Fig. 3. Vascular defects in Mib1^–/– embryos. (A-D) Whole-mount Flk1 antibody staining of E9.0 wild-type (A,C) and Mib1^–/– (B,D) embryos. Mib1^–/– embryos have relatively thin and disorganized blood vessels. (A,B) Lateral view of the head; (C,D) Dorsal view of the trunk. (E-N) Transverse sections of E9.0 wild-type (E,G,I,K,M) and Mib1^–/– (F,H,J,L,N) embryos, stained with an anti-PECAM antibody (E,F) or labeled by in situ hybridization with specific probes for ephrin B2 (G,H), Sm22 (I,J), Dll4 (K,L) and Hey1 (M,N). The PECAM-stained sections revealed the marked reduction or loss of the dorsal aorta (da) in the Mib1^–/– embryos. The lack of vascular ephrin B2 expression (H; inset), smooth muscle cell recruitment (J; inset), and Hey1 expression (N; inset) in the Mib1^–/– embryos is evident, whereas the Dll4 expression is normal (L; inset). (O,P) Expression of vascular Notch target genes (Hey1 and Hey2) and genes for vasculogenesis (ephrin B2, EphB4, Vegf and Shh). Total RNA from E9.0 wild-type and Mib1^–/–yolk sacs was analyzed by semi-quantitative RT-PCR (O). The expression of Hey1 and Hey2 was analyzed by real-time quantitative RT-PCR (P). The numbers on each bar indicate the mean fold of induction, and the error bars indicate the standard deviation. ß-actin was used for normalization. The results are representative of three independent experiments. ***P<0.0001, *P<0.01.