FIGURE 5

Cell proliferation in the palate was restored by combinatorial treatment with the teratogen and Wnt agonists. (A) Immunofluorescence images of proliferative cells of the plate at 96 hpf. Experimental time course is the same as in Figure 4A. WAF (30 μM) exposure induced cleft palate and decreased the number of pH3-positive cells in the palate. The number of pH3-positive cells was restored by combinatorial treatment with BIO (100 nM), CHIR99021 (300 nM) or WAY-262611 (250 nM). Green indicates cartilage cells double stained with anti-coll2 antibody and lectin PNA. White dotted lines trace the shape of the palate. Magenta indicates proliferative cells stained with anti-pH3 antibody. (B) Quantification of the number of pH3-positive cells in the palate. Numerical value was normalized to 10⁴ μm². (C) VPA (20 μM) exposure induced cleft palate and the number of pH3-positive cells was decreased. The number of pH3-positive cells was restored by combinatorial treatment with BIO (100 nM), CHIR99021 (300 nM) or WAY-262611 (250 nM). (D) Quantification of the number of pH3-positive cells in the palate. Numerical value was normalized to 10⁴ μm². n = 12 (Control), 15 (WAF), 12 (WAF + BIO), 16 (WAF + CHIR), 15 (WAF + WAY) in (B), n = 12 (Control), 14 (VPA), 14 (VPA + BIO), 12 (VPA + CHIR), 13 (VPA + WAY) in (D), *P < 0.05, **P < 0.01, ***P < 0.001 (one-way ANOVA followed by Dunnett’s multiple comparison test). Scale bar: 50 μm.