Coactivation of Rac and Rho by Wnt/Frizzled signaling is required for vertebrate gastrulation

Wnt/Frizzled (Fz) signaling controls cell polarity/movements during vertebrate gastrulation via incompletely defined mechanisms. We demonstrated previously that Wnt/Fz activation of Rho, a GTPase and regulator of cytoskeletal architecture, is essential for vertebrate gastrulation. Here we report that in mammalian cells and Xenopus embryos, Wnt/Fz signaling coactivates Rho and Rac, another GTPase and distinct regulator of cytoskeletal architecture. Wnt/Fz activation of Rac is independent of Rho and mediates Wnt/Fz activation of Jun N-terminal kinase (JNK). Dishevelled (Dvl), a cytoplasmic protein downstream of Fz, forms a Wnt-induced complex with Rac independent of the Wnt-induced Dvl-Rho complex. Depletion or inhibition of Rac function perturbs Xenopus gastrulation without affecting Wnt/Fz activation of the Rho or beta-catenin pathway. We propose that parallel activation of Rac and Rho pathways by Wnt/Fz signaling is required for cell polarity and movements during vertebrate gastrulation.     

PKC delta is essential for Dishevelled function in a noncanonical Wnt pathway that regulates Xenopus convergent extension movements

Protein kinase C (PKC) has been implicated in the Wnt signaling pathway; however, its molecular role is poorly understood. We identified novel genes encoding delta-type PKC in the Xenopus EST databases. Loss of PKC delta function revealed that it was essential for convergent extension during gastrulation. We then examined the relationship between PKC delta and the Wnt pathway. PKC delta was translocated to the plasma membrane in response to Frizzled signaling. In addition, loss of PKC delta function inhibited the translocation of Dishevelled and the activation of c-Jun N-terminal kinase (JNK) by Frizzled. Furthermore, PKC delta formed a complex with Dishevelled, and the activation of PKC delta by phorbol ester was sufficient for Dishevelled translocation and JNK activation. Thus, PKC delta plays an essential role in the Wnt/JNK pathway by regulating the localization and activity of Dishevelled.     

Numb and Numbl are required for maintenance of cadherin-based adhesion and polarity of neural progenitors

The polarity and adhesion of radial glial cells (RGCs), which function as progenitors and migrational guides for neurons, are critical for morphogenesis of the cerebral cortex. These characteristics largely depend on cadherin-based adherens junctions, which anchor apical end-feet of adjacent RGCs to each other at the ventricular surface. Here, we show that mouse numb and numb-like are required for maintaining radial glial adherens junctions. Numb accumulates in the apical end-feet, where it localizes to adherens junction-associated vesicles and interacts with cadherins. Numb and Numbl inactivation in RGCs decreases proper basolateral insertion of cadherins and disrupts adherens junctions and polarity, leading to progenitor dispersion and disorganized cortical lamination. Conversely, overexpression of Numb prolongs RGC polarization, in a cadherin-dependent manner, beyond the normal neurogenic period. Thus, by regulating RGC adhesion and polarity, Numb and Numbl are required for the tissue architecture of neurogenic niches and the cerebral cortex.     

Association of ASIP/mPAR-3 with adherens junctions of mouse neuroepithelial cells.

Polarity proteins play fundamental roles in asymmetric cell division, which is essential for the production of different types of cells in multicellular organisms. Here, we explore the localization of atypical PKC isotype-specific interacting protein (ASIP), a mammalian homologue of the Caenorhabditis elegans polarity protein PAR-3, in embryonic neural tissues. Although ASIP is localized on tight junctions in cultured epithelial cells, it localizes on adherens junctions outlined by beta-catenin and afadin at the luminal surface, an apical end of the neuroepithelium in developing mouse central nervous systems. Mammalian homologues of other C. elegans polarity proteins, mPAR-6 and aPKC, also localize in the adherens junctions. In dorsal root ganglia of the peripheral nervous system, ASIP is found predominantly in the cytoplasm of ganglion cells. In dividing preneural cells at the ventricular (luminal) surface of the embryonic telencephalon, ASIP localize in adherence junctions of luminal surface regardless of the axis of cell division. Therefore, only the daughter cell facing the lumen (apical daughter) may inherit ASIP when the division plate is oriented parallel to the surface. Given the roles of Bazooka, a Drosophila homologue of ASIP/PAR-3, in the asymmetric division of the Drosophila neuroblast, these observations suggest that ASIP, along with other polarity proteins and adherens junction proteins, plays an important role in neural cell differentiation by means of asymmetric cell division.     

