The flt-1^-/- phenotype is partially rescued non-cell-autonomously and by an sflt-1 transgene. (A) Coculture analysis. Day-3 EBs were plated in wells so that (i) 100% were flt-1^-/- mutant EBs or (ii) 75% were WT and 25% were flt-1^-/- mutant EBs, and after attachment and incubation for a further 5 days, cultures were fixed and stained for β-galactosidase activity. Original magnification × 10. (B) Day-8 WT (Bi-ii), flt-1^-/- transfected with a PECAM-sflt transgene (Biii-iv), and flt-1^-/- (Bv-vi) ES differentiation cultures were fixed and reacted with an anti-PECAM antibody. Original magnifications × 10 (Bi,iii,v) and × 20 (Bii,iv,vi). Note that branching in flt-1^{-/-PECAM-sflt} cultures more closely resembles WT than flt-1^-/- cultures. (C) The average number of branch points per mm of vessel length was determined using multiple fields of each genotype. ^**P < .01, flt-1^-/- versus flt-1^{-/-PECAM-sflt}; ^***P < .001, WT versus flt-1^-/-.