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First published online 5 January 2006
doi: 10.1242/dev.02217

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Hedgehog lipid modifications are required for Hedgehog stabilization in the extracellular matrix

Ainhoa Callejo^*, Carlos Torroja^*,, Luis Quijada and Isabel Guerrero

Centro de Biología Molecular `Severo Ochoa', C.S.I.C., Universidad Autónoma de Madrid, Cantoblanco, E-28049 Madrid, Spain.

View larger version (103K): [in a new window]   Fig. 1. The large punctate structures of Hh, HhN and Hhc85s are endocytic vesicles and only the lipid-modified Hh forms oligomers in Drosophila. (A) Glycerol gradients (50-15%) used to fractionate Hh-GFP, HhN-GFP, Hhc85s-GFP and Hhc85sN-GFP obtained from salivary glands expressing the four Hh forms. We observed that wild-type Hh fractionated as two different density gradients: one associated with the high molecular weight fraction (9-14), which corresponded to oligomers; the other associated with the low molecular weight fraction (17-23), corresponding to monomers. HhN-GFP, Hhc85s-GFP and Hhc85sN-GFP elute with the low-density fractions (monomers). (B-D,b-d) Wing imaginal discs expressing the different Hh-GFP forms with the hh-Gal4 driver. Hh-GFP forms a short gradient of around six or seven cell diameters (blue line in B), while HhN-GFP and Hhc85s-GFP proteins are unable to generate a proper gradient (blue line in C and D). HhN-GFP and Hhc85s-GFP vesicles are present far from the Hh-expressing cells. Internalized dextran red-positive vesicles (24 minutes of incubation) co-localized with Hh-GFP (b), HhN-GFP (c) and Hhc85s-GFP (d) in both A and P compartments of the wing imaginal cells that expressed the three forms of Hh in their own expression domain using the Hh-Gal4 driver (arrowheads). Some GFP labeled particles do not co-localize with the internalized dextran (arrows). These particles are nevertheless intracellular (see Fig. S1D in the supplementary material). These results indicate that, in all cases, the punctate structures are endocytic vesicles. The A compartment is orientated towards the left; the P compartment is orientated towards the right.

View larger version (139K): [in a new window]   Fig. 2. Differential accumulation of Hh-GFP versus HhN-GFP and Hhc85s-GFP in the plasma membrane of shits1 mutant clones. (A-E) shits1mutant clones induced in UAS-Hh-GFP/hhGal4 (A,D), UAS-HhN-GFP/hhGal4 (B,E) and Hhc85s-GFP/hhGal4 (C) wing imaginal discs. Hh-GFP accretions were only observed in the basolateral plasma membrane of the A compartment cells (arrowheads in A), while HhN-GFP (B) and Hhc85s-GFP (C) were detected only in the apical part of the A compartment cells. (D,E) Transverse sections of the shits1mutant clones in A,B, respectively. Arrows indicate the apical parts and arrowheads indicate the basolateral parts of the cells. There is basolateral accumulation of Hh (D) and apical accumulation of HhN (E). The AP boundary is indicated with a broken line.

View larger version (137K): [in a new window]   Fig. 3. Differential localization of Hh-GFP, HhN-GFP and Hhc85s-GFP in the plasma membrane. (A-C) Extracellular labeling of Hh-GFP (A), HhN-GFP (B) and Hhc85s-GFP (C) showing their distribution at the apical versus basolateral plane. (a-c) AP z sections of the same wing discs in A-C. (a') AP z sections of a wing disc double-stained with anti-Hh antibody (green) and anti-DE-cadherin (purple) as an apical region marker. The three Hh forms occur in the extracellular space on the apical side of the producing cells (asterisk), while only lipid-unmodified forms of Hh appear in the apical extracellular space in the A compartment (arrows in apical planes and Z sections). Wild-type Hh forms an extracellular gradient in the basolateral A cells next to the AP compartment, while the lipid-unmodified forms are poorly represented and do not form a gradient (arrowheads in basolateral panels and z sections).

View larger version (84K): [in a new window]   Fig. 4. Internalization and degradation of HhN and Hhc85s independent of Ptc. (A-C) dor8 clones induced in UAS-Hh /HhGal4 (A), HhN-GFP/HhGal4 (B) and Hhc85s-GFP/HhGal4 (C) wing discs. The graphs represent a normalized pixel intensity of Ptc, Hh and ß-Gal staining, measured in the area represented by white frames in A-C. The three forms of Hh accumulate in dor8 clones, indicating an active process of degradation. There is precise direct correlation between the amounts of accumulated wild type Hh and Ptc in A (see graph), but not in the case of the lipid-unmodified Hh forms, where Ptc is not acculumated (see graphs in B,C). (D-F) dor8- clones in ap-Gal4/UAS-Hh-GFP (D), ap-Gal4/UAS-HhN-GFP (E) and ap-Gal4/UAS-Hhc85s-GFP (F) wing imaginal discs. There is increased accumulation of HhN-GFP and Hhc85s-GFP in dor- clones, induced both in the A and P compartments, even when they are located far from their expressing domains (arrows in E,F). However, wild-type Hh-GFP accumulates over a very short range in A compartment dor- clones (arrows in D) and is never accumulated in P compartment dor- clones (arrowheads in D). The accumulation of HhN-GFP and Hhc85s-GFP (green) in the P compartment dor- cells, where Ptc (red) is not present, indicates a process of internalization and degradation independent of Ptc (arrowheads in E,F). This is reinforced by the absence of Ptc accumulation in A compartment dor- clones in the case of Hhc85s-GFP (arrows in F). In the case of HhN-GFP, the small amount of Ptc accumulation in dor- clones induced in the A compartment reveals some interaction between HhN and the Ptc receptor (arrows in E). The diagrams on the right-hand side of the figure represent the areas of the disc shown in the confocal panels and the interpretation of the results shown.

