Drosophila Twins regulates Armadillo levels in response to Wg/Wnt signal

doi:10.1242/dev.00980

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First published online February 18, 2004
doi: 10.1242/10.1242/dev.00980

Development 131, 1007-1016 (2004)
Published by The Company of Biologists 2004

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Drosophila Twins regulates Armadillo levels in response to Wg/Wnt signal

Ruchi Bajpai^*^,, Kalpana Makhijani^*, Prashanth Ramesh Rao and L. S. Shashidhara

Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad 500 007, India

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Fig. 1. Effect of tws mutation on Wg, DV-Vg and Ct expression patterns. (A-C) Tws expression levels (detected by anti-Tws antibody staining) in wild-type (A), tws^P/+ (B) and tws^P/tws^P (C) wing discs. Homozygous mutants barely have any gene product. (D,E) Wild-type (D) and tws^P/tws^P (E) wing discs stained for Ci, which is expressed only in the anterior compartment. Note complete mirror-symmetry duplication in tws^P/tws^P disc as reported by Uemura et al. (Uemura et al., 1993

). (F,G) Wild-type (F) and tws^P/tws^P (G) wing discs stained for Wg. (H,I) Higher magnification images of wild-type (H) and tws^P/tws^P (I) wing discs showing Wg expression in the DV boundary in more detail. Note that Wg levels in the DV boundary of tws^P/tws^P discs are marginally lower, while the expression pattern itself is unchanged compared with wild type. (J,K) Wild-type (J) and tws^P/tws^P (K) wing discs stained for vg-BE. There is no change in the expression pattern of vg-BE between normal and mutant discs. (L,M) Wild type (L) and tws^P/tws^P (M) wing discs stained for Ct. Note reduction in Ct expression levels in the mutant disc.

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Fig. 2. Downregulation of targets of Wg signaling in tws^P/tws^P wing discs. Wild-type wing discs (A,C,E,G) and tws^P/tws^P wing discs (B,D,F,H) showing the expression patterns of Ac (A,B), Sca (C,D), Dll (E,F) and vg-QE (G,H). Note that, compared with their wild-type counterparts, levels of all the targets of Wg are lower in tws^P background. Control and experimental images were digitized at identical fluorescence and camera settings.

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Fig. 3. Loss-of-function clones of tws^P affect wing margin. (A,B) Cuticle of phenotypes displayed by the mitotic clones of tws^P. We observed characteristic serrated-margin phenotypes in both anterior (A) and posterior (B) compartments. (C,D) Wing discs with tws^P/tws^P clones. tws⁺/tws⁺ cells are marked with GFP. Note large clusters of tws⁺/tws⁺ cells with smaller or no associated tws^P/tws^P spots (C). Occasionally larger clusters of tws^P/tws^P cells were also seen (D). (E-H) Wing discs with tws^P/tws^P clones stained for Wg (E), Ct (F), Dll (G) and vg-QE (H). Arm-lacZ (red) was used to mark tws^P/tws^P clones in E-G, and GFP was used in H. For all the discs, the viability of tws^P/tws^P clones were confirmed by DAPI staining (data not shown). We did not observe any change in Wg expression levels, while Ct showed marginal reduction in its levels. Partial to complete loss of Dll and vg-QE was observed in tws^P/tws^P clones.

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Fig. 4. Arm is not stabilized in the DV boundary of tws^P/tws^P wing discs. (A) Wild-type wing disc stained with anti-Arm (red) and anti-Wg (green) antibodies. Note higher levels of Arm in cells adjacent to Wg-expressing DV boundary compared with distant non-DV cells. (B) tws^P/tws^P wing disc stained with anti-Arm (red) and anti-Wg (green) antibodies. Note reduction in the levels of Arm in cells adjacent to the Wg-expressing DV boundary. Also seen in B are large peripodial cells. In wild-type wing discs, they normally are not in the same focal plane as disc cells. A' and B' show x-z sections of the cells around the DV boundary of wild type (A') and tws^P/tws^P (B') wing discs. Although Arm is localized apically, owing to its stabilization in the cytoplasm of DV cells, it is seen all along the cross-section of the cells. However, in mutant cells, Arm is localized only to the apical adherens junctions. (C,D) Wild-type wing disc double-stained (but shown in single channels) with anti-Arm (C) and FITC-conjugated actin-phalloidin (D). Note that both Arm and Actin-phalloidin show intense staining in the cells surrounding the DV boundary (arrows in C,D). (E,F) tws^P/tws^P wing disc double-stained (shown in single channels) with anti-Arm (E) and FITC-conjugated actin-phalloidin (F). Note reduced levels of Arm in the DV boundary (arrow in E). However, actin-phalloidin staining is as intense as in the wild-type disc (arrow in F). (G) hsFLP; P[FRT]82 tws^P/P[FRT]82 arm-lacZ wing disc stained for Arm (green). tws^P/tws^P mitotic clones are marked with arm-lacZ (red), which is shown in single channel in G'. G'' is a merge of G and G'. A large clone abutting the DV boundary is shown. Note that levels of Arm are lower in tws^P/tws^P cells compared with neighboring tws⁺ cells. The viability of tws^P/tws^P cells were confirmed by DAPI staining (data not shown). (H,I) vg-GAL4; UAS-arm^S2 (H) and vg-GAL4; UAS-arm^S2 tws^P/tws^P (I) wing discs stained for Myc tag, which marks expression of only the overexpressed Arm. Note that Myc staining in I is significantly lower than in H, suggesting enhanced degradation of Arm in tws^P/tws^P discs. (J,K) UAS-arm^S10; vg-GAL4 (J) and UAS-arm^S10; vg-GAL4; tws^P/tws^P (K) wing discs stained for Myc tag. Levels of Myc in K are comparable with those in J, suggesting that tws^P/tws^P has no effect on degradation-resistant form of Arm.

