1Molecular and Vascular Medicine Unit and Renal Unit, Beth Israel Deaconess Medical Center, 3Hematology Division, Brigham and Women's Hospital, 4Hematology Division and 7Howard Hughes Medical Institute, Children's Hospital, 5Renal Unit, Massachusetts General Hospital, and Departments of 2Medicine and 6Pediatrics, Harvard Medical School, Boston, Massachusetts
Submitted 28 March 2005 ; accepted in final form 16 May 2005
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ABSTRACT |
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In the course of systematic characterization of anemia mutants of zebrafish (49), the retsina mutant was mapped to the slc4a1/ae1 gene. The loss of function associated with mutant retsina alleles of slc4a1/ae1 causes dyserythropoietic anemia, associated with erythroid-specific defects in cytokinesis (40). Because SLC4A1/AE1 is part of a large multigene family of bicarbonate transporters in mammals and other organisms, it seemed likely that the zebrafish genome would harbor additional slc4 genes and that some would be expressed in the zebrafish pronephric kidney.
The developmental function of ion transporters in zebrafish kidney has only recently come under study (19, 20). Among the few ion transporters of zebrafish kidney studied functionally are two Na-phosphate cotransporters (25, 38) and one proton-peptide cotransporter (59). As mammalian kidneys express multiple SLC4 anion exchangers, including AE1, AE2, and AE3, we screened a zebrafish kidney cDNA library by hybridization at low stringency with a zebrafish slc4a1/ae1 probe to identify zebrafish cDNAs homologous to slc4a1/ae1. In this study, we present the cDNA sequence, genomic structure analysis, map position, functional characterization, and subcellular localization of zebrafish slc4a2/ae2.
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METHODS |
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Radiation hybrid mapping of the slc4a2/ae2 gene. The genomic location of the slc4a2/ae2 gene was determined using the Goodfellow T51 radiation hybrid (RH) panel (24, 31). Specific PCR amplification of part of the ae2 gene on this panel used forward primer 5'-TGTTGTGTGTTTTGTGTAGGGTTA-3' (4,1874,210 nt) and reverse primer 5'-CGATTGTTTTGCTGCTAGTTGCTA-3' (4,4504,427 nt). These primers amplify a fragment of the ae2 3'-untranslated region (UTR) with no identity to any other sequence in the current genomic databases. Amplifications were performed using protocols described at http://zfrhmaps.tch.harvard.edu/ZonRHmapper/protocol.htm and at http://wwwmap.tuebingen.mpg.de (65). Following initial denaturation at 94°C for 5 min, PCR included 35 cycles of 94°C for 30 s, annealing at 60°C for 30 s, and extension at 72°C for 1 min, with a final 72°C extension for 10 min. Raw RH (65) mapping scores were analyzed using Instant RH mapping software at http://zfrhmaps.tch.harvard.edu/ZonRHmapper/instantMapping.htm based on the current T51 RH panel map calculated using SAMapper 1.0 (53, 65).
In situ hybridization. ae2 probes generated from engineered plasmid 254-in_situ were made in a backbone of pCRII (Invitrogen, Carlsbad, CA) and encoded 175 nt of 5'-UTR plus the 5'-most 747 nt of the coding sequence, a region absent from zAE1 and with low similarity to known SLC4A3/AE3 genes, including that of skate (28). Plasmid 254-in_situ was linearized with SpeI and transcribed from the T7 promoter to generate antisense cRNA probe, or linearized with XhoI and transcribed from the SP6 promoter to generate sense cRNA probe. Probes were labeled with digoxigenin-UTP using the DIG RNA Labeling Mix (Roche) and purified with G-50 Sephadex Quick Spin Columns (Roche).
