Epiblast diversification and blood formation in a human pregastrula

a. UMAP visualization of all cells from CS7 human embryo. b. UMAP plots showing the expression of extra-embryonic mesoderm, hematopoietic and endothelial-related genes in cells from CS7 human embryo. c. UMAP plot showing the cell types in integrative analysis of human CS6 and CS7 scRNA-seq data. 4 epiblast clusters, hypoblast, yolk sac endoderm, yolk sac extraembryonic mesoderm and blood cells in CS6 as well as their counterpart cell populations in human CS7 are included, with all cells (left), CS6 (upper right) and CS7 (lower right) displayed. Colors indicate cell types and shapes encode sampling stages. d. Dot plot showing the expression of endothelial cell-related genes in cell clusters of Fig. 2a. e. UMAP visualization of endothelial cells and newly identified five hematopoietic cell clusters in CS7 human embryo. f. Dot plot displays normalized expression of marker genes in identified cell types in panel e. Note that two hematopoietic progenitor populations were identified. One with high expression of YSMP signature genes such as CD34, CD44, MYCN and SPINK2, and the other with high expression of MPO, a signature gene of pMPs defined in CS6 human embryo, which were thus annotated as YSMP and pMP, respectively. Furthermore, all three types of mature blood cells belonging to primitive hematopoiesis have been identified, including primitive erythrocytes (KLF1 and GYPA), primitive megakaryocytes (PF4 and GP9), and primitive macrophages (C1QA and SPP1). g. Dot plot shows the normalized expression of Heme biosynthesis-related genes in pEry cells from CS6 and CS7. h. Dot plot shows scaled expression of top 15 DEGs between pEry from CS6 and CS7. i. Bar plots show representative GO terms enriched in CS6 pEry (top) and CS7 pEry (bottom). BH-adjusted one-sided hypergeometric test was used. Note that erythrocyte development associated pathways such as heme synthesis were significantly enhanced at CS7 compared to CS6, revealing the process of hematopoietic maturation from CS6 to CS7. j. Violin plots displaying signature scores of gas transport and erythrocyte differentiation in pEry clusters from CS6 and CS7 (P < 1e−16). The P value was calculated using two-sided Wilcoxon test and **** indicates P value < 0.0001. k. Dot plot showing normalized expression of representative megakaryocyte maturation-related genes in pMeg clusters from CS6 and CS7. l. Dot plot showing scaled expression of top 15 DEGs for pMeg clusters from CS6 and CS7. m. Bar plots show the representative GO terms enriched in CS6 pMeg (top) and CS7 pMeg (bottom). BH-adjusted one-sided hypergeometric test was used. Note that megakaryocyte development related pathways such as coagulation and platelet formation were significantly enhanced at CS7 compared to CS6, revealing the process of hematopoietic maturation from CS6 to CS7. n. Violin plots displaying signature scores of coagulation and megakaryocyte development in pMeg clusters from CS6 and CS7 (P < 1e−16). The P value was calculated using two-sided Wilcoxon test and **** indicates P value < 0.0001. o. Dot plot showing scaled expression of top 15 DEGs for pMP_CS6, pMP_CS7 and YSMP. p. Heatmap showing the enriched hallmark pathways among pMP_CS6, pMP_CS7 and YSMP. q. Violin plots display oxidative phosphorylation (top) and glycolysis (bottom) signature scores in pMP_CS6, pMP_CS7 and YSMP. The P value was calculated using Kruskal-Wallis test across multiple groups. * indicates P value < 0.05, **** indicates P value < 0.0001. r. Bar plots show the representative GO terms enriched respectively in pMP_CS6 (top), pMP_CS7 (middle) and YSMP (bottom). BH-adjusted one-sided hypergeometric test was used. Note that CS6 pMPs enrich pathways associated with cholesterol transport and cell adhesion, while YSMPs at CS7 show higher levels of genes associated with ribosome synthesis and cell mitosis. s. Bar plot shows the cell proportions of cell cycle phases in pMP_CS6, pMP_CS7 and YSMP. t. Bar plot shows the representative hallmark pathways enriched in pMP (from both CS6 and CS7) and YSMP. Bar color indicates the cell type enriched for the pathway. Two-sided t test was used. u. UMAP visualization of major myeloid cell types in CS10 and CS11 yolk sac (top) with indicated myeloid cell clusters from CS6 and CS7 mapped on (bottom). v. Heatmap showing scaled expression of transcription factors in each gene pattern along macrophage specification. w. Four distinct gene expression patterns along the developmental pseudotime of macrophage specification from pMP_CS6, pMP_CS7 to macrophage with representative GO terms for each gene pattern are listed on the right. The fitted solid curve represents the GAM-predicted mean expression trend along pseudotime. The error bands around the line indicate the 95% confidence interval of the fitted mean, calculated as mean±1.96×standard error of the GAM fit.