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

This repository contains the scripts to reproduce the DNMT KO analysis of our study. It covers most of Figures 1-2 as well as Supplementary Figures 1-4. The source code for the second part of the paper, where we explore the Tet TKO embryos, is available in this repository

Experimental design

We generated Dnmt1^-/-, Dnmt3a^-/- and Dnmt3b^-/- embryos together with matching wildtypes from heterozygous matings. We collected embryos at E8.5, when progenitor cells for all major organs have formed and methylation mutants are not yet lethal, and performed scRNA-seq. To increase the statistical power of our analysis we combined our data set of KO embryos with a published data set where Dnmt1, Dnmt3a and Dnmt3b were disrupted using CRISPR-Cas9 and also profiled using scRNA-seq at E8.5. In total, our analysis comprises 51,811 cells from 17 WT embryos, 45,579 cells from 14 Dnmt3a^-/- embryos, 55,237 cells from 12 Dnmt3b^-/- embryos and 25,185 cells from 15 Dnmt1^-/- embryos. We assigned celltype labels by mapping the RNA expression profiles to a reference atlas that spans E6.5 to E8.5

Key results

• Dnmt3a^-/- and Dnmt3b^-/- embryos show relatively minor defects in cell type proportions. In contrast, Dnmt1^-/- embryos show widespread defects in cell type proportions, including a relative overrepresentation of ExE ectoderm and immature embryonic cell types such as rostral neuroectoderm and caudal epiblast. We also observe a relative underrepresentation of some mature embryonic cell types, including Neural crest, NMPs, Brain, Spinal cord and Gut cells.

• We find a small number of DE genes when comparing Dnmt3a^-/- and Dnmt3b^-/- to WT samples. In contrast, we find a large number of DE genes in the Dnmt1^-/- KO across most cell types, but particularly in the Neural crest, Caudal mesoderm and Blood progenitors.

• Posterior Hox genes (for example Hoxc9, Hoxc8, Hoxb9 and Hoxa9) are downregulated in posterior cell types of Dnmt1^-/- KO cells, including NMPs, somitic mesoderm, intermediate mesoderm and ExE mesoderm.

• Among the genes that are upregulated in the Dnmt1^-/- KO we observe primed pluripotency markers (Pou5f1, Utf1, Slc7a3, Fgf5 and Pim2) and ExE marker genes (Rhox5, Krt8, Apoe, Ascl2, Trap1a and Xlr3a) across most cell types.

Data

Raw data is available at GEO.
Parsed data can be downloaded in progress....

R Shiny app

Interactive R shiny app is available here.

Contact

For questions on the computational analysis: Ricard Argelaguet (ricard.argelaguet@gmail.com). For questions on the experimental work: Tim Lohoff (tlohoff431@gmail.com) or Stephen Clark (sclark@altoslabs.com)