Fig. 2

Zebrafish igfbp5a mRNA is specifically expressed in NaR cells and its levels are correlated with NaR cell number. (a and b) Low [Ca²⁺] treatment results in similar increases in the number of igfbp5a mRNA-expressing cells and trpv5/6 mRNA-expressing cells. Zebrafish larvae (72 hpf) were transferred to artificial freshwater with the indicated [Ca²⁺], raised to 120 hpf, and analyzed by in situ hybridization for the indicated genes. Representative views are shown in (a). Inserts are higher magnification views. The number of igfbp5a mRNA- and trpv5/6 mRNA-labeled cells on the yolk sac was manually counted and shown in (b). Each dot represents an individual fish. Mean±S.D., n=6. Groups labeled with different letters are significantly different from each other (P<0.05). (c) Double-label in situ hybridization analysis of igfbp5a mRNA and trpv5/6 mRNA in zebrafish larvae. Upper panel shows a representative result. Lower panel is a representative result of igfbp5a mRNA (green) and atp6v1al mRNA (red). (d) Relationship between NaR cell number and [Ca²⁺]. Zebrafish embryos were raised in artificial freshwater with the indicated varying [Ca²⁺]. NaR cells were analyzed by in situ hybridization of igfbp5a mRNA, and their number counted manually. Data shown are mean±S.D., n=6–8. (e) Relationship between igfbp5a mRNA levels and [Ca²⁺]. Total RNA was isolated from 120 hpf larvae raised as in (d). The igfbp5a mRNA levels were measured by qRT-PCR and normalized by the β-actin levels. Mean±S.D., n=3. (f) Correlation analysis of NaR cell number and igfbp5a mRNA levels