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First published online 17 January 2006
doi: 10.1242/jcs.02761

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Cytoskeletal dynamics and cell signaling during planar polarity establishment in the Drosophila embryonic denticle

Meredith H. Price^1,*,, David M. Roberts^2,*, Brooke M. McCartney³, Erin Jezuit¹ and Mark Peifer^1,2,4,

¹ Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-3280, USA
² Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-3280, USA
³ Department of Biological Sciences, Carnegie Mellon University, Pittsburgh PA 15213, USA
⁴ Curriculum in Genetics and Molecular Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-3280, USA

View larger version (138K): [in a new window]   Fig. 1. Denticles. Embryos, anterior at top. Unless noted, embryos here and in other Figures are wild type. (A,B) Cuticles. Six rows of cells per segment produce cuticle-covered cell projections called denticles. (C,D) F-actin (red). Arm (green, outlining cells). Denticles are actin-based and initially point posteriorly. Scale bars, 15 µm.

View larger version (169K): [in a new window]   Fig. 2. Denticles develop from polarized actin accumulations. Embryos, anterior left or upper left, F-actin (green or single label; A-C,E-H=phalloidin; D=anti-actin) and P-Tyr (B,E-H) or Arm (D) to outline cells (red). Insets=close-ups. (A) Extended germband. (B,C) Cells of presumptive denticle belts accumulate apical actin (brackets, B inset), beginning laterally (B, bottom left). (C,D) Actin concentrates along posterior cell margins (arrows). Insets B,D are close-ups. (E) Actin condenses into foci along the posterior margin (arrows). Inset, rare error in denticle positioning in row 1 (arrowhead). (F,G) Stages in denticle elongation (arrows, arrowheads, multiple denticles/cell). (H) Dorsal hairs. Condensations form and hairs (inset) elongate at the posterior-margin (blue arrowheads) or anterior-margin (white arrowheads) in different cell rows. (I-P) Stills, movie of Moesin-GFP-expressing embryo. Times in hours:minutes. (I) Before denticle formation. (J,K) Actin accumulates apically in presumptive denticle belts (brackets). (L) Actin foci along the posterior margin (arrows). (M-O) Foci condense and brighten. White arrows, red arrowheads – foci merging. (P) Denticles begin elongation. Scale bars, 10 µm.

View larger version (113K): [in a new window]   Fig. 3. MT and P-Tyr localization during denticle development. (A-D) Embryos, anterior left (A,D) or upper left (B,C). F-actin (phalloidin, green), P-Tyr (A,B red) or MTs (C,D red). (A) P-Tyr (red) accumulates with and around actin (green) in condensations (arrows). (B) P-Tyr (red) is enriched at denticle base (white arrow) but less prominent at the tip (blue arrow). (C) MTs (red) form circumferential arrays without enrichment near condensations (green, arrows). (D) MTs enriched at the denticle base (blue arrows) and in the core (white arrows) during elongation. (E,F) Deconvolved confocal images. (E) F-Actin (phalloidin). (E1) Early condensations. (E2) Late condensations with `donut holes'. (E3,E4) U- or V-shaped denticles. (F) MTs (red). F-actin (Phalloidin; green). (F1) MTs encircling a condensation. (F2,F3) MTs in the core of V-shaped denticles. (F4) Fully elongated denticle. MTs at base and in core. Scale bars, 5 µm.

View larger version (120K): [in a new window]   Fig. 4. Dia in denticles. Embryos, anterior to top. Dia (red), F-actin (phalloidin; green). (A) Before denticle development. (B) As denticles elongate, Dia becomes enriched in denticles (white arrowheads) while remaining cortical (blue arrowheads) in non-denticle-producing cells. (C-E) Deconvolved confocal images. (C) No substantial enrichment in early actin condensations (arrowhead). (D) Dia begins accumulating in elongating denticles (arrowheads). (E) Dia accumulates in a punctate fashion in elongated denticles (arrowheads). Scale bars, 5 µm.

View larger version (79K): [in a new window]   Fig. 5. Arp3 and Ena in denticles. Embryos, anterior to top. (A-C) Deconvolved confocal images, Arp3 (red), F-actin (phalloidin; green). (A) Arp3 puncta within and surrounding actin condensations (arrowhead). (B) Arp3 enrichment as denticles elongate (arrowheads). (C) Arp3 in elongated denticles (arrowhead). (D-I) Ena (red or single channel), F-actin (phalloidin; green). (D) Denticle-producing cells accumulate cortical Ena (brackets); in other cells Ena remains at tricellular junctions (arrowheads). (E,F) Ena enriched in elongating denticles (brackets). Arrowheads, tricellular junctions in cells that do not make denticles. (G-I) Deconvolved confocal images. Blue arrowheads – Ena in tricellular junctions. (G) Actin condensations without Ena enrichment (yellow arrowhead). (H) Ena in elongating denticles (yellow arrowhead). (I) Ena in elongated denticles (yellow arrowhead). Inset, elongated denticle in cross section – Ena in denticle and at its base (white arrowhead). Scale bars, 5 µm.