Role of beta-catenin in the developing cortical and hippocampal neuroepithelium

beta-Catenin plays a pivotal role in Wnt signaling during embryogenesis and is a component of adherens junctions. Since targeted disruption of the beta-catenin gene is lethal at gastrulation we have used a D6-Cre mouse line for conditional inactivation of beta-catenin in the mouse cerebral cortex and hippocampus after embryonic day (E) 10.5. In D6-Cre floxed beta-catenin mice, hippocampal CA1-CA2 fields are disrupted in similar manner as in Wnt-3a and LEF-1 mutants. The cortex of D6-Cre floxed beta-catenin mutants is strongly affected which contrasts with the normal cortex observed in Wnt-3a and LEF-1 mutants. Severe abnormalities in the organization of the neuroepithelium are observed that include disrupted interkinetic nuclear migration, loss of adherens junctions, impaired radial migration of neurons toward superficial layers and decreased cell proliferation after E15.5. At newborn stage, a premature disassembly of the radial glial scaffold and increased numbers of astrocytes are found in the cortex.     

Interaction of Frizzled 7 and Dishevelled in Xenopus.

Frizzled proteins act as putative Wnt receptors and depending on Wnt/Frizzled interactions distinct intracellular pathways can be activated. The canonical Wnt pathway, triggered by Wnt-1-type ligands, is the best characterized and involves the activation of the cytoplasmic protein Dishevelled (Dsh). The Xenopus frizzled 7 receptor (Xfz7) can act in the canonical (Wnt-1-type) as well as in a non-canonical Wnt pathway which involves the activation of protein kinase C (PKC). In order to analyze the interaction between Xfz7 and Xdsh protein, we tested the effect of overexpression of Xfz7 on the subcellular distribution of Xdsh-myc protein. We demonstrate that Xfz7 can recruit Xdsh-myc to the plasma membrane and target genes of the Wnt-1-type pathway such as siamois and nodal related 3 (Xnr-3) are only activated in the presence of exogenous Xdsh-myc. Xfz7 in combination with Xwnt-8b, however, is able to induce siamois even in the absence of Xdsh-myc. Similar results were obtained after expression of Human frizzled 5 (Hfz5) together with Xwnt-5a but this receptor-ligand combination recruits Xdsh-myc only very poorly to the membrane. These results suggests that the endogenous Xdsh pool and exogenous Xdsh-myc differ in their ability to be recruited by Frizzled receptors.     

Intracellular Golgi Complex organization reveals tissue specific polarity during zebrafish embryogenesis

Background: Cell polarity is essential for directed migration of mesenchymal cells and morphogenesis of epithelial tissues. Studies in cultured cells indicate that a condensed Golgi Complex (GC) is essential for directed protein trafficking to establish cell polarity underlying directed cell migration. Dynamic changes of the GC intracellular organization during early vertebrate development remain to be investigated. Results: We used antibody labeling and fusion proteins in vivo to study the organization and intracellular placement of the GC during early zebrafish embryogenesis. We found that the GC was dispersed into several puncta containing cis‐ and trans‐Golgi Complex proteins, presumably ministacks, until the end of the gastrula period. By early segmentation stages, the GC condensed in cells of the notochord, adaxial mesoderm, and neural plate, and its intracellular position became markedly polarized away from borders between these tissues. Conclusions: We find that GC is dispersed in early zebrafish cells, even when cells are engaged in massive gastrulation movements. The GC accumulates into patches in a stage and cell‐type specific manner, and becomes polarized away from borders between the embryonic tissues. With respect to tissue borders, intracellular GC polarity in notochord is independent of mature apical/basal polarity, Wnt/PCP, or signals from adaxial mesoderm. Developmental Dynamics 245:678–691, 2016. © 2016 Wiley Periodicals, Inc.     

