ROSA26-targeted sFlt-1 isoform transgene preferentially rescues the mosaic endothelial expression of pFlk (Tyr-1173). Day 8 differentiated cultures were fixed and stained with antibodies to total Flk-1 and pFlk (Tyr-1173/1175). (A) ES-derived vessels were visualized by staining for pFlk in green (a, e, I, and m), total Flk in red (b, f, j, and n), and DRAQ 5 in blue to visualize nuclei (c, g, k, and o). In a–c, e–g, i–k, and m-o, red asterisks denote endothelial cells with moderate to high levels of pFlk, and yellow asterisks denote endothelial cells with none to low levels of pFlk. Note that pFlk-positive endothelial cells are mosaic in WT (a–d), and flt-1^−/− endothelial cells have more uniform high levels of pFlk (e–h). Expression of the sFlt-1 transgene (i–l) recreates the heterogeneous pattern of pFlk vessel staining, whereas expression of the mFlt-1 transgene (m–p) does not lead to heterogeneity of staining. (B) Stained cultures were visualized for total Flk, and the same cells were scored as positive or negative for pFlk. Percentages of a representative experiment are shown on the y axis. (C) Individual endothelial cells were outlined and analyzed for the ratio of pFlk/total Flk using imaging software. The numbers were graphed in order of descending ratios for each genotype. In each graph, the darker areas show the number of cells that had pFlk/total Flk ratios between 0.2 and 0.3. Bar, 10 μm.