HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Supplemental Table and Figure All Versions of this Article: 292/1/R470    most recent 00200.2006v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Web of Science (16) Citing Articles via Google Scholar Google Scholar Articles by Esaki, M. Articles by Hirose, S. Search for Related Content PubMed PubMed Citation Articles by Esaki, M. Articles by Hirose, S.

DEVELOPMENTAL PHYSIOLOGY AND PREGNANCY

Visualization in zebrafish larvae of Na⁺ uptake in mitochondria-rich cells whose differentiation is dependent on foxi3a

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

Submitted 19 March 2006 ; accepted in final form 25 August 2006

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
Uptake of Na⁺ from the environment is an indispensable strategy for the survival of freshwater fish, as they easily lose Na⁺ from the plasma to a diluted environment. Nevertheless, the location of and molecules involved in Na⁺ uptake remain poorly understood. In this study, we utilized Sodium Green, a Na⁺-dependent fluorescent reagent, to provide direct evidence that Na⁺ absorption takes place in a subset of the mitochondria-rich (MR) cells on the yolk sac surface of zebrafish larvae. Combined with immunohistochemistry, we revealed that the Na⁺-absorbing MR cells were exceptionally rich in vacuolar-type H⁺-ATPase (H⁺-ATPase) but moderately rich in Na⁺-K⁺-ATPase. We also addressed the function of foxi3a, a transcription factor that is specifically expressed in the H⁺-ATPase-rich MR cells. When foxi3a was depleted from zebrafish embryos by antisense morpholino oligonucleotide injection, differentiation of the MR cells was completely blocked and Na⁺ influx was severely reduced, indicating that MR cells are the primary sites for Na⁺ absorption. Additionally, foxi3a expression is initiated at the gastrula stage in the presumptive ectoderm; thus, we propose that foxi3a is a key gene in the control of MR cell differentiation. We also utilized a set of ion transport inhibitors to assess the molecules involved in the process and discuss the observations.

chloride cell; Sodium Green; Na⁺/H⁺ exchanger; vacuolar-type H⁺-ATPase

Since the first suggestion by Krogh (28) that Na⁺ uptake is likely coupled to the excretion of H⁺ to maintain an electroneutral state, two different molecular mechanisms have been proposed. In the first model, the Na⁺/ H⁺ exchanger (NHE) is considered a key molecule for the incorporation of ambient Na⁺ by electroneutrally exchanging Na⁺ with H⁺ inside the cells (61). However, in the gill epithelium, this mechanism has been questioned on thermodynamic grounds, as the gill epithelium is surrounded by an extremely dilute environment (2, 25, 26, 32, 44). An alternative model postulates Na⁺ uptake through the epithelial Na⁺ channel (ENaC) that is electrogenically coupled to vacuolar-type H⁺-ATPase (H⁺-ATPase) (2, 31). In this model, energy consumptive excretion of H⁺ by H⁺-ATPase creates an electrical gradient that provides the driving force to draw in Na⁺ through ENaC from the diluted environment. This model has been supported by several pharmacological studies with selective inhibitors of ENaC and H⁺-ATPase (9, 11, 31, 32, 45, 46). Currently, identification of an ENaC homologue from fish has not been successful. However, immunolocalization of ENaC with a heterologous antibody has implied its existence in fish (17, 52, 56). Studies of the immunolocalization of H⁺-ATPase have revealed a subcellular localization to the apical membranes and have also shown that it is expressed in MR cells of mudskipper (Periophthalmodon schlosseri) (57), coho salmon (Oncorhynchus kisutch) (58), and zebrafish (Danio rerio) (33); in pavement cells of tilapia (O. mossambicus) (17, 56); and in both cell types of rainbow trout (O. mykiss) (30, 56). Although MR cells have been assumed to be the primary sites of Na⁺ absorption, these observations suggest that pavement cells may also have the potential to absorb Na⁺, at least in some species.

Notably, Kaneko and colleagues (22) have demonstrated that the H⁺-ATPase is localized to the basolateral, but not apical membrane of MR cells in killifish (Fundulus heteroclitus), suggesting that molecules other than H⁺-ATPase may be primarily involved in the Na⁺ uptake process in this species. On the other hand, isoforms of NHE have been shown to be localized to the apical side of MR cells in mudskipper (57) and Osorezan dace (Tribolodon hakonensis) (16). Moreover, Choe et al. (6) found that in an elasmobranch, the Atlantic stingray (Dasyatis sabina), NHE3 is localized to the apical membrane of the MR cells and expression is enhanced by acclimation to freshwater. In addition, selective inhibitors of NHE block Na⁺ uptake in goldfish (Carassius auratus) (45). These observations suggest that NHE, rather than ENaC coupled to H⁺-ATPase, may be critically involved in Na⁺ uptake in some species or in some circumstances, although the thermodynamic problem has yet to be resolved.

Zebrafish is a stenohaline freshwater fish. It has been extensively utilized as a model vertebrate for studies of embryogenesis and organogenesis, but is also increasingly recognized as a useful model for the investigation of physiological traits (3, 33, 35, 41, 47). During zebrafish development, the MR cells first appear on the body surface, especially on the yolk sac, prior to formation of the gills. The larval MR cells seem to be functionally equivalent to those on the gills at later stages (17, 18, 21, 33, 34). Furthermore, their localization to the spherical yolk sac, a much simpler structure than the gills, provides better opportunities to analyze morphology, gene expression, and functions of the MR cells in vivo.

In this study, we addressed the location of, and molecules involved in, Na⁺ absorption in zebrafish larvae. First, using Sodium Green, a Na⁺-dependent fluorescent reagent, we succeeded in visualizing the Na⁺-absorbing cells and obtained direct evidence that Na⁺ absorption takes place in a subset of the MR cells on the body surface, especially those on the yolk sac. Notably, this cell population was not so rich in Na⁺-K⁺-ATPase, but contained abundant H⁺-ATPase. Second, we examined the function of foxi3a, a transcription factor that is sporadically expressed on the yolk sac surface postgastrulation (51). We found that it was specifically expressed in the H⁺-ATPase-rich MR cells. Furthermore, when foxi3a was depleted from embryos by injection of an antisense morpholino oligonucleotide, differentiation of MR cells was inhibited and Na⁺ absorption was severely reduced, indicating that MR cells are the primary sites for Na⁺ absorption. Finally, we examined molecules involved in the process by using of a set of ion transport inhibitors.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
Zebrafish culture. Zebrafish were maintained as described previously (55). The wild-type zebrafish TL line was generously provided by Dr. A. Kawakami (Yokohama, Japan). Fertilized eggs were incubated in 1x freshwater [1x FW: 60 mg ocean salt (Rohtomarine) per 1 liter of distilled water] at 28.5°C unless otherwise mentioned. Here 1x FW was used as our standard freshwater and corresponded to "Fish Water" as described in "The Zebrafish Book" (55). The concentration of major ions, pH, and osmolarity in 1x FW are listed in Supplemental Table S1 (the online version of this article contains the supplemental table). 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).

