Cell-cycle progression is initiated before IAHC formation. (A) Force directed graph (FDG) visualization of all cells in the EHT continuum. (B) Density plot showing the distribution of cells across inferred trajectory (pseudotime: scaled from 0 to 100). (C) Left: Heatmap illustrating the differentially expressed genes (DEGs) between the heterozygous cells in C5:pre-HE and C6:HE based on 1.4 Log₂ fold change and false discovery rate <0.05. Right: Panther pathway analysis of the upregulated genes in each cluster. (D) Distribution of the cell-cycle phase in indicated clusters. (E,G) A magnified image of immunofluorescent staining on frozen E10.5 dorsal aorta sections for CD31 (red), Gfi1:Tomato (cyan), and CCNB1 (E, yellow) or KI67 (G, yellow). (F,H) Quantification of CCNB1 (F) and KI67 (H) signal intensity in CD31 endothelial cells lining the entire dorsal aorta. Individual cells and medians are shown; gray boxes indicate the upper (75%) and lower (25%) percentile; whiskers indicate ±1.5 times the interquartile range (IQR). Two-tailed t test. *P < .05, ***P < .001.