Fig. 8. Ccm1 lies genetically upstream of Notch signaling and downstream of Vegf. (A-F) Whole-mount in situ hybridization of Ccm1^+/+ and Ccm1^-/- tissues at E8.5, before the mutant phenotype can be distinguished grossly. (A-D) Disruption of arterial Notch gene expression is demonstrated using probes for Dll4 and Notch4. (A,B) Hybridization with a probe for the Notch ligand Dll4 shows marked downregulation of Dll4 transcript throughout the dorsal aorta of Ccm1^-/- embryos, compared with normal controls. (C,D) A decrease in Notch4 transcript is evident in the branchial arch artery and proximal aorta of a Ccm1^-/- embryo. The Notch4 signal intensity was similar, although weak, for the caudal aorta of both the wild type and the mutant. (E,F) In situ hybridization with a probe for Vegfa shows a similar intensity of ubiquitous signal from both Ccm1^+/+ and Ccm1^-/- embryos. (G) Quantification of transcript levels by real-time quantitative PCR at E8.8. Comparison of three pairs of Ccm1^+/+ and Ccm1^-/- embryo cDNA samples confirmed downregulation of Efnb2 expression (despite intact extravascular domains of expression shown in Fig. 7A-D). Marked downregulation of Dll4 with modest downregulation of Notch4 was also observed, in agreement with the in situ hybridization data. Transcript levels for Vegfa were similar between genotypes.