Single-cell multi-omics profiling links dynamic DNA methylation to cell fate decisions during mouse early organogenesis

Abstract

Background: Perturbation of DNA methyltransferases (DNMTs) and of the active DNA demethylation pathway via ten-eleven translocation (TET) methylcytosine dioxygenases results in severe developmental defects and embryonic lethality. Dynamic control of DNA methylation is therefore vital for embryogenesis, yet the underlying mechanisms remain poorly understood.

Results: Here we report a single-cell transcriptomic atlas from Dnmt and Tet mutant mouse embryos during early organogenesis. We show that both the maintenance and de novo methyltransferase enzymes are dispensable for the formation of all major cell types at E8.5. However, DNA methyltransferases are required for silencing of prior or alternative cell fates such as pluripotency and extraembryonic programmes. Deletion of all three TET enzymes produces substantial lineage biases, in particular, a failure to generate primitive erythrocytes. Single-cell multi-omics profiling moreover reveals that this is linked to a failure to demethylate distal regulatory elements in Tet triple-knockout embryos.

Conclusions: This study provides a detailed analysis of the effects of perturbing DNA methylation on mouse organogenesis at a whole organism scale and affords new insights into the regulatory mechanisms of cell fate decisions.

Fig. 1

scRNA-seq of Dnmt3a^-/-, Dnmt3b^-/- and Dnmt1^-/- mutant embryos during mouse early organogenesis. a Table with the numbers of E8.5 embryos and cells of each genotype analysed in this study. KO refers to the mouse models used in this study, CRISPR indicates published data which was generated using zygotic CRISPR-Cas9 injections [23]. b Dimensionality reduction (UMAP) of the wildtype reference dataset used for assigning cell types in this study. Cells are coloured by cell type as in the original publication [25]. c RNA expression of Dnmt1, Dnmt3a and Dnmt3b for each cell type in the reference atlas (quantified at the pseudobulk level). d Mapping of the KO cells to the reference atlas using the matching nearest neighbours (MNN) algorithm [26]. Each plot shows the UMAP of the reference atlas as in b, but cells are coloured by whether they are a nearest neighbour to a cell in our wildtype (blue) or mutant (red) embryos. e Box plots display the log2 difference in cell type proportions between WT and KO E8.5 embryos. Each point represents a comparison of cell type proportions between a KO embryo and the average proportions in WT embryos. f Polar bar plots display the number of differentially expressed genes for each KO and cell type. In the top panel bar plots are coloured by cell type identity and in the bottom panel, they are coloured by whether genes are up or downregulated. g Bar plots display the number of downregulated (top) or upregulated (bottom) genes in the Dnmt1-/- mutants. Shown are only genes which are markers for embryonic versus extra-embryonic (ExE) tissues. h Bar plots display the number of DE genes in the Dnmt1^-/- mutants for each cell type. Genes are grouped and coloured by the cell type that they mark in the reference atlas. Note that a gene might be a marker of multiple cell types, thus the y-axis is not directly comparable to f

Fig. 2

DNMT1 is required for the repression of pluripotency and extra-embryonic programmes and for the up-regulation of posterior Hox genes. a Polar bar plots display the number of differentially expressed genes in Dnmt1-/- cells, split by whether genes are downregulated (left) or upregulated (right). Each bar corresponds to a different cell type. Shown are all Hox genes (top) primed pluripotency markers (middle) and markers of extra-embryonic (ExE) tissues (bottom), according to the reference atlas. b Heatmaps display the log fold change in gene expression between mutant and wildtype. Shown are Hox genes (top), primed pluripotency markers (middle) and markers of ExE lineages (bottom). c Gene expression levels quantified at the pseudobulk level, where each data point corresponds to a different embryo and cell type. Shown are Hox genes (top), primed pluripotency markers (middle) and ExE tissue markers (bottom)

Fig. 3

scRNA-seq of Tet-TKO mutant embryos during mouse early organogenesis reveals that TET enzymes are required for the specification of primitive erythrocytes. a Schematic summarising the chimaera assay. Fluorescently labelled Tet-TKO ESCs are injected into wild type blastocysts, transferred into pseudopregnant hosts then collected at E7.5 or E8.5. FACS is used to isolate labelled KO cells (red) and non-labelled WT host cells (blue) which are processed and sequenced using scRNA-seq. b Mapping of the KO cells to the reference atlas using the matching nearest neighbours (MNN) algorithm [26]. UMAP plot of wildtype reference atlas [25] with cells coloured whether they are a nearest neighbour to a WT host (red) or Tet-TKO (blue) cell. c Box plots display the log2 difference in cell type proportions between WT and Tet-TKO E8.5 embryos. Each point represents a comparison of proportions between a Tet-TKO sample and the corresponding proportions in the matching WT host embryo. d Polar bar plots display the number of differentially expressed genes for each KO and cell type. In the right panel bar plots are coloured by cell type identity and in the left panel, they are coloured by whether genes are up or downregulated. e Bar plots display the number of DE genes for each cell type. Genes are grouped and coloured by the cell type that they mark in the reference atlas. Note that a gene might be a marker of multiple cell types, thus the values in the y-axis are not directly comparable to d. f RNA expression levels of the haemoglobin X alpha-like embryonic chain gene (Hba-x) in WT to Tet-TKO cells. Shown are different cell types grouped from the haematoendothelial trajectory

Fig. 4

scNMT-seq of Tet-TKO cells reveals impaired DNA demethylation of erythroid enhancers during primitive erythro- poiesis. a Schematic summarising the scNMT-seq chimaera assay. Fluorescently labelled Tet-TKO ESCs are injected into wild type blastocysts, transferred into pseudopregnant hosts then collected at E8.5. FACS is used to isolate specific populations (CD41+, erythroid; KDR+, Haematoendothelial progenitors; CD41+ KDR+, blood progenitors and CD41−, KDR−) of both labelled KO cells (red) and non-labelled WT host cells (blue) which are processed and sequenced using scNMT-seq. b Scatter plot displaying expression levels of the haemoglobin alpha adult chain 1 gene (Hba-a1) in cells ordered along a reconstructed primitive erythropoiesis trajectory. Cells are coloured by genotype, WT (N=301, top) and Tet-TKO (N=221, bottom). The line displays the LOESS curve. c As b for Dnmt and Tet genes, and Uhrf1. To avoid cluttering the LOESS curves are shown without the corresponding data points. d Scatterplot displaying global CpG methylation in cells ordered along the same pseudotime trajectory as b and coloured by genotype. The line displays the LOESS curve. e DNA methylation (yellow) and chromatin accessibility (green) profiles quantified over multiple genomic contexts in WT (N=67,top) and Tet-TKO (N=57, bottom) erythroid cells. Each column corresponds to a different genomic context: promoters (N=18,329), surface ectoderm enhancers (N=2138), haematoendothelial progenitors enhancers (N=3616), and erythroid enhancers (N=4319). Shown is the mean +/− 1 standard deviation in running averages of 50bp windows around the centre of the genomic annotation (2kb upstream and downstream). f Boxplots showing the distribution of DNA methylation (top) and chromatin accessibility (bottom) in erythroid cells in WT (N=67, blue) and Tet-TKO (N=57, red) at different genomic annotations