ROSA26-targeted Flt-1 isoforms differentially rescue flt-1^−/− mutant vessel parameters. (A and B) Day 8 differentiated ES cell cultures were reacted with the PECAM antibody, and representative areas were analyzed for vessel area (A) or branch point frequency (B). (A) The mean vessel area of all rescue clones was significantly different from WT and flt-1^−/− mutant clones (flt-1^−/− vs. sFlt and mFlt, P ≤ 0.0005; WT vs. sFlt and mFlt, P ≤ 0.02) but did not differ between sFlt-1 and mFlt-1 rescue clones (sFlt vs. mFlt, P = 0.86). (B) The mean branch points/millimeter vessel length was significantly different between sFlt-1 and mFlt-1 rescue clones (sFlt vs. mFlt, **, P ≤ 0.03). Branch points/millimeter vessel length was not significantly different between flt-1^−/− mutant and mFlt clones (flt-1^−/− vs. mFlt, P = 0.97; WT vs. mFlt, P ≤ 0.01). Branch points/millimeter vessel length was significantly different between flt-1^−/− mutant and sFlt isoform clones (flt-1^−/− vs. sFlt, P ≤ 0.01; WT vs. sFlt, P = 0.16). Error bars represent SEM.