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Am J Physiol Regul Integr Comp Physiol 293: R1743-R1753, 2007. First published August 8, 2007; doi:10.1152/ajpregu.00248.2007
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WATER AND ELECTROLYTE HOMEOSTASIS

Localization of ammonia transporter Rhcg1 in mitochondrion-rich cells of yolk sac, gill, and kidney of zebrafish and its ionic strength-dependent expression

Tsutomu Nakada,1 Kazuyuki Hoshijima,1 Masahiro Esaki,1 Saori Nagayoshi,2 Koichi Kawakami,2 and Shigehisa Hirose1

1Department of Biological Sciences, Tokyo Institute of Technology, Yokohama; and 2Division of Molecular and Developmental Biology, National Institute of Genetics, Mishima, Shizuoka, Japan

Submitted 11 April 2007 ; accepted in final form 3 August 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Members of the Rh glycoprotein family have been shown to be involved in ammonia transport in a variety of species. Here we show that zebrafish Rhcg1, a member of the Rh glycoprotein family, is highly expressed in the yolk sac, gill, and renal tubules. Molecular cloning and characterization indicate that zebrafish Rhcg1 shares 82% sequence identity with the pufferfish ortholog fRhcg1. RT-PCR, combined with in situ hybridization, revealed that Rhcg1 is first expressed in vacuolar-type H+-ATPase/mitochondrion-rich cells (vH-MRC) on the yolk sac of larvae at 3 days postfertilization (dpf) and later in vH-MRC-like cells in the gill at 4–5 dpf. Ammonia excretion from zebrafish larvae increased in parallel with the expression of Rhcg1. At larval stages, Rhcg1 mRNA was detected only on the yolk sac and gill; however, the kidney, as well as the gill, becomes a major site of Rhcg1 expression in adults. Using a zebrafish Tol2 transgenic line whose vH-MRC are labeled with green fluorescent protein (GFP) and an antibody against zebrafish Rhcg1, we demonstrate that Rhcg1 is located in the apical regions of 1) vH-MRC on the yolk sac and vH-MRC-like cells (cell population with the expression of Rhcg1 and GFP) in the gill and 2) cells in the renal distal tubule and intercalated cell-like cells in the collecting duct of the kidney. Remarkably, expression of Rhcg1 mRNA at the larval stage was changed by environmental ionic strength. These results suggest that roles of zebrafish Rhcg1 are not solely ammonia secretion to eliminate nitrogen from the gill.

osmoregulation; salinity; nitrogen metabolism; mitochondria-rich cell

TELEOST FISHES HAVE SEVERAL ammonia1 transport systems to maintain homeostasis. Most teleost fish excrete nitrogenous waste as ammonia from the gill to environmental water without converting it to urea or uric acid although freshwater fish, in particular, can also excrete ammonia into urine for maintenance of the acid-base balance of the body fluid under the conditions of low external pH (26, 64). The molecular mechanism of ammonia transport across the gill epithelial and renal tubule cells remains controversial (11, 60, 63, 66).

Members of the ammonia transporter/methylammonium permease/Rhesus glycoprotein family are involved in ammonia transport in a broad range of organisms (35, 36, 44). Ammonia transport activity has been demonstrated with three human Rhesus (Rh) glycoproteins, Rh-associated glycoprotein (RhAG), Rh type B glycoprotein (RhBG), and Rh type C glycoprotein (RhCG) by using the Saccharomyces cerevisiae and Xenopus oocyte heterologous expression systems (3, 30, 33, 35, 43, 58, 59). RhAG is associated with major blood group antigens on the surface of red blood cells in humans (51). RhBG and RhCG have been localized to the basolateral and apical membranes, respectively, in the connecting segment and collecting duct of the kidney, which is a major site of transepithelial ammonia transport (9, 50, 56).

We previously reported that four members of the pufferfish Rh glycoprotein family (fRhag, fRhbg, fRhcg1, and fRhcg2) are ideally located for ammonia excretion in the pillar cells, pavement cells, and mitochondrion-rich cells (MRCs) of the gill. Furthermore, functional analysis using the Xenopus oocyte expression system showed that all of the four Rh glycoproteins are capable of transporting methylammonium, an analog of ammonia (41). These data suggest that Rh glycoproteins of seawater fish have important roles in ammonia excretion from the gill. However, involvement of Rh glycoproteins in ammonia excretion in freshwater fish is totally unknown. Freshwater and seawater fish need opposite ion transport mechanisms in their osmoregulatory organs (e.g., gill, intestine, and kidney) to compensate for the loss of ions or water. Therefore, they have different types of MRCs, which are responsible for ion absorption or secretion, in their gills (11, 14, 19, 48, 49, 63). Drinking rate, intestinal fluid absorption, and structures of the nephron in the kidney are also different between freshwater and seawater fish (16, 37, 45, 55, 61).

Recently, the zebrafish has been used for physiological studies as a model freshwater fish (7, 10, 29, 46). The value of zebrafish as a model species has been further increased by the development of a new methodology, namely a highly efficient transgenesis method using the Tol2 transposable element (24). By performing an enhancer trap screen using Tol2, we have created a variety of zebrafish lines with tissue-specific green fluorescent protein expression (S. Nagayoshi and K. Kawakami, unpublished observation). One of the transgenic zebrafish lines, HG9B, which exhibits green fluorescent protein expression in MRC-like cells was characterized and used in this study for monitoring Rh protein expression in MRCs.

