafish (Danio rerio Hamilton; 400–800 mg) were obtained commercially from Big Al's Aquarium Services (Ottawa, Canada). The fish were kept in plastic aquaria supplied with recirculating, filtered and dechlorinated fresh water. Fish were maintained at 28°C on a 14 h:10 h light:dark photoperiod and were fed daily using a commercial fish diet(Westerfield, 2007). Groups of five to eight fish were acclimatized to differing environmental conditions for 7 days prior to experimentation. The different conditions were as follows:dechlorinated fresh water (hereafter referred to as control), low and high chloride water, with final chloride concentrations of 0.02 and 2.0 mmol l^–1, respectively, and base water (control water supplemented with either 2 or 5 mmol l^–1 NaHCO₃). The ionic composition of control water was 0.8 mmol l^–1 Na⁺,0.4 mmol l^–1 Cl^–, 0.25 mmol l^–1 Ca²⁺, 0.03 mmol l^–1K⁺. Low and high Cl^– media were prepared by combining appropriate quantities of NaCl and with other salts as required(CaSO₄·2H₂O, NaSO₄,K₂HPO₄) in reverse osmosis water.

Embryos were obtained using standard techniques for zebrafish breeding(Westerfield, 2007) and newly spawned eggs were collected from random groups of adult breeders and kept in rearing tanks at 28°C until needed. All procedures involving animal use were carried out according to institutional guidelines and in accordance with those of the Canadian Council on Animal Care (CCAC).

Measurements of net acid excretion were performed in Miami. Fish used for breeding were obtained from Aquatica tropical, Miami, FL, USA and were maintained in dechlorinated city of Miami tap water [for water composition see Bielmyer et al. (Bielmyer et al.,2007)]. Fertilized eggs were obtained according to standard protocols for zebrafish breeding(Westerfield, 2007). All procedures on zebrafish were in accordance with University of Miami's Institutional Animal Care and Use Committee.

Putative zebrafish orthologs of SLC26A3, A4 and A6c were obtained by using BLAST analysis of nucleotide or amino acid sequences of human SLC26 genes(GenBank Accession numbers NM_000111, NM_000441 and AB102713) against the Ensembl zebrafish genome(www.ensembl.org). Putative full length orthologs for zA3 (si:dkey-31f5.2), zA4 (si:dkey-31f5.9)and zA6c (zgc:175226) genes were located on chromosomes 4, 4 and 6,respectively. The two putative orthologs for A3 and A4 were fully annotated whereas the presumptive A6c ortholog was assigned a designation of secondary active sulfate transmembrane transporter. It should be noted that at the time of the initial data mining (2003–2004), only a single zA6 gene was identified; only recently was zA6c determined to be one of several zA3 isoforms (see Fig. 1).

A series of phylogenetic analyses of the vertebrate solute carrier 26(SLC26) proteins was performed. The SLC26 sequences used in the analyses are as follows: Homo sapiens A1 (NP_998778), A2 (NP_000103), A3(NP_000102), A4 (NP_000432), A5 (AAI00834), A6a (NP_075062), A6b (NP_599025),A6c (NP_602298), A6d (NP_001035544), A7 (AAH94730), A7b (NP_599028), A8a(NP_443193), A8b (NP_619732), A9a (NP_443166), A9b (NP_599152), A11 (Q86WA9); Mus musculus A1 (AAH32151), A2 (NP_031911), A3 (AAI39274), A4(NP_035997), A5 (AAI08988), A6 (NP_599252), A7 (NP_666059), A8 (NP_666188), A9(NP_796217), A11 (NP_848858); Xenopus laevis A1 (AAP45002), A3(AAP37475), A4 (AAQ11740), A6 (AAN85411); Xenopus tropicalis A4(AAI57509), A6 (AAI61524), A6b (NP_001072916), A9 (AAI35994); Gallus gallus A5 (NP_001072945); Takifugu obscurus A3 (BAE75794), A5(BAE75795), A6a (BAE75796), A6b (BAE75797), A6c (BAE75798), A11 (BAE75799); Oncorhynchus mykiss A1 (NP_001117958); Opsanus beta A6(ABQ01444), A6b (ACF75333); Anguilla japonica A1 (BAD22606), A6(BAC16761), A6b (BAD22607); Tetraodon nigroviridis A1 (CAG04906), A2(CAF95371); Danio rerio A1 (NM_001080667), A2 (XP_685114), A3(FJ170816), A4 (FJ170817), A5a (AAP32789), A5b (NP_958881), A6 (XP_687043), A6(XP_685992), A6b (XP_001344243), A6c (FJ170818), A11 (NP_956061), unknown(XP_001921766). Amino acid sequence alignments were used for phylogenetic analyses, and were performed using ClustalX (version 1.8). Phylogenetic hypotheses were constructed using both neighbor joining (NJ) and maximum parsimony (MP) techniques using the PAUP^* [beta test version 4.0b10] software package. MP analysis consisted of a heuristic search with TBR branch swapping, ACCTRAN character state optimization enforced, with random stepwise additions and 1000 random addition replicates. NJ was performed on a matrix of mean character distances. Support for nodes in both cases was obtained through bootstrap analyses with 1000 pseudoreplicates. In both analyses, gaps were included and treated as missing data, and the urochordate Ciona intestinalis SLC26A5 (AAP57206) sequence was used as a an outgroup.

