Localization of ammonia transporter Rhcg 1 in mitochondrion-rich cells of yolk sac, gill, and kidney of zebrafish and its ionic strength-dependent expression

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 Rhcgl, 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 RhcgI 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 Rhcgl. At larval stages, RhcgI mRNA was detected only on the yolk sac and gill; however, the kidney, as well as the gill, becomes a major site of RhcgI expression in adults. Using a zebratish Tol2 transgenic line whose vH-MRC are labeled with green fluorescent protein (GFP) and an antibody against zebrafish Rhcgl, we demonstrate that RhcgI 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 RhcgI 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 Rhcgl mRNA at the larval stage was changed by environmental ionic strength. These results suggest that roles of zebrafish Rhcgl are not solely ammonia secretion to eliminate nitrogen from the gill.