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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.
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