Extended Data Fig. 2: Characteristics of haematopoietic progenitors in human embryos.

a, Heat map showing differential regulon expression between haematopoietic progenitor clusters (YSMP, n = 116; ErP, n = 72; MkP, n = 30; GMP, n = 45; myeloblast, n = 100; CD7^lo progenitor (CD7^loP), n = 140; CD7^hi progenitor (CD7^hiP), n = 104; HSPC, n = 33) generated by SCENIC and clear sets of cell-type specific regulons that may play critical roles in the development of each progenitor population. The number of genes associated with each regulon is listed in parentheses. ErP and MkP signatures were very similar, although MkP appeared to have downregulated expression of KLF1and up-regulated expression of other platelet-related transcription factors such as TAL1 and NFIB. CD7^loP had overlapping modules with myeloblast and GMP, sharing the myeloid-restricted TFEC pathways, but lacked expression of more myeloid-committed CEBPs. CD7^hiP showed many signatures typical of lymphoid potential, such as the activation of LEF1 and TCF4 signals. HSPC were characterized by activation of the HOXA10 module, as well as higher levels of lymphoid-associated BCL11A when compared to YSMP. b, GSEA plots of the top two differentially expressed regulons between YSMP (n = 116 cells) and HSPC (n = 33 cells). GSEA analysis revealed that YSMPs highly enriched the SPR and ZFP64 regulons, while HSPCs had higher expression of the EOMES and POU3F2 modules. P value was calculated using permutation test (one-sided) based on phenotype by GSEA 3.0 software, representing the statistical significance of enrichment score. c, Volcano plot of DEGs between YSMP (n = 116 cells) and HSPC (n = 33 cells), with the top 10 genes for each cluster indicated. Although the regulon landscape was similar between these two groups, we identified 110 DEGs (Supplementary Table 3). There were more upregulated genes in HSPC (86, red) than in YSMP (24, blue), with HSPC expressing genes related to antigen presentation including CD74 and HLA-DRA as well as lymphoid-related genes including IGLL1. DEGs were detected using FindAllMarkers function in Seurat (one-sided Wilcoxon rank-sum test, with P value adjusted for multiple testing using Bonferroni correction), and genes with fold change >1.25 and adjusted P < 0.05 were selected. d, Proportion changes of YSMPs and HSPCs in the haematopoietic progenitor populations of yolk sac and liver between CS11 and CS23 (n = 8 biologically independent embryo samples). The proportion of the YSMP population peaked at CS11 before steadily decreasing, while that of the HSPC population expanded between CS17 and CS20 before reducing to about 10% at CS23. e, Proportion changes of different haematopoietic progenitor clusters from CS12 to CS23 in the liver (n = 6 biologically independent embryo samples and 259 cells), and CS13 to CS20 in the blood (n = 5 biologically independent embryo samples and 131 cells). f, Heat map showing expression levels of the top five differentially expressed transcription factors between YSMP, ErP, MkP, myeloblast, HSPC, CD7^loP and CD7^hiP cells. DEGs were detected using FindAllMarkers function in Seurat (one-sided Wilcoxon rank-sum test, with P value adjusted for multiple testing using Bonferroni correction), and genes with fold change >1.5 and adjusted P < 0.05 were selected.

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