Fig. 1. Fgf4^-/- ES cells are deficient in neural induction. (A-E) Immunostaining (left; phase-contrast, right) for Oct4 and nestin (A,D) and Oct4 and TuJ1 (B,C,E) in wild-type (A,B), Fgf4^+/- (C) and Fgf4^-/- mouse ES cells on day 6 (A,D) and day 10 (B,C,E) of the monolayer neural differentiation protocol. (F) RT-PCR for neural markers Sox1 and nestin in Fgf4^+/- and Fgf4^-/- ES cells in self-renewing conditions on day 2 of the neural induction protocol. ß-actin was used as a loading control. (G) Quantitative RT-PCR for nestin on day 5 of the neural differentiation assay. TBP, TATA-binding protein. (H,I) Immunostaining for Oct4 and nestin (H) and Oct4 and TuJ1 (I) of Fgf4^-/- ES cells cultured in FGF4 (5 ng/ml) for the first 24 hours of a 6 day (H) and 10 day (I) neural monolayer assay. Scale bar: 100 µm. (J) Cell counts of Oct4-positive, nestin-positive, and double-negative cells for wild-type, Fgf4^-/- ES cells, and Fgf4^-/- ES cells treated with FGF4 after 6 days culture in N2B27 (n=3 for each sample).