Figure 4. Mef2c interaction is required for SHF-specific expression of mNkx2.5-SHF enhancer in OFT and RV.

Shown in A–D are representative transient whole mount and section expression data for mNkx2.5-SHF-lacZ reporter transgenes combining the conserved 5’ enhancer from mouse Nkx2.5 and the chicken Nkx2.5 proximal promoter. Expression at E11.0 is observed in branchial arch ectoderm and mesoderm, and in the SHF derivatives of OFT and RV in the wild-type transgene mNkx2.5-SHF-lacZ (mSHF WT). Planes of section in C and D are shown in A by white dashed lines c and d. E. EMSA demonstrates preferential binding of Mef2c to the CArG consensus in the conserved 5’ enhancer of mouse Nkx2.5 over SRF (compare lane 3 to lane 4 and to binding of Mef2c to control Mef2c site in control lanes 8 and 9). Binding is lost upon mutation of the central AT residues in the 5’ mouse enhancer site (lane 4–6; 10–11). F. ChIP experiments show that Mef2c is associated with the 5’ mNkx2.5 SHF enhancer region and mNkx2.5 promoter proximal region in E10.5 heart tissue (Hrt) vs. pharyngeal arch tissue. G. While overexpression of Mef2c alone has a modest impact on mNkx2.5-SHF lux activity, much greater enhancement of basal and BMP-activated expression is observed with combined expression of Mef2c with SRF, Myocardin, p300 and cardiogenic GATAs. Increased activation is lost with Mef2c CArG box mutation (right-most columns). H. Mutation of Mef2c binding CArG box consensus results in loss of mNkx2.5-SHF enhancer activity. Representative Xgal staining pattern of E10.5 mouse embryo transgenic for mNkx2.5-SHF m2c-lacZ transgenic reporter bearing a point mutation of the consensus Mef2c binding site (mSHF m2c). LacZ expression is lost in OFT and RV while expression in PA SHF mesoderm is relatively maintained (arrow). Embryonic stages are shown in lower left. Abbreviations are as in previous figures.