Fig. 3. Analysis of the tie-1 promoter for endothelial specificity in EBs and 2D culture. (A) tie-1-EGFP ES cells were aggregated into EBs and plated on gelatin in the presence of 10 ng/ml of rVEGF. Whole 10-day-old EBs were stained for CD31 (red) to visualize pseudovascular structures. Analysis of EGFP fluorescence showed localization of EGFP⁺ cells within the CD31-stained vascular network in EBs derived from the A1TG clone (top panel). The two photographs on the bottom represent staining of EBs derived from a neomycin-selected control ES-cell clone, which does not carry the tie-1-EGFP transgene. Bar, 100 µM. (B) 10-day-old EBs were enzymatically dissociated and single cells were plated on gelatin for 1 day. Immunofluorescence staining for CD31 confirmed that all EGFP⁺ cells were also CD31⁺. Note the presence of CD31⁺EGFP^- cell clumps, that possibly represent endothelial cell progenitor colonies. Bar, 50 µM. (C) tie-1-EGFP ES cells were plated on gelatin-coated slides in the presence of 10 ng/ml rVEGF. After 10 days of differentiation, slides were fixed and stained for CD31. In addition, cell nuclei were stained with DAPI. Bar, 50 µM.