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The Armadillo family is formed by proteins which possess an Arm domain comprising multiple copies of a 42-amino-acid motif, the Arm repeat, initially described for the Drosophila segment polarity gene product Armadillo. The Arm domain serves in protein-protein interactions which are required for the family members Armadillo, β-catenin and plakoglobin to mediate cell-cell adhesion and Wnt/Wingless signalling. Similarily, p120 cas, the Arm domain containing src substrate, also binds to cadherins and becomes tyrosine phosphorylated in response to a variety of stimuli. However, a putative function of p120 cas in adhesion or signalling has not yet been demonstrated. It has also not been shown until now that an Arm domain is a common signal transduction motif. Using Xenopus embryos we show by expression of murine p120 cas1B (mp120 cas1B) in ventral blastomeres that this catenin cannot replace β-catenin function in dorsal axis formation. Thus, the presence of an Arm domain per se is not sufficient to activate the Wnt/Wg pathway. Indeed, injection of mp120 cas1B into dorsal blastomeres led instead to delayed blastopore closure and posteriorized phenotypes with malformed head structures indicative of disturbed gastrulation movements. Because neither convergent extension behaviour nor adhesion to fibronectin was altered in the injected embryos we assume that mp120 cas1B influences motility or orientation of migrating mesodermal cells. [ABSTRACT FROM AUTHOR]

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/β-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.

The cranial neural crest (CNC) is a highly motile and multipotent embryonic cell population, which migrates directionally on defined routes throughout the embryo, contributing to facial structures including cartilage, bone and ganglia. Cadherin-mediated cell-cell adhesion is known to play a crucial role in the directional migration of CNC cells. However, migrating CNC co-express different cadherin subtypes, and their individual roles have yet to be fully explored. In previous studies, the expression of individual cadherin subtypes has been analysed using different methods with varying sensitivities, preventing the direct comparison of expression levels. Here, we provide the first comprehensive and comparative analysis of the expression of six cadherin superfamily members during different phases of CNC cell migration in Xenopus. By applying a quantitative RT-qPCR approach, we can determine the copy number and abundance of each expressed cadherin through different phases of CNC migration. Using this approach, we show for the first time expression of E-cadherin and XB/C-cadherin in CNC cells, adding them as two new members of cadherins co-expressed during CNC migration. Cadherin co-expression during CNC migration in Xenopus, in particular the constant expression of E-cadherin, contradicts the classical epithelial-mesenchymal transition (EMT) model postulating a switch in cadherin expression. Loss-of-function experiments further show that E-cadherin is required for proper CNC cell migration in vivo and also for cell protrusion formation in vitro. Knockdown of E-cadherin is not rescued by co-injection of other classical cadherins, pointing to a specific function of E-cadherin in mediating CNC cell migration. Finally, through reconstitution experiments with different E-cadherin deletion mutants in E-cadherin morphant embryos, we demonstrate that the extracellular domain, but not the cytoplasmic domain, of E-cadherin is sufficient to rescue CNC cell migration in vivo., (Copyright © 2016 Elsevier Inc. All rights reserved.)

A complex network of transcription factors regulates specification of neural crest cells at early neurula stage by stabilizing neural crest identity and activating neural crest effector genes so that distinct subpopulations evolve. In this network, c-myc acts on top of the gene hierarchy controlling snail2, AP2 and prohibitin1 (phb1) expression. While snail2 and AP2 are well studied neural crest specifier genes little is known about the role of phb1 in this process. To identify phb1 regulated genes we analyzed the transcriptome of neural crest explants of phb1 morphant Xenopus embryos. Among 147 phb1 regulated genes we identified the membrane-associated protein-tyrosine phosphatase PRP4A3 (prl3) and the atypical cadherin and Wnt-PCP component van gogh like1 (vangl1). Gain of function, loss of function and epistasis experiments allowed us to allocate both genes in the neural crest specification network between phb1 and twist. Interestingly, both, vangl1 and prl3 regulate only a small subset of neural crest marker genes. The identification of two membrane-associated proteins as novel neural crest specifiers indicates that in addition to gene regulation by combinatory effects of transcription factors also post-translational modifications (prl3) and cell-cell adhesion and/or regulation of cell-polarity (vangl1) specify the identity of neural crest cell populations. genesis 53:627-639, 2015. © 2015 Wiley Periodicals, Inc., (© 2015 Wiley Periodicals, Inc.)

AFM-based force spectroscopy in combination with optical microscopy is a powerful tool for investigating cell mechanics and adhesion on the single cell level. However, standard setups featuring an AFM mounted on an inverted light microscope only provide a bottom view of cell and AFM cantilever but cannot visualize vertical cell shape changes, for instance occurring during motile membrane blebbing. Here, we have integrated a mirror-based sideview system to monitor cell shape changes resulting from motile bleb behavior of Xenopus cranial neural crest (CNC) cells during AFM elasticity and adhesion measurements. Using the sideview setup, we quantitatively investigate mechanical changes associated with bleb formation and compared cell elasticity values recorded during membrane bleb and non-bleb events. Bleb protrusions displayed significantly lower stiffness compared to the non-blebbing membrane in the same cell. Bleb stiffness values were comparable to values obtained from blebbistatin-treated cells, consistent with the absence of a functional actomyosin network in bleb protrusions. Furthermore, we show that membrane blebs forming within the cell-cell contact zone have a detrimental effect on cell-cell adhesion forces, suggesting that mechanical changes associated with bleb protrusions promote cell-cell detachment or prevent adhesion reinforcement. Incorporating a sideview setup into an AFM platform therefore provides a new tool to correlate changes in cell morphology with results from force spectroscopy experiments.

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.

E-cadherin is a key cell-cell adhesion molecule but the impact of receptor density and the precise contribution of individual cadherin ectodomains in promoting cell adhesion are only incompletely understood. Investigating these mechanisms would benefit from artificial adhesion substrates carrying different cadherin ectodomains at defined surface density. We therefore developed a quantitative E-cadherin surface immobilization protocol based on the SNAP-tag technique. Extracellular (EC) fragments of E-cadherin fused to the SNAP-tag were covalently bound to self-assembled monolayers (SAM) of thiols carrying benzylguanine (BG) head groups. The adhesive functionality of the different E-cadherin surfaces was then assessed using cell spreading assays and single-cell (SCSF) and single-molecule (SMSF) force spectroscopy. We demonstrate that an E-cadherin construct containing only the first and second outmost EC domain (E1-2) is not sufficient for mediating cell adhesion and yields only low single cadherin-cadherin adhesion forces. In contrast, a construct containing all five EC domains (E1-5) efficiently promotes cell spreading and generates strong single cadherin and cell adhesion forces. By varying the concentration of BG head groups within the SAM we determined a lateral distance of 5-11 nm for optimal E-cadherin functionality. Integrating the results from SCMS and SMSF experiments furthermore demonstrated that the dissolution of E-cadherin adhesion contacts involves a sequential unbinding of individual cadherin receptors rather than the sudden rupture of larger cadherin receptor clusters. Our method of covalent, oriented and density-controlled E-cadherin immobilization thus provides a novel and versatile platform to study molecular mechanisms underlying cadherin-mediated cell adhesion under defined experimental conditions.