RNA sequence analysis to identify markers of pre-HSCs and embryonic HSCs. (A) Schematic showing the 4 cell populations that were compared. (B) Venn diagram of differentially expressed genes (DEGs) from pairwise comparisons. DEGs were identified by using a false discovery rate (FDR) cutoff <0.05 and a fold change cutoff >2. (C) Expression clusters defined by using consensus clustering and DEGs. Color indicates row-wise normalized expression levels. Numbers in parentheses indicate the number of genes in each cluster. (D) Gene ontology (GO) terms associated with genes in cluster 3. Level 5 biological process terms were used for enrichment analysis. Enrichment P values were computed by using the hypergeometric distribution. Nominal P values were corrected for multiple testing by using the Benjamini-Hochberg method. (E) List of cell surface marker genes in cluster 3 with more than twofold changes in gene expression in Ly6a-GFP⁺ HCCs relative to Ly6a-GFP^– HCCs or Ly6a-GFP^+/− endothelial cells. (F) Quantitative polymerase chain reaction analysis of Tnfrsf7 messenger RNA in all 4 populations. n = 6, whiskers represent the 5th to 95th percentile. Significance is by 1-way analysis of variance and Dunnett’s multiple comparison test with Ly6a-GFP⁺ HCCs as a comparator (#). **P < .01. E, endothelial cells; Endo, endothelial cells (CD31⁺CD144⁺ESAM⁺Kit^–); FC, fold change; H, hematopoietic clusters.