Similar to mammalian proximal tubular cells, H+-ATPase rich (HR) cells in zebrafish skin and gills are also responsible for Na+ uptake and acid secretion functions. However, the basolateral transport pathways in HR cells are still unclear. In the present study, we tested the hypothesis if there are specific slc4 members involved in basolateral ion transport pathways in HR cells. Fourteen isoforms were identified in the zebrafish(z) slc4 family, and the full-length cDNAs of two novel isoforms, zslc4a1b (anion exchanger, zAE1b) and zslc4a4b (Na+/HCO3− cotransporter, zNBCe1b), were sequenced. mRNA signals of zslc4a1b and zslc4a4b were mainly detected in certain groups of ionocytes in zebrafish skin/gills. Further double immunocytochemistry or in situ hybridization demonstrated that zAE1b, but not zNBCe1b, was localized to basolateral membranes of HR cells. Acclimation to low-Na+ or acidic environments stimulated the mRNA expression of zslc4a1b in zebrafish gills, and loss-of-function of zslc4a1b with specific morpholinos caused significant decreases in both the whole body Na+ content and the skin H+ activity in the morphants. On the basis of these results, it was concluded that zAE1b, but not zNBCe1b, is involved in the basolateral transport pathways in Na+ uptake/acid secretion mechanisms in zebrafish HR cells.
- acid-base regulation
- ion regulation
- mitochondrion-rich cell
- Na uptake
bicarbonate (HCO3−) is a single-carbon molecule that plays crucial roles in diverse animal physiological processes, including whole body and cellular pH regulation, cellular volume mediation, and NaCl absorption (1, 4, 34). The solute carrier family 4 (SLC4) was identified as the transporters responsible for the transmembrane movement of HCO3−, and the family contains 10 members in mammals (34). In mammalian nephrons, about 70–80% of filtered sodium and bicarbonate is reabsorbed in proximal tubules (43), and the electrogenic Na+-HCO3− cotransporter (NBCe1/SLC4A4) is present in the basolateral membrane of proximal tubular cells for the epithelial transport of bicarbonate from the lumen to the blood (34). Human mutations in NBCe1 are associated with proximal renal tubular acidosis (pRTA), which results in increased renal HCO3− levels due to a failure of bicarbonate reabsorption (4). On the other hand, in α-intercalated cells of the collecting duct, 5% filtered bicarbonate is reabsorbed, and the major basolateral bicarbonate transporter is the kidney form of the anion exchanger (kAE1/SLC4A1) (1). Different sets of AE1 mutations, such as AE1R589H (21) and AE1901X (6, 40), result in distal RTA, which is due to the failure of acid secretion by α-intercalated cells of the cortical collecting duct in the kidney. In proximal tubular cells and collecting duct α-intercalated cells, cytosolic carbonic anhydrase generates protons and bicarbonate, protons are excreted by the apical Na+/H+ exchanger (NHE) and/or H+-ATPase (HA), and the generated HCO3− is reabsorbed into the blood by the basolateral NBCe1 (proximal tubular cells) or kAE1 (α-intercalated cells) (1, 4, 34, 43).
Similar to mammalian kidneys, freshwater (FW) fish gills are the main organ responsible for ion uptake and acid/base regulation, and specialized ionocytes in the gills were suggested to achieve these functions via the transport of H+ and/or HCO3− in exchange for the uptake of Na+ and/or Cl− from the hypotonic environment. To date, in the slc4 family only two members, AE1 and NBCe1, have been proposed to play roles in these ionoregulatory pathways in fish gills (8–9, 17–19). NBCe1 has repeatedly been proposed as the major candidate for a basolateral transporter in fish gill Na+ uptake/acid secretion mechanisms, mostly on the basis of gene expression and pharmacological data after acid or hypercapnic treatment (27, 29). NBCe1 is localized in certain types of ionocytes in fish gills (11, 27); however, there has been no direct evidence of the colocalization of NBCe1 with either NHE or HA in the same gill ionocytes to achieve the epithelial transport pathways. On the other hand, most previous studies focused on the role of apical Cl−/HCO3− exchange in Cl− uptake/base secretion mechanisms in FW fish gills (9, 17–19, 41). It was not until recently that AE1 was proposed to mediate the basolateral transport pathways in FW fish gill cells based on the basolateral localization of AE1 (18, 39). However, there is no molecular physiological evidence to support the role of AE1 in basolateral transport pathways in fish gills.
