Destruction complex dynamics: Wnt/ß-catenin signaling alters Axin-GSK3ß interactions in vivo.

The central regulator of the Wnt/ß-catenin pathway is the Axin/APC/GSK3ß destruction complex (DC), which, under unstimulated conditions, targets cytoplasmic ß-catenin for degradation. How Wnt activation inhibits the DC to permit ß-catenin-dependent signaling remains controversial, in part because the DC and its regulation have never been observed in vivo Using bimolecular fluorescence complementation (BiFC) methods, we have now analyzed the activity of the DC under near-physiological conditions in Drosophila By focusing on well-established patterns of Wnt/Wg signaling in the developing Drosophila wing, we have defined the sequence of events by which activated Wnt receptors induce a conformational change within the DC, resulting in modified Axin-GSK3ß interactions that prevent ß-catenin degradation. Surprisingly, the nucleus is surrounded by active DCs, which principally control the degradation of ß-catenin and thereby nuclear access. These DCs are inactivated and removed upon Wnt signal transduction. These results suggest a novel mechanistic model for dynamic Wnt signal transduction in vivo.

Preparation of Precisely Oriented Cryosections of Undistorted Drosophila Wing Imaginal Discs for High Resolution Confocal Imaging.

The combination of immunofluorescence and laser scanning confocal microscopy (LSM) is essential to high-resolution detection of molecular distribution in biological specimens. A frequent limitation is the need to image deep inside a tissue or in a specific plane, which may be inaccessible due to tissue size or shape. Recreating high-resolution 3D images is not possible because the point-spread function of light reduces the resolution in the Z-axis about 3-fold, compared to XY, and light scattering obscures signal deep in the tissue. However, the XY plane of interest can be chosen if embedded samples are precisely oriented and sectioned prior to imaging (Figure 1). Here we describe the preparation of frozen tissue sections of the Drosophila wing imaginal disc, which allows us to obtain high-resolution images throughout the depth of this folded epithelium.

Regulation of Dpp activity by tissue-specific cleavage of an upstream site within the prodomain.

BMP4 is synthesized as an inactive precursor that is cleaved at two sites during maturation: initially at a site (S1) adjacent to the ligand domain, and then at an upstream site (S2) within the prodomain. Cleavage at the second site regulates the stability of mature BMP4 and this in turn influences its signaling intensity and range of action. The Drosophila ortholog of BMP4, Dpp, functions as a long- or short-range signaling molecule in the wing disc or embryonic midgut, respectively but mechanisms that differentially regulate its bioactivity in these tissues have not been explored. In the current studies we demonstrate, by dpp mutant rescue, that cleavage at the S2 site of proDpp is required for development of the wing and leg imaginal discs, whereas cleavage at the S1 site is sufficient to rescue Dpp function in the midgut. Both the S1 and S2 sites of proDpp are cleaved in the wing disc, and S2-cleavage is essential to generate sufficient ligand to exceed the threshold for pMAD activation at both short- and long-range in most cells. By contrast, proDpp is cleaved at the S1 site alone in the embryonic mesoderm and this generates sufficient ligand to activate physiological target genes in neighboring cells. These studies provide the first biochemical and genetic evidence that selective cleavage of the S2 site of proDPP provides a tissue-specific mechanism for regulating Dpp activity, and that differential cleavage can contribute to, but is not an absolute determinant of signaling range.

In vivo analysis in Drosophila reveals differential requirements of contact residues in Axin for interactions with GSK3beta or beta-catenin.

Proper regulation of the Wingless/Wnt signaling pathway is essential for normal development. The scaffolding protein Axin plays a key role in this process through interactions with Drosophila Shaggy and Armadillo. In the current studies, we used a yeast two-hybrid assay to identify ten amino acids in Axin that are critical for in vitro interaction with Shaggy and two for interaction with Armadillo. We then generated five Axin variants in which individual putative contact amino acids were mutated and compared their activity, as assayed by rescue of axin null mutant flies, to that of Axin lacking the entire Shaggy (AxinDeltaSgg) or Armadillo (AxinDeltaArm) binding domain. Although we expected these mutants to function identically to Axin in which the entire binding domain was deleted, we instead observed a spectrum of phenotypic rescue. Specifically, two point mutants within the Shaggy binding domain showed loss of activity similar to that of AxinDeltaSgg and dominantly interfered with complex function, whereas a third mutant allele, AxinK446E, retained most function. Two Axin point mutants within the Armadillo binding domain were weak alleles and retained most function. These findings demonstrate the importance of in vivo verification of the role of specific amino acids within a protein.

