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First published online 12 January 2005
doi: 10.1242/dev.01636

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Drosophila glypicans Dally and Dally-like shape the extracellular Wingless morphogen gradient in the wing disc

Chun Han^1,2,*, Dong Yan^1,2,*, Tatyana Y. Belenkaya² and Xinhua Lin^1,2,

¹ Division of Developmental Biology, Cincinnati Children's Hospital Medical Center, Cincinnati, OH 45229, USA
² The Graduate Program in Molecular and Developmental Biology, University of Cincinnati College of Medicine, Cincinnati, OH 45229, USA

View larger version (128K): [in a new window]   Fig. 1. Dally and Dlp are required for the proper distribution of extracellular Wg. All wing discs are oriented dorsal top-right, anterior top-left. All mutant clones are marked by the absence of GFP and outlined by broken lines. (A-C') Extracellular Wg staining in discs bearing large clones mutant for dally. Extracellular Wg is significantly reduced in the clones in a cell non-autonomous manner. The yellow arrows indicate the wild-type cells that have reduced extracellular Wg level. (D-E') Extracellular Wg staining in discs bearing large clones mutant for dlp. Within the clones, extracellular Wg is reduced outside of a zone that is 7-10 cell diameters wide and centered at the DV boundary. (F-G') Extracellular Wg staining in discs bearing large clones mutant for both dally and dlp. Extracellular Wg level is greatly reduced in these clones, and the reduction is stronger than that of dally or dlp alone.

View larger version (80K): [in a new window]   Fig. 8. Wg movement is impeded by HSPG-deficient cells. Extracellular Wg staining in discs bearing narrow stripes of clones mutant for sfl (A-B') or dally-dlp (C-D'). All discs are oriented anterior left, dorsal up. The mutant clones are marked by the absence of GFP in the merged images, and outlined by broken lines. The extracellular Wg is reduced not only within these mutant clones, but also in the wild-type cells behind them (pointed by blue arrows). For comparison, the yellow arrows indicate wild-type cells at similar distances from the DV boundary. In each disc, Wg distribution is averaged and plotted (A'',B'',C'',D'') from an area including the narrow clone and the cells behind it and from a control area (boxed in the merged images). The colors of profiles are specified at the bottom of the figure. The left-right axis of the plot profiles corresponds to the ventrodorsal axis of the boxed regions. The drops of GFP intensity indicate the position of the mutant clones.

View larger version (122K): [in a new window]   Fig. 2. The expression of Dally and Dlp in the wing disc. All discs are oriented dorsal upwards, anterior leftwards. (A-B') Dally and Dlp expression in third instar wing imaginal discs visualized by dally-lacZ expression (green in A and A') and Dlp staining (green in B and B'), respectively. The AP and DV compartment boundaries are visualized by Ci staining (blue in A' and B') and Wg or wg-lacZ expression (red in A' and B'), respectively. (C-D') Dlp staining (green) in discs carrying random clones expressing either the active form of Arm (Armact) (C,C') or the dominant-negative form of TCF (TCFDN) (D,D'). The clones are marked by GFP (red) and outlined with broken lines. The expression of Dlp is reduced in clones expressing Armact, and increased in clones expressing TCFDN. (E-F') Dlp staining (red) in discs carrying dsh clones (E,E') or a large notum clone covering the entire A compartment (F,F'). Both dsh and notum mutant cells are marked by the absence of GFP (green) (E',F'). Dlp levels are increased in dsh clones, but not altered in the absence of notum function.

View larger version (147K): [in a new window]   Fig. 3. Notum suppresses Wg signaling mainly through Dally. (A-D) Hnt staining (red) in a wild-type disc (A), a homozygous dally disc (C) and discs carrying clones mutant for notum (B) or dally-notum (D). Wg-dependent Hnt expression marks sense organ precursor (SOP) cells, which will give rise to bristles on the future wing margin. In the wild-type disc, SOP cells are in two rows abutting Wg-expressing cells. Each row contains 20-22 cells. In the clone mutant for notum, Hnt is ectopically expressed such that the rows are doubled, while in the dally disc, the number of SOP cells in each row is reduced to 9-11. In the clone mutant for dally-notum, the number of SOP cells is also reduced. (E-H) Anterior wing margins of wild-type adult (E), notum escaper (F), dally escaper (G) and a wing carrying large clones of dally-notum double mutant (marked by yellow colour) (H). The dorsal view of the wild-type wing margin reveals two rows of sensory bristles: densely packed stout mechanosensory bristles and interspersed chemosensory bristles. Three chemosensory bristles are indicated by red arrows to show the spacing. The wing of the notum escaper has many ectopic bristles of both types, while that of dally has a reduced number of chemosensory bristles, as visualized by the increased spacing (red arrows). The dally-notum clone does not have ectopic bristles but has increased spacing between chemosensory bristles (red arrows). (I,J) Hnt staining in discs carrying large clones mutant for either dlp (I) or dlp-notum (J). Occasionally, few ectopic SOP cells can be seen in dlp clones (white arrows), while the dlp-notum clone has many ectopic SOP cells. (K,L) Anterior wing margins of dlpA187/A203 escaper (K) and a wing carrying a large clone of dlp-notum double mutant (L). The dlp wing has a few ectopic stout mechanosensory bristles (black arrows), while many ectopic bristles of both types were produced in the dlp-notum clone.