Taken together, whether NBCe1 or AE1 is involved in Na+ uptake/acid secretion pathways in fish gills as in mammalian kidneys, and how the two transporters are involved, is still puzzling. Recent studies identified a novel type of ionocyte, H+-ATPase-rich (HR) cells, which express apical NHE and HA and two carbonic anhydrases as mammalian proximal tubular cells do and are responsible for Na+ uptake/acid secretion in the skin and gills of zebrafish (13, 24–25, 46). In the present study, the zebrafish was used as an in vivo model to test whether specific slc4 members are involved in Na+ uptake/acid-base regulatory pathways in HR cells. Experiments were designed for the following specific aims: 1) to identify members of the slc4 gene family, 2) to investigate expression patterns of slc4 family members in various tissues and embryos, 3) to determine the cellular localization of slc4 members in specific ionocytes, 4) to assay the effects of environmental pH values and Na+ concentrations on mRNA expressions of slc4 members, and 4) to perform a functional analysis of slc4 members by a loss-of-function approach.
MATERIALS AND METHODS
The AB strain of zebrafish, obtained from stocks of the Institute of Cellular and Organismic Biology, Academia Sinica, were reared in circulating FW (local tap water) at 28.5°C under a 14:10-h light-dark photoperiod. Fish were fed artificially bred brine shrimp. Embryos were collected within 30 min after fertilization and incubated in petri dishes until the required developmental stages. The experimental protocols were approved by the Academia Sinica Institutional Animal Care and Utilization Committee (approval no. RFiZOOHP2009086).
Following a previous study (25), high- (10 mM) and low-Na+ (0.04 mM) FWs were prepared with double-deionized water (Milli-RO60; Millipore, Temecula, CA) with sufficient CaSO4·2H2O, MgSO4·7H2O, Na2SO4, NaCl, K2HPO4, and KH2PO4 added. Other ion concentrations ([Cl−] = 0.4–0.6 mM; [Ca2+] = 0.18–0.20 mM; [Mg2+] = 0.18–0.21 mM; and [K+] = 0.15–0.17 mM) and the pH (6.7–6.9) of the media were similar to those in the local tap water. FW (control, pH 6.7–6.9) and acidic FW (pH 4.00–4.05, adding H2SO4 to FW) were also prepared. Adult zebrafish were acclimated for 14 days to high-Na+, low-Na+, and acidic FW and to control FW, and all showed normal behaviors with no mortality during the acclimation period. During the experiments, high- and low-Na+ FWs were replaced every 2 days to maintain the proper ion concentrations, while acidic FW was continually pumped into the experimental tank bottom with an electrical pump to maintain a stable pH. The pH values of all experimental media were checked with a pH meter (Mettler Toledo MP225, Schwerzenbach, Switzerland), and ion concentrations were determined with an atomic absorption spectrophotometer (Z-8000, Hitachi, Tokyo, Japan). After 14 days of acclimation, gills were collected for the subsequent analysis.
Molecular cloning and sequence analysis.
All members of the zslc4 family were predicted from the Ensembl and NCBI databases. Full-length or partial complementary (c)DNAs of 10 zslc4 members were cloned and sequenced from zebrafish. Amino acid sequences of slc4 from organisms representing different taxa were aligned and analyzed for phylogenetic and molecular evolution with the ClustalW2 program and MEGA (version 3). A rooted phylogenetic tree was built using a neighbor-joining (NJ) method with bootstrap analysis for 1,000 cycles.
Preparation of total RNA.
Zebrafish embryos and adult tissues were homogenized in TRIzol reagent (Ambion, Woodward, TX). Total RNA was purified following the manufacturer's protocol. The total amount of RNA was determined at absorbances of 260 and 280 nm by spectrophotometry (ND-1000, Nano Drop Technologies, Wilmington, DE). All RNA pellets were stored at −20°C.
Reverse-transcription polymerase chain reaction (RT-PCR) and 5′ and 3′ rapid amplification of cDNA ends.
Total RNA extracted from zebrafish tissues and embryos was treated with DNase I (Qiagene, Hilden, Germany) to remove genomic DNA contamination, and then phenol-chloroform extraction and purification were performed to stop the reaction. 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)20, 250 ng of random primers, 5 mM dithiothreitol, 40 units of an RNase inhibitor, and 200 units of SuperScript III RT (Invitrogen, Carlsbad, CA) for 1 h at 50°C, followed by incubation at 70°C for 15 min. For the PCR amplification, 1 μ1 of cDNA (<500 ng) was used as a template in a 50-μl final reaction volume containing 0.25 mM dNTPs, 2.5 units of ExTaq polymerase (Takara, Shiga, Japan), and 0.2 μM of each primer. Thirty cycles were performed for each reaction. The primer sets for the PCR analysis of the expression patterns in different tissues are listed in Supplemental Table S1 (Supplemental Material for this article is available online at the Journal website). Specific primers of the 5′ and 3′ rapid amplification of cDNA ends (RACE) were designed from partial sequences obtained from the cloned PCR products. The RACE PCR program followed the manufacturer's protocol, and RACE PCR products were also subcloned into the pGEM-T Easy vector and sequenced.
In situ hybridization.
