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.

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.