Na⁺ accumulation analyses. Na⁺-dependent fluorescent reagent, Sodium Green tetra-acetate cell permeant, was purchased from Invitrogen and a 10-mM stock solution was prepared in DMSO. Sodium Green has been well characterized as a reagent for measuring intracellular Na⁺ concentration, especially in cell culture (1a, 29, 49, 59). On binding Na⁺, it exhibits an increase in fluorescence intensity in the range of 0 to 135 mM Na⁺ (13). At 55 h and 7 days postfertilization (hpf or dpf), living larvae were incubated in 10 µM Sodium Green diluted in 1x FW for 1 h. Subsequently, larvae were washed briefly with 1x FW, anesthetized in 0.2 mg/ml tricaine (3-amino benzoic acid ethylester, Nacalai Tesque), 1 mM Tris·HCl, pH 7.0, and embedded in 2% methylcellulose in 1x FW for photography with an Axiovert 200 inverted fluorescent microscope (Zeiss). Sodium Green was excited by a mercury vapor light source through an excitation filter with maximum transmission at 485 nm and a 20-nm bandwidth. The fluorescence was collected using a 515- to 565-nm band-pass emission filter. We have confirmed that the tricaine treatment did not noticeably affect illumination of Sodium Green. Single focused images were created from a series of partially focused images using software from HeliconFocus (Helicon, Ukraine). Mitochondrial-staining reagent, MitoTracker (Invitrogen), was used to illuminate the MR cells (12, 14, 33, 38). For double staining with Sodium Green and MitoTracker, 55-hpf larvae were first incubated for 30 min in 10 µM Sodium Green and then in 10 µM Sodium Green and 500 nM MitoTracker Orange CMTMRos for an additional 30 min. In double stains of Sodium Green with concanavalin A (ConA; a lectin protein capable of selectively binding -mannopyranosyl and -glucopyranosyl residues), larvae were first incubated in 10 µM Sodium Green for 30 min and subsequently in 10 µM Sodium Green and 50 µg/ml of Alexa Fluor 594-conjugated ConA (Invitrogen) for 30 min.

To examine the effects of Na⁺ and K⁺ ionophores on Sodium Green illumination, we used monensin (WAKO) as the Na⁺ ionophore and valinomycin (Invitrogen) as the K⁺ ionophore. Larvae (55 hpf) were incubated for 40 min in 10 µM Sodium Green diluted in 1x FW in the presence of 1 µM monensin or 1 µM valinomycin. The larvae were subsequently anesthetized and embedded in 1.5% methylcellulose in the presence of the ionophore.

To analyze Na⁺ accumulation in zebrafish larvae reared in a range of Na⁺ concentrations, we prepared 100x FW (6 g ocean salt per liter of distilled water, pH 6.1–6.8), 1x FW (as described above), 1/20x FW (3 mg ocean salt per liter, pH 7.2) and distilled water. Larvae were reared in the different mediums from fertilization. At 55 hpf, larvae were incubated for 1 h in 10 µM Sodium Green diluted in each medium. They were washed with each medium and anesthetized in tricaine to be photographed. We note that the zebrafish larvae were able to grow in distilled water at least until 4 dpf without any malformation (K. Hoshijima, unpublished observations).

Blockage of Na⁺ accumulation with ion transport inhibitors. EIPA (Sigma) is used as a selective inhibitor of the NHE (3, 27, 45). Amiloride (Sigma) targets both the ENaC and the NHE, but ENaC is more efficiently inhibited than NHE at low concentrations (3, 27, 60). Bafilomycin A1 (Wako) is an inhibitor of H⁺-ATPase (4) and has been shown to effectively inhibit H⁺ efflux from the MR cells on the yolk sac of zebrafish larvae at 10 µM (33). Metolazone (WAKO), an analog of thiazide, is used as an inhibitor of Na⁺-K⁺-2Cl^– cotransporter (NKCC) (54). Ouabain (WAKO) is an inhibitor of Na⁺-K⁺-ATPase. Ethoxzolamide (Sigma) is an inhibitor of carbonic anhydrase (CA) (3). It is noteworthy that the inhibitory effects of these reagents have been tested in zebrafish or other fish (3, 11, 33, 45, 46, 54, 60, 62). The test concentrations used in this study were determined according to the references.

To examine blockage of Na⁺ absorption by the ion transport inhibitors, larvae were reared in 20-fold diluted FW, which reduces Na⁺ in the MR cells. At 55 hpf, larvae were preincubated for 1.5 h in 1x FW in the presence of the inhibitors, followed by incubation in 1x FW for 1 h with the inhibitors and 10 µM Sodium Green. The Sodium Green reaction was terminated by washing with 1x FW containing the inhibitors. The inhibitors were also added during anesthesia.

Immunohistochemistry. At 55 hpf, larvae were fixed with 4% paraformaldehyde in PBS (in mM: 137 NaCl, 27 KCl, 10 Na₂HPO₄, 2 KH₂PO₄, pH 7.4) at 4°C overnight and dehydrated in 100% methanol. For double staining with 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) for 1 h, the larvae were incubated at 25°C overnight with an antibody against the B subunit of dace (Tribolodon hakonensis) H⁺-ATPase (diluted 1:500 with PBT containing 10% sheep serum) (16) or with an antibody against the -subunit of eel (Anguilla japonica) Na⁺-K⁺-ATPase (diluted 1:1,000) (39). Following a wash with PBT, the larvae were further incubated for 2 h at 25°C with donkey anti-rabbit IgG conjugated with Alexa Fluor 488 (diluted 1:1,000, Invitrogen) and then washed with PBT and embedded in 100% glycerol to photograph. We note that the peptide sequence of the B subunit of dace H⁺-ATPase (GenPept accession no. BAC75967; amino acid residues 1–160) used for antibody preparation exhibits 99% identity to that of zebrafish (NP_878299 [GenBank] ; residues 201–360) and that the peptide sequence of the -subunit of eel Na⁺-K⁺-ATPase (2122241A; residues 469–773) used for antibody preparation exhibits 92% identity to that of zebrafish (NP_571761 [GenBank] ; residues 473–779).

