Activated c-Kit signaling is functionally required for the enhanced hemogenesis in Tie2-Cre;Smad4^fl/fl embryos. (A) Representative FACS analysis of the caudal half cells from Tie2-Cre;Smad4^fl/fl and control embryos, showing obviously increased proportion of CD41⁻CD45^-CD31⁺c-Kit⁺ cells (red quadrant) in the Smad4 mutants (left). The graphs to the right show the percentages of the putative endothelium (CD31⁺CD41⁻CD45⁻) and relative c-Kit⁺ and c-Kit⁻ proportions within the CD31⁺CD41⁻CD45⁻ population (n = 8). (B) Real-time PCR analysis of c-Kit expression in the sorted Tie2⁺ populations derived from the caudal half of E9.5 embryos (n = 3). (C) Real-time PCR analysis of c-Kit expression in the immortalized endothelial cells derived from Smad4 mutant and control embryos (n = 3). Relative expression fold to that of control embryos is shown. (D) Real-time PCR analysis of c-Kit expression in E9.5 primary embryonic cells treated with BMP4 for the indicated periods (n = 3). (E) Real-time PCR analysis of c-Kit expression in the immortalized embryonic endothelium line treated by the indicated regents for 24 hours (n = 3). Relative expression fold to that of control group is shown. (F) AGM cultures derived from E9.5 embryos were performed and BMP4 and/or imatinib were added as indicated. Graph showing the percentages of different subpopulations in the cultures determined by FACS analysis (n = 3). (G) Graph showing the hemogenic colonies generated in the AGM cultures from Tie2-Cre;Smad4^fl/fl and control embryos with or without imatinib (n = 4). (H) Tie2⁺ cells from the caudal half of Tie2-Cre;Smad4^fl/fl and control embryos were cultured on OP9 with or without imatinib for 7 to 9 days. Representative FACS analysis of cells generated in the cultures is shown (left). The graph to the right denotes the numbers of CD45⁺ cells generated in the cultures (n = 3). Data are mean ± standard deviation. h, hours; NS, not significant. *P < .05; **P < .01.