Fig. 3

Focal adhesions are assembled in a specific spatiotemporal pattern, and are differentially disrupted by loss of lama1. (A-H) Antibody staining for the focal adhesion protein vinculin at 14 hpf (A-D) or 24 hpf (E-H), in control (A,E) or lama1^UW1 mutant (B-D, F-H) embryos. (H’) No primary anti-vinculin antibody control. Orange arrowheads, location of optic stalk furrow; white arrows, lens-retina interface; dashed line, outline of optic vesicle. (I-P) Single confocal slices from 4D datasets of EGFP-vinculin (grayscale) localization during optic cup morphogenesis, ~12–24 hpf. (I-L) EGFP-vinculin recruitment in control embryo is apparent at the optic stalk furrow (J, orange arrowhead), and lens-retina interface (L, white arrow). (m-P) EGFP-vinculin recruitment in lama1^UW1 mutant embryo at the optic stalk furrow (N, orange arrowhead), and lens-retina interface (P, white arrow). Dorsal views; scale bar, 50 µm. A, anterior; P, posterior; M, medial; L, lateral. (Q-Y) Quantification of EGFP-vinculin recruitment at the optic stalk furrow and lens-retina interface. (Q,R,U,V) mCherry-CAAX (membranes, gray) localization in same single confocal slices as J,N,L,P, respectively. At the optic stalk furrow or lens-retina interface (Q,R,U,V; yellow regions), EGFP-vinculin fluorescence intensity was normalized to mCherry-CAAX. Enrichment was measured by comparing to normalized EGFP-vinculin fluorescence intensity at the midline (Q,R,U,V; cyan regions). A control medial optic cup domain (U,V; magenta regions) was quantified as a location where EGFP-vinculin recruitment was not noted; this domain was also compared to normalized EGFP-vinculin fluorescence intensity at the midline (U,V; cyan regions). (S) Quantification of EGFP-vinculin recruitment indicates decreased ECM adhesion at the optic stalk furrow in the lama1^UW1 mutant. (T) Average basal endfoot width at the optic stalk furrow, based on counting the number of cells within the quantified region; no significant difference indicates that differences in cell morphology at that position (e.g. basal constriction) cannot strictly account for differences in apparent EGFP-vinculin recruitment. (W) Quantification of EGFP-vinculin recruitment indicates increased ECM adhesion at the lens-retina interface in the lama1^UW1 mutant. (X) Average basal endfoot width at the lens-retina interface, based on counting the number of cells within the quantified region; lama1^UW1 mutants have fewer cells with wider basal surfaces, indicating that increased cell number or constriction cannot account for apparent increased EGFP-vinculin recruitment at the mutant basal surface. (Y) Quantification of EGFP-vinculin recruitment at a control site in the medial optic cup where no enrichment of vinculin recruitment occurred in control or lama1^UW1 mutants. Quantifications were performed on 10 control embryos and 6 mutant embryos (one eye scored per embryo); n.s., not significant;*P<0.005, using a two-tailed t-test of unequal variance.