The SLC26 sequences obtained from GenBank were used to design PCR primers for real-time reverse transcriptase PCR and to generate probes for in situ hybridization; these various primers are summarized in Table 1. Additionally, for each gene, primers were designed to amplify PCR products with expected sizes of 1577 (a3), 1857 (a4) and 1807 (a6c) base pairs(Table 1). Analysis of the genomic sequence of si:dkey-31f5.9 (putative SLC26A4) revealed a possible alternative start codon 301 nucleotides upstream of the predicted start codon. Using this region to blast the zebrafish EST database it was confirmed that this region is expressed and indeed contains several highly conserved amino acids. Thus, the forward primer for a4 was designed from the genomic sequence to potentially amplify a longer cDNA. This resulted in a final cDNA sequence for A4 which was 39 nucleotides longer than si:dkey-31f5.9. Following 3′- and 5′-RACE, sequencing of multiple overlapping clones and contig assembly, final sequences were assembled for SLC26A3 (accession no. FJ170816), SLC16 A4 (accession no. FJ170817) and SLC26A6c (accession no. FJ170818) that were 99–100% identical to the `in silico' sequences when compared over comparable regions.

Embryonic (<2 d.p.f.), larval (3–30 d.p.f.) and adult zebrafish were killed by anesthetic overdose (0.05 mg ml^–1 MS-222;ethyl 3-aminobenzoate methanesulfonate; Sigma, St Louis, MO, USA) in control water. Pooled embryos or larvae were rapidly frozen in liquid nitrogen and stored at –80°C. For adults, complete fish were frozen in liquid nitrogen, ground on dry ice using a mortar and pestle and stored at–80°C until the preparation of total RNA.

Whole body total RNA was isolated from zebrafish using TRIzol reagent(Invitrogen, Burlington, Ontario, Canada) according to the instructions of the manufacturer and 5 μg were reverse transcribed using oligo(dT) primers(Sigma Genosys, Oakville, Ontario, Canada) and Superscript II reverse transcriptase (Invitrogen). PCR products were amplified using the following cycling parameters: 2 min at 94°C followed by 30 cycles of 30 s at 92°C, 30 s at 58°C and 1 min 30 s at 72°C. The final extension of the amplified products was at 72°C for 10 min. The final constitution of the PCR mix was: 1×PCR buffer, 1.5 mmol l^–1MgCl₂, 0.2 mmol l^–1 dNTP, 2 pmol each of forward and reverse primers, 2.5 i.u. of Taq polymerase (Invitrogen), and 100 ng of zebrafish gill cDNA. The PCR products were run on a 1.25% agarose gel and purified using a PCR purification kit (Sigma). A PCR cloning kit was used to clone the purified PCR products into a pCR II vector (Invitrogen). The desired clone was purified using a GelElute plasmid miniprep kit (Sigma). Purified plasmids were sequenced using M13 Forward (–20) and M13 Reverse primers.

Total RNA was extracted from aliquots of powdered tissue samples using the Absolutely RNA RT-PCR Miniprep Kit (Stratagene, Mississauga, Ontario, Canada). To remove any remaining genomic DNA, the RNA was treated on-column using RNase-free DNase (5 μl) for 20 min at room temperature. The RNA was eluted in 50 μl of nuclease-free H₂O and its quality was assessed by gel electrophoresis and spectrophotometry (Eppendorf Biophotometer). The cDNA was synthesized from 1 μg of RNA using random hexamer primers(Boehringer-Mannheim, Laval, Quebec, Canada) and Stratascript reverse transcriptase (Stratagene). Relative mRNA levels were assessed by real-time PCR on samples of cDNA (0.5 μl) using a Brilliant SYBR Green QPCR Master Mix Kit (Stratagene) and a Stratagene MX-4000 multiplex quantitative PCR system. ROX (Stratagene) was used as reference dye. The PCR conditions (final reaction volume of 25 μl) were as follows: 0.5 μl cDNA template, 300 nmol l^–1 forward and reverse primer, 12.5 μl 2×Master Mix, 1:30000 final dilution of ROX. The annealing and extension temperatures over 40 cycles were 58°C (30 s) and 72°C (30 s),respectively. All the primers used for real-time PCR (including the reference gene β-actin) were designed using Primer3 software(http://www-genome.wi.mit.edu/cgi-bin/primer/primer3_www.cgi; Table 1). The specificity of the primers was verified by cloning (TOPO TA cloning kit; Invitrogen) and sequencing of amplified products. The construction of SYBR Green dissociation curves after completion of 40 PCR cycles revealed the presence of single amplicons for each primer pair. To ensure that residual genomic DNA was not being amplified, control experiments were performed in which reverse transcriptase was omitted during cDNA synthesis. Relative expression of mRNA levels was determined (using β-actin as an endogenous standard; GenBank accession no. BC063950.1) by a modification of the ΔΔCt method(Pfaffl, 2001). Amplification efficiencies were determined from standard curves generated by serial dilution of plasmid DNA, and varied between ∼90 and ∼110%.

