Two streams of E cells converge before hemogenic endothelium. (A) UMAP of EHT trajectory (from Figure 1C, with FL-HSC removed) showing the 7 clusters identified by Louvain clustering in supplemental Figure 5A, with Wnthi E subdivided into Wnthi AE and Wnthi VE, plus Wntlo E subdivided into Wntlo AE and Wntlo VE based on the arterial/venous score determined as shown in panels B and C. (B) Enlarged UMAP highlighting the 2 streams of endothelial cells converging to conflux AE. Numbers in yellow circles represent pseudotime bins up to the point of convergence. The dotted gray line represents the boundary between AE and VE. (C) Arterial score vs venous score over pseudotime bins. Cluster VE from panel A is used as the first pseudotime bin. Curves are fitted for AE score and VE score of each branch using a generalized additive model. (D) Violin plots of expression of cluster-specific genes, including venous marker Nr2f2, arterial marker Sox17, Wntlo AE-specific gene Tmem255a, Wnthi AE-specific genes Foxq1 and Nkd1, and Notch ligand Dll4. (E) Average expression of Wntlo E, Wnthi E, and pre-HE-specific genes over pseudotime. Differentially expressed genes were derived by pairwise expression analysis between Wntlo E and Wnthi E. Pre-HE specific genes were derived by comparing pre-HE with Wntlo plus Wnthi E. (F) Heatmap showing stream-specific Reactome pathway activity over pseudotime. AUCell package63 was used to compute a pathway activity score for each cell. One vs the rest Student t test was used to identify group-specific pathways and the top 6 most significant pathways were plotted.