View larger version (170K): [in a new window]   Fig. 6. APC2 co-localizes with actin throughout denticle development. Embryos, anterior left. (A,B) APC2 (red), actin (green). Late condensations. (C-H) Stills, movie of embryo expressing UAS-APC2-GFP driven throughout the epidermis using 69B-GAL4. Times in hours:minutes. (C,D) APC2-GFP localizes apically in cells of presumptive denticle belts (brackets). (E-H) APC2-GFP localizes to posterior margin (E, arrows), and condensations focus and brighten (F-H). Colored arrowheads – condensations merging. (I-L) APC2. Wild type (I) and dsh75 (J,K) or armXM19 (L) maternal and zygotic mutants. APC2 (red, single channel in L) localizes to denticle primordia even when they arise at the cell apex (arrowheads, J-L). (I,J) Coracle (green; outlining cells). (K) F-actin (phalloidin; green). Scale bars, 10 µm.

View larger version (118K): [in a new window]   Fig. 7. Asymmetric localization of polarity proteins. Embryos, anterior left. (A) DE-cadherin (green), P-Tyr (red). (B) Arm (green), F-actin (red). (A,B) Neither DE-cadherin nor Arm localize to denticles (white arrows). Cortical Arm levels are higher in denticle-producing cells (brackets). Arm levels are somewhat higher at dorsal/ventral cell borders (blue arrowheads) than at anterior/posterior cell borders (red arrows). (C-E) Fmi (single channel or green). (C) Prior to denticle development Fmi localizes uniformly to cortex. (D,E) F-actin (red), Fmi (green), Arm (violet). Cells just anterior to denticle belts accumulate less Fmi (bracket). Cells just posterior to denticle belts accumulate Fmi uniformly on all cell interfaces (arrows). (E) Close-up, yellow-boxed region in D. Denticle-secreting cells accumulate more Fmi at anterior/posterior margins (red arrows) than on dorsal/ventral margins (blue arrowheads). (F) Stills, movie of Fz-GFP. Times in hours:minutes:seconds. (G) False-color closeup of F; pixel intensity color-coded from blue to yellow. (H-K) Still images, Dsh-GFP. Red arrows – asymmetric accumulation at anterior/posterior boundaries. (F-K) Blue arrowheads=presumptive vesicles, red arrows/arrowheads=asymmetric accumulation along anterior-posterior boundaries, red brackets=presumptive denticle belts, blue arrows=presumed actin condensations. Scale bars, 5 µm.

View larger version (133K): [in a new window]   Fig. 8. Canonical Wg and Hh signaling are required for denticle polarity. Embryos, anterior left. F-actin (green or single label; phalloidin, A-H,L-M; anti-actin, J,K,N-P). Cell outlines (red; P-Tyr, E-H,M; anti-Arm, J,K,N-P). B-H. Actin condensation and denticle formation are not restricted to the posterior margin. Some denticles form at the cell apex (red arrowheads), while others form at anterior or posterior cell junctions (yellow and white arrowheads, respectively). A-C,E,F, wgIG22. D, wg; Df(3L)H99. (A) All cells remain cuboidal and accumulate apical actin (beginning laterally; brackets). (B,C) Actin condensations form (B) and sharpen (C). (D) Denticle polarity is lost even if cell death is blocked. (E) Denticle elongation. (F) Elongated denticles. (G,H) Embryos maternally and zygotically dsh75 (G) or armXM19 (H). (I) Stills, movie of wgIG22 embryo expressing Moesin-GFP. Time in hours: minutes. Colored arrows – condensations. (J-L) armXP33 zygotic mutants. Many cells retain normal polarity (white arrows). A subset have reversed polarity (yellow arrows) or lose polarity (blue arrows). (K) Closeup, bracketed area in J. (L) Polarity reversals remain in elongated denticles. (M) UAS-Fz2-GPI expressed using Rho-GAL4. Occasional polarity reversals (red arrows). (N,O) hhAC. (N) Condensation polarity is lost (white arrow). Many cells produce multiple denticles (blue arrows). (O) Denticles elongating; no segmental periodicity. (P) ptc9. Many cells form apical condensations (arrows). Scale bars, 5 µm.

View larger version (161K): [in a new window]   Fig. 9. Roles for PCP proteins.. Embryos, anterior upper left. F-actin (green; phalloidin). Cell outlines (red; P-Tyr). White arrowheads=mispositioned denticles, blue arrowheads are correctly-positioned denticles. (A,B) Wild type, (C) Maternal and zygotic stbm6. (D-E) Zygotic fmi192. (F) Maternally dsh75, zygotically dsh1. (G) Maternal and zygotic pkpk1. (H,I) Maternal and zygotic fzD21/fzR52. (J-L) Zygotic fzD21 Df(3L)Dfz2/fzR52 Df(3L)Dfz2.

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