In situ hybridization was carried out as previously described (47), with modifications. Embryos were fixed with 4% paraformaldehyde, postfixed in methanol, rehydrated through steps of progressively decreasing methanol solutions, treated with proteinase K for no longer than 35 min [only if embryos were older than 24 h postfertilization (hpf)], and fixed a second time in 4% paraformaldehyde. Embryos were then washed, prehybridized, and hybridized overnight with labeled RNA probe. After further washing and blocking, embryos were incubated with alkaline phosphatase-coupled anti-digoxigenin Fab fragments (Roche), followed by color development with BM Purple AP substrate (Roche) and storage in 4% paraformaldehyde.
cRNA expression in Xenopus laevis oocytes. Capped zebrafish ae2 cRNA (MEGAscript, Ambion, Austin, TX) was transcribed with T3 polymerase from XbaI-linearized plasmids 254130 or 175-A22 and purified with the RNeasy kit (Qiagen). Female X. laevis anesthetized with 0.17% tricaine were subjected to a partial ovariectomy in accordance with protocols approved by the Institutional Animal Care and Use Committee of Beth Israel Deaconess Medical Center. Excised, minced ovarian segments were incubated for 1 h with gentle shaking at room temperature in 2 mg/ml type A collagenase (Roche) in ND-96, containing (in mM) 96 NaCl, 2 KCl, 1.8 CaCl2, 1 MgCl2 5 HEPES, and 2.5 sodium pyruvate, pH 7.4, supplemented with 5 mg/100 ml gentamicin. Stage V-VI oocytes were washed, manually defolliculated, and maintained at 19°C. On the same or next day, oocytes were manually microinjected (Drummond) with 50 nl water or solution containing 2.7, 3, or 10 ng cRNA, as indicated. Oocytes were maintained in ND-96 plus gentamicin at 19°C with a daily change of medium until use for ion transport assays 34 days later.
36Cl flux assays in X. laevis oocytes. 36Cl influx assays were performed as 30-min uptakes in ND-96 lacking gentamicin and pyruvate and containing 10 µM bumetanide, as described previously (15). Total bath [Cl] was 103 mM. For 36Cl efflux assays, individual oocytes were injected with 50 nl of 260 mM Na36Cl (10,00020,000 cpm). Following a 5- to 10-min recovery period in Cl-free ND-96 containing (in mM) 96 Na isethionate, 2 K gluconate, 1.8 Ca gluconate, 1 Mg gluconate, and 5 HEPES, pH 7.4, the efflux assay was initiated by transfer of single oocytes into borosilicate tubes containing 1 ml ND-96 or other efflux solution as described (52). At defined intervals, 0.95 ml of this efflux solution was removed for scintillation counting and replaced with an equal volume of fresh solution. Following completion of the assay with a final efflux period in Cl-free medium or in the presence of the inhibitor DIDS to confirm oocyte integrity, each oocyte was lysed in 100 µl of 1% SDS. Samples were counted for 4 min, such that the magnitude of two SD was <5% of the sample mean. In some efflux experiments, Na+ in ND-96 was substituted with K+ or with N-methyl-D-glucamine (NMDG). In some experiments, 20 mM NH4Cl was added in place of 20 mM NaCl. Hypertonic solutions were made with added NaCl.
Measurement of Cl/HCO3 exchange.
Oocytes previously injected with water or with 3 or 10 ng cRNA were incubated for 30 min at room temperature with 5 µM BCECF-AM, rinsed, then mounted in a 0.8-ml superfusion chamber on a microscope stage. Cl/HCO3 exchange was measured by BCECF fluorescence ratio imaging of oocyte pHi changes during removal and restoration of superfusate [Cl] (72 mM) in the presence of 5% CO2/24 mM HCO3, with gluconate as the substituting anion (15). Data acquisition and analysis were done using Metamorph software (Universal Imaging, W. Chester, PA). Initial rates of dpHi/dt were measured from the least squares linear fit of initial slopes. Buffer capacity (T) was calculated as the sum of intrinsic buffer capacity (
i) plus CO2 buffer capacity (
CO2). Data were expressed as proton flux (JH+), calculated as dpHi/dt x
T.
Two-electrode voltage clamp of X. laevis oocytes. Two-electrode voltage clamp was performed as previously described (15), with modifications. Oocytes injected 3 days previously with 2.7 ng cRNA were mounted in an ND-96 bath and subjected to 800-ms voltage-clamp steps from 100 mV to +40 mV in 20-mV increments.
