scRNA-seq analysis reveals the transitions along the EHT trajectory affected by MEKK3 loss in endothelial cells. (A) Projection of combined EC MEKK3^KO and control endothelial, HEC, and IAHC cells (Ter119⁻ CD41^lo/− CD31⁺CD144⁺ ESAM⁺; gating strategy in supplemental Figure 5A) onto the EHT trajectory from Zhu et al⁵¹ labeled based on cell types. The inferred EHT trajectory from Zhu et al (cells represented by gray dots) was generated by uniform manifold approximation and projection and validated by several approaches, including scVelo.⁷⁸ Cell types were annotated using a κ-nearest-neighbor classifier (see Materials and methods). AE cells are defined by positive arterial score (supplemental Figure 5B). (B) Number of DEGs uniquely up- or downregulated in MEKK3^KO for each cell type, excluding the global DEGs (see Materials and methods). (C) Heatmap showing top Reactome pathways that are altered in pre-HE cells comparing MEKK3^KO vs control. AUCell package⁷⁹ was used to compute a pathway activity score for each cell. A 2-sided Student t test was used to identify cell type–specific pathways, and the global differentially regulated pathways were excluded (see Methods). (D) Separate projections of E10.0 EC MEKK3^KO and control cells on the EHT trajectory. (E) Bar charts depicting the distribution of cells in Wnt^hi/lo AE, conflux AE (cAE), pre-HE, HEC, and IAHC cell populations in E10.0 EC MEKK3^KO and control littermates. For each transition, the differences in the distributions of cells in EC MEKK3^KO vs control cells were tested using a proportion test. HEC+IAHC cells were combined in the analysis due to the low cell numbers in these populations in the E10.0 EC MEKK3^KO sample. ****P < .0001. (F) The proportion of AE, HEC, and IAHC cells within Ter119⁻CD61^low/−CD31⁺CD144⁺CD44⁺ cells in E10.0 EC MEKK3^KO AGM regions compared with littermate controls as determined by flow cytometry. (See supplemental Figure 5D for gating strategy.) Error bars represent mean ± SD. N = 4 experiments using pooled embryos. ****P < .0001; **P < .01.