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First published online 16 November 2005
doi: 10.1242/dev.02145

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Arrow (LRP6) and Frizzled2 cooperate to degrade Wingless in Drosophila imaginal discs

Eugenia Piddini^*, Francis Marshall^*, Laurence Dubois, Elizabeth Hirst and Jean-Paul Vincent

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

View larger version (73K): [in a new window]   Fig. 1. Wingless is degraded in a lysosomal compartment. (A) HRP-wingless (dark stain) is detected in recognizable multi-vesicular bodies and lysosomes in wing imaginal discs. HRP-wingless was driven by dpp-Gal4, which is expressed in a broad stripe that lies perpendicular to the Wingless expression domain. This micrograph was taken outside the domain of dpp expression (in receiving cells). (B,C) Wingless protein (shown in C) accumulates in deep-orange (VPS18) mutant cells, which are recognized by the absence of ß-galactosidase (shown in B). (D) Accumulation of Wingless protein in cells that express shi[ts] under the control of the dpp-Gal4 driver, upon incubation for 3 hours at 32°C. (E,F). Imaginal discs expressing shi[ts] under the control of apterous-Gal4 (in the dorsal compartment; top half) fixed after a 5-hour incubation at 32°C. distalless expression is reduced in E and engrailed expression is reduced in F. (G,H) Increased stabilization of Armadillo upon reduction of dynamin-mediated endocytosis. Both panels show a disc overexpressing armadillo under the control of engrailed-Gal4 (in the posterior compartment). Both discs were kept at 32°C for 3 hours prior to fixation and staining with anti-Armadillo. (G) A shi[ts] heterozygote. (H) A shi[ts] hemizygote. Armadillo staining is increased in H.

View larger version (135K): [in a new window]   Fig. 2. Frizzled2 stimulates Wingless internalization. (A-A''',B-B''') Colocalization of Wingless and Frizzled2 or Frizzled2GPI in an endocytic compartment. (A) Imaginal discs were isolated from dpp-Gal4 UAS-Frizzled2-FLAG and bathed in 3 kDa dextran for 10 minutes. After a 20-minute chase, they were fixed and stained. (A) Low magnification of imaginal disc. Boxed area shown at high magnification in A',A'''. Anti-Wingless is shown in A and A', anti-FLAG (Frizzled2) in A'' and Texas-red Dextran in A'''. (B) Imaginal disc expressing Frizzled2GPI under the control of dpp-Gal4 and labelled with anti-Wingless (B,B'), anti-Myc (B'') and dextran as described (B'''). Wingless is strongly stabilized (compare the extent of stabilization, indicated by double-headed arrows in A,B). (B'-B''') High magnification of the boxed area in B. There is reduced colocalization of Dextran, Frizzled2GPI and Wingless. (C) Binding of GFP-Wingless to S2R+ cells transfected with Frizzled2-FLAG. The transfected cells in this field of view are indicated by white arrows. Binding was performed at 4°C, hence the round appearance of the cells. GFP-Wingless in this and all subsequent experiments is detected with an anti-GFP antibody. (D,D') Upon shifting to 25°C, GFP-Wingless (D') is internalized in Frizzled2-FLAG-positive structures (D, detected with anti-Flag). Wingless internalization is also seen in untransfected cells (asterisk), although at a much reduced level (total fluorescence of the untransfected cell is 36% that of the transfected cell). In D' (and F'), residual extracellular staining was stripped so that only internalized epitopes are revealed. (E) Binding at 4°C of GFP-Wingless to cells transfected with Frizzled2GPI. (F,F') In cells transfected with Frizzled2GPI (F, detected with anti-Myc), little internalization of GFP-Wingless (F') is seen upon shifting to 25°C (the difference in mean fluorescence between transfected and untransfected cells is over fivefold higher in the Frizzled2 cells shown than in the corresponding Frizzled2GPI cells). Weakly expressing cells do internalize GFP-wingless (not shown) perhaps because this fusion protein can cooperate with endogenous proteins that contain an internalization signal. Scale bars: 10 µm.

View larger version (197K): [in a new window]   Fig. 3. Arrow provides a degradation signal. In all experiments described here, dpp-Gal4 was used as a driver and staining was with anti-Wingless. (A) Wingless stabilization by Frizzled2 as shown in Fig. 2A (in an imaginal disc of the genotype dpp-Gal4 UAS-Frizzled2-FLAG). (B) Wingless is reduced to a range shorter than in wild-type tissue by co-expression of Arrow with Frizzled2. The range is shorter in the dpp-Gal4 domain (marked with a double-headed arrow) than in neighbouring tissue. (C) Wingless accumulates if endocytosis is blocked in the otherwise same genotype as in B. Here, endocytosis was blocked by culturing the larvae at 32°C for 3 hours in a shi[ts] hemizygous background. No accumulation was seen in the identical genotype cultured at 25°C throughout (not shown). (D) Activated Armadillo (Arm*) does not affect the distribution of Frizzled2-stabilized Wingless.

