Figure 5

Quantitative analysis of Hh-GFP distribution: Cholesterol is required to restrict distribution but endocytosis is not required for distribution. (A-C) Schematic illustration of quantitative analysis. (A) Three dimensional reconstruction of a confocal z-stack with Hh-GFP (green) and dsRed (red) marking the expressing cells. (B) Generation of isosurfaces. DsRed isosurfacing was used to generate a distance map used to measure distances of Hh-GFP particles. Hh-GFP particles were isosurfaced to identify particles using an intensity threshold and size criteria. (C) Depiction of particle distance measurements. Particles were measured for the shortest distance to the expressing cells (lines depict manual measurements but all measurements were calculated in an automated fashion). Scale bar: 5 μm. (D-F) Mean of normalized HhNp-GFP (green) versus HhN-GFP (red) distribution profiles in a wild-type background. All samples were normalized to generate percentages of particles at the distances. Normalized data was then averaged to generate distribution profiles. Enlargement of the distribution near the x-axis shows more HhN-GFP is detected further from the A/P boundary (0 on the x-axis) at 8 (D; HhNp-GFP n = 5, HhN-GFP n = 4) and 24 hr (E; HhNp-GFP n = 16, HhN-GFP n = 7). The same is seen at 72 hr (F; HhNp-GFP n = 5, HhN-GFP n = 6). (G-H) Mean of normalized HhNp-GFP distribution profiles in wild-type background (green) versus shi^ts1 mutant background (blue). At 8 (G; shi^ts1 n = 4) and 24 hr (H; shi^ts1 n = 7), HhNp-GFP in the mutant background is less restricted and found further away from the A/P boundary than in the wild-type background. The same HhNp-GFP distribution profiles in the wild-type background from D and E are used for G and H, respectively.