Primers were designed to produce probes of 748, 486 and 805 base pairs for za3, za4 and za6c, respectively(Table 1) using plasmid DNA as template. An aliquot of the PCR products was run on a 1.25% gel and the rest was purified using a PCR purification kit (Sigma). The purified product was cloned in PCR II vector (Invitrogen) and the desired clone was purified using a plasmid miniprep purification kit (Sigma). The purified plasmid DNA was sequenced using M13 forward (–20) and M13 reverse primers to confirm identity and determine the orientation of the cloned sequence within the vector. To obtain linear DNA flanked by SP6 and T7 promoters, M13 forward(–20) and M13 reverse primers were used to amplify the inserted probes. Sense and antisense DIG-labeled RNA probes were synthesized using 1 μg of purified PCR product in the in vitro transcription reaction using the appropriate SP6 or T7 RNA polymerase (New England Biolabs) for 1 h at 37°C.

Larvae were placed in 4% PFA (pH 7.4) at 4°C overnight before being washed (2× 5 min) with PBS and dehydrated by methanol washes(CH₃OH): 2× 5 min at room temperature followed by 1× 60 min at –20°C. Following dehydration, embryos were rehydrated in a graded series of CH₃OH–PBS washes: 1× 5 min in 75%CH₃OH–25% PBS, 1× 5 min in 50%CH₃OH–50% PBS, 1× 5 min in 25%CH₃OH–75% PBS, 3× 5 min in 100% PBST (1× PBS,0.1% Tween 20).

Embryos were rinsed in 4 ml of 20 mg ml^–1 of Proteinase K(Invitrogen) for 10 min at room temperature (RT) before they were washed 1× 5 min in PBST and fixed for 20 min in 4% PFA–PBS. The hybridization mix (Hyb-mix) for use in prehybridization, hybridization and washes was prepared as follows: 5.0 ml formamide, 2.5 ml 20× SSC, 50μl of 20% Tween 20, 5 mg ml^–1 heparin, 92 μl 1 mol l^–1 citric acid, H₂O to final volume of 10 ml. For prehybridization and hybridization, 100 mg ml^–1 yeast tRNA was added to the Hyb-mix.

Larvae were prehybridized for 2–3 h in 200 ml Hyb-mix at 65°C. For hybridization, 200 ml of Hyb-mix containing 1 ml of probe was added to the embryos and they were left overnight at 65°C. Embryos were then washed as follows: 10 min in 75% Hyb-mix–25% 2× SSC at 65°C; 10 min in 50% Hyb-mix–50% 2× SSC at 65°C; 10 min in 25%Hyb-mix–75% 2× SSC at 65°C; 10 min in 100% 2× SSC at 65°C; 2×30 min in 0.2× SSC at 60°C; 5 min in 75%0.2× SSC–25% PBST at RT; 5 min in 50% 0.2× SSC–50%PBST at RT; 5 min in 25% 0.2× SSC–75% PBST at RT; 5 min in PBST at RT.

Following washes, the larvae were pre-incubated for 1 h in PBST containing 2% calf serum and 2 mg ml^–1 BSA and were then incubated with 1:2500 anti-DIG antibody (Roche) for 2–4 h at RT with gentle shaking. Following incubation, embryos were washed (6× 15 min in PBST) and placed in staining buffer (100 mmol l^–1 Tris pH 9.5, 50 mmol l^–1 MgCl₂, 100 mmol l^–1 NaCl,0.1% Tween 20, 1 mmol l^–1 levamisol) for 5 min. 14 μl of 5-bromocresyl-3-indolyl phosphate (BCIP; Fisher, Ottawa, Ontario, Canada; BCIP stock solution was 50 mg ml^–1 in dimethylformamide) and 27μl of nitroblue tetrazolium (NBT; Sigma; NBT stock solution was 50 mg in 0.7 ml dimethylformamide + 0.3 ml H₂O) were added to the staining buffer and embryos were stained for up to 2 h or until satisfactory coloration occurred. The embryos were then washed 2× 5 min in PBST at RT.

The larvae were examined using a Nikon Eclipse E600 light microscope combined with a Nikon Plan Fluor 20× dry objective lens (numerical aperture 0.50). Images were taken using QImaging MicroPublisher 5.0 digital microscope camera and QCapture v2.68.

