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First published online 11 August 2004
doi: 10.1242/dev.01317

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Diego interacts with Prickle and Strabismus/Van Gogh to localize planar cell polarity complexes

¹ Brookdale Department of Molecular, Cell and Developmental Biology, Mount Sinai School of Medicine, One Gustave L. Levy Place, New York, NY 10029, USA
² Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauer Strasse 108, 01307 Dresden, Germany

View larger version (92K): [in a new window]   Fig. 1. The dgo eye phenotype. (A) Schematic drawing of 3rd instar larval eye imaginal disc, with the morphogenetic furrow (MF; yellow) and the DV midline (the equator; gray) indicated. Anterior is leftwards and dorsal upwards in this and all subsequent figures. Initially, ommatidial preclusters are symmetrical and organized in the AP axis. Subsequently, they rotate 90° with respect to the equator; at the end of this process chirality is established by the positions of R3 and R4. (Right) Schematic presentation of chiral organization of dorsal and ventral adult ommatidia; in addition to the chiral forms, symmetrical clusters with R3/R3 or R4/R4 cell pairs as found in PCP mutants are shown. R3 cells are highlighted in green and R4s in magenta. (B) Partial view of a developing eye imaginal disc demonstrating the regularity of polarity establishment. Ommatidial clusters are marked with anti-Elav (green; labeling all photoreceptors) and svp-lacZ [magenta: svp is expressed initially in R3/R4 (see left side of panel) and later also in R1/R6 at weaker levels]. The MF is on left side adjacent to field shown. Orientation of some dorsal ommatidial preclusters is highlighted with yellow arrows; white line marks the equator. (C,D) Tangential sections of adult eyes with the respective schematic presentations of the genotypes indicated. Wild-type dorsal and ventral ommatidial arrangement is represented by black and red arrows, respectively; symmetrical R3/R3 and R4/R4 ommatidia are represented by green and magenta arrows, respectively. (C) Wild-type eye with regular arrangement of dorsal and ventral ommatidia around equator. (D) dgo380 eye (null allele). The equatorial arrangement is disturbed with a random arrangement of both chiralities (black and red arrows), and the presence of many symmetrical clusters of both R3/R3 and R4/R4 types (green and magenta arrows). (E,E') Confocal microscopy images of mosaic 3rd instar eye disc, with dgo– tissue marked by absence of green (GFP); anti-Bar labeling R1/R6 (magenta) highlights orientation of clusters. (E') Single channel showing Bar staining. Orientation defects of the preclusters are visible from early stages in mutant tissue. Examples with abnormal orientation are indicated with white arrows; yellow arrows indicate wild-type orientation for comparison.

View larger version (75K): [in a new window]   Fig. 2. Localization of Dgo during PCP establishment in the eye. Anterior is leftwards and dorsal upwards in all panels except F and I, where proximal is leftwards. (A) Single apical confocal microscopy section showing ommatidial rows 1-7 stained with anti-Dgo (green) and phalloidin (highlighting F-actin, red) to mark the center of each cluster. Morphogenetic furrow (anterior) is on the left of the panel and the equator is at the bottom. (Right panels) Monochrome higher magnifications of single clusters from row 3, row 5 and row 6 (as indicated). Dgo is first detected apical at the membrane in all cells ahead of and posterior to MF (up to row 2). More posteriorly, the initial uniform apical localization changes (row 3) within five cell preclusters, and Dgo is present at higher levels in R3/4 cells within the equatorial-polar (DV) axis. (Row 5) Dgo is maintained at the border between the R3/R4 cells, but it is now detected less in parts of R3 and at higher levels in R4. (Row 6) The R4 specific enrichment is now complete and persists through row 10. Whereas there is no Dgo detected at membranes where R3/R4 abut the R2/R5 pair, Dgo is enriched at the opposing R8, R2/R5 membranes. (B-D) Mosaic analysis of GFP-Dgo to determine the precise localization of Dgo at the R3/R4 cell boundary. GFP-Dgo is in green, anti-DE Cadherin is in red. Right side panels show a schematic of actual clusters on the left. Cells are numbered according to their position; cells shown in white express GFP-Dgo in the given cluster; GFP-Dgo at localization membranes is highlighted with green lines. (B,C) Mosaic clusters in row 3 with either R4 (B) or R3 (C) expressing GFP-Dgo. Dgo localization reflects a horseshoe-like pattern in both R3 and R4 cells. (D,E) Mosaic clusters in rows 5-6, with (again) either R4 (D) or R3 (E) expressing GFP-Dgo. Dgo is now enriched only at the R3 side of the R3/R4 cell boundary and the polar side of R4 (very similar to the localization of Fz). (F) GFP-Dgo (green) clones in pupal wings at 60-80 hours at 18°C. lacZ (blue) marks the cells that express GFP-Dgo; F-actin (phalloidin, red) labels the growing actin hairs; proximal is leftwards. GFP-Dgo localizes to the distal membrane of each cell that expresses it. (G-I) Schematic summary presentation of the Dgo localization patterns in developing eye (G, row 3; H, row 6) and pupal wing (I) cells; again, Dgo shows the same localization pattern as Fz and Dsh.

