Endothelial cell divisions are oriented perpendicular to the vessel long axis in ES cell–derived vessels. Mouse ES cells (WT; Tg PECAM-H2B-GFP) differentiated to day 7 to 8 were imaged for several hours prior to fixation and staining for PECAM-1. (A) A confocal image showing H2B-GFP signal in green (i), the same field with PECAM-1 stain in red after fixation (ii), and the overlay of the 2 images (iii), showing the H2B-GFP signal and PECAM-1 stain in the same cells. (B) The top portion of the images in panel A, showing time lapse images from 0 minute (i) to 292 minutes (vi). Panel iv was used to calculate the angle of division relative to the vessel axis, according to the drawn yellow lines. The numbers in the lower right represent elapsed time in minutes. (C) Calculation of division angles relative to the vessel long axis. Top panel was colorized as in Figure 1, except the chromosomes are blue. The 90-degree division angle is perpendicular to the vessel long axis, and the 0-degree division angle is parallel to the vessel long axis; n = 125 divisions. (D) Representation of endothelial division angles, with the vessel long axis diagrammed by the long horizontal lines. Each shorter line represents 3 angle measurements that were close or equivalent to each other. For a video of panel B, see Video S1 (available on the Blood website; see the Supplemental Videos link at the top of the online article).