Fig. 11. (A) FRL-1 enhances MAPK activity to cause neural induction in animal caps. Animal caps injected with 1 ng of FRL-1 were dissected at stage 8, together with an uninjected control (lane 1). FRL-1-injected animal caps were cultured in 100% Steinberg's solution (SS) with 0.1% bovine serum only (0.1% BSA in SS) (lane 2), or with LY294002 (20 µM), a specific inhibitor of phosphatidylinositol 3 kinase, in 0.1% BSA in SS (lane 3) or with PD98059 (20 µM), a inhibitor of MEK (MAPKK), in 0.1% BSA in SS (lane 4) or with dimethyl sulfoxide (DMSO) in 0.1% BSA in SS (lane 5) until sampling, then harvested at stage 25 with the whole embryos for RT-PCR. Since PD98059 and LY294002 were dissolved in DMSO, DMSO treatment is a control to exclude DMSO effects. FRL-1 induced the expression of N-CAM (lane 2), and this was inhibited by treatment with PD98059 but not LY294002, compared with DMSO alone. (B) FRL-1 inhibition of BMP signaling is required for MAPK activation. FRL-1 or uninjected animal caps were dissected at stage 8 and treated with or without PD98059 or DMSO only. The animal caps and control embryos were harvested at stage 12. The expression of BMP responsive genes, Xvent-1 and Xmsx-1 were inhibited by injection of FRL-1 whereas the inhibition of Xvent-1 and Xmsx-1 was rescued by treatment with PD98059.