Proteins were prepared from fresh tissues by homogenization on ice in 1:5 w/v of extraction buffer containing 50 mmol l^–1 Tris-HCl, 150 mmol l^–1 NaCl, 1% NP-40, 0.5% sodium deoxycholate, 2 mmol l^–1 sodium fluoride, 2 mmol l^–1 EDTA, 0.1%SDS, and protease inhibitor cocktail (Roche). The samples were incubated on ice for 10 min and briefly sonicated to break up any DNA that might have been extracted. The samples were centrifuged at 16,000 g for 20 min at 4°C and the supernatants were stored at –20°C before use. Samples were size fractionated by reducing SDS-PAGE using 10% separating and 4% stacking polyacrylamide gels and transferred to nitrocellulose membranes(Bio-Rad, Mississauga, ON, Canada). After transfer, each membrane was blocked for 2 h in 5% milk powder–TBS-T (1× TBS, 0.1% Tween 20) and then incubated (1:5000 dilution) for 3 h at RT with a polyclonal affinity-purified rabbit primary antibody custom produced (Abgent; San Diego, CA, USA) against a synthetic peptide (AEQHERINRKRKTLR) corresponding to amino acids 21–35 of zSLC26A3 (accession no. ACI05561). The membranes were washed (4× 5 min) in PBS and incubated for 1 h at RT with peroxidase-conjugated secondary anti-rabbit Ig (1:25000). The specific bands were detected by enhanced chemiluminescence (ECL; Pierce; SuperSignal West Pico Chemiluminescent Substrate, Rockford, IL, USA). The protein size marker used was obtained from Fermentas Life Sciences. Antibodies were also produced against zSLC26A4 and A6c but these did not produce satisfactory results.

Larvae were killed with an anesthetic (MS-222) overdose and incubated for 20 min at 4°C in a solution of 4% paraformaldehyde (PFA; prepared in PBS,pH 7.4). They were then washed with PBS (3× 5 min) and treated with 100%ethanol at –20°C for 10 min. Larvae were rinsed again with PBS(3× 5 min) and subsequently blocked with 3% bovine serum albumin (BSA)in PBS for 1 h before being washed again with PBS (3× 5 min). Larvae were incubated for 2 h at RT with 1:100 PBS-diluted primary ZA3 antibody. Several of the larvae were co-incubated with 1:100 α5, a mouse monoclonal antibody directed against Na⁺/K⁺-ATPase of chicken, or ZN-12, a zebrafish-derived neuron-specific marker(Trevarrow et al., 1990). ZN-12 and α5 were obtained from the Developmental Studies Hybridoma Bank(University of Iowa, USA).

Larvae were washed (3× 5 min) with PBS before being incubated for 1 h with 1:400 Alexa-Fluor-546 anti-rabbit and 1:400 Alexa-Fluor-488 anti-mouse(Molecular Probes, Burlington, ON, Canada). The larvae were then washed again(3× 5 min) in PBS and whole-mount preparations were examined with a confocal scanning system (Olympus BX50WI, Melville, NY, USA) equipped with an argon laser. Images were collected using Fluoview 2.1.39 graphics software(Fluoview, Melville, NY, USA).

Morpholino oligonucleotides (MOs) complementary to the translational start site of za3, za4 and za6c were microinjected into one-cell stage embryos. Injection needles were pulled from filamented 1.0 mm borosilicate glass (Sutter Instrument, Novato, CA, USA) and the injections were controlled with an IM 300 programmable microinjector (Narishige, East Meadow, NY, USA). MOs (GeneTools) used were as follows: za3,[5′-ATGCAGCCTTTCGGCAGACACTATG-3′]; za4,[5′-ATGGCGATACATCTTGTGGAGTACG-3′]; za6c,[5′-GAGAGAATGGATTCAAGACATGAAG-3′]; and a standard control,[5′-CCTCTTACCTCAGTTACAATTTATA-3′]. MOs were diluted to working standards of 4 ng ml^–1 in Danieau buffer (58 mmol l^–1 NaCl, 0.7 mmol l^–1 KCl, 0.4 mmol l^–1 MgSO₄, 0.6 mmol l^–1Ca(NO₃)₂, 5 mmol l^–1 Hepes, pH 7.6) and 0.05% Phenol Red. Embryos were injected with approximately 1 nl of the MO–Danieau buffer solution.

The gene-specific MOs were tagged with carboxyfluorescein at the 3′ends, allowing the injected embryos to be screened for MO uptake with a Nikon SMZ1500 (Nikon, Mississauga, Ontario, Canada) fluorescence dissecting microscope. Any embryos not exhibiting widespread fluorescence 24 h post-injection were discarded.

To test for efficacy of the za3, za4 and za6cmorpholinos, fusion constructs were made in which the za3, za4 and za6c MO target sequences were separately introduced upstream of and in frame with the red fluorescent protein dTomato(Shaner et al., 2004) coding sequence. Embryos were injected with in vitro-synthesized zA-dTomato mRNA alone or together with each of the zA MOs (only one MO was co-injected into each embryo).