Immunoblot analysis of zebrafish Ae2 polypeptide in X. laevis oocytes. Ten oocytes were suspended at 4°C in 1% Triton oocyte lysis buffer (10 µl/oocyte) containing (in mM) 50 Tris·HCl, pH 7.4, 150 NaCl, and 1 EDTA, and protease inhibitor cocktail (Roche). After 30-min intermittent vigorous shaking, the extract was centrifuged for 30 min in a microfuge. Clarified lysate was fractionated by SDS-PAGE (8% gels), and protein was transferred to nitrocellulose. Immobilized protein was subjected to immunoblot with affinity-purified rabbit polyclonal antibodies to mouse AE2 COOH-terminal aa 12241237 (4) and visualized on X-OMAT X-ray film (Kodak) by enhanced chemiluminescence (PerkinElmer).
Immunofluorescence detection of zebrafish Ae2 polypeptide in transiently transfected HEK-293 cells. HEK-293 cells were transfected with zebrafish ae2 cDNA (plasmids 254130 or 175-A22) using Lipofectamine 2000 (Invitrogen), then plated onto glass coverslips. Next, 2472 h later, cells were fixed with 2% paraformaldehyde, quenched, and immunostained with affinity-purified rabbit anti-mAE2a antibody to aa 12241237 or antibody to mAE2a aa 330352, in the presence of irrelevant peptide or of a specific peptide antigen at 24 µg/ml. After being washed, sections were incubated with fluorophor-conjugated secondary anti-rabbit Ig (Jackson Immunochemicals, Westport, PA). Images were recorded with a Bio-Rad MRC-1024 laser-scanning confocal fluorescence microscope.
Immunofluorescence detection of Ae2 polypeptide in zebrafish embryos and adults. Zebrafish embryos at 24 and 48 hpf were immersion-fixed in 4% paraformaldehyde, quenched, rinsed, and embedded as a lightly suspended pellet in 3% agar. Embedded embryos were cryoprotected in 30% sucrose in PBS, mounted in Tissue-Tek OCT mounting medium (Ted Pella, Redding, CA), and frozen with liquid nitrogen. Cryosections (10 µm, Reichert-Jung) were incubated 2 h with the indicated affinity-purified anti-mouse AE2 antibodies in the presence of either irrelevant peptide or of a specific peptide antigen at 24 µg/ml. After being washed, sections were incubated for 45 min with secondary fluorophor-coupled anti-Ig. Mouse monoclonal antibody to chicken Na+-K+-ATPase was previously described (4). FITC-coupled wheat germ agglutinin was from Vector Laboratories (Burlingame, CA). Stained sections were visualized, and images were acquired by confocal immunofluorescence microscopy.
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RESULTS |
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The four completely sequenced cDNA clones encode two variant alleles of zebrafish Ae2 polypeptide. Allele 1 is encoded by overlapping clones 175-A22 (nt 14536), 254-G4 (nt 12782), and 136-F19 (nt 24556641) and supported by six partially sequenced clones. Allele 2 is represented by clone 130-G21 (2,6594,536 nt), and supported by one partially sequenced clone. Allele 3 is present in the slc4a2/ae2 genomic sequence in BAC CH211160I2 (GenBank BX663499 [GenBank] ), identified by database search after completion of our cDNA sequencing. The nucleotide sequences of the three ae2 alleles encode three nonsynonymous single-nucleotide polymorphisms (SNPs), L24V, V58L, and L966F, and 22 synonymous SNPs in the Ae2 coding region (Supplementary Table 1), and additional polymorphisms in the 3'-UTR.