View larger version (92K): [in a new window]   Fig. 5. Inductive effect of HhN but not of Hh from peripodial cells upon disc proper cells. (A-C') Expression of Hh-GFP (A,a,A',a'), HhN-GFP (B,b,B',b') and Hhc85s-GFP (C,c,C',c') in peripodial cells using the Ubx-Gal4 driver. The Ubx is expressed at high levels in the part of the peripodial membrane corresponding to the P compartment (arrowheads in A,B,C). HhN-GFP and Hhc85s-GFP, but not wild-type Hh-GFP, are observed in internalized vesicles in the cells of the disc proper (arrows in posterior and arrowheads in anterior compartment in A',B',C'). The internalized vesicles of HhN-GFP are more clearly visualized in the A than in the P compartment, unlike Hhc85s-GFP vesicles, which are visible in both A and P compartments. Dpp (red) is ectopically induced in the disc proper cells in HhN-GFP/Ubx-Gal4 disc (B,B'). (D-G) Wing and notum phenotypes of the Hh-GFP/Ubx-Gal4 and Hhc85s-GFP/Ubx-Gal4 (D,F), HhN-GFP/Ubx-Gal4 flies (E,G). The notum is enlarged and contains extra-macrochaetae (G) and there is modification of the wing pattern (E) in HhN-GFP/Ubx-Gal4 flies. (H) Diagrams showing the organization of the disc proper cells and peripodial cells in a front- and transverse view of a wing imaginal disc.

View larger version (98K): [in a new window]   Fig. 6. Signaling activity of Hh, HhN and Hhc85s. (A) A wing imaginal disc with the posterior P compartment cells expressing Hh (green) and the anterior A compartment cell expressing Ptc (red) in response to Hh. The red frame indicates the territory represented in the adjacent panel, showing the real staining of the wing disc with Hh (green) and Ptc (red) antibodies. The right panel shows the expression of different Hh target genes in A compartment cells. (B) The different thresholds of Hh signaling required for the activation of the different targets. Ectopic Hh-GFP (C-F), HhN-GFP (G-J) and Hhc85s-GFP (K-N) clones induce activation of En (C,G,K), Ptc (D,H,L), Dpp (E,I,M) and Iro (F,J,N) in the anterior compartment (labeled in red). Hh-expressing cells are marked by b-Gal (blue) in most of the panels, with the exception of panels E, I and M, where they are labeled by the absence of CD2 (blue). Arrows indicate the autonomous response and arrowheads indicate the non-autonomous response to various forms of Hh. (O-Q) The activation of Hh targets by ectopic Hh-GFP (O), HhN-GFP (P) and Hhc85s-GFP (Q) clones in the A compartment. Light green represents the levels and graded distribution of Hh protein and the dark green represents the signaling activity of wild-type Hh, HhN-GFP and Hhc85s-GFP. In O, but not in P and Q, Hh protein distribution is the same as its signaling activity gradient.

View larger version (94K): [in a new window]   Fig. 7. Diffusion properties and signaling activity of Hh-GFP, HhN-GFP and Hhc85s-GFP in ttv- mutant cells. (A-K) ttv- clones labeled by the absence of b-gal staining (blue) in wing discs expressing Hh-GFP (A-C), HhN-GFP (E-G) and Hhc85s-GFP (I-K) with the hh-Gal4 driver (green) and stained for Ptc (A,E,I), Col (B,F,J) and Iro (C,G,K) in red. HhN-GFP and Hhc85s-GFP, but not wild-type Hh-GFP, appear inside the ttv- cells and in the cells located anterior to the clone (arrows in E,I). Ptc and Col are expressed only in the first row of ttv- cells next to the AP border (arrowheads in A,B,E,F,I,J) and Hh-GFP vesicles co-localize with Ptc in all cases (arrowheads in A). Iro is expressed only in the first row of cells in the wild-type Hh-GFP- and Hhc85s-GFP-expressing discs (arrows in C,K). In HhN-GFP-expressing discs, Iro is activated throughout the ttv- clone (arrowheads in G, clone 1 in H). However, Iro expression decreases in a clone far from the HhN-GFP-producing cells because HhN-GFP levels are lower than at the AP compartment border (arrow in G, clone 2 in H). (D,H,L) Graphs showing the activation of Ptc, Col and Iro in ttv- clones abutting the AP compartment border (clone 1) or located distant to the AP border (clone 2) by wild-type Hh-GFP (D), HhN-GFP (H) and Hhc85s-GFP (L). The light green represents Hh levels and the graded distribution of the protein produced in the P compartment and secreted to the A compartment; the dark green represents the signaling activity induced by the different Hh forms. The broken lines represent the Hh gradients and signaling activity in a disc that does not contain ttv- clones.

View larger version (56K): [in a new window]   Fig. 8. Signaling activity of Hh-GFP, HhN-GFP and Hhc85s-GFP in cells lacking tout-velu function and simultaneously expressing Hh. (A-D) Wing imaginal discs with clones mutant for ttv that also express wild-type Hh ectopically. All clones in the anterior compartment shown in the figure are located outside the expression domain of the different target genes tested. Clones are marked with GFP (A-D) and outlined with a red line (A'-D'). The discs are stained for different Hh targets (red): En (A,A'), Ptc (B,B'), Iro (C,C') and Ci (D,D'). (E) The results of the clone analysis. Hh triggers the response of low-threshold responses (Ci and Iro) in ttv- cells, while failing to activate high-threshold response genes (Ptc, En and Col) apart from the first row of cells within the clone.