Larval (4 d.p.f.) zebrafish (3–5 larvae per experiment for N=1) were placed in 15 ml HCO₃^– free control water (reconstituted from reverse osmosis water) in a titration vessel(Radiometer Analytical, London, Ontario, Canada; PP.22–45 ml). The water was constantly aerated with atmospheric air using PE50 tubing to aid in mixing. A pH electrode (model PHC4000.8; Radiometer) and a microburette tip,both of which were attached to a pH-stat titration system (model TIM 854 or 856; Radiometer), were positioned just below water level into the titration vessel. The pH-stat titrations were performed at pH 6.800 throughout all experiments, with pH values and rate of acid addition logged on personal computers using Titramaster software (versions 1.3 and 2.1 OR 85). Generally,water pH was maintained within ±0.003 pH unit around the set point throughout the experiments. Base secretion rates were calculated from the rate of addition and concentration of titrant (0.0005 mol l^–1HCl). Water samples were taken for determination of ammonia excretion rates using a colorimetric assay (Verdouw et al., 1978). Net acid excretion(⁠

Data are shown in figures as means ±1 standard error of the mean. Real-time PCR data were analyzed using a one-sample t-test to determine whether relative mRNA levels were greater than 1. All other data were analyzed by two-way analysis of variance (ANOVA) or one-way ANOVA (base excretion data; Fig. 11). In cases where the ANOVA indicated statistical differences, the data were further analyzed using a Bonferroni multiple comparison test (all pairwise comparisons). In all cases, the fiducial limit of significance was set at 5%.

A phylogenetic analysis of the SLC26 gene family produced well supported trees of similar topology from both the NJ(Fig. 1) and MP methods. Zebrafish SLC26A3 grouped with other vertebrate SLC26A3 sequences, with the closest relative being the T. obscurus SLC26A3. Similarly, SLC26A4 grouped with other vertebrate SLC26A4 sequences. Interestingly, a X. laevis sequence that had been denoted as an SLC26A3 protein grouped within the SLC26A4 group, suggesting this protein is actually a second X. laevis SLC26A4 protein. The zebrafish SLC26A6c sequence obtained in the current study also grouped with other vertebrate SLC26A6 sequences,specifically with the fish SLC26A6c proteins, and has been denoted as such. Interestingly, the various fish SLC26A6 isoforms (A6a–A6c) all group together, and independently of the tetrapod SLC26A6 isoforms (A6a–A6d). This suggests that the common vertebrate ancestor had only a single SLC26A6 gene, and evolved multiple copies after the divergence of fish and tetrapods. It is unclear how many SLC26A6 isoforms are found in tetrapod vertebrates,with as many as four being found in humans, but fish species appear to contain at least three, although four are found in zebrafish. It also appears that O. beta may be unique in that it has a duplication of the A6a isoform. Furthermore, it should be noted that a single zebrafish SLC26A6 sequence (no additional isoform designation) is basal to both the tetrapod and fish SLC26A6 groups, which may represent an ancestral SLC26A6 ortholog.

Representative micrographs obtained from in situ hybridization on whole-mount larvae (7 d.p.f.) are depicted in Fig. 2. slc26a3 mRNA appeared to be most abundant in the heart, mesonephros, neuromasts and gill(Fig. 2A). For slc26a4mRNA, the predominant sites of expression appeared to be the heart and mesonephros (Fig. 2B). A similar pattern of expression was observed for slc26a6c with obvious localization to the mesonephros and mesonephric duct(Fig. 2C). No staining was observed using a sense zA4 probe (Fig. 2D).

Although polyclonal antibodies were generated for the three SLC26 proteins described in this paper, only the one raised against zSLC26A3 proved to be useful for immunocytochemistry. On western blots, the antibody yielded a single band of approximately 80 kDa (Fig. 3D) which was not observed when using the pre-immune serum or after pre-absorption of antibody with excess peptide antigen. The estimated molecular mass of 80 kDa is similar to the predicted mass of the ALC26A3 protein (83 kDa). Overall however, the antibody proved difficult to work with and required the use of extremely sensitive chemiluminescence detection kits. An analysis of numerous whole-mount specimens, ranging from 3–9 d.p.f.,revealed several patterns of SLC26A3 immunoreactivity that are depicted in Fig. 3. The neural masts of the lateral line system exhibited marked immunoreactivity(Fig. 3A). Aside from the neural masts, the most common observation was the presence of a modest number(less than 10%) of SLC26A3-positive cells that were scattered among the more prevalent NKA-positive cells dispersed along the developing gill arches(Fig. 3B). In some instances,SLC26A3 was colocalized with Na⁺/K⁺-ATPase(Fig. 3C) whereas in some cells expressing SLC26A3, there was no apparent Na⁺/K⁺-ATPase immunoreactivity (Fig. 3D). SLC26A3-positive cells were never observed on the yolk sac or skin of the body. In contrast to the results of in situ hybridization, zA3 protein immunoreactivity was not apparent in the heart or mesonephros,possibly a reflection of insufficient antibody penetration into these deeper lying tissues (e.g. compared to epithelial cells).

The effects of developmental age (1 hpf–30 d.p.f.) on the relative expression of SLC26 mRNAs are depicted in Fig. 4. There was an obvious transient increase in mRNA expression of za3, za4 and za6cat 5–10 d.p.f.; additionally, zA4 was significantly elevated at 4 d.p.f. (Fig. 4). The changes in abundance of SLC26 mRNAs were not a result of marked changes in the reference gene β-actin because average Ct values for this gene varied little between 1 hpf and 15 d.p.f. (20.1±0.5–21.8±0.4) in comparison with the genes of interest. At 30 d.p.f. and in adults (>3 months), the Ct values for β-actin were reduced to 18.5±0.5 and 17.8±0.3, respectively, which may have influenced the relative mRNA expression data.