View larger version (123K): [in a new window]   Fig. 3. Dgo membrane association requires fz. Panels show single confocal sections; anterior is leftwards and the equator is at the bottom (MF is at the left edge of each panel, except in A where it is on the right, marked by the red arrowhead). Mutant tissue is marked by absence of ß-gal staining or GFP (blue). White/yellow arrows indicate examples of clusters with PCP protein localization in R3/R4 cell pair (equivalent to row 3 or 4, see also Fig. 2A) and arrowheads in C-E indicate the asymmetric R4-like enrichment (around row 5-6). Green channel: anti-Dgo (in A-E). Red channel: Fz-GFP in C and D; Pk in panels E and F. Clones of null alleles of the respective genes are shown. (A) fz– clone anterior to MF, which is marked with red arrowhead. Strikingly, also anterior to the MF, Dgo is delocalized in fzR52 tissue [Dgo is apically localized in all wd-type cells (blue), ahead of the MF]. (B) fz– tissue posterior to MF; apical membrane-associated Dgo localization is absent in fz– cells (examples in wild-type area are indicated by white arrows). Dgo is enriched at membranes between fz+ and fz– cells (e.g. vertical arrowhead). (C) dgo– clone anterior to MF. There is no change in apical Fz-GFP localization in mutant tissue [compare with wild-type cells (blue)]. (D) dgo– tissue posterior to MF. Apical membrane-associated Fz-GFP localization is not affected in dgo– cells (examples of preclusters in wild-type and mutant areas are indicated by white arrows). (E) Pk localization in fz– tissue posterior to MF. While apical levels of Pk localization are unaffected, the characteristic asymmetric PCP localization pattern is not observed. (F) Pk localization in dgo–: apical localization pattern of Pk in mutant tissue is unchanged. (G,H) Dgo localization in pk– and stbm– clones: the Dgo pattern is as in wild type. Although Dgo displays the R4 asymmetry as in wild type, the R4-like enrichment to either equatorial or polar side is randomized [e.g. in the equatorial cell (red arrowhead), reflecting chirality flips], mimicking the adult phenotype of random chirality. (I) pk–, stbm– tissue: apical Dgo localization is reduced and characteristic R4-like pattern is not resolved.

View larger version (128K): [in a new window]   Fig. 4. Dgo, with Pk and Stbm, promotes the apical localization of Fmi and Fz. Apical confocal sections are shown; anterior is leftwards and the equator at bottom. MF is at left margin of panels, except in F where it is indicated by red arrowhead. Mutant tissue is marked by absence of ß-gal staining or GFP (blue). White/yellow arrows indicate PCP protein localization in R3/R4 pair (row 3 or 4) and arrowheads indicate the R4-like enrichment (row 5 and posterior), except in F where it indicates clones ahead of MF. Fmi (green) and Fz-GFP (red) localization is analyzed in mutant tissue of null alleles of the respective PCP genes. (A) pk–: no significant abnormalities in the apical localization of Fmi. (B) pk–, dgo–: loss of apical Fmi localization. (C) pk–, dgo–: reduction in apical Fz localization. (D) dgo–, stbm–: loss of apical Fmi localization (compare with A and B). (E) dgo–, stbm–: loss of apical Fz-GFP localization. The vertical arrow indicates a mosaic in which the R3 specific Fz-GFP staining is missing. (F) pk–, stbm– mutant clones behind and ahead of MF (marked by red arrowhead): apical Fmi localization is lost in mutant tissue ahead of MF, similar to pk–, dgo– and pk–, stbm– double mutants (compare with B and C), but apical localization of Fmi is observed posterior to MF, although no clear pattern is detected. In pk–, stbm– double mutant tissue there is a different effect on Fmi anterior and posterior to MF. (F) pk–, stbm–: apical Fz-GFP localization behaves like Fmi: it is unaffected posterior to MF (but lost anterior to MF, not shown).

