Regulation of distinct branches of the non-canonical Wnt-signaling network in Xenopus dorsal marginal zone explants.

BACKGROUND: A tight regulation of the Wnt-signaling network, activated by 19 Wnt molecules and numerous receptors and co-receptors, is required for the establishment of a complex organism. Different branches of this Wnt-signaling network, including the canonical Wnt/ß-catenin and the non-canonical Wnt/PCP, Wnt/Ror2 and Wnt/Ca(2+) pathways, are assigned to distinct developmental processes and are triggered by certain ligand/receptor complexes. The Wnt-signaling molecules are closely related and it is still on debate whether the information for activating a specific branch is encoded by specific sequence motifs within a particular Wnt protein. The model organism Xenopus offers tools to distinguish between Wnt-signaling molecules activating distinct branches of the network. RESULTS: We created chimeric Wnt8a/Wnt11 molecules and could demonstrate that the C-terminal part (containing the BS2) of Wnt8a is responsible for secondary axis formation. Chimeric Wnt11/Wnt5a molecules revealed that the N-terminus with the elements PS3-1 and PS3-2 defines Wnt11 specificity, while elements PS3-1, PS3-2 and PS3-3 are required for Wnt5a specificity. Furthermore, we used Xenopus dorsal marginal zone explants to identify non-canonical Wnt target genes regulated by the Wnt5a branch and the Wnt11 branch. We found that pbk was specifically regulated by Wnt5a and rab11fip5 by Wnt11. Overexpression of these target genes phenocopied the overexpression of their regulators, confirming the distinct roles of Wnt11 and Wnt5a triggered signaling pathways. Furthermore, knock-down of pbk was able to restore convergent extension movements in Wnt5a morphants. CONCLUSIONS: The N-terminal part of non-canonical Wnt proteins decides whether the Wnt5a or the Wnt11 branch of the Wnt-signaling network gets activated. The different non-canonical Wnt branches not only regulate cellular behavior, but, surprisingly, also regulate the expression of different target genes. One of these target genes, pbk, seems to be the relevant target gene executing Wnt5a-mediated regulation of convergent extension movements.

Live imaging of Xwnt5A-ROR2 complexes.

Secreted molecules of the Wnt family regulate key decisions in embryogenesis and adult tissue homeostasis by activating a complex network of Wnt signaling pathways. Although the different branches of Wnt signaling have been studied for more than 25 years, fluorophore tagged constructs for live cell imaging of Wnt molecules activating the Wnt/ß-catenin pathway have become available only recently. We have generated a fluorophore tagged Wnt construct of the Xenopus Wnt5a protein (Xwnt5A) with the enhanced green fluorescent protein (EGFP), Xwnt5A-EGFP. This construct activates non-canonical Wnt pathways in an endocytosis dependent manner and is capable of compensating for the loss of endogenous Xwnt5A in Xenopus embryos. Strikingly, non-canonical Wnt pathway activation was restricted to short-range signaling while an inhibitory effect was observed in transwell cell cultures taken as long-range signaling model sytem. We used our Xwnt5A-EGFP construct to analyze in vivo binding of Wnt5A to its co-receptor ROR2 on the microscopic and on the molecular level. On the microscopic level, Xwnt5A-EGFP clusters in the membrane and recruits ROR2-mCherry to these clusters. Applying dual-colour dual-focus line-scanning fluorescence correlation spectroscopy on dorsal marginal zone explants, we identified membrane tethered Xwnt5A-EGFP molecules binding to ROR2-mCherry molecules. Our data favour a model, in which membrane-tethered Wnt-5A recruits ROR2 to form large ligand/receptor clusters and signals in an endocytosis-dependent manner.

Molecular dissection of Wnt3a-Frizzled8 interaction reveals essential and modulatory determinants of Wnt signaling activity.

BACKGROUND: Wnt proteins are a family of secreted signaling molecules that regulate key developmental processes in metazoans. The molecular basis of Wnt binding to Frizzled and LRP5/6 co-receptors has long been unknown due to the lack of structural data on Wnt ligands. Only recently, the crystal structure of the Wnt8-Frizzled8-cysteine-rich-domain (CRD) complex was solved, but the significance of interaction sites that influence Wnt signaling has not been assessed. RESULTS: Here, we present an extensive structure-function analysis of mouse Wnt3a in vitro and in vivo. We provide evidence for the essential role of serine 209, glycine 210 (site 1) and tryptophan 333 (site 2) in Fz binding. Importantly, we discovered that valine 337 in the site 2 binding loop is critical for signaling without contributing to binding. Mutations in the presumptive second CRD binding site (site 3) partly abolished Wnt binding. Intriguingly, most site 3 mutations increased Wnt signaling, probably by inhibiting Wnt-CRD oligomerization. In accordance, increasing amounts of soluble Frizzled8-CRD protein modulated Wnt3a signaling in a biphasic manner. CONCLUSIONS: We propose a concentration-dependent switch in Wnt-CRD complex formation from an inactive aggregation state to an activated high mobility state as a possible modulatory mechanism in Wnt signaling gradients.

Synthesis of novel inhibitors blocking Wnt signaling downstream of ß-catenin.

Large scale screening of libraries consisting of natural and small molecules led to the identification of many small molecule inhibitors repressing Wnt/ß-Catenin signaling. However, targeted synthesis of novel Wnt pathway inhibitors has been rarely described. We developed a modular and expedient way to create the aromatic ring system with an aliphatic ring in between. Our synthesis opens up the possibility, in principle, to substitute all positions at the ring system with any desired substituent. Here, we tested five different haloquinone analogs carrying methoxy- and hydroxy-groups at different positions. Bona fide Wnt activity assays in cell culture and in Xenopus embryos revealed that two of these compounds act as potent inhibitors of aberrant activated Wnt/ß-Catenin signaling.

Live imaging of active fluorophore labelled Wnt proteins.

For almost 30 years, Wnt proteins have been known as key regulators of many developmental decisions, including the formation of the embryonic axes, patterning of the CNS, limb bud outgrowth and segment polarity. However, live cell imaging of active Wnt proteins was rarely reported. Here, we have generated a Wnt2b-EGFP fusion protein that retains functionality in bona fide Wnt activity assays, although the secreted protein is rapidly cleaved by extracellular proteases. We can show with this new tool that Wnt2b-EGFP moves along the microtubules of Wnt producing cells and that this directed movement is essential for the secretion of active Wnt protein.