In this study, we isolated the full-length cDNA of zebrafish Rhcg1 and examined its localization and expression pattern at different developmental stages by RT-PCR and in situ hybridization. Furthermore, we carried out immunohistochemistry with a specific polyclonal antibody against the COOH terminus of zebrafish Rhcg1. The results, when compared with our previous study on pufferfish Rhcg1 (41), indicate that Rhcg1 is expressed in MRCs of both seawater and freshwater fish; however, the MRC cell populations expressing Rhcg1 are quite different; in the case of seawater fish (pufferfish), Rhcg1 is expressed in MRCs rich in Na+-K+-ATPase (NaK-MRC), while in freshwater fish (zebrafish) it is expressed in MRCs rich in vacuolar-type H+-ATPase (vH-MRC). Unexpectedly, and distinct from pufferfish orthologs, the kidney also becomes a major site of Rhcg1 expression in adult zebrafish. Immunohistochemistry demonstrates that Rhcg1 is located in apical regions of the distal tubule and collecting duct cells. Moreover, our study showed that expression of Rhcg1 mRNA was influenced by environmental ionic strength. These results suggest that the physiological roles of zebrafish Rhcg1 include not only nitrogen elimination but also orchestration of nitrogen metabolism and ion homeostasis.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Zebrafish culture. Zebrafish were maintained as described (10, 57). The wild-type zebrafish Tubingen long fin (TL) line was generously provided by Dr. A. Kawakami (Yokohama, Japan). Fertilized eggs were incubated in 1x freshwater (1x FW; 60 mg artificial ocean salt; Rohtomarine; Rei-sea, Tokyo, Japan) per 1 liter of distilled water at 28.5°C. Here 1x FW was used as the standard freshwater and corresponds to "Fish Water" as described in The Zebrafish Book (57). The pH and osmolarity of 1x FW are 6.7–7.4 and 1.64 mOsmol/l, respectively. The concentrations of major ions of 1x FW are as follows (in µM): 686 Na+, 14.6 K+, 16.2 Ca2+, 78.3 Mg2+, 804 Cl–, 41.6 SO42–, and 1.25 Br–. In some experiments, 1/20x FW (20-fold diluted 1x FW) and 100x FW (100-fold concentrated 1x FW) were used. To prepare acidic water, 1x FW was adjusted to pH 5.0 by the addition of HCl. No change in the pH of the bathing water after the experiments was confirmed with a pH meter. For tests for acclimation to ammonia, NH4Cl was added to 1x FW at final concentrations of 5, 50, and 500 µM. Hatched or dechorionated larvae were used for the following experiments without feeding. The animal protocols and procedures were approved by the Institutional Animal Care and Use Committee of Tokyo Institute of Technology and conform to the American Physiological Society's "Guiding Principles in the Care and Use of Laboratory Animals" (1).

Whole mount in situ hybridization. Whole mount in situ hybridization was performed as previously described with minor modifications (53), using a drRhcg1a probe corresponding to a 828-bp fragment of cDNA (GenBank accession no. AF398238; nucleotides 384-1212) (20).

Total RNA isolation and RT-PCR. Total RNA was isolated from whole embryos/larvae with RNeasy Lipid Tissue Kit (Qiagen, Tokyo, Japan) and RNase-Free DNase Set (Qiagen) following the manufacturer's instruction. Total RNA of various tissues of adult zebrafish was isolated with Isogen reagent (Nippon Gene, Toyama, Japan) (54). The isolated RNA was dissolved in diethyl pyrocarbonate-treated water, and the concentration was measured spectrophotometrically at 260 nm. For cDNA synthesis, 1 µg of total RNA was reverse-transcribed with oligo(dT)20 primer and Superscript III (Invitrogen, Carlsbad, CA). PCR was performed with 0.5 µl of the above-mentioned cDNA, ExTaq polymerase (Takara, Shiga, Japan), and 0.2 mM each primer. To assess expression level, PCR was performed with 28 and 20 cycles for the Rh family members and beta-actin, respectively. All primer sets used were listed in Table 1. The PCR products were sequenced to ensure that they were the desired gene fragments. Full-length cDNA of drRhcg1 was obtained by 5'-rapid amplification of cDNA ends (RACE), 3'-RACE and RT-PCR with RLM-RACE kit (Ambion, Austin, TX) as previously described (42).


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  		Table 1. List of primers used for PCR amplification

 
Measurement of ammonia excretion rate. Ammonia excretion rates were measured as previously described with slight modification (65). At each time point [0, 1, 2, 3, 4, 5, and 6 days postfertilization (dpf)], 40 embryos/larvae were randomly selected and placed into four microtubes containing 500 µl of 1x FW. After 3 h, the bathing media (FW) were collected and immediately frozen at –20°C. Ammonia concentration was determined by the colorimetric indophenol blue method with Ammonia Test Wako (Wako Pure Chemicals, Osaka, Japan).

