Cardiomyocyte differentiation by GATA-4-deficient embryonic stem cells

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Abstract
In situ hybridization studies, promoter analyses and antisense RNA experiments 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 factor is essential for cardiomyocyte lineage commitment. First, we assessed the ability of Gata4−/− ES cells form cardiomyocytes during in vitro differentiation of embryoid bodies. Contracting cardiomyocytes were seen in both wild-type and Gata4−/− embryoid bodies, although cardiomyocytes were observed more often in wild type than in mutant embryoid bodies. Electron microscopy of cardiomyocytes in the Gata4−/− embryoid bodies revealed the presence of sarcomeres and junctional complexes, while immunofluorescence confirmed the presence of cardiac myosin. To assess the capacity of Gata4−/− ES cells to differentiate into cardiomyocytes in vivo, we prepared and analyzed chimeric mice. Gata4−/− ES cells were injected into 8-cell-stage embryos derived from ROSA26 mice, a transgenic line that expresses β-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 endocardium, myocardium and epicardium. In situ hybridization showed that myocardium derived from Gata4−/− ES cells expressed several cardiac-specific transcripts, including cardiac α-myosin heavy chain, troponin 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 for terminal differentiation of cardiomyocytes and suggest that additional GATA-binding proteins known to be in cardiac tissue, such as GATA-5 or GATA-6, may compensate for a lack of GATA-4.