Sprout formation and migration are reduced in the absence of flt-1 Day-8 WT-eGFP (+/+; Ai-iii, Ci-iii) or flt-1^-/--eGFP (-/-; Aiv-vi, Civ-vi) ES differentiation cultures were imaged by time-lapse at 1-minute intervals for the indicated times. (A) Over a 10-hour period, numerous new WT sprouts (i-iii) formed (white arrowheads) and remained or extended and/or fused at later times (black arrowheads), whereas very few flt-1^-/- sprouts (iv-vi) formed during the same time period. Scale bar is 20 μm (Supplemental Movies S2-S3). (B) Movie frames were analyzed to determine a sprout index (no. of sprouts/mm/h) for multiple movies on day 6 (d6) or day 8 (d8) of differentiation. Gray dots indicate WT-eGFP sprout indices; black dots, flt-1^-/--eGFP sprout indices; and squares with lines, the average of each group. ^*P < .03; ^**P < .01. (C) Over a 40-minute period, a WT sprout formed and extended between 30 and 40 μm, while over a 100-minute period a flt-1^-/- mutant sprout formed and extended only 20 μm. White arrowheads point to sprout tips, and time in minutes is at bottom left. Scale bar = 20 μm (Supplemental Movies S4-S5). (D) Movie frames were analyzed to determine average sprout velocity (μm/h) for multiple sprouts from different movies on day 8 (d8) of differentiation. Gray dots indicate WT-eGFP average sprout velocities; black dots, flt-1^-/--eGFP average sprout velocities; and squares with lines, the average of each group. ^***P < .001.