The fragments of zslc4 isoforms (Supplemental Tables S3 and S4) were obtained by PCR and inserted into the pGEM-T easy vector (Promega, Madison, WI). Digoxigenin (Dig)- (Roche, Penzberg, Germany) and dinitrophenol (DNP)-labeled (Perkin-Elmer, Boston, MA) RNA probes were synthesized by in vitro transcription with T7 and SP6 RNA polymerase (Takara). 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). Zebrafish embryos were anesthetized on ice and fixed with 4% paraformaldehyde in a phosphate-buffered saline (PBS) solution (1.4 mM NaCl, 0.2 mM KCl, 0.1 mM Na2HPO4, and 0.002 mM KH2PO4; pH 7.4) at 4°C overnight. Afterward, samples were washed with diethylpyrocarbonate-PBST (PBS with 0.1% Tween 20) several times (for 10 min each). After PBST washing, samples were incubated with hybridization buffer [HyB, 50% formamide, 5× saline sodium citrate buffer (SSC), and 0.1% Tween 20] at 65°C for 5 min and with HyB containing 500 μg/ml yeast transfer RNA at 65°C for 4 h before hybridization. After overnight hybridization with 100 ng/ml Dig-labeled antisense or sense RNA probes, embryos were serially washed with 50% formamide-2 × SSC (at 65°C for 20 min), 2× SSC (at 65°C for 10 min), 2× SSC (at 65°C for 10 min), 0.2× SSC (at 65°C for 30 min, 2 times), and PBST at room temperature for 10 min. Afterward, embryos were immunoreacted with an alkaline phosphatase-coupled anti-Dig antibody (1:8,000) and were then treated with nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate for the alkaline phosphatase reaction. For double fluorescence in situ hybridization, two probes (1 Dig-labeled and the other DNP-labeled) for two target genes were simultaneously subjected to hybridization treatment with similar protocols as described above. Fluorescence detection was conducted with a commercial kit of tyramide signal amplification (TSA) Plus Fluorescence Systems (Perkin-Elmer, Waltham, MA). The hybridization signals detected by the Dig-labeled RNA probes were amplified through fluorescein-TSA, while cyanine 3-TSA was used for the DNP-labeled probes. Images were obtained with a LeicaZ16 macroscope (Leica, Heidelberg, Germany).
Whole mount immunohistochemistry.
For double-labeling of zAE1b and H+-ATPase, zebrafish samples were fixed with 4% paraformaldehyde in a PBS solution at 4°C for 2 h. After being washed with PBS, the samples were incubated with 3% bovine serum albumin for 2 h to block nonspecific binding. Samples were then incubated overnight at 4°C with an anti-zAE1b polyclonal antibody (labeled with Zenon Alexa Fluor 405; Molecular Probes, Invitrogen) and a polyclonal antibody against the A subunit of killifish H+-ATPase (13). After being washed with PBS for 20 min, samples were further incubated in Alexa Fluor 488 goat anti-rabbit immunoglobulin G (IgG; Molecular Probes; diluted 1: 200 with PBS) for 2 h at room temperature. For double-labeling with concanavalin A (ConA, a marker of apical membranes of HR cells) (24) and zAE1b, live embryos were first preincubated in FW containing 0.5 mg/ml Texas red-conjugated ConA (Molecular Probes) for 10 min. After being washed, ConA-labeled embryos were fixed and immunolabeled with the anti-zAE1b polyclonal antibody. The anti-zAE1b antibody was generated in rabbits injected with a 21-residue synthetic peptide (RLDVKPRKPSKSSGPPPEDPL) of zAE1b, used at a dilution of 1:50. Images were acquired with a Leica TCS-SP5 confocal laser-scanning microscope (Leica Lasertechnik, Heidelberg, Germany). For z-plan image, 30 serial sections (0.5 μm/section; total thickness, 15 μm) of confocal microscopic images were acquired and subjected to image reconstruction and analysis.
Western blot analysis.
Proteins at 50 µg/well were loaded onto a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) at 100 V for 2 h. After separation, proteins were transferred onto polyvinylidene difluoride membranes (Millipore) at 100 V for 2 h. After being blocked for 1.5 h in 5% nonfat milk, the blots were incubated with the anti-zAE1b antibody (overnight, 4°C, diluted 1:250) and with an alkaline-phosphatase-conjugated goat anti-rabbit IgG antibody (diluted 1:5,000, at room temperature; Jackson Laboratories, West Grove, PA) for another 2 h. The blots were developed with 5-bromo-4-chloro-3-indolylphosphate/nitro-blue tetrazolium.
mRNA expressions of target genes were measured by a quantitative real-time (qRT)-PCR with an ABI Prism 7000 sequence analysis system (Applied Biosystems, Foster City, CA) or a Roche Lightcycler 480 (Roche, Penzberg, Germany) in a final volume of 10 μl, containing 5 μl of 2× SYBR green master mix (ABI), 3.2 ng of cDNA, and 50 nM of the primers pairs. The standard curve of each gene was checked in a linear range with β-actin as the internal control. The primer sets for the qRT-PCR are given in Supplemental Table S4.