Unexpectedly robust assembly of the Axin destruction complex regulates Wnt/Wg signaling in Drosophila as revealed by analysis in vivo.

Secreted proteins in the Wnt family regulate gene expression in target cells by causing the accumulation of the transcriptional activator beta-catenin. In the absence of Wnt, a protein complex assembled around the scaffold protein Axin targets beta-catenin for destruction, thereby preventing it from transducing inappropriate signals. Loss of Axin or its binding partners APC and GSK3 results in aberrant activation of the Wnt signaling response. We have analyzed the effects of mutant forms of Drosophila Axin with large internal deletions when expressed at physiological levels in vivo, either in the presence or absence of wild type Axin. Surprisingly, even deletions that completely remove the binding sites for fly APC, GSK3 or beta-catenin, though they fail to rescue to viability, these mutant forms of Axin cause only mild developmental defects, indicating largely retained Axin function. Furthermore, two lethal Axin deletion constructs, AxinDeltaRGS and AxinDeltabeta cat(DeltaArm), can complement each other and restore viability. Our findings support a model in which the Axin complex is assembled through cooperative tripartite interactions among the binding partners, making the assembly of functional complexes surprisingly robust.

Wingless/Wnt signal transduction requires distinct initiation and amplification steps that both depend on Arrow/LRP.

Members of the Wg/Wnt family provide key intercellular signals during embryonic development and in the maintenance of homeostatic processes, but critical aspects of their signal transduction pathways remain controversial. We have found that canonical Wg signaling in Drosophila involves distinct initiation and amplification steps, both of which require Arrow/LRP. Expressing a chimeric Frizzled2-Arrow protein in flies that lack endogenous Wg or Arrow showed that this construct functions as an activated Wg receptor but is deficient in signal amplification. In contrast, a chimeric Arrow protein containing the dimerization domain of Torso acted as a potent amplifier of Wg signaling but could not initiate Wg signaling on its own. The two chimeric proteins synergized, so that their co-expression largely reconstituted the signaling levels achieved by expressing Wg itself. The amplification function of Arrow/LRP appears to be particularly important for long-range signaling, and may reflect a general mechanism for potentiating signals in the shallow part of a morphogen gradient.

Endocytic trafficking of Wingless and its receptors, Arrow and DFrizzled-2, in the Drosophila wing.

During animal development, Wnt/Wingless (Wg) signaling is required for the patterning of multiple tissues. While insufficient signal transduction is detrimental to normal development, ectopic activation of the pathway can be just as devastating. Thus, numerous controls exist to precisely regulate Wg signaling levels. Endocytic trafficking of pathway components has recently been proposed as one such control mechanism. Here, we characterize the vesicular trafficking of Wg and its receptors, Arrow and DFrizzled-2 (DFz2), and investigate whether trafficking is important to regulate Wg signaling during dorsoventral patterning of the larval wing. We demonstrate a role for Arrow and DFz2 in Wg internalization. Subsequently, Wg, Arrow and DFz2 are trafficked through the endocytic pathway to the lysosome, where they are degraded in a hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs)-dependent manner. Surprisingly, we find that Wg signaling is not attenuated by lysosomal targeting in the wing disc. Rather, we suggest that signaling is dampened intracellularly at an earlier trafficking step. This is in contrast to patterning of the embryonic epidermis, where lysosomal targeting is required to restrict the range of Wg signaling. Thus, signal modulation by endocytic routing will depend on the tissue to be patterned and the goals during that patterning event.

Wg/Wnt signal can be transmitted through arrow/LRP5,6 and Axin independently of Zw3/Gsk3beta activity.

Activation of the Wnt signaling cascade provides key signals during development and in disease. Here we provide evidence, by designing a Wnt receptor with ligand-independent signaling activity, that physical proximity of Arrow (LRP) to the Wnt receptor Frizzled-2 triggers the intracellular signaling cascade. We have uncovered a branch of the Wnt pathway in which Armadillo activity is regulated concomitantly with the levels of Axin protein. The intracellular pathway bypasses Gsk3beta/Zw3, the kinase normally required for controlling beta-catenin/Armadillo levels, suggesting that modulated degradation of Armadillo is not required for Wnt signaling. We propose that Arrow (LRP) recruits Axin to the membrane, and that this interaction leads to Axin degradation. As a consequence, Armadillo is no longer bound by Axin, resulting in nuclear signaling by Armadillo.