Antibody production. A cDNA fragment encoding a part of the COOH terminus of drRhcg1 (amino acid residues 425-488) was subcloned into the BamHI/EcoRI site of the bacterial expression vector pGEX4T2 (GE Healthcare Bioscience, Piscataway, NJ). The recombinant protein was purified with glutathione Sepharose 4B (GE Healthcare) following the manufacturer's instruction. Briefly, BL21 cells transformed with the expression vectors were used to inoculate 1.5 liters of Luria-Bertani's broth containing 100 µg/ml ampicillin. The cultures were grown to an A600 of 0.5 at 37°C, and protein expression was induced by adding isopropyl-1-thio-D-galactopyranoside to a final concentration of 0.3 mM for 4 h at 37°C. The cells were harvested from the cultures by centrifugation, resuspended in 20 ml of PBS, and then disrupted by freezing-thawing and sonication. After centrifugation (10,000 g at 4°C), supernatants were saved and purified with glutathione Sepharose 4B. After purification, recombinant proteins were dialyzed against saline at 4°C. Polyclonal antibodies were prepared in Japanese white rabbits by injecting ~200 µg of purified recombinant proteins, emulsified with the adjuvant TiterMax Gold (CytRx) (1:1) intramuscularly at multiple sites. The rabbits were injected three times at 1-mo intervals and bled 7 days after the third immunization.

Immunohistochemistry. Immunohistochemistry with zebrafish larvae was performed as previously described (10). Larvae were fixed with 4% paraformaldehyde in PBS comprising 137 mM NaCl, 27 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, pH 7.4 at 4°C overnight and dehydrated in 100% methanol. For double staining with concanavalin A (ConA), the larvae were incubated with 50 µg/ml Alexa Fluor 594-conjugated ConA for 30 min prior to fixation. After rehydration in PBS with 0.1% Tween-20 (PBT) and incubation in PBT containing 10% sheep serum (Sigma, St. Louis, MO) for 1 h, the larvae were incubated at 4°C overnight with an antiserum against the drRhcg1 (diluted 1:2,000 with PBT containing 10% sheep serum) or with a preimmune serum. In the case of whole mount immunohistochemistry of transgenic zebrafish HG9B, anti-GFP monoclonal antibody (diluted 1:100, Clontech, Palo Alto, CA) was used. Following a wash with PBT, the larvae were further incubated for 2 h at RT with Alexa Fluor 488-conjugated anti-rabbit IgG, Alexa Fluor 594-conjugated anti-rabbit IgG, or Alexa Fluor 594-conjugated anti-mouse IgG (diluted 1:1,000, Invitrogen) and then washed with PBT and embedded in 100% glycerol to photograph.

Immunohistochemistry of gill and kidney sections was performed as previously described (41). Gills and kidneys from adult zebrafish were fixed with 4% paraformaldehyde in PBS at 4°C overnight. After incubation in PBS containing 20% sucrose for 16 h at 4°C, specimens were frozen in Tissue Tek OCT Compound on a cryostat holder. Sections (6 µm) were prepared in the cryostat at –20°C and mounted on APS-coated glass slides and air dried for 1 h. After being washed with PBS, sections were first incubated in PBS with 0.1% Triton X-100 for 10 min and then incubated for 1 h at room temperature with 2.5% normal goat serum or 5% fetal bovine serum. After blocking, the sections were incubated with anti-drRhcg1 antiserum (1:2,000) and/or rat anti-Na+-K+-ATPase antiserum (1:1,000) (40) overnight at 4°C. Sections were then washed with PBS and treated with Alexa Fluor 488-conjugated anti-rabbit IgG, Alexa Fluor 594-conjugated anti-rabbit IgG (1:2,000, Invitrogen), and/or Cy3-conjugated anti-rat IgG (1:2,000, Jackson ImmunoResearch Laboratories, West Grove, PA) for 1 h at RT. Hoechst 33342 (100 ng/ml; Invitrogen) was also added in the secondary antibody solutions for nuclei staining. Fluorescence images were acquired by using Axiovert 200M epifluorescence microscope (Carl Zeiss, Thornwood, NY) equipped with an ApoTome optical sectioning device (Carl Zeiss).


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Isolation and characterization of zebrafish Rhcg1. To identify the zebrafish Rhcg orthologs, a homology search in the NCBI zebrafish genome database was performed. The amino acid sequence of fRhcg1, which is expressed in the MRCs on the gill of pufferfish (41), was used as query. We found three Rhcg-related sequences (accession no: AF398238, XM_687189, XM_690091) in the database. Since the sequence of AF398238 [GenBank] has the highest homology to fRhcg1, we named it drRhcg1 (dr for Danio rerio) and isolated the complete cDNA sequence by 5'- and 3'-RACE and RT-PCR from 5-dpf larval RNA as previously described (42). The isolated cDNA was ~1.7 kb including 30 bp of 5' UTR and 200 bp of 3' UTR and contains a polymorphism of 15 bases (7-amino acid replacement) compared with the deposited sequence AF39828. The sequence information for drRhcg1 has been deposited in the DDBJ/EMBL/GenBank database (accession no. AB286865). The coding region predicts a protein of 488 amino acid residues with a calculated molecular mass of 54 kDa (Fig. 1A). Hydrophobicity analysis of the zebrafish Rhcg1 protein predicts a membrane topology with 12 putative transmembrane spans (Fig. 1, B and C). We also isolated the complete cDNA sequences of other two Rhcg-related genes, and named them drRhcg2 (accession no. AB286866) and drRhcg3 (accession no. AB286867).