Translational knockdown with antisense morpholino oligonucleotides (MOs).
The morpholino-modified antisense oligonucleotides were purchased from Gene Tools (Philomath, OR). MOs against zslc4a1b, which began at −1 bp and spanned the ATG ending at the 25th nucleotide position, 5′-CTTCGCAGACTCATTCATCTCCATG-3′, were prepared with sterile water. The MO solution (1 ng per embryo) containing 0.1% phenol red (as a visualizing indicator) was injected into zebrafish embryos at the one- to two-cell stage using an IM-300 microinjection system (Narishigi Scientific Instrument Laboratory, Tokyo, Japan).
Surface pH of zebrafish embryos.
A noninvasive scanning H+-selective electrode technique (SIET) was used to measure extracellular H+ activity (pH) at the surface of zebrafish embryos as previously described (13, 24). Briefly, microelectrodes with a tip diameter of 3–4 μm were pulled from glass capillary tubes using a P-97 Flaming Brown pipette puller (Sutter Instruments, San Rafael, CA), then baked at 200°C overnight, and vapor-silanized with dimethyl chlorosilane (Fluka, Buchs, Switzerland) for 30 min. The microelectrodes were backfilled with a 1-cm column of 100 mM KCl/H2PO4 (pH 7.0) and then frontloaded with a 20- to 30-μm column of liquid ion exchanger cocktail (hydrogen ionophore I-cock-tail B; Fluka). The H+ microelectrode was positioned with a step-motor-driven three-dimensional (3D) positioner (Applicable Electronics, East Falmouth, MA) via an Ag/AgCl wire electrode holder (World Precision Instruments, Sarasota, FL), and the circuit was completed by placing a salt bridge (3 M KCl in 3% agarose connected to an Ag/AgCl wire). Data acquisition, preliminary processing, and control of the 3D electrode positioner were performed with ASET software (Science Wares, East Falmouth, MA). The microelectrode system was attached to an Olympus upright microscope (BX-50WI; Tokyo, Japan). The Nernstian properties of each microelectrode were measured by placing the microelectrode in a series of standard pH solutions (pH 6, 7, and 8). By plotting the voltage output of the probe against the log H+ concentration, a linear regression yielded a Nernstian slope of 57.65 with a SD of 2.3 (n = 6).
To detect the surface H+ activities of zebrafish embryos, a scanning ion-selective electrode technique was performed at room temperature (24–26°C) in a small plastic recording chamber filled with 1 ml of recording solution which contained artificial medium ([Na+] = 0.4–0.6 mM; [Cl−] = 0.4–0.6 mM; [Ca2+] = 0.18–0.20 mM; [Mg2+] = 0.18–0.20 mM; and [K+] = 0.15–0.17 mM), 300 μM MOPS buffer (Sigma, St. Louis, MO), and 0.1 mg/l Tricaine (3-aminobenzoic acid ethyl ester; Sigma, pH 6.8). An anesthetized embryo was positioned in the center of the chamber with its lateral side contacting the base of the chamber; it remained in that position for 3 min. Then the probe was moved to the target positions on the skin surface of the yolk sac and recorded for 30 s and moved ∼1 cm away from the embryo to record the background values of the medium. The voltage outputs were converted to H+ concentrations according to the 3-point calibration curve (described above), and Δ[H+] was used to represent the measured H+ gradients between the target point on the skin surface and the background.
Measurement of whole body Na+ content.
Twenty zebrafish were anesthetized with 100–200 mg/l of buffered MS222 (3-aminobenzoic acid ethyl ester; Sigma), briefly rinsed in deionized water and then pooled as one sample. HNO3 at 13.1 N was added to the sample for digestion at 60°C overnight. Digested solutions were diluted with double-deionized water, and the total Na+ content was measured with an atomic absorption spectrophotometer. Standard solutions of Na+ measurements from Merck (Darmstadt, Germany) were used to make the standard curve.
Values are presented as means ± SD and were compared using Student's t-test.
Identification, molecular cloning, and sequencing of the slc4 gene family by database mining.
From the NCBI and Ensembl genome databases (release 56 version of the Ensembl genome database), 14 genes (zslc4a1a, zslc4a1b, zslc4a2a, zslc4a2b, zslc4a3, zslc4a4a, zslc4a4b, zslc4a5a, zslc4a5b, zslc4a7, zslc4a8, zslc4a10a, zslc4a10b, and zslc4a11) of the slc4 family in zebrafish were predicted. A phylogenetic tree was generated based on an NJ analysis (Fig. 1). According to the phylogenetic tree, the zebrafish and human slc4 members were grouped into two subfamilies which contained three Cl-HCO3− anion exchangers (slc4a1–3), five Na+-coupled HCO3− transporters (slc4a4–10), and one borate transporter (slc4a11) that formed an additional branch. However, slc4a9 was reported to be a fourth Cl−/HCO3− exchanger that was not found in zebrafish. In addition, the subphylogenetic trees of AE and electrogenic NBC (Supplemental Figs. S1 and S2) consisted of other teleostean species including fugu (Takifugu rubripes), medaka (Oryzias latipes), and tetraodon (Tetraodon nigroviridis). In each of the AE and NBC trees, two subgroups were clustered and annotated as a and b (Supplemental Figs. S1 and, S2).
