Figure 1

Retinoic acid inhibits cardiomyogenesis and enhances skeletal myogenesis in P19 cells. P19 cells were aggregated with 0.8% dimethylsulfoxide (DMSO) in the presence and absence of 30 nM RA. Panel I: Cells were fixed on day 9 for immunofluorescence with MF20 antibody (A-D) and counter stained with Hoechst dye (E-H). Magnification is 160x. Panel II: Cardiac (n = 3) and skeletal (n = 4) myogenesis were quantified by counting the number of MHC+ve myocytes as a percentage of the total. Average +/- standard error of mean (SEM) is shown and statistics were Student's t-test, *P < 0.05. Panel III: Total RNA was harvested for northern blot analysis on the days indicated and hybridized to the cDNAs on the right. Lanes are spliced from the same autoradiogram. Panel IV: Quantitative polymerase chain reaction (PCR) analysis was performed on day 4 of differentiation for Pax3 and Wnt3a transcript levels (n = 2) and on day 9 for MyoD levels (n = 4). Results were expressed as fold change of transcript levels in the presence compared to the absence of RA treatment. Panel V: Chromatin immunoprecipitation experiments were performed on day 2 P19 aggregates treated with DMSO/retinoic acid and analysed by real-time PCR using primers for sites within regulatory regions of the genes indicated. Average +/-SEM is shown, relative to IgG, and statistics were Student's t-test of each region compared to IgG, n = 3-4, *P < 0.05. Panel VI: The position and conservation of the Meox1-2 and the Pax3-2 retinoic acid response elements are shown.