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1.
Figure 3

Figure 3. From: naked cuticle targets dishevelled to antagonize Wnt signal transduction.

Cuticle phenotypes of embryos injected with nkd dsRNA. (A) Wild-type (wt) embryo shortly before hatching. The ventral side is characterized by denticle belts separated with naked cuticle. (B) nkd RNAi mimics the nkd phenotype. Embryos are shorter and lack denticle belts, resembling nkd−/− embryos (see Fig. 1K). nkd RNAi embryos also show the characteristic defects in the Filzkörper (FK).

Raphaël Rousset, et al. Genes Dev. 2001 March 15;15(6):658-671.
2.
Figure 6

Figure 6. From: naked cuticle targets dishevelled to antagonize Wnt signal transduction.

Effects of overproduced Nkd on planar cell polarity. (A) Wild-type (WT) wing pattern in region distal to posterior cross vein; same area shown in B–E. (B) Effect of overproduction of Nkd, which is similar to the phenotype shown in C, but different to the phenotypes shown in D and E. (C) Phenotype of loss of dsh function (dsh1 allele). (D) Phenotype of loss of frizzled function (fzJ22 allele). (E) Phenotype of loss of prickle function (pk30 allele). Arrows indicate hair orientation. (F) Wild-type wing pattern in the area shown in G and H: region posterior to vein 5. (G) Phenotype of heat shock promoter-driven frizzled expression. (H) Phenotype of heat shock promoter–driven fz expression in the presence of UAS-nkd. Almost complete suppression of polarity defects by Nkd overexpression is observed. High magnification shows that fz overexpression also induces double hair cells (G inset) and that their number decreases in the presence of Nkd (H inset).

Raphaël Rousset, et al. Genes Dev. 2001 March 15;15(6):658-671.
3.

Figure 5. From: naked cuticle targets dishevelled to antagonize Wnt signal transduction.

Nkd interacts with the basic/PDZ region of Dsh. (A) Interaction of different Dsh deletion mutants, fused to GAD, with GB or GB-Nkd in the yeast two-hybrid system. Yeast growth was evaluated using the ADE reporter gene present in the strain: Yeast colonies expressing the different combinations of fusion proteins, as indicated, were grown on medium containing (+) or lacking (−) adenine. (B,C) Using the GST pull-down assay, [35S]methionine-Dsh D1 to D6 were tested for their capacity to bind GST-Nkd (B), whereas in (C), GST-DshD6PDZ and GST-DshPDZ proteins were incubated with [35S]methionine-Nkd. The curved bands observed with D1 and D6 are due to comigration with a byproduct of GST-Nkd protein (data not shown). (D) Summary of domain mapping results from the yeast two-hybrid system (Y2H) and the GST pull-down assay (GST). (+) Positive interaction, (−) no interaction, (+/−) weak interaction, (n.d.) not determined. The different domains of Dsh, DIX, basic region (b), PDZ, and DEP, are indicated.

Raphaël Rousset, et al. Genes Dev. 2001 March 15;15(6):658-671.
4.
Figure 2

Figure 2. From: naked cuticle targets dishevelled to antagonize Wnt signal transduction.

nkd autonomously regulates interommatidial bristle formation. (A) Wild-type eye margin at dorsum of head shows bristle suppression 0–1 ommatidial rows (blue brackets) from eye margin. (B) nkd7E89 mutant eye shows suppression of bristles 3–5 ommatidial rows from eye margin. Occasional bristles are present closer to eye margin (yellow arrow). No bristle phenotype was seen with the weaker nkd9G33 allele, whereas h1 nkd7H16 eyes are small and rough (not shown), possibly due to h/nkd interactions (Zeng et al. 2000) and hence could not be scored for this phenotype. (C,D) Nkd/GFP misexpression clones in sev-wg adult eyes. (C) Bristles are restored only in the region of the clone marked by GFP. (D) Cartoon depiction of nine adult eye clones (green color); vertical black lines represent bristles. (E) Cut nuclear localization in bristle cells of a wild-type pupal eye disc. (F,G) Restoration of bristle cells in a sev-wg pupal eye, revealed by Cut localization (red color), is confined to the region of the Nkd/GFP misexpression clone (green color); the clone begins at the edge of the disc (left) and continues inward (right). (H) Merged image of F and G shows that GFP and Cut colocalize in bristle cell precursor nuclei present in the clone.