Several members of the zebrafish slc4 family were previously reported (28, 35–36, 38). In the present study, 10 other members of the family were successfully cloned and sequenced: zslc4a1a, zslc4a1b, zslc4a3, zslc4a4b, zslc4a5a, zslc4a5b, zslc4a7, zslc4a8, zslc4a10a, and zslc4a11. Among them, zAE1b (zslc4a1b) and zNBCe1b (zslc4a4b) were cloned and sequenced from the full-length cDNAs (Figs. 2 and 3), which contained the 5′ and 3′ untranslated regions. The zebrafish zAE1b gene (accession no. FJ 211592) is located on chromosome 12 (genome database Zv8), and the coding region contains 851 amino acids with 13 putative transmembrane segments. zAE1a and zAE1b have 59.1% identity (Fig. 2). The zebrafish zNBCe1b gene (accession no. EF634453) is located on chromosome 21. The coding region of zNBCe1b contains 1,074 amino acids with 10 putative transmembrane segments and is more similar to pancreatic NBCe1 (NBCe1-B; 78.3%) than the alternative spliced isoform, kidney NBCe1 (NBCe1-A; 73.4%). zNBCe1a and zNBCe1b show 82% identity (Fig. 3). In comparison with their human counterparts, zebrafish AE1b and NBCe1b had some conserved amino acid sequences such as DIDS binding sites, transmembrane domains, and carbonic anhydrase II binding sites (Figs. 2 and 3).
RT-PCR and in situ hybridization analysis of mRNA expression patterns of the zebrafish slc4 gene family.
mRNA expressions of the zebrafish zslc4 gene family in various tissues were examined by RT-PCR, and β-actin was used as the internal control. As shown in Fig. 4, zslc4 members revealed different expression patterns in the adult tissues examined. zslc4a1a and zslc4a7 were expressed in the examined tissues including brain, eyes, gills, hearts, intestines, kidneys, liver, muscles, spleen, and ovaries. On the other hand, zslc4a1b, zslc4a2a, zslc4a5b, and zslc4a10a were expressed in two or three tissues. Among the 12 isoforms, 7 of them (zslc4a1a, zslc4a1b, zslc4a3, zslc4a4b, zslc4a7, zslc4a8, and zslc4a11) were expressed in the gills. zslc4a1b was mainly expressed in the gills, with a little in the eyes, while zslc4a4b was expressed in the eyes and gills and faintly expressed in the intestine, kidneys, and ovaries. The kidneys mainly expressed zslc4a1a, zslc4a2a, zslc4a3, zslc4a4a, zslc4a7, and zslc4a11. Furthermore, the expression patterns of zslc4a5b and zslc4a10a were similar and mainly expressed in brain, eye, and spleen, while zslc4a5b also expressed a little signal in heart and ovaries.
Whole mount in situ hybridization was used to detect zslc4 gene family mRNA expressions in zebrafish embryos at different developmental stages. Among the family members that were cloned in the present study, only zslc4a1b (zAE1b) and zslc4a4b (zNBCe1b) showed a salt-and-pepper pattern (i.e., ionocyte pattern) (15) of mRNA expression in the embryonic skin of zebrafish, and thus the following experiments focused on these two isoforms. As shown in Figs. 5 and 6, the results suggest that zslc4a1b and zslc4a4b are expressed in some types of ionocytes in the skin and gills of zebrafish. zslc4a1b began to be expressed in skin ionocytes at 1 day postfertilization (dpf) (Fig. 5A). Following development, zslc4a1b signals increased to cover the entire yolk and yolk tube (Fig. 5, A–E), and the branchial region began to show clear zslc4a1b mRNA signals at 4 dpf (Fig. 5F). On the other hand, at 1 dpf, zslc4a4b signals were detected only in the ear region, not in skin ionocytes (Fig. 6A). mRNA signals of zslc4a4b were first detected in ionocytes at 2 dpf (Fig. 6B). Following development, zslc4a4b signals increased and extended to skin areas covering the yolk, yolk tube, head, and gills (Fig. 6, B–F). Similar to zsl4a1b, zslc4a4b began to show evident expression in the branchial region at 4 dpf (Fig. 6F).
