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Godzilla-dependent transcytosis promotes Wingless signalling in Drosophila wing imaginal discs

Nature Cell Biology volume 18, pages 451–457 (2016)Cite this article

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Abstract

The apical and basolateral membranes of epithelia are insulated from each other, preventing the transfer of extracellular proteins from one side to the other1. Thus, a signalling protein produced apically is not expected to reach basolateral receptors. Evidence suggests that Wingless, the main Drosophila Wnt, is secreted apically in the embryonic epidermis2,3. However, in the wing imaginal disc epithelium, Wingless is mostly seen on the basolateral membrane where it spreads from secreting to receiving cells4,5. Here we examine the apico-basal movement of Wingless in Wingless-producing cells of wing imaginal discs. We find that it is presented first on the apical surface before making its way to the basolateral surface, where it is released and allowed to interact with signalling receptors. We show that Wingless transcytosis involves dynamin-dependent endocytosis from the apical surface. Subsequent trafficking from early apical endosomes to the basolateral surface requires Godzilla, a member of the RNF family of membrane-anchored E3 ubiquitin ligases. Without such transport, Wingless signalling is strongly reduced in this tissue.

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Figure 1: Apical accumulation of intracellular Wingless and basolateral enrichment of extracellular Wingless.
Figure 2: Tracking the progress of Wingless along the apico-basal axis.
Figure 3: Loss of Godzilla or Synaptobrevin interferes with Wingless signalling and trafficking.
Figure 4: Knockdown of godzilla prevents Wingless transcytosis.
Figure 5: Basolateral Notum originating from the haemolymph inhibits Wingless signalling in wing imaginal disc.

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Acknowledgements

This work was supported by the MRC (U117584268 to J.-P.V.), the European Union (ERC grant WNTEXPORT; 294523 to J.-P.V.), the Swedish Cancer Society (15/391 to R.H.P.), the Swedish Children Cancer Foundation (2015-0096 to R.H.P.), the Swedish Research Council (2015-04466 to R.H.P.) and the SSF Programme Grant (RB13-0204 to R.H.P.). We thank the Centre for Cellular Imaging (CCI) at the University of Gothenburg for providing confocal imaging support. We are indebted to L.-A.B.-Lopez for devising the idea of tag switching, to C. O’Kane for providing the syb144 allele, to H. Nojima for help with generating Fig. 5c and Supplementary Fig. 5 and Y. Bellaiche for discussion. We are also grateful to SWEDBO for organizing the conference where our collaboration began.

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Author notes

  1. Yasuo Yamazaki and Lucy Palmer: These authors contributed equally to this work.

  2. Ruth H. Palmer and Jean-Paul Vincent: These authors jointly supervised this work.

Authors and Affiliations

  1. Department of Medical Biochemistry and Cell Biology, Institute of Biomedicine, The Sahlgrenska Academy at the University of Gothenburg, Medicinaregatan 9A, 40539 Gothenburg, Sweden

    Yasuo Yamazaki & Ruth H. Palmer

  2. The Francis Crick Institute, Mill Hill Laboratory, The Ridgeway, Mill Hill, London NW7 1AA, UK

    Lucy Palmer, Cyrille Alexandre, Satoshi Kakugawa, Karen Beckett & Jean-Paul Vincent

  3. Division and Morphogenesis Team, Polarity, Institut Curie, CNRS UMR 3215, INSERM U934, 26 rue d’Ulm, 75248 Paris Cedex 05, France

    Isabelle Gaugue

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Contributions

Most of the experiments with imaginal discs were performed by L.P. and Y.Y. Genome engineering was performed by C.A. with help from I.G. S.K. contributed to the experiment with Notum. The project was conceived and elaborated by Y.Y., L.P., K.B., C.A., R.P. and J.-P.V. The first draft of the paper was written by J.-P.V. with substantial subsequent contributions from R.P., L.P., Y.Y. and C.A. These authors also contributed to the design and interpretation of experiments.

Corresponding authors

Correspondence to Ruth H. Palmer or Jean-Paul Vincent.