Exon-intron organization of the zebrafish ae2 gene. The exon-intron organization of the ae2 gene was revealed by nucleotide sequence alignment of the ae2 cDNA (AY876015) with the ae2 gene encoded in BAC CH211160I2 (BX663499 [GenBank] , extending from mid-intron 1 to the second polyadenylation site) and BAC DKEY-270K22 (BX323804 [GenBank] , containing exon 1 and proximal intron 1). Table 1 compares the exon-intron organization of the genes for zebrafish slc4a2/ae2 with those of mouse and rat. Each of these three orthologous genes consists of 23 exons and 22 introns, and all intron donor and acceptor splice sites of the zebrafish ae2 gene exhibit GT/AG consensus sequences. Nearly all the exon-intron boundaries of the three orthologous genes match perfectly in exon partitioning and in positions of split codons. Only for introns 2 and 5, where polypeptide alignment was not straightforward (see Fig. 1) might the intron-exon junctions differ among the species.
In contrast, 15 of 22 intron lengths in the zebrafish ae2 gene were much longer than the corresponding introns of the mouse and rat genes. This difference is largely responsible for the 60-kb length of the zebrafish ae2 gene compared with lengths of 16.717 kb for the mammalian AE2 genes (32, 54, 62). The length of ae2 intron 1 remains uncertain, because BAC CH211160I2 lacks the exon 1 sequence, and BAC DKEY-270K22 has only exon 1 sequence.
Genomic mapping of the ae2 gene. The ae2 gene was assigned linkage group 2 (LG2) by RH mapping on the Goodfellow TH51 RH panel (24, 31). The LG2 markers zc64c8.zc and fc74f03 exhibited linkage to the ae2 gene with LOD scores of 10.2, and marker fj85c11.x1 showed linkage with a LOD score of 11.1. BAC CH211160I2 (BX663499 [GenBank] ) contained the LG2 marker bz2c8.z and most of the ae2 gene, establishing physical linkage between this LG2 marker and the ae2 gene.
The ae2 region on LG2 is syntenic with human chromosome 7q3536 (8). The syntenic region includes, in addition to the zebrafish and human AE2 genes, the syntenic genes fc74f03 and human ABCF2 (ATP-binding cassette subfamily F member 2), eng2b and human engrailed homolog 2, and twhh and human sonic hedgehog preproprotein. This synteny strengthened the validity of the ae2 gene location based on RH mapping and physical linkage and strengthened the identity of ae2 as an SLC4A2/AE2 ortholog.
Deduced Ae2 polypeptide sequence.
The 1,228-aa zebrafish Ae2 polypeptide is predicted by Kyte-Doolittle hydropathy analysis to have a hydrophilic NH2-terminal cytoplasmic domain of 708 aa, followed by a hydrophobic polytopic membrane domain of 520 aa containing 1214 predominantly
-helical transmembrane (TM) spans (Supplementary Fig. 1). This topography is conserved among the SLC4/AE polypeptides. The hydrophilic COOH-terminal cytoplasmic tail of
37 aa contains a conserved putative binding site at 1203LDADD1207 for carbonic anhydrase 2, and another potential site at 1212LDDKD1216. Two extracellular consensus N-glycosylation sequences are located in the predicted extracellular loop between TM5 and TM6, in contrast to three N-glycosylation sites in this loop in other AE2 polypeptide orthologs (Fig. 1). Ae2 conserves 843KLGKIF848, the covalent binding site for the isothiocyanate group of the stilbene disulfonate inhibitor DIDS.
Pairwise alignment of the Ae2 allele 1 aa sequence with those of other SLC4/AE family members (Supplementary Table 2) revealed greater percent identity with AE2 polypeptides of other species than with AE1 or AE3 polypeptides, further supporting the identity of this cloned cDNA as an AE2 ortholog. Multiple sequence alignment of zebrafish Ae2 with skate AE2 (28), chicken AE21 (17), mouse AE2a (2), and mouse bAE3(30) emphasized the highly conserved TM region (with the notable exception of the exofacial loop between TM 5 and TM6), whereas the cytoplasmic domain exhibited several stretches of much lower similarity (Fig. 1). Therefore, individual subdomains of zebrafish Ae2 (cytoplasmic stretches aa 132, 3396, 97234, and 235708; TM stretches 709853, 854883, and 8841228) were subjected to pairwise comparison with the corresponding regions of other SLC4/AE anion exchangers (Supplementary Table 2). Each region of Ae2 exhibited higher similarity to corresponding regions of AE2 from other species than to those of AE1 or AE3. Interestingly, zebrafish Ae2 shared slightly greater identity with chicken AE2.1 than with skate AE2.
