GFP expression allows for visualization of mammalian sprouting angiogenesis. Stable ES cell lines expressing PECAM-GFP were generated (see “Materials and methods”) and subjected to time-lapse image analysis of vascular processes for 4.5 to 9.5 hours. (A) Day-8 WT-eGFP (+/+, i-iii) or flt-1^-/--eGFP (-/-, iv-ix) ES differentiation cultures were imaged by time-lapse at 1-minute intervals for 4.5 to 9.5 hours, then fixed and reacted with an anti-PECAM antibody. The last frame captured (i,iv,vii) was overlayed (iii,vi,ix) with the PECAM-stained image (ii,v,viii). Original magnification × 20. (B) Time-lapse frames of a day-8 WT-eGFP ES differentiation culture showing the formation of a vascular sprout and its fusion with another sprout. Time in minutes is at bottom left of each frame. The white arrowhead indicates sprout formation and extension; the white arrow, the fusion of the sprout tip with the second sprout. Scale bar in panel B is 20 μm (Supplemental Movie S1).