Fig. 2: EphB4 and ephrin-B2 modulate HUVEC heterogeneity and cell fate plasticity.
From: Eph-ephrin signaling couples endothelial cell sorting and arterial specification
a, b Reciprocal regulation of EphB4 and ephrin-B2. Immunoblot analysis of EphB4, ephrin-B2, transmembrane Notch1 (NOTCH1TM), cleaved Notch1 intracellular domain (N1ICD), Dll4, Sox17, VEGFR2 and GAPDH in siControl, siEPHB4, siEFNB2 and siEPHB4 + siEFNB2 HUVEC lysates (n = 3 experiments). c RT-qPCR for EPHB4 and EFNB2 gene expression after EPHB4 and EFNB2 KD in HUVECs (n = 3 experiments). d EphB4 upregulation in Efnb2iΔEC retinal vascular front. e EphB4 immunosignal quantitation (n = 3 control and 5 Efnb2iΔEC mice) and quantitation of EphB4/AP signal (n = 5 control and 7 Ephb4iΔEC mice). f Ephrin-B2 increase in Ephb4iΔEC P6 retina. Indirect ephrin-B2 detection using EphB4/AP protein. g 2D UMAP representation of unbiased Leiden clustering of merged siControl, siEPHB4 and siEFNB2 HUVEC scRNA-seq. h Z-normalized gene expression of top 5 marker genes per Leiden cluster. i Z-normalized gene expression of key cell type markers in Mitotic, Venous-like and Tip cell-like clusters. j 2D UMAP representation of unbiased Leiden sub-clustering. k Z-normalized gene expression of key cell type markers in Mitotic, Venous-like 1-3 and Tip cell-like 1-2 subclusters. l UMAP representation showing increased proportion of siEPHB4 cells in the Tip cell-like clusters relative to siControl. m Fraction distribution of siControl, siEPHB4 and siEFNB2 cells in HUVEC subclusters. n Upregulation of arterial and downregulation of venous genes in siEPHB4 cells. Arterial and venous markers selected from pseudobulk DGE analysis comparing siControl and siEPHB4 subpopulations. o Z-normalized gene expression comparing siControl, siEPHB4 and siEFNB2 HUVECs. p Arterial and venous markers selected from pseudobulk DGE analysis comparing siControl and siEFNB2 subpopulations. P values were calculated using one-way ANOVA (b, c) and two-tailed unpaired t test (e). In vivo experiments were performed with tamoxifen injections at P1-P3 with analysis at P6 (d, f). Data are presented as mean values ± SEM. Source data are provided as a Source Data file.