Figure 1
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  		Fig. 1. Structural features of zebrafish Rhcg1. A: nucleotide and deduced amino acid sequences of Rhcg1 cDNA. Numbers on left refer to the first amino acid residue on the line, and numbers on right to the last nucleotide on the line. The asterisk (*) indicates a stop codon. The circled amino acid residues are potential N-glycosylation sites. Twelve transmembrane-spanning regions predicted by hydrophobicity are indicated by solid underlines. B: Kyte-Doolittle hydropathy plot (27) of zebrafish Rhcg1. The plot was constructed using the GENETYX-MAC computer program. C: secondary structure model of Rhcg1 with putative 12 transmembrane spans. The region used as antigen is indicated by the bold line. The potential N-glycosylation sites are also shown.

 
A phylogenetic tree was constructed based on the amino acid sequences of zebrafish and other vertebrate Rhcgs including those of human, pufferfish, chicken, and frog. As expected, the sequence of drRhcg1 is most closely related to Rhcg1 of pufferfish (82% identity in amino acids). The two other zebrafish Rhcg orthologs, drRhcg2 and drRhcg3, are more closely related to pufferfish Rhcg2 than Rhcg1 (Fig. 2).


Figure 2
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  		Fig. 2. Phylogenetic analysis of zebrafish Rhcg1 protein. The phylogenetic tree was constructed by the neighbor-joining method with 1,000 bootstrap replicates using Clustal W software. The scale bar represents a genetic distance of 0.05 amino acid substitutions per site. The abbreviations are as follows: dr, zebrafish (Danio rerio); f, fugu pufferfish (Takifugu rubrips); m, mouse (Mus musculus); and h, human (Homo sapience). The accession nos. are as follows: drRhcg1 (AB286865), drRhcg2 (AB286866), drRhcg3 (AB286867), fRhcg1 (AB218981), fRhcg2 (AB218982), hRHCG (NP_057405), fRhbg (AB218980), drRhbg (AAQ09527), hRhBG (NP_065140), fRhag (AB218979), drRhag (NP_998010), hRhAG (AAF78209), fRhced (Rh30-like protein, AAM48580), drRhced (Rh30-like protein, NP_001019990), mRhced (NP_035400), hRhCE (P18577), hRhD (CAC10191), fRhP2a (fRhag-like1, Rhag1, AAM48575), fRhP2b (fRhag-like2, Rhag2, AAM48576), and drRhP2 (Rh50-like protein, NM_131547).

 
Expression of zebrafish Rhcg1 mRNA in different developmental stages and adult tissues. We examined the tissue distribution of Rhcg1 mRNA in adult zebrafish by RT-PCR. The Rhcg1 message was detected in the gill and kidney (Fig. 3A). Expression in the kidney is in marked contrast to that of pufferfish Rhcg1 whose expression is confined only to the gill (41); this difference may reflect the varied strategies of freshwater and seawater fish for maintaining body fluid homeostasis and will be discussed later. Expression and tissue distribution of other members, Rhag, Rhbg, Rhcg2, and Rhcg3, were also determined. As expected from our previous study of pufferfish Rh proteins (41), zebrafish Rhag, Rhbg, and Rhcg2 were specifically expressed in the gill; Rhcg3 expression was not observed in the tissues examined (Fig. 3A). Rhag mRNA was also detected in the heart and kidney, tissues that contain abundant hematopoietic cells. This result is consistent with our previous study on pufferfish Rhag, which is strongly expressed in red blood cells. Next we examined expression of Rhcg1 mRNA at different time points from 12 h postfertilization (hpf) to 6 dpf by RT-PCR. Total RNA of whole zebrafish embryos/larvae was extracted at each developmental stage. The expression of Rhcg1 mRNA became detectable at 3 dpf and was expressed in all subsequent stages we examined (Fig. 3B).


Figure 3
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  		Fig. 3. Expression of Rhcg1 mRNA in larval and adult zebrafish. A: expression of Rh family members in different tissues of adult zebrafish. RT-PCR was performed with total RNA isolated from the indicated tissues of adult zebrafish. beta-actin was used as an internal control. B: expression of Rhcg1 at different developmental stages of zebrafish. RT-PCR was performed with total RNA isolated from the indicated stages of whole embryos/larvae. beta-actin was used as an internal control.

 
Rhcg1 is expressed in MRC on the yolk sac surface and gill in zebrafish larvae. To elucidate localization of Rhcg1 mRNA at the cellular level, we carried out in situ hybridization using 1–6 dpf zebrafish larvae. Consistent with RT-PCR results (Fig. 3B), Rhcg1 signal became visible at 3 dpf on the surface of the yolk sac (Fig. 4A). At 4 dpf and thereafter, signal was also detectable in the gill (Fig. 4, B–D), and the numbers of Rhcg1-positive cells on the yolk sac surface and in the gill increased substantially. The distribution of Rhcg1-positive cells was highly reminiscent of the distribution of mitochondrion-rich cells (MRCs; or ionocytes). No signals were observed in other tissues, including the kidney, at the larval stages we analyzed.


