CARDIOMYOCYTE DIFFERENTIATION BY GATA-4-DEFICIENT EMBRYONIC STEM-CELLS

In situ hybridization studies, promoter analyses and antisense RNA exp eriments have implicated transcription factor GATA-4 in the regulation of cardiomyocyte differentiation. In this study, we utilized Gata4(-/ -) embryonic stem (ES) cells to determine whether this transcription f actor is essential for cardiomyocyte lineage commitment, First, we ass essed the ability of Gata4(-/-) ES cells form cardiomyocytes during in vitro differentiation of embryoid bodies, Contracting cardiomyocytes were seen in both wildtype and Gata4(-/-) embryoid bodies, although ca rdiomyocytes were observed more often in wild type than in mutant embr yoid bodies, Electron microscopy of cardiomyocytes in the Gata4(-/-) e mbryoid bodies revealed the presence of sarcomeres and junctional comp lexes, while immunofluorescence confirmed the presence of cardiac myos in, To assess the capacity of Gata4(-/-) ES cells to differentiate int o cardiomyocytes in vivo, we prepared and analyzed chimeric mice, Gata 4(-/-) ES cells were injected into 8-cell-stage embryos derived from R OSA26 mice, a transgenic line that expresses beta-galactosidase in all cell types, Chimeric embryos were stained with X-gal to discriminate ES cell- and host-derived tissue, Gata4(-/-) ES cells contributed to e ndocardium, myocardium and epicardium. In situ hybridization showed th at myocardium derived from Gata4(-/-) ES cells expressed several cardi ac-specific transcripts, including cardiac alpha-myosin heavy chain, t roponin C, myosin light chain-2v, Nkx-2.5/Csx, dHAND, eHAND and GATA-6 , Taken together these results indicate that GATA-4 is not essential f or terminal differentiation of cardiomyocytes and suggest that additio nal GATA-binding proteins known to be in cardiac tissue, such as GATA- 5 or GATA-6, may compensate for a lack of GATA-4.