Fig. 5
From: Glycogen synthase kinase 3 controls migration of the neural crest lineage in mouse and Xenopus
GSK3 is required to establish polarity and to form lamellipodia in migrating neural crest cells. a, b GSK3 inhibition increases nuclear RAC1 and reduces cytoplasmic RAC1 in neural crest cells. a In control explants, RAC1 (red) is high in the nucleus while in the cytoplasm it is enriched at cell protrusions (arrowheads). Actin was labelled with phalloidin (green). b In BIO-treated explants, nuclear RAC1 is high but cytoplasmic staining is lost (white arrowheads). c Relative levels of Rac1 fluorescence in the nucleus or the cytoplasm (n = 9) **p ≤ 0.001 and ***p ≤ 0.0001, two-tailed unpaired t-test. d–e GSK3 inhibition reduces the expression of cdc42 in neural crest cells. d Cdc42 is cytoplasmic and perinuclear in neural crest cells. e BIO-treated explants lose cdc42 staining. f–g In controls, anti-lamellipodin (green) stains the ruffled edge of migrating cells. g In contrast, BIO-treated cells show increased total lamellipodin throughout the cell, losing specific localization at the cell edge. Scale bar, 20 μm. h, i Bright-field still images from controls (h) or BIO-treated samples (i) (n = 3 explants with ≥10 cells per explant). Control images show cells form fan-shaped stable lamellipodia and ready to migrate away from the cluster (h, white arrowheads). In treated cells, despite some cells form stable lamellipodia as found in controls (i, light blue arrowheads), cells predominantly formed an irregular protrusion that tend to retract (i, green arrowheads). Scale bar, 500 μm (see corresponding Supplementary Movies 6, 7). j Graph showing the number of cells delaminating from the premigratory neural crest cell clusters in 2 h. *p ≤ 0.05 and **p ≤ 0.001, two-tailed unpaired t-test. k Percentage of delaminating cells that show stable (persistent) lamellipodia or unstable (short-lived) lamellipodia. l, m Still images of time-lapse showing control mouse neural crest cells (l, supplementary movies 3,5) and pCAAG::creERtm ;GSK3αfl/fl ; GSK3βfl/fl; Rosamtmg/+ deleted cells (m, supplementary movie 4, 5). Upon tamoxifen-induced knockout of GSK3, the neural crest cells are unable to migrate and the cell edge does not form lamellipodia
