RA facilitates emergence of neural cells. ESCs were cultured under monolayer differentiation conditions with RA (N2B27 + 10-8 M RA) or without RA (N2B27 + 0 or N2B27 + ethanol). A, Expression of neural markers during differentiation under monolayer cultures at different time points. RNA was isolated from ESCs (day 0), monolayer cultures (days 1, 3, 5 and 10) and adult mouse brain, and analyzed by RT-PCR for expression of markers of neural progenitors/stem cells and neurons. To normalize their expression to the amount of cDNA present in the sample, the cDNA for endogenous GAPDH was amplified; B, C, Q-PCR for the neural marker Pax6 and Nestin during monolayer differentiation; D, F-G, Cultures at different time points of monolayer differentiation, shown in phase contrast or stained for markers as indicated; D, Cultures at 72 h of monolayer differentiation showed that in RA-treated cultures, Sox1GFP+ and Nestin+ cells were found, while nearly no Oct4+ cells were detected; E, Western blot analysis of Tuj1 at day 6, of NeuN at day 8 and day 10, and of GFAP at day 10 under monolayer cultures; F, At day 9, though Tuj1+ cells were also found in control cultures, the marker of mature neurons MAP2 was only detected in RA-treated cultures; G, At day 11, neurons (MAP2+ cells) emerged in all cultures, however, the marker of glia cells GFAP was only found in RA-treated cultures with a very low percentage. In control cultures, cells were dominantly Nestin+, while in RA-treated cultures, the percentage of Nestin+ cells was much lower. Scale bars: D (a, b, e, f, i, j): 40 μm; D (c, g, k): 80 μm; D (d, h, l), F-G: 25 μm.