The interactive effects of selective SLC26 gene knockdown and ambient Cl^– levels on rates of J^Cl–_in are depicted in Fig. 7. The embryos injected with zA–dTomato mRNA alone exhibited ubiquitous expression. However, no dTomato expression was observed when the corresponding SLC26 MO was injected into the embryos along with or after zA–dTomato mRNA injection, which indicates that the SLC26 morpholinos can effectively block the translation of zA-dTomato and are, therefore, able to recognize and block translation of the target sequence in vivo.

Although successful knockdown appeared to be achieved for each gene (based on the presence or absence of red fluorescence; see above), a significant reduction of

Raising fish in water containing high levels of NaHCO₃(Fig. 8A, Fig. 9) or exposing them to elevated NaHCO₃ for 24 h at 7 d.p.f.(Fig. 8B) resulted in significant increases in the rates of

Whole body base excretion in 5–7 d.p.f. larvae was significantly reduced by morpholino knockdown of zA3 or zA6c(Fig. 11).

The use of morpholino antisense oligonucleotides to achieve transient gene knockdown in developing zebrafish(Nasevicius and Ekker, 2000)has emerged as an important tool to assess physiological functions, including mechanisms of transepithelial ion and ammonia transport(Pan et al., 2005; Horng et al., 2007; Lin et al., 2008; Shih et al., 2008; Braun et al., 2009; Tseng et al., 2009; Chang et al., 2009). In their recent review on the use of antisense technologies in gene knockdown experiments, Eisen and Smith (Eisen and Smith, 2008) highlighted three important issues that should be considered in such studies. First, evidence should be provided that targeted gene knockdown is indeed occurring. Second, when using multiple morpholinos targeted against several paralogs of a multi-gene family, their specificity needs to be established and third, the possibility that off target effects might be accounting for observed phenotypes should be considered.

The most convincing method to verify knockdown is to directly assess protein levels in sham- and morpholino-injected fish by western blotting. Unfortunately, this technique requires the procurement of appropriate antibodies able to recognize zebrafish-specific proteins. In the course of the present study, we developed three homologous antibodies against the three SLC26 gene products under investigation (zA3, zA4 and zA6c). However, because only one of the antibodies (to zA3) proved useful, we chose another strategy commonly used to assess the efficacy of knockdown, in which embryos are co-injected with a fluorescent fusion construct(Eisen and Smith, 2008). The results of these experiments revealed a complete absence of fluorescence for up to 7 d.p.f. in fish co-injected with morpholinos. Moreover, using this approach we were able to confirm specificity of the morpholino oligonucleotides because fluorescence was prevented only in those embryos that were co-injected with the corresponding morpholino (e.g. co-injection of SLC26A3 fusion construct and SLC26A3 morpholino). The specificity of the morpholinos was not surprising given that the maximum number of identical nucleotides between any two morpholinos was 7/24. Two other strategies employed to minimize the chances of off target effects are the use of two or more morpholinos against any single gene target and RNA rescue in which a form of the target RNA (not recognizable by the morpholino) is co-injected in an attempt to re-establish the control phenotype. Neither of these approaches was used in the present study owing, in part, to diminishing isotope resources. Indeed, we are unaware of any isotope provider currently able to supply ³⁶Cl to North American researchers.

The previous evidence implicating Cl^–/HCO₃^– exchange as a mechanism for Cl^– uptake in freshwater fish is overwhelming(Krogh, 1937; Krogh, 1938; Maetz and Garcia Romeu, 1964; De Renzis and Maetz, 1973; De Renzis, 1975; Braun et al., 2009) (reviewed by Marshall, 1995; Claiborne, 1998; Evans et al., 1999; Marshall, 2002; Perry et al., 2003a; Perry et al., 2003b; Evans et al., 2005; Perry and Gilmour, 2006; Marshall and Grosell, 2006; Tresguerres et al., 2006; Hwang and Lee, 2007; Evans, 2008). Prior to the demonstration of pendrin (SLC26A4)-like immunoreactivity on the elasmobranch gill (Piermarini et al.,2001), it was generally considered that branchial Cl^–/HCO₃^– exchange was accomplished by one or more members of the SLC4 gene family. This conclusion was reinforced by the results of studies utilizing immunocytochemistry(Wilson et al., 2000), in situ hybridization (Sullivan et al.,1996) and data obtained from pharmacological studies employing the Cl^–/HCO₃^– exchange blockers SITS/DIDS or SCN^–(Epstein et al., 1973; Perry and Randall, 1981; Perry et al., 1981a; Chang and Hwang,2004; Preest et al.,2005). With the benefit of hindsight, however, the results of these former studies are subject to reinterpretation. For example, the 24-mer in situ probe used by Sullivan et al.(Sullivan et al., 1996),although homologous with rat AE1 (SLC4A1)(Kudrycki and Shull, 1989)shares 63–71% sequence identity with zslc26a3, za4 and za6c and thus may have hybridized to multiple mRNA targets including members of the SLC26 gene family. In addition, disulfonic stilbene derivatives including SITS and DIDS are not specific blockers of SLC4 anion exchangers but also block members of the SLC26 gene family(Soleimani et al., 2001). Finally, the antibody used by Wilson et al.(Wilson et al., 2000) was generated against denatured rainbow trout (Oncorhynchus mykiss) red blood cell SLC4A1 protein (Cameron et al.,1996) and thus its specificity for SLC4A1 versus other SLC4 and SLC26 proteins cannot be easily confirmed by sequence analysis.

