Soluble Frizzled-related proteins promote exosome-mediated Wnt re-secretion.

Wnt proteins are thought to be transported in several ways in the extracellular space. For instance, they are known to be carried by exosomes and by Wnt-carrier proteins, such as sFRP proteins. However, little is known about whether and/or how these two transport systems are related. Here, we show that adding sFRP1 or sFRP2, but not sFRP3 or sFRP4, to culture medium containing Wnt3a or Wnt5a increases re-secretion of exosome-loaded Wnt proteins from cells. This effect of sFRP2 is counteracted by heparinase, which removes sugar chains on heparan sulfate proteoglycans (HSPGs), but is independent of LRP5/6, Wnt co-receptors essential for Wnt signaling. Wnt3a and Wnt5a specifically dimerize with sFRP2 in culture supernatant. Furthermore, a Wnt3a mutant defective in heterodimerization with sFRP2 impairs the ability to increase exosome-mediated Wnt3a re-secretion. Based on these results, we propose that Wnt heterodimerization with its carrier protein, sFRP2, enhances Wnt accumulation at sugar chains on HSPGs on the cell surface, leading to increased endocytosis and exosome-mediated Wnt re-secretion. Our results suggest that the range of action of Wnt ligands is controlled by coordination of different transport systems.

Dissection of N-deacetylase and N-sulfotransferase activities of NDST1 and their effects on Wnt8 distribution and signaling in Xenopus embryos.

Wnt is a family of secreted signaling proteins involved in the regulation of cellular processes, including maintenance of stem cells, carcinogenesis, and cell differentiation. In the context of early vertebrate embryogenesis, graded distribution of Wnt proteins has been thought to regulate positional information along the antero-posterior axis. However, understanding of the molecular basis for Wnt spatial distribution remains poor. Modified states of heparan sulfate (HS) proteoglycans are essential for Wnt8 localization, because depletion of N-deacetylase/N-sulfotransferase 1 (NDST1), a modification enzyme of HS chains, decreases Wnt8 levels and NDST1 overexpression increases Wnt8 levels on the cell surface. Since overexpression of NDST1 increases both deacetylation and N-sulfation of HS chains, it is not clear which function of NDST1 is actually involved in Wnt8 localization. In the present study, we generated an NDST1 mutant that specifically increases deacetylation, but not N-sulfation, of HS chains in Xenopus embryos. Unlike wild-type NDST1, this mutant did not increase Wnt8 accumulation on the cell surface, but it reduced canonical Wnt signaling, as determined with the TOP-Flash reporter assay. These results suggest that N-sulfation of HS chains is responsible for localization of Wnt8 and Wnt8 signaling, whereas deacetylation has an inhibitory effect on canonical Wnt signaling. Consistently, overexpression of wild-type NDST1, but not the mutant, resulted in small eyes in Xenopus embryos. Thus, our NDST1 mutant enables us to dissect the regulation of Wnt8 localization and signaling by HS proteoglycans by specifically manipulating the enzymatic activities of NDST1.

Understanding and manipulating extracellular behaviors of Wnt ligands.

Wnt, a family of secreted signaling proteins, serves diverse functions in embryogenesis, organogenesis, cancer, and stem cell functions. In the context of development, Wnt has been considered a representative morphogen, forming concentration gradients to give positional information to cells or tissues. However, although gradients are often illustrated in schemata, the reality of concentration gradients, or in other words, actual spatial distribution of Wnt ligands, and their behaviors in the extracellular space still remain poorly known. To understand extracellular behavior of Wnt ligands, quantitative analyses such as fluorescence correlation spectroscopy (FCS) and fluorescence recovery after photobleaching (FRAP) are highly informative because Wnt dispersal involves physical and biochemical processes, such as diffusion and binding to or dissociation from cell surface molecules, including heparan sulfate proteoglycans (HSPGs). Here, I briefly discuss representative methods to quantify morphogen dynamics. In addition, I discuss molecular manipulations of morphogens, mainly focusing on use of protein binders, and synthetic biology of morphogens as indicators of current and future directions in this field.

The Helicobacter pylori CagA oncoprotein disrupts Wnt/PCP signaling and promotes hyperproliferation of pyloric gland base cells.

