Introduction activation of the RXR gene resulted in embryonal death as a consequence of cardiac hypoplasia be-Mouse embryonic stem (ES) cells have been shown to differentiate into the cardiogenic (Wobus et al., cause of ventricular chamber defects (Sucov et al., 1994; Dyson et al., 1995). Ventricular hypoplasia 1991; Miller-Hance et al., 1993; Maltsev et al., 1993, 1994), myogenic (Rohwedel et al., 1994), was also observed after vitamin A deficiency (Wilson and Warkany, 1949; Wilson et al., 1953). However, neurogenic (Strü bing et al., 1995), haematopoietic (Doetschman et al., 1985; Wiles, 1993) and epi- dramatic abnormalities of the normal embryonic development, including those of the heart and outthelial lineage (Bagutti et al., 1996) when cultivated in embryo-like aggregates, the so-called embryoid flow tract, were obtained by combining mouse strains with mutant RAR and RXR subtypes (Kastbodies (eb). The cardiogenic differentiation of ES cells into cardiomyocytes of atrium-, ventricle-and ner et al., 1994; Lohnes et al., 1994; Mendelssohn et al., 1994; Luo et al., 1995; Sucov and Evans, sinusnodal-like cell types is accompanied by the developmentally controlled expression of cardiac- 1995). In vitro studies with ES cells (Wobus et al., 1994) specific genes, ie-and-cardiac MHC, atrial natriuretic factor (ANF), ventricle-specific MLC-2v and earlier studies with embryonic carcinoma (EC) cells (Edwards and McBurney, 1983; Rudnicki and (Maltsev et al., 1994; Wobus et al., 1995; Fässler et al., 1996), of proteins (-cardiac MHC, troponin- McBurney, 1987) demonstrated that all-trans RA in a time-and concentration-dependent manner T) and of ion channels (Ca2+, Na+, K+; Maltsev et al., 1994). In vitro differentiated cardiomyocytes influenced the efficiency of cardiogenic differentiation. Treatment with high concentrations reacted with characteristic positive or negative effects to chronotropic agents (Wobus et al., 1991, of RA (10− 7 and 10− 8 RA) during the first 2 days or between day 2 and 5 of ES cell-derived embryoid 1995). The process of cardiac specialization during ES cell-derived cardiogenic differentiation is ac- body formation significantly inhibited cardiogenesis, whereas treatment between day 5 and companied by an upregulation of MLC-2v gene expression (Fässler et al., 1996). 7 resulted in an increased cardiomyocyte differentiation (Wobus et al., 1994). The ventricular isoform of the regulatory myosin lightchain (MLC-2v) wasusedasavaluablemarker To characterize the differentiation and specialization of ES cells into distinct cardiac cell types, considerably elucidating the molecular mechanisms that underlie the regulation of muscle gene ex- MLC-2v gene expression was used as a marker for ventricular differentiation. In addition, ventricular pression during cardiac growth and development (Chien et al., 1991, 1993). The ventricle-specific cells were characterized by functional analyses based on electrophysiological properties. Here, we activity of the 2.1 kb MLC-2v promoter has previously been demonstrated in embryonic, fetal and show that treatment of embryonic stem (ES) cellderived embryoid bodies with all-trans and 9-cis adult myocardium of transgenic mice (Franz et al., 1993). In situ analysis of day 8 murine embryos RA, respectively, accelerated the differentiation of ES cells into cardiomyocytes, enhanced the level of showed that MLC-2v was expressed exclusively in the ventricle region of the heart tube, with negligible-cardiac MHC and MLC-2v mRNA at an early developmental stage and resulted in an increased expression in the atrial primordium, suggesting that ventricular specification occurs prior to septation number of …