Fig. 5

Cytoskeletal abnormalities in surface ectoderm of Grhl2 mutant embryos. a, b, m, n Surface-subtracted images of dorsal view of phalloidin stained PNP regions (asterisk indicates closure point) of Grhl2^−/− and Axd/Axd mutants and equivalent wild types at E9.5. F-actin localises at cell boundaries in the surface ectoderm of wild-type controls (a, m), and shows profoundly disturbed organisation in Grhl2^−/− (box in b) and greater intensity of staining in the midline in Axd/Axd (arrow in n). c, d High-resolution images of the PNP closure point (boxed in a, b), show enrichment in an ‘actin cable’ at the margins of the open neural folds in wild type (arrow in c), which is disrupted in Grhl2^−/− (d). e, f, o, p Transverse sections of phalloidin (grey) stained Grhl2^−/− and Axd/Axd embryos at E9.5 (nuclei stained blue with DAPI). Disturbed F-actin organisation in the surface ectoderm at the PNP closure point in Grhl2^−/− embryos is indicated (arrow in f). i–l Immunostaining for p-MLCII (green) at E9.5. In wild-type embryos, surface-subtracted images of dorsal views of the PNP show notable enrichment in a ‘cable’ at the surface ectoderm (se)/neuroepithelium (ne) boundary in the PNP (arrow in magnified image in g), as well as in the midline overlying the most recently closed neural tube (arrows in g, I). Like F-actin, pMLCII is disorganised in Grhl2^−/− embryos at the PNP closure point (arrow in h) and at discrete regions of the open neural folds (boxed in h). In contrast to pMLCII localisation at cell–cell junctions in the surface ectoderm of wild-type embryos (k, q), pMLCII appeared uniformly distributed around the margins of surface ectoderm cells and excluded from cell–cell junctions in Grhl2^−/− embryos (l). m–r The localisation of F-actin (m–p) and pMLCII (q, r) in Axd/Axd was comparable to wild-type embryos at E9.5 but consistently exhibited greater intensity of staining in the midline (arrows in n and r). Scale bars: 10 μm in c, d, k, l and magnified areas of g–h, k–l, q, r; 25 μm in a, b, g, h, m, n, q, r; 50 μm in i, j. Images are representative of a minimum of three embryos per genotype