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Fig. 3. Identification of an evolutionarily conserved minimal enhancer element sufficient to direct expression in heart and smooth muscle. (A) Representative expression pattern of F0 transgenic embryos with the two minimal transcriptional enhancers (MyE6 and MyE8). Whereas MyE6 directed lacZ expression to the heart, the 350 bp MyE8 fragment was sufficient to direct expression in heart and developing dorsal aorta and head vessels at E12.5. (B) Transgenic construct used to identify minimal elements of the myocardin enhancer (MyE). The construct numbers (MyE1-MyE9) are indicated on the left and fractions of F0 transgenic embryos showing cardiac expression at E12.5 are indicated in the right column. The 10 kb enhancer fragment MyE1 is identical to construct 5 in Fig. 1. An evolutionary conservation track shows the overall conservation score of MyE1 across all species, as well as pairwise alignment of rat, human, dog and chicken, each aligned to the mouse genome (Markov model, http://genome.ucsc.edu) (Kent et al., 2002). By transgenic analysis two evolutionarily conserved minimal enhancer elements, indicated in red, were identified (MyE6 and MyE8; 2 kb and 350 bp, respectively). Note that MyE8, but not MyE6, is conserved in the chicken. (C) Alignment of conserved mouse and human sequences in the myocardin enhancer MyE8. The conserved Mef2-binding site (blue shading), five candidate Foxo sites (yellow shading) and a conserved Tead-binding site (green shading) are noted.

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