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First published online 13 August 2008
doi: 10.1242/dev.025205

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Planar polarity genes in the Drosophila wing regulate the localisation of the FH3-domain protein Multiple Wing Hairs to control the site of hair production

MRC Centre for Developmental and Biomedical Genetics and Department of Biomedical Science, University of Sheffield, Western Bank, Sheffield S10 2TN, UK.

View larger version (105K): [in this window] [in a new window]   Fig. 1. Core polarity gene function and the site of prehair initiation. Drosophila 32-hour pupal wings immunolabelled for actin [Ph; magenta in B-H, white in E'-H'], clonal marker (lacZ, fz-EGFP or dsh expression; green) and Fmi or Stbm (blue). Arrowheads indicate prehairs initiating at edges of mutant cells touching a mitotic clone boundary. Distal is to the right in this and subsequent figures. Diagrams illustrate sites of core polarity protein localisation (Fz/Dsh distal complex in green, Stbm/Pk proximal complex in orange) in wild type or on edges of clones, and observed sites of prehair initiation; first row of mutant cells in each clone marked with asterisks. Sites of prehair initiation in mutant cells away from clone edges are not illustrated but are located at the cell centre (except as noted in the text for the first rows of cells within stbm tissue). (A) Diagram of normal localisation of distal and proximal core polarity proteins and site of prehair initiation in wild-type wings. (B,B') fz21 clone. Note that prehair initiation is delayed in mutant cells away from clone edge. Normal polarity is reversed in non-mutant cells owing to the influence of the clone (Vinson and Adler, 1987). (C,C') stbm6 clone. Note that prehair initiation is less delayed than in fz clones and is more likely to be towards a cell edge. (D,D') stbm6/fz21 twin clone. (E,E') sha1/fz21 twin clone. (F,F') stbm6/sha fz21 twin clone. (G,G') stbm6 clone in a fz21 background. (H,H') fz21 clone in a stbm6 background. Note that fz mutant cells are marked by loss of Dsh junctional recruitment (Shimada et al., 2001). Scale bar: 10 µm.

View larger version (115K): [in this window] [in a new window]   Fig. 2. Frtz is proximally localised under control of polarity gene activity. Drosophila 28-hour pupal wings immunolabelled for Frtz (magenta in A-G, white in A'-G') and/or Fmi (A, blue; H, magenta), and clonal marker (lacZ or GFP, green) or EGFP-Fy (green in H, white in H'). Arrowheads indicate proximally enriched localisation of protein. (A,A') frtz33 clone. (B,B') fy2 clone. (C,C') inIH56 clone. (D,D') mwh1 clone. (E,E') stbm6 clone. (F,F') pkpk-sple- 13 clone. (G,G') fz21 clone. (H,H') Mosaic expression of EGFP-Fy induced from a P{w+, ActP-FRT-PolyA-FRT-EGFP-Fy} transgene using hsFLP expression. Scale bar: 10 µm.

View larger version (98K): [in this window] [in a new window]   Fig. 3. Mwh is an FH3-domain protein that localises more strongly proximally in cells. Adult (A-C) or pupal (E-G'') Drosophila wings. (A) mwh1 adult wing between veins 3 and 4, showing trichomes with characteristic multiple wing hair phenotype. (B) Wing expressing CG13913-IR under control of ptc-GAL4 between veins 3 and 4 showing a multiple wing hair phenotype. (C) mwh1 wing expressing EGFP-CG13913 from a P{w+, ActP-EGFP-CG13913} transgene showing rescue of the multiple wing hair phenotype. (D) Diagram of the CG13913 coding sequence, showing the extent of the Interpro GBD/FH3 (IPR014768) and FH3 (IPR010472) domains, and the point at which the inversion in mwh1 breaks the coding sequence (367, arrow). (E-E'') mwh1 clone in a 32-hour wing, labelled for Mwh (magenta), clonal marker (lacZ, green) and Fmi (blue). (F-F'') mwh1 clone in a 32.5-hour wing, labelled for Mwh (green) and actin (Ph, magenta). Arrowheads indicate trichomes in which Mwh does not colocalise strongly with actin bundles. (G-G'') mwh1 clone in 32.5-hour wing, labelled for Mwh (green) and -tubulin (magenta). Arrowheads indicate trichomes in which Mwh does not colocalise with -tubulin. Scale bars: 50 µm in A; 10 µm in G''.