Helicobacter pylori strains that deliver the oncoprotein CagA into gastric epithelial cells are the major etiologic agents of upper gastric diseases including gastric cancer. CagA promotes gastric carcinogenesis through interactions with multiple host proteins. Here, we show that CagA also disrupts Wnt-dependent planar cell polarity (Wnt/PCP), which orients cells within the plane of an epithelium and coordinates collective cell behaviors such as convergent extension to enable epithelial elongation during development. Ectopic expression of CagA in Xenopus laevis embryos impaired gastrulation, neural tube formation, and axis elongation, processes driven by convergent extension movements that depend on the Wnt/PCP pathway. Mice specifically expressing CagA in the stomach epithelium had longer pyloric glands and mislocalization of the tetraspanin proteins VANGL1 and VANGL2 (VANGL1/2), which are critical components of Wnt/PCP signaling. The increased pyloric gland length was due to hyperproliferation of cells at the gland base, where Lgr5+ stem and progenitor cells reside, and was associated with fewer differentiated enteroendocrine cells. In cultured human gastric epithelial cells, the N terminus of CagA interacted with the C-terminal cytoplasmic tails of VANGL1/2, which impaired Wnt/PCP signaling by inducing the mislocalization of VANGL1/2 from the plasma membrane to the cytoplasm. Thus, CagA may contribute to the development of gastric cancer by subverting a Wnt/PCP-dependent mechanism that restrains pyloric gland stem cell proliferation and promotes enteroendocrine differentiation.

Intercellular exchange of Wnt ligands reduces cell population heterogeneity during embryogenesis.

Wnt signaling is required to maintain bipotent progenitors for neural and paraxial mesoderm cells, the neuromesodermal progenitor (NMP) cells that reside in the epiblast and tailbud. Since epiblast/tailbud cells receive Wnt ligands produced by one another, this exchange may average out the heterogeneity of Wnt signaling levels among these cells. Here, we examined this possibility by replacing endogenous Wnt3a with a receptor-fused form that activates signaling in producing cells, but not in neighboring cells. Mutant mouse embryos show a unique phenotype in which maintenance of many NMP cells is impaired, although some cells persist for long periods. The epiblast cell population of these embryos increases heterogeneity in Wnt signaling levels as embryogenesis progresses and are sensitive to retinoic acid, an endogenous antagonist of NMP maintenance. Thus, mutual intercellular exchange of Wnt ligands in the epiblast cell population reduces heterogeneity and achieves robustness to environmental stress.

Alignment of the cell long axis by unidirectional tension acts cooperatively with Wnt signalling to establish planar cell polarity.

Planar cell polarity (PCP) is the aligned cell polarity within a tissue plane. Mechanical signals are known to act as a global cue for PCP, yet their exact role is still unclear. In this study, we focused on PCP in the posterior neuroectoderm of Xenopus laevis and investigated how mechanical signals regulate polarity. We reveal that the neuroectoderm is under a greater tension in the anterior-posterior direction and that perturbation of this tension causes PCP disappearance. We show that application of uniaxial stretch to explant tissues can control the orientation of PCP and that cells sense the tissue stretch indirectly through a change in their shape, rather than directly through detection of anisotropic tension. Furthermore, we reveal that PCP is most strongly established when the orientation of tissue stretch coincides with that of diffusion of locally expressed Wnt ligands, suggesting a cooperative relationship between these two PCP regulators.

Optogenetic relaxation of actomyosin contractility uncovers mechanistic roles of cortical tension during cytokinesis.

Actomyosin contractility generated cooperatively by nonmuscle myosin II and actin filaments plays essential roles in a wide range of biological processes, such as cell motility, cytokinesis, and tissue morphogenesis. However, subcellular dynamics of actomyosin contractility underlying such processes remains elusive. Here, we demonstrate an optogenetic method to induce relaxation of actomyosin contractility at the subcellular level. The system, named OptoMYPT, combines a protein phosphatase 1c (PP1c)-binding domain of MYPT1 with an optogenetic dimerizer, so that it allows light-dependent recruitment of endogenous PP1c to the plasma membrane. Blue-light illumination is sufficient to induce dephosphorylation of myosin regulatory light chains and a decrease in actomyosin contractile force in mammalian cells and Xenopus embryos. The OptoMYPT system is further employed to understand the mechanics of actomyosin-based cortical tension and contractile ring tension during cytokinesis. We find that the relaxation of cortical tension at both poles by OptoMYPT accelerated the furrow ingression rate, revealing that the cortical tension substantially antagonizes constriction of the cleavage furrow. Based on these results, the OptoMYPT system provides opportunities to understand cellular and tissue mechanics.

