Fig. 3

Wdr5-mediated H3K4me3 is required for digestive organ differentiation.

a Two transgenic lines of Tg(hsp70:HA-Wdr5^WT) and Tg(hsp70:HA-Wdr5^{S91K,F133A,Y191F}) were treated with heat shock. Two transgenes of Tg(hsp70:HA-Wdr5^WT) and Tg(hsp70:HA-Wdr5^{S91K,F133A,Y191F}) were generated in wdr5^+/− background. The upper diagram showing how the heat shock treatment was performed. The embryos crossed from each transgenic line were treated with four times of heat shock (39 °C for half an hour) at 1, 1.5, 2, and 2.5 dpf, respectively. The treated embryos were sampled at 3 dpf. Representative images were taken before the genotyping. b Co-IP analysis of the interaction between HA-Wdr5 and H3, H3K4me3 or H4K16ac in two transgenetic lines: Tg(hsp70:HA-Wdr5^WT) and Tg(hsp70:HA-Wdr5^{S91K,F133A,Y191F}) under heatshock condition. HA beads were used for immunoprecipitation. β-Actin and β-Tubulin were used as the interaction negative and positive controls, respectively. More than 300 embryos of 1 dpf from each line were treated with heat shocks as described in Fig. 3a. c Co-IP of the interaction between Flag-N-Setd1a or Flag-Rbbp5 with the HA-Wdr5^WT or HA-Wdr5^{S91K,F133A,Y191F} in 293 T cells. The upper diagram showing Flag-tagged N terminal of zebrafish Setd1a protein used in Co-IP. Different plasmids were transfected or co-transfected into 293 T cells as indicated. HA beads were used for immunoprecipitation. A zebrafish Wdr5 antibody was used to detect HA-Wdr5^WT or HA-Wdr5^{S91K,F133A,Y191F}. Flag antibody was used to detect Flag-Rbbp5 and Flag-N-Setd1a. d Western blots detected by anti-Wdr5, anti-HA, anti-H3K4me3 or anti-H3 antibody in different samples as indicated. Two transgenic lines Tg(hsp70:HA-Wdr5^WT) and Tg(hsp70:HA-Wdr5^{S91K,F133A,Y191F}) were generated in wdr5^+/− background. More than 200 embryos of 1 dpf from each line were treated with heat shocks as described in Fig. 3a. Treated embryos were genotyped before protein extraction. At least 30 embryos in each group were pooled together for protein extraction. e WISH of fabp10a in transgenic wdr5^−/− and sibling embryos treated with heat shocks as described in Fig. 3a. 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.