Fig. 2

DNA methylation is required for HSPC generation. (A) Expression of HSPC markers runx1 and cmyb (arrowheads) at 36?hpf, erythroid marker gata1 (arrowheads) at 4 dpf and lymphoid marker rag1 (arrowheads) at 4 dpf in siblings and dnmt1 mutants by WISH. n?3 replicates. (B) Quantification of WISH results in A. (C) Confocal imaging showing the number of kdrl⁺/cmyb⁺ HECs in AGM at 36?hpf (white arrowheads), and cmyb⁺ HSPCs in the caudal hematopoietic tissue (CHT) region at 2 dpf in siblings and dnmt1 mutants (left panels, n?3 replicates), with quantification (right panels). n=5 embryos. (D) qPCR analysis of runx1 and cmyb expression in sibling and dnmt1 mutant embryos at 36?hpf. n=3 replicates. (E) Expression of runx1 and cmyb in sibling, dnmt1 mutant and dnmt1 mutant embryos injected with hsp70: mismatch-dnmt1-EGFP constructs. The arrowheads indicate the expression of HSPC markers runx1 and cmyb. n?3 replicates. (F) Quantification of WISH results in E. (G) qPCR analysis of runx1 and cmyb expression in control, dnmt1 morphants and embryos co-injected with dnmt1-atgMO and hsp70: mismatch-dnmt1-EGFP constructs. n=3 replicates. (H) Transplantation results showing HSPC reconstitution in the CHT region of recipient embryos at 36?hpf. Green, cmyb⁺ EGFP cells; red, Rhodamine. White arrowheads show EGFP⁺ HSPCs contributed by donor cells; yellow arrowheads show donor-derived EGFP^? hematopoietic cells in CHT region. (I) Summary of transplantation results in H. Data are mean±s.d. ***P<0.001, ****P<0.0001 (unpaired two-tailed Student's t-test). Scale bars: 100??m (A,E); 50??m (C,H).