Cadherin-11 Ectodomain Signaling in Cranial Neural Crest Migration

During development, a multi-potent group of cells known as the cranial neural crest (CNC) migrate to form craniofacial structures. Proper migration of these cells requires proteolysis of cell adhesion molecules, such as cadherins. In Xenopus laevis, preventing extracellular cleavage of cadherin-11 impairs CNC migration. However, overexpression of the soluble cleavage product (EC1-3) is capable of rescuing this phenotype. The mechanism by which EC1-3 promotes CNC migration has not been investigated until now. Here we show that EC1-3 stimulates phosphorylation of Akt, a target of PI3K, in X.laevis CNC. Through immunoprecipitation experiments, we determined that EC1-3 interacts with all ErbB receptors, PDGFRα, and FGFR1. Of these receptors, only ErbB2 was able to produce an increase in Akt phosphorylation upon treatment with a recombinant EC1-3. This increase was abrogated by mubritinib, an inhibitor of ErbB2. We were able to recapitulate this decrease in Akt phosphorylation in vivo by knocking down ErbB2 in CNC cells. Knockdown of the receptor also significantly reduced CNC migration in vivo. We confirmed the importance of ErbB2 and ErbB receptor signaling in CNC migration using mubritinib and canertinib, respectively. Mubritinib and the PI3K inhibitor LY294002 significantly decreased cell migration while canertinib nearly prevented it altogether. These data show that ErbB2 and Akt are important for CNC migration and implicate other ErbB receptors and Akt-independent signaling pathways. Our findings provide the first example of a functional interaction between the extracellular domain of a type II classical cadherin and growth factor receptors in an animal model

The ectodomain of cadherin-11 binds to erbB2 and stimulates Akt phosphorylation to promote cranial neural crest cell migration

<div><p>During development, a multi-potent group of cells known as the cranial neural crest (CNC) migrate to form craniofacial structures. Proper migration of these cells requires proteolysis of cell adhesion molecules, such as cadherins. In <i>Xenopus laevis</i>, preventing extracellular cleavage of cadherin-11 impairs CNC migration. However, overexpression of the soluble cleavage product (EC1-3) is capable of rescuing this phenotype. The mechanism by which EC1-3 promotes CNC migration has not been investigated until now. Here we show that EC1-3 stimulates phosphorylation of Akt, a target of PI3K, in <i>X</i>.<i>laevis</i> CNC. Through immunoprecipitation experiments, we determined that EC1-3 interacts with all ErbB receptors, PDGFRα, and FGFR1. Of these receptors, only ErbB2 was able to produce an increase in Akt phosphorylation upon treatment with a recombinant EC1-3. This increase was abrogated by mubritinib, an inhibitor of ErbB2. We were able to recapitulate this decrease in Akt phosphorylation <i>in vivo</i> by knocking down ErbB2 in CNC cells. Knockdown of the receptor also significantly reduced CNC migration <i>in vivo</i>. We confirmed the importance of ErbB2 and ErbB receptor signaling in CNC migration using mubritinib and canertinib, respectively. Mubritinib and the PI3K inhibitor LY294002 significantly decreased cell migration while canertinib nearly prevented it altogether. These data show that ErbB2 and Akt are important for CNC migration and implicate other ErbB receptors and Akt-independent signaling pathways. Our findings provide the first example of a functional interaction between the extracellular domain of a type II classical cadherin and growth factor receptors.</p></div

Mubritinib and canertinib perturb CNC migration <i>ex vivo</i>.

<p>(A-C) Lateral views of tailbud stage <i>X</i>.<i>laevis</i> embryos after <i>in situ</i> hybridization with <i>Sox10</i> and <i>Twist</i> to visualize CNC positioning. Anterior is to the left, dorsal is up. Embryos treated with 40 μM of the ErbB2 inhibitor, mubritinib (N = 4, n = 72), or 25 μM of the pan-ErbB inhibitor, canertinib (N = 4, n = 68), show no difference in CNC migration compared to DMSO controls (N = 6, n = 100; N = 4, n = 69, respectively). (D-E) The distance of migration within each branchial arch was measured and normalized to the head size. CNC migration as observed in the mandibular (M), hyoid (H), 3^rd and 4^th branchial arches. (F-U) Time-lapse images of CNC explants migrating on fibronectin substrate and treated with 0.5% DMSO (N = 8, n = 24), 6 μM mubritinib (N = 4, n = 12), 10 μM canertinib (N = 4, n = 12), and 20 μM LY294002 (N = 3, n = 9) for indicated lengths of time. (V) Fold change in explant surface area over time. Areas were normalized to measurements calculated at initial time points (t = 0). All inhibitors significantly decreased CNC migration <i>ex vivo</i>. Error bars represent the 95% confidence interval. One-tailed, Student’s t-tests were performed to determine statistical significance. ** p<0.01, ***p<0.001. N, number of experiments; n, number of embryos or explants.</p

Overexpression of EC1-3-myc and treatment with recombinant EC1-3 increases Akt phosphorylation.

