Fig. 1: scRNA-seq profiling of Dnmt3a^−/− and Dnmt3b^−/− differentiating EBs.

a Overview of the experimental design and visualisation of collected time points. The colour scheme for WT, 3AKO and 3BKO samples will be kept throughout the manuscript. b UMAP embedding of 487 WT, 3AKO and 3BKO single cell transcriptomes. Cells are coloured by cluster (top panel) and genotype/time of cells’ collection (bottom panel). 3AKO = lightblue/darkblue, 3BKO = light orange (3Days)/dark orange (9Days), WT = light grey (3Days)/dark grey (9Days). c Gene expression levels distribution of representative epiblast (Lefty1, Pou5f1), meso-endoderm (Gata4, T) and ectoderm markers (Sox2, Tubb3) in the five identified cell clusters. d Pseudotime analysis of single cell differentiation trajectories with reverse graph embedding³³. The line plot on the UMAP represents the embedded trajectory graph. Cells are coloured according to pseudotime (left panel) and differentiation paths (i.e. Epi-ME, Epi-Ect, right panels). e UMAP visualisation of the reconstructed differentiation trajectories for each source cell type (i.e. WT, 3AKO and 3BKO genotype). f Heatmap showing the expression patterns of genes differentially regulated in pseudotime along the two differentiation branches, performed via the graph-autocorrelation analysis method³³. Genes are grouped according to the branch in which they show significant variation (FDR < 0.01): Epi-to-Ect specific (top cluster), Epi-to-ME (bottom cluster) specific or regulated in both branches (mid cluster). g Barplots showing the differential cell type abundance in terms of the genotype of origin (i.e. WT, 3AKO and 3BKO) in each of the five identified cell clusters. For each cluster, the relative proportion of mutant cells (3AKO, 3BKO) was compared with WT cells using Fisher’s exact test. Top panels report the percentage of cells in each cluster for the indicated comparisons (i.e. 3AKO vs WT, 3BKO vs WT). Bottom panels report the odds ratio from Fisher’s exact test, coloured for their significance (enriched = red, depleted = blue, non-significant = white, ****p value < 0.0001, one-sided).