28 hour pupal wings immunolabelled for Frtz (A-G, magenta or white) and/or Fmi (A, blue; H, magenta), and clonal marker (lacZ or GFP, green) or EGFP-Fy (H, green or white). Arrowheads indicate proximally enriched localisation of protein. Scale bar in H’ represents 10μm.
(A) frtz³³ clone.
(B) fy² clone.
(C) in^IH56 clone.
(D) mwh¹ clone.
(E) stbm⁶ clone.
(F) pk^pk-sple-13 clone.
(G) fz²¹ clone.
(H) Mosaic expression of EGFP-Fy induced from a P{w+, ActP-FRT-PolyA-FRT-EGFP-Fy} transgene using hsFLP expression.

32 hour pupal wings immunolabelled for Mwh (magenta or white), and clonal marker (frtz expression, GFP or lacZ, green). Scale bar in E’ represents 10μm.
(A) frtz³³ clone.
(B) fy² clone.
(C) in^IH56 clone.
(D) fz²¹ clone.
(E) stbm⁶ clone.

Pupal wings (A-D) immunolabelled for Mwh (green), actin (Ph, magenta or white) and Arm or Fmi (blue), or S2 cells transfected with EGFP-Mwh (E,F). Scale bars in D’, E” and F” represent 10μm.
(A) mwh¹ clone in wing aged 65 hour at 18°C. Arrowheads indicate examples of cells with actin bundles radiating across entire apical surface, prior to formation of clear prehair structures.
(B) mwh¹ clone in wing aged 32.25 hour at 25°C. Prehair formation more advanced than in wing shown in (A), but some cells still show actin bundles radiating across entire apical surface (arrowheads).
(C) mwh¹ clone in wing aged 32.5 hour at 25°C. All mutant cells now showing multiple prehairs forming at cell periphery.
(D) Same clone as in (C), but sectioned ∼1 μm deeper, showing excess actin bundles below apical surface.
(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) 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.

32 hour pupal wings immunolabelled for actin (magenta or white), 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. Scale bar in panel H’ represents 10μm. Diagrams (A, B’-D’) illustrate sites of core polarity protein localisation (Fz/Dsh distal complex in green, Stbm/Pk proximal complex in orange) in wildtype 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 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 wildtype wings.
(B) fz²¹ clone. Note prehair initiation is delayed in mutant cells away from clone edge. Normal polarity is reversed in non-mutant cells due to the influence of the clone (Vinson and Adler, 1987).
(C) stbm⁶ clone. Note prehair initiation is less delayed than in fz clones, and more likely to be towards a cell edge.
(D) stbm⁶/fz²¹ twinclone.
(E) sha¹/fz²¹ twinclone.
(F) stbm⁶/sha fz²¹ twinclone.
(G) stbm⁶ clone in a fz²¹ background.
(H) fz²¹ clone in a stbm⁶ background. Note that fz mutant cells are marked by loss of Dsh junctional recruitment (Shimada et al., 2001).

Adult (A-C) or pupal wings (E-G). Scale bar in A represents 50μm, in G“ 10μm.
(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 shows a multiple wing hair phenotype.
(C) mwh¹ wing expressing EGFP-CG13913 from a P{w+, ActP-EGFP-CG13913} transgene shows rescue of the multiple wing hair phenotype.
(D) Diagram of the CG13913 coding sequence, showing the extent of the Interpro GBD/FH3 domain (IPR014768) and FH3 domain (IPR010472), and the point at which the inversion in mwh¹ breaks the coding sequence (arrow).
(E) mwh¹ clone in 32 hr wing, labelled for Mwh (magenta), clonal marker (lacZ, green) and Fmi (blue).
(F) mwh¹ clone in 32.5 hr wing, labelled for Mwh (green) and actin (Ph, magenta), arrowheads indicate trichomes, in which Mwh does not colocalise strongly with actin bundles.
(G) mwh¹ clone in 32.5 hr wing, labelled for Mwh (green) and α-tubulin (magenta), arrowheads indicate trichomes in which Mwh does not colocalise with tubulin.

Immunoblots using anti-Mwh on extracts from pupal wings of the indicated genotypes, with w¹¹¹⁸ as wildtype 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 varies in intensity. Blots 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).

Diagram represents a Z-section of apical regions of a cell, showing distal complexes (green) and proximal complexes (orange). Proximal complex recruits In, Fy and Frtz (red), which in turn recruit or activate an unknown kinase (pink) which modifies Mwh (magenta), which consequently is stabilised in apical/proximal regions. Mwh acts as an inhibitor of actin filament formation in electron dense pimples on 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 lack of Mwh activity. Distal complex promotes pimple activation, either via repression of Mwh activity or via an independent mechanism.