Whole mount in situ hybridization. Whole mount in situ hybridization was performed as previously described with brief modifications (53), using a digoxigenin-labeled RNA probe for foxi3a corresponding to a 543-bp fragment of cDNA (51) or a probe for CA isoform 2 (ca2) corresponding to a 583-bp fragment of the cDNA (GenBank accession no. NM_199215; nt 57–639). We note that the ca2 is different from the paralog cahz (GenBank accession no. NM_131110) that is expressed in blood. To combine with immunohistochemistry, larvae hybridized with digoxigenin-labeled RNA probe were incubated with anti-digoxigenin Fab fragment conjugated with alkaline phosphatase (diluted 1:4,000; Roche) and anti-dace H⁺-ATPase antibody (diluted 1:500) simultaneously. After incubation with donkey anti-rabbit IgG conjugated with Alexa Fluor 488, larvae were photographed as described above. Larvae were then washed with PBT and incubated in BCIP/NBT color development substrate (Promega) to visualize the in situ hybridization. In double stains with Sodium Green, living larvae were first stained with Sodium Green and photographed as described above. Subsequently, they were fixed with 4% paraformaldehyde in PBS at 4°C overnight and dehydrated in 100% methanol and were then processed according to the in situ hybridization protocol.

Measurement of Na⁺ influx with ²²Na⁺. Ten 55-hpf larvae were transferred into a 1.5-ml microcentrifuge tube. After being rinsed with 1 ml of 1x FW, the larvae were incubated in 200 µl of 1x FW containing 7.4 kBq ²²Na⁺ for 1 h. Subsequently, the larvae were rinsed three times with 1 ml of high-salt buffer containing 112 mM of nonradioactive Na⁺, pH 7.7, and then placed into 500 µl of 1x FW and transferred into test tubes to analyze incorporated ²²Na⁺ with a gamma counter (model ARC-300; Aloka). To examine the effects of ion transport inhibitors on Na⁺ influx, 10 larvae in a microcentrifuge tube were preincubated for 1.5 h in 1x FW in the presence of the inhibitors and then incubated for 1 h in 1x FW with the inhibitors and 7.4 kBq ²²Na⁺. We note that because Na⁺ influx activity varied among larvae laid by different parents, we prepared experimental and control larvae from siblings in any series of experiments and calculated the activity relative to the control; comparisons across experiments were then made using the relative activities rather than absolute values of the incorporated Na⁺ amounts.

Morpholino injection. Morpholinos are chemically modified oligonucleotides that are more stable in, and less toxic to, living cells. Antisense morpholino oligonucleotides specific for a target gene are designed to complementarily anneal to the mRNA around the translation start site and knockdown gene function by repressing translation (40). Morpholino injection was performed as previously described (20). Two antisense morpholino oligonucleotides against foxi3a were synthesized (Gene Tools): FOXI3A-MO#1 (5'-ATGCTTTCTTCCCGTTTCTCTTTGT-3') and FOXI3A-MO#2 (5'-GAGAAAATGCCTCCTGCTCTGCTGC-3'). Approximately 1 nl of 0.5 mg/ml FOXI3A-MO#1 or 2 mg/ml FOXI3A-MO#2 in 1x Danieau buffer [in mM: 58 NaCl, 0.7 KCl, 0.4 MgSO₄, 0.6 Ca(NO₃)₂, 5 HEPES at pH 7.6] was injected into the yolk of one-cell embryos. The injected embryos were reared in 1x FW and analyzed as described above.

Analysis of Sodium Green intensity with imageJ. Fluorescent intensities with Sodium Green were analyzed with imageJ software version 1.36 (http://rsb.info.nih.gov/ij/index.html). Prior to the analysis, single-focused images with Sodium Green were converted to grayscale but not modified at all. Within imageJ, a Sodium Green-positive MR cell was encircled completely with the peripheral area and the maximum (max) and the minimum (min) intensity values in the circle were measured. We considered the minimum value to be background in the circle and defined the MR cell intensity as (max – min). We analyzed 40 positive cells in the middle of each image or all cells if there were < 40 detectable Sodium Green-positive cells. We note that for any series of experiments, we prepared individual images under identical conditions, i.e., identical excitation light intensity, exposure time, and magnification.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
Na⁺ accumulation occurs in a subset of the MR cells on the zebrafish larvae. To examine Na⁺ metabolism in zebrafish larvae, we stained the body with Sodium Green, a Na⁺-dependent fluorescent reagent. We observed bright fluorescence in round-to-oval shaped cells that were sporadically distributed on the yolk sac surface (Fig. 1A). The fluorescence in these cells was severely reduced in the presence of 1 µM monensin, a Na⁺ ionophore (Fig. 2, A and B, and Table 1), but was much less affected in the presence of valinomycin, a K⁺ ionophore (Fig. 2, A and C, and Table 1). These results indicate that the fluorescence in these cells is sensitive to intracellular Na⁺. Sporadic staining also occurred on the developing gills at a later stage (Fig. 1B). The distribution of these cells resembled that of the larval MR cells that first differentiate on the body surface, especially the yolk sac and then on the gills coincident with their development. To examine whether these Na⁺ accumulating cells correspond to the MR cells, we used MitoTracker, a mitochondrial-staining reagent that has previously been shown to illuminate the MR cells (12, 14, 33, 38). In a double stain of Sodium Green with MitoTracker, we found that Na⁺ accumulation occurred only in a subset of the MR cells [Sodium Green⁺/MR cell = 37.6%, calculated from a total of 218 cells in four larvae (Fig. 1, C–E)].

View larger version (62K): [in this window] [in a new window]   Fig. 1. Na+ accumulation in a subset of the mitochondria-rich (MR) cells on the yolk sac of zebrafish larvae. A and B: Sodium Green, a Na+-dependent fluorescent reagent, predominantly stains round-to-oval shaped cells distributed sporadically on the yolk sac surface of 55-hpf larva (A, lateral view) and on the gills of 7-dpf larva (B, ventral view). Scale bars in A, 0.5 mm; in B, 200 µm. C–E: high magnification view of the yolk sac surface of a 55-hpf larva costained with Sodium Green (C); and MitoTracker (D); merge of C and D (E). Sodium Green-positive cells (arrowheads in C) overlap with a subset of the MR cells (arrowheads in D). Scale bar in C, 50 µm.

View larger version (33K): [in this window] [in a new window]   Fig. 2. Fluorescence of Sodium Green in MR cells is severely reduced in the presence of a Na+ ionophore but not a K+ ionophore. A–C: lateral views of the yolk sac of 55-hpf larva stained with Sodium Green in the absence of any ionophore (A), or in the presence of 1 µM monensin (B) or 1 µM valinomycin (C). Scale bars, 200 µm.