Localization of ae2 mRNA.
ae2 mRNA was localized in embryos by whole-mount in situ hybridization (Figs. 2 and 3). Antisense probe predominantly stained the anterior pronephric duct of embryos at 24, 48, and 72 hpf (Fig. 2A, B, D, E, G, H). The specificity of this staining pattern was shown by the lack of staining with sense probe (Fig. 2C). The glomerular and pronephric tubule segments remained mostly unstained. The pronephric duct region of the embryo consists of hematopoietic tissue as well as epithelial tubules and developing vasculature destined to serve both tissue types. Four observations supported the conclusion that ae2 mRNA is expressed in pronephros. First, ae2 mRNA expression and location were not modified in the mutant cloche (Fig. 2F), in which hematopoiesis and vasculogenesis are absent (56) but kidney development is maintained (33). Second, ae2 mRNA expression and location were similarly unchanged in the mutant spadetail (not shown), in which erythropoiesis is absent but vasculogenesis is maintained (56). Third, the distribution of ae2 mRNA resembled that reported for Na+-K+-ATPase 1-subunit mRNA (33). Fourth, ae2 mRNA colocalized with the pronephric distribution of the renal developmental marker pax2.1 (34) but not with pax2.1 expression in neural structures (Fig. 2I). pax2.1 was also expressed in the optic vesicle, the midbrain-hindbrain boundary, otic placode, and spinal cord (data not shown) as previously described (34, 41).
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Functional characterization of zebrafish Ae2. ae2 cRNA was injected into X. laevis oocytes to compare the functional properties of zebrafish Ae2 polypeptide with those of the extensively studied mouse AE2a. Zebrafish Ae2 mediated 36Cl influx in X. laevis oocytes, with higher quantities of injected cRNA leading to higher influx activity (Fig. 4A). Ae2 also mediated 36Cl efflux that was trans-anion dependent and DIDS sensitive (Fig. 4, B and C). Zebrafish Ae2 required higher concentrations of DIDS for inhibition than did mAE2a. (Fig. 4, C and D). These data demonstrate the Cl/Cl exchange activity of zebrafish Ae2.1
Zebrafish Ae2-mediated Cl/Cl exchange was Na+ independent, because substitution of bath Na+ with equimolar NMDG or K+ produced minimal difference in 36Cl efflux (Fig. 5, AC). The electrical properties of zebrafish Ae2 were of interest, because expression in X. laevis oocytes of trout AE1 leads to large anion currents (22). Despite parallel increases in oocyte cation currents (27) and taurine flux (22), mutagenesis results to date support the hypothesis that these anion currents are mediated by the trout AE1 polypeptide (11). However, the lack of effect of K+ substitution suggested electroneutrality of transport by zebrafish Ae2. This electroneutrality was further supported by two-electrode voltage-clamp measurements of currents in oocytes expressing Ae2 (Fig. 5D). The small ohmic control conductance of 1.3 ± 0.04 µS (n = 5) was not increased by Ae2 expression but rather decreased slightly to 0.99 ± 0.07 µS (n = 5). The control reversal potential of 44 ± 3 mV remained unchanged at 47 ± 4 mV. Thus, as true for mouse AE1 (14, 22), expression of zebrafish Ae2 produced no detectable anion current. Therefore, Ae2 mediates electroneutral anion exchange without induction of any apparent current mediated by Ae2 or by endogenous oocyte ion channel(s).
