Fig. 10. (A) FRL-1 caused neural induction via FGF signaling in animal caps. The animal caps that were injected with 1 ng of FRL-1 alone or co-injected with 2 ng of XFD, were dissected at stage 9, together with an uninjected control and harvested at stage 25 with the whole embryos for RT-PCR analysis. FRL-1 induced the expression of N-CAM without induction of ms-actin, which was inhibited by XFD. (B) FRL-1 stimulated the MAPK activity. 1 ng of FRL-1 was injected into each animal blastomere of 4-cell-stage embryos, which were dissected at stage 9 with an uninjected control in Ca²⁺- and Mg²⁺-free MBS and harvested at stage 10.5 for western blotting. bFGF treatment (100 ng/ml) served as a positive control. FRL-1 induced MAPK activation to the same extent as bFGF, and to a greater extent than in uninjected caps. Total MAPK protein (ERK) was detected and used as a loading control. (C) Injection of FRL-1MO downregulated MAPK activity. Animal caps injected with 20 ng of FRL-1-MO or FRL-1-4misMO were dissected at stage 9 with the uninjected control and harvested at stage 10.5 for western blotting. (D,E) MAPK activity was enhanced in FRL-1-injected embryos. Activation of MAPK was detected by whole-mount immunohistology. 500 pg of lacZ mRNA was injected with (D) and without (E) 1 ng of FRL-1 into one blastomere of 2-cell-stage embryos. Injected embryos were grown to stage 9. The injected regions were visualized by red gal staining and the MAPK activated-region was visualised by blue staining. (F,G) Activated MAPKK rescued the phenotype caused by FRL-1MO. Head structure defects induced by 15 ng of FRL-1MO were rescued by co-injection of 2 ng of SESE-MAPKK (constitutively-activated MAPKK).

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