Fig. 1. EC-specific gain-of-function allele of Notch4 elicits DA enlargement and CV underdevelopment. (A,B) Whole-mount CD31 staining shows enlarged DA and underdeveloped CV in the trunk region of embryos expressing int3 in ECs at E9.0 (18 ss). Arrows, DA; arrowheads, anterior CV (ACV). (C,D) Higher magnifications of A, B, respectively. Arrows and white brackets, DA; arrowheads and blue brackets, ACV. (E,F) CD31 staining (red) of cross-sections of A, B, respectively, confirms enlarged DA and underdeveloped CV in embryos expressing EC-specific int3. Arrows, DA; arrowheads, ACV. (G,H) Quantitative analysis of EC distribution. Total ECs, including those in the DA, primordial CV and capillaries, were counted from cross-sections of the anterior region of E8.75 (15-16 ss) embryos. A total of 3328 and 3334 ECs were counted in control and mutant embryos, respectively. Total EC number between mutant and control is comparable (n=5, P=0.94). The proportion of ECs in DA (da, red) to primordial ACV including capillaries (p-acv, blue) is significantly increased (n=5; ^*P=0.02) in mutants (H) when compared with controls (G). (I,J) Whole-mount lacZ staining of the Tie2-lacZ reporter identifies head vessels at E10.5. Females were treated with tetracycline water (500 µg/ml) until E7.5, and embryos were collected at E10.5. Internal carotid arteries (yellow arrows) are enlarged, and head veins are reduced (yellow arrowheads) in embryos expressing EC-specific int3 (J). Scale bars: 600 µm in B; 200 µm in D,F,J.