View larger version (87K): [in this window] [in a new window]   Fig. 3. Na+ accumulation occurs in MR cells that are rich in H+-ATPase. A–C: high magnification view of the yolk sac surface of 55-hpf larva costained with Sodium Green (A); and Alexa Fluor 594-conjugated ConA (B); merge of A and B (C). The cells positive for Sodium Green (arrowheads in A) are also positive for ConA signals (arrowheads in B). Scale bar in A, 50 µm. D–I: high magnification views of the yolk sac (D–F) and the region posterior to the yolk sac (yolk extension: G–I) of 55-hpf larva stained with Alexa Fluor 594-conjugated ConA (D and G) followed by immunohistochemistry with anti-H+-ATPase antibody (E) or with anti-Na+-K+-ATPase antibody (H). F: merge of D and E. I: merge of G and H. ConA signals (arrowheads in D and G) overlap with the MR cells that are rich in H+-ATPase (arrowheads in E) but only moderately express Na+-K+-ATPase (arrowheads in H). Scale bars in D, 20 µm and in G, 50 µm.

View larger version (69K): [in this window] [in a new window]   Fig. 4. Differentiation of H+-ATPase-rich MR cells is dependent on foxi3a. A and B: whole mount in situ hybridization with foxi3a probe at 55 hpf (A, lateral view) and 9 hpf (B, lateral view). The animal pole and the dorsal side are up and right, respectively in B. Arrowheads in B indicate the yolk plug at the vegetal pole. C–E: high magnification views of the yolk extension costained by in situ hybridization with foxi3a probe (C) and by immunohistochemistry with anti-H+-ATPase antibody (D). E: merge of C and D. Purple in C and green in D show foxi3a signals entirely overlap with H+-ATPase-positive cells. F–K: 2-ng FOXI3A-MO#2-injected larvae (F, H, and J) and noninjected control larvae (G, I, and K) were analyzed at 55 hpf by immunohistochemistry with anti-H+-ATPase antibody (F and G), or by staining with Alexa Fluor 594-conjugated ConA (H and I), and Sodium Green (J and K). Cells were counted on one side of the larvae only. We note that all MO-injected larvae (n = 5) showed <5 positive cells on one side by immunohistochemistry with anti-H+-ATPase antibody or by ConA staining. On the other hand, all control larvae (n = 5) showed >70 positive cells by both methods. Scale bars, 200 µm.

View larger version (8K): [in this window] [in a new window]   Fig. 5. Na+ influx is blocked in foxi3a-depleted larvae. Na+ influx was measured with 22Na+ in 0.5-ng FOXI3A-MO#1-injected (MO-injected) larvae and noninjected control larvae at 55 hpf. Influx activity in the MO-injected larvae is represented as the means ± SE relative to control (n = 4). Significant difference from noninjected control larvae was evaluated by a Mann-Whitney U-test (*P < 0.05).

View larger version (134K): [in this window] [in a new window]   Fig. 6. Na+ accumulation is dependent on Na+ concentration in the environment. A–D: 55-hpf larvae were stained with Sodium Green after rearing in 100x FW (freshwater; A), 1x FW (B), 1/20x FW (C) and distilled water (D). The intensities of Sodium Green signals are similar between in 100x FW and in 1x FW (see also Table 1), but substantially reduced in 1/20x FW. The signals are barely detectable in distilled water. Scale bars, 200 µm.

View larger version (123K): [in this window] [in a new window]   Fig. 7. Blockage of Na+ accumulation with ion transport inhibitors. A–G: 55-hpf larvae were stained with Sodium Green in the absence of any inhibitors (A), or in the presence of 10 µM EIPA (B), 10 µM amiloride (C), 100 µM amiloride (D), 10 µM bafilomycin A1 (E), 100 µM metolazone (F), 500 µM ouabain (G) and 10 µM ethoxzolamide (H). Lateral views of the yolk sac. Scale bars, 200 µm.

View larger version (44K): [in this window] [in a new window]   Fig. 8. Expression of carbonic anhydrase (CA) in Na+-accumulating MR cells. A and B: magnified lateral views of the yolk sac of 55-hpf larva stained with Sodium Green (A) followed by in situ hybridization with CA isoform 2 (ca2) probe (B). Sodium Green signals (white arrowheads in A) entirely overlap with ca2-expressing cells (black arrowheads in B). Scale bar, 50 µm.

View larger version (12K): [in this window] [in a new window]   Fig. 9. Effects of ion transport inhibitors on Na+ influx in zebrafish larvae. Na+ influx was measured with 22Na+ in 55-hpf larvae in the presence of ion transport inhibitors at the indicated concentrations. The influx activities (black bars) were represented as the means ± SE (n 3 for all points) relative to those of control larvae in the absence of any inhibitors (white bar). Significant difference from influx in control larvae was evaluated by a Mann-Whitney U-test (*P < 0.05). BAF, bafilomycin A1; MET, metolazone.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

The correlation between Na⁺ accumulation in the MR cells and MR cell-dependent Na⁺ influx also indicates that accumulation reflects a Na⁺ absorption process through these cells. This hypothesis is further supported by our observations. First, Na⁺ accumulation was also observed in the gills, which are primary tissues for ion uptake in freshwater fish (Fig. 1B). Second, Na⁺ accumulation was dependent on ambient Na⁺ concentration (Fig. 6). Third, the H⁺-ATPase-rich MR cells, where Na⁺ accumulation occurred, have previously been shown to excrete H⁺ (33), consistent with models for Na⁺ uptake coupled to H⁺ excretion. Finally, we also confirmed that Na⁺ accumulation was not the result of apoptotic cells and that Sodium Green did not induce apoptosis (Supplemental Fig. 1; the online version of this article contains supplemental Fig. 1). Taken together, we conclude that Na⁺ absorption occurs in the H⁺-ATPase-rich MR cells on the zebrafish larvae.

The MR cells in which we observed Na⁺ accumulation were not only rich in H⁺-ATPase but also expressed Na⁺-K⁺-ATPase (Fig. 3). This observation is in contrast to the findings from Hwang and colleagues (33) in which Na⁺-K⁺-ATPase was not detectable in the H⁺-ATPase-rich MR cells. We assume that this difference reflects different staining conditions, including antibodies and detection systems. On the other hand, we observed a similar localization of H⁺-ATPase (33). Expression was very abundant near the apical surface, i.e., in a "subapical" region (as revealed by costaining with ConA), but also weakly distributed throughout the cells. As H⁺-ATPase is a transmembrane protein complex, the cytoplasmic distribution likely reflects the localization to tubular structures invaginated from the basolateral membrane.

