Figure 4.

Reduction of ADAM function decreases cadherin-11 cleavage and CNC migration. (A) Cos-7 cells overexpressing Cad-11 and ADAM13 were treated with 0 μM, 1 μM, or 10 μM of marimastat. Cad-11 cleavage was determined by Western blot analysis of Cad-11 (top panel). ADAM13 levels were also detected by Western blot (bottom panel). M, mature-form ADAM13; P, proform of ADAM13. (B) Lateral view of tailbud stage embryos treated by whole-mount in situ hybridization using slug to label neural crest cells. Embryos at stage 17 were injected under the epidermis with 10 nl of 10% DMSO (left) or the same amount of 1 mM marimastat in 10% DMSO (right). At tailbud stage the CNC in control embryos have migrated in the hyoid, branchial, and mandibular segments (100%, n = 24). In contrast, 87.5% of the embryos injected with the marimastat inhibitor have severe inhibition of CNC migration (n = 24). (C) Western blot analysis detecting ADAM and Cad-11 expression in control noninjected embryos (NI) or injected with morpholinos directed against ADAM9 (MO9), ADAM13 (MO13), or ADAM19 (MO19). Each lane represents the glycoproteins from five embryos equivalent. PACSIN2 and the β1-integrin protein levels are unaffected by MO injection. In contrast, the uncleaved cadherin-11 protein level is increased twofold with each MO. (D) ADAM9, 13, and 19 protein expression was knocked down using a cocktail of all three specific MO. Embryos were extracted at stage 15 (premigration) or at stage 21(mid-migration), and were immunoprecipitated for Cad-11. Cad-11 was then detected by Western blot (20 embryos/lane). At stage 21, the cadherin-11 cleavage fragments are reduced in embryos injected with the 3MO. (E) In vivo migration analysis of embryos injected at the 16-cell stage with mRNA encoding GFP alone (0.5 ng/injection) or combined with 1 ng of the 3MO cocktail (0.33 ng of each MO/injection). The CNC in GFP mRNA injected embryos migrated in 21 of 21 embryos. The CNC in GFP mRNA combined with 3MOs migrated in only eight of 33 embryos (24%).