View larger version (118K): [in a new window]   Fig. 4. The roles of Wg receptors in extracellular Wg distribution. Extracellular Wg staining in wing discs carrying mutant clones for fz (A,A'), fz2 (B,B'), fz-fz2 (C,C') and arr (D,D'). Discs are oriented dorsal top-right, anterior top-left (A-B'), and dorsal right, anterior up (C-D'). The mutant clones are marked by the absence of GFP and outlined by broken lines, except in D. Extracellular Wg is not altered in mutant clones for fz or fz2 alone. By contrast, it is increased in clones mutant for fz-fz2 and arr. The yellow arrows indicate the presumptive ectopic Wg-expressing cells, and the blue arrows indicate the mutant cells accumulating Wg on the cell surface (C,D).

View larger version (141K): [in a new window]   Fig. 5. Accumulated Wg in fz-fz2 or arr clones are mainly resulted from upregulated Dlp. Extracellular Wg staining in wing discs carrying mutant clones of varying genotypes. Discs are oriented dorsal right, anterior up (A-D'), and dorsal top-right, anterior top-left (E-F'). The mutant clones are marked by the absence of GFP and outlined by broken lines except in C,C'. In F,F', only the clone far away from DV boundary is outlined. (A,A') A wing disc carrying clones mutant for fz-fz2. Dlp levels are enhanced in these clones. (B,B') A wing disc carrying clones mutant for sfl-fz-fz2. Extracellular Wg levels are reduced in the clones. (C,C') A wing disc carrying clones mutant for arr. Dlp levels are enhanced in the clones. (D,D') A wing disc carrying clones mutant for botv-arr. Extracellular Wg is reduced in the clones. (E-F') Wing discs carrying clones mutant for dsh. Extracellular Wg levels are increased in the clones. This effect can be clearly seen even the clones are located far away from Wg-expressing cells. Some of the mutant cells are located more than 20 cells away from the DV boundary.

View larger version (115K): [in a new window]   Fig. 6. The effects of overexpressing Dally and Dlp on extracellular Wg distribution. Extracellular Wg staining in a wild-type wing disc (A,A'), and discs overexpressing Dlp (B,B') and Dally (C,C') in the posterior compartment. The posterior compartment is marked by either GFP (green in A' and C') or the absence of Ci (green in B'). Signal profiles of extracellular Wg from selected areas in the anterior and posterior compartments of each wing disc are plotted and compared (A'',B'',C''). In the inset in each plot profile, the areas used for analysis are shown in box. The wing discs are oriented dorsal top-right, anterior top-left. The left-right axis of the plot profiles corresponds to the dorsoventral axis of the boxed regions.

View larger version (127K): [in a new window]   Fig. 7. The effects of overexpressing Fz, Fz2 and Arr on extracellular Wg distribution. Extracellular Wg staining in discs overexpressing Fz (A,A'), Fz2 (B,B') and Arr-GFP (C,C') in the posterior compartment. The posterior compartments are marked by GFP. The images and plot profiles are displayed as in Fig. 6.

View larger version (9K): [in a new window]   Fig. 9. A model of restricted Wg movement along cell surface. Secreted extracellular Wg is bound and stabilized by glypicans Dally and Dlp on the cell surface. The differential concentration of Wg from producing cells to receiving cells drives Wg diffusion along cell surface to more distal cells through disassociation/re-association with HSPGs. In the absence of Dally and Dlp, the Wg protein can no longer move further. It either stays on cell membrane or diffuses into disc lumen, or is degraded. The thin black arrows at the top of this diagram indicate the displacement of Wg from one GAG chain to another. The double-headed arrow indicates the lateral movement of glypicans on the cell membrane.