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First published online 21 April 2004
doi: 10.1242/dev.01102

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Patched controls the Hedgehog gradient by endocytosis in a dynamin-dependent manner, but this internalization does not play a major role in signal transduction

Centro de Biología Molecular `Severo Ochoa', CSIC, Universidad Autónoma de Madrid, Cantoblanco, E-28049 Madrid, Spain

View larger version (95K): [in a new window]   Fig. 1. The HhGFP chimera behaves as the wild-type Hh protein. (A) Scheme of the predicted HhGFP fusion protein processing. HhGFP-F, full-length protein; HhGFP-U, the unprocessed protein without the signal peptide; HhC, the C-terminal region of the processed protein; HhGFP-Np, the N-terminal region of the processed protein with the GFP fragment and the palmitic acid and cholesterol modifications. (B) Western blot of protein extracts from AB1-Gal4/UAS-Hh and AB1-Gal4/UAS-HhGFP salivary glands stained with anti-Hh or anti-GFP. U, unprocessed protein (HhU); N, processed protein (HhNp); C, catalytic fragment (HhC). (C) Ectopic clones of HhGFP (green) in A cells (labeled by ß-Gal staining, blue) of a wing imaginal disc stained with anti-En (red). Ectopic expression of HhGFP in A wing pouch cells is able to induce En expression inside the clone and non-autonomously in a region of three to four cell diameters around the clone (arrowheads in red panel) as wild-type Hh. Note that HhGFP vesicles are detected in cells far from the source (arrows in green panel). (D) hh-Gal4/UAS-HhGFP wing imaginal disc stained with anti-Ptc (red). (d) Magnification of the boxed area in D. HhGFP signal is observed in A cells co-localizing with Ptc protein in vesicles (arrowheads). (E) disp- clones (labeled by the lack of ß-gal staining, red) in a en-Gal4/UASHhGFP (green) wing imaginal disc. There is an accumulation of Hh-GFP in the disp- mutant cells (arrowheads) with respect to the wild-type region (asterisk). (F) en-Gal4/UASHhGFP; hhts2 pharate raised at restrictive temperature during larvae development. All the structures were rescued except for some head structures (arrow) where the expression of en depends on Hh expression. In this and all other figures, the A compartment of the wing disc is towards the left and the P is towards the right. A broken line indicates the AP compartment border. Scale bars: 15 µm in C,d; 50 µm in D,E.

View larger version (93K): [in a new window]   Fig. 2. The PtcWTGFP chimera behaves as the wild-type Ptc protein. (A) Scheme of the PtcGFP fusion protein. The GFP is tagged to the C-terminal cytoplasmic tail of Ptc. (B) Western blot of protein extracts from AB1-Gal4/UAS-PtcWTGFP or AB1-Gal4/UAS-Ptc14GFP salivary glands stained with anti-GFP and anti-Tubulin. Both PtcWTGFP and Ptc14GFP proteins migrate with an apparent molecular weight of 180-200 kDa. (C) Collier (Col) expression pattern in a wild-type wing imaginal disc. (D) A large ectopic PtcWTGFP clone in the P compartment causes a reduction of Col expression (arrowhead) at the AP compartment border because of the sequestration of Hh by the ectopic Ptc (see also Fig. 7A). (E) An ectopic PtcWTGFP clone in the P compartment of a wing imaginal disc stained with anti-Smo (red). Note the autonomous downregulation of Smo because of the repressive activity of Ptc (arrowheads).

View larger version (125K): [in a new window]   Fig. 7. Ptc14 does not internalize Hedgehog. (A,B) Ectopic wild type PtcWTGFP (A) and Ptc14GFP (B) clones in the wing imaginal disc stained with anti-Hh antibody (red). (a,b) Detail of the clones shown in A and B. Only a few vesicles of Hh are observed in the clones expressing the Ptc14 mutant protein (Ptc14GFP) (b) compared with those that express the wild-type Ptc protein (PtcWTGFP, arrows) (a). (C) Ptc (green) and Hh (red) expression in a wing imaginal disc containing a ptc14 clone (labeled by the absence of ß-gal staining, blue). Ptc staining inside the clone (outlined in pale blue) has the same intensity levels than in the wild-type area (arrow, green panel). (c,c') Magnification of the boxed areas in C. Ptc and Hh colocalize in spots in the wild-type area (circles) (c). Ptc does not accumulate in spots (green) in the ptc14 clone. However, Hh vesicles are still present in the ptc14 clone (red, arrowheads) (c'). (D) ptc14 clone at the AP border (shown by the absence of green, GFP) in a shits1 wing disc after 3 hours at the restrictive temperature. In the absence of dynamin-dependent internalization, Hh (red) accumulates in the PtcWT territories at the AP border and also in the PtcWT cells anterior to the mutant clone (arrowhead). However, Hh protein is not accumulated in the ptc14 mutant cells (asterisk). Note that the accumulation of Hh anterior to the ptc14 clone (arrowhead) is higher than the accumulation in wild-type territory at the same distance from the AP border (arrow). This indicates that Hh diffuses further through the ptc14 mutant cells than through wild-type cells. Scale bars: 50 µm.