Although we cannot exclude the possibility that SLC4 genes are also involved, the results of the present study using a combination of approaches,including gene knockdown, provide evidence for the involvement of SLC26 gene family members in Cl^– uptake in zebrafish larvae. Indirect evidence was provided by the increased expression of SLC26 mRNA in larvae exposed to water containing low ambient [Cl^–] or elevated[HCO₃^–], conditions which markedly increased rates of whole body Cl^– uptake. Direct evidence was provided by the demonstration of significantly reduced rates of Cl^– uptake and base excretion in fish experiencing za3 knockdown while being raised in control water and a significant attenuation of the stimulatory effects of prior exposure to low ambient [Cl^–] or elevated ambient [HCO₃^–] (za3 and za6conly) on Cl^– uptake. The effects of low ambient[Cl^–] on increasing Cl^– uptake capacity was reported previously for adult zebrafish(Boisen et al., 2003) and tilapia [Oreochromis mossambicus(Chang et al., 2003)]. We(S.F.P. and M.B., unpublished data) recently demonstrated that adult zebrafish exposed to water of low ambient [Cl^–] exhibit increased branchial expression of SLC26A3, A4 and A6c mRNA thereby suggesting that at least in zebrafish, the increase in Cl^– transport capacity[as indicated by a threefold increase in

The results of previous studies examining ionic uptake in developing zebrafish embryos and larvae have implicated ionocytes of the yolk sac and skin as sites of Ca²⁺ (Pan et al., 2005) and Na⁺ uptake(Yan et al., 2007) (reviewed by Hwang and Lee, 2007). For Ca²⁺ uptake, the participating ionocyte subtypes are believed to be mitochondrion rich (MR) cells enriched with Na⁺/K⁺-ATPase (NaR cells) whereas for Na⁺uptake, the participating subtypes are thought to be MR cells enriched with apical V-type H⁺-ATPase [HR cells(Hwang and Lee, 2007)]. Thus,we had anticipated that the results of the in situ hybridization and immunocytochemistry (SLC26A3 only) experiments would also reveal the presence of SLC26 mRNA or protein within ionocytes of the yolk sac and skin. However,in contrast to expectations, we were unable to detect SLC26 mRNA on the yolk sac or skin up to 9 d.p.f. Instead, SLC26 mRNA appeared to be localized to the mesonephros and/or mesonephric ducts, heart (za3 and za4),neuromasts (za3) and gills (Fig. 2). The immunocytochemistry results demonstrated that a subset of branchial MR cells exhibited SLC26A3 immunoreactivity. The apparent absence of SLC26 Cl^–/HCO₃^– transporters on the yolk sac and skin noted in this study obviously is inconsistent with the prevailing view that ionic uptake in developing fish occurs predominantly across the skin prior to maturation of the gills (reviewed by Varsamos et al., 2005). However, ionocytes are clearly present within the gill arches of larval zebrafish (Fig. 3) and as in other species (Varsamos et al.,2005), they appear well before the development of lamellae(Rombough, 2002; Rombough, 2007). Thus, it is conceivable that Cl^– uptake in larval zebrafish is mediated by SLC26 Cl^–/HCO₃^– exchangers restricted to a subset of ionocytes of the gill. Some of these ionocytes were also enriched with Na⁺/K⁺-ATPase but others were not.

Previous experiments have provided indirect evidence that branchial ionocytes (also referred to as MR cells or FW chloride cells) are responsible for Cl^– uptake in adult FW fish. For example, in brown trout(Salmo trutta), the intracellular concentration of chlorine within branchial MR cells is affected by ambient Cl^– levels(Morgan et al., 1994) or external application of the Cl^– uptake inhibitor thiocyanate(Morgan and Potts, 1995); the pavement cells were unaffected. In rainbow trout(Goss and Perry, 1993; Perry and Goss, 1994), brown bullhead (Ictalurus nebulosus)(Goss et al., 1992; Goss et al., 1994) and tilapia(Oreochromis mossambicus) (Chang et al., 2003), the rates of Cl^– uptake (and in some cases base excretion) are significantly correlated with the numbers of MR cells in contact with the water. In light of the finding of specific sub-types of MR cells (Pisam et al.,1987; Chang et al.,2001; Goss et al.,2001; Hiroi et al.,2005; Laurent et al.,2006), models have been developed in which specific functions are assigned to the various MR cell types. By analogy with the β-intercalated cell of the mammalian renal collecting duct, one of the branchial MR cell subtypes is thought to function as a base-secreting cell with an apical membrane Cl^–/HCO₃^– exchanger and a basolateral V-type H⁺-ATPase(Piermarini et al., 2002). Thus, we propose that the SLC26 Cl^–/HCO₃^– exchangers in the zebrafish gill also are localized to base-secreting MR cells, a proposition that will require further testing.

