FIG. 6.

The MEF2 site in the HRC enhancer is required for expression in cardiac, skeletal, and arterial smooth muscle in vivo. Wild-type MEF2 site (A to D and I to L) and mutant MEF2 site (E to H and M to P) HRC-lacZ transgenic mice were analyzed for expression in vivo. Representative X-Gal-stained, transgenic embryos are shown at 9.0 dpc (A and E), 11.5 dpc (B and F), 13.5 dpc (I and M), and 16.5 dpc (J and N). X-Gal-stained hearts are shown from transgenic embryos dissected at 16.5 dpc (K and O) or transgenic adults dissected at 16 weeks of age (L and P). The embryos in panels J and N have been skinned to help visualize the underlying skeletal muscle. Panels C, D, G, and H show transverse sections of transgenic embryos collected and X-Gal stained at 11.5 dpc. Bar, 100 μm. The 2,726-bp wild-type (wt) HRC enhancer construct directed lacZ expression to the heart throughout embryonic development (A and B). By 11.5 dpc, expression was restricted to the ventricles (C), and the ventricle-restricted cardiac expression continued in the fetal (K) and adult (L) heart. The mutant MEF2 2,726-bp HRC enhancer construct (mMEF2) failed to direct expression to the heart at any stage in the embryo (E to G), fetus (O), or adult (P). The wild-type construct directed strong expression to arterial smooth muscle, including the dorsal aorta, beginning at 11.5 dpc (D). Arterial smooth muscle expression was also evident in the fetus (K) and adult (L). Smooth muscle expression was restricted to arteries. Note that the esophagus staining in the adult (L) represents skeletal muscle in the adult esophagus. The MEF2 mutant enhancer failed to drive lacZ expression in smooth muscle at all stages, including 11.5 dpc (G and H), 16.5 dpc (O), and adult (P). The wild-type transgene was robustly expressed in skeletal muscle in both the hypaxial and epaxial domains at 11.5 dpc (B and C). The MEF2 mutant transgene was also expressed in both hypaxial and epaxial myotomes, but the level of expression was much weaker at 11.5 dpc (F and G). Both the wild-type (B and C) and mutant MEF2 (F and G) enhancers directed expression to the dorsal limb muscles at 11.5 dpc. The wild-type enhancer drove strong lacZ expression in all skeletal muscles at 13.5 dpc (I) and 16.5 dpc (J). The mutant MEF2 enhancer directed only very weak skeletal muscle expression at 13.5 dpc (M), and expression was essentially absent by 16.5 dpc (N). Ao, aorta; Ctd, carotid artery; CV, cardinal vein; DA, dorsal aorta; Es, esophagus; hrt, heart; LA, left atrium; LV, left ventricle; NT, neural tube; S, somite (myotome); RA, right atrium; RV, right ventricle; SubCL, subclavian artery; Tr, trachea. Arrowheads denote expression in dorsal limb muscles.