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. Author manuscript; available in PMC: 2020 Sep 23.
Published in final edited form as: Nat Genet. 2020 Mar 23;52(4):378–387. doi: 10.1038/s41588-020-0595-4

Figure 3. Erythroid-to-myeloid committed progenitor frequency changes are concordant with skewed HSC transcriptional priming.

Figure 3.

a) UMAP highlighting the selected HSC clusters (HSC 1–3, left panel). Differential gene expression between WT (n = 2,150 cells) and Tet2 KO (n = 2,989 cells, central panel) or WT and Dnmt3a KO (n = 1,325 cells, right panel) HSC 1–3 clusters. Red dots represent differentially expressed genes (permutation test followed by Benjamini-Hochberg (BH) correction, FDR < 0.05, see online methods) with an absolute log2 fold change higher than 0.5. Pathway enrichment was performed with EnrichR57. b) Top panel: heatmap showing single cells from HSC 1–3 clusters. Bottom panel: Generalized additive model fit for erythroid, myelo-monocytic and stem scores from WT (n = 7 mice) HSC 1–3 clusters (n = 7,648 cells). Grey areas represent the 95% confidence interval. c) For each genotype, 1,225 cells from the HSC 1–3 clusters were randomly sampled and density plots were generated. The percentage of cells with either erythroid or myelo-monocytic priming is shown. d) Transcriptional priming scores for HSC 1–3 cells for Tet2 KO (2,989 cells; n = 7 mice), WT (2,150 cells; n = 7 mice) and Dnmt3a KO (1,225 cells, n = 4 mice) progenitors (two-sided Wilcoxon rank sum test followed by Bonferroni correction). e) Posterior probabilities of Gaussian mixture model fit for myelo-monocytic transcriptional priming for 1,225 randomly sampled cells from the HSC 1–3 clusters for WT (n = 4), Tet2 KO (n = 3) or Dnmt3a KO (n = 4) from Chromium samples (Binomial test). f) In vitro colony-forming assay using purified LT-HSCs from WT (n = 282 colonies), Tet2 KO (n = 391 colonies) or Dnmt3a KO (n = 209 colonies; two-sided Fisher exact test; CFU-GM = colony-forming unit granulocytic/monocytic; BFU-E = burst-forming unit erythroid; CFU-GEMM = colony-forming unit granulocytic/erythroid/monocyte/megakaryocyte, see online methods). g) Schematic representation of the procedure for visualization of the differentiation topology. h) Differentiation topologies derived from scRNA-seq data.