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Overactivation of Wnt/β-Catenin signal is responsible for the increase of cell proliferation in wdr5−/− mutant digestive organs. a Western blots of pH3, Wdr5, β-Actin, H3 in WT and wdr5−/− mutant embryos at 3 and 5 dpf. Relative intensity of pH3 was normalized with H3. b Cryosections of WT and wdr5−/− mutant embryos at 3 and 5 dpf were immunostained by anti-pH3 (in red) anti-Bhmt (a liver specific marker in green). The nuclear was stained with DAPI (in blue). L: liver; I: intestine. Framed area was magnified in bottom panel. Scale bar: 40 μm. The percentage of pH3 positive cells in each sample was calculated as the number of pH3 positive cells divided by total cell number in different organs from continuous cryosections. Also see Supplementary Fig. 6b. c Transcript TPM of β-Catenin targeted genes (myca, mycb, mycn and ccnd1) in RNA-seq from WT and wdr5−/− mutant embryos at 3 dpf. d Western blots of β-Catenin, Wdr5, β-Actin, in WT and wdr5−/− mutant embryos at 3 dpf. Relative intensity of β-Catenin was normalized with β-Actin. e Western blots of β-Catenin, pH3, Wdr5, β-Actin and H3 in WT and wdr5−/− mutant embryos with different treatment as indicated. The wdr5−/− mutant and WT embryos at 2.3 or 4 dpf were treated with SAL or DMSO. The protein was extracted from treated embryos at 3 and 5 dpf. Also see Supplementary Fig. 6d, e. f Relative expression level of myca and ccnd1 in WT and wdr5−/− mutant embryos at 3 dpf with SAL or DMSO. The treatment was described in Fig. 4e. g Cryosections of WT and wdr5−/− mutant embryos at 3 dpf treated with SAL or DMSO were immunostained by anti-pH3 (in red) and anti-Bhmt (in green). The nuclear was stained with DAPI (in blue). L: liver; I: intestine. Framed area was magnified in bottom panel. Scale bar: 40 μm. Statistical analysis on the percentage of pH3 positive cells in the liver or intestine of wdr5−/− embryos at 3 dpf treated with SAL or DMSO was showed in the right panel. The treatment was described in Fig. 4e. Each experiment was repeated for three times with similar results and a representative was showed here. n indicates the number of zebrafish embryos in each group. Data are mean±S.D. Two-tailed t-test was applied for each individual comparison (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; n.s no significance).
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