Mouse embryonic stem (mES) cells differentiate into skeletal muscle in response to retinoic acid (RA). mES cells were aggregated in hanging drops for 2 days, cultured in suspension for a further 5 days with increasing concentrations of RA, and transferred to tissue culture dishes. Panel I: RNA was harvested from day 7 cultures and subjected to reverse transcriptase- polymerase chain reaction (PCR) followed by Southern blot analysis with the probes indicated on the left. Panel II: RNA was harvested from days 7 and 15 after differentiation with or without 25nM retinoic acid and examined by quantitative PCR analysis. The expression levels are expressed as fold increase in the presence, compared to the absence of RA, as the mean and standard error, n = 3. Statistics were Student's t-test, *P < 0.05. Panel III: On day 20 of differentiation, cells were fixed and reacted with Hoechst dye to detect nuclei (A and C) and with MF20 antibody to detect muscle (B and D). Magnification is 400×.