Experimental design and overview of single cell RNA-Seq data. (A) The caudal part of embryos was isolated (boundaries are illustrated with scissors), and then organs and gut tube removed. VU were isolated and included in the sample. The tissue was dissociated and cells were isolated by fluorescence-activated cell sorting, then analyzed by scRNA-Seq, scATAC-Seq, or in functional assays. All cell populations purified and sequenced are listed in supplemental Table 2; sort plots are shown in supplemental Figures 1 and 2. (B) The number of cells sequenced (x-axis) and genes per cell detected for representative samples. (C) UMAP of continuous EHT trajectory and FL-HSCs, with selected cell populations labeled. (D) Distribution of cells from each dataset in the UMAP reflecting EHT trajectory. (E) UMAP illustrating the 2 streams of E cells expressing high levels of the arterial marker Efnb2 that converge to form the stem leading to HE and IACs. (F) E+HE+IAC cells separately purified from the VU arteries, and from the DA within the caudal half of the embryo, highlighted on the global UMAP plot. (G) Cell count along the pseudotime trajectory. Bar graph quantifies results from a single sort of 10.5 dpc E+HE+IAC cells; heat maps below the graph show distribution of cells in all sorted cell populations.