Fig. 5

Loss of Trpv5/6 results in IGF-PI3K-Akt activation and increased NaR cell proliferation under normal [Ca²⁺]. (a and b) Elevated NaR cell number and density in the mus (loss-of-function trpv5/6) mutant larvae under normal [Ca²⁺]. Homozygous mus embryos and their siblings were raised in 0.2 mM [Ca²⁺] water. DMSO or 0.3 μM BMS-754807 was added at 24 hpf. At 120 hpf, fish were fixed and NaR cells were labeled by in situ hybridization for igfbp5a mRNA. Representative views are shown in (a) and quantitative results from three independent experiments in (b). (c and d) Elevated Akt signaling in the mus mutant larvae. The experimental groups were the same as described in (a). pAkt-positive cells were labeled and scored according to Supplementary Figure S1d. Representative views are shown in (c) and quantitative results are shown in (d). (e) Elevated NaR cell proliferation in the mus mutant fish. Homozygous mus embryos and their siblings were raised in 0.2 mM [Ca²⁺] water. At 120 hpf, they were fixed and analyzed by in situ hybridization for igfbp5a mRNA and BrdU staining after a 30 min pulse of BrdU (10 mM) prior to the sampling. (f and g) Knockdown of Trpv5/6 results in increased pAkt-positive cells (f) and igfbp5a mRNA-expressing NaR cells (g). Embryos injected with control or Trpv5/6 targeting MOs were raised in 0.2 mM [Ca²⁺] water. pAkt-positive cells (f) and NaR cells (g) were labeled and scored at 72 and 120 hpf as described above