View larger version (140K): [in a new window]   Fig. 3. Ptc is required for dynamin-dependent internalization of Hh. (A) shits1 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) shits1 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 shits1 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 shits1 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 PtcWT area, just anterior to the clone (arrowheads). (D) shits1 clones (absence of GFP, green) in a wing imaginal disc after 10 hours at restrictive temperature. In shits1 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.

View larger version (98K): [in a new window]   Fig. 4. Hh signaling is unaffected in dor mutant cells. (A) A confocal section of a Rab-7GFP/c765-Gal4 wing imaginal disc, stained with anti-Ptc (blue) and anti-Hh (red) antibodies. (a) Magnification of boxed area in A. The staining shows co-localization of Hh, Ptc and Rab-7 at the AP compartment border (circles in a). There are also spots of Ptc and Hh colocalization that do not colocalize with Rab7 (arrowheads in a); these are probably early endosomes. The inset in a shows detail of the co-localization of the three proteins at some of these spots. Rab-7 shows doughnut-shaped GFP emission (arrow) surrounding the Ptc and Hh spots (arrowheads). (B) dor8 mutant clones (labeled by the lack of ß-gal staining, blue) at the AP compartment border showing high accumulation of Hh (green) and Ptc (red) proteins because of the inhibition of lysosomal degradation. (C) Plot showing the green color pixel intensity of the cells in clone 1 (blue line) and clone 2 (red line) [corresponding to boxed areas in B (green panel)] with respect to the distance of the cells to the AP border. The same intensity (Hh accumulation) is seen in the A cells located at the same distance from the AP compartment border (arrowhead in B and C), whether Hh crosses (from the P compartment) a wild type (clone 1) or mutant (clone 2) area. (D) dor8 mutant clones (labeled by the lack of GFP staining, blue) close to the AP boundary showing no alteration in activation of the Hh targets [as shown by the expression of dpp-lacZ (red) (arrowhead) compared with its expression in the wild-type area (arrow)]. Scale bars: 50 µm in A,B,D; 15 µm in a.

View larger version (88K): [in a new window]   Fig. 5. Hh spreading is not impeded in ptc14 mutant clones but signaling is normal. (A,C,E) Wild-type expression of the Hh targets En (red) and Ci (green) (A), Col (red) (C) and Dpp (red) (E) in the third instar wing imaginal disc. (B,D,F) Activation of En, Col and Dpp (red) in ptc14 mutant clones abutting the AP compartment border (labeled by the lack of green GFP staining) (clones 2, 4 and 6) but not in clones outside the AP border (clones 1, 3 and 5). Note that Hh target gene activation occurs not only inside the clone touching the AP compartment border but also in cells located more anterior to the clone (arrowheads). The plots (right) show the fluorescence intensity for each target gene with respect to the distance of the cells to the AP border. The activation of the target genes occurs inside and anterior to the clone (arrowheads). Of 62 clones in the A compartment with A sister clones, 39 were located outside of the AP compartment border and did not show ectopic expression of En, Col and Dpp, and 23 were touching the compartment border showing activation of the targets inside and also in cells located anterior to the clone. (b) GFP signal and Ci staining of the disc in B. The clone touching the AP border has its twin in the A compartment (red arrow). The presence of the Ci staining sets the position of the AP border (red arrowhead). Scale bar: 50 µm.