Figure 4
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  		Fig. 4. Expression patterns of Rhcg1 mRNA on the surface of the yolk sac and gill of zebrafish larvae. Whole mount in situ hybridization with drRHcg probe at 3 dpf (A), 4 dpf (B), 5 dpf (C), and 6 dpf (D). High-magnification views of boxed areas (gill, E–H; yolk sac, I–L) in corresponding images of A–D. Arrows indicate typical signals for drRHcg1. Bars = 200 µm.

 
Coincidental increase in the number of Rhcg1-positive cells and ammonia excretion rate. In situ hybridization showed that the number of Rhcg1-expressing cells apparently increased during development (Fig. 4). To verify this result quantitatively, we counted the number of Rhcg1-positive cells on one side of yolk sac surface at different developmental stages. The number of Rhcg1-positive cells increased between 3 and 4 dpf and then plateaued (Fig. 5A). The ammonia excretion rate was also measured at different developmental stages and was found to increase in parallel with the number of Rhcg1-expressing cells (Fig. 5B).


Figure 5
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  		Fig. 5. Coincident increase in the number of Rhcg1-expressing cells and the ammonia excretion rate. A: number of Rhcg1-expressing cells at different developmental stages (n = 4; 10 larvae each). The Rhcg1-expressing cells identified by in situ hybridization were counted on one side of the yolk sac of individual larvae (n = 9–12). B: ammonia excretion rate of zebrafish larvae at different developmental stages. Ammonia content in the media was determined by the indophenol blue method.

 
Colocalization of Rhcg1 protein and ConA-binding sites on the surface of the larval yolk sac membranes. Lin et al. (29) and we (10) previously showed that there are two populations of MRCs on the surface of the yolk sac of zebrafish larvae: NaK-MRCs and vH-MRCs. To determine in which MRCs Rhcg1 is expressed, we next carried out immunohistochemistry of 4-dpf zebrafish larvae with a specific antiserum against zebrafish Rhcg1 and fluorescence-labeled ConA, a marker of the apical regions of vH-MRCs (10, 29). As expected, the immunolocalization of Rhcg1 on the yolk sac surface (Fig. 6A) was very similar to that of in situ hybridization histochemistry (Fig. 4). Interestingly, most of Rhcg1 signals were colocalized with ConA fluorescence, indicating that Rhcg1 is localized to the apical regions of vH-MRCs (Fig. 6A). No signals were observed with preimmune serum instead of anti-Rhcg1 antiserum (data not shown).


Figure 6
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  		Fig. 6. Localization of Rhcg1 protein in the apical region of mitochondrion-rich cells (MRCs). A: immunostaining with anti-drRhcg1 antiserum (green) and concanavalin A (ConA; magenta) at 4 days postfertilization (dpf) wild-type zebrafish larvae. The surface of the yolk sac membrane (top) and high-magnification views of boxed areas in corresponding images of top panels (bottom) are shown. The anti-drRhcg1 antiserum was raised in a rabbit by injection of recombinant protein that includes the COOH terminus of drRhcg1. Alexa Fluor 594-conjugated ConA was used as an apical surface marker of MRCs. Colocalization of Rhcg1 and ConA signals (white) are shown in the merged images. Bars = 100 µm. B: apical localization of Rhcg1 protein on green fluorescent protein (GFP)-expressing cells of HG9B transgenic zebrafish line. HG9B transgenic zebrafish (4 dpf) showed GFP expression (green) on the yolk sac surface (top). Bar = 200 µm. ConA signals (magenta) were localized to apical regions of GFP-expressing cells (middle). Immunostaining of Rhcg1 (magenta) and GFP (green) were performed with 4-dpf HG9B zebrafish (bottom). Bars = 20 µm.

 
Expression of GFP in vH-MRC in transgenic zebrafish HG9B. Although fluorescence-labeled ConA and H+-ATPase antibody are powerful tools for identification of vH-MRCs, there are some limitations in their usage. In our preliminary studies, ConA can bind to apical regions of MRCs only of living fish. Immunostaining with anti-H+-ATPase antibody did not show clear staining on gill sections of adult zebrafish (data not shown). To overcome these limitations, we performed an enhancer trap screen using the Tol2 transposable element (S. Nagayoshi and K. Kawakami, unpublished observation) and identified a zebrafish line HG9B that expressed GFP in a pattern very similar to the distribution of MRCs. We found that this transgenic zebrafish line shows definitive GFP expression in the ConA positive MRCs (Fig. 6B), which were previously shown to be vH-MRC (10, 29). This GFP expression on the yolk sac surface begins to appear before 24 hpf (data not shown). Immunohistochemistry of HG9B with the anti-Rhcg1 antiserum also confirmed apical localization of Rhcg1 on the GFP-expressing vH-MRC (Fig. 6B).

Expression of Rhcg1 in the gill and kidney of adult zebrafish. As the results of RT-PCR (Fig. 3) indicated relatively high expression of Rhcg1 in the gill and kidney of adult zebrafish, we performed immunohistochemistry of these tissues. To identify the cell type in which Rhcg1 is expressed, we used the HG9B transgenic zebrafish line. As expected, GFP-positive cells were distributed in the basal region of lamella in the gill of HG9B, and differed from the population of NaK-MRCs (Fig. 7, A and B). In this paper, the branchial cell population with the GFP expression in the HG9B line is referred to vH-MRC-like cells, because high expressions of V-ATPase were not experimentally confirmed in the cells. Immunohistochemical analysis using the anti-Rhcg1 antiserum also revealed apical localization of Rhcg1 protein on the vH-MRC-like cells (Fig. 7, C and D).


