Fig. 4

Local Igfbp5a facilitates [Ca²⁺]-induced IGF signaling activation and IGF signaling is required for low [Ca²⁺]-induced NaR cell proliferation. (a and b) Igfbp5a knockdown inhibits low [Ca²⁺]-induced Akt signaling. Embryos injected with control MO or Igfbp5a targeting MOs were transferred to water with the indicated [Ca²⁺] at 72 hpf and fixed 2 h later for pAkt staining. Representative views are shown in (a) and quantitative results in (b). Scale bar=50 μm. The total number of fish analyzed from three independent experiments is shown on the top of each column. (c) Zebrafish igfbp5a encodes a secreted protein that binds IGFs. Conditioned media were prepared from HEK293 cells transfected with expression plasmids encoding the indicated proteins. They were analyzed by ligand blot with DIG-labeled IGF-1 or IGF-2, and by western blot using the indicated antibody. LBD, ligand binding domain mutant. (d and e) Inhibition of IGF1R abolishes low [Ca²⁺]-induced increase in NaR cells. Larvae (72 hpf) were transferred to 0.2 mM or 0.001 mM [Ca²⁺] water containing DMSO, BMS-754807 (BMS, 0.3 μM), or NVP-AEW541 (NVP, 2 μM), and raised to 120 hpf. NaR cells were labeled by in situ hybridization for igfbp5a mRNA. Representative views are shown in (d). The animals were scored according to the scoring system shown in Supplementary Figure S1c and the results are shown in (e). (f) Effect of IGF1R inhibition on igfbp5a mRNA levels. The experimental groups were the same as described in (d). The mRNA levels of igfbp5a were measured by qRT-PCR and normalized by the β-actin levels. Values shown are mean±S.D., n=3. *P<0.001 compared with the corresponding 0.2 mM [Ca²⁺] control group. (g) Effects of PI3K, Akt, and MEK inhibitors. Larvae (72 hpf) were transferred to 0.2 mM or 0.001 mM [Ca²⁺] water containing DMSO, Wortmannin (0.06 μM), LY294002 (5 μM), U0126 (U, 10 μM), PD98059 (PD, 10 μM), Akti-1/2 (Akti, 5 μM), or MK2206 (MK, 2 μM), and raised to 120 hpf. NaR cell density was determined as described in (e). (h) Effects of blocking PI3K, Akt, and MEK on igfbp5a mRNA levels. The experimental groups were the same as described in (g). The igfbp5a mRNA levels were measured by qRT-PCR and normalized by the β-actin levels. Mean±S.D., n=3. *P<0.001 compared to the corresponding 0.2 mM [Ca²⁺] control group