Paraxial protocadherin coordinates cell polarity during convergent extension via Rho A and JNK

Convergent extension movements occur ubiquitously in animal development. This special type of cell movement is controlled by the Wnt/planar cell polarity (PCP) pathway. Here we show that Xenopus paraxial protocadherin (XPAPC) functionally interacts with the Wnt/PCP pathway in the control of convergence and extension (CE) movements in Xenopus laevis. XPAPC functions as a signalling molecule that coordinates cell polarity of the involuting mesoderm in mediolateral orientation and thus selectively promotes convergence in CE movements. XPAPC signals through the small GTPases Rho A and Rac 1 and c-jun N-terminal kinase (JNK). Loss of XPAPC function blocks Rho A-mediated JNK activation. Despite common downstream components, XPAPC and Wnt/PCP signalling are not redundant, and the activity of both, XPAPC and PCP signalling, is required to coordinate CE movements.     

Antagonistic regulation of convergent extension movements in Xenopus by Wnt/beta-catenin and Wnt/Ca2+ signaling

Convergent extension movements are the main driving force of Xenopus gastrulation. A fine-tuned regulation of cadherin-mediated cell-cell adhesion is thought to be required for this process. Members of the Wnt family of extracellular glycoproteins have been shown to modulate cadherin-mediated cell-cell adhesion, convergent extension movements, and cell differentiation. Here we show that endogenous Wnt/beta-catenin signaling activity is essential for convergent extension movements due to its effect on gene expression rather than on cadherins. Our data also suggest that XLEF-1 rather than XTCF-3 is required for convergent extension movements and that XLEF-1 functions in this context in the Wnt/beta-catenin pathway to regulate Xnr-3. In contrast, activation of the Wnt/Ca2+ pathway blocks convergent extension movements, with potential regulation of the Wnt/beta-catenin pathway at two different levels. PKC, activated by the Wnt/Ca2+ pathway, blocks the Wnt/beta-catenin pathway upstream of beta-catenin and phosphorylates Dishevelled. CamKII, also activated by the Wnt/Ca2+ pathway, inhibits the Wnt/beta-catenin signaling cascade downstream of beta-catenin. Thus, an opposing cross-talk of two distinct Wnt signaling cascades regulates convergent extension movements in Xenopus.     

Ror2 Receptor Mediates Wnt11 Ligand Signaling and Affects Convergence and Extension Movements in Zebrafish

The receptor-tyrosine kinase Ror2 acts as an alternative receptor or co-receptor for Wnt5a and mediates Wnt5a-induced convergent extension movements during embryogenesis in mice and Xenopus as well as the polarity and migration of several cell types during development. However, little is known about whether Ror2 function is conserved in other vertebrates or is involved in other non-canonical Wnt ligands in vivo. In this study we demonstrated that overexpression of dominant-negative ror2 (ror2-TM) mRNA in zebrafish embryos resulted in convergence and extension defects and incompletely separated eyes, which is consistent with observations from slb/wnt11 mutants or wnt11 knockdown morphants. Moreover, the co-injection of ror2-TM mRNA and a wnt11 morpholino or the coexpression of ror2 and wnt11 in zebrafish embryos synergetically induced more severe convergence and extension defects. Transplantation studies further demonstrated that the Ror2 receptor responded to the Wnt11 ligand and regulated cell migration and cell morphology during gastrulation. DnRor2 inhibited the action of Wnt11, which was revealed by a decreased percentage of Wnt11-induced convergence and extension defects. Ror2 physically interacts with Wnt11. The intracellular Tyr-647 and Ser-863 sites of Ror2 are essential for mediating the action of Wnt11. Dishevelled and RhoA act downstream of Wnt11-Ror2 to regulate convergence and extension movements. Overall, our data suggest an important role of Ror2 in mediating Wnt11 signaling and in regulating convergence and extension movements in zebrafish.     

