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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.
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