Fig. 4. Proximal narrowing limits flow into dilated distal vessels of Ccm1^-/- mice. (A-D) Confocal immunofluorescent detection of the endothelial antigen Pecam. (A,B) Composite whole-mount images of E8.5 Ccm1^+/+ and Ccm1^-/- embryos. The amnion and yolk sac have been removed, allowing the embryos to be extended (some kinking of the midpoint of the Ccm1^+/+ embryo resulted). Note the enlarged diameter of the dorsal aorta of the caudal Ccm1^-/- embryo (arrow in B). The intersomitic arteries that extend dorsally from the aorta are dilated and more prominent in Ccm1^-/- embryos. (C,D) Higher magnification views of the first branchial arch artery and proximal aorta (see boxes in A and B). The wild-type embryo has a uniform, broad dorsal aorta (double arrow in C). By contrast, the Ccm1^-/- embryo shows narrowing of the branchial arch artery and adjacent dorsal aorta (single arrows in D). Distally, the aorta restores to a more normal diameter (double arrow in D). (E-H) Injection of India ink into the ventricle of embryonic hearts. (E,F) At E8.5, ink fills the first branchial arch artery and dorsal aorta of a wild-type embryo, eventually entering the head veins. Ink fails to enter the dorsal aorta of a Ccm1^-/- embryo, and instead flows in a retrograde manner through the sinus venosus and into the common cardinal vein (arrow in F). Yolk sacs were removed following injection to improve visualization. (G,H) At E9.5, injected ink flows primarily through the second and third branchial arch arteries to fill the dorsal aorta (arrowheads in G). Ink also fills the venous system of the embryo. By contrast, injection of a Ccm1^-/- embryo fails to opacify the dorsal aorta. A small amount of ink is observed in the first and second branchial arch arteries (arrowheads in H) with a minimal amount entering the adjacent aorta. Scale bars: 200 µm.