Fig. 2. (A) Both the EGF and CFC domain are necessary for FRL-1-mediated induction of expression of neural markers. 1 ng of FRL-1-6myc, which encodes for FRL-1 tagged with six c-myc-epitope (FRL-1), the EGF domain-deleted form of FRL-1-6myc (FRL-1EGF domain) or the CFC domain-deleted form of FRL-1-6myc (FRL-1CFC domain) was injected into each animal blastomere of 4-cell-stage embryos. Animal caps were dissected at stage 9 with an uninjected control and harvested at stage 25 for RT-PCR. Western blotting analysis showed FRL-1-6myc, FRL-1EGF domain-6myc and FRL-1CFC domain-6myc proteins were generated in animal caps at stage 10.5. Although FRL-1 induced the expression of N-CAM, it was not detected in the FRL-1EGF domain and FRL-1CFC domain mRNA-injected animal caps. (B) Activity of neural induction by FRL-1 was conserved among other members of the EGF-CFC family in vertebrates. Animal caps injected with 2 ng of zebrafish oep, mouse cripto or chick CFC (chick cripto) were dissected at stage 9 with an uninjected control and harvested at stage 25 with whole embryos for RT-PCR. The data showed that not only FRL-1 but also oep, chick CFC, and mouse cripto induced the expression of N-CAM without ms-actin.

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