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Wg and Wnt4 provide long-range directional input to planar cell polarity orientation in Drosophila

Nature Cell Biology volume 15, pages 1045–1055 (2013)Cite this article

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Abstract

Planar cell polarity (PCP) is cellular polarity within the plane of an epithelial tissue or organ. PCP is established through interactions of the core Frizzled (Fz)/PCP factors and, although their molecular interactions are beginning to be understood, the upstream input providing the directional bias and polarity axis remains unknown. Among core PCP genes, Fz is unique as it regulates PCP both cell-autonomously and non-autonomously, with its extracellular domain acting as a ligand for Van Gogh (Vang). We demonstrate in Drosophila melanogaster wings that Wg (Wingless) and dWnt4 (Drosophila Wnt homologue) provide instructive regulatory input for PCP axis determination, establishing polarity axes along their graded distribution and perpendicular to their expression domain borders. Loss-of-function studies reveal that Wg and dWnt4 act redundantly in PCP determination. They affect PCP by modulating the intercellular interaction between Fz and Vang, which is thought to be a key step in setting up initial polarity, thus providing directionality to the PCP process.

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Figure 1: Wg/Wnt expression and PCP patterning.
Figure 2: Ectopic dWnt4 or Wg expression reorients PCP direction in neighbouring cells.
Figure 3: Ectopic Wnt4 expression reorients Fz–PCP factors in pupal wings.
Figure 4: Wnt4 misexpression reorients PCP during early pupal development.
Figure 5: dWnt4, wg double mutant LOF wings show PCP defects.
Figure 6: dWnt4, wg double mutants show early loss of PCP orientation.
Figure 7: Wg and Wnt4 inhibit intercellular Fz recruitment of Vang to the cell membrane.
Figure 8: In vivo co-expression of Wnt4 inhibits non-autonomous effects of Fz.

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Acknowledgements

We are grateful to P. Adler, K. Choi, D. Strutt, G. Struhl, J-P. Vincent, S. Cohen, H. Bellen, the Bloomington Drosophila Stock Center and the DSHB for fly strains and reagents, and Benoit Aigouy for providing the ‘Packing Analyser V2.0’ software. We thank all Mlodzik laboratory members for helpful discussions and suggestions, G. Struhl and P. Lawrence for sharing unpublished results and discussion and Paul Wassarman, P. Olguin, W. Gault, G. Collu, L. Kelly and R. Krauss for helpful comments and suggestions on the manuscript. Confocal microscopy was carried out at the Microscopy Shared Resource Facility of the Icahn School of Medicine at Mount Sinai. This work was supported by a National Institutes of Health (NIGMS) grant to M.M.

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  1. Department of Developmental and Regenerative Biology, Graduate School of Biomedical Sciences, Icahn School of Medicine at Mount Sinai, One Gustave L. Levy Place, New York, New York 10029, USA

    Jun Wu, Jose Maria Carvajal-Gonzalez & Marek Mlodzik

  2. Instituto Cajal, CSIC, Av. Doctor Arce 37, 28002 Madrid, Spain

    Angel-Carlos Roman

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J.W. and M.M. planned the project, designed and carried out the experiments, analysed the data and wrote the paper; A.R. and J.C. analysed the data and designed quantitative analytical tools for the project.

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Correspondence to Marek Mlodzik.

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Supplementary Figure 1 Wnt4 PCP phenotypes are not mediated through canonical Wnt signalling.

a,a’, Illustration of wg, dWnt4 and dWnt6 expression patterns in wing (a) and eye discs (a’), relative to the proposed Fz activity gradient (arrows indicate direction of PCP). b–b”, A late third instar larval wing disc containing Wnt4 expressing clones (labelled by GFP, green) and stained for fj–lacZ (blue) and anti-Dll (red) expression, both defining canonical Wnt/ β-catenin signalling targets. Note that their expression is not affected by Wnt4 misexpression. b’,b”, Individual channels of fj–lacZ and Dll staining. These data demonstrate that canonical Wnt/ β-catenin signalling is not affected by Wnt4 misexpression. c,c’, 30–35 h APF pupal wing with ectopic Wnt4 expressing clones (marked by GFP, green) stained for fj–lacZ (blue, single channel in c’). fj-lacZ expression is not affected by Wnt4-expressing clones. Dll is also not affected, showing high expression at the wing margin and low flat expression across the wing blade. Wnt4 clones were induced during first and early second larval instar. d, Adult wing containing an ArmS10 (stabilized active form of β-catenin)-expressing clone (labelled by GFP co-expression). ArmS10 represents activated canonical Wg signalling. Although ArmS10 expressing cells induced ectopic anterior wing margin fate (as expected for high level canonical Wg signalling), indicated by margin bristles and veinlike appearance, these cells did not alter the cellular polarity of neighbouring cells. Clones were induced during the second larval instar. These data document that the GOF PCP effects of dWnt4 (or Wg) are mediated not through canonical Wnt signalling and thus probably through direct effects on Fz–PCP signalling. e,e’, Two examples of ectopic Wnt4-expressing clones (green) in fzP21 mutant wings. Ectopic Wnt4 expression did not alter PCP (as determined by cellular hair orientation) of surrounding cells in the fzP21 mutant background.

