Increased activation of ERK is responsible for the enhanced hemogenesis in Tie2-Cre;Smad4^fl/fl embryos. (A) Graphs showing the absolute number of recovered cells, the cell viability determined by 7-AAD staining, and the frequencies and total numbers of CD45⁺ cells generated in the AGM cultures derived from E9.5 embryos (n = 4). BMP4 and/or PD 98059 were added as indicated. (B) Caudal half of E9.5 embryos were cultured as explants in the presence of BMP4 and/or PD 98059. Representative FACS analysis for CD31⁺c-Kit^high expression is shown (left). The graphs to the right denote the cellularity recovered and the percentages of the gated populations in the explant cultures (n = 3). (C) Immunohistochemistry staining of pERK in the aorta region of Tie2-Cre;Smad4^fl/fl and control embryos, showing apparently increased expression of endothelial pERK in the mutants. Arrowheads indicate the pERK-positive aortic endothelium in the control embryos. The diagram on the left illustrates locations analyzed. Scale bars: 50 μm. (D) AGM cultures derived from E9.5 embryos were performed and PD 98059 was added as indicated. Representative FACS analysis for CD45 expression is shown (upper). The graphs to the lower denote the frequencies and the total numbers of CD45⁺ cell generated in the cultures (n = 4). Data are mean ± SD. DA, dorsal aorta; NS, not significant. *P < .05; **P < .01.