The prickle-related gene in vertebrates is essential for gastrulation cell movements

Involving dynamic and coordinated cell movements that cause drastic changes in embryo shape, gastrulation is one of the most important processes of early development. Gastrulation proceeds by various types of cell movements, including convergence and extension, during which polarized axial mesodermal cells intercalate in radial and mediolateral directions and thus elongate the dorsal marginal zone along the anterior-posterior axis [1,2]. Recently, it was reported that a noncanonical Wnt signaling pathway, which is known to regulate planar cell polarity (PCP) in Drosophila [3,4], participates in the regulation of convergent extension movements in Xenopus as well as in the zebrafish embryo [5-8]. The Wnt5a/Wnt11 signal is mediated by members of the seven-pass transmembrane receptor Frizzled (Fz) and the signal transducer Dishevelled (Dsh) through the Dsh domains that are required for the PCP signal [6-8]. It has also been shown that the relocalization of Dsh to the cell membrane is required for convergent extension movements in Xenopus gastrulae. Although it appears that signaling via these components leads to the activation of JNK [9,10] and rearrangement of microtubules, the precise interplay among these intercellular components is largely unknown. In this study, we show that Xenopus prickle (Xpk), a Xenopus homolog of a Drosophila PCP gene [11-13], is an essential component for gastrulation cell movement. Both gain-of-function and loss-of-function of Xpk severely perturbed gastrulation and caused spina bifida embryos without affecting mesodermal differentiation. We also demonstrate that XPK binds to Xenopus Dsh as well as to JNK. This suggests that XPK plays a pivotal role in connecting Dsh function to JNK activation.     

Wnt5a induces homodimerization and activation of Ror2 receptor tyrosine kinase

Wnts are secreted glycoproteins that control vital biological processes, including embryogenesis, organogenesis and tumorigenesis. Wnts are classified into several subfamilies depending on the signaling pathways they activate, with the canonical subfamily activating the Wnt/beta-catenin pathway and the non-canonical subfamily activating a variety of other pathways, including the Wnt/calcium signaling and the small GTPase/c-Jun NH2-terminal kinase pathway. Wnts bind to a membrane receptor Frizzled and a co-receptor, the low-density lipoprotein receptor related protein. More recently, both canonical and non-canonical Wnts were shown to bind the Ror2 receptor tyrosine kinase. Ror2 is an orphan receptor that plays crucial roles in skeletal morphogenesis and promotes osteoblast differentiation and bone formation. Here we examine the effects of a canonical Wnt3a and a non-canonical Wnt5a on the signaling of the Ror2 receptor. We demonstrate that even though both Wnt5a and Wnt3a bound Ror2, only Wnt5a induced Ror2 homo-dimerization and tyrosine phosphorylation in U2OS human osteoblastic cells. Furthermore, Wnt5a treatment also resulted in increased phosphorylation of the Ror2 substrate, 14-3-3beta scaffold protein, indicating that Wnt5a binding causes activation of the Ror2 signaling cascade. Functionally, Wnt5a recapitulated the Ror2 activation phenotype, enhancing bone formation in the mouse calvarial bone explant cultures and potentiating osteoblastic differentiation of human mesenchymal stem cells. The effect of Wnt5a on osteoblastic differentiation was largely abolished upon Ror2 down-regulation. Thus we show that Wnt5a activates the classical receptor tyrosine kinase signaling cascade through the Ror2 receptor in cells of osteoblastic origin.     

Activation of Gbetagamma signaling downstream of Wnt-11/Xfz7 regulates Cdc42 activity during Xenopus gastrulation

Wnt-11/Xfz7 signaling plays a major role in the regulation of convergent extension movements affecting the dorsal marginal zone (DMZ) of gastrulating Xenopus embryos. In order to provide data concerning the molecular targets of Wnt-11/Xfz7 signals, we have analyzed the regulation of the Rho GTPase Cdc42 by Wnt-11. In animal cap ectoderm, Cdc42 activity increases as a response to Wnt-11 expression. This increase is inhibited by pertussis toxin, or sequestration of free Gbetagamma subunits by exogenous Galphai2 or Galphat. Activation of Cdc42 is also produced by the expression of bovine Gbeta1 and Ggamma2. This process is abolished by a PKC inhibitor, while phorbol esther treatment of ectodermal explants activates Cdc42 in a PKC-dependent way, implicating PKC downstream of Gbetagamma. In activin-treated animal caps and in the embryo, interference with Gbetagamma signaling rescues morphogenetic movements inhibited by Wnt-11 hyperactivation, thus phenocopying the dominant negative version of Cdc42 (N(17)Cdc42). Conversely, expression of Gbeta1gamma2 blocks animal cap elongation. This effect is reversed by N(17)Cdc42. Together, our results strongly argue for a role of Gbetagamma signaling in the regulation of Cdc42 activity downstream of Wnt-11/Xfz7 in mesodermal cells undergoing convergent extension. This idea is further supported by the observation that expression of Galphat in the DMZ causes severe gastrulation defects.     