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Immunodetection of Ae2 polypeptide. Figure 9A shows that two antibodies raised against conserved peptide sequences of mAE2 are cross-reactive and immunospecific as immunocytochemical reagents detecting zebrafish Ae2. Antibody to mAE2a COOH-terminal aa 12241237 recognizes a sequence identical in 9 of the COOH-terminal 12 aa of zebrafish Ae2. Antibody to mAE2a aa 330352 recognizes a region critical for acute regulation by pHi and extracellular pH (52), with 20 of 23 identical residues in Ae2 and highly conserved among all SLC4 polypeptides. Both antibodies detect Ae2 in the peripheral membrane of transfected HEK 293 cells. Figure 9B shows that antibody to mAE2a aa 12241237 also detects Ae2 polypeptide on immunoblots of lysates of cRNA-injected oocytes, although with lower signal intensity than mAE2a. The other anti-mAE2 antibody did not reliably detect Ae2 by immunoblotting (not shown). Ae2 was also not recognized by antibodies to Slc4a1/Ae1 COOH-terminal peptide (9 of 14 aa identical residues) or to Ae1 aa 90105 (9 of 16 identical residues) (40) by either immunoblot or immunofluorescence techniques (not shown).
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DISCUSSION |
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Genetic polymorphism of the ae2 gene. The zebrafish Ae2 polypeptide sequence has greater aa identity with AE2 polypeptides than with AE3 polypeptides of other species. Synteny of ae2 with human AE2 further supports the identity of ae2 as an AE2 ortholog. Thus the separation of the SLC4 genes SLC4A1/AE1, SLC4A2/AE2, and SLC4A3/AE3 appears to have preceded the evolutionary divergence of the teleosts. Although zebrafish are diploid, many orthologs of mammalian genes have undergone duplication at some point following divergence of the ray fin fish lineage from the lobe fin lineage that gave rise to birds and mammals. An example is the multiple genes encoding subunits of Na+-K+-ATPase (13). This fact suggests the possibility that additional SLC4A2/AE2 orthologs might be present in the zebrafish genome, as well as one or more AE3 orthologs. The presence of alternately initiated ae2 transcripts remains to be determined. The ae2 sequence presented in this paper has a length and exon composition similar to those of mouse AE2a (1, 45). However, the Ae2 NH2-terminal aa sequence of zebrafish is different from those of mammalian AE2a polypeptides.
The high degree of polymorphism among three Ae2 alleles detected in a pooled zebrafish kidney library emphasizes the need for cautious design of oligonucleotide primers when genomic or cDNA sequences based on any single sequence entry in the zebrafish dataset are amplified. Such polymorphisms may influence the outcome of experiments involving antisense morpholino oligonucleotide knockdown, RNAse protection, or RT-PCR.
Functional characterization of recombinant Ae2 polypeptide. This work represents one of the few functional characterizations of a zebrafish ion transporter in the pronephric duct. Others include two Na+-phosphate cotransporter isoforms (25, 38), a H+-peptide cotransporter (59), and a zinc transporter from the gill also expressed in the kidney (43). Functional genetic characterization has been initiated for the multiple genes encoding subunits of the Na+-K+-ATPase (13), and differential localization of two vH+-ATPase B subunit gene products has been described (48). In contrast, functional analysis of recombinant trout erythroid AE1 has been extensive (11, 22, 27), and initial functional analyses of recombinant skate erythroid AE1, AE2, and AE3 have also been reported (28). The function of trout AE1 expressed in X. laevis oocytes has differed from that of mammalian AE1 in its insensitivity to DIDS and its association with Cl and cation conductances as well as with increased taurine transport. The trout AE1 polypeptide has been proposed to mediate all these activities, and directed mutagenesis experiments to date are consistent with this hypothesis (11). It was therefore of interest to test the electrogenicity of zebrafish Ae2 function.
As shown in Fig. 4, Ae2 mediates Cl/Cl exchange, which is slightly less sensitive than mAE2a to inhibition by DIDS. The small outward currents of Ae2-expressing oocytes are not consistent with the electrochemical gradient driving net Cl efflux at membrane potentials negative to the reversal potential. The 11.7 nmol/h 36Cl influx attributable to Ae2 in oocytes previously injected with 3 ng cRNA (Fig. 4A) corresponds to a rate of 0.75 nmol/h predicted by the outward difference current attributable to Ae2 expression at resting potential (20 nA). In contrast, CFTR expression mediated cAMP-activated inward current at negative holding potential (not shown). The bath Na+ and K+ substitution experiments also fail to support the idea of simultaneous activation of anion and cation current. These data together suggest that Ae2 mediates neither Cl current nor electrogenic Cl/anion exchange.