View larger version (89K): [in a new window]   Fig. 4. The degrading activity of Arrow is specific to Frizzled2. (A,A') Wingless stabilized by Dally-like, as shown in A, is only weakly reduced by further addition of Arrow, as shown in A'. (B-B') Likewise, Arrow does not degrade Wingless stabilized by Frizzled2GPI. (C-C') Expression of a form of Frizzled2 that lacks most of the intracellular tail (Frizzled2C) causes Wingless accumulation at the cell surface (C, main panel) and appears to reduce Wingless internalization (as seen at high magnification in the inset; compare with Fig. 2A'). As shown in C', such accumulation is abrogated by Arrow overexpression. This result provides indirect evidence that Arrow contains an endocytic (in addition to a degradation) signal as it can promote the removal of Frizzled2C-complexed Wingless from the cell surface. (D,D') A form of Arrow that lacks amino acids 1477 to 1612 (ArrC) is deficient in degradation activity. This is shown in an imaginal disc expressing ArrC in the posterior compartment. A pulse of Frizzled2 expression (from hs-Frizzled2) was induced 1.5 hours prior to fixation. (D) ArrC expression detected with anti-HA; (D') the distribution of Wingless. Double-headed arrows mark the extent of the Wingless range in the two halves of the disc. In the posterior compartment, the domain where Wingless vesicles are detected is expanded.

View larger version (77K): [in a new window]   Fig. 5. Arrow captures Wingless but less efficiently than Frizzled2. (A,A') Transfection of Frizzled2 leads to strong accumulation (compare the GFP signal on transfected cells with that on untransfected cells). (A) Anti-HA, which reveals transfected Frizzled2; (A') GFP-Wingless. (B,B') Transfection of Arrow in S2 cells causes weak (but significant) accumulation of exogenously added GFP-Wingless at the cell surface. Arrow is labelled in red (with anti-HA) and GFP-Wingless is shown in green. (A',B') Transfected cells are marked with an arrowhead and untransfected cells by an asterisk. Cells were kept at 4°C throughout to prevent endocytosis. Anti-HA was used to detect both transfected receptors to ensure that cells with similar expression levels would be compared. Samples were processed in parallel. (C) GFP-Wg binding to S2 cells transfected with Frizzled2 (top) or Arrow (bottom), as quantified by fluorescence intensity after immunocytochemistry using an anti-GFP antibody. Histograms show the percentage of cells (y-axis) with a given fluorescence intensity expressed in arbitrary units (x-axis). The maximum fluorescence recorded in Arrow-overexpressing cells is almost threefold less than that in Frizzled2-overexpressing cells. (D) Increased accumulation of Wingless onto Arrow-expressing (driven by dpp-Gal4) cells in a disc compromised for dynamin activity (shi[ts] hemizygote at 32°C for 3 hours). There is a faint stripe of Wingless accumulation in the dpp expression domain.

View larger version (61K): [in a new window]   Fig. 6. Wingless accelerates Frizzled2 internalization. (A,B) Imaginal disc of the genotype hs-FLAG-Frizzled2 fixed 1.5 hours after a pulse of uniform expression of tagged Frizzled2 (A) and Wingless (B). Note the relative increase in vesicular Frizzled2 around the area of Wingless expression. As many of these vesicles also contain Wingless (insets), it appears that Wingless stimulates Frizzled2 internalization. (C,D) Imaginal discs of the same genotype (hs-FLAG-Frizzled2) fixed 50 minutes (C) or 3 hours (D) after heat-shock and stained with anti-FLAG. Fifty minutes after the pulse, Frizzled2 is found throughout the disc, predominantly outlining cell membranes, whereas after 3 hours, the level of Frizzled2 appears preferentially reduced in the area surrounding the source of Wingless (the central region of the pouch) suggesting, possibly, increased degradation. (E) Imaginal disc 1 hour after uniform induction of Patched expression. The protein (as detected with an anti-Patched antibody) is seen uniformly throughout the disc. (F) Three hours after induction, the Patched protein is preferentially reduced in the posterior compartment, where its ligand, Hedgehog is expressed. Staining along the anteroposterior boundary could originate from endogenous expression as an anti-Patched antibody was used. However, deeper into the anterior and posterior compartments, Patched is not normally expressed and protein turnover can be compared in these two domains. (G,H) Division of labour between Frizzled2 and Arrow. (G) Frizzled2 provides most of the capturing activity, while Arrow brings a degradation signal. Both receptors provide an internalization signal. (H) When Frizzled2 is in relative excess at the cell surface, Wingless is captured and put on hold until Arrow becomes available to signal and trigger degradation.

View larger version (109K): [in a new window]   Fig. 7. Loss of frizzled frizzled2 affects intracellular Wingless. (A,A') Distribution of Wingless in frizzled frizzled2 mutant clones (outlined in white, indicated by -/-). This disc has been stained using a protocol that distinguishes extracellular (A) from intracellular (A') Wingless. Extracellular accumulation at the surface of mutant cells can be seen in A. Note in A' the specific depletion of large vesicles within the clone when compared with wild-type tissue (one such vesicle is marked by an arrowhead). Breaks in the stripe of expression are due to occasional loss of wingless transcription in the absence of signalling. (B,B') The same staining protocols were applied to discs containing arrow[2] mutant clones (outlined in white). Extracellular accumulation is seen (B). Intracellularly (B'), large vesicles are still seen in arrow mutant cells, consistent with the suggestion that Wingless continues to be internalized normally.