Noncanonical Wnt signaling regulates midline convergence of organ primordia during zebrafish development

Several components of noncanonical Wnt signaling pathways are involved in the control of convergence and extension (CE) movements during zebrafish and Xenopus gastrulation. However, the complexity of these pathways and the wide patterns of expression and activity displayed by some of their components immediately suggest additional morphogenetic roles beyond the control of CE. Here we show that the key modular intracellular mediator Dishevelled, through a specific activation of RhoA GTPase, controls the process of convergence of endoderm and organ precursors toward the embryonic midline in the zebrafish embryo. We also show that three Wnt noncanonical ligands wnt4a, silberblick/wnt11, and wnt11-related regulate this process by acting in a largely redundant way. The same ligands are also required, nonredundantly, to control specific aspects of CE that involve interaction of Dishevelled with mediators different from that of RhoA GTPase. Overall, our results uncover a late, previously unexpected role of noncanonical Wnt signaling in the control of midline assembly of organ precursors during vertebrate embryo development.     

Nectin and junctional adhesion molecule are critical cell adhesion molecules for the apico‐basal alignment of adherens and tight junctions in epithelial cells

Tight junctions (TJs) and adherens junctions (AJs) form an apical junctional complex at the apical side of the lateral membranes of epithelial cells, in which TJs are aligned at the apical side of AJs. Many cell adhesion molecules (CAMs) and cell polarity molecules (CPMs) cooperatively regulate the formation of the apical junctional complex, but the mechanism for the alignment of TJs at the apical side of AJs is not fully understood. We developed a cellular system with which epithelial‐like TJs and AJs were reconstituted in fibroblasts and analyzed the cooperative roles of CAMs and CPMs. We exogenously expressed various combinations of CAMs and CPMs in fibroblasts that express negligible amounts of these molecules endogenously. In these cells, the nectin‐based cell–cell adhesion was formed at the apical side of the junctional adhesion molecule (JAM)‐based cell–cell adhesion, and cadherin and claudin were recruited to the nectin‐3‐ and JAM‐based cell–cell adhesion sites to form AJ‐like and TJ‐like domains, respectively. This inversed alignment of the AJ‐like and TJ‐like domains was reversed by complementary expression of CPMs Par‐3, atypical protein kinase C, Par‐6, Crb3, Pals1 and Patj. We describe the cooperative roles of these CAMs and CPMs in the apico‐basal alignment of TJs and AJs in epithelial cells.     

Reorganization of actin cytoskeleton by FRIED, a Frizzled-8 associated protein tyrosine phosphatase.

Frizzled receptors transduce signals from the extracellular Wnt ligands through multiple signaling pathways that affect cytoskeletal organization and regulate gene expression. Direct intracellular mediators of Frizzled signaling are largely unknown. We identified FRIED (Frizzled interaction and ectoderm defects) by its association with the C-terminal PDZ-binding motif of Xenopus Frizzled 8. FRIED contains an N-terminal KIND domain, a FERM domain, six PDZ domains, and a tyrosine phosphatase domain, being similar in structure to the protein tyrosine phosphatase PTP-BAS/PTP-BL. We report that FRIED proteins with the FERM domain localize to the apical cortex and can inhibit Wnt8-mediated, but not beta-catenin-mediated, secondary axis induction in Xenopus embryos, suggesting a specific interaction with Wnt signaling. A FRIED construct containing the FERM domain induced reorganization of pigment granules and cortical actin in Xenopus ectoderm. Wnt5a suppressed the depigmentation of ectoderm triggered by FRIED, demonstrating that Wnt5a and FRIED functionally interact to regulate the cytoskeletal organization. Our data are consistent with the possibility that FRIED functions by modulating Rac1 activity. We propose that FRIED is an adaptor protein that serves as a molecular link between Wnt signaling and actin cytoskeleton.     

Dickkopf-1 regulates gastrulation movements by coordinated modulation of Wnt/beta catenin and Wnt/PCP activities, through interaction with the Dally-like homolog Knypek