Figure 7
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  		Fig. 7. Rhcg1 is localized to the apical region of MRCs in the gill of adult zebrafish. The dotted lines in the pictures indicate the borders of tissues. A: localization of Na+-K+-ATPase and GFP in the gill of HG9B transgenic zebrafish. Immunohistochemistry was carried out by using frozen sections of the gill from adult HG9B fish. Na+-K+-ATPase was stained with antiserum (red) (40), and nuclei were stained with Hoechst 33342 (blue). B: high-magnification view of the Na+-K+-ATPase- and GFP-positive cells in same series of gill sections as A. C: localization of Rhcg1 and GFP in the gill of HG9B transgenic zebrafish. Rhcg1 was stained with a specific antiserum (red) and nuclei were stained with Hoechst 33342 (blue). D: high-magnification view of the Rhcg1- and GFP-positive cells in same series of gill sections as C. Bars = 50 µm.

 
We also performed immunohistochemistry of the kidney to localize Rhcg1 expression. To identify the type of renal tubules, Na+-K+-ATPase was used as a marker protein that localized in different patterns among the segment of tubules (23, 28). Anti-Na+-K+-ATPase antiserum was able to strongly stain the basolateral surface of the proximal tubules and entire cells in distal tubules. Costaining with anti-Rhcg1 antiserum and anti-Na+-K+-ATPase antiserum showed that Rhcg1 localized to the apical region of cells in a number of the distal tubules (Fig. 8, A and B). Furthermore, apical regions of the collecting ducts (tubules not stained with anti-Na+-K+ATPase antiserum) also express Rhcg1 (Fig. 8, A and C).


Figure 8
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  		Fig. 8. Apical localization of Rhcg1 in the renal distal tubule and collecting duct. Immunohistochemistry was carried out by using frozen sections of the kidney from adult wild-type zebrafish. Na+-K+-ATPase (red) and Rhcg1 (green) were stained with antisera, and nuclei were stained with Hoechst 33342 (blue). A: low-magnification view of the kidney. d, distal tubule; p, proximal tubule; cd, collecting duct. B: high-magnification view of an Rhcg1-positive and Rhcg1-negative distal tubule. C: high-magnification view of the collecting duct. Bars = 20 µm.

 
Expression of Rhcg1 mRNA is regulated by environmental ionic strength. To clarify the physiological roles of Rhcg1, we investigated the environmental factors that affect the expression of Rhcg1. We first compared, by RT-PCR, Rhcg1 expression in zebrafish that acclimated to normal FW and ammonium-containing FW (5, 50, and 500 µM NH4Cl). However, the expression level of Rhcg1 was not significantly different at either larval or adult stages, when animals were acclimated to the different media (data not shown). As several reports have indicated that metabolic acidosis enhances the ammonia excretion of fish, we next determined the Rhcg1 expression in the gill and kidney of adult fish that acclimated to acidic FW (pH 5.0, adjusted with HCl) for 3 days. However, this treatment did not change the expression level of Rhcg1 either (data not shown). Finally, we examined the effect of ambient ionic strength on Rhcg1 expression of 6 dpf larvae. To our surprise, a significant increase in the expression of Rhcg1 was observed in 1/20x FW-acclimated larvae. Furthermore, acclimation to 100x FW reduced the expression of Rhcg1 (Fig. 9).


Figure 9
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  		Fig. 9. Ionic strength-dependent expression of Rhcg1 mRNA in zebrafish larvae. Total RNA was isolated from 6 dpf larvae acclimated to 1/20x, 1x, and 100x freshwater (FW). Each acclimation was started at 1 dpf and extended to 6 dpf. GAPDH was used as an internal control. PCR reactions performed: 32 and 29 cycles (for Rhcg1) and 25 cycles (for GAPDH).

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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In this study, we identified five zebrafish Rh-related genes (Rhag, Rhbg, Rhcg1, Rhcg2, and Rhcg3), and have isolated the full-length cDNA of Rhcg1. We previously reported that pufferfish have only two Rhcg homologs (fRhcg1 and fRhcg2) that are expressed in the branchial pavement cells and MRCs, respectively (41). Phylogenetic analysis revealed that zebrafish and pufferfish Rhcg1 belong to a different branch from drRhcg2 and drRhcg3 (Fig. 2). RT-PCR using total RNA derived from multiple tissues of adult zebrafish revealed that as seen in pufferfish, zebrafish Rhcg2 is specifically expressed in the gill, further confirmation that drRhcg2 is a zebrafish ortholog of pufferfish fRhcg2. In contrast, no expression of drRhcg3 mRNA was observed in the tissues examined in this study (Fig. 3A).

