Fig. 6. Identification of Tead proteins as specific regulators of myocardin expression in branchial arch arteries and aorta. (A) A eukaryotic expression screen, in which we co-transfected MyE8 luciferase along with pools of 100 cDNA clones from a mouse E10.5 expression library revealed Tead2 as an activating cDNA of the minimal myocardin enhancer (well C12). (B) Transient transfections in COS cells show that Tead2 activates the MyE8-luciferase reporter but was less effective in activating the MyE8 reporter in which the predicted Tead-binding site (see Fig. 3C) was mutated. (C) Binding of Tead1 to the Tead-binding site in minimal enhancer element MyE8. A ³²P-labeled oligonucleotide containing the conserved Tead site and Tead1 translated in reticulocyte lysate was used for electrophoretic mobility shift assays. DNA binding was seen only in reactions containing lysates with Tead1. The DNA-Tead complex was supershifted using a Tead1-specific antibody and unlabeled wild-type (WT) oligonucleotide efficiently competed for DNA binding, whereas unlabeled mutant (Mut) oligonucleotide did not. (D) Requirement of the Tead-binding site for activity of the myocardin enhancer (MyE4) specifically in branchial arch arteries at E10.5. Two independent F0 transgenic embryos generated with construct MyE4, and two independent F0 embryos with MyE4 in which the Tead-binding site was mutated, are shown. Note that lacZ expression is absent in the branchial arch arteries of the MyE4-Tead mutant embryos indicated by arrows. (E) Transverse cross-sections of E10.5 transgenic embryos show that cardiac lacZ expression, which is highest in the ventricles, is very similar in wild-type and Tead mutants. LV, left ventricle; RV, right ventricle. (F) A cross-section through the outflow tract region shows lacZ expression in cells (asterisk) surrounding the aortic sac (as) and third branchial arch artery (baa3) of wild-type but not Tead mutant embryos. (G) lacZ expression can be appreciated in the dorsal aorta of wild-type embryos but not in the Tead mutant embryos.