Fig. 6.

Loss of Fz4 affects ADAM13 processing. (A) Western blot of ADAM13 at various stages of embryo development (stage 19 to 30). Embryos were injected with the Fz4 morpholino (+) or the ADAM13 morpholino (MO13, last lane) to visualize the specific bands. In control embryos the pro-form (P, 120 kDa), the mature form (M, 100 kDa) and the cytoplasmic domain (Cyto, 17 kDa) are visible, whereas these are absent in MO13 embryos. There is no difference of processing at stage 19. At stage 24, the mature form of ADAM13 decreases while the cytoplasmic domain increases in MOFz4 injected embryos (red line). At stage 30 the decrease of the mature form is still obvious, whereas there is no visible difference for the cytoplasmic domain fragment. The β1 integrin subunit was used as a loading control. (B) Western blot for ADAM13 in embryos injected with RFP, Fz4 or Fz4-v1 mRNA. Embryos injected with the morpholino to ADAM13 (MO13) serve as a control for the antibody specificity. At stage 19 embryos expressing Fz4 or Fz4-v1 show less ADAM13 cytoplasmic domain fragments (Cyto, 47% and 23%, respectively). At this stage Fz4 also reduces the mature form of ADAM13 (M, 58%), whereas Fz4-v1 does not. At stage 30, Fz4-v1 injected embryos have almost no cytoplasmic domain fragment, whereas the mature form is not affected. (C) Representative example of dissected CNC expressing ADAM13-GFP and mb-Cherry (total of eight explants for each condition). ADAM13 GFP is clearly present in control CNC, whereas it is undetectable in CNC injected with MO Fz4. The morpholino has no effect on mb-Cherry expression. Scale bar: 50 µm.