Figure 1
Figure 1. Enhanced hemogenic activity of Smad4-deficient endothelium in vivo and in vitro. (A) Sections of β-D-galactosidase (LacZ)-stained Tie2-Cre;ROSA-LacZ double transgenic embryos at 22 sp. Scale bars: 50 μm. (B) Whole mount immunostaining of c-Kit (red) and CD31 (green) at 18 sp. Confocal images of the embryonic vitelline arteries (VA, upper, three-dimensional reconstructions with totally 15 μm thickness) and dorsal aortas (DA) (lower, representative single images) are shown. c-Kit+CD31+ clusters (upper, arrowheads) or cluster cells (lower, arrowheads) attached to the ventral side of CD31+ endothelial layer are shown. The arrow indicates a c-Kit+CD31− circulating cell in the aorta. Scale bars: 20 μm. (C) Immunohistochemistry staining of Runx1 in the aorta region of Tie2-Cre;Smad4fl/fl and control embryos at 20 sp. Continuous sections for each genotype are shown. Arrowheads indicate the Runx1+ intra-aortic clusters with more than 5 cells exclusively in the Tie2-Cre;Smad4fl/fl embryos. The diagram on the left illustrates the location analyzed. Scale bars: 50 μm. (D) Representative FACS analysis of caudal half cells for c-Kit and CD31 expression (left). The graph to the right shows percentages of the gated populations (n = 8). (E) Real-time PCR analysis of RNA extracts from E9.5 caudal half. Relative expression fold to that of control embryos is shown (n = 4). (F) LacZ stained AGM cultures derived from E9.5 ROSA-LacZ transgenic embryos without (left) or with (middle and right) Tie2-Cre transgene. Blue cycles indicate individual colonies. Magnification (right) of the red boxed region in the macroscopic view (middle) is shown. Scale bars: left and middle, 200 mm; right, 100 μm. (G) Representative FACS analysis of the cells recovered from the E9.5 AGM culture. (H) CD31 (red) and CD45 (green) double immunostaining of an E9.5 AGM culture. Arrowheads denote the “budding” cells co-expressed CD31 and CD45 with the CD45 signals enriched in the detaching parts. Scale bars: left, 100 μm; right, 50 μm. (I-J) Graphs showing the numbers of colonies generated in the AGM cultures from Tie2-Cre;Smad4fl/fl and control embryos (n = 4). (K) Representative FACS analysis of cells derived from the AGM cultures (left). The graph to the right shows the percentages of different subpopulations (n = 3). Data are mean ± standard deviation. DA, dorsal aorta; ee, embryo equivalent; me, mesenchymal cells surrounding the vitelline artery; nt, neural tube; SR, serum replacement; VA, vitelline artery. **P < .01, compared with control embryos.

Enhanced hemogenic activity of Smad4-deficient endothelium in vivo and in vitro. (A) Sections of β-D-galactosidase (LacZ)-stained Tie2-Cre;ROSA-LacZ double transgenic embryos at 22 sp. Scale bars: 50 μm. (B) Whole mount immunostaining of c-Kit (red) and CD31 (green) at 18 sp. Confocal images of the embryonic vitelline arteries (VA, upper, three-dimensional reconstructions with totally 15 μm thickness) and dorsal aortas (DA) (lower, representative single images) are shown. c-Kit+CD31+ clusters (upper, arrowheads) or cluster cells (lower, arrowheads) attached to the ventral side of CD31+ endothelial layer are shown. The arrow indicates a c-Kit+CD31− circulating cell in the aorta. Scale bars: 20 μm. (C) Immunohistochemistry staining of Runx1 in the aorta region of Tie2-Cre;Smad4fl/fl and control embryos at 20 sp. Continuous sections for each genotype are shown. Arrowheads indicate the Runx1+ intra-aortic clusters with more than 5 cells exclusively in the Tie2-Cre;Smad4fl/fl embryos. The diagram on the left illustrates the location analyzed. Scale bars: 50 μm. (D) Representative FACS analysis of caudal half cells for c-Kit and CD31 expression (left). The graph to the right shows percentages of the gated populations (n = 8). (E) Real-time PCR analysis of RNA extracts from E9.5 caudal half. Relative expression fold to that of control embryos is shown (n = 4). (F) LacZ stained AGM cultures derived from E9.5 ROSA-LacZ transgenic embryos without (left) or with (middle and right) Tie2-Cre transgene. Blue cycles indicate individual colonies. Magnification (right) of the red boxed region in the macroscopic view (middle) is shown. Scale bars: left and middle, 200 mm; right, 100 μm. (G) Representative FACS analysis of the cells recovered from the E9.5 AGM culture. (H) CD31 (red) and CD45 (green) double immunostaining of an E9.5 AGM culture. Arrowheads denote the “budding” cells co-expressed CD31 and CD45 with the CD45 signals enriched in the detaching parts. Scale bars: left, 100 μm; right, 50 μm. (I-J) Graphs showing the numbers of colonies generated in the AGM cultures from Tie2-Cre;Smad4fl/fl and control embryos (n = 4). (K) Representative FACS analysis of cells derived from the AGM cultures (left). The graph to the right shows the percentages of different subpopulations (n = 3). Data are mean ± standard deviation. DA, dorsal aorta; ee, embryo equivalent; me, mesenchymal cells surrounding the vitelline artery; nt, neural tube; SR, serum replacement; VA, vitelline artery. **P < .01, compared with control embryos.

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