Effect: 40% chance of receiving an extra egg from a clutch if one or more parents are below level 5.
METHOD 1 – HIDDEN – 10% chance of encountering during a Breeder Job quest or Promotion image.
METHOD 2 – HIDDEN – 5% chance of encountering when a Breeder is depicted with a pair of dragons.
First published online 23 May 2006
doi: 10.1242/jcs.02973
1 Howard Hughes Medical Institute and Department of Developmental Biology, Stanford University School of Medicine, Stanford, CA 94305, USA
2 Abteilung Stammzellbiologie, DFG Research Center for Molecular Physiology of the Brain (CMPB), Georg-August-Universität Göttingen, Justus-von-Liebig-Weg 11, 37077 Göttingen, Germany
3 Department of Biological Sciences and Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA 94305, USA
* Author for correspondence (e-mail: awodarz{at}gwdg.de)
Accepted 8 March 2006
Key words: Wingless, Wnt, Cadherin, Catenin, Cell adhesion
In addition to its essential role in the structure of the adhesion complex, ß-catenin has a major function in the regulation of cell-cell adhesion (Nelson and Nusse, 2004
). ß-catenin can be modified by phosphorylation on either serine/threonine or tyrosine residues (Behrens et al., 1993
; Bek and Kemler, 2002
; Brembeck et al., 2004
; Hoschuetzky et al., 1994
; Nelson and Nusse, 2004
; Piedra et al., 2001
; Shiozaki et al., 1995
; Sommers et al., 1994
). In v-src-transformed MDCK cells, loss of cell-cell adhesion and increased invasiveness of the cells correlate with an increase in tyrosine phosphorylation of ß-catenin (Behrens et al., 1993
). Similar observations have been made in a human esophageal cancer cell line that shows increased tyrosine phosphorylation of ß-catenin after treatment with EGF (Shiozaki et al., 1995
). Recently, Brembeck et al. have found evidence for a switch between the adhesive and transcriptional functions of ß-catenin that is modulated by phosphorylation of Tyr 142 of ß-catenin (Brembeck et al., 2004
). Phosphorylation leads to binding of the BCL9-2 protein to ß-catenin, which in turn interferes with
-catenin contact. These dynamic interactions suggest that Wnt signaling, by virtue of using ß-catenin as a transcriptional effector, may also change the adhesive role of ß-catenin.
In Drosophila, adherens junctions consist of the same components as in vertebrates: E-cadherin (DE-cadherin), ß-catenin, which is encoded by the segment polarity gene armadillo (arm) and
-catenin (Cox et al., 1996
; McCartney et al., 2001
; Oda and Tsukita, 1999
; Pai et al., 1996
; Tepass et al., 1996
; Uemura et al., 1996
). Arm is also an essential component of the Wingless (Wg) signaling pathway. Wg is very pleiotropic and is involved in a large number of developmental processes, including segmentation of the embryo, development of the gut and the heart, and morphogenesis of wings, legs and eyes (Klingensmith and Nusse, 1994
; Seto and Bellen, 2004
).
In this work, we have addressed the question of whether there is any functional connection between Wg signaling and cadherin-mediated cell adhesion, or whether these two processes are independent of each other. Mammalian cells overexpressing Wnt1, the vertebrate ortholog of Wg, show effects of Wnt1 expression on cadherin-mediated cell adhesion (Bradley et al., 1993
; Hinck et al., 1994
; Seto and Bellen, 2004
). These include an increase in the amount of ß-catenin and plakoglobin (another member of the Armadillo protein family), an increase in cadherin protein levels and an overall strengthening of cell adhesion in the Wnt1-expressing cells. However, these observations were made in cells that constitutively express transfected Wnt genes, raising the possibility that the observed effects might not be the primary response of the cells to the Wnt signal. Therefore, we have used a Drosophila tissue culture system that is based on cl-8 wing imaginal disc cells (Peel et al., 1990
). cl-8 cells respond to stimulation with Wg by an increase in the level of a cytosolic, hypophosphorylated form of Arm, which is caused by a posttranscriptional stabilization of the Arm protein (Van Leeuwen et al., 1994
). The same response can be obtained by transfection of the cells with a cDNA encoding a temperature-sensitive allele of Wg or by overexpression of Dishevelled (Dsh), an intracellular component of the Wg signaling pathway (Van Leeuwen et al., 1994
; Yanagawa et al., 1995
). cl-8 cells have proved useful for Drosophila developmental and cell biological studies, in particular in recent RNAi screens for genes controlling signaling responses (Lum et al., 2003
). We find that Wg signaling leads to an initial downregulation of the E-cadherin-Arm complex at cell-cell contacts, followed by transcriptional upregulation of DE-cadherin expression. We suggest that Wg signaling facilitates cell movement and division in epithelial tissues by transiently reducing cell-cell adhesion.