Depletion of foxi3a from the zebrafish embryo caused a blockage of MR cell differentiation and severe reduction of Na⁺ influx (Figs. 4 and 5). These results indicate not only that the MR cells are the primary sites for Na⁺ influx, but also that foxi3a plays a critical role in MR cell differentiation. The foxi3a was first reported to be expressed in mucous cells (51), but here we demonstrate that it is specifically expressed in H⁺-ATPase-rich MR cells. Interestingly, foxi3a expression is initiated on the ventral side of the animal hemisphere at the late gastrula stage (Fig. 4B) (51), a region where the epidermis differentiates (24). Even at first appearance, the expression was already sporadic, suggesting that it is expressed in the MR cell precursors. Thus, we propose that foxi3a functions as a master control gene of MR cell differentiation.

Blockage by bafilomycin A1 of Na⁺ accumulation in the MR cells and Na⁺ influx supports the hypothesis that H⁺-ATPase is critically involved in Na⁺ absorption by creating an electrical gradient that provides the driving force (Figs. 7 and 9). EIPA, a selective inhibitor of NHE, also showed blockage of Na⁺ accumulation in the MR cells, suggesting that the protein is also involved in the Na⁺ absorption process (Fig. 7). However, the effect of the inhibitor on Na⁺ influx was not as strong as the effect on Na⁺ accumulation (Fig. 9). Likewise, ouabain, an inhibitor of Na⁺-K⁺-ATPase, had little effect on Na⁺ accumulation in the MR cells but obviously reduced Na⁺ influx (Figs. 7 and 9). We assume that the discrepancy between the results is likely because radioisotope influx and Sodium Green monitor different aspects of Na⁺ absorption. The former reflects total incorporated Na⁺ in larvae. The latter, however, should represent Na⁺ accumulated transiently in the MR cells during transport from the environment to the plasma and should reflect a limited portion of total Na⁺ incorporated in the body. We also anticipate that inhibition of Na⁺ uptake from the environment at the apical surface of MR cells gives different results from inhibition of Na⁺ export to the plasma at the basolateral membrane. For example, if ouabain blocked Na⁺ export to the plasma from the MR cells but not Na⁺ uptake at the apical surface, Sodium Green would reveal the Na⁺ absorbed into the MR cells even though the excess Na⁺ would soon leak out; however, in this case, total Na⁺ incorporated in the plasma would be significantly reduced. We should note that Grosell and colleagues (3) first examined the effects of ion transport inhibitors on Na⁺ (and Cl^–) uptake in zebrafish and found that the effectiveness was different in fish acclimated to different ambient salinities. In this study, we have used younger larvae that were acclimated to different ambient salinities than those used in the Grosell report. With some of the inhibitors, our results are different than those reported by Grosell and colleagues (3), suggesting that zebrafish may use different Na⁺ (and Cl^–) uptake mechanisms depending on the environmental salinity, as well as the developmental stage.

Further understanding of the mechanism of Na⁺ uptake through the MR cells will require identification of the genes expressed in these cells and subsequent functional analyses. In the present study, we have shown that ca2 as well as H⁺-ATPase and Na⁺-K⁺-ATPase are expressed in the MR cells. Currently, we have not identified MR cell-specific nhe genes. Although nhe2, nhe3a/b, and nhe5 are expressed in 55-hpf larvae, the expression level is too low to identify the cell-specific localization by whole mount in situ hybridization (K. Hoshijima, unpublished observations). The heterologous NHE antibodies we used did not recognize the zebrafish homologs. To identify MR cell-specific nhe genes, we would need to improve the sensitivity of in situ hybridization and/or develop NHE antibodies specific to the zebrafish proteins. In addition, preparation of transgenic zebrafish in which GFP is expressed under the control of nhe gene promoters could be a useful approach. To analyze gene function, gene-specific depletion in the zebrafish larvae would be powerful. As described here for foxi3a, depletion of candidate genes by antisense morpholino oligonucleotides and subsequent analyses with Sodium Green in combination with measurement of Na⁺ influx with the radioisotope ²²Na⁺ could clarify their involvement in the Na⁺ uptake process.

Perspectives

Identification by this study of a Na⁺-absorbing site in H⁺-ATPase-rich MR cells simultaneously opens a new question as to the function of the other population of MR cells, i.e., the Na⁺-K⁺-ATPase-rich MR cells. Because this population of MR cells possess typical features of MR cells (see INTRODUCTION), they likely absorb ambient ions, possibly major ions other than Na⁺, such as Ca²⁺ and Cl^–. Indeed, Hwang and colleagues (41) have recently demonstrated the presence of the epithelial Ca²⁺ channel (ECaC), which is considered to be involved in Ca²⁺ uptake, in a subpopulation of the Na⁺-K⁺-ATPase-rich MR cells. The rest of the population may be involved in Cl^– uptake (5). These possibilities may also be explored as described here, i.e., by immunohistochemical localization of key molecules and by their depletion with morpholinos, followed by analysis with fluorescent dye sensitive to target ions, and with radioisotopes. We also predict that foxi3b (51), a paralog of foxi3a, might play an important role in MR cell differentiation in combination with foxi3a.

	GRANTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
This work was supported by Ministry of Education, Culture, Sport, Science and Technology of Japan (MEXT) Grants-in-Aid for Scientific Research 14104002, 17570003, and 18059010 and the 21st Century Center of Excellence Program of MEXT and National Institute of Genetics Cooperative Research Program Grant 2005-B3.

	ACKNOWLEDGMENTS

 
We thank Drs. Atsushi Kawakami and Makoto Kobayashi for providing zebrafish and advising on management of the zebrafish facility and Dr. Lisa Goering for copyediting the manuscript.

	FOOTNOTES

 

Address for reprint requests and other correspondence: Shigehisa Hirose, Dept. of Biological Sciences, Tokyo Institute of Technology, 4259-B-19 Nagatsuta-cho, Midori-ku, Yokohama 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.

^* M. Esaki and K. Hoshijima contributed equally to this work.

