Figure 3
Dnmt1 mRNA rescue and anti-phosphorylated histone H3 (pH3) immunostaining assays in dnmt1-deficient embryos. (A-H) Dnmt1 mRNA rescue assay at 36 hpf (A-D) and 5 dpf (E-H). Dnmt1 ATG morpholino (4 ng) and splicing-specific morpholino (8 ng) effectively reproduced decreased cmyb expression phenotype in wild-type embryos (B, C, F, G). Dnmt1 mRNA rescued the defects of dnmt1 morphants. (I-N) Rescue assay of wild-type dnmt1 mRNA in ldd794 dnmt1 mutants at 36 hpf (I-K) and 3 dpf (L-N). Wild-type dnmt1 mRNA effectively restored cmyb expression in ldd794 dnmt1 mutants at 36 hpf (K) and 3 dpf (N). (A’-N’) Magnified images of the boxed regions in A to N, respectively. Red arrows indicate cmyb-positive HSPCs in the AGM or CHT. (O-T) The proliferation of HSPCs was reduced in dnmt1−/− mutants. Double immunostaining of cmyb-EGFP (O-P) and anti-PH3 (Q-R) in the CHT of Tg(cmyb-EGFP) line at 72 hpf. The bottom panel shows merged images (S, T). (U) Statistics of PH3 and cmyb-EGFP double positive cells.