<p>(A, upper) Injections of 200 pg mRNA for UGP alone or together with 800 pg of EC1-3-myc mRNA were performed on single blastomeres of 2-cell <i>X</i>.<i>laevis</i> embryos. CNC expressing UGP were dissected at neurula stages, lysed and analyzed for phospho-Akt (pAkt), phospho-MAPK (pMAPK) and GAPDH via western blot. (A, lower) Non-injected CNC were dissected and treated for 5 minutes with 10 ng/mL of recombinant EC1-3 (bacEC) before being processed as above. (B) As a control, bacEC was depleted from treatment solution using a specific bacEC antibody conjugated to agarose beads and then applied to CNC. Lysates were then analyzed as above. Immunodepletion using unconjugated beads was used as a control. (C) Significant increases in phospho-Akt were observed in CNC overexpressing EC1-3-myc (N = 5, p = 0.013) and those treated with bacEC (N = 5, p = 0.021). There were not significant (N.S.) increases in the phosphorylation of MAPK in CNC overexpressing EC1-3-myc (N = 5, p = 0.09), nor those treated with bacEC (N = 5, p = 0.178). GAPDH was used as a loading control for western blots and normalizer for calculation of phospho-Akt and phospho-MAPK. Error bars are one standard deviation to the mean. One-tailed, Student’s t-tests were performed to determine statistical significance. * p<0.05. N, number of experiments.</p

ErbB2-ErbB3 heterodimers bind PI3K p85 in the presence of recombinant EC1-3 (bacEC).

<p>(A) Quantitative real-time PCR analysis of ErbB receptors from dissected CNC and stage 16 embryos. The relative gene expression in the CNC is compared to that of whole embryos at the same stage. ErbB2, 3, and 4 are enriched in the CNC compared to whole embryos. <i>Slug</i> was used as a positive control for CNC enrichment. <i>GAPDH</i> was used for normalization. (B) Western blot of co-immunoprecipitation experiments with ErbB3-HA and p85 in the presence or absence of bacEC and ErbB2-flag. Hek293T cells were transfected with ErbB3-HA alone or together with ErbB2-flag, serum starved, and treated with 10 ng/mL bacEC for 5 minutes before being lysed for co-immunoprecipitation experiments. ErbB3-HA modestly binds p85 when transfected alone. Co-transfection of ErbB3-HA with ErbB2-flag noticeably increases this interaction. Association of p85 with ErbB3-HA is further elevated in the presence of bacEC. (C) Co-precipitation of p85 with ErbB3-HA and ErbB2-flag significantly increases (p = 0.03) in the presence of bacEC. Immunoprecipitated ErbB3-HA was used for normalization. Results are representative of three independent experiments.</p

EC1-3 stimulates Akt and CNC migration.

<p>During migration of cranial neural crest (CNC) cells, ADAM13 (blue) cleaves the extracellular domain of cadherin-11 (red) at the cell surface to release a soluble fragment (EC1-3) capable of promoting CNC migration. EC1-3 can induce cell migration by binding to ErbB receptors: EGFR/ErbB1 (teal), ErbB2 (yellow), ErbB3 (green) and ErbB4 (purple). In particular, binding of EC1-3 with ErbB2-ErbB3 dimers increases docking of the regulatory (p85) subunits of PI3K. Recruitment of PI3K to the cell membrane ultimately leads to the phosphorylation of Akt, which is important for CNC migration. Because inhibition of ErbB2 is less detrimental to cell migration than inhibition of all ErbB receptors, it is possible that EC1-3 utilizes Akt-independent pathways downstream of ErbB dimers that do not include ErbB2. Whether EC1-3 signaling first requires receptors to bind their cognate ligand still needs to be investigated.</p

Inhibition of PI3K perturbs CNC migration.

<p>(A,B) Lateral views of tailbud stage <i>X</i>.<i>laevis</i> embryos after <i>in situ</i> hybridization with <i>Sox10</i> and <i>Twist</i> to visualize CNC positioning. Anterior is to the left, dorsal is up. Migration of CNC cells into the branchial arches is perturbed in embryos treated with 30 μM LY294002 (N = 4, n = 52). (C) Western blot of embryos treated with DMSO or 30 μM LY294002. Embryos treated from stage 17–18 to stage 25–26 with LY294002 had reduced levels of phosphorylated Akt (pAkt). One-embryo equivalents were loaded per well. (D,E) The length of CNC migration (shown in A) in each branchial arch was quantified and normalized to head size. LY294002 significantly reduced CNC migration into all branchial arches compared to DMSO controls (N = 4, n = 66). Treatment with 40 μM rapamycin (N = 4, n = 37) did not noticeably alter CNC positioning compared to DMSO controls (N = 4, n = 35, respectively). CNC cells migrated into the mandibular (M), hyoid (H), 3^rd and 4^th branchial arches. Ruler bars denote how CNC segments were measured. Error bars are one standard deviation to the mean. One-tailed, Student’s t-tests were performed to determine statistical significance. *** p<0.001. N, number of experiments; n, number of embryos.</p

EC1-3-myc and non-adhesive EC1-3-myc interact with growth factor receptors.

<p>Western blots of EC1-3-myc (A) and non-adhesive EC1-3-myc (B; naEC1-3-myc) co-immunoprecipitations (coIPs). Receptors were immunoprecipitated using anti-HA or anti-flag antibodies. Both EC1-3 constructs co-IPed with all receptors: HA-tagged EGFR/ErbB1, HA-tagged ErbB3 and flag-tagged ErbB2, ErbB4, PDGFRα and FGFR1. (C) Western blot of EC1-3-myc coIP with cytosolic proteins ARID3a-flag and importinβ1-flag. To test if EC1-3 could interact non-specifically with proteins following extraction we tested Flag-tagged constructs overexpressed in Hek293T cells. EC1-3-myc did not associate with ARID3a-flag, nor importinβ1-flag. ErbB2-flag was used as a positive control. Results are representative of three independent experiments.</p