Surprisingly, we detected strong expression of Rhcg1 mRNA and protein not only in the gills but also the kidney of adult zebrafish (Figs. 3A and 8). Our previous study showed no expression of Rhcg1 in the kidney of the pufferfish (41). This difference between zebrafish and pufferfish could be related to the nephron structures of these species. Most seawater fish including pufferfish (Takifugu rubripes) lack the distal tubular segment of the nephron (16, 23, 37, 45). Thus, the lack of Rhcg1 in pufferfish kidney might be due to the absence of the distal segment of the nephron. Additionally, zebrafish Rhcg1 was expressed in a number of distal tubules, which may correspond to the mammalian connecting tubule, and collecting ducts where studies in other organisms have shown Rhcg is expressed (9, 50, 56).

It is well known that the cortical collecting duct of the mammalian kidney consists of two major cell types: principal and intercalated cells (21, 31). The intercalated cells are further divided into at least two groups, {alpha}- and beta-intercalated cells, based on the localization of the proton pump and other features (4, 5). The {alpha}-intercalated cells express H+-ATPase on the apical plasma membrane and mediate net H+ secretion to maintain the acid-base balance. The mammalian Rhcg is distributed in the apical plasma membrane of the {alpha}-intercalated cells and is thought to mediate ammonia excretion (3, 9, 33, 50, 56, 67). In contrast to mammalian studies, no report has established the presence of intercalated cells in the kidney of freshwater teleost fish. However, in this study, we have elucidated the cellular localization of Rhcg1 in the kidney, which is likely identical or very similar to that of the mammalian ortholog (9), suggesting the presence of intercalated cell-like cells in the renal collecting duct of a freshwater teleost.

Although Rhcg1 is expressed in the adult zebrafish kidney, we found no expression of Rhcg1 mRNA in the kidney of 3–6 dpf larvae (Figs. 4 and 8). In teleost embryos/larvae, the first kidney is called the pronephros and is replaced by the mesonephros (mature kidney in teleost) at a later developmental stage (2). Our result therefore suggests that expression of Rhcg in the kidney may occur at a later stage of maturation, likely after replacement of the pronephros with mesonephros.

In this study, a coincident increase in ammonia excretion rates and the number of Rhcg1-expressing cells on the yolk sac surface was observed (Fig. 5). This result suggests that Rhcg1 may be involved in ammonia excretion at these developmental stages.

In our preliminary studies, high concentration of environmental ammonia (5–500 µM NH4Cl) did not influence Rhcg1 expression in larval or adult zebrafish (data not shown). These data suggest that expression of Rhcg1 mRNA is not regulated by the environmental ammonia concentration, at least in this species. Ammonia is also known to be important as urinary buffer in mammals (15, 22). Several reports indicated that ammonia secretion from the kidney of freshwater teleosts also has a significant role in acid-base balance (26, 39, 64). In particular, a study of rainbow trout showed that metabolic acidosis increases the urinary ammonia concentration and the renal activities of ammoniagenic enzymes (e.g., phosphate-dependent glutaminase, glutamate dehydrogenase, {alpha}-ketoglutarate dehydrogenase, alanine aminotransferase, and phosphoenolpyruvate carboxykinase) for net acid excretion (64). Although we examined whether Rhcg1 is involved in ammonia excretion in acidosis, 3 days exposure to acidic water (pH 5.0) did not influence expression level of Rhcg1 in the gill and kidney of adult zebrafish. We should note that the expression level of glutamate dehydrogenase, a key enzyme for ammonia production, is not changed under this condition either (data not shown). These results may suggest that pH 5.0 is not enough to induce the acidosis response for zebrafish. However, this acidic water is of the lowest pH that we could test because our wild-type zebrafish line does not survive longer than 24 h in water at pH 4.5 or lower. Thus, in future studies, it will be informative to do experiments using other acid-resistant fish, such as Osorezan dace (17), to elucidate the role of Rhcg1 in acidosis. Several studies have suggested that the renal acid-base regulatory capacity of seawater fish is low because they produce only a small amount of concentrated urine to prevent dehydration (8, 34, 38). This functional difference between the freshwater and seawater fish kidney might also be one of the reasons for the presence and absence of the Rhcg1 regulatory system.

Seawater and freshwater fish have different types of MRCs with opposite functions. The MRCs of seawater fish account for secretion of Na+ and Cl– into the environment, and are thought to consist of one single type of cell that are rich in mitochondria and Na+-K+-ATPase, reflecting their extraordinary power of active ion transport (19). In contrast, in the case of freshwater fish, the presence of at least two types of MRCs has been demonstrated, and they can be distinguished by their morphology, affinities for lectins, and gene expression patterns (10, 12, 13, 18, 29). In particular, previous studies indicated that the MRCs of the zebrafish larvae can be classified into two groups based on their characteristic expression of major ATPases: 1) NaK-MRC and 2) vH-MRC. The vH-MRC is also distinguished by the affinity of its apical regions for ConA (10, 29). In this study, we demonstrated localization of Rhcg1 on the apical regions of vH-MRCs of zebrafish larvae (Fig. 6). Colocalization of the Rhcg protein and H+-ATPase was also observed in the intercalated cells of mammalian renal distal tubule (9). Although the role of H+-ATPase for ammonia excretion is still unknown, H+-ATPase might enhance the ammonia excretion rate by establishing a pH gradient across the cell membrane. Indeed, using the Xenopus oocyte expression system, Mak et al. (33) showed that the mammalian Rhcg-mediated methylammonium uptake into oocytes was pH sensitive, being enhanced by an alkaline extracellular pH and inhibited by an acidic extracellular pH. The colocalization of zebrafish Rhcg1 and H+-ATPase in the same manner as in mammals may suggest functional coupling of both molecules for transepithelial ammonia secretion.

