Introduction The establishment and maintenance of transcriptional diversity during organogenesis is a key feature of embryonic development. Within the developing heart, myocardial cells in different cardiac chambers exhibit differences in gene expression, which reflect functional compartmentalisation (Christoffels et al., 2000). These transcriptional differences are established during early heart development and prefigure the formation of the specialised left and right atrial and ventricular chambers, which direct separate systemic and pulmonary blood flows. Molecular analysis of the regulatory circuits controlling cardiomyocyte diversity has identified a small number of cis- acting motifs and trans-acting factors involved in atrial versus ventricular identity and in differential gene expression between cells of the ventricular and atrioventricular canal myocardium (Wang et al., 2001; Habets et al., 2002). In addition, several cis- acting elements active in cardiomyocytes of either the left or right ventricle have been defined in transgenic mice (Schwartz and Olson, 1999; Kelly et al., 1999). Although the trans-acting factors that regulate such transgenes remain unknown, mutational analysis of transcription factors expressed throughout the heart has in some cases revealed compartment- restricted roles during early development (Lyons et al., 1995; Lin et al., 1997). A small number of cardiac transcription factors, in particular the basic helix-loop-helix (bHLH) proteins Hand1 and Hand2, and the T-box-containing regulatory factor Tbx5, show left/right differences in expression pattern in the embryonic ventricles, and the generation of null alleles in the Hand1, Hand2 and Tbx5 genes has shown that these factors are important in chamber morphogenesis (Srivastava et al., 1997; Firulli et al., 1998; Riley et al., 1998; Bruneau et al., 2001). Despite these studies, the molecular mechanisms that initiate and maintain left versus right ventricular specific gene expression are poorly understood. In particular, little is known about the factors regulating transcriptional differences between left and right ventricular chambers at later developmental stages. How left/right ventricular transcriptional differences are maintained is of major interest, as the fundamentally different roles of the two ventricles become apparent only on the separation of pulmonary and systemic circulatory systems at birth. Furthermore, in the adult, cardiac hypertrophy elicits changes in gene expression that differ in the left versus right ventricular free walls (Vikstrom et al., 1998). 5005 The molecular mechanisms that establish and maintain transcriptional differences between cardiomyocytes in the left and right ventricular chambers are unkown. We have previously analysed a myosin light chain 3f transgene containing an nlacZ reporter gene, which is transcribed in left but not right ventricular cardiomyocytes. In this report we examine the mechanisms involved in maintaining regionalised transgene expression. Primary cardiomyocytes prepared from left and right ventricular walls of transgenic mice were found to maintain transgene expression status in culture. However, similar cultures prepared from nontransgenic mice or rats show uniform expression after transient transfection of Mlc3f constructs, suggesting that the mechanism responsible for differential expression of the transgene between left and right ventricular cells does not operate on transiently introduced molecules. These data suggest that developmental cell history determines transgene expression status. Maintenance of transgene expression status is regulated by a cell-autonomous mechanism that is independent of DNA methylation, trichostatin A-sensitive histone deacetylation and miss-expression of transcription factors that are expressed in the left or right ventricles of the embryonic heart. Parallels between Mlc3f transgene repression in right ventricular cardiomyocytes and polycomb-mediated silencing in Drosophila suggest that Mlc3f regulatory sequences included on the transgene may contain a cellular memory module that is switched into an on or off state during early cardiogenesis. Epigenetic mechanisms may therefore be involved in maintaining patterning of the mammalian myocardium. Key words: Cardiomyocytes, Transcriptional repression, Transgenic mice Summary Cell history determines the maintenance of transcriptional differences between left and right ventricular cardiomyocytes in the developing mouse heart Robert G. Kelly* ,‡ , Marguerite Lemonnier, Stephane Zaffran, Andrew Munk and Margaret E. Buckingham ‡ CNRS URA 2578, Department of Developmental Biology, Pasteur Institute, 25 Rue du Dr Roux, Paris 75015, France *Present address: Department of Genetics & Development, Columbia University, 701 West 168th Street, New York, NY 10032, USA ‡ Authors for correspondence (e-mail: margab@pasteur.fr; rkelly@pasteur.fr) Accepted 6 August 2003 Journal of Cell Science 116, 5005-5013 © 2003 The Company of Biologists Ltd doi:10.1242/jcs.00824 Research Article