Expression of β-galactosidase from the flt-1 locus reveals heterogenous endothelial cell expression in developing vessels. Day 8 differentiated cultures were fixed and stained with antibodies to β-galactosidase and PECAM-1. (A–H) flt-1^−/− mutant vessels. (I–P) flt-1^+/− heterozygous (phenotypically normal) vessels. ES-derived vessels were visualized by staining for β-galactosidase in red (A, E, I, and M), PECAM-1 in green (B, F, J, and N), and DRAQ 5 in blue to visualize nuclei (C, G, K, and O). In A, E, I, and M, red arrows denote endothelial cells with moderate to high levels of β-galactosidase, and red arrowheads denote endothelial cells with none to low levels of β-galactosidase. All panels are z-stack compilations of 12-μm thickness to avoid sampling heterogeneity, with the exception of K and O, which are single confocal images from the stack. Bars, 10 μm.