Expression of the MFps protein partially restores migration and sprouting response of Flk1 null progenitors in vitro. (A) Schematic depiction of chicken–β-actin promoter-based construct used to overexpress the fps^MF cDNA in ES cells. (B) Western blot analysis of Flk1^lacZ/lacZ null ES cell clones demonstrated a 2- to 3-fold increase in MFps in 10 of 11 puro-resistant clones compared with endogenous wild-type Fps protein levels in Flk1 null clones transfected with the empty puromycin construct (protein concentrations were standardized against β-actin levels [lower blot in B]). (C-D) Flk1^lacZ/+ embryoid bodies showed endothelial migration and vascular channel formation, indicated by arrows. (E) Flk1^GFP/+ embryoid bodies demonstrated angiogenic sprouting activity, indicated by arrows. (F-G) Flk1^lacZ/lacZ embryoid bodies demonstrated dramatically reduced numbers of X-gal–stained cells and little migration or sprouting (arrowheads). (H) Similarly, Flk1^GFP/GFP embryoid body–derived cells showed little EGFP expression and no migration or sprouting activity (arrowheads). (I-J) MFps-expressing Flk1^lacZ/lacZ embryoid bodies showed an increased number of X-gal–stained cells (asterisk in I) and increased sprouting activity (arrow in J). (K) MFps-expressing Flk1^GFP/GFP embryoid body–derived cells displayed increased EGFP expression, increased sprouting (arrow), and evidence of aberrant cellular morphology (asterisk). (L) Control Flk1^lacZ/lacZ null EB-derived cells incubated with no primary antibody. Indirect PECAM immunofluorescence staining of Flk1^lacZ/lacZ EB-derived cells (M) and MFps-expressing, Flk1^lacZ/lacZ EB-derived cells (N).