AN HF-1A HF-1B/MEF-2 COMBINATORIAL ELEMENT CONFERS CARDIAC VENTRICULAR SPECIFICITY AND ESTABLISHES AN ANTERIOR-POSTERIOR GRADIENT OF EXPRESSION/

The molecular determinants that direct gene expression to the ventricl es of the heart are for the most part unknown. Additionally, little da ta is available on how the anterior/posterior axis of the heart tube i s determined and whether the left and right atrial and ventricular cha mbers are assigned as part of this process. Utilizing myosin light cha in-2 ventricular promoter/beta-galactosidase reporter transgenes, we h ave determined the minimal cis-acting sequences required for ventricul ar-specific gene expression. In multiple independent transgenic mouse lines, we found that both a 250 base pair myosin light chain-2 ventric ular promoter fragment, as well as a dimerized 28 bp sub-element (HF-1 ) containing binding sites for HF1a and HF1b/MEF2 factors, directed ve ntricular-specific reporter expression from as early as the endogenous gene, at day 7.5-8.0 post coitum. While the endogenous gene is expres sed uniformly throughout both ventricles, the transgenes were expresse d in a right ventricular/conotruncal dominant fashion, suggesting that they contain only a subset of the elements which respond to positiona l information in the developing heart tube. Expression of the transgen e was cell autonomous and its temporospatial characteristics not affec ted by mouse strain/methylation state of the genome. To determine whet her ventricular-specific expression of the transgene was dependent upo n regulatory genes required for correct ventricular differentiation, t he 250 base pair transgene was bred into both retinoid X receptor alph a and Nkx2-5 null backgrounds. The transgene was expressed in both mut ant backgrounds, despite the absence of endogenous myosin light chain- 2 ventricular transcript in Nkx2-5 null embryos. Ventricular specifica tion, as judged by transgene expression, appeared to occur normally in both mutants. Thus, the HF-1 element, directs chamber-specific transc ription of a transgene reporter independently of retinoid X receptor a lpha and Nkx2-5, and defines a minimal combinatorial pathway for ventr icular chamber gene expression. The patterned expression of this trans gene may provide a model system in which to investigate the cues that dictate anterior-posterior (right ventricle/left ventricle) gradients during mammalian heart development.