Under control conditions, Cl^– uptake was significantly reduced only in those fish experiencing knockdown of zA3, a result which suggests that zA3-mediated Cl^–/HCO₃^– exchange may be the most significant route of Cl^– entry when considering the three SLC26 paralogs investigated in this study. Additionally, we suggest that zA6c is playing a role; whereas Cl^– uptake was not statistically reduced after za6c knockdown, there was an obvious downward trend (P=0.084; Fig. 7) and similar to za3 knockdown, base efflux was significantly decreased in the zA6c morphants(Fig. 11). Given the likelihood of multiple routes of Cl^– uptake, the results of single knockdown experiments should be interpreted cautiously. Thus, while knockdown of za4 did not decrease Cl^– uptake under control conditions, this may simply reflect compensatory increases in Cl^– uptake via other routes including zA3- and zA6c-mediated Cl^–/HCO₃^– exchange. Finally, because other isoforms of zA6 exist (see Fig. 1), further studies will be required to fully elucidate the relative roles of the various zA6 genes.

The effects of SLC26 gene knockdown on Cl^– uptake were more pronounced (especially for za4 and za6c) in fish previously exposed to either low environmental [Cl^–] or elevated ambient [HCO₃^–]. These findings, together with the observation of increased levels of SLC26 mRNA, suggest that the increased rates of Cl^– uptake associated with these treatments (see also Boisen et al.,2003) probably arise, in part, from transcriptionally mediated increases in zA3, zA4 and zA6c expression. Thus, Cl^– uptake,although unresponsive to za4 knockdown under control conditions, was markedly reduced by this treatment in fish raised in water containing low Cl^– or 5 mm NaHCO₃ (Figs 7 and 10). Interestingly, SLC26 gene knockdown was completely without effect on Cl^– uptake in fish raised in water with a high [Cl^–] (but assayed in control water). These finding suggest that expression of the SLC26 exchangers (zA3,zA4 and zA6c) was decreased in these fish (note that mRNA levels, however,were unaffected) and that Cl^– uptake may have been occurring via other routes.

Similar to the fish gill, which absorbs Cl^– from water,the mammalian kidney reabsorbs Cl from tubular urine using a variety of transport proteins including members of the SLC4 and SLC26 gene families(Romero et al., 2004; Mount and Romero, 2004; Alper, 2006; Pushkin and Kurtz, 2006; Soleimani and Xu, 2006; Sindic et al., 2007). The SLC26 genes that have been implicated are A4 (localized to the apical membrane of β-intercalated cells) and A6 (localized to apical membrane of proximal tubule cells). Additionally, SLC26A6 together with basolateral SLC26A1 play integral roles in oxalate, formate and sulfate reabsorption at the proximal tubule. Similarly, SLC26A1 has been implicated in renal sulfate transport in eel (Anguilla japonica) (Nakada et al., 2005) and rainbow trout(Katoh et al., 2006). Little is known about the role of SLC4 Cl^–/HCO₃^– exchangers in the fish kidney although SLC4A2 (AE2) has been identified in the kidney of larval zebrafish (Shmukler et al.,2005; Shmukler et al.,2008). Interestingly, SLC26A3, shown in this study as a route of Cl^– absorption in zebrafish larvae, is not expressed in the mammalian kidney. Another major difference between the kidney and gill models is that the fish gill can absorb Cl^– from very dilute water where there is no obvious favorable chemical gradient for Cl^–to allow electroneutral Cl^–/HCO₃^–exchange. Two possible scenarios may account for the ability of FW fish to absorb Cl^– from dilute media. First, the intracellular levels of HCO₃^–, although too low at the macroscopic level to fuel Cl^–/HCO₃^– exchange,might be enriched within micro-domains near the apical membrane by the integrated actions of carbonic anhydrase and V-type H⁺-ATPase(Tresguerres et al., 2006). Second, one or more of the anion transporters may be operating in an electrogenic mode. For example, the SCL26A6 teleost fish paralogs that have been characterized to date appear to be electrogenic operating in an nHCO₃^–/Cl^– mode(Grosell et al., 2009; Kurita et al., 2008). This transport stoichiometry would be favorable for Cl^–/HCO₃^– exchange across the apical membrane because the inside negative membrane potential would fuel the activity of nHCO₃^–/Cl^– exchange by SLC26A6.

Zebrafish exhibit an unusually high affinity for Cl^–uptake (K_m 8 μmol l^–1) when acclimated to soft water (43 μmol l^–1 Cl^–)(Boisen et al., 2003). This raises the obvious question as to how the SLC26 exchangers might be involved in branchial Cl^– uptake given that they normally exhibit K_m values for Cl^–/HCO₃^– exchange in the low millimolar range (e.g. Shcheynikov et al.,2006). The simplest explanation (also see Grosell et al., 2009) is that one or more of the zebrafish SLC26 Cl^– transporters exhibit unusually low K_m values in comparison to orthologous mammalian genes. Clearly this is an area that warrants further research.