Although most SNPs found in the ae2 coding region were synonymous, three nonsynonymous cSNPs were found among three distinct ae2 alleles. The functional importance of the L24V and V58L polymorphisms is unclear, as functional properties intrinsic to the far NH2-terminal region of mammalian AE2a have not been described. However, the L966F polymorphism resides in the exofacial loop between putative TM 7 and 8, two residues beyond G964, conserved in AE1 and AE2 of multiple species. This glycine has been implicated (as G767) in the binding of carbonic anhydrase IV to human SLC4A4/NBCe1b, associated with increased HCO3 transport activity (5). It is thus intriguing to speculate that the Ae2 cSNP L966F distinguishing allele 1 from alleles 2 and 3 might control Ae2 sensitivity to stimulation by carbonic anhydrase IV, a gene amply represented among zebrafish ESTs.
Localization of ae2 mRNA in the zebrafish embryo. The zebrafish larval kidney, the pronephros, is composed of two glomeruli fused at the midline, two pronephric tubules, and paired bilateral pronephric ducts that modify the composition of the blood filtrate before delivering it to the cloaca for excretion. The pronephros interacts closely with endothelial cells for blood filtration. The subsequently appearing mesonephric kidney (96 hpf) plays an additional role as the primary organ of hematopoiesis (19, 20, 63). Together with vasculature and blood, the pronephros derives from ventral mesoderm (63).
Whole mount in situ hybridization revealed ae2 expression evident as early as the five-somite stage in the prospective midbrain, with expression later evident first in nephrogenic cell populations of the intermediate mesoderm, and later in the pronephric ducts. ae2 expression was preceded by and partially colocalized with expression of the neural and renal patterning factor pax2.1, a marker of the prospective midbrain and developing pronephros. A developmental function of AE2 in the mouse is evident from the absence of maxillary incisors, gastric mucosal atrophy, and peri-weaning mortality (23), but mAE2 expression patterns in the early mouse embryo remain unreported.
Because the fish kidney also contains hematopoietic and endothelial tissue, it was important to identify the cell types expressing ae2 mRNA. Maintenance of ae2 mRNA expression in the pronephric duct in the absence of both primitive and definitive hematopoietic precursors and vasculogenic cells in the cloche mutant (Fig. 2F) and in the absence of erythropoiesis in the spadetail mutant supports pronephric expression of ae2 mRNA. The sinewy deformation of the pronephric duct in the cloche mutant may reflect the developmental consequence of the absence of trophic factors, matrix, and cytokines normally secreted by adjacent hematopoietic cells. Alternatively, the deformation might reflect consequences of decreased expression of vasculogenic genes in cloche (56), perhaps secondary to elimination of bipotential hemangioblasts.
Localization of Ae2 polypeptide in the zebrafish embryo. Mammalian AE2 is a basolateral membrane protein in all AE2-expressing renal epithelial cells and in most other epithelial cells. AE2 in the basolateral membrane serves either to load cells with Cl for apical secretion and/or to defend cells against alkaline load. In the context of acid secretion by the gastric parietal cell, basolaterally localized AE2 may fulfill both functions (55). Basolateral AE2 in the choroid plexus epithelial cell (4), in the colonic surface epithelial cell (3), and in the inner medullary collecting duct cell (54) may also serve as both Cl loader and pH regulator. Recent data on regulation of renal AE2 polypeptide levels by dietary Na+ loading suggest a possible role in transepithelial NaCl transport (44).
In mammalian epithelial cells in tissue culture, AE2 is almost uniformly basolateral. In intact tissue, possible apical AE2 immunostaining has been noted in a restricted area of mouse duodenum Brunner's glands (3). Before the recent appearance of data on apical localization of several SLC26 Cl/HCO3 exchangers (37), AE2 was proposed as the major, cAMP-stimulated, apical Cl/HCO3 exchanger of biliary epithelium (50), but recombinant rodent AE2 is not stimulated by cAMP (29; Chernova MN, Jiang L, and Alper SL, unpublished observations). Nonetheless, AE2 expression in the apical membranes of human biliary canaliculi and small bile ducts has been detected with a single IgM monoclonal antibody (35) and has also been suggested by immunogold electron microscopic localization of rat AE2 to the subapical region of cholangiocytes in rat bile duct (57). More recently, apical targeting of human AE2-GFP fusion proteins in biliary canaliculus of polarized primary hepatocytes (6) was shown to contrast to their basolateral targeting in polarized Madin-Darby canine kidney cells (6, 12).
