Effects of cortisol (20 mg/l) in 3-dpf zebrafish larvae. The effects of cortisol on mRNA expression (A), sodium content (B), ncc signal, (C), and the density of ncc-expressing cells (D) are shown. The expression of mRNA was analyzed by qPCR, and the values were normalized to those for β-actin. Values are the mean ± SD (n = 6). Student′s t-test, *P < 0.05; **P < 0.01, ***P < 0.001. Scale bar: 100 µm.

Effects of gr and mr knockdown in 3-dpf zebrafish larvae. The effects of injection with gr or mr MO on ncc mRNA expression (A, E), sodium content (B), ncc mRNA signals (C), and the density of ncc-expressing cells (D) are shown. Rescue experiments were performed by co-injecting embryos with gr MO and gr cRNA,and examining ncc expression (E). The expression of mRNA was analyzed using qPCR with β-actin as the internal control. Different letters indicate significant differences (p < 0.05), as determined using one-way ANOVA followed by Tukey′s multiple-comparison test. Value are the mean ± SD (n = 6). Scale bar: 100 µm.

Effect of ncc and/or gcm2 MO on sodium content in zebrafish larvae; and effects of gr and mr knockdown and cortisol treatment on ncc mRNA expression in zebrafish larvae injected with gcm2 MO. The sodium content (A) and expression of ncc mRNA (B), ncc signals (C), and the density of ncc-expressing cells (D) in the indicated larvae are shown. The expression of mRNA was analyzed by qPCR using β-actin as the internal control. Different letters indicate significant differences (p < 0.05), as determined using one-way ANOVA followed by Tukey′s multiple-comparison test. Values are the mean ± SD. (n = 5–6). Scale bar: 100 µm.

Co-localization of gr mRNA with NCC cells in zebrafish larvae. (A) In situ hybridization againstgr mRNA. (B) Immunocytochemical staining of NCC. Arrows indicate co-localization of gr mRNA and NCC protein signals in the same cells. Scale bar: 20 µm.