Abstract
Wnts are evolutionarily conserved secreted signalling proteins that, in various developmental contexts, spread from their site of synthesis to form a gradient and activate target-gene expression at a distance. However, the requirement for Wnts to spread has never been directly tested. Here we used genome engineering to replace the endogenous wingless gene, which encodes the main Drosophila Wnt, with one that expresses a membrane-tethered form of the protein. Surprisingly, the resulting flies were viable and produced normally patterned appendages of nearly the right size, albeit with a delay. We show that, in the prospective wing, prolonged wingless transcription followed by memory of earlier signalling allows persistent expression of relevant target genes. We suggest therefore that the spread of Wingless is dispensable for patterning and growth even though it probably contributes to increasing cell proliferation.
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Acknowledgements
This work was supported by the UK Medical Research Council (U117584268), an ERC grant (WNTEXPORT) from the European Union to JPV and a Sir Henry Wellcome post-doctoral fellowship to L.A.B-L. (082694/Z/07/Z). We are grateful to U.-M. Fiuza for discussion and A. Mitchell for help in generating the first wingless knockout allele. Discussions with G. Struhl have led to significant improvement of the manuscript. We thank colleagues listed in the Methods Summary, as well as the Developmental Studies Hybridoma Bank and the Bloomington Stock Center for providing antibodies and fly strains.
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All the experiments were performed jointly by L.A.B.-L. and C.A. C.A., L.A.B.-L., and J.-P.V. contributed equally to the conception of the work, the interpretation of results, and manuscript preparation.
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Extended data figures and tables
Extended Data Figure 1 Engineering the wg locus to express membrane-tethered Wg.
a, Structure of the wingless locus before targeting, after targeting, and after Cre-mediated excision. The wg{KO} allele was used as a founder line for subsequent reintegration. b, Cherry expression in wing, leg and haltere imaginal discs of larvae carrying one copy of wg{KO; Cherry}. c, Cuticle preparation of a homozygous wg{KO}larva at low and high magnification (black arrow). The phenotype is identical to that of wgCX4 homozygous embryos39. d, Diagram showing the reintegration of a wild type wingless cDNA in the wg{KO} to generate wg{KO; Wg} (note presence of mini-white). e, Diagram showing the reintegration of the NRT–Wg cDNA in wg{KO}. This was achieved using either pax-Cherry or mini-white as a genetic marker, as indicated. f, Wing of a wild-type fly. g, Wing of a wg{KO; Wg} homozygous fly. h, Overlay of the wings shown in Fig. 1b to illustrate the mild wing size reduction in NRT–Wg flies. i, Wing size of wg{KO; NRT–Wg} homozygous (n = 14) and control (wg{KO; NRT–Wg}/GlaBC) flies (n = 16, ***P < 0.001). j–l, High-magnification view of the wing margin of wild-type, homozygous wg{KO; Wg}, and homozygous wg{KO; NRT–Wg}. They are barely distinguishable. m–o, Views of the dorsal thorax illustrate the normal arrangement of pattern elements such as microchaetes and macrochaetes in the genotypes indicated. Error bars represent s.d. Statistical significance was assessed using Student’s t-test.
Extended Data Figure 2 Senseless expression and growth in wingless-null patches surrounded by wild-type or Neurotactin–Wingless-expressing cells.
a, b, Expression of Senseless (red) is lost in patches of wingless mutant cells (GFP-negative; wgCX4 homozygotes) except in the cells located within one cell diameter of surrounding GFP-positive cells, which are wild-type cells (a) or homozygous wg{KO; NRT–Wg} (b) (white arrows). Mosaics were created by mitotic recombination in a way that generates approximately the same number progenitors for the two genotypes, as described in Methods. c, Example of mosaic imaginal discs generated as above to measure the growth of wingless mutant territory (wgCX4 homozygous; GFP-negative) relative to that of wild type (c) or wg{KO; NRT–Wg} homozygous (d) tissue. Wg and NRT–Wg, detected with anti-Wg, are shown in red. e, Outline of the territory where the surface areas were assessed. f, Quantification of the areas colonised by wingless mutant cells (GFP-negative) in the two genetic backgrounds. On average, the wingless-null territory was smaller in the wg{KO; NRT–Wg} homozygous background (n = 24) than in the wild type (n = 20, ***P < 0.001). Error bars represent s.d. Statistical significance was assessed using Student’s t-test.