View larger version (81K): [in this window] [in a new window]   Fig. 4. Mwh localisation is regulated by in, fy and frtz. Drosophila 32-hour pupal wings immunolabelled for Mwh (magenta in A-E, white in A'-E') and clonal marker (frtz expression, GFP or lacZ, green). (A,A') frtz33 clone. (B,B') fy2 clone. (C,C') inIH56 clone. (D,D') fz21 clone. (E,E') stbm6 clone. Scale bar: 10 µm.

View larger version (57K): [in this window] [in a new window]   Fig. 5. Mwh gel-mobility is modified by frtz activity and phosphorylation. Immunoblots using anti-Mwh on extracts from Drosophila pupal wings of the indicated genotypes, with w1118 as wild-type control. Positions of molecular weight markers shown on left; arrow on right indicates Mwh signal; arrowhead points to non-specific band that is not altered in mutant extracts. Some extracts also show a weak higher-molecular-weight band which appears to be specific (asterisk), but which varies in intensity. Blots were reprobed for actin or tubulin as loading controls. (A) Extracts from 32-hour pupal wings at 25°C. (B) Extracts from 32-hour pupal wings at 25°C, either treated with the phosphatase inhibitor microcystin (control, w1118) or with lambda phosphatase (P'ased). (C) Extracts from 28-hour pupal wings raised at 29°C or 64-hour pupal wings raised at 18°C (both equivalent to 32 hours at 25°C).

View larger version (83K): [in this window] [in a new window]   Fig. 6. Loss of mwh activity results in excess apical actin. Drosophila (A-D') pupal wings immunolabelled for Mwh (green), actin (Ph, magenta or white) and Arm or Fmi (blue), and (E-F'') S2 cells transfected with EGFP-Mwh. (A,A') mwh1 clone in wing aged 65 hours at 18°C. Arrowheads indicate examples of cells with actin bundles radiating across the entire apical surface, prior to formation of distinct prehair structures. (B,B') mwh1 clone in wing aged 32.25 hours at 25°C. Prehair formation is more advanced than in the wing shown in A, but some cells still show actin bundles radiating across the entire apical surface (arrowheads). (C,C') mwh1 clone in wing aged 32.5 hours at 25°C. All mutant cells now showing multiple prehairs forming at the cell periphery. (D,D') Same clone as in C, but sectioned 1 µm deeper, showing excess actin bundles below the apical surface. (E-E'') S2 cell transfected with EGFP-Mwh (green), labelled for actin (Ph, magenta) and plated on Concanavalin A for 1 hour, shows altered morphology with projections extending from cell edges and a loss of actin bundles at cell periphery. (F-F'') DIC image of an S2 cell transfected with EGFP-Mwh (green) and plated on Concanavalin A for 24 hours, showing more extreme phenotype with long filopodia-like extensions around the cell periphery. Scale bars: 10 µm.

View larger version (16K): [in this window] [in a new window]   Fig. 7. Model for placement of prehair initiation in Drosophila. Diagram representing a z-section of apical regions of a cell, showing distal complexes (green) and proximal complexes (orange). The proximal complex recruits In, Fy and Frtz (red), which in turn recruit or activate an unknown kinase (pink) that modifies Mwh (magenta), which is consequently stabilised in apical/proximal regions. Mwh acts as an inhibitor of actin filament formation in electron-dense pimples on the surface of cells (dark grey), possibly by interfering with the activity of conventional formins. Distally in the cell, pimples become activated as a consequence of a lack of Mwh activity. The distal complex promotes pimple activation, either by repression of Mwh activity or via an alternative mechanism.

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