Fig. 2

Fig. 2 The DNA methyltransferase activity of Dnmt3ba is required for HSPC development.

A WISH at 36 hpf showing the embryos treated with Nanaomycin A at 24 hpf impaired the expression of HSPC marker cmyb (arrowheads) in the VDA region. Three independent experiments were conducted. B Quantification of cmyb⁺ cells (n = 32 (DMSO), 20 (Nanaomycin A) embryos). C Confocal imaging shows the kdrl⁺runx1⁺ HECs (white arrowheads) in the VDA region of DMSO or Nanaomycin A-treated embryos with Tg(kdrl:mCherry/runx1:en-GFP) background at 36 hpf. Three independent experiments were conducted. D Quantification of the number of kdrl⁺runx1⁺ HECs (n = 25 (DMSO), 28 (Nanaomycin A) embryos). E WISH showing the expression of HSPC marker cmyb (arrowheads) in the CHT region of DMSO or Nanaomycin A-treated embryos at 72 hpf. Three independent experiments were conducted. F Quantification of cmyb⁺ cells (n = 24 (DMSO), 19 (Nanaomycin A) embryos). G Schematic representation of heat-shock strategy to temporally and transiently express dnmt3ba or dnmt3ba^△DCM at the beginning of EHT (24–32 hpf). H WISH showing the expression of cmyb (arrowheads) at 36 hpf in WT, dnmt3ba mutants, and dnmt3ba mutants injected with hsp70l:dnmt3ba-EGFP or hsp70l:dnmt3ba^△DCM-EGFP construct, respectively. Three independent experiments were conducted. I Quantification of cmyb⁺ cells (n = 26 (dnmt3ba^+/+), 26 (dnmt3ba^−/−), 22 (dnmt3ba^−/−+hsp70l:dnmt3ba), 25 (dnmt3ba^−/−+hsp70l:dnmt3ba^△DCM) embryos). J WISH showing the expression of cmyb (arrowheads) at 36 hpf in WT, dnmt3ba mutants, and dnmt3ba mutants injected with fli1a:dnmt3ba-EGFP or fli1a:dnmt3ba^△DCM-EGFP construct, respectively. Three independent experiments were conducted. K Quantification of cmyb⁺ cells (n = 23 (dnmt3ba^+/+), 19 (dnmt3ba^−/−), 16 (dnmt3ba^−/−+fli1a:dnmt3ba), 21 (dnmt3ba^−/−+fli1a:dnmt3ba^△DCM) embryos). Scale bars, 100 µm. Statistical significance was determined by a Two-tailed Student’s t-test in (B, D, F) and a One-way ANOVA in (I, K), respectively. Data are presented as mean ± SD.