Several members of the sarcomeric myosin heavy chain (MHC) gene family have been mapped in the human genome but many of them have not yet been identified. In this study we report the identification of two human skeletal MHC genes as fast IIa and IIx MHC based on pattern of expression and sequence homology with the corresponding rat genes in the 3'-translated and untranslated regions. The distribution of these two gene products as well as that of the beta/slow MHC gene was analyzed in human skeletal muscles by in situ hybridization. The distribution of beta/slow, IIa, and IIx MHC transcripts defines three major muscle fiber types expressing a single MHC mRNA, i.e., either beta/slow, IIa, or IIx MHC mRNA, and two populations of hybrid fibers coexpressing beta/slow with IIa or IIa with IIx MHC mRNA. Fiber typing by ATPase histochemistry shows that IIa MHC transcripts are more abundant in histochemical type IIa fibers, whereas IIx MHC transcripts are more abundant in histochemical type IIb fibers.

During heart development, chamber myocardium forms locally from the embryonic myocardium of the tubular heart. The atrial natriuretic factor (ANF) gene is specifically expressed in this developing chamber myocardium and is one of the first hallmarks of chamber formation. We investigated the regulatory mechanism underlying this selective expression. Transgenic analysis shows that a small fragment of the ANF gene is responsible for the developmental pattern of endogenous ANF gene expression. Furthermore, this fragment is able to repress cardiac troponin I (cTnI) promoter activity selectively in the embryonic myocardium of the atrioventricular canal (AVC). In vivo inactivation of a T-box factor (TBE)- or NK2-homeobox factor binding element (NKE) within theANF fragment removed the repression in the AVC without affecting its chamber activity. The T-box family member Tbx2, encoding a transcriptional repressor, is expressed in the embryonic myocardium in a pattern mutually exclusive to ANF, thus suggesting a role in the suppression of ANF. Tbx2 formed a complex with Nkx2.5 on the ANF TBE–NKE, and was able to repress ANF promoter activity. Our data provide a potential mechanism for chamber-restricted gene activity in which the cooperative action of Tbx2 and Nkx2.5 inhibits expression in the AVC.

Specific regions of the embryonic heart tube differentiate into atrial and ventricular chamber myocardium, whereas the inflow tract, atrioventricular canal, inner curvatures, and outflow tract do not. These regions express Tbx2, a transcriptional repressor. Here, we tested its role in chamber formation. The temporal and spatial pattern of Tbx2 mRNA and protein expression in mouse hearts was found to be complementary to that of chamber myocardium‐specific genes Nppa, Cx40, Cx43, and Chisel, and was conserved in human. In vitro, Tbx2 repressed the activity of regulatory fragments of Cx40, Cx43, and Nppa. Hearts of transgenic embryos that expressed Tbx2 in the prechamber myocardium completely failed to form chambers and to express the chamber myocardium‐specific genes Nppa, Cx40, and Chisel, whereas other cardiac genes were normally expressed. These findings provide the first evidence that Tbx2 is a determinant in the local repression of chamber‐specific gene expression and chamber differentiation. Developmental Dynamics 229:763–770, 2004.