Volume 7, Issue 2, Pages 367-375 (February 2001) Asymmetric Localization of Frizzled and the Establishment of Cell Polarity in the Drosophila Wing  David I Strutt  Molecular Cell  Volume 7, Issue 2, Pages 367-375 (February 2001) DOI: 10.1016/S1097-2765(01)00184-8

Figure 1 Fz Function and Trichome Polarity in the Wing Distal is to the right and anterior is up. (A)–(C) show photomicrographs of adult wings. (A), dorsal surface of wild-type wing; (B), high-magnification view of a wild-type wing between longitudinal veins 3 and 4; and (C), high-magnification of a similar region in a fz mutant wing. (D)–(F) show confocal sections through the apical regions of the cells in pupal wings stained for actin distribution using phalloidin. (D) Approximately 31 hr after prepupa formation (APF), actin is distributed at the apical cell periphery. (E) Thirty-two hours APF, actin staining reveals a trichome forming at the distal vertex of each cell. (F) Thirty-three hours APF, each cell now shows an emerging trichome with definite polarity Molecular Cell 2001 7, 367-375DOI: (10.1016/S1097-2765(01)00184-8)

Figure 2 Fz-GFP Localizes at the Distal Boundary of Pupal Wing Cells Except where otherwise stated, all images are distal to the right. (A)–(D) show Fz-GFP (green, upper and lower panels) and actin (red, upper panels) in wild-type (A and B) and fz (C and D) wings. (A), Thirty-two hours APF, actin is first visible at distal cell vertex (arrowheads); (B), 33 hr APF; (C), 31 hr APF; and (D), 38 hr APF. Note Fz-GFP rescues polarity defect in fz wing (except at extreme distal tip; data not shown). (E)–(H) show Fz-GFP (green, upper and middle panels) and Stan (red, upper and lower panels) in wild-type wings. (E), 19 hr APF; (F), 29 hr APF; (G), 39 hr APF. (H) shows xz confocal section with apical up, showing apical colocalization of Fz-GFP and Stan at PD cell boundaries at 29 hr APF. (I) shows wild-type wing mosaic for cells expressing Fz-GFP (green) and not expressing (lacZ marker, red). Fz-GFP is present on distal cell boundaries (white arrowheads) but not proximal cell boundaries (white arrows, note absence of yellow fluorescence that would indicate overlap of Fz-GFP and lacZ staining); we find that mosaic low-level Fz-GFP expression does not affect polarity signaling as assayed by normal trichome polarity Molecular Cell 2001 7, 367-375DOI: (10.1016/S1097-2765(01)00184-8)

Figure 3 Fz-GFP Distribution Is Altered by Several Planar Polarity Mutations All images are apical confocal sections, distal to the right. (A), (B), (G), and (H) are all shortly prior to trichome initiation (30–32 hr APF). (C)–(F) are all shortly after trichome initiation (32–33 hr APF). Fz-GFP distribution is shown in green (A, B, E–H) or white (C and D). (A) shows stanE59 mutant cells (not expressing lacZ, red). Note that Fz-GFP is lost from all stan+/stan− cell boundaries, in particular at the proximal edge of the clone (arrowheads), and that in the adjacent wild-type cells, Fz-GFP shows a preference for lying perpendicular to the clone boundary. (B), dsh3 mutant cells (not expressing lacZ, red); (C), pkpk-sple-13; (D), VangA3; (E), in1 (costained for actin, red); (F), mwh1 (costained for actin, red); (G), Fz-GFP (green, left and middle panel) and Stan (red, left and right panel) in dsh3 mutant cells (not expressing lacZ, red, left and right panels); and (H), Fz-GFP (green, left and middle panel) and Stan (red, left and right panel) in in1. All alleles used are reported to be amorphic (see FlyBase) with the exception of mwh1, which is a strong allele Molecular Cell 2001 7, 367-375DOI: (10.1016/S1097-2765(01)00184-8)

Figure 4 Fz-GFP Distribution Prefigures Trichome Polarity All images are apical confocal sections, distal to the right. (A) High-level Fz-GFP (green) (generated using UAS-Fz-GFP/armadillo-GAL4) and actin (red) at 32 hr APF. (B) Clone of cells overexpressing Fz (marked with lacZ, red) at 30 hr APF. On lateral and proximal clone edges, wild-type cells have abnormal Fz-GFP (green) distribution (white arrowheads). (C) Clone of cells overexpressing Fz (marked with lacZ, blue) at 32 hr APF. Trichomes (actin staining, red) point away from the boundary of the clone (white arrowheads). (D) The same clone as in (C) also labeled for Fz-GFP (green). (E) Fz-GFP (green) distribution around clones of VangA3 mutant cells (not expressing lacZ, red) at 31 hr APF. Note strong localization of Fz-GFP on boundary between Vang+ and Vang− cells (white arrowheads). (F) Clone of VangA3 mutant cells (not expressing lacZ, blue) at 32 hr APF labeled for actin (red) and Fz-GFP (green) Molecular Cell 2001 7, 367-375DOI: (10.1016/S1097-2765(01)00184-8)

Figure 5 Fz Localization Is Not Required for Nonautonomous Polarity Signaling Distal is to the right, anterior is up. (A), (B), and (G) show photomicrographs of adult wings; (C)–(F) and (H) show apical confocal sections of pupal wings at about 32 hr APF (C) or 29 hr APF (D–F and H). (A) and (B) show mwh1 fz1 clones lying just below longitudinal vein 5; the approximate clone outline is indicated by the red line. In (A), fz nonautonomous phenotype is manifest, with trichomes outside the clone pointing towards the clone boundary. In (B), nonautonomous phenotype is rescued by the presence of an Actin-FzP278L-GFP transgene. Note that Actin-FzP278L-GFP acts as a weak dominant negative for fz autonomous function, leading to a weak polarity phenotype at the distal tip of the wing in flies carrying the transgene. (C) shows a clone of cells overexpressing FzP278L (marked with lacZ, blue) labeled for actin (red) and Fz-GFP (green). (D) shows FzP278L-GFP (driven by the Actin5C promoter, green, left and middle panels) costained for actin (red, middle and right panels). (E) shows Stan (red) in a clone of a strong fz allele (fz25, marked by lack of GFP, green). Note accumulation of Stan on clone boundary (arrowheads). (F) shows Stan (red) in a clone of a fz allele deficient in only autonomous polarity signaling (fz17, marked by lack of GFP, green). Stan in cells immediately outside the clone shows a tendency to accumulate on cell boundaries lying perpendicular to the clone boundary (arrowheads). (G) shows a clone of cells overexpressing FzΔIntra-GFP marked by absence of forked function; the approximate clone outline is indicated by a red line. Trichomes outside the clone point towards the clone boundary. (H) shows FzΔIntra-GFP (driven by the Actin5C promoter, green, left and middle panels) costained for Stan (red, middle and right panels) Molecular Cell 2001 7, 367-375DOI: (10.1016/S1097-2765(01)00184-8)

Figure 6 Model for Role of Fz in Cell Polarization Panels from left to right represent a timecourse, showing Fz distribution (green) and putative Fz activity gradient (green) and Stan distribution (red). Polarity signal results in shallow gradient of Fz activity across cells, which is amplified by localization and anchoring of Fz to distal cell edge. See text for more details Molecular Cell 2001 7, 367-375DOI: (10.1016/S1097-2765(01)00184-8)