The Rho guanine nucleotide exchange factor Trio is required for neural crest cell migration and interacts with Dishevelled.

Directional migration during embryogenesis and tumor progression faces the challenge that numerous external signals need to converge to precisely control cell movement. The Rho guanine exchange factor (GEF) Trio is especially well suited to relay signals, as it features distinct catalytic domains to activate Rho GTPases. Here, we show that Trio is required for Xenopus cranial neural crest (NC) cell migration and cartilage formation. Trio cell-autonomously controls protrusion formation of NC cells and Trio morphant NC cells show a blebbing phenotype. Interestingly, the Trio GEF2 domain is sufficient to rescue protrusion formation and migration of Trio morphant NC cells. We show that this domain interacts with the DEP/C-terminus of Dishevelled (DVL). DVL - but not a deletion construct lacking the DEP domain - is able to rescue protrusion formation and migration of Trio morphant NC cells. This is likely mediated by activation of Rac1, as we find that DVL rescues Rac1 activity in Trio morphant embryos. Thus, our data provide evidence for a novel signaling pathway, whereby Trio controls protrusion formation of cranial NC cells by interacting with DVL to activate Rac1.

E-cadherin is required for cranial neural crest migration in Xenopus laevis.

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.

Evolution of the Rho guanine nucleotide exchange factors Kalirin and Trio and their gene expression in Xenopus development.

Guanine nucleotide exchange factors (GEFs) activate Rho GTPases by accelerating their GDP/GTP exchange. Trio and its paralog Kalirin (Kalrn) are unique members of the Rho-GEFs that harbor three catalytic domains: two functional GEF domains and a serine/threonine kinase domain. The N-terminal GEF domain activates Rac1 and RhoG GTPases, while the C-terminal GEF domain acts specifically on RhoA. Trio and Kalrn have an evolutionary conserved function in morphogenetic processes including neuronal development. De novo mutations in TRIO have lately been identified in patients with intellectual disability, suggesting that this protein family plays an important role in development and disease. Phylogenetic and domain analysis revealed that a Kalrn/Trio ancestor originated in Prebilateria and duplicated in Urbilateria to yield Kalrn and Trio. Only few taxa outside the vertebrates retained both of these highly conserved proteins. To obtain first insights into their redundant or distinct functions in a vertebrate model system, we show for the first time a detailed comparative analysis of trio and kalrn expression in Xenopus laevis development. The mRNAs are maternally transcribed and expression increases starting with neurula stages. Trio and kalrn are detected in mesoderm/somites and different neuronal populations in the neural plate/tube and later also in the brain. However, only trio is expressed in migrating neural crest cells, while kalrn expression is detected in the cranial nerves, suggesting distinct functions. Thus, our expression analysis provides a good basis for further functional studies.

Lef1 regulates caveolin expression and caveolin dependent endocytosis, a process necessary for Wnt5a/Ror2 signaling during Xenopus gastrulation.

The activation of distinct branches of the Wnt signaling network is essential for regulating early vertebrate development. Activation of the canonical Wnt/ß-catenin pathway stimulates expression of ß-catenin-Lef/Tcf regulated Wnt target genes and a regulatory network giving rise to the formation of the Spemann organizer. Non-canonical pathways, by contrast, mainly regulate cell polarization and migration, in particular convergent extension movements of the trunk mesoderm during gastrulation. By transcriptome analyses, we found caveolin1, caveolin3 and cavin1 to be regulated by Lef1 in the involuting mesoderm of Xenopus embryos at gastrula stages. We show that caveolins and caveolin dependent endocytosis are necessary for proper gastrulation, most likely by interfering with Wnt5a/Ror2 signaling. Wnt5a regulates the subcellular localization of receptor complexes, including Ror2 homodimers, Ror2/Fzd7 and Ror2/dsh heterodimers in an endocytosis dependent manner. Live-cell imaging revealed endocytosis of Ror2/caveolin1 complexes. In Xenopus explants, in the presence of Wnt5a, these receptor clusters remain stable exclusively at the basolateral side, suggesting that endocytosis of non-canonical Wnt/receptor complexes preferentially takes place at the apical membrane. In support of this blocking endocytosis with inhibitors prevents the effects of Wnt5a. Thus, target genes of Lef1 interfere with Wnt5a/Ror2 signaling to coordinate gastrulation movements.

Cadherin-11 localizes to focal adhesions and promotes cell-substrate adhesion.

Cadherin receptors have a well-established role in cell-cell adhesion, cell polarization and differentiation. However, some cadherins also promote cell and tissue movement during embryonic development and tumour progression. In particular, cadherin-11 is upregulated during tumour and inflammatory cell invasion, but the mechanisms underlying cadherin-11 stimulated cell migration are still incompletely understood. Here, we show that cadherin-11 localizes to focal adhesions and promotes adhesion to fibronectin in Xenopus neural crest, a highly migratory embryonic cell population. Transfected cadherin-11 also localizes to focal adhesions in different mammalian cell lines, while endogenous cadherin-11 shows focal adhesion localization in primary human fibroblasts. In focal adhesions, cadherin-11 co-localizes with ß1-integrin and paxillin and physically interacts with the fibronectin-binding proteoglycan syndecan-4. Adhesion to fibronectin mediated by cadherin-11/syndecan-4 complexes requires both the extracellular domain of syndecan-4, and the transmembrane and cytoplasmic domains of cadherin-11. These results reveal an unexpected role of a classical cadherin in cell-matrix adhesion during cell migration.

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.