Extended Data Figure 3 Activity of the wingless promoter during imaginal disc development.
a, Timing of key developmental stages at 25 °C. b, Developmental timing at 18 °C as it relates to the results illustrated in c. c, Permanent labelling of wg-expressing cells and their descendants at different stages of development. Genotype was wg{KO; Gal4}, tubulin-gal80ts/UAS-Flp; Actin FRT stop FRT LacZ so that the stop cassette was only excised in cells that express wingless at the time of shifting to 29 °C to activate Flp expression and hence excision of the stop cassette. Discs were shifted from 18 °C to 29 °C at different stages (shown in b) but they were fixed and stained at the same stage, just before puparation.
Extended Data Figure 4 Tissue-specific allele switching to determine the anatomical origin of organismal developmental delay in Neurotactin–Wingless animals.
a, Cumulative pattern of vestigial-gal4 activity in various organs precursors. Expression of vestigial-gal4 at any stage or place leads to excision of the stop cassette in Actin FRT stop FRT LacZ thus marking permanently the corresponding cells. As expected, nearly the whole wing and haltere discs were labelled at the end of larval development. In wing imaginal discs, only a few cells were β-Galactosidase-negative that did not overlap with the domain of Wg expression (anti-Wg, red). In the eye antennal disc, the patterns of Wg (white arrowhead) and β-Galactosidase expression are also non-overlapping. Therefore, in combination with UAS-Flp, vestigial-gal4 is expected to excise an FRT cassette throughout the domain of wingless expression. Examination of the brain and CNS shows that vestigial-gal4 is unexpectedly active in these tissues. b, In larvae of genotype vestigial-gal4, UAS-Flp, wg{FRT Wg FRT NRT–Wg}/Cyo wg, most of the wg-expressing cells in leg, haltere and wing imaginal discs, but not in the brain and CNS, were converted to expressing NRT-HA-Wg (anti-HA; green). c, Developmental timing in wg{KO; WTBody; NRT–WgDisc} (vestigial-gal4, UAS-Flp, wg{FRT Wg FRT NRT–Wg}) and control (vestigial-gal4, UAS-Flp, wg{FRT Wg FRT NRT–Wg}/GlaBC) larvae (80 animals, 4 experiments). The two data sets cannot be statistically distinguished (P > 0.05). d, Adult wing size for three genotypes: wg{KO; WTBody; NRT–WgDiscs}/GlaBc obtained from selfed vestigial-gal4, UAS-Flp, wg{FRT Wg FRT NRT–Wg}/GlaBc (n = 16, shown in black); wg{KO; WTBody; NRT–WgDiscs}, obtained from homozygous vestigial-gal4, UAS-Flp, wg{FRT Wg FRT NRT–Wg} (n = 15, shown in purple); and wg{KO; NRT–WgBody;WTDiscs}, obtained from homozygous vestigial-gal4, UAS-Flp, wg{FRT NRT–Wg FRT Wg} (n = 13, shown in grey). e–g, Extent of Distal-less expression in wg{KO; WTBody; NRT–WgDiscs} heterozygotes (over Cyo; e) and homozygotes (f). All the discs were obtained from immobile larvae at the time of anterior spiracle eversion, an event that marks the onset of pupariation. The extent of the Distal-less domain was estimated from the surface area of a polygon drawn around the zone of immunoreactivity, as shown. The results, plotted in panel g, show a mild reduction in wg{KO; WTBody; NRT–WgDiscs}discs (n = 13) compared to controls (n = 20; ***P < 0.001; n.s., not significantly different). Error bars represent s.d. Statistical significance was assessed using Student’s t-test.
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Alexandre, C., Baena-Lopez, A. & Vincent, JP. Patterning and growth control by membrane-tethered Wingless. Nature 505, 180–185 (2014). https://doi.org/10.1038/nature12879
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DOI: https://doi.org/10.1038/nature12879
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