View larger version (145K): [in a new window]   Fig. 6. Ptc14 activates the Hh pathway only in the absence of Hh. (A-D) The upregulation of Ptc (A) and Col (C) expression (green) at the AP compartment border observed in a wild-type wing disc is not produced in ptc14 clones (shown by the lack of ß-gal staining, red) in an hhts2 disc after 30 hours at the restrictive temperature (B,D; arrowheads). Of 20 clones in the A compartment with A sister clones, eight were touching the AP compartment border and did not showed activation of Ptc or Col at the restrictive temperature. (E) Cuticle preparation of wild-type, ptc14 and ptc14; hhts2 embryos at the restrictive temperature. Note that the double mutant ptc14; hhts2 embryo is like an hh- embryo, and not like a ptc- embryo, which would normally be the case with ptc-; hh- double mutant embryos. (F) Wg expression pattern in wild-type, ptc14 and ptc14; hhts2 embryos at the restrictive temperature. In the ptc14 embryo, Wg stripes are broadened compared with the wild-type embryo, but Wg is not expressed in ptc14; hhts2 embryos at the restrictive temperature. Scale bars: 50 µm.

View larger version (157K): [in a new window]   Fig. 8. Ptc14 does not accumulate in endosomes. (A,B) Internalized Red-dextran (5 minutes pulse) at the AP border of a wild-type wing imaginal disc (A) and in a ptc14 clone (B). (a) Details of the colocalization of Red-dextran positive vesicles and Ptc (green). (b) There is no colocalization of the internalized Red-dextran vesicles with Ptc protein in ptc14 clone cells. There is reduced Ptc staining in vesicles labeled with Red-dextran (green, arrowheads). Very few Red-dextran vesicles contain appreciable accumulation of Ptc (B, circles) compared with wild-type Ptc (A, circles). (C,D) Extracellular staining of Ptc in a wing disc containing ectopic PtcWTGFP (C) and Ptc14GFP (D) clones using the anti-Ptc antibody, which recognizes one of the extracellular domains of Ptc. Staining is observed in both clones (arrowheads), indicating that a certain amount of PtcWTGFP and Ptc14GFP reaches the plasma membrane. (E,F) PtcWTGFP (E) and Ptc14GFP (F) ectopic clones in the A compartment far from the AP border in wing discs incubated with Red-dextran for 10 minutes. (e) Magnification of E showing the extensive co-localization between PtcWTGFP and internalized Red-dextran (circles), indicating that PtcWTGFP is endocytosed independently of the presence of Hh. (f) Magnification of F showing very little colocalization between Ptc14GFP and internalized Red-dextran (arrowheads), indicating that Ptc14GFP is not efficiently endocytosed. Circles indicate some Red-dextran vesicles with Ptc14GFP accumulation. The analysis of the PtcWTGFP protein content in endocytic vesicles compared with the Ptc14GFP mutant protein (E,F, columns) shows at least eight times more wild-type Ptc protein in endosomes than Ptc mutant protein (see Materials and methods). Scale bars: 15 µm in A,B; 50 µm in C-F.

View larger version (205K): [in a new window]   Fig. 9. Co-expression of PtcS2 and Ptc14 recovers the normal subcellular distribution of Ptc14 protein. (A-C) The distinct staining patterns of PtcWTGFP (A), PtcS2GFP (B) and Ptc14GFP (C) in salivary glands cells (using the AB1-Gal4 driver). (D) The subcellular distribution of Ptc14GFP protein in salivary gland cells changes to the wild-type pattern when it is co-expressed with non-labeled PtcS2. Scale bar: 30 µm.

View larger version (139K): [in a new window]   Fig. 10. Hh internalization in Hh-receiving cells in the absence of Ptc. (A) ptc14 clone (labeled by the lack of ß-gal staining, blue) abutting the AP compartment border in an hh-Gal4/UAS-HhGFP background stained with anti-Ptc antibody (red). (a) Magnification of the same ptc14 clone in A. There is no colocalization between HhGFP punctuate structures and Ptc14 (arrowheads). Only a few of the Hh-GFP vesicles show colocalization with Ptc staining, though not as spots (circles). Compare with HhGFP staining in a wild-type background (Fig. 1D). (B) ptc14 clone in an hh-Gal4/UAS-HhGFP wing disc with internalized Red-dextran. (b) Magnification of the ptc14 clone in B. HhGFP vesicles do not colocalize with Ptc but most of them colocalize with internalized Red-dextran (circles). (C) ptc16 clone (indicated by the lack of ß-gal staining, blue) abutting the AP compartment border in an hh-Gal4/UASHhGFP background stained with anti-Ptc antibody (red). (c) Magnification of the ptc16 clone in C. (D) ptc16 clone in an hh-Gal4/UAS-HhGFP wing disc showing the internalized Red-dextran. (d) Magnification of the ptc16 clone in D. Observe the HhGFP punctuate structures (green, arrowheads) in A cells close to the AP border with no Ptc protein (c). These structures colocalize with Red-dextran (circles; d). Scale bars: 50 µm in A-D; 15 µm in a-d.

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