A subsequent RT-PCR analysis was conducted to examine zslc4a1b and zslc4a4b mRNA expressions at various developmental stages of zebrafish embryos. As shown in Fig. 7, zslc4a1b was initially expressed in tissues of embryos from 21 h postfertilization (hpf), and the expression level was maintained during development (Fig. 7). The expression of zslc4a4b began much later than that of zslc4a1b. It was not until 3 dpf that a clear signal of the zslc4a4b transcript was found in zebrafish embryos (Fig. 7). These data support results of the above whole mount in situ hybridization (Figs. 5 and 6), i.e., the different timings of the initial expressions of the two slc4 isoforms in zebrafish embryos.
Localization of zAE1b (zslc4a1b) and zslc4a4b mRNA in zebrafish ionocytes.
To identify the cell types of ionocytes expressing zAE1b (zslc4a1) and zNBCe1b (zslc4a4b), double-fluorescence immunocytochemistry (Fig. 8, A–C) and double-fluorescence in situ hybridization (Fig. 8, D–F) in the yolk sac of 5-dpf zebrafish embryos were conducted. As shown in Fig. 8, A–C, protein signals of zAE1b were colocalized with H+-ATPase (a marker for HR cells) (24) in HR cells. On the other hand, mRNA signals of zslc4a4b (zNBCe1b) were colocalized with zslc12a10.2 (Fig. 8, D–F), a marker for the Na+-Cl−-cotransporter (NCC) type of ionocytes in zebrafish (44), indicating HR cells do not express zNBCe1b. Taken together, zAE1b and zNBCe1b were respectively expressed in HR and NCC types of ionocytes in zebrafish.
Cellular localization of zAE1b in zebrafish ionocytes.
To further identify the cellular localization of the zAE1b protein in zebrafish HR cells, double-fluorescence immunocytochemistry for zAE1b and lectin ConA (a marker for apical membranes of HR cells) (24) with confocal microscopic analysis was conducted. As shown in Fig. 9, ConA and zAE1b signals were colocalized in HR cells (Fig. 9, A–C); ConA showed an apical pattern (Fig. 9, A and D), while zAE1b was distributed in the basolateral portion of the cells (Fig. 9, B and D).
Effects of environmental acidities and Na+ concentrations on zAE1b and zNBCe1b mRNA expressions in zebrafish gills.
Acclimation to a low-pH or low-Na+ environment, which was previously reported to stimulate the acid secretion or Na+ uptake function of zebrafish HR cells (13, 25, 46), was hypothesized to also stimulate the mRNA expression of zAE1b if the transporters were involved in the functions of HR cells. As shown in Fig. 10, after acclimation to an acidic or low-Na+ environment, the mRNA expression (measured by real-time PCR, with β-actin as internal control) of zAE1b in zebrafish adult gills was upregulated 2.8- and 2.2-fold, respectively (Fig. 10, A and B). On the contrary, transcription of zNBCe1b was downregulated 2.3- and 2.6-fold, respectively, in acidic and low-Na+ environments (Fig. 10, C and D), similar to the previous results for the zNCC (44). This implies that zNBCe1b may be involved in the functions of NCC cells but not in those of HR cells.
Effects of zAE1b morpholinos on protein expressions in zebrafish embryos.
To further characterize the functional roles of zAE1b in acid secretion and Na+ uptake functions of zebrafish HR cells, loss-of-function experiments with specific zAE1b MOs were conducted. In a preliminary experiment, 1.0, 2.0, and 4.0 ng/embryo of the zAE1b MO were injected into zebrafish embryos, which respectively showed 14.3%, 14.7%, and 17.75% mortalities, compared with the wild-type (WT) which had a 90% survival rate. The phenotypes and behaviors of the zAE1b morphants appeared normal compared with those of the WT. Immunocytochemical (Fig. 11, A–F) and Western blot (Fig. 11G) experiments were conducted to examine the effectiveness and specificity of the zAE1b MO (at a dose of 1.0 ng/embryo). Compared with the WT, zAE1b morphants still showed the existence of HR cells (apically labeled with ConA) (Fig. 11D) but without zAE1b protein signals (Fig. 11E). As shown in Fig. 11G, the protein signals (with a size of ∼100 kDa in the Western blot) in MO-injected embryos were much decreased compared with those in the WT.
Effects of zAE1b knockdown on whole body Na+ contents in zebrafish embryos.
Whole body ion contents in zebrafish embryos are the ultimate results of ion net fluxes. Therefore, in this experiment, the whole body Na+ contents at different stages of zebrafish embryos injected with the zAE1b MO at 1.0 ng/embryo were measured. As shown in Fig. 12, the Na+ content in 1- and 3-dpf zAE1b morphants were significantly lower than that in WT embryos. However, the Na+ content in morphants had recovered to the level of the WT at 4 dpf, and this was probably due to either the degradation of the MOs or other compensatory pathways.
Effects of zAE1b knockdown on the surface H+ activity in the yolk sac skin of zebrafish embryos.