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Fig. 5. Tws functions downstream of Dsh to inhibit Sgg activity. (A,B) vg-GAL4/UAS-Dsh (A) and vg-GAL4/+; UAS-Dsh/tws^P (B) wing blades. Inset in A shows expression pattern of vg-GAL4. Dsh-induced phenotype (ectopic bristles along the posterior margin) is suppressed by one copy of tws^P mutation. (C) vg-GAL4;UAS-Sgg fly showing underdeveloped wing blades owing to loss of Wg signaling. (D) vg-GAL4/+; UAS-Sgg/tws^P fly showing enhanced phenotype. Note total loss of wings and wing-to-notum transformation (shown at higher magnification in D'), a characteristic loss-of-wg phenotype. (E,F) vg-GAL4/+; UAS-Dsh tws^P/tws^P (E) and vg-GAL4/UASDNGSK-3ß; tws^P/tws^P (F) wing discs stained for Dll expression. Overexpression of dominant-negative Sgg/GSK3ß, but not Dsh, causes the rescue of tws^P/tws^P discs at the level of Dll expression (refer to Fig. 2F for Dll expression in tws^P/tws^P discs). (G) vg-GAL4/UAS-APC/CBD wing disc stained for Arm. Note very high levels of Arm in the DV boundary (compare with Fig. 4C) because of its sequestration by APC. In these cells, APC sequesters Arm, because Sgg (thereby the degradation machinery) is inhibited by Wg. (H) vg-GAL4/UAS-hAPC/CBD; tws^P/tws^P wing disc stained for Arm. In tws mutant background, APC fails to sequester Arm, suggesting that Sgg is active in all cells including the DV boundary.

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Fig. 6. Rescue of tws mutant phenotypes by UAS-Tws. (A) tws⁶⁰/tws^{60 pupa.} Homozygous tws^P and tws⁶⁰ mutants die at early pupal stages and do not show any signs of appendage development. (B,C) Ventral (B) and dorsal (C) views of two different tws⁶⁰/tws⁶⁰ pharate adults rescued by the expression of UAS-Tws using hs-GAL4 driver. Note normal development of all appendages. Eye in of the pharate adult in B is white because of prolonged preservation of the sample in ethanol prior to photography. (D) Partial rescue of tws^P/tws^P flies by en-GAL4 mediated UAS-Tws expression. Note that development of all appendages is partially rescued as Tws is expressed only in the posterior compartment. Overexpression of Tws in tws⁶⁰/tws⁶⁰ background induces gain-of-Wg phenotype. (E,F) Wild-type wing blades showing posterior hinge (E) and anterior margin (F) at higher magnification. (G) UAS-arm^S10; vg-GAL4 wing blade with tufts of ectopic bristles (arrow) at the posterior hinge. (H) 405-GAL4/UAS-arm^S10 wing blade with anterior margin-specific bristles in ectopic positions (arrow). Inset shows 405-GAL4 expression pattern. (I,J) Similar phenotypes (arrows) are observed when Tws is overexpressed in tws⁶⁰/tws⁶⁰ background using hs-GAL4.

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Fig. 7. Dominant-negative phenotypes caused by the overexpression of Tws in wild-type background. (A-C and E) vg-GAL4/UAS-Tws wing discs stained with Ct (A), Sca (B), Dll (C) and Vg (E) antibodies. (D) Wild-type wing disc stained with anti-Vg antibodies. Tws overexpression caused the downregulation of both short-range (Ct expression in the DV boundary and Sca expression in the presumptive margin SMCs; arrow in B) and long-range (Dll and Vg) targets of Wg signaling. Refer to Fig. 1L, Fig. 2C and Fig. 2E, for wild-type expression patterns of Ct, Sca and Dll, respectively. (F-G) Wild-type (F) and vg-GAL4/UAS-Tws (G) wing discs stained with Arm. Note overexpression of Tws causes reduction in cytoplasmic Arm levels. (H) vg-GAL4/UAS-Tws wing blade showing serrated posterior margin. (I) UAS-arm^S10; vg-GAL4/UAS-Tws wing blade. Note suppression of Tws-induced serrated margin phenotype. This wing blade is indistinguishable from UAS-arm^S10; vg-GAL4 wing blades (data not shown).

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