DEV02217 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1. Hh-GFP, HhN-GFP, Hhc85s-GFP and Hhc85sN-GFP are correctly processed and recognized by anti-Hh antibodies. (A) Upper panel: western blot using anti-Hh antibody of Hedgehog-GFP fusion protein and its mutant forms expressed in the salivary glands. This blot indicates that the Hh forms are correctly processed and are expressed at equivalent levels. Lower panel: Coomasie staining of the SDS-PAGE gel as a control of the amount of protein extract loaded for each sample. (B,C) Ectopic HhN-GFP (B,b) and Hhc85s-GFP (C,c) clones showing the co-localization of protein-GFP and anti-Hh antibody (Takei et al., 2004). They are co-localized in the punctate structures in both the A and P compartments. White frames in B and C indicate the territory represented in b and c, respectively. (D) Hh-GFP/hh-Gal4 wing disc incubated with dextran red to label the endocytic compartment and subsequently incubated with anti-GFP antibody at 4°C to label the extracellular Hh-GFP (see Materials and methods). Some Hh punctate structure (green), but not all, co-localize with dextrans (arrowheads), indicating that they are internalized vesicles. The punctate structures that do not co-localize with dextrans are also intracellular vesicles (arrows), because they are not labeled by extracellular staining.

• Supplemental Figure 2 -

  Fig. S2. Long-range diffusion and signaling of a single HhN-GFP ectopic clone induced in the disc proper cells.  (A,B) A wing imaginal disc containing a single HhN-GFP (green) ectopic clone. (A) This clone was induced in the disc proper cells and shows a long-range activation of Iro (red). (B) Peripodial cells of the same disc, marked by Ubx expression (blue), do not contain ectopic clones. (C,D) A comparison of the diffusion range of HhN-GFP (the same ectopic clone shown in A,B) with that of Hh-GFP ectopic clone. Both ectopic clones, HhN-GFP (C) and Hh-GFP (D) are of equivalent size, position and expression levels. The gradient produced by HhN-GFP is much extended than the one produced by Hh-GFP. These data together with the ones shown in Figs 5 and 6 indicate that Hh-N forms a very extended gradient.

• Supplemental Figure 3 -

  Fig. S3. ptc^–, ttv^–, double mutant clones do not internalized Hh.  (A) Apical view of confocal images of a ttv^– clone (labeled by the absence of b-gal in red) abutting the AP border in a UAS-Hh-GFP/hh-Gal4 wing imaginal disc showing the internalization of Hh-GFP in the first row of cells inside the clone touching the AP border. (B) ptc^–, ttv^– double clones abutting the AP border in a UAS-Hh-GFP/hh-Gal4 wing imaginal disc. Hh-GFP vesicles are absent inside the ptc^–, ttv^– clone.