The predominantly apical localization of Ae2 in some portions of the zebrafish pronephric duct is the first histological report of apical AE2 localization in the kidney. The hints of axial variation of Ae2 apical localization along the pronephric duct may offer new opportunities for definition of membrane protein-sorting signals and study of their regulation. This axial variation of apparent Ae2 polarity may represent Ae2 polypeptide variants with distinct targeting signals, or antibody cross-reactivity with other (possibly related) zebrafish polypeptide gene products. Basolateral membrane accumulation of human kidney AE1 (kAE1) is controlled in part by a Yxx motif in the COOH-terminal tail (18, 58) conserved in zebrafish Ae2, and in part by a yet undefined region of the NH2-terminal 360 cytoplasmic amino acids (58). The human distal renal tubular acidosis AE1 mutation G609R leads to loss of polarity of kAE expression in tissue culture (46), but this Gly residue is also conserved in zebrafish Ae2.
The apical predominance of Ae2 in the pronephric duct at 24 and 48 hpf alternatively may reflect a developmental immaturity, perhaps similar to the nonpolarized distribution of collecting duct Na+-K+-ATPase in embryonic rabbit kidney (7) or its variably apicolateral distribution in early postnatal mouse collecting duct (36). Study of zebrafish Ae2 may contribute to a further understanding of the differences in apical sorting of membrane proteins distinguishing hepatocytes from renal and intestinal epithelial cells in mammals (61).
In freshwater fish such as zebrafish, the predominantly apical localization of Ae2 in pronephric duct epithelial cells, if it persists in the adult, should aid in reclamation of filtered luminal Cl originally taken up from the surrounding water by the gill and skin (10, 42), or perhaps in secretion of Cl as changing external conditions require (9). Electroneutral Cl/HCO3 exchange has been measured in brush-border membrane vesicles isolated from the kidney of the freshwater eel A. anguilla (60). SLC4 homologs related to NBCe1 and to SLC4A11/BTR1 have been respectively localized to proximal and distal tubular segments of the X. laevis embryonic nephron by in situ hybridization (64). The ability to achieve gene inactivation using antisense N-morpholino oligomers in zebrafish embryos (39) will aid in defining the role of Ae2 in zebrafish development and in pronephric duct function and may provide further insights into the Cl reabsorptive and secretory functions of mammalian kidney tubules (26).
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FOOTNOTES |
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Supplementary Fig. 1 and Supplementary Tables 1 and 2 can be accessed at http://ajprenal.physiology.org/cgi/content/full/00122.2005/DC1).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 All functional experiments in Figs. 48 were performed with cDNA encoding the full-length zebrafish Ae2 open reading frame reconstructed from clones 254-G4 and 130-G21 before discovery of the three ae2 alleles (Supplemntary Table 1). Ae2 allele 1 encodes aa L24, V58, and L966, whereas allele 3 encodes V24, L58, and F966. Allele 2 also encodes F966. The reconstructed ae2 cDNA proved to be a hybrid of alleles 1 and 2, encoding L24, V58, and F966. The functional data presented in Figs. 48 represent this Ae2 hybrid allele. As clone 175-A22 was subsequently found to encode full-length Ae2 polypeptide allele 1, cRNA from this allele 1 clone was also tested in X. laevis oocytes. Allele 1 polypeptide was indistinguishable from the fusion allele in assays of 36Cl influx (n = 20) and of bath Cl-dependent 36Cl efflux (n = 9, data not shown).
* B. E. Shmukler and C. E. Kurschat contributed equally to this work.
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REFERENCES |
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