(A-C’’) Representative transverse sections taken through the midbrain region of 8–9ss embryos expressing L-NTF-HA (A–A’’), S-NTF-HA (B-B’’), or GFP (C-C’’), followed by immunostaining for HA or GFP (green), laminin (red), and Snail2 (violet). (A-C, left column) Low magnification (20×) images display merged triple-label immunohistochemistry with nuclear DAPI stain (blue). (A’-C’, middle column) Higher magnification images of dorsal neural tubes from section images in (A-C) demonstrating HA (or GFP) and laminin co-localization. (A’’-C’’, right column) The same images in (A’-C’) but showing laminin and Snail2 co-localization. Dotted lines in (A’, A’’, B’, B’’, C’, C’’) mark dorsal neural tube midlines created by fusing neural folds. Arrowheads in (A’, B’) indicate marked reductions in laminin immunoreactivity as well as laminin “patchiness.” The scale bar in (A) is 50 μm and applicable to (B, C), while the scale bar in (A’) is 20 μm and applicable to (A’’, B’, B’’, C’, C’’). (D) Representative line scans of laminin immunostaining intensity along the basement membranes of the L-NTF-HA-expressing side in (A-A’’, red) and the unelectroporated contralateral side in (A-A’’, blue) in a medial (0 μm) to lateral (45 μm) direction. (E) Basement membrane levels of laminin and fibronectin (for fibronectin images, see Fig. S3) for each section (treatment and control sides) were quantified by generating ratios of the areas under the curve between treatment (L-NTF-HA, S-NTF-HA, GFP, or wildtype (WT)) line scans versus the unelectroporated, contralateral side (see D). Ratios lower than one indicate a reduction in laminin or fibronectin. At least 15 symmetric sections (see Section 2) originating from at least three embryo midbrains were analyzed per treatment group. Both S-NTF-HA and L-NTF-HA overexpression caused significant reductions in both laminin (33% and 32% reductions, respectively, p < 0.05) and fibronectin (23% and 21% reductions, respectively, p < 0.05) levels versus GFP controls. GFP ratios were not significantly different from wildtype controls. Treatment levels were analyzed by ANOVA, and asterisks denote treatments that are significantly different to GFP controls (p < 0.05).