ER71 regulates Flk-1⁺PDGFRα⁻ hemangiogenic and Flk-1⁺PDGFRα⁺ mesoderm development. (A) PCR genotyping is shown for Er71^+/+, Er71^+/−, and Er71^−/− ES cells and tail DNA from Er71^+/− mice. (B) Er71^+/+ and Er71^−/− EBs were subjected to blast colony-forming cells (day 3) and primitive erythroid colony (Eryp) replating (day 4). (C-D) Day 6 Er71^+/+ and Er71^−/− EBs were analyzed for the expression of hematopoietic and endothelial cell markers. Numbers indicate the percentage of live cells gated within each quadrant. (E) Er71^+/+ and Er71^−/− EBs were analyzed for Flk-1 and PDGFRα expression at the indicated times. Representative results from 9 experiments are shown. (F-G) Flk-1⁺PDGFRα⁺ and Flk-1⁺PDGFRα⁻ cell populations were sorted from day 3.5 A2 EBs. Expression of T/Brachyury (T/Bry) and Er71 (F) as well as Isl1, Mesp1, Scl, and Lmo2 (G) were analyzed by qRT-PCR. Unsorted day 3.5 EBs were used for comparison. (H) Differentiated progeny cells of the sorted Flk-1⁺PDGFRα⁺ and Flk-1⁺PDGFRα⁻ cell populations cultured on OP9 cells were analyzed by qRT-PCR. (I) Flk-1 and PDGFRα expression in iEr71 EBs (± DOX) were analyzed as in panel E. Data are presented as the means ± SD of at least 4 independent experiments.