Fig. 4.

Overexpression of Fz4 inhibits CNC cell migration. (A,B) Histogram representing the percentage of embryos with no fluorescent CNC cells in the migration pathway following targeted injection at the 8-cell stage in one dorsal animal blastomere (% inhibition of CNC cell migration). Injection of RFP alone is used to normalize to zero and account for any mis-targeting. (A) Injection of Fz4 inhibits migration and can be rescued by either ADAM13 or the cadherin-11 extracellular fragment EC1-3. Student's t-test was performed to compare % inhibition to Fz4 alone. (B) Similarly, injection of Fz4-v1 inhibits migration, and can be partially rescued with ADAM13, the extracellular domain of cadherin-11 (EC1-3) and ADAM13 lacking the cytoplasmic domain (Δcyto). Student t-tests were used to compare values to Fz4-v1. (C) Fluorescently labeled CNC cells were grafted to determine whether the inhibition of CNC cell migration was cell autonomous. CNC cells overexpressing Fz4-v1 do not migrate. Wild type CNC cells grafted into embryos overexpressing Fz4-v1 in the pathway migrated similar to control. (D) Western blot from HEK293T cells transfected with ADAM13, or the non-proteolytic mutant of ADAM13 (E/A) and PAPC. These cells were mixed with cells transfected with Fz4-v1 or RFP and incubated together for 24 h. The conditioned supernatant, which contains Fz4-v1, does not inhibit ADAM13 cleavage of PAPC. *P<0.05, ***P<0.001. n = number of embryos scored from three or more independent experiments. Error bars represent ±s.e.m. ns, not significant.