In this study, we performed a transposon-mediated enhancer trap screen and identified a transgenic zebrafish line, HG9B, that specifically expresses GFP in the vH-MRCs on the yolk sac and gill. As GFP expression became noticeable at a very early stage of development (before 24 hpf), we think that the GFP expression is regulated by an enhancer that regulates a gene important for differentiation of vH-MRCs. At present, we are searching candidate genes around the integration site of the transposon construct in the HG9B line.

We observed that low ionic strength (1/20x FW) enhances the expression of Rhcg1, and high ionic strength (100x FW) reduces expression (Fig. 9). Using a fluorescent indicator, Na-Green, and radioactive tracer 22Na, Esaki et al. (10) showed that, in zebrafish larvae, Na+ uptake into vH-MRC occurs as early as 2 dpf when Rhcg1 is not yet expressed as far as we examined. These results indicate that zebrafish Rhcg1 is not likely involved in Na+ uptake at early larval stages. However, the increase in the mRNA expression of Rhcg1 under the 1/20x FW (and the reduction in 100x FW) suggests that Rhcg1 may be involved in adaptation mechanisms for the environment of low ionic strength. It is possible that ammonia gradient across the plasma membrane made by Rhcg1 indirectly influences the osmoregulatory systems (e.g., ion absorption from environment) at later developmental stages. Several studies have shown indirect evidence for a correlation between Na+ uptake and NH4+ excretion in the teleost fish gill (25, 32, 47, 62), and Rhcg1 may contribute to this. However, at present, it is very difficult to clarify the roles of Rhcg1 in maintaining ion homeostasis because the nature of ammonia transport by Rh glycoproteins is unclear and still controversial (3, 6, 30, 33, 52, 58, 67). Further studies are necessary for clarifying the roles of Rh glycoproteins in uptake of ions and osmoregulation of freshwater fish. On the other hand, it is possible that changes in the expression of Rhcg1 mRNA in the different ionic strength media was secondary phenomenon reflecting changes in water chemistry of the gill boundary layers or to altered expressions of other ion transport proteins.

In this study, changes in Rhcg1 expression were examined at the mRNA level by varying several environmental factors (ammonia concentrations, pH, and ionic strength). Unfortunately, Western blotting of Rhcg1 using the raised antibody did not give clear signals because of impediment by nonspecific smear bands that may derive from yolk proteins. Currently, therefore, we cannot rule out the possibilities that accumulation and/or targeting to the plasma membrane of the Rhcg1 protein were affected without changes in the mRNA levels under the conditions employed here. Conversely, increase in Rhcg1 mRNA expression in the low ionic strength does not necessarily indicate upregulations of transport activity of the Rhcg1 protein. Further studies are necessary to clarify the detailed regulatory mechanisms of Rhcg1 expression, localization, and activity under the extreme conditions.

Perspective

Fish have evolved several types of ammonia transport systems. In this study, we have shown that the expression pattern and localization of zebrafish Rhcg1 is different from pufferfish. In particular, localization in the renal tubules suggested that Rhcg1 might be involved in ammonia transport systems in the kidney besides the nitrogen elimination from the gill. This difference in localization of Rhcg1 between seawater and freshwater fish suggests that teleost fish use the Rh glycoproteins in various ways to adapt to different water environments. Clarification of roles of Rh glycoproteins would help to better understand the diversity in nitrogen metabolism and transport systems.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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REFERENCES
 
This work was supported by Grant-in-Aid for Scientific Research 14104002 (to S. Hirose), 18059010 (to S. Hirose), 17570003 (to K. Hoshijima), 18-5667 (to T. Nakada), 17018034 (to K. Kawakami) and 18101008 (to K. Kawakami) from the Ministry of Education, Culture, Sport, Science and Technology of Japan (MEXT), JSPS Research Fellowships for Young Scientists, the 21st Century Centers of Excellence Program of MEXT, National Institute of General Medical Sciences grant R01-GM-069382, the National BioResource Project, and National Institute of Genetics Cooperative Research Program Grant 2005-B3.


   ACKNOWLEDGMENTS
 
We thank Akira Kato, Sayako Kobayashi, Yukihiro Kurita, and Taku Fukuzawa for discussion and technical assistance, Setsuko Sato for secretarial assistance, and Dr. Kazuki Horikawa for initial characterization of the enhancer trap lines. We also thank Dr. Lisa M. Goering for copyediting the manuscript.


   FOOTNOTES
 

Address for reprint requests and other correspondence: Shigehisa Hirose, Dept. of Biological Sciences, Tokyo Institute of Technology, 4259-B19 Nagatsuta-cho, Midori-ku, Yokohama-shi, Kanagawa-ken, 226-8501 Japan (e-mail: shirose{at}bio.titech.ac.jp)

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 Ammonia exists in aqueous solutions in two molecular forms, NH3 and NH4+, which are in equilibrium with each other. In this paper, the term "ammonia" refers to the combination of both forms. The term "ammonium" specifically refers to the molecular species NH4+. When referring to NH3, we specifically state "NH3". Back


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