SIET was used to assay the surface H+ activity of the yolk sac skin of zebrafish embryos injected with the zAE1b MO (at a dose of 1.0 ng/embryo). An external pH gradient (ΔpH) at the surface of intact embryos was measured at a location near the lower part of the yolk sac which was previously reported to exhibit the highest H+ activity (i.e., the lowest ΔpH) (13). As shown in Fig. 13, the H+ activity in zAE1b morphants at 48 hpf was significantly lower than that in the WT.
On the basis of the phylogenetic tree analysis, the zebrafish slc4 family, similar to its mammalian counterpart, can be classified into two groups, although the ortholog of slc4a9 has not yet been found. The zebrafish slc4 family has 14 members, more than the 10 in humans, because some members in the zebrafish family are composed of 2 isoforms (zslc4a1a, zslc4a1b, zslc4a2a, zslc4a2b, zslc4a4a, zslc4a4b, zslc4a5a, zslc4a5b, zslc4a10a, and zslc4a10b). This phenomenon is commonly observed in zebrafish (2).
In the present study, we successfully cloned and sequenced the partial cDNAs of 10 slc4 isoforms from zebrafish and identified the isoforms that are expressed in ionocytes of gills/skin by in situ hybridization and RT-PCR. Hence, subsequent experiments were focused on two novel isoforms, zslc4a1b (zAE1b) and zslc4a4b (zNBCe1b). Previously reported zAE1a and zNBCe1a (NP_938152.1 and NP_001030156.1; reannotated in the present study, Fig. 1), were found to have dominant expressions in kidneys of zebrafish (28, 38) as do their human counterparts, AE1 and NBCe1 (34). On the contrary, the present RT-PCR and in situ hybridization analysis revealed that the novel zAE1b and zNBCe1b were not expressed (or only very little) in the kidneys but were mainly expressed in some groups of cells in zebrafish gills/skin, implying their existence in ionocytes and their roles in fish gill ionoregulatory mechanisms. Sussman et al. (38) used an anti-daceNBCe1 antibody to detect protein signals in a group of gill cells in zebrafish, but there were no mRNA data either in situ or by RT-PCR to support that. Moreover, compared with zNBCe1a (79%), zNBCe1b was more similar to daceNBCe1 with 92% identity in the antigen sequence, and thus zNBCe1b was clustered with daceNBCe1 in a clade (Supplemental Fig. S2). The protein signals in zebrafish gills reported by Sussman et al. (38) may merely reflect the present in situ hybridization data of zNBCe1b (but not zNBCe1a) (Figs. 6 and 8).
The roles of AE1 in transepithelial secretion/reabsorption of acid-base equivalents and Cl− and related diseases in mammalian kidneys are well studied (1, 33). However, the expression and function of AE1 in fish are still a puzzling issue. In early studies, AE1 was proposed to be involved in apical Cl−/HCO3− exchange in fish gill ionocytes (37, 45). Pharmacological studies showed apical Cl−/HCO3− activity in fish gill cells (5, 30); however, the molecular evidence for AE1 at either the mRNA or protein level in fish gill ionocytes (37, 45) is still debatable due to the specificity of the antibody or probe used (17, 41). Current models of apical Cl−/HCO3− transport in fish gill cells tend to favor a role of another Cl−/HCO3− transporter, pendrin (SLC26) (3, 31). The present study used zebrafish embryos as the model to provide convincing molecular physiological evidence for the expression and possible functional roles of AE1 in fish skin/gills.
In the zebrafish slc4 family, zslc4a2a and zslc4a2b were previously cloned, and the whole mount in situ hybridization of the two genes (35, 36) does not show a salt-and-pepper pattern (i.e., ionocyte pattern). Among the other 10 members cloned in the present study, only zAE1b and zNBCe1b were found to be respectively expressed in specific groups of ionocytes, HR cells and NCC cells, based on double immunocytochemical or in situ hybridizational analysis. Further double-labeling experiments demonstrated the cellular localization of apical ConA (an apical marker for HR cells) and basolateral zAE1b in HR cells. A recent study using an anti-tilapia AE1 antibody also reported the basolateral localization of AE1-like protein in a group of ionocytes in the spotted green pufferfish (39). Since HR cells are responsible for Na+ uptake/acid secretion functions in zebrafish (7, 13, 17, 24), zAE1b in basolateral membranes of cells could play a part in these transport functions. Acclimation to low-Na+ and acidic FWs, which was reported to respectively enhance the functions of Na+ uptake and acid secretion (14, 25, 46), was also found to stimulate the mRNA expression of zAE1b in zebrafish, supporting the notion that zAE1b is responsible for the basolateral HCO3− efflux that achieves the epithelial acid-base transport function and also provides an intracellular pH gradient for operations of the apical zNHE3b in HR cells (17, 25, 46). This is further supported by the results of the zAE1b knockdown experiments, in which both the Na+ content and surface H+ activities were reduced in morphants injected with the zAE1b MOs. Taken together, it is reasonable for zAE1b to be located in basolateral membranes, for linking with other transporters and enzymes (see below) to achieve the transepithelial H+ secretion/base uptake/Na+ uptake function in zebrafish HR cells.