Asymmetric requirement of Dpp/BMP morphogen dispersal in the Drosophila wing disc.

How morphogen gradients control patterning and growth in developing tissues remains largely unknown due to lack of tools manipulating morphogen gradients. Here, we generate two membrane-tethered protein binders that manipulate different aspects of Decapentaplegic (Dpp), a morphogen required for overall patterning and growth of the Drosophila wing. One is "HA trap" based on a single-chain variable fragment (scFv) against the HA tag that traps HA-Dpp to mainly block its dispersal, the other is "Dpp trap" based on a Designed Ankyrin Repeat Protein (DARPin) against Dpp that traps Dpp to block both its dispersal and signaling. Using these tools, we found that, while posterior patterning and growth require Dpp dispersal, anterior patterning and growth largely proceed without Dpp dispersal. We show that dpp transcriptional refinement from an initially uniform to a localized expression and persistent signaling in transient dpp source cells render the anterior compartment robust against the absence of Dpp dispersal. Furthermore, despite a critical requirement of dpp for the overall wing growth, neither Dpp dispersal nor direct signaling is critical for lateral wing growth after wing pouch specification. These results challenge the long-standing dogma that Dpp dispersal is strictly required to control and coordinate overall wing patterning and growth.

Regulation of Wnt/PCP signaling through p97/VCP-KBTBD7-mediated Vangl ubiquitination and endoplasmic reticulum-associated degradation.

The four-pass transmembrane proteins Vangl1 and Vangl2 are dedicated core components of Wnt/planar cell polarity (Wnt/PCP) signaling that critically regulate polarized cell behaviors in many morphological and physiological processes. Here, we found that the abundance of Vangl proteins is tightly controlled by the ubiquitin-proteasome system through endoplasmic reticulum-associated degradation (ERAD). The key ERAD component p97/VCP directly binds to Vangl at a highly conserved VCP-interacting motif and recruits the E3 ligase KBTBD7 via its UBA-UBX adaptors to promote Vangl ubiquitination and ERAD. We found that Wnt5a/CK1 prevents Vangl ubiquitination and ERAD by inducing Vangl phosphorylation, which facilitates Vangl export from the ER to the plasma membrane. We also provide in vivo evidence that KBTBD7 regulates convergent extension during zebrafish gastrulation and functions as a tumor suppressor in breast cancer by promoting Vangl degradation. Our findings reveal a previously unknown regulatory mechanism of Wnt/PCP signaling through the p97/VCP-KBTBD7-mediated ERAD pathway.

Quantitative analyses reveal extracellular dynamics of Wnt ligands in Xenopus embryos.

The mechanism of intercellular transport of Wnt ligands is still a matter of debate. To better understand this issue, we examined the distribution and dynamics of Wnt8 in Xenopus embryos. While Venus-tagged Wnt8 was found on the surfaces of cells close to Wnt-producing cells, we also detected its dispersal over distances of 15 cell diameters. A combination of fluorescence correlation spectroscopy and quantitative imaging suggested that only a small proportion of Wnt8 ligands diffuses freely, whereas most Wnt8 molecules are bound to cell surfaces. Fluorescence decay after photoconversion showed that Wnt8 ligands bound on cell surfaces decrease exponentially, suggesting a dynamic exchange of bound forms of Wnt ligands. Mathematical modeling based on this exchange recapitulates a graded distribution of bound, but not free, Wnt ligands. Based on these results, we propose that Wnt distribution in tissues is controlled by a dynamic exchange of its abundant bound and rare free populations.

Improvement of Phycocyanobilin Synthesis for Genetically Encoded Phytochrome-Based Optogenetics.