Dickkopf-1 (Dkk1) is a secreted protein that negatively modulates the Wnt/beta catenin pathway. Lack of Dkk1 function affects head formation in frog and mice, supporting the idea that Dkk1 acts as a "head inducer" during gastrulation. We show here that lack of Dkk1 function accelerates internalization and rostral progression of the mesendoderm and that gain of function slows down both internalization and convergence extension, indicating a novel role for Dkk1 in modulating these movements. The motility phenotype found in the morphants is not observed in embryos in which the Wnt/beta catenin pathway is overactivated, and that dominant-negative Wnt proteins are not able to rescue the gastrulation movement defect induced by absence of Dkk1. These data strongly suggest that Dkk1 is acting in a beta catenin independent fashion when modulating gastrulation movements. We demonstrate that the glypican 4/6 homolog Knypek (Kny) binds to Dkk1 and that they are able to functionally interact in vivo. Moreover, Dkk1 regulation of gastrulation movements is kny dependent. Kny is a component of the Wnt/planar cell polarity (PCP) pathway. We found that indeed Dkk1 is able to activate this pathway in both Xenopus and zebrafish. Furthermore, concomitant alteration of the beta catenin and PCP activities is able to mimic the morphant accelerated cell motility phenotype. Our data therefore indicate that Dkk1 regulates gastrulation movement through interaction with LRP5/6 and Kny and coordinated modulations of Wnt/beta catenin and Wnt/PCP pathways.     

Inhibition of endocytosis blocks Wnt signalling to beta-catenin by promoting dishevelled degradation

Aim: The Wnt/Frizzled signalling pathway is highly conserved through evolution. Frizzled, the receptors for Wnts, have the topology of seven transmembrane spanning domain receptors. An important means of regulation of these receptors is internalization and desensitization through clathrin‐mediated endocytosis. Therefore, we investigated the effects of endocytosis inhibition on Frizzled4‐green fluorescent protein (FZD₄‐GFP) localization, dishevelled levels and Wnt‐3a signalling to β‐catenin. Methods: Experiments were performed in the mouse neuronal cell line SN4741 that has previously proven to be valuable for the investigation of Wnt/Frizzled signalling. FZD₄‐GFP distribution has been examined using confocal laser scanning microscopy. Dishevelled protein expression levels and the activation of β‐catenin upon treatment with endocytosis inhibitors (hyperosmolaric sucrose and K⁺ depletion), kinase inhibitors and Wnt‐3a were analysed by immunoblotting. Results: Hyperosmotic sucrose and K⁺ depletion increased the membrane localization of FZD₄‐GFP, and in parallel triggered fast (1–2 h) and almost complete (approx. 95%) degradation of endogenous dishevelled, which was independent of Wnt‐induced, CK1‐mediated phosphorylation of dishevelled. In addition, dishevelled depletion induced by endocytosis inhibition completely prevented canonical signalling by Wnt‐3a to β‐catenin even when osmotic conditions and endocytosis were reverted to normal. Conclusions: The data provide evidence for a molecular mechanism that could be a basis for a novel negative feedback loop within the Wnt/Frizzled pathway depending on dishevelled degradation. The identification of molecular details of regulatory mechanisms for the Wnt/Frizzled signalling pathway increases our understanding of pathway regulation, which might be of special physiological significance for embryonic development, cancer and neurological disorders.     

Establishment of hair bundle polarity and orientation in the developing vestibular system of the mouse

The morphological development of the vestibular maculae in the mouse was studied in order to identify elements that may determine how hair-bundle polarity is established. Utricles and saccules develop in parallel. Hair-bundles first appear at embryonic day (E) 13.5. They are initially not polarised and have a kinocilium located at the centre of the cell surface surrounded by stereocilia. Polarisation is rapidly established as the kinocilium becomes eccentrically positioned. The orientation of these polarised bundles is initially not random. It varies systematically across the maculae and the general orientation in utricles is the opposite of that in saccules. At E15.5, in both maculae, hair-bundle orientation angles fall into two populations that differ by approximately 180 degrees defining a line of orientation reversal, the position of which varies little during subsequent maturation. Many more immature hair bundles appear at E15.5 suggesting a second wave of hair cell differentiation is initiated. Otoconial membrane is produced simultaneously across the entire width of both maculae, indicating directional growth of the overlying extracellular matrix is unlikely to influence hair-bundle orientation. Growth of both maculae occurs asymmetrically, essentially outwards from the striola, but it is most pronounced after orientation is defined. Microtubules are prominent in hair cells at the earliest stages of their differentiation, but are oriented parallel to the long axis of the cell and, thus, may not have a role in directing hair-bundle polarity. Microfilament assemblies that are aligned parallel to the apical surface and connect to the adherens junctions in supporting cells could provide a "framework" for hair-bundle orientation. The striated rootlets of ciliary centrioles that are aligned parallel to the cell surface with their tips associated with microfilament assemblies at adherens junctions were the only structural asymmetry identified that might influence the development of hair-bundle polarity.     