Role of glypican 4 in the regulation of convergent extension movements during gastrulation in Xenopus laevis

Coordinated morphogenetic cell movements during gastrulation are crucial for establishing embryonic axes in animals. Most recently, the non-canonical Wnt signaling cascade (PCP pathway) has been shown to regulate convergent extension movements in Xenopus and zebrafish. Heparan sulfate proteoglycans (HSPGs) are known as modulators of intercellular signaling, and are required for gastrulation movements in vertebrates. However, the function of HSPGs is poorly understood. We analyze the function of Xenopus glypican 4 (Xgly4), which is a member of membrane-associated HSPG family. In situ hybridization revealed that Xgly4 is expressed in the dorsal mesoderm and ectoderm during gastrulation. Reducing the levels of Xgly4 inhibits cell-membrane accumulation of Dishevelled (Dsh), which is a transducer of the Wnt signaling cascade, and thereby disturbs cell movements during gastrulation. Rescue analysis with different Dsh mutants and Wnt11 demonstrated that Xgly4 functions in the non-canonical Wnt/PCP pathway, but not in the canonical Wnt/beta-catenin pathway, to regulate gastrulation movements. We also provide evidence that the Xgly4 protein physically binds Wnt ligands. Therefore, our results suggest that Xgly4 functions as positive regulator in non-canonical Wnt/PCP signaling during gastrulation.     

Ryk cooperates with Frizzled 7 to promote Wnt11-mediated endocytosis and is essential for Xenopus laevis convergent extension movements

The single-pass transmembrane protein Ryk (atypical receptor related tyrosine kinase) functions as a Wnt receptor. However, Ryk's correlation with Wnt/Frizzled (Fz) signaling is poorly understood. Here, we report that Ryk regulates Xenopus laevis convergent extension (CE) movements via the β-arrestin 2 (βarr2)-dependent endocytic process triggered by noncanonical Wnt signaling. During X. laevis gastrulation, βarr2-mediated endocytosis of Fz7 and dishevelled (Dvl/Dsh) actually occurs in the dorsal marginal zone tissues, which actively participate in noncanonical Wnt signaling. Noncanonical Wnt11/Fz7-mediated endocytosis of Dsh requires the cell-membrane protein Ryk. Ryk interacts with both Wnt11 and βarr2, cooperates with Fz7 to mediate Wnt11-stimulated endocytosis of Dsh, and signals the noncanonical Wnt pathway in CE movements. Conversely, depletion of Ryk and Wnt11 prevents Dsh endocytosis in dorsal marginal zone tissues. Our study suggests that Ryk functions as an essential regulator for noncanonical Wnt/Fz-mediated endocytosis in the regulation of X. laevis CE movements.     

Filopodia formation mediated by receptor tyrosine kinase Ror2 is required for Wnt5a-induced cell migration

The receptor tyrosine kinase Ror2 plays important roles in developmental morphogenesis. It has recently been shown that Ror2 mediates Wnt5a-induced noncanonical Wnt signaling by activating the Wnt-JNK pathway and inhibiting the beta-catenin-TCF pathway. However, the function of Ror2 in noncanonical Wnt signaling leading to cell migration is largely unknown. We show, using genetically different or manipulated cultured cells, that Ror2 is critical for Wnt5a-induced, but not Wnt3a-induced, cell migration. Ror2-mediated cell migration requires the extracellular cysteine-rich domain (CRD), which is the binding site for Wnt5a, and the cytoplasmic proline-rich domain (PRD) of Ror2. Furthermore, Ror2 can mediate filopodia formation via actin reorganization, irrespective of Wnt5a, and this Ror2-mediated filopodia formation requires the actin-binding protein filamin A, which associates with the PRD of Ror2. Intriguingly, disruption of filopodia formation by suppressing the expression of either Ror2 or filamin A inhibits Wnt5a-induced cell migration, indicating that Ror2-mediated filopodia formation is essential for Wnt5a-induced cell migration.     

