Fig. 3. Notch4 gain-of-function mutation promotes arterial marker expression in venous ECs. (A) CD31 (green) and BrdU (red) staining of DA cross-sections in E9.0 (13-15 ss) embryos prior to any apparent gross abnormalities in the mutant. Arrows, BrdU-labeled ECs. (B) Rate of BrdU incorporation in DA ECs at 13-15 ss suggests int3 does not affect proliferation in the enlarged DA. Data were analyzed by t-test and results are reported as mean±s.d. (n=3). A total of 2320 and 2074 ECs were counted in control and mutant embryos, respectively. (C,D) Cross-sections of E9.0 (16 ss) embryos expressing Efnb2-tauLacZ. lacZ-stained sections (blue) through the heart were counterstained with Eosin (pink). Ephrin B2 is expressed in the ACV (arrowheads) in the mutant (D) but not in the control (C). Arrows, DA. (E,F) CCV of 15 ss embryos stained for expression of Efnb2-tauLacZ (red) and EphB4 (green). Both markers are co-expressed in the mutant EC (F). (G,H) Cross-sections of 15 ss embryos after Dll4 in situ hybridization staining. Dll4 is expressed in the ACV (black arrowheads) and the CCV (blue arrowheads) in the mutant (H). Note the atretic anterior DA on the right side of the mutant, one of the occasional cases where the DA on one side was small. Scale bars: 10 µm in A; 25 µm in D,F; 100 µm in H.