Fig. 6: Dnmt1 de novo methylation and its effects in the early embryo.

a, Representative images of IAPEz-gag expression assayed by RNA-FISH in dissociated EBs after 10 days of differentiation from WT, TKO_L and DKO₀ cells. n = total counted nuclei. IAPEz-gag-positive cells out of total counted nuclei: WT 99/3,767; TKO_L 3,374/5,916; DKO₀ P5 315/1,018; DKO₀ P15 53/416; DKO₀ P25 7/676. b, Experimental design for the generation of zygotic Dnmt3a/b DKO embryos and subsequent Dnmt1i (GSK3484862) treatment (from the two-cell stage until day E3.5 blastocyst) and retransfer for in vivo post-inhibitor recovery until E6.5. WGBS sample collections are indicated and include E3.5 (n = 8 WT, 12 Dnmt1 KO, 20 DKO, 8 DKO^DMSO, 8 DKO^Dnmt1i embryos) and E6.5 (n = 3 epiblast samples). Epi, epiblast; ExE, extraembryonic ectoderm. c, Morphological and viability assessment of DKO^Dnmt1i E3.5 blastocysts. Left: representative images of DKO blastocysts treated with DMSO or different concentrations of Dnmt1i from the two-cell stage until day E3.5. Pie charts show the proportions of normal and delayed/abnormal blastocysts. n = number of normal blastocysts/total number of DKO 2-cell embryos included in each treatment. Right: representative images of WT and Dnmt1i-treated E6.5 embryos. Scale bars, 100 µm. d, Heatmap representation of mean methylation over IAPEz-int (n = 5,362) in the respective E3.5 blastocyst samples compared with E6.5 epiblasts. e, Profile plot of methylation changes between an E3.5 blastocyst and E6.5 epiblast. Shaded area represents standard deviation. f, RT-PCR quantification of IAPEz-gag expression relative to β-actin in WT, Dnmt1 KO, DKO^DMSO and DKO^Dnmt1i in E3.5 and retransferred E6.5 embryos. n = 3 replicates, with a single replicate consisting of a pool of 10 blastocysts from multiple mice or one E6.5 epiblast. Bars, mean; error bars, standard deviation. Quantification for f is provided in Supplementary Table 4. Additional source data can be found at https://doi.org/10.6084/m9.figshare.14555250.