	REFERENCES

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 

1. American Physiological Society. Guiding principles for research involving animals and human beings. Am J Physiol Regul Integr Comp Physiol 283: R281–R283, 2002.[Free Full Text]
1. Amorino GP, Fox MH. Intracellular Na⁺ measurements using sodium green tetraacetate with flow cytometry. Cytometry 21: 248–256, 1995.[CrossRef][Web of Science][Medline]
2. Avella M, Bornancin M. A new anaylsis of ammonia and sodium transport through the gills of the freshwater rainbow trout (Salmo gairdneri). J Exp Biol 142: 155–176, 1989.[Abstract/Free Full Text]
3. Boisen AM, Amstrup J, Novak I, Grosell M. Sodium and chloride transport in soft water and hard water acclimated zebrafish (Danio rerio). Biochim Biophys Acta 1618: 207–218, 2003.[Medline]
4. Bowman EJ, Siebers A, Altendorf K. Bafilomycins: a class of inhibitors of membrane ATPases from microorganisms, animal cells, and plant cells. Proc Natl Acad Sci USA 85: 7972–7976, 1988.[Abstract/Free Full Text]
5. Chang IC, Hwang PP. Cl^– uptake mechanism in freshwater-adapted tilapia (Oreochromis mossambicus). Physiol Biochem Zool 77: 406–414, 2004.[CrossRef][Web of Science][Medline]
6. Choe KP, Kato A, Hirose S, Plata C, Sindic A, Romero MF, Claiborne JB, Evans DH. NHE3 in an ancestral vertebrate: primary sequence, distribution, localization, and function in gills. Am J Physiol Regul Integr Comp Physiol 289: R1520–R1534, 2005.[Abstract/Free Full Text]
7. Choe KP, Verlander JW, Wingo CS, Evans DH. A putative H⁺-K⁺-ATPase in the Atlantic stingray, Dasyatis sabina: primary sequence and expression in gills. Am J Physiol Regul Integr Comp Physiol 287: R981–R991, 2004.[Abstract/Free Full Text]
8. Claiborne JB, Edwards SL, Morrison-Shetlar AI. Acid-base regulation in fishes: cellular and molecular mechanisms. J Exp Zool 293: 302–319, 2002.[CrossRef][Web of Science][Medline]
9. Clarke AP, Potts WTW. Sodium, net acid and ammonia fluxes in freshwater-adapted European flounder (Platichthys flesus L.). Pharmacological inhibition and effects on gill ventilation volume. J Zool 246: 427–432, 1998.[CrossRef]
10. Evans DH, Piermarini PM, Choe KP. The multifunctional fish gill: dominant site of gas exchange, osmoregulation, acid-base regulation, and excretion of nitrogenous waste. Physiol Rev 85: 97–177, 2005.[Abstract/Free Full Text]
11. Fenwick JC, Wendelaar Bonga SE, Flik G. In vivo bafilomycin-sensitive Na⁺ uptake in young freshwater fish. J Exp Biol 202: 3659–3666, 1999.[Abstract]
12. Galvez F, Reid SD, Hawkings G, Goss GG. Isolation and characterization of mitochondria-rich cell types from the gill of freshwater rainbow trout. Am J Physiol Regul Integr Comp Physiol 282: R658–R668, 2002.[Abstract/Free Full Text]
13. Haugland RP. The Handbook: A Guide to Fluorescent Probes and Labeling Technologies. Eugene, OR: Molecular Probes, 2005.
14. Hawkings GS, Galvez F, Goss GG. Seawater acclimation causes independent alterations in Na⁺/K⁺- and H⁺-ATPase activity in isolated mitochondria-rich cell subtypes of the rainbow trout gill. J Exp Biol 207: 905–912, 2004.[Abstract/Free Full Text]
15. Heijden A, Verbost P, Eygensteyn J, Li J, Bonga S, Flik G. Mitochondria-rich cells in gills of tilapia (Oreochromis mossambicus) adapted to fresh water or sea water: quantification by confocal laser scanning microscopy. J Exp Biol 200: 55–64, 1997.[Abstract]
16. Hirata T, Kaneko T, Ono T, Nakazato T, Furukawa N, Hasegawa S, Wakabayashi S, Shigekawa M, Chang MH, Romero MF, Hirose S. Mechanism of acid adaptation of a fish living in a pH 3.5 lake. Am J Physiol Regul Integr Comp Physiol 284: R1199–R1212, 2003.[Abstract/Free Full Text]
17. Hiroi J, Kaneko T, Seikai T, Tanaka M. Developmental sequence of chloride cells in the body skin and gills of Japanese flounder (Paralichthys olivaceus) larvae. Zoolog Sci 15: 455–460, 1998.
18. Hiroi J, McCormick SD, Ohtani-Kaneko R, Kaneko T. Functional classification of mitochondrion-rich cells in euryhaline Mozambique tilapia (Oreochromis mossambicus) embryos, by means of triple immunofluorescence staining for Na⁺/K⁺-ATPase, Na⁺/K⁺/2Cl^– cotransporter and CFTR anion channel. J Exp Biol 208: 2023–2036, 2005.[Abstract/Free Full Text]
19. Hirose S, Kaneko T, Naito N, Takei Y. Molecular biology of major components of chloride cells. Comp Biochem Physiol B Biochem Mol Biol 136: 593–620, 2003.[CrossRef][Medline]
20. Hoshijima K, Metherall JE, Grunwald DJ. A protein disulfide isomerase expressed in the embryonic midline is required for left/right asymmetries. Genes Dev 16: 2518–2529, 2002.[Abstract/Free Full Text]
21. Kaneko T, Shiraishi K, Katoh F, Hasegawa S, Hiroi J. Chloride cells during early life stages of fish and their functional differentiation. Fisheries Sci 68: 1–9, 2002.
22. Katoh F, Hyodo S, Kaneko T. Vacuolar-type proton pump in the basolateral plasma membrane energizes ion uptake in branchial mitochondria-rich cells of killifish Fundulus heteroclitus, adapted to a low ion environment. J Exp Biol 206: 793–803, 2003.[Abstract/Free Full Text]
23. Kerstetter TH, Kirschner LB, Rafuse DD. On the mechanisms of sodium ion transport by the irrigated gills of rainbow trout (Salmo gairdneri). J Gen Physiol 56: 342–359, 1970.[Abstract/Free Full Text]
24. Kimmel CB, Ballard WW, Kimmel SR, Ullmann B, Schilling TF. Stages of embryonic development of the zebrafish. Dev Dyn 203: 253–310, 1995.[Web of Science][Medline]
25. Kirschner LB. The mechanism of sodium chloride uptake in hyperregulating aquatic animals. J Exp Biol 207: 1439–1452, 2004.[Abstract/Free Full Text]
26. Kirschner LB. Sodium chloride absorption across the body surface: frog skins and other epithelia. Am J Physiol Regul Integr Comp Physiol 244: R429–R443, 1983.[Abstract/Free Full Text]
27. Kleyman TR, Cragoe EJ Jr. Amiloride and its analogs as tools in the study of ion transport. J Membr Biol 105: 1–21, 1988.[CrossRef][Web of Science][Medline]
28. Krogh A. Osmotic regulation in freshwater fishes by active absorption of chloride ions. Z Vergl Physiol 24: 656–666, 1937.[CrossRef]
29. Li F, Sugishita K, Su Z, Ueda I, Barry WH. Activation of connexin-43 hemichannels can elevate [Ca²⁺]_i and [Na⁺]_i in rabbit ventricular myocytes during metabolic inhibition. J Mol Cell Cardiol 33: 2145–2155, 2001.[CrossRef][Web of Science][Medline]
30. Lin H, Pfeiffer D, Vogl A, Pan J, Randall D. Immunolocalization of H⁺-ATPase in the gill epitthelia of rainbow trout. J Exp Biol 195: 169–183, 1994.[Abstract]
31. Lin H, Randall D. Evidence for the presence of an electrogenic proton pump on the trout gill epithelium. J Exp Biol 161: 119–134, 1991.[Abstract/Free Full Text]
