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Figure 1. Cadherin-11 is cleaved in vivo during cranial neural crest migration. (A) Wild-type embryos were extracted at stage 19, stage 21, and stage 23 representative of the different phases of CNC migration (20 embryos/lane). Western blot analysis for Cad-11 shows an increase in full-length protein (120 kDa) as migration proceeds, as well as the appearance of one 80-kDa cleavage product. (B) Schematic representation of full-length Cad-11. The EC1 domain contains a QAV homophilic binding motif consistent with type II cadherins (Hadeball et al., 1998). The cleavage site (*) is determined by calculating the relative molecular mass of the C-terminal fragment taking into account the N-glycosylation sites. The cytoplasmic region of Cad-11 can bind to β-catenin (Kawaguchi et al., 1999).
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Figure 2. Binding of endogenous ADAM13 and cadherin-11 occurs during CNC migration and corresponds with cadherin-11 cleavage. Wild-type embryos (50 embryos/stage) were extracted at stages 7, 10.5, 19, and 23. Embryo extracts were immunoprecipitated for Cad-11 and detected by Western blot for ADAM13 (A) and Cad-11 (B). (C) Glycoproteins from five embryos were purified using Con-A agarose, separated by SDS PAGE, and blotted using ADAM13 antibodies. Both pro- (P) and mature (M) forms are detected.
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Figure 3. ADAM9 and 13 rescue CNC migration in cells overexpressing cadherin-11. (A) In situ hybridization was performed using a combination of CNC markers xTwist and Sox10. Embryos were injected into one blastomere at the two-cell stage with synthetic mRNA for either Cad-11 alone (top left) or in combination with ADAM13 (bottom left). The site of injection was determined by coinjecting mRNA for β-galactosidase. The right panels correspond to the noninjected side of each embryo. Disruption of CNC migration was determined by comparing the distance migrated on the injected side (left panels) versus the noninjected side (right panels) of the same embryo. (B) Quantification of three independent rescue experiments. n = 30 for GFP-injected embryos, n = 79 for Cad-11–injected embryos, and n = 80 for Cad-11– and ADAM13-injected embryos. (C) Visualization of CNC cell migration in vivo using RFP as a lineage tracer. One dorsal animal cell at the eight-cell stage was injected with mRNA encoding RFP and cadherin-11 to inhibit CNC migration. Synthetic RNA (0.25 ng) encoding ADAM9, ADAM13, ADAM19, and ADAM13-E/A were each coinjected with cadherin-11 to determine their ability to rescue migration. (D) Histograms representing the percentage of embryos in which the RFP-labeled cells migrated. Significance was determined by students t test (p <0.05). The number of embryo analyzed was as follows: RFP = 51, Cad-11 = 61, Cad-11+ADAM9 = 67, Cad-11+ADAM13 = 59, Cad-11+ADAM13-E/A = 70, and Cad-11+ADAM19 = 56.
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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%).
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Figure 5. The Cad-11 extracellular cleavage fragment binds to full-length cadherin-11 and promotes CNC cell migration. (A) EC1-3 binding experiment performed in cell culture. Conditioned media from EC1-3-mt–transfected cells was incubated with live cells transfected with full-length Cad-11 (left) or an extracellular truncated form (ΔEC1-3, right). After 20 min, the cells were washed, fixed, and processed for immunofluorescence using mAb 9E10 (myc). The green fluorescence represent EC1-3-mt bound to cells, whereas DAPI was used to stain all cell nuclei. Bars represent the average number of cells positive for EC1-3 that were counted in 19 frames. We found no fluorescence associated with cells expressing the cadherin-11 lacking the EC1-3 domain. (B) Lateral view of embryos (St 26) that were injected in one CNC precursor cell at the 16-cell stage with synthetic mRNA for GFP alone, GFP (top left) and Cad-11 (top right), or GFP, Cad-11 and EC1-3 (bottom). GFP mRNA, 0.5 ng, 1 ng of Cad-11 mRNA, and 1 ng of EC1-3 mRNA was used. The extent of CNC migration was determined by GFP fluorescence, and results from three independent experiments are plotted. Bars represent the percentage of embryos in which CNC migration was observed. The total number of embryos counted was GFP alone (n = 40), GFP+Cad-11 (n = 75), or GFP+Cad-11+EC1-3 (n = 82). *Statistically significant at p < 0.05.
