Chimera and flow cytometry analysis of MFps-expressing Flk1^GFP/GFP ES cell–derived embryos confirmed the endothelial rescue potential of the MFps protein. (A) E10.5 Flk1^GFP/+ chimera showing strong ES cell contribution to the vascular endothelial lineage as judged by EGFP expression analysis by direct fluorescence microscopy. (B) E11.5 Flk1^GFP/GFP null chimera demonstrates a lack of contribution of Flk1 null ES cells to vascular lineage; however, this chimera shows strong contribution of Flk1 null cells to the heart as observed by the high levels of EGFP expression (asterisk in B). (C-H) E11.5 MFps-expressing Flk1^GFP/GFP chimeras show signs of vascular hemorrhage in head and neck (arrows in C) and hemangioma formation in the yolk sac (arrowheads in G-H). (F) Higher-power magnification of head region in panel E, showing EGFP-positive vascular-like channel formation. (I-J) Flow cytometry analysis of single-cell suspensions obtained from the embryo (I) and yolk sac (J) of control non-PECAM–stained embryos (left panels), PECAM-labeled Flk1^GFP/GFP chimeras (middle panels), and MFps-expressing Flk1^GFP/GFP chimeras (right panels). Substantial numbers of EGFP/PECAM double-positive endothelial cells were detected in the MFps-expressing Flk1^GFP/GFP-derived chimeric embryo and yolk sacs (circles in upper right quadrant of right panels) but not in the Flk1^GFP/GFP-derived populations (middle panels). Original magnification × 12 (A, C, G, H); × 20 (B, D); × 40 (E); and × 200 (F).