Figure 1.

Low [Ca²⁺] treatment increases NaR cell number and density on the larval yolk sac by reactivating a mitotic program in pre-existing NaR cells. (a and b) Low [Ca²⁺] treatment increases NaR cell density and number on the larval yolk sac skin. Zebrafish larvae (72 hpf) were transferred to artificial freshwater containing low [Ca²⁺], low [Na⁺], or low [Cl⁻], raised to 120 hpf, and analyzed by in situ hybridization for the indicated genes. Representative views are shown in (a). Shown here and in all following figures are lateral views of the yolk sac region. Anterior to the left and dorsal up. Scale bar=50 μm. The total number of ionocytes on one side of 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) Low [Ca²⁺] treatment increases cell proliferation. Larvae (72 hpf) were transferred to artificial freshwater with the indicated [Ca²⁺]. BrdU (10 mM) was added at 96 hpf. The larvae were raised to 120 hpf and stained using a BrdU antibody. Tr, trunk; Yo, yolk sac. (d) The mitotic cells under low [Ca²⁺] are NaR cells. 72 hpf larvae were transferred to artificial freshwater with the indicated [Ca²⁺] and raised to 120 hpf. BrdU (10 mM) was added 30 min before sampling. The larvae were analyzed by trpv5/6 mRNA in situ hybridization (green) and BrdU staining (red)