Fig. 5. Efnb2 loss-of-function mutation elicits enlarged DA and underdeveloped CV, resembling Notch gain-of-function mutant morphology. (A,B) Whole-mount CD31 staining of E9.0 (17 ss) embryos. Arrows, DA; arrowheads, CV. (C,D) Higher magnifications of A, B, respectively. Note the enlarged DA (arrows and white brackets) and underdeveloped CV (arrowheads and blue brackets) in the Efnb2-deficient embryo (B,D). (E,F) Quantitative analysis of EC distribution. Total ECs, including those in the DA, CV and capillaries, were counted from the cross-sections of the anterior region of E8.75 (15-17 ss) embryos. A total of 2122 and 1714 ECs were counted in control and mutant embryos, respectively. Total EC number between mutants and controls is decreased (n=3, P=0.02). The proportion of ECs in DA (da, red) over primordial ACVs (p-acv, blue) is significantly increased (n=3; ^*P=0.02) in mutants (F), when compared with controls (E). (G,H) CD31 (red) and EphB4 (green) staining of cross-sections of E8.75 (15 ss) embryos. EphB4⁺ ECs are present in the DA (arrow) of the enlarged Efnb2-deficient DA (H). (I,J) Whole-mount CD31 staining shows enlarged DA and reduced CV in E8.75 (16 ss) embryos with Tie1-cre-mediated deletion of Efnb2 (J). (K,L) Higher magnifications of I, J, respectively. Arrows and white brackets, DA; Arrowheads and blue brackets, ACV. Scale bars: 200 µm.