Wnt5a/Ror2-induced upregulation of xPAPC requires xShcA

Ror receptor-tyrosine kinases act as Wnt-5a receptors in beta-catenin independent Wnt-signaling pathways. In Xenopus, expression of xPAPC is regulated by a Wnt-5a/Ror2 pathway, which resembles typical signaling cascades downstream of receptor-tyrosine kinases. Here, we have identified the phospho-tyrosine binding protein ShcA as an intracellular binding partner of Ror2. ShcA binds to a conserved motif in Ror2 via its SH2-domain. Wnt-5a induces clustering of Ror2 in the cell membrane and recruitment of ShcA to the Ror2 receptor complex. We further show that ShcA is co-expressed with Ror2 in developing Xenopus embryos and ShcA is required for Wnt-5a/Ror2 mediated upregulation of xPAPC, demonstrating the functional relevance of this interaction.     

Frizzled receptors activate a novel JNK-dependent pathway that may lead to apoptosis.

Extracellular Wnt ligands and their receptors of the Frizzled family control cell fate, proliferation, and polarity during metazoan development. Frizzled signaling modulates target gene expression through a beta-catenin-dependent pathway, functions to establish planar cell polarity in Drosophila epithelia, and activates convergent extension movements and intracellular Ca(2+) signaling in frog and fish embryos. Here, we report that a Frizzled receptor, Xenopus Frizzled 8 (Xfz8), activates c-Jun N-terminal kinases (JNK) and triggers rapid apoptotic cell death in gastrulating Xenopus embryos. This activity of Xfz8 required the cytoplasmic tail of the receptor and was blocked by a dominant inhibitor of JNK. Moreover, the cytoplasmic tail of Xfz8 targeted to the membrane was sufficient for activation of JNK and apoptosis. The apoptotic signaling was shared by a specific subset of Frizzled receptors, was inhibited by Wnt5a, and occurred in a Dishevelled- and T cell factor (TCF)-independent manner. Thus, our experiments identify a novel Frizzled-dependent signaling pathway, which involves JNK and differs from the beta-catenin-dependent and convergent extension pathways.     

A novel role for a nodal-related protein; Xnr3 regulates convergent extension movements via the FGF receptor

Convergent extension behaviour is critical for the formation of the vertebrate body axis. In Xenopus, components of the Wnt signaling pathway have been shown to be required for convergent extension movements but the relationship between cell fate and morphogenesis is little understood. We show by loss of function analysis that Xnr3 activates Xbra expression through FGFR1. We show that eFGF activity is not essential in the pathway, and that dishevelled acts downstream of Xnr3 and not in a parallel pathway. We provide evidence for the involvement of the EGF-CFC protein FRL1, and suggest that the pro-domain of Xnr3 may be required for its activity. Since Xnr3 is a direct target of the maternal betacatenin/XTcf3 signaling pathway, it provides the link between the initial, maternally controlled, allocation of cell fate, and the morphogenetic movements of cells derived from the organizer.     

Increasingly complex: new players enter the Wnt signaling network

Wnt proteins can activate different intracellular signaling cascades in various organisms by interacting with receptors of the Frizzled family. The first identified Wnt signaling pathway, the Wnt/beta-catenin pathway, has been studied in much detail and is highly conserved among species. As to non-canonical Wnt pathways, the current situation is more nebulous partly because the intracellular mediators of this pathway are not yet fully understood and, in some cases, even identified. However, there are increasing data that prove the existence of non-canonical Wnt signaling and demonstrate its involvement in different developmental processes. In vertebrates, Wnt-11 and Wnt-5A can activate the Wnt/JNK pathway, which resembles the planar cell polarity pathway in Drosophila. The Wnt/Ca(2+)-pathway has only been described in Xenopus and zebrafish so far and it is unclear whether it also exists in other organisms. Two recent papers provide us with new insight into non-canonical Wnt signaling by (1) presenting a new intracellular mediator of non-canonical signaling in Xenopus1 and (2) implicating the existence of an additional non-canonical Wnt signaling pathway in flies.     