32. Lin H, Randall DJ. H⁺-ATPase activity in crude homogenates of fish gill tissue-inhibitor sensitivity and environmental and hormonalregulation. J Exp Biol 180: 163–174, 1993.[Abstract]
33. Lin LY, Horng JL, Kunkel JG, Hwang PP. Proton pump-rich cell secretes acid in skin of zebrafish larvae. Am J Physiol Cell Physiol 290: C371–C378, 2006.[Abstract/Free Full Text]
34. Lin LY, Hwang PP. Mitochondria-rich cell activity in the yolk-sac membrane of tilapia (Oreochromis mossambicus) larvae acclimatized to different ambient chloride levels. J Exp Biol 207: 1335–1344, 2004.[Abstract/Free Full Text]
35. Liu NA, Liu Q, Wawrowsky K, Yang Z, Lin S, Melmed S. Prolactin receptor signaling mediates the osmotic response of embryonic zebrafish lactotrophs. Mol Endocrinol 20: 871–880, 2006.[Abstract/Free Full Text]
36. Maren TH. Carbonic anhydrase: chemistry, physiology, inhibition. Physiol Rev 47: 595–781, 1967.[Free Full Text]
37. Marshall WS. Na⁺, Cl^–, Ca²⁺ and Zn²⁺ transport by fish gills: retrospective review and prospective synthesis. J Exp Zool 293: 264–283, 2002.[CrossRef][Web of Science][Medline]
38. Marshall WS. Rapid regulation of NaCl secretion by estuarine teleost fish: coping strategies for short-duration freshwater exposures. Biochim Biophys Acta 1618: 95–105, 2003.[Medline]
39. Mistry AC, Honda S, Hirata T, Kato A, Hirose S. Eel urea transporter is localized to chloride cells and is salinity dependent. Am J Physiol Regul Integr Comp Physiol 281: R1594–R1604, 2001.[Abstract/Free Full Text]
40. Nasevicius A, Ekker SC. Effective targeted gene ’knockdown’ in zebrafish. Nat Genet 26: 216–220, 2000.[CrossRef][Web of Science][Medline]
41. Pan TC, Liao BK, Huang CJ, Lin LY, Hwang PP. Epithelial Ca²⁺ channel expression and Ca²⁺ uptake in developing zebrafish. Am J Physiol Regul Integr Comp Physiol 289: R1202–R1211, 2005.[Abstract/Free Full Text]
42. Payan P, Maetz J. Branchial Sodium Transport Mechanisms in Scyliorhinus canicula: evidence for Na⁺/NH₄⁺ and Na⁺/H⁺ exchanges and for a role of carbonic anhydrase. J Exp Biol 58: 509–522, 1973.[Abstract/Free Full Text]
43. Perry SF, Shahsavarani A, Georgalis T, Bayaa M, Furimsky M, Thomas SL. Channels, pumps, and exchangers in the gill and kidney of freshwater fishes: their role in ionic and acid-base regulation. J Exp Zoolog A Comp Exp Biol 300: 53–62, 2003.[Medline]
44. Potts WTW. Kinetics of sodium uptake in freshwater animals: a comparison of ion-exchange and proton pump hypotheses. Am J Physiol Regul Integr Comp Physiol 266: R315–R320, 1994.[Abstract/Free Full Text]
45. Preest MR, Gonzalez RJ, Wilson RW. A pharmacological examination of Na⁺ and Cl^– transport in two species of freshwater fish. Physiol Biochem Zool 78: 259–272, 2005.[CrossRef][Web of Science][Medline]
46. Reid SD, Hawkings GS, Galvez F, Goss GG. Localization and characterization of phenamil-sensitive Na⁺ influx in isolated rainbow trout gill epithelial cells. J Exp Biol 206: 551–559, 2003.[Abstract/Free Full Text]
47. Rombough P. Gills are needed for ionoregulation before they are needed for O₂ uptake in developing zebrafish, Danio rerio. J Exp Biol 205: 1787–1794, 2002.[Abstract/Free Full Text]
48. Sakamoto T, Uchida K, Yokota S. Regulation of the ion-transporting mitochondrion-rich cell during adaptation of teleost fishes to different salinities. Zoolog Sci 18: 1163–1174, 2001.[CrossRef][Web of Science][Medline]
49. Sasaki S, Siragy HM, Gildea JJ, Felder RA, Carey RM. Production and role of extracellular guanosine cyclic 3',5' monophosphate in sodium uptake in human proximal tubule cells. Hypertension 43: 286–291, 2004.[Abstract/Free Full Text]
50. Shepard JL, Amatruda JF, Stern HM, Subramanian A, Finkelstein D, Ziai J, Finley KR, Pfaff KL, Hersey C, Zhou Y, Barut B, Freedman M, Lee C, Spitsbergen J, Neuberg D, Weber G, Golub TR, Glickman JN, Kutok JL, Aster JC, Zon LI. A zebrafish bmyb mutation causes genome instability and increased cancer susceptibility. Proc Natl Acad Sci USA 102: 13194–13199, 2005.[Abstract/Free Full Text]
51. Solomon KS, Logsdon JM Jr, Fritz A. Expression and phylogenetic analyses of three zebrafish FoxI class genes. Dev Dyn 228: 301–307, 2003.[CrossRef][Web of Science][Medline]
52. Sullivan G, Fryer J, Perry S. Immunolocalization of proton pumps (H⁺-ATPase) in pavement cells of rainbow trout gill. J Exp Biol 198: 2619–2629, 1995.
53. Thisse C, Thisse B. High resolution whole-mount in situ hybridization. Zebrafish Sci Monitor 5: 8–9, 1998.
54. Vazquez N, Monroy A, Dorantes E, Munoz-Clares RA, Gamba G. Functional differences between flounder and rat thiazide-sensitive Na-Cl cotransporter. Am J Physiol Renal Physiol 282: F599–F607, 2002.[Abstract/Free Full Text]
55. Westerfield M. The Zebrafish Book: A Guide to the Laboratory Use of Zebrafish (Danio rerio). Eugene, OR: University of Oregon Press, 1995.
56. Wilson JM, Laurent P, Tufts BL, Benos DJ, Donowitz M, Vogl AW, Randall DJ. NaCl uptake by the branchial epithelium in freshwater teleost fish: an immunological approach to ion-transport protein localization. J Exp Biol 203: 2279–2296, 2000.[Abstract]
57. Wilson JM, Randall DJ, Donowitz M, Vogl AW, Ip AK. Immunolocalization of ion-transport proteins to branchial epithelium mitochondria-rich cells in the mudskipper (Periophthalmodon schlosseri). J Exp Biol 203: 2297–2310, 2000.[Abstract]
58. Wilson JM, Whiteley NM, Randall DJ. Ionoregulatory changes in the gill epithelia of coho salmon during seawater acclimation. Physiol Biochem Zool 75: 237–249, 2002.[CrossRef][Web of Science][Medline]
59. Winslow JL, Cooper RL, Atwood HL. Intracellular ionic concentration by calibration from fluorescence indicator emission spectra, its relationship to the K_d, F_min, F_max formula, and use with Na-Green for presynaptic sodium. J Neurosci Methods 118: 163–175, 2002.[CrossRef][Web of Science][Medline]
60. Wood CM, Matsuo AY, Gonzalez RJ, Wilson RW, Patrick ML, Val AL. Mechanisms of ion transport in Potamotrygon, a stenohaline freshwater elasmobranch native to the ion-poor blackwaters of the Rio Negro. J Exp Biol 205: 3039–3054, 2002.[Abstract/Free Full Text]
61. Wright PA, Wood C. An analysis of branchial ammonia excretion in the freshwater rainbow trout: effects of environmental pH change and sodium uptake blockage. J Exp Biol 114: 329–353, 1985.[Abstract/Free Full Text]
62. Yuan S, Joseph EM. The small heart mutation reveals novel roles of Na⁺/K⁺-ATPase in maintaining ventricular cardiomyocyte morphology and viability in zebrafish. Circ Res 95: 595–603, 2004.[Abstract/Free Full Text]

