|
Display additional annotations [+]
|
| |
Fig 2. Inhibition of PI3K perturbs CNC migration.
(A,B) Lateral views of tailbud stage X.laevis embryos after in situ hybridization with Sox10 and Twist 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), 3rd and 4th 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.
https://doi.org/10.1371/journal.pone.0188963.g002 |
|
Display additional annotations [+]
|
| |
Fig 6. Knockdown of ErbB2 decreases phosphorylation of Akt and CNC migration.
(A) Western blot of non-injected (NI) and injected embryo extracts probed for flag. Single-cell embryos were injected with 800 pg DN-ErbB2-Flag mRNA with or without 25 ng of ErbB2 morpholino (MO ErbB2). Translation of DN-ErbB2-flag was hindered in embryos co-injected with MO ErbB2. GAPDH is shown as a loading control. One-embryo equivalents are loaded per lane. (B) Western blot of CNC extracts probed for phospho-Akt, total Akt and GAPDH. Eight-cell embryos were either injected with 200 pg RFP mRNA alone (N = 4, n = 32) or with 3.1 ng MO Erb2 (N = 3, n = 43) into CNC progenitor cells. CNC cells were then dissected from embryos once they reached stage 17–18. Injection of MO ErbB2 into CNC cells precursors dramatically reduced phosphorylation of Akt without altering the levels of control proteins. Ten CNC explants are loaded in each lane. (C-E) Early tailbud stage embryos labeled for Twist and Sox10 CNC markers using in situ hybridization. Embryos were injected as in part (B) along with a set injected with 300 pg rat ErbB2-flag and 1.6 ng MO ErbB2 (E; N = 2, n = 20). Embryos injected with RFP mRNA and MO ErbB2 showed defects in CNC migration (D) compared to RFP-injected controls (C). Migration was partially rescued by co-injecting the morpholino with rat ErbB2-flag mRNA (E). (F) CNC migration was measured in each branchial arch and normalized to head size from embryos in part (C-E). Co-injection of MO ErbB2 with RFP mRNA significantly reduced CNC migration in the mandibular (M), hyoid (H), 3rd and 4th branchial arches. Co-injection of MO ErbB2 with rat ErbB2 mRNA partially rescued migration. Error bars are one standard deviation to the mean. 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.
https://doi.org/10.1371/journal.pone.0188963.g006 |
|
Display additional annotations [+]
|
| |
Fig 7. Mubritinib and canertinib perturb CNC migration ex vivo.
(A-C) Lateral views of tailbud stage X.laevis embryos after in situ hybridization with Sox10 and Twist 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), 3rd and 4th 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 ex vivo. 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.
https://doi.org/10.1371/journal.pone.0188963.g007
|