Supplementary Figure 2 Reduction of Wg expression at the wing margin in wgCX3 clones.

a,a’, wgCX3 mutant clones labelled by lack of β-gal staining (green; white lines indicate clonal borders in a’). Anti-Wg is in red (monochrome in a’). The clone on the left (yellow arrow) affects the Wg-expression domain on both sides of the dorsoventral boundary with Wg expression levels reduced in the mutant area (yellow area), comparef with adjacent wild-type tissues. The red arrow marks Wg levels in a clone that affects only expression in the dorsal compartment; Wg is again reduced but to a lesser extent (red arrow), as Wg expressed on the ventral side can diffuse into the (dorsal) area.

Supplementary Figure 3 Planar cell polarity phenotypes in wg, dWnt4 adult escaper animals.

a–c, PCP phenotypes associated with wnt4−, wg−, wnt6−, wnt10− LOF clones. The red area in a indicates the region shown in b and c. a’, Schematic illustration of how cells (green area) can receive Wnt signals from two sides of the remaining wing margin, when part of the wing margin is lost owing to either wg clones or other manipulations. b, Wild-type wing: cells orient towards the wing margin as indicated by cellular hairs (represented by red arrows). c, Wings containing Df(2L)NL mutant clones (Df(2L)NL removes dWnt4, wg, dWnt6 and dWnt10; dWnt4, wg and dWnt6 are all expressed at the wing margin). Loss of wg in wing margin cells causes loss of margin bristles, a high threshold target of canonical Wg signalling, and thus loss of margin bristles serves as a marker for mutant clones in margin cells. The arrows indicate cellular orientation of cells surrounding the wild-type area (showing wing margin fate), which express the Wnt genes. Areas with no margin are mutant and have no local Wnt expression. Note that cells orient locally towards the patches of wild-type margin (Wnt-expressing cells), and parallel to areas that have mutant (missing) “margin”, consistent with the notion that cells orient towards Wg/Wnt-expression domains. d–f, wnt4−/−, wgCX3/IL114 double mutant phenotypes in wings of rare adult escapers. The red box in d indicates the area of interest in e and f. Wings are oriented such that vein 2 is at 0°. b,b’, In wild-type wings cellular hairs are oriented along wing vein 2 (near 0°). f,f’, In wnt4−/−, wgCX3/IL114 double mutant wings cellular hairs often point away from the wing margin (green arrows). Polarity distribution quantification reveals that wing hairs orient at minus 15–45° in the double mutant background (p = 10−6 as compared with wild type; n, number of cells; statistical analysis was carried out with the Kolmogorov–Smirnov test designed to compare two independent populations/patterns of cells).

Supplementary Figure 4 Wg and Wnt4 affect intercellular Fz–Vang recruitment in S2+R cells.

a, Quantification of Wg effect on intercellular Fz–Vang recruitment; note the dosage dependent inhibition of the Vang membrane stabilization event (see also Fig. 7 and main text). Fz/DE-cad cells were co-transfected with increasing amounts of Wg DNA (as indicated), and subsequently mixed with Vang/DE-cad-transfected cells. The inhibitory effect of Wg on Fz–Vang recruitment is dosage dependent and highly reproducible (n, number of contacting cell pairs between Fz/DE-cad-expressing cells and Vang/DE-cad-expressing cells). Statistical analysis: Fischer’s exact test (two tailed) and p values are as indicated (n, number of cell pairs in contact). b–d, Examples of Fz–Vang recruitment in the presence of CM of Upd-V5 (b–b”; note the normal Vang stabilization, red in b,b’, at contact membranes), Wnt4 (c–c”; note the loss of Vang at cell membranes) or Wg (d–d”). Whereas Fz–Vang membrane recruitment is not affected in the presence of Upd CM (b), it is largely lost from cell contacts when cells are exposed to Wnt4 (c) or Wg (d) CM.

Supplementary Figure 5 Quantification of Wnt4 effect on non-autonomous Fz gain of function in vivo.

a, Illustration of area of interest for quantification (example in wild-type wing). All wings are oriented anterior up and distal to the right (photographed with ×20 objectives at 1280 pixels × 1024 pixels: the grey box is about 550600 pixels away from the margin). The low edge of the grey area is in the middle between veins 3 and 4, reflecting the border of the dpp-expression domain in pupal wings. The green box (600 pixels × 70 pixels) includes about four rows of cells adjacent to the dpp-expression domain, quantified in b–d. b–d, Examples of wings of the indicated genotypes: b, dppGal4/UAS-Wnt4; c, UAS-LacZ/+; dppGal4, UAS–fz; d, dppGal4, UAS–fz/UAS-Wnt4. Green boxes are drawn as in a. b’–d’ Orientation angle distributions (five wings were quantified for each genotype). Co-expression of Wnt4 with Fz reduced the ‘pointing away’ orientation from the Fz overexpression domain as compared with control (c’ versus d’; p = 10−14; n, numbers of cellular wing hairs, equivalent to cells; statistical analysis was carried out with the Kolmogorov–Smirnov test designed to compare two independent populations/patterns of cells.

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Wu, J., Roman, AC., Carvajal-Gonzalez, J. et al. Wg and Wnt4 provide long-range directional input to planar cell polarity orientation in Drosophila. Nat Cell Biol 15, 1045–1055 (2013). https://doi.org/10.1038/ncb2806

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