This article has been cited by other articles:

				Y.-C. Tseng, R.-D. Chen, J.-R. Lee, S.-T. Liu, S.-J. Lee, and P.-P. Hwang Specific expression and regulation of glucose transporters in zebrafish ionocytes Am J Physiol Regulatory Integrative Comp Physiol, August 1, 2009; 297(2): R275 - R290. [Abstract] [Full Text] [PDF]

				P.-P. Hwang Ion uptake and acid secretion in zebrafish (Danio rerio) J. Exp. Biol., June 1, 2009; 212(11): 1745 - 1752. [Abstract] [Full Text] [PDF]

				B.-K. Liao, R.-D. Chen, and P.-P. Hwang Expression regulation of Na+-K+-ATPase {alpha}1-subunit subtypes in zebrafish gill ionocytes Am J Physiol Regulatory Integrative Comp Physiol, June 1, 2009; 296(6): R1897 - R1906. [Abstract] [Full Text] [PDF]

				Y.-F. Wang, Y.-C. Tseng, J.-J. Yan, J. Hiroi, and P.-P. Hwang Role of SLC12A10.2, a Na-Cl cotransporter-like protein, in a Cl uptake mechanism in zebrafish (Danio rerio) Am J Physiol Regulatory Integrative Comp Physiol, May 1, 2009; 296(5): R1650 - R1660. [Abstract] [Full Text] [PDF]

				A. S. Flynt, E. J. Thatcher, K. Burkewitz, N. Li, Y. Liu, and J. G. Patton miR-8 microRNAs regulate the response to osmotic stress in zebrafish embryos J. Cell Biol., April 6, 2009; 185(1): 115 - 127. [Abstract] [Full Text] [PDF]

				M. Inokuchi, J. Hiroi, S. Watanabe, P.-P. Hwang, and T. Kaneko Morphological and functional classification of ion-absorbing mitochondria-rich cells in the gills of Mozambique tilapia J. Exp. Biol., April 1, 2009; 212(7): 1003 - 1010. [Abstract] [Full Text] [PDF]

				W.-J. Chang, J.-L. Horng, J.-J. Yan, C.-D. Hsiao, and P.-P. Hwang The transcription factor, glial cell missing 2, is involved in differentiation and functional regulation of H+-ATPase-rich cells in zebrafish (Danio rerio) Am J Physiol Regulatory Integrative Comp Physiol, April 1, 2009; 296(4): R1192 - R1201. [Abstract] [Full Text] [PDF]

				J. Hiroi, S. Yasumasu, S. D. McCormick, P.-P. Hwang, and T. Kaneko Evidence for an apical Na-Cl cotransporter involved in ion uptake in a teleost fish J. Exp. Biol., August 15, 2008; 211(16): 2584 - 2599. [Abstract] [Full Text] [PDF]

				T.-Y. Lin, B.-K. Liao, J.-L. Horng, J.-J. Yan, C.-D. Hsiao, and P.-P. Hwang Carbonic anhydrase 2-like a and 15a are involved in acid-base regulation and Na+ uptake in zebrafish H+-ATPase-rich cells Am J Physiol Cell Physiol, May 1, 2008; 294(5): C1250 - C1260. [Abstract] [Full Text] [PDF]

				J.-J. Yan, M.-Y. Chou, T. Kaneko, and P.-P. Hwang Gene expression of Na+/H+ exchanger in zebrafish H+-ATPase-rich cells during acclimation to low-Na+ and acidic environments Am J Physiol Cell Physiol, December 1, 2007; 293(6): C1814 - C1823. [Abstract] [Full Text] [PDF]

				T. Nakada, K. Hoshijima, M. Esaki, S. Nagayoshi, K. Kawakami, and S. Hirose Localization of ammonia transporter Rhcg1 in mitochondrion-rich cells of yolk sac, gill, and kidney of zebrafish and its ionic strength-dependent expression Am J Physiol Regulatory Integrative Comp Physiol, October 1, 2007; 293(4): R1743 - R1753. [Abstract] [Full Text] [PDF]

				J.-L. Horng, L.-Y. Lin, C.-J. Huang, F. Katoh, T. Kaneko, and P.-P. Hwang Knockdown of V-ATPase subunit A (atp6v1a) impairs acid secretion and ion balance in zebrafish (Danio rerio) Am J Physiol Regulatory Integrative Comp Physiol, May 1, 2007; 292(5): R2068 - R2076. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Supplemental Table and Figure All Versions of this Article: 292/1/R470    most recent 00200.2006v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Web of Science (16) Citing Articles via Google Scholar Google Scholar Articles by Esaki, M. Articles by Hirose, S. Search for Related Content PubMed PubMed Citation Articles by Esaki, M. Articles by Hirose, S.