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Updated: Apr 22, 2026
Modeling Paracrine Noncanonical Wnt Signaling In Vitro
Published on: December 10, 2021
Live imaging of Xwnt5A-ROR2 complexes
Veronika Wallkamm1, Rene Dörlich2, Karolin Rahm1
1Zoological Institute, Department of Cell and Developmental Biology, Karlsruhe Institute of Technology, Karlsruhe, Germany.
Abstract:
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/β-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.
Insights
Researchers developed a fluorescent Xenopus Wnt5a protein (Xwnt5A-EGFP) construct to study Wnt signaling. This tool revealed that Wnt5A activates non-canonical pathways via short-range, endocytosis-dependent signaling and binds to its co-receptor ROR2.
Area of Science:
- Developmental Biology
- Cell Signaling
- Molecular Imaging
Background:
- Wnt family proteins are crucial for embryonic development and tissue maintenance, acting through complex signaling pathways.
- Live imaging of Wnt pathway activation, particularly the Wnt/β-catenin pathway, has been limited until the recent development of tagged Wnt constructs.
- Understanding Wnt5a's role in non-canonical signaling and its interaction with co-receptors is essential for deciphering developmental processes.
Purpose of the Study:
- To generate a functional, fluorescently tagged Xenopus Wnt5a (Xwnt5A-EGFP) construct for live cell imaging of Wnt signaling.
- To investigate the range and mechanism of non-canonical Wnt pathway activation by Xwnt5A.
- To analyze the in vivo interaction between Wnt5a and its co-receptor ROR2 at the molecular and microscopic levels.
Main Methods:
- Generation of a fluorophore-tagged Xenopus Wnt5a construct (Xwnt5A-EGFP).
- Assessing Xwnt5A-EGFP functionality in Xenopus embryos and transwell cell cultures.
- Utilizing dual-color fluorescence correlation spectroscopy (FCS) for in vivo molecular interaction analysis.
Main Results:
- Xwnt5A-EGFP activates non-canonical Wnt pathways in an endocytosis-dependent manner and can rescue endogenous Xwnt5A function.
- Non-canonical Wnt pathway activation by Xwnt5A was observed to be short-range signaling, with inhibitory effects noted in long-range models.
- Xwnt5A-EGFP forms membrane clusters that recruit ROR2-mCherry, indicating direct binding and co-localization.
Conclusions:
- The Xwnt5A-EGFP construct is a valuable tool for studying Wnt5a signaling dynamics in live systems.
- Wnt5a signaling operates via short-range, endocytosis-dependent mechanisms, involving the formation of ligand/receptor clusters with ROR2.
- These findings support a model where membrane-tethered Wnt5a recruits ROR2 to initiate signaling.
