Smad4 deficiency in the endothelium results in a gain-of-BMP4 function in hemogenesis. (A) Sorted Tie2⁺ and Tie2⁻ cells from the caudal half of Tie2-Cre;Smad4^fl/fl and control embryos, respectively, were reciprocally mixed in the AGM culture. Colonies generated (left, n = 4) and different subpopulations detected by FACS (right, n = 2) are shown. fl/+, Tie2-Cre;Smad4^fl/+; fl/fl, Tie2-Cre;Smad4^fl/fl. (B) Real-time PCR analysis of gene expression in the caudal half of Tie2-Cre;Smad4^fl/fl and control embryos. Relative expression fold to that of control embryos is shown (n = 3). (C) Immunofluorescence staining of BMP4 (green) and endomucin (red) in the aorta region of Tie2-Cre;Smad4^fl/fl and control embryos, showing the increased expression of BMP4 around the aorta of mutant embryo. Nuclei were stained with DAPI (blue). Scale bars: 50 μm. (D) Immunohistochemistry staining of pSmad1/5/8 in the aorta region of Tie2-Cre;Smad4^fl/fl and control embryos. Arrowheads indicate the pSmad1/5/8-positive aortic endothelium. Scale bars: 50 μm. (E) Real-time PCR analysis of gene expression in the sorted Tie2⁺ (left) and Tie2⁻ (right) populations derived from the caudal half of Tie2-Cre;Smad4^fl/fl and control embryos. Relative expression fold to that of control embryos is shown (n = 3). (F) Graph showing the hemogenic colonies generated in the AGM cultures derived from E9.5 embryos. BMP4 addition significantly increases the colony generation in the AGM cultures (n = 6). (G) Caudal half Tie2⁺ cells from E9.5 embryos were cultured on OP9-DL1 with or without BMP4 for 7 to 9 days. Representative FACS analysis of cells generated in the cultures is shown. Data are mean ± standard deviation. DA, dorsal aorta; nt, neural tube. *P < .05; **P < .01.