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Fig. 3. Ptc is required for dynamin-dependent internalization of Hh. (A) shi^ts1 A clone (labeled by the absence of ß-Gal staining, blue) after 3 hours at the restrictive temperature that accumulates high levels of Hh (green) and Ptc (red) (arrowheads). (B) shi^ts1 A clone after 3 hours at the restrictive temperature in a hh-Gal4/UAS-HhGFP wing disc. (b) Magnification of boxed area in B. The accumulation of Hh (green) in shi^ts1 clone cells is extracellular, as shown by `in vivo' incubation of the wing imaginal disc in a cold medium with anti-GFP antibody (red) (arrowheads, red panel in B and b). Note that the intracellular HhGFP (green and not red in the P compartment) is not labeled by this method (arrows in b). (C) ptc¹⁶ clone (labeled by absence of GFP, green) at the AP border in a shi^ts1 wing disc after 3 hours at the restrictive temperature. Hh protein (red panel) does not accumulate in a shi background when Ptc protein is not present (blue panel) (arrow). Note that Hh crosses the Ptc^- territory and accumulates when it reaches the Ptc^WT area, just anterior to the clone (arrowheads). (D) shi^ts1 clones (absence of GFP, green) in a wing imaginal disc after 10 hours at restrictive temperature. In shi^ts1 clones there is not increase or decrease in Col expression after 10 hours at restrictive temperature (asterisk) compared with wild-type territory (arrow). There is not extension of signaling either (arrowhead). Scale bars: 50 µm in A-D; and 15 µm in b.

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