In the current models of fish gill Na+ uptake/acid-base regulatory mechanisms, NBC was repeatedly proposed as a major player because it is in mammalian kidney (8–9, 17, 19, 27, 29). These models are mostly derived from gene expression data after acid or hypercapnic treatment, and very few data are available on the localization of NBC in fish gill ionocytes. In the Osorezan dace, NBC1 was localized with a homologous antibody in a portion of Na+-K+-ATPase-labeled gill ionocytes (11). In rainbow trout, an anti-rat kidney NBC was also used to detect signals in the basolateral region of a group of gill cells (27). However, so far there is no direct evidence of the colocalization of NBC with either NHE or H+-ATPase in the same gill ionocytes, which is physiologically important for accomplishing Na+ uptake/acid secretion functions by ionocytes. Using double in situ hybridization, the present study demonstrated colocalization of zNBCe1b with related transporters in fish skin/gill ionocytes. Surprisingly, zNBCelb is not in HR cells, the ionocytes responsible for Na+ uptake/acid secretion, but is expressed in another type of ionocyte, NCC cells, which are mainly involved in Cl− and/or Na+ uptake functions in zebrafish (44) and tilapia (12, 20). The exact role of zNBCe1b in the transepithelial transport mechanism in NCC cells remains to be explored in the future; however, subsequent experiments suggested that the physiological function of zNBCe1b differs from that of zAE1b. Acclimation to low-Na+ and acidic FWs caused exactly the opposite effects on the mRNA expression of the two slc4 isoforms: suppressing zNBCe1b while stimulating zAE1b. The timing of the initial expressions of the two isoforms in developing zebrafish embryos also suggested their different functions. The transcripts of zAE1b and zNBCe1b were first detected at 1 and 2 dpf, respectively, when HR cells and NCC cells sequentially began to differentiate in zebrafish embryos (13, 44), reflecting the initial involvement of the 2 slc4 isoforms in the distinct functions of HR and NCC types of ionocytes to meet physiological requirements during zebrafish development.
Interestingly, zebrafish HR cells are analogous to mammalian proximal tubular cells based on the apical expressions of H+-ATPase (24) and zNHE3b (46) as well as two carbonic anhydrases (the membrane-bound zCA15a and the cytosolic zCA2-like a) and their functional roles in Na+ uptake/acid secretion (24–25, 46). The basolateral expression and functional roles of zAE1b in zebrafish HR cells, a notable and novel finding from the present study, appear to be analogous to those of the mammalian α-intercalated cells, which are mainly responsible for acid secretion (32). Compared with mammalian kidney cells which face the lumen, zebrafish HR cells are exposed to a hypotonic environment (i.e., FW) with much more challenging ion gradients. From an evolutionary physiological point of view, it is an interesting and challenging issue to see whether the expression and function of zAE1, instead of zNBCe1b, in HR cells is more advantageous for overcoming harsh ion gradients by aquatic zebrafish.
By integrating the results of previous studies with our results, we could more comprehensively interpret the model of Na+ uptake/acid-base regulatory mechanisms in zebrafish HR cells (17–18). In apical membranes of HR cells, there are two acid transporters, zHNE3b and H+-ATPase, that transport H+ out of cells (7, 13, 24). The H+ and the environmental HCO3− react to generate H2O and CO2 through the membrane-bound zCA15a (25). The CO2 enters HR cells and is hydrated by the cytosolic zCA2-like a to form H+ and HCO3− (25). The basolateral zAE1b extrudes cytosolic HCO3− out of cells to fulfill the epithelial acid secretion function, and this also provides an intracellular pH gradient favorable for the operation of apical NHE3b to achieve the apical Na+ uptake mechanism. The basolateral Na+-K+-ATPase (zatp1a1a.5), another major player in the overall mechanism, is responsible for the excretion of Na+ and also provides an intracellular negative potential (22). In the mammalian α-intercalated cells, the basolateral electroneutral K+-Cl− cotransporter (KCC) is involved in the recycling of Cl− that is accumulated by the basolateral AE1 and thus maintains low intracellular Cl− (42). Whether ortholog(s) of KCC is also expressed and involved in the Cl− recycling in zebrafish HR cells is an important issue for future study.
This study was financially supported by grants (to P. P. Hwang) from the National Science Council, and Academia Sinica, Taiwan, ROC.
No conflicts of interest, financial or otherwise, are declared by the author(s).
We thank Y. C. Tung and J. Y. Wang for assistance during the experiments and the Core Facility of the Institute of Cellular and Organismic Biology.
- Copyright © 2011 the American Physiological Society