The effects of exposure to acidic water on Na⁺ uptake in GR morphants. GR knockdown did not have any effect on Na⁺ uptake by zebrafish larvae maintained under normal pH (7) conditions (Fig. 4A, N = 6). However, while not eliminated entirely, the stimulation of Na⁺ uptake following 24-h exposure to acidic water was significantly attenuated in the GR morphants (Fig. 4A; N = 6). Data are presented as means ± SEM; different letters denote significant difference among treatment groups. The effectiveness of GR knockdown was confirmed by demonstrating via immunocytochemistry a marked reduction in the number of GR-positive cells on the yolk sac in the GR-MO’s (Fig. 4C) in comparison to the control MO’s (4C). Scale bars = 200 μm (4B and 4C).

Localization of GR on yolk sack ionocytes in larval zebrafish. 4 dpf zebrafish larvae were stained with conA (green, a vital dye for HR cells) and GR (red, denoted with asterisks). The majority of GR-positive cells were also stained with conA, suggesting that HR-cells are enriched with GR (A and B). On the other hand, no obvious expression of GR (red, denoted with asterisks) was observed in NaR cells (green; stained with Na⁺/K⁺-ATPase antibody, alpha 5, denoted with arrow; (C and D). This antibody successfully recognized an appropriately sized band (<90 kDa) on western blotting (E). Scale bars: 200 μm for Fig. 6A, 6C and 50 μm for (B and D). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

The lack of an effect of cortisol on Na⁺ uptake via NCC cells. The microinjection of morpholino against a transcription factor glial cell missing 2 led to a significant reduction in conA positive HR cells (11A, B). mRNA expression of GR was significantly lower in gcm-2 morphants than in sham injected larvae (N = 4–6; 11C). The Na⁺ uptake was significantly elevated in gcm-2 morphants, but no further stimulation was observed following the cortisol treatment (11D; N = 5–6). Data are presented as means ± SEM and an asterisk/different letter denotes significant difference among treatment groups. Scale bars = 200 μm.