Raphaël Rousset, et al. Genes Dev. 2001 March 15;15(6):658-671.
5.
Figure 1

Figure 1. From: naked cuticle targets dishevelled to antagonize Wnt signal transduction.

Epistasis study in the eye and embryo. (A) Schematic diagram of the Wg pathway and the planar cell polarity pathway. Arrows and bars show positive and negative actions, respectively. P represents the phosphorylated state of Dsh. See text for details. (B–J) Nkd can suppress Wg and Dsh misexpression eye phenotypes, but not the ArmS10misexpression eye phenotype. Ventral is to the left and anterior up. (B) The wild-type adult eye consists of an array of ommatidia and interommatidial bristles. (C,E) The sev-wg or UAS-dsh eyes lack bristles and/or ommatidia. (G) The UAS-armS10 eye has disrupted ommatidia and loss of bristles (average number of bristles/eye = 11.6; n = 8). (D,F) Co-misexpression of UAS-nkd dramatically suppressed the sev-wg (n = 8) and the UAS-dsh (n = 8) eye phenotypes. (H) Co-misexpression of UAS-nkd did not suppress the UAS-armS10 phenotype (average number of bristles/eye = 5.4; n = 8). UAS-GPI-Dfz2 suppresses the sev-wg loss of bristle phenotype (I) but does not suppress the UAS-dsh phenotype (J). (K,L) Injection of Xenopus GSK3β mRNA into nkd mutant embryos can restore denticles to the nkd embryos. Anterior is up. (K) nkd mutant embryos lack ventral denticle belts. (L) Injection of GSK3β into nkd7H16 embryos restored ventral denticles (brackets). nkd7H16 mutant embryos were identified by a Ubx mutation resulting in the transformation of the first abdominal segment A1 to the third thoracic segment T3 (arrow).

Raphaël Rousset, et al. Genes Dev. 2001 March 15;15(6):658-671.
6.

Figure 4. From: naked cuticle targets dishevelled to antagonize Wnt signal transduction.

Nkd and Dsh directly interact in the yeast two-hybrid system, coimmunoprecipitation and GST pull-down assays. (A) Interaction between Nkd and Dsh in the yeast two-hybrid system using a liquid culture β-galactosidase assay. Yeast cells were cotransformed with plasmids expressing the GAL4 DNA-binding domain (GB, amino acids [aa] 1–147) either alone (−) or in fusion with Nkd, along with plasmids expressing the GAL4 activation domain (GAD, aa 768–881) alone (−) or in fusion with Dsh. The interaction between GB-Nkd and GAD-Zw3, GAD-Arm, or GAD-Dfz2CT (intracellular portion of Dfz2) was also tested. Murine p53 (aa 72–390) fused to GB and SV40 large T-antigen (T-Ag, aa 87–708) fused to GAD were used as positive controls. For each transformation, results corresponding to the mean of 2-independent yeast colonies are shown. The values are expressed in Miller units. The activation was confirmed using a second reporter gene (ADE2) present in the yeast strain and protein blots of yeast extracts showed that the different fusion proteins accumulated to similar levels (data not shown). (B) Coimmunoprecipitation of Nkd and Dsh in COS-7 cells. Extracts from COS-7 cells expressing Nkd-myc and Dsh-myc were subjected to coimmunoprecipitation with anti-Dsh antibody (α Dsh Ab) (+). As a control, the anti-Dsh antibody was omitted (−). The eluted protein from the beads (P) and one-tenth of the supernatant (S), as well as proteins corresponding to one-tenth of the input (I) were analyzed by Western blot using anti-c-Myc antibody. (C,D) Interaction between Nkd and Dsh in the GST pull-down assay. Bacterially expressed GST-Nkd protein was incubated with [35S]methionine-Dsh, produced and labeled by in vitro translation (C), whereas GST-Dsh was incubated with [35S]methionine-Nkd (D). As controls, GST-Nkd or GST-Dsh proteins were also tested for their interaction with [35S]methionine-Luciferase (Luc). The eluted proteins from the beads (P) and one-tenth of the supernatant (S) were separated by SDS-PAGE and viewed using a phosphorimager (Molecular Dynamics).

Raphaël Rousset, et al. Genes Dev. 2001 March 15;15(6):658-671.

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