Csk^–/– embryos display branching defects during vascular development. (A-B) X-gal–stained yolk sac membranes from Csk^+/+, Flk-1^+/– and Csk^–/–, Flk-1^+/– embryos at E8.2. Black arrowheads in panel B show examples of unsuccessful sprouting. Note that blood vessels in panel B are, in general, larger than those in panel A. White arrowhead points to a large vessel. To ensure that the observed defects in Csk^–/–, Flk-1^+/– embryos were not due to Flk-1^+/– heterozygosity, immunohistochemical (IHC) staining was also performed with anti–Flk-1 monoclonal antibody (BD-Pharmingen, San Diego, CA) on Csk^+/+ and Csk^–/– embryos resulting from Csk^+/– × Csk^+/– mating. Furthermore, Csk^+/– mice were also crossed with Flt-1^+/– mice to label endothelial cells with a different marker. Results from these experiments (not shown) were consistent with those shown in this figure. (C-D) Yolk sac membranes from E8.5 embryos. Note Csk^–/– yolk sacs have fewer but larger blood vessels than the healthy control. Arrowhead points to one blood vessel with increased caliber. (E-F) Paraffin sections of anti-Csk–stained yolk sacs. These 2 panels illustrate 2 points: (1) vascular lumens are enlarged in Csk^–/– yolk sacs; and (2) Csk is expressed in endothelial cells (arrows in E). (G-H) E8.5 embryo proper, showing dorsal aortae and intersomitic vessels (arrowhead). In the mutant, intersomitic vessels do not successfully sprout from the dorsal aortae. The arrowhead in panel H indicates unsuccessful sprouting. (I-J) show the ventricle of E8.5 embryos. In healthy embryos (I), ventricular endocardium (blue) form branches (arrowhead). In Csk^–/– mutant, the endocardium (blue staining pointed by arrowhead) does not branch. (K-L) Whole mount, anti-Csk IHC-stained yolk sac membranes. Sections shown in panels E and F were derived from K and L, respectively. Bars in panels A-D and G-L represent 200 μm; bars in panels E-F represent 50 μm.