View larger version (160K): [in a new window]   Fig. 5. Dsh, and Stbm localization in PCP mutants. Apical confocal sections with MF (anterior) at left edge and equator at bottom. MF is at the left margin of all panels, except in G, where it is indicated by red arrowheads. Mutant tissue is marked by absence of ß-gal staining or GFP (blue). White/yellow arrows indicate PCP protein localization in R3/R4 pairs (rows 3 or 4) and arrowheads mark clusters with R4-like enrichment (row 5 onwards). Localization of Dsh and Fz-GFP (red) and Strabismus (green) are shown. Clones of null alleles of the respective PCP genes (as indicated) are shown. (A) dgo–: no differences in Dsh localization are detected between wild-type and mutant tissue. (B) pk–, dgo–: loss of apical localization of Dsh. (C) dgo–: no difference in Stbm localization between wild-type and mutant tissue. (D) pk–: apical localization of Stbm is observed (although it is slightly reduced). (E) pk–, dgo–: loss of apical Stbm localization. (F) fmi–: loss in apical localization of Stbm. (G) fmi–: loss of apical Fz-GFP localization is observed.

View larger version (22K): [in a new window]   Fig. 6. Dgo physically interacts with Pk and Stbm in vitro. (A) Dgo Ankyrin repeats bind to a 131 amino acid fragment of Pk (white box in scheme). GST fusion proteins with fragments of the C terminus of Pk indicated at the top (color coded below, compare with scheme) were tested for binding to full-length Dgo (35S-Dgo in vitro translated; marked yellow in the scheme on the right) or to its Ankyrin repeat region (35S-Dgo Ank). An unrelated control (35S-Control) does not bind to Pk. As a standard, 10% of the in vitro translated protein used for the binding reaction was loaded directly (10% input). The Pk region interacting with Dgo comprises residues 820-908. Purple boxes in Dgo scheme on right indicate the Ankyrin repeats and light and dark green boxes in scheme underneath indicate the PET and the three LIM domains of Pk, respectively. (B) Dgo Ankyrin repeats interact with Stbm. In vitro translated fragments of the C-terminal cytoplasmic region of Stbm (indicated on left and marked in yellow in scheme on right) were tested for their binding to a GST fusion protein of the Ankyrin repeats of Dgo (G-Dgo Ank) or to an unrelated fusion protein (G-Control). 35S-Stbm CtermN2 and 35S-Stbm CtermC2 define a region of 85 amino acids required for binding of Stbm to Dgo. Standard is as in A. Light green boxes in scheme on right indicate position of the predicted four transmembrane domains of Stbm.

View larger version (56K): [in a new window]   Fig. 7. (A) Model for maintenance of the apical PCP protein complex (prior to PCP signaling, e.g. anterior to MF). Known physical interactions are highlighted in blue (this work) (Tree et al., 2002; Jenny et al., 2003). The predicted Fmi-mediated complex, containing also Fz, Stbm and Dsh, is stable when either Dgo or Pk are removed, but unstable in the double mutant. (B) Proposed model for PCP signaling circuitry during PCP signaling, e.g. in R3/R4 cells. Fmi is maintained through Dgo function in R3 and Stbm/Pk in R4. Known physical interactions are in blue (Jenny et al., 2003; Tree et al., 2002). Factors depicted in gray are downregulated through PCP signaling at this stage. See text for details.

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