Regulation of endothelial cell cytoskeletal reorganization by a secreted frizzled-related protein-1 and frizzled 4- and frizzled 7-dependent pathway: role in neovessel formation

Consistent with findings of Wnt pathway members involved in vascular cells, a role for Wnt/Frizzled signaling has recently emerged in vascular cell development. Among the few Wnt family members implicated in vessel formation in adult, Wnt7b and Frizzled 4 have been shown as involved in vessel formation in the lung and in the retina, respectively. Our previous work has shown a role for secreted Frizzled-related protein-1 (sFRP-1), a proposed Wnt signaling inhibitor, in neovascularization after an ischemic event and demonstrated its role as a potent angiogenic factor. However the mechanisms involved have not been investigated. Here, we show that sFRP-1 treatment increases endothelial cell spreading on extracellular matrix as revealed by actin stress fiber reorganization in an integrin-dependent manner. We demonstrate that sFRP-1 can interact with Wnt receptors Frizzled 4 and 7 on endothelial cells to transduce downstream to cellular machineries requiring Rac-1 activity in cooperation with GSK-3beta. sFRP-1 overexpression in endothelium specifically reversed the inactivation of GSK-3 beta and increased neovascularization in ischemia-induced angiogenesis in mouse hindlimb. This study illustrates a regulated pathway by sFRP-1 involving GSK-3beta and Rac-1 in endothelial cell cytoskeletal reorganization and in neovessel formation.     

Domain-specific early and late function of Dpatj in Drosophila photoreceptor cells.

The formation and maintenance of cell polarity is essential for epithelial morphogenesis. Dpatj (Drosophila homolog of mammalian Patj) is a multi-PDZ domain protein that localizes to the apical cell membrane and forms a protein complex with cell polarity proteins, Crumbs (Crb) and Stardust (Sdt). Whereas Crb and Sdt are known to be required for the organization of adherens junctions (AJs) and rhabdomeres in differentiating photoreceptors, the in vivo function of Dpatj as a member of the Crb complex in developing eye has been unclear due to the lack of loss-of-function mutations specifically affecting the dpatj gene. Our genetic analysis of hypomorph, null, and RNA interference reveals distinct dual functions of Dpatj in developing and mature photoreceptors. The C-terminal region (PDZ domains 2-4) of Dpatj is not essential for development of the animal but is required to prevent late-onset photoreceptor degeneration. In contrast, the N-terminal region of Dpatj is essential for animal viability and photoreceptor morphogenesis during development. The localization and maintenance of Crb and Sdt in the apical photoreceptor membrane are strongly affected by reduced levels of Dpatj. Dpatj is necessary for proper positioning of AJs and the integrity of photoreceptors in the developing retina as well as for the maintenance of adult photoreceptors. Our study provides evidence that Dpatj has domain-specific early and late functions in regulating the localization and stability of the Crb-Sdt complex in photoreceptor cells.     

Unipolar membrane association of Dishevelled mediates Frizzled planar cell polarity signaling

Drosophila epithelia acquire a planar cell polarity (PCP) orthogonal to their apical-basal axes. Frizzled (Fz) is the receptor for the PCP signal, and Dishevelled (Dsh) transduces the signal. Here, I demonstrate that unipolar relocalization of Dsh to the membrane is required to mediate PCP, but not Wingless (Wg) signaling. Dsh membrane localization reflects the activation of Fz/PCP signaling, revealing that the initially symmetric signal evolves to one that displays unipolar asymmetry, specifying the cells' ultimate polarity. This transition from symmetric to asymmetric Dsh localization requires Dsh function, and reflects an amplification process that generates a steep intracellular activity gradient necessary to determine PCP.     

Axis determination by inhibition of Wnt signaling in Xenopus

The Wnt family of secreted polypeptides participate in a variety of developmental processes in which embryonic polarity is established. To study a role for Wnt ligands in vertebrate axis determination, we interfered with Wnt signaling in the embryo using the extracellular domain of Xenopus Frizzled 8 (ECD8), which blocks Wnt-dependent activation of a target gene in Xenopus ectodermal explants. Expression of ECD8 in ventral blastomeres resulted in formation of secondary axes containing abundant notochord and head structures. These results suggest that Wnt signaling is required to maintain ventral cell fates and has to be suppressed for dorsal development to occur.