Developmental bottleneck between pre-HE and HE cells. (A) UMAP of E10.5 E+HE+IAC cells showing 9 cell types from Figure 2A. (B) Expression of key markers of clusters, including Hey2 in conflux AE and pre-HE, Cd44 in conflux AE, pre-HE, HE, and IACs; Ptprc in IACs; Gfi1 and Runx1 in HE and IACs; and high levels of Sox17 in conflux AE and pre-HE, with downregulation in HE. Note Runx1 is expressed at low levels in all subsets of endothelial cells. (C) Velocyto analysis revealing different differentiation dynamics along the EHT in ED10.5 E+HE+IAC cells. To the right is an enlargement velocity of pre-HE cells that have accumulated at the bottleneck between pre-HE and HE. (D) Activity of pathways from Kyoto Encyclopedia of Genes and Genomes database, computed for each cell using the AUCell method.⁶³  (E) UMAP of E+HE+IAC cells from 10.5 dpc Runx1^+/+ and Runx1^+/− littermates. Bars at bottom depict the distribution of cells between conflux AE, pre-HE, combined HE, and IAC populations in 10.5 dpc Runx1^+/+ and Runx1^+/− littermates. P values indicate significant differences in the distributions of cells in pre-HE and HE in Runx1^+/+ vs Runx1^+/− samples based on proportion test. ***P < .001. (F) UMAP of E+HE+IAC cells from 10.5 dpc control embryos (cR1/⁺) and littermates ectopically expressing RUNX1 in all endothelial cells from the Rosa26 locus (Cre;cR1/⁺).¹¹  Bars at bottom as in panel E. ***P < .001. (G) Limiting dilution assay to determine the frequency of HE in the CD44⁺ fraction of E+HE+IAC cells isolated from 10.5 dpc embryos (see supplemental Figure 2G for fluorescence-activated cell sorting plots). Shown are frequencies of cells that yielded hematopoietic cells (B220⁺, CD19⁺, Mac1⁺, Gr1⁺, and/or CD41 and CD45) ex vivo. Frequencies were calculated by ELDA.⁶⁴  Data represent 3 independent cell purifications and limiting dilution assays (mean ± standard deviation [SD], unpaired 2-tailed Student t test).