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

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.