Optogenetics is a powerful technique using photoresponsive proteins, and the light-inducible dimerization (LID) system, an optogenetic tool, allows to manipulate intracellular signaling pathways. One of the red/far-red responsive LID systems, phytochrome B (PhyB)-phytochrome interacting factor (PIF), has a unique property of controlling both association and dissociation by light on the second time scale, but PhyB requires a linear tetrapyrrole chromophore such as phycocyanobilin (PCB), and such chromophores are present only in higher plants and cyanobacteria. Here, we report that we further improved our previously developed PCB synthesis system (SynPCB) and successfully established a stable cell line containing a genetically encoded PhyB-PIF LID system. First, four genes responsible for PCB synthesis, namely, PcyA, HO1, Fd, and Fnr, were replaced with their counterparts derived from thermophilic cyanobacteria. Second, Fnr was truncated, followed by fusion with Fd to generate a chimeric protein, tFnr-Fd. Third, these genes were concatenated with P2A peptide cDNAs for polycistronic expression, resulting in an approximately 4-fold increase in PCB synthesis compared with the previous version. Finally, we incorporated the PhyB, PIF, and SynPCB system into drug inducible lentiviral and transposon vectors, which enabled us to induce PCB synthesis and the PhyB-PIF LID system by doxycycline treatment. These tools provide a new opportunity to advance our understanding of the causal relationship between intracellular signaling and cellular functions.

Heparan Sulfate Proteoglycan Clustering in Wnt Signaling and Dispersal.

Wnt, a family of secreted signal proteins, serves diverse functions in animal development, stem cell systems, and carcinogenesis. Although Wnt is generally considered a morphogen, the mechanism by which Wnt ligands disperse is still debated. Heparan sulfate proteoglycans (HSPGs) are extracellular regulators involved in Wnt ligand dispersal. Drosophila genetics have revealed that HSPGs participate in accumulation and transport of Wnt ligands. Based on these findings, a "restricted diffusion" model, in which Wnt ligands are gradually transferred by repetitive binding and dissociation to HSPGs, has been proposed. Nonetheless, we recently found that HSPGs are not uniformly distributed, but are locally clustered on cell surfaces in Xenopus embryos. HSPGs with N-sulfo-rich HS chains and those with N-acetyl-rich unmodified HS chains form different clusters. Furthermore, endogenous Wnt8 ligands are discretely accumulated in a punctate fashion, colocalized with the N-sulfo-rich clusters. Based on these lines of evidence, here we reconsider the classical view of morphogen spreading controlled by HSPGs.

Assembly of protein complexes restricts diffusion of Wnt3a proteins.

Members of the Wnt protein family play roles in many aspects of embryogenesis and homeostasis. Despite their biological significance, characteristics of Wnt proteins still remain unclear, mainly due to their insolubility after the removal of serum. Here we examine Wnt proteins in serum-containing media by using analytical ultracentrifugation with a fluorescence detection system. This analysis reveals that Wnt3a assembles into high-molecular-weight complexes that become dissociable by interaction with the extracellular domain of the Frizzled8 receptor or secreted Wnt-binding protein sFRP2. Cross-linking and single-particle analyses of Wnt3a fractionated by gel filtration chromatography show the homo-trimer to be the smallest form of the assembled Wnt3a complexes. Fluorescence correlation spectroscopy and immunohistochemistry reveal that the assembly of Wnt3a complexes restricted their diffusion and signaling range in Xenopus laevis embryos. Thus, we propose that the Wnt diffusion range can be controlled by a balance between the assembly of Wnt complexes and their dissociation.

Ripply3 is required for the maintenance of epithelial sheets in the morphogenesis of pharyngeal pouches.

During tissue development, the morphogenesis of epithelial sheets is regulated by many factors, including mechanical force, although the underlying mechanisms remain largely unknown. In the pharyngeal region of the vertebrate embryo, endodermal epithelium is reiteratively folded outward to form pharyngeal pouches, making partitions between the pharyngeal arches. Ripply3, encoding a member of the Ripply family of adaptor proteins, is required for the pouch formation posterior to the 2nd pharyngeal pouch. In this study, we found that the expression of mouse Ripply3 was specifically activated in accordance with the bending of the endodermal epithelium during the pouch formation. In Ripply3-deficient embryos, a continuous monolayer of the endodermal epithelium was not maintained posterior to the 2nd pharyngeal pouch. Corresponding to the endodermal region of the deformed epithelium, the activated form of Integrin ß1, which was localized at the basal side of the epithelial cells in the wild-type embryos, was not persistently observed in the mutants. On the other hand, cell proliferation and apoptotic cell death in the endoderm were not obviously affected by the Ripply3 deficiency. Significantly, Ripply3 expressed in cultured cells was found to be preferentially accumulated in the focal adhesions, which are Integrin-mediated adhesive contact sites transmitting mechanical force between the extracellular matrix and attached cells. Furthermore, Ripply3 promoted the maturation of focal adhesions in these cells. Thus, Ripply3 appears to have been activated to enhance the connection between the extracellular matrix and endodermal epithelial cells, as a mechanism to resist the mechanical stress generated during the bending of the epithelial sheets.

