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First published online 12 January 2005
doi: 10.1242/dev.01639

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Glypicans shunt the Wingless signal between local signalling and further transport

National Institute for Medical Research, The Ridgeway Mill Hill, London NW7 1AA, UK

View larger version (25K): [in a new window]   Fig. 1. Molecular lesions in dlpMH20 and dallyMH32 and organisation of the tagged glypicans used. (A) Diagram of the genomic regions of dlp and dally. The position of the original P-elements is shown with an inverted triangle. The deletions in dlpMH20 and dallyMH32 are indicated as small black bars (to scale). The exact break points relative to the predicted start of transcription are -42 to +467 for dlp (ATG is at 454) and -472 to +1410 for dally (ATG is at 728). The deficiencies used in complementation assays uncover the whole region with break points located far away and are represented as long black lines [Df(3L) ED4413 dally was generated for this study while Df(3L) ED4543 dlp was obtained from Drosdel]. (B) The tagged glypicans used in this study. The location and nature of the tag is shown. HA-Dally was only expressed in cultured cells while Flag-Dally and Dlp-HA were expressed in transgenic flies and cells.

View larger version (190K): [in a new window]   Fig. 2. Glypicans are required for Wingless activity in embryos. (A-D) Expression of rhomboid in various mutant embryos at about stage 13. (A) Expression of rhomboid in wingless mutants is characterised by a `tramtrack pattern' (this is seen in all embryos, n=100). (B) No such pattern is seen in hedgehog mutants, which express rhomboid in broadened stripes compared with wild type (stripes become occasionally split as shown on the right hand side). (C) In wingless hedgehog mutants, rhomboid stripes collapse into ventral rings (this is true for most segments in all embryos, n=53). Such rings are never seen in wingless mutants, n=100, and rarely so in hedgehog mutants (12% of hedgehog mutants have one or two ventral rings, none have more n=25). (D) The phenotype of embryos lacking maternal and zygotic dally and dlp is similar to - though slightly more variable than - that of wingless hedgehog mutants (a majority of abdominal rhomboid stripes collapse into rings in 69% of embryos and all embryos have at least one collapsed stripe, n=26). (E) Cuticle phenotype of wingless hedgehog double mutant. The epidermis is covered with denticles (the lawn phenotype) and two mid-ventral `whorls' can be seen (arrows). Whorls are sometimes seen in hedgehog mutants but not in the mid-ventral region. (F) The phenotype of embryos lacking maternal and zygotic dally and dlp is similar although more variable with fewer less marked mid-ventral whorls. This suggests that weak residual signalling could occur in the absence of the glypicans.

View larger version (79K): [in a new window]   Fig. 3. Signal transduction in dally dlp mutants expressing exogenous Wingless. (A) Lawn phenotype of an embryo lacking maternal and zygotic dally and dlp, as shown in Fig. 2E. (B) Naked cuticle is induced along the ventral region by expression of GFP-Wingless under the control of sim-gal4 in dally dlp-deficient embryos [as used by Desbordes and Sanson (Desbordes and Sanson, 2003)]. This shows that signal transduction can take place in the absence of the glypicans if sufficient Wingless expression is sustained.

View larger version (90K): [in a new window]   Fig. 4. Wing phenotypes in mutant flies. (A) Wild type. Homozygous dally mutant flies survive and sometimes (5%) display notches in the margin, which are symptomatic of reduced Wingless signalling (B). Distal truncation of vein 5 is also frequent but we have not attempted to characterise this further. The few homozygous dlp mutants that survive to adulthood (around 1%) have wings characterised by two fully penetrant phenotypes: a narrowing of the space between veins 3 and 4 (C), which suggests reduced hedgehog signalling (Crozatier et al., 2002) and (D) the formation of ectopic bristles on either side of the margin (arrowheads, compare with wild type shown in E), an indication of excess Wingless signalling. The same phenotypes are seen in surviving flies carrying the mutation (dlpMH20 and dallyMH32) over a large deficiency (Df(3L) ED4543 dlp and Df(3L) ED4413 dally, respectively).

