Visualization in zebrafish larvae of Na+ uptake in mitochondrion-rich cells whose differentiation is dependent on foxi3alpha

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 mitochondrion-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 foxi3alpha transcription factor that is specifically expressed in the H(+)-ATPase-rich MR cells. When foxi3alpha 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, foxi3alpha expression is initiated at gastrula stage in the presumptive ectoderm; thus we propose that foxi3alpha 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. Key words: chloride cell, mitochondria-rich cell, Sodium Green, Na+/H+ exchanger (NHE), vacuolar-type H+-ATPase.