The planar polarity gene strabismus regulates convergent extension movements in Xenopus

The signaling mechanisms that specify, guide and coordinate cell behavior during embryonic morphogenesis are poorly understood. We report that a Xenopus homolog of the Drosophila planar cell polarity gene strabismus (stbm) participates in the regulation of convergent extension, a critical morphogenetic process required for the elongation of dorsal structures in vertebrate embryos. Overexpression of Xstbm, which is expressed broadly in early development and subsequently in the nervous system, causes severely shortened trunk structures; a similar phenotype results from inhibiting Xstbm translation using a morpholino antisense oligo. Experiments with Keller explants further demonstrate that Xstbm can regulate convergent extension in both dorsal mesoderm and neural tissue. The specification of dorsal tissues is not affected. The Xstbm phenotype resembles those obtained with several other molecules with roles in planar polarity signaling, including Dishevelled and Frizzled-7 and -8. Unlike these proteins, however, Stbm has little effect on conventional Wnt/beta-catenin signaling in either frog or fly assays. Thus our results strongly support the emerging hypothesis that a vertebrate analog of the planar polarity pathway governs convergent extension movements.     

Wnt signalling regulates paxillin ubiquitination essential for mesodermal cell motility

Gastrulation movements are critical for establishing the three germ layers and the architecture of vertebrate embryos. During Xenopus laevis gastrulation, mesodermal tissue migrates on the blastocoel roof and elongates along the antero-posterior axis. During this process, cells in the dorsal mesoderm are polarized and intercalate with each other, which is defined as convergent extension and is known to be regulated by the non-canonical Wnt pathway. Here, we show that paxillin plays an essential role in this process. Paxillin is a focal-adhesion associated protein implicated in the regulation of actin cytoskeletal organization and cell motility, but its role in Xenopus embryogenesis has not yet been clarified. We demonstrate that the Wnt pathway controls the ubiquitination and stability of paxillin, and that this regulatory mechanism is essential for convergent extension movements. We identified a RING finger protein XRNF185, which physically binds to paxillin and the proteasome. XRNF185 destabilizes paxillin at focal adhesions and promotes mesodermal cell migration during convergent extension. We propose a mechanism to regulate gastrulation movements that involves paxillin ubiquitination and stability controlled by Wnt signalling.     

Syndecan-4 regulates non-canonical Wnt signalling and is essential for convergent and extension movements in Xenopus embryos

Early shaping of Xenopus laevis embryos occurs through convergent and extension movements, a process that is driven by intercalation of polarized dorsal mesodermal cells and regulated by non-canonical Wnt signalling. Here, we have identified Xenopus syndecan-4 (xSyn4), a cell-surface transmembrane heparan sulphate proteoglycan. At the gastrula stage, xSyn4 is expressed in the involuting dorsal mesoderm and the anterior neuroectoderm. Later, it is found in the pronephros, branchial arches, brain and tailbud. Both gain- and loss-of-function of xSyn4 impaired convergent extension movements in Xenopus embryos and in activin-treated ectodermal explants. xSyn4 interacts functionally and biochemically with the Wnt receptor Frizzled7 (xFz7) and its signal transducer Dishevelled (xDsh). Furthermore, xSyn4 is necessary and sufficient for translocation of xDsh to the plasma membrane - a landmark in the activation of non-canonical Wnt signalling. Our results suggest that the ability of xSyn4 to translocate xDsh is regulated by fibronectin, a component of the extracellular matrix required for proper convergent extension movements. We propose a model where xSyn4 and fibronectin cooperate with xFz7 and Wnt in the specific activation of the non-canonical Wnt pathway.