Fig. 6
From: Glycogen synthase kinase 3 controls migration of the neural crest lineage in mouse and Xenopus
Inhibition of ALK leads to decreased levels of pY-GSK3 in mouse neural crest. a–f ALK is expressed in the neural crest. (a, d) mRNA in situ hybridization for Alk in e8.5 and e9.5 mouse embryos. b, c, e, f Antibody staining for activated ALK protein shows expression at the neural plate border (red arrows) and in the branchial arches. g Staining for total ALK (green). g Very little total ALK is present in the neural plate with some present at the edge of the premigratory neural crest (white arrowheads). h Migratory neural crest cells express higher levels of ALK. i In fully migratory NC cells, ALK appears to be nuclear. Underneath each panel, ALK merged with Hoechst. j Co-immunostaining of pY-GSK3 (green) and ALK (red) show that ALK is expressed in all cells that express pY-GSK3. k Treatment with the ALK inhibitor crizotinib (CTB) for 24 h reduces the levels of pY-GSK3. l, m, n, o Quantitation of loss of pY-GSK3 and ALK fluorescence using the alternative inhibitor NVP-TAE also results in a loss of mean fluorescence intensity. p–s Bright-field images of neural crest explants treated with vehicle control or three different ALK inhibitors: 1.5 µM CTB, 1.5 µM AZD-3463, or 1 µM NVP-TAE-684. All three treatments showed a loss of migratory neural crest (red dotted lines) (n = 3 explants with ≥10 cells per explant, scale bar = 500 µm). t Area quantification of the premigratory neural crest (pNC, area depicted by blue dotted lines covered in p–s) and the migratory neural crest population (mNC, red dotted lines). A significant reduction in mNC was seen in AZD and NVP treated explants. u–y Phalloidin staining shows F-actin structure in explants. Hoechst marks nuclei. Note the loss of lamellipodial structures upon ALK inhibition (w, x) is comparable to BIO treatment (v) (scale bar = 25 µm). y Percentage of cells with lamellipodial formations at the leading edge upon treatment with GSK3 inhibitor BIO or with ALK inhibitors (n = 10–20 cells per explant). Bars, mean ± SD. One-way ANOVA and unpaired t-test *P < 0.05, **P < 0.01