View larger version (119K): [in a new window]   Fig. 5. Cell-autonomous and non-cell-autonomous effects of Dlp overexpression. (A,B) Overexpression of Dlp in broad domains causes the loss of margin tissue. Here, overexpression was activated in the posterior compartment with engrailed-gal4 (A) or in the central region of the wing with dpp-gal4 (B). Notching is localised to where the margin overlaps with the region of overexpression (arrowheads). Details of the margin are shown on the right-hand side. (C,D) Overexpression of Dlp in scattered clones (Flp-on Dlp) leads to the formation of ectopic bristles (also to loss of margin tissue, not shown here). (C) Ectopic bristles (arrows) caused by random unmarked clones. (D) Margin area near a clone marked with the pwn mutation (outlined). An ectopic bristle (pwn+, i.e. outside the clone) is seen at the edge of the clone (arrow). (E,F) Overexpression of Dlp causes local accumulation of Wingless at the cell surface. Overexpression was induced in clones marked by ß-galactosidase (E). Wingless accumulation is seen in F (visualised by anti-Wingless antibody) (white arrows).

View larger version (16K): [in a new window]   Fig. 6. Model that reconciles the opposite phenotypes seen as a result of dlp overexpression. Wingless can bind to several receptors as it reaches a target cell. Binding to Dlp would prevent access to the signalling receptors and favour presentation to a neighbouring cell. By contrast, binding to the signalling receptors would not only lead to signalling but also to trapping and degradation, thus preventing subsequent transport.

View larger version (57K): [in a new window]   Fig. 7. Dlp overexpression blunts the Wingless gradient while loss of dlp sharpens it. (A) Overexpression of Dlp with engrailed-gal4 eliminates expression of senseless (a `high Wingless target') in the posterior compartment (on the right of the broken line where engrailed-gal4 is expressed). (B) At the same time, expression of distal-less, a `low Wingless target' is broadened (arrowhead) specifically in the posterior compartment. (C,D) A similar broadening of distal-less expression is seen in large Dlp misexpression clones (right-hand clone, marked with GFP in C). There is slight upregulation of distal-less at the edge of the clone within its normal domain of expression (arrowhead in D) consistent with increased presentation activity as a result of Dlp overexpression. (E,F) Mutant dlp cells have reduced levels of Wingless protein. Mutant clones are marked by the absence of GFP (green in E). Reduction of Wingless protein (shown in F) is subtle but unambiguous [see, for example, the reduction in the number of vesicles in the mutant area (arrowhead)]. (G,H) Reduction of Wingless protein at the surface of dlp mutant cells. Extracellular staining (shown in H) was performed as described previously (Strigini and Cohen, 2000). Again, mutant cells are marked by the absence of GFP (green in G). (I,J) Expression of distal-less in wild type (I) and homozygous dlp (J) discs. Both panels are from discs processed and photographed under identical conditions. The domain of distal-less expression is clearly narrower in wing discs obtained from dlp homozygous larvae than in the wild type. Thus, in dlp mutants, a low level target is activated over a reduced range. (K,L) Expression of senseless in wild-type (K) and homozygous dlp (L) discs. Again, both panels are from discs processed and photographed under identical conditions. The domain of senseless expression is slightly wider in the dlp mutant, consistent with the formation of ectopic bristles near the adult wing margin.

View larger version (79K): [in a new window]   Fig. 8. Binding activity and subcellular localisation of Dally and Dlp. (A-D) Transfection of Dally-HA or Dlp-HA in S2 cells causes accumulation of exogenous GFP-Wingless at the cell surface. Transfected cells are recognised with anti-HA (in A and C). HA immunoreactivity is reproducibly lower for Dally than for Dlp (compare A with C) but we do not know whether this is due to differences in expression levels or epitope accessibility. Nevertheless, Dally transfected cells reproducibly accumulate more GFP-Wingless (compare B with D). (E,F) Subcellular distribution of exogenous Dally and Dlp in wing imaginal discs. FLAG-tagged Dally expressed under the control of dpp-gal4 is present both at the cell surface and in vesicles (E), while HA-tagged Dlp is almost exclusively seen at the cell surface. Inset in E shows the detail of a disc expressing FLAG-Dally that was briefly stained with Texas Red Dextran to label the endocytic pathway. The disc was immersed live in a solution of Texas Red dextran for 10 minutes. This was followed by a 20 minute chase and subsequent fixation. Partial colocalisation of Dally (green) with dextran (red) shows that some Dally is in endocytic structures.