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.

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.

Mitochondrion-rich cells (MRCs), or ionocytes, play a central role in aquatic species, maintaining body fluid ionic homeostasis by actively taking up or excreting ions. Since their first description in 1932 in eel gills, extensive morphological and physiological analyses have yielded important insights into ionocyte structure and function, but understanding the developmental pathway specifying these cells remains an ongoing challenge. We previously succeeded in identifying a key transcription factor, Foxi3a, in zebrafish larvae by database mining. In the present study, we analyzed a zebrafish mutant, quadro (quo), deficient in foxi1 gene expression and found that foxi1 is essential for development of an MRC subpopulation rich in vacuolar-type H+-ATPase (vH-MRC). foxi1 acts upstream of Delta-Notch signaling that determines sporadic distribution of vH-MRC and regulates foxi3a expression. Through gain- and loss-of-function assays and cell transplantation experiments, we further clarified that (1) the expression level of foxi3a is maintained by a positive feedback loop between foxi3a and its downstream gene gcm2 and (2) Foxi3a functions cell-autonomously in the specification of vH-MRC. These observations provide a better understanding of the differentiation and distribution of the vH-MRC subtype.

Freshwater (FW) fishes actively absorb salt from their environment to tolerate low salinities. We previously reported that vacuolar-type H+-ATPase/mitochondrion-rich cells (H-MRCs) on the skin epithelium of zebrafish larvae (Danio rerio) are primary sites for Na+ uptake. In this study, in an attempt to clarify the mechanism for the Na+ uptake, we performed a systematic analysis of gene expression patterns of zebrafish carbonic anhydrase (CA) isoforms and found that, of 12 CA isoforms, CA2a and CA15a are highly expressed in H-MRCs at larval stages. The ca2a and ca15a mRNA expression were salinity-dependent; they were upregulated in 0.03 mM Na+ water whereas ca15a but not ca2a was down-regulated in 70 mM Na+ water. Immunohistochemistry demonstrated cytoplasmic distribution of CA2a and apical membrane localization of CA15a. Furthermore, cell surface immunofluorescence staining revealed external surface localization of CA15a. Depletion of either CA2a or CA15a expression by Morpholino antisense oligonucleotides resulted in a significant decrease in Na+ accumulation in H-MRCs. An in situ proximity ligation assay demonstrated a very close association of CA2a, CA15a, Na+/H+ exchanger 3b (Nhe3b), and Rhcg1 ammonia transporter in H-MRC. Our findings suggest that CA2a, CA15a, and Rhcg1 play a key role in Na+uptake under FW conditions by forming a transport metabolon with Nhe3b.

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