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Integrated supplementary information

Supplementary Figure 1 Basolateral distribution of extracellular Wingless and tools to track Wingless along the apical-basal axis.

a, Cross-sectional view of a wild type imaginal disc stained with mouse anti-Wingless (red) in the absence of detergent, followed by staining with rat anti-E-Cadherin (green) in the presence of detergent. Most Wingless immunoreactivity is along the basal half of the cell surface; no signal can be detected above (apical to) adherens junctions. Scale bar represents 24 μm. b,c, Characterization of the strain used to track Wingless protein. Two cDNAs were inserted in the locus, as illustrated in Fig. 2a. In the resulting flies (wgKO(F-WgOLLAS-F-WgHA)/wgCX4), without Flp, only WinglessOLLAS is produced and this is sufficient to sustain normal development (b). In separate experiments, we have shown that WinglessHA is also fully functional6. Expression of Flp from UAS-Flp and hedgehog-Gal4 in the posterior compartment (panel c; right hand side of disc) converts the locus to expressing WinglessHA (green) while the control anterior compartment continues to express WinglessOLLAS (red). This image is representative of >10 discs from three independent experiments. d-k, Computationally reconstructed cross sections of the imaginal discs shown in Fig. 2k–r (hemizygous shibirets). d,Steady-state distribution of extracellular Wingless along the apico-basal axis (A → B). e-g,Upon blocking endocytosis, extracellular Wingless accumulates at the apical surface and becomes depleted from the basolateral surface. h, Steady state distribution of extracellular Wingless at normal level of endocytosis activity. i, apical accumulation and basolateral depletion upon endocytosis blockade for 60 min. j,k, The pre-blockade distribution is progressively restored upon resumption of endocytosis by transfer to 18 °C. Note that panels d-k were generated from the same preparations as those shown in Fig. 2k-r. For information on number of samples analysed, see legend of Fig. 2. Scale bars = 24 μm in a, 500 μm in b, 24 μm in c, and 16 μm in d-k.

Supplementary Figure 2 Removal of Dally and Dlp does not cause apical accumulation of extracellular Wingless in wing imaginal discs.

a, dally dlp mutant clones, generated by FRT-mediated mitotic recombination and hs-Flp are depleted of Dlp protein, as expected. Mutant cells (arrows; marked by the absence of GFP) lack anti-Dlp immunoreactivity. b-e, Distribution of extracellular Wingless in and around a dally dlp double mutant clone (labeled by the absence of GFP). The same clone is shown in panels b-e with the ‘prime’ panels showing magnification of the area flanking the Wingless strip. Lack of glypicans leads to a mild reduction of extracellular Wingless but does not cause accumulation at the apical surface (b, c), as seen in >10 discs from 3 experiments. Scale bars represent 24 μm.

Supplementary Figure 3 Loss of Godzilla or Synaptobrevin does not alter Wingless expression in wing imaginal discs.

a,b, Godzilla RNAi (hedgehog-Gal4; UAS-gzlRNAi) depletes endogenous Godzilla protein, as detected by immunofluorescence. The domain of RNAi expression is marked with GFP. Panel a shows a subapical plane and panel b an optical cross section. Scale bars represent 10 μm. c, RNAi-mediated depletion of Godzilla protein in the territory marked with GFP does not affect wingless-lacZ reporter expression (red; detected with anti-β-galactosidase). d, Depletion of syb does not alter wingless-lacZ reporter expression (hedgehog-Gal4; UAS-sybRNAi). The discs shown in c and d are representative of >10 discs from 2 experiments. Scale bars in c and d = 50 μm.

Supplementary Figure 4 Godzilla likely interacts with Wingless at rab5-positive endosomes.

a,b, YFP-tagged constitutively active Rab5 (YFP.Rab5.Q88L) was expressed in the wing pouch with the MS1096-Gal4driver. Most of the resultant enlarged apical endosomes contain Wingless and Godzilla. (b) Close-up view of Wingless-Godzilla colocalisation on the enlarged endosomes. These pictures are representative of >5 discs. Scale bars represent 20 μm (a) or 5 μm (b). c, Godzilla overexpression produces enlarged endosomes where Wingless accumulates. This disc is representative from >10 discs from 2 experiments. Scale bars represent 10 μm. d,e, Expression of GFP-tagged ligase-dead Godzilla (Gzl.LD.GFP) in the posterior compartment (hedgehog-Gal4; UAS-Gzl.LD.GFP) leads to loss of margin tissue in the posterior wing (d) Scale bar represents 500 μm. Senseless immunoreactivity is lost in the posterior compartment of 3rd instar imaginal discs of the same genotype (e). These images are representative of >10 discs from 2 independent experiments. Scale bar represents 50 μm.

Supplementary Figure 5 Distribution of Wingless and Frizzled2.

Imaginal disc from a frizzled2. GFP−KI larva stained with anti-GFP (green) and anti-Wingless (red). A single focal plane (basolateral, see diagram in b’) is shown. Panels b’-b”’ show enlarged white box from panel a. Representative of >5 imaginal discs of the same genotype. Scale bars represent 25 μm.

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Yamazaki, Y., Palmer, L., Alexandre, C. et al. Godzilla-dependent transcytosis promotes Wingless signalling in Drosophila wing imaginal discs. Nat Cell Biol 18, 451–457 (2016). https://doi.org/10.1038/ncb3325

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