Roles of two types of heparan sulfate clusters in Wnt distribution and signaling in Xenopus.

Wnt proteins direct embryonic patterning, but the regulatory basis of their distribution and signal reception remain unclear. Here, we show that endogenous Wnt8 protein is distributed in a graded manner in Xenopus embryo and accumulated on the cell surface in a punctate manner in association with "N-sulfo-rich heparan sulfate (HS)," not with "N-acetyl-rich HS". These two types of HS are differentially clustered by attaching to different glypicans as core proteins. N-sulfo-rich HS is frequently internalized and associated with the signaling vesicle, known as the Frizzled/Wnt/LRP6 signalosome, in the presence of Wnt8. Conversely, N-acetyl-rich HS is rarely internalized and accumulates Frzb, a secreted Wnt antagonist. Upon interaction with Frzb, Wnt8 associates with N-acetyl-rich HS, suggesting that N-acetyl-rich HS supports Frzb-mediated antagonism by sequestering Wnt8 from N-sulfo-rich HS. Thus, these two types of HS clusters may constitute a cellular platform for the distribution and signaling of Wnt8.

Differences in the secretion and transport of Wnt proteins.

During the last three decades, our understanding about Wnt signaling has progressed greatly, especially with regards to the molecular mechanism of intracellular transmission of this signaling, as well as its physiological roles. In parallel, the molecular nature of Wnt proteins has gradually but surely been clarified. Wnt proteins are post-translationaly modified with fatty acid and glycosaminoglycans, resulting in constraint of the 3D structure and behavior of the proteins. Specific binding proteins or extracellular vesicles, which appear to shield the lipid moiety from the aquatic environment, enable Wnt proteins to be transported in the extracellular space. Equally, Wnt-interacting proteins in the extracellular space, including heparan sulfate proteoglycan, are also involved in its spreading. Recent studies also show that intercellular transmission of Wnt proteins occurs by cell migration and extension of cell protrusions. Here, we will show the molecular and cellular bases of the trafficking of Wnt proteins and discuss questions that remain to be answered.

Secreted Wnt "inhibitors" are not just inhibitors: regulation of extracellular Wnt by secreted Frizzled-related proteins.

Gradient formation and signaling ranges of secreted proteins are crucial problems to understand how morphogens work for positional information and patterning in animal development. Yet, extracellular behaviors of secreted signaling molecules remain unexplored compared to their downstream pathways inside the cell. Recent advances in bioimaging make it possible to directly visualize morphogen molecules, and this simple strategy has, at least partly, succeeded in uncovering molecular behaviors of morphogens, such as Wnt (wingless-type MMTV integration site family member) and BMP (bone morphogenetic protein) as well as secreted Wnt binding proteins, sFRPs (secreted Frizzled-related proteins), in embryonic tissues. Here, we review the regulation of Wnt signaling by sFRPs, focusing on extracellular regulation of Wnt ligands in comparison with other morphogens. We also discuss evolutionary aspects with comprehensive syntenic and phylogenetic information about vertebrate sfrp genes. We newly annotated several sfrp genes including sfrp2-like 1 (sfrp2l1) in frogs and fishes and crescent in mammals.

Secreted Frizzled-related proteins enhance the diffusion of Wnt ligands and expand their signalling range.

Secreted Frizzled-related proteins (sFRPs) are thought to negatively modulate Wnt signalling. Although Wnt proteins are thought to diffuse extracellularly and act as morphogens, little is known about the diffusibility of either Wnts or sFRPs. Here we show that Frzb and Crescent (Cres), which are members of the sFRP family, have the ability to regulate the diffusibility and signalling areas of the Wnt ligands Wnt8 and Wnt11. We found, using the Xenopus embryo, that Wnts do not diffuse effectively, whereas Frzb and Cres spread very widely. Interestingly, Frzb and Cres substantially promoted the diffusion of Wnt8 and Wnt11 through extracellular interactions. Importantly, we show that Wnt8 conveyed by sFRPs can activate canonical Wnt signalling despite the function of sFRPs as Wnt inhibitors, suggesting a novel regulatory system for Wnts by sFRPs.