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First published online 7 January 2004
doi: 10.1242/dev.00958

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Drosophila glypicans control the cell-to-cell movement of Hedgehog by a dynamin-independent process

Chun Han, Tatyana Y. Belenkaya^*, Bei Wang^* and Xinhua Lin

Division of Developmental Biology, Children's Hospital Medical Center, Cincinnati, OH 45229, USA

View larger version (64K): [in a new window]   Fig. 1. dly is required for Hh signalling in embryogenesis. All cuticles are oriented anterior towards the left. (A-C) Ventral views are shown. Cuticle preparation of the wild-type first instar larva (A), hhIJ35 homozygous embryo (B) and dlyA187 mutant embryo derived from a dlyA187 germline clone (C). (D-I) All embryos are oriented anterior towards the left and dorsal upwards. En staining in stage 10 wild-type (D) and dlyA187 mutant (E) embryos. (F,G) wg transcription in stage 10 wild-type (F), and dlyA187 mutant (G) embryos visualized by in situ hybridization. (H,I) bap transcription in stage 10 wild-type (H) and dlyA187 mutant (I) embryos visualized by in situ hybridization.

View larger version (128K): [in a new window]   Fig. 2. dally and dly are required for Hh signalling in wing development. (A-C) Wings are oriented proximal towards the left and anterior upwards. (A) wild-type wing with labeled longitudinal veins. (B) A wing carrying clones of dally80 showing a partial L5 vein loss (arrow) (80%, n=100). (C) A wing carrying clones of dally80-dlyA187 mutant cells showing L3-L4 vein fusion (arrow) (10%, n=80). (D-J') All the wing discs were derived from third instar larvae. Anterior is towards left and dorsal is upwards in all the wing disc images. (D-F') Ptc expression (red) in a wild-type wing disc (D) and a disc carrying a dally80-dlyA187 clone (E,E',F,F'). In wild-type disc, Ptc is expressed in a stripe of cells abutting the AP boundary in response to Hh from the P compartment. Ptc expression is reduced in dally80-dlyA187 mutant cells (marked by the absence of GFP in E' and F'). (G-H') En (red) staining in a wild-type wing disc (G) and a disc bearing dally80-dlyA187 clones (H,H'). En expression in the A compartment is induced by Hh in a three to four cell diameter stripe (bracket) anterior to the AP boundary (marked by a broken line in G). This expression is eliminated in the dally80-dlyA187 clone (marked by the absence of GFP in H'). (I-J') Col (red) staining in a wild-type wing disc (I) and a disc bearing dally80-dlyA187 clones (J,J'). Col is expressed in four cell diameters anterior to the AP boundary. In dally80-dlyA187 clones (marked by the absence of GFP in J'), Col is restricted to two cell diameters with a lower level in the more anterior row of cells.

View larger version (83K): [in a new window]   Fig. 4. Hh moves from cell to cell. All the wing discs were derived from third instar larvae. Anterior is towards left and dorsal is upwards in all the wing disc images (A-H). (A-C') dpp-lacZ staining (red) in a wild-type wing disc (A), and discs carrying sfl9B4 mutant clones (B,B',C,C'). In the wild-type wing disc, dpp-lacZ is expressed in eight to ten cells anterior to the AP boundary in response to Hh signal (A). dpp-lacZ expression was only observed in a maximum of three cells at the posterior edge of sfl9B4 clones (marked by broken lines in B and C, and by the absence of GFP in B' and C'), and high levels of lacZ expression were only detected in one or two cells adjacent to posterior wild-type cells. (D-H) GFP-Dpp expression under the control of dppGal4 in a wild-type wing disc (D) and discs carrying mutant clones of sfl9B4 (E,F), ttv63 (G), and dally80-dlyA187 (H). The mutant clones are marked by the absence of Myc (E,F,H) or ß-gal (G) staining (red) and are outlined with broken lines. In all cases, GFP-Dpp expression is restricted to the posterior-most row of cells in the mutant clones. Hh movement towards anterior cells is blocked by even one cell diameter of mutant clones (arrows in B,C,E,H) as indicated by dpp-lacZ or GFP-Dpp expression.

View larger version (92K): [in a new window]   Fig. 5. Hh movement is independent of dynamin-mediated endocytosis. All the wing discs were derived from third instar larvae. Anterior is towards left and dorsal is upwards in all the wing disc images (A-E'). In all discs, the shits1 clones are marked by the absence of ß-gal staining (green) and outlined with broken lines. The AP boundaries are determined by Ci staining (not shown) and are marked by solid lines. (A-C) Hh staining in a wild-type wing disc (A) and a disc carrying a shits1 clone in the A compartment (B,C). Hh protein appears to be graded membrane staining and is concentrated in large punctate particles (indicated by arrows) in three or four cell diameters abutting AP boundary in wild-type disc (A). These large punctate particles are absent inside the shits1 clone and can only be observed at the boundary between mutant and wild-type cells (arrows in B). (B') The merged image stained with Hh and lacZ. (C) A basal optical section of the same disc shown in B where Hh accumulation along the cell membrane in the clone is more prominent. (D,D') Ptc staining in a disc carrying shits1 clones at AP boundary. Ptc expression is expanded anteriorly within the lower larger clone. Noticeably, the wide-type cells anterior to the upper small clone still express Ptc, suggesting that shits1 cells cannot block Hh movement. (E,E') Ci staining in a disc carrying shits1 clones. Ci expression pattern is not significantly altered in the clones.

View larger version (103K): [in a new window]   Fig. 6. Dally colocalizes with and stabilizes Hh. (A,B) Hh staining (red) in wing discs expressing GFP-Dly (green) under the control of hhGal4 (A) or ptcGal4 (B). Hh staining and GFP-Dly in boxed areas are shown separately in small panels. (C-C') Hh staining in a wing disc carrying a sfl9B4 mutant clone (marked by the absence of GFP in C'). The AP boundary is determined by Ci staining (not shown) and marked by solid lines. The clone is outlined with broken lines. (D,E) Hh staining (green) in a stage 9 wild-type (D) or dlyA187 (E) embryo. The Hh expressing cells are determined by En expression (red).

View larger version (68K): [in a new window]   Fig. 3. Ttv affects HS GAG modifications in Dally and Dly. Total protein from third-instar larvae was analyzed by 4-20% gradient SDS-PAGE followed by western blotting with rabbit anti-Dly (Baeg et al., 2001) and rabbit anti-Dally (Lin and Perrimon, 1999) antibodies. In wild-type larvae, both Dly and Dally migrate as high molecular masses (Mr), characteristic of HS GAG-modified Dly and Dally (shown by arrows). In homozygous ttvl(2)00681 larval extracts, the high Mr HS GAG-modified Dly is shifted to relative low Mr smear and the high Mr HS GAG-modified Dally is significantly reduced.

View larger version (15K): [in a new window]   Fig. 7. Model of Hh movement. The orange arrow indicates that Disp releases Hh proteins from the cell membrane of the Hh-producing cells. The purple arrow indicates that glypicans can present Hh to Ptc. The thin black arrows at the top of this diagram indicate the displacement of Hh from one GAG chain to another GAG chain. The double-headed arrow indicates the lateral movement of glypicans on cell membrane.