QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 4 August 2004
doi: 10.1242/dev.01296

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Related articles in Development Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Hoffmans, R. Articles by Basler, K. Search for Related Content PubMed PubMed Citation Articles by Hoffmans, R. Articles by Basler, K.

Identification and in vivo role of the Armadillo-Legless interaction

Institut für Molekularbiologie, Universität Zürich, Winterthurerstrasse 190, 8057 Zürich, Switzerland

View larger version (50K): [in a new window]   Fig. 5. arm-D164A animals closely resemble lgs mutants. (A) The cuticle of a wild-type (wt) embryo shown in dark-field. (B) Embryo maternally and zygotically mutant for lgs20F. This allele carries a premature stop codon and is hence considered a null allele (Kramps et al., 2002). (C) Embryo representing class II (see Materials and methods), derived from an arm2a9 germline clone with maternal (mat) and zygotic (zyg) Arm-D164A function. Such embryos display a lawn of denticles similar to that of embryos that are maternally and zygotically mutant for the lgs null allele (B). (D) Embryo representing class III (see Materials and methods), derived from an arm2a9 germline clone with only maternal (mat) Arm-D164A function. Such embryos display a phenotype which is more severe than that of class II and lgs null embryos. Note that the reduced contrast in the denticle patterns of the embryos shown in A and B versus those in C and D stems from the marker gene yellow, which, for technical reasons (Kramps et al. 2002), is mutant in embryos A and B, but wild type in C and D.

View larger version (54K): [in a new window]   Fig. 1. Identification of mutations in ß-catenin that affect Lgs binding. (A) Schematic representation of the ß-catenin protein. The Arm repeats are marked by different colours and numbered 1-12. Black lines represent the binding domains of ß-catenin interaction partners. P marks the phosphorylation sites used by the degradation complex. The red line indicates the protein product of the arm2a9 allele, which contains an X-ray induced frame shift in Arm repeat 3 and results at best in a truncated protein. (B) Electrostatic surface of ß-catenin. Blue and red surfaces represent regions of positive (basic) and negative (acidic) potential, respectively. White arrow indicates the acidic knob that is essential for Lgs binding (amino acids 162 to 164). The broken line indicates the basic groove in which E-cadherin, TCF4 and APC make multiple contacts with ß-catenin (reviewed by Daniels et al., 2001). (C,D) Space filling models of Arm repeats 1-12. The mutations are indicated in the colour of the Arm repeat that contains the mutation (same colour scheme as in A). The model in D is turned by 90° along the horizontal axis compared with that in C. (E) Interaction of mutant ß-catenin proteins with human LGS1 tested by yeast two-hybrid analysis. Mutations D162A, D164A and R386A show an effect on Lgs binding. Bars are colour-coded to match the colour scheme of the Arm repeats in A. The protein Huckebein (Hkb) served as a negative control, as it is a transcription factor (Bronner et al., 1994) that plays no role in Wnt/Wg signalling. (F) A subset of the ß-catenin mutants was tested for binding to APC, E-cadherin, TCF4 and -catenin. D162A and D164A do not have a negative effect on binding of ß-catenin to APC, E-cadherin and TCF4. The mutations that affected Lgs binding also reduced -catenin binding by 50%. R386A affected Lgs binding to variable degree (compare E with F), but led to a reproducible reduction in the binding to APC, E-cadherin and TCF. Results are presented as the percentage of binding compared with wild-type ß-catenin.

View larger version (149K): [in a new window]   Fig. 2. Constitutively active forms of Arm depend on Lgs binding for signalling activity. (A) The cuticle of a wild-type (wt) embryo. (B) Ubiquitous expression of a constitutively active form of Arm (ArmS10-wt) results in a naked cuticle phenotype. (C,D) Ubiquitous expression of ArmS10 carrying the D164A or the K435E mutations to impair the binding to Lgs or Pan, respectively, no longer causes a naked cuticle phenotype. Occasional ectopic denticles in areas where Wg is active (and which are normally naked) can be observed and indicate that these two mutant forms may exhibit slight dominant-negative activities, possibly by titrating away Pan and Lgs, respectively, from wild-type Arm. (E) The cuticle of an embryo containing the daughterless-Gal4 (DaG4) driver is indistinguishable from that of wild-type embryos (A). (F-H) Ubiquitous expression of a constitutively active, membrane-targeted form of Arm (Arm-wt) results in a naked cuticle. Mutations in Arm that affect binding to Lgs (Arm-D164A) or Pan (Arm-K435E) still result in a naked cuticle phenotype, most probably because in this situation endogenous Arm, and not membrane-targeted Arm, mediates the signalling output (Tolwinski and Wieschaus, 2001; Tolwinski and Wieschaus, 2004). All transgenes in these experiments were controlled by UAS-promoters driven by DaG4 (Wodarz et al., 1995).

View larger version (109K): [in a new window]   Fig. 3. Arm-D164A fails to restore wild-type levels of Dll expression in arm-null clones. Confocal sections of wing discs are shown, stained for either Lgs or Dll expression. Loss of GFP (green) marks clones lacking Arm (left panels). Merged images are shown towards the right. (A) arm2a9 clones express Lgs, indicating that these cells are still viable and capable of expressing proteins. Not only the overall levels, but also the subcellular distribution of Lgs is unaffected by the loss of Arm. The magnification in this panel is sixfold higher than those in B-D. (B) arm2a9 clones do not express Dll. (C) Ubiquitous expression of Arm-wt rescues Dll expression in arm2a9 clones. (D) Ubiquitous expression of Arm-D164A has only weak rescuing activity and most arm2a9 clones exhibit severely reduced levels of Dll expression.

View larger version (163K): [in a new window]   Fig. 4. Arm-D164A can restore functional adherens junctions in arm clones. Confocal sections of wing disc cells are shown, stained for E-cadherin expression (central panels) marking the adherens junctions. Loss of GFP (green) marks clones lacking Arm (left panels). Merged images are shown to the right. (A) arm2a9 clones are small, have an abnormally round shape, and show diffuse E-cadherin staining. (B) arm2a9 clones expressing Arm-wt exhibit the typical irregular outline of wild-type clones in wing discs and show restored E-cadherin staining. (C) arm2a9 clones expressing Arm-D164A behave like those expressing Arm-wt; they exhibit irregular outlines and restored E-cadherin staining.