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Figure 6. The cadherin-11 extracellular cleavage fragment rescues CNC migration in embryos with reduced ADAM13 expression. (A) Schematic representation of the experimental method. Embryos were injected at the one-cell stage with MO13 and MO19 (5 ng each) and then again at the 16-cell stage, with a lineage tracer and mRNA encoding the various constructs, in D1.2 to target CNC. In this case we are testing the ability of the mRNA to rescue CNC migration. (B) In situ hybridization using Twist and Sox10 to label the CNC. The left panels represent the control side where migration was inhibited by the MO. The right panels represent the experimental side injected either with β-Gal or the EC1-3 mRNA. The black lines represent the extent of migration of the most posterior segment. After injection of the EC1-3 migration is rescued. (C) Quantification of three individual experiments described above (2MO is MO13 + 19). The total number of embryos for each injection set was n = 77 (2MO + β-gal), n = 96 (2MO + β-gal + EC1-3), n = 82 (2MO + β-gal + R13), n = 66 (2MO + β-gal + C11), and n = 91 (noninjected + β-gal + EC1-3).
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Figure 7. Combinations of MO to the different meltrins result in a reduction of CNC migration. (A) Embryos were injected at the 16-cell stage in D1-2 with the MO (0.33 ng each) and GFP as a lineage tracer to test their ability to prevent CNC migration. (B) Lateral views of representative embryos at tailbud stage. Migration was determined by the presence of GFP-labeled cells in the CNC pathways as evident in the GFP control. (C) Histogram representing the percentage of migration in embryos injected with the various MO (0.5 ng of each A9, A13, and A19) with or without the EC1-3 mRNA.
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Figure 8. The cadherin-11 extracellular fragment (EC1-3) is not cell-autonomous. (A) Schematic representation of the experimental design. The EC1-3 mRNA was coinjected with GFP mRNA at the 32-cell stage in the a2 cell. The 3MO cocktail (0.5 ng ADAM9, 13 and 19) was injected with RFP mRNA in the b2 cell of the same embryo (all mRNA were at 0.25 ng). Embryos were grown to stage 26 before imaging the GFP and RFP fluorescence (B). The percentage of embryos with migrating CNC cells expressing RFP was then counted and is presented in C. Asterisks indicate statistical significance as determined by Student's t test (p < 0.05). (D) Late stage (stages 45–47) analysis of RFP localization in differentiated facial structures in the dual injected embryos from above. Embryos were scored for having strong, little, or trace to no expression in the developing facial cartilage. (E) Histogram representing scoring data from late stage embryo analysis.
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Figure 9. Why cleave cadherin-11? (A) Cadherin-11 (red) is expressed at the surface of CNC throughout their migration and is associated with ADAM13 (blue). Cleavage of cadherin-11 could promote CNC cell migration via three possible molecular mechanisms. (1) Removal of the cadherin-11 extracellular adhesive (HAV) sequence prevents its ability to bind to full-length cadherin-11 molecules on neighboring cells. (2) The extracellular cleavage fragment retains the adhesive domain and could act as a competitive inhibitor by binding to full-length cadherin-11 and preventing its interaction with other full-length cadherin-11 molecules. (3) The extracellular cleavage fragment may also interact with unknown receptors and activate promigratory signaling cascade. We have also shown that the cleaved cadherin-11 fragment that remains in the plasma membrane can still interact with cytoplasmic protein such as β-catenin and possibly p120 maintaining cytoskeletal organization and controlling β-catenin signaling. (B) The decrease in cell adhesion may increase the fluidity of the CNC tissue allowing for the migration of a cohesive sheet of cells during the first phase of CNC migration. Further decrease of cell adhesion would promote the separation of CNC and the migration of single cells during phase 2.
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