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-1
EGF 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.