Expression of E-cadherin in cl-8 cells not only affected the levels of Arm and
-catenin proteins but also their subcellular localization. In untransfected cl-8 cells, Arm was about equally abundant in the cytosolic supernatant fraction and in a Triton X-100-insoluble pellet fraction, whereas
-catenin was present mostly in the cytosolic fraction (Fig. 1C). In cl8mEcad cells, almost all Arm and
-catenin were present in the insoluble fraction (Fig. 1C). This redistribution was caused by the formation of a protein complex containing E-cadherin, Arm and
-catenin, which could be immunoprecipitated from Triton X-100 lysates of cl8mEcad cells with antibodies against each of these three proteins (data not shown). cl8mEcad cells treated with Cu2+, to further increase levels of E-cadherin, showed a significant amount of Arm and
-catenin in the Triton X-100 insoluble pellet fraction, indicating a stable association of the cadherin-catenin complex with the cytoskeleton (Fig. 1C).
In untransfected cl-8 cells, Arm was hardly detectable by immunofluorescence microscopy and did not show any plasma membrane localization (Fig. 2). In cl8mEcad cells, by contrast, Arm was highly concentrated at cell-cell contact sites together with E-cadherin (Fig. 2).
-catenin accumulated at similar cell-cell contact sites (Fig. 2). These data indicate that expression of mouse E-cadherin in cl-8 cells initiates the formation of a functional cadherin-catenin complex at cell-cell contact sites.
Activation of the Wingless signaling pathway leads to a redistribution of Armadillo and to reduced E-cadherin levels at the cell surface
We have shown previously that Wg signaling in cl-8 cells can be activated either by treatment with Wg-conditioned medium, or by transfection of wgIL114, a temperature-sensitive allele (Van Leeuwen et al., 1994
), or by overexpression of Dsh, an intracellular component of the Wg signaling pathway (Yanagawa et al., 1995
). We generated stable cell lines that expressed Cu2+-inducible E-cadherin, combined with either wgIL114 with a constitutive promoter (cl8Wgts/mEcad) or Dsh cDNA under control of the hsp70 heat shock promoter (cl8HSDsh/mEcad). Wingless signaling could then be activated by changing the temperature from the restrictive temperature (25°C) to the permissive temperature (16°C) in the case of the cl8Wgts/mEcad cells, or by a 30-minute heat shock at 37°C in the case of the cl8HSDsh/mEcad cells. In addition, the level of E-cadherin expression was controlled independently by addition of Cu2+ to the medium.
We first examined the distribution of Arm by cell fractionation. The cells were lysed in hypotonic buffer, followed by centrifugation, giving rise to a cytosolic supernatant fraction and a pellet fraction containing membranes, membrane-associated proteins and nuclei. In uninduced cl8Wgts/mEcad cells (Fig. 3, lanes 1 and 2) and cl8HSDsh/mEcad cells (Fig. 3, lanes 9 and 10) Arm was present almost exclusively in the pellet fraction and was absent from the cytosolic fraction, just as in cl8mEcad cells. Elevation of E-cadherin expression by addition of Cu2+ led to a further increase in the level of membrane-associated Arm, and to the accumulation of E-cadherin and Arm in the cytosolic fraction (Fig. 3, lanes 3, 4, 11 and 12). In the absence of Wg, the distribution of Arm was very similar to the distribution of E-cadherin, suggesting that most of the Arm was associated with E-cadherin. As in cl8mEcad cells, expression of E-cadherin led to the accumulation of two forms of Arm, as a result of a difference in phosphorylation on serine and threonine residues, as shown previously (Peifer et al., 1994a
