ACE (CD143) is an HE enrichment marker. (A) Violin plots showing the expression of shortlisted cell surface markers. Cd31 expression is shown for reference. The mean expression is indicated by the horizontal black line. The percentage of cells with non-zero expression (read count >0) is shown above each plot. (B) Representative immunofluorescence (IF) staining of formalin-fixed paraffin-embedded (FFPE) sections of E10.5 embryo sections for CD31 (red) and ACE (gray). D, dorsal; DA, dorsal aorta; NT, neural tube; V, ventral. (C) Left: IF staining of E10.5 AGM FFPE sections in panel B. CD31 (red), RUNX1 (green), and ACE (gray). Example IAHC cells are indicated with magenta arrow. White arrows indicate example RUNX1⁺ cells in the lining of the aorta. Right: Percentage of ACE⁺ cells in CD31⁺ E10.5 DA. (D) FACS plots showing the enrichment of E10.5 HE with CDH5⁺ alone (left), CDH5 and CD44 (middle), CDH5 and ACE (right). (E) Experimental design of hanging drop reaggregation AGM cultures with LIN (CD41⁺CD45⁺TER119⁺) depleted or LIN and ACE (LIN⁻CDH5⁺ACE⁺) depleted cells. CFU assays were performed after 4 days of culture. The numbers of replicates are indicated at the bottom of each column. Two-tailed Student t test was used to assess statistical significance. Error bars represent SEM. **P < .01. (F) Numbers of wells with proliferating cells following 10 days of coculture of FACS sorted LIN⁻KIT⁻CDH5⁺ACE⁺ or LIN⁻KIT⁻CDH5⁺ACE⁻ cells (100 cells/well) on vascular niche cells. Exp, experiment. (G) UMAP computed from integrating cells from cluster C1-C8 (excluding KO cells and KO clusters) with scRNA-seq data obtained from sorted ACE⁺ and ACE⁻ E10.5 AGM cells. Left: UMAP representation of the previously determined in silico clusters C1-C8 from Figure 2C (excluding KO cells and KO clusters). Right: scRNA-seq data obtained from sequencing ACE⁺ and ACE⁻ cells. (H) Heatmap displaying the percentage of cells from the ACE scRNA-seq data mapped to the in silico clusters based on k-nearest-neighbor classifier approach. AE, arterial endothelial; EHT, endothelial-to-hematopoietic transition; ENDO, endothelial; HE, hemogenic endothelium; IAHC, intra-aortic hematopoietic clusters; UC, unclassified; VE, venous endothelial.