Fig. 5. OLP induction by SHH depends on activation of the MAPK pathway by FGFR1. (A) Neocortical neuroepithelial cells cultured for 24 hours in the presence of the MEK1/2 inhibitor U0126 and either SHHAg or FGF2 fail to develop Olig2-positive cells. The inhibitor of PI 3-kinase, LY294002, has no effect on the inducing activities of either SHHAg or FGF2. (B) To assess whether FGF2 and/or SHH activate the MAPK pathway we cultured E13.5 cortical cells in the absence (a-f) or presence of either SHHAg (g-j) or FGF2 (k-n), together with PD173074 (c,i) or cyclopamine (e,m) for 1 hour prior to immunolabelling with an anti-phospho-ERK1/2 antibody and Hoechst dye (b,d,f,h,j,l,n). FGF2 by itself caused strong activation of MAPK. SHH failed to activate MAPK above endogenous levels (compare a, g) and all MAPK activity was abolished by PD173074 (c,i). (C) Protein lysates from cortical cultures incubated with FGF2 or SHHAg and PD173074 or cyclopamine for 1 hour or 18 hours were separated by PAGE, and analysed for the presence of phosphorylated ERK1/2 (p42/p44) by western blot. SHHAg failed to activate MAPK above control levels, and PD173074 abolished all MAPK activity even in the presence of SHH.