Viable mice with compound mutations in the Wnt/Dvl pathway antagonists nkd1 and nkd2

Gradients of Wnt/beta-catenin signaling coordinate development and physiological homeostasis in metazoan animals. Proper embryonic development of the fruit fly Drosophila melanogaster requires the Naked cuticle (Nkd) protein to attenuate a gradient of Wnt/beta-catenin signaling across each segmental anlage. Nkd inhibits Wnt signaling by binding the intracellular protein Dishevelled (Dsh). Mice and humans have two nkd homologs, nkd1 and nkd2, whose encoded proteins can bind Dsh homologs (the Dvl proteins) and inhibit Wnt signaling. To determine whether nkd genes are necessary for murine development, we replaced nkd exons that encode Dvl-binding sequences with IRES-lacZ/neomycin cassettes. Mutants homozygous for each nkd(lacZ) allele are viable with slightly reduced mean litter sizes. Surprisingly, double-knockout mice are viable, with subtle alterations in cranial bone morphology that are reminiscent of mutation in another Wnt/beta-catenin antagonist, axin2. Our data show that nkd function in the mouse is dispensable for embryonic development.     

Nkd1 Functions as a Passive Antagonist of Wnt Signaling

Wnt signaling is involved in many aspects of development and in the homeostasis of stem cells. Its importance is underscored by the fact that misregulation of Wnt signaling has been implicated in numerous diseases, especially colorectal cancer. However, how Wnt signaling regulates itself is not well understood. There are several Wnt negative feedback regulators, which are active antagonists of Wnt signaling, but one feedback regulator, Nkd1, has reduced activity compared to other antagonists, yet is still a negative feedback regulator. Here we describe our efforts to understand the role of Nkd1 using Wnt signaling compromised zebrafish mutant lines. In several of these lines, Nkd1 function was not any more active than it was in wild type embryos. However, we found that Nkd1’s ability to antagonize canonical Wnt/β-catenin signaling was enhanced in the Wnt/Planar Cell Polarity mutants silberblick (slb/wnt11) and trilobite (tri/vangl2). While slb and tri mutants do not display alterations in canonical Wnt signaling, we found that they are hypersensitive to it. Overexpression of the canonical Wnt/β-catenin ligand Wnt8a in slb or tri mutants resulted in dorsalized embryos, with tri mutants being much more sensitive to Wnt8a than slb mutants. Furthermore, the hyperdorsalization caused by Wnt8a in tri could be rescued by Nkd1. These results suggest that Nkd1 functions as a passive antagonist of Wnt signaling, functioning only when homeostatic levels of Wnt signaling have been breached or when Wnt signaling becomes destabilized.     

A targeted mutation of Nkd1 impairs mouse spermatogenesis

Nkd1 is an antagonist of the canonical Wnt/beta-catenin signaling pathway. The EF-hand motif of Nkd1 is required for its inhibitory function. Early studies suggested that Nkd1 might play important roles in mouse embryonic development and tumorigenesis. We constructed Nkd1(-/-) mice whose Nkd1 protein lacked the EF-hand and was unable to inhibit Wnt/beta-catenin signaling. The homozygotes were viable and grew normally, but their fertility in males was reduced. In wild-type adult testes, Nkd1 mRNA was expressed more abundantly in the elongating spermatids than in the round spermatids. Lack of EF-hand caused reductions in the testis weight and sperm count by 30 and 60%, respectively. During testis development, Nkd1 mRNA expression started at the 25th day after birth, coincident with the onset of Wnt1 expression. Nuclear localization of beta-catenin increased in the elongating spermatids, suggesting that the mutant Nkd1 failed to inhibit the Wnt/beta-catenin pathway. These results suggest that deletion of the EF-hand from Nkd1 reduces the number of the elongating spermatids at haploid stage. In contrast, the mutant Nkd1 did not affect intestinal polyposis in Apc(Delta716) mice.     

Stable anterior anchoring of the oocyte nucleus is required to establish dorsoventral polarity of the Drosophila egg

Wnt signals control cell fate decisions and orchestrate cell behavior in metazoan animals. In the fruit fly Drosophila, embryos defective in signaling mediated by the Wnt protein Wingless (Wg) exhibit severe segmentation defects. The Drosophila segment polarity gene naked cuticle (nkd) encodes an EF hand protein that regulates early Wg activity by acting as an inducible antagonist. Nkd antagonizes Wg via a direct interaction with the Wnt signaling component Dishevelled (Dsh). Here we describe two mouse and human proteins, Nkd1 and Nkd2, related to fly Nkd. The most conserved region among the fly and vertebrate proteins, the EFX domain, includes the putative EF hand and flanking sequences. EFX corresponds to a minimal domain required for fly or vertebrate Nkd to interact with the basic/PDZ domains of fly Dsh or vertebrate Dvl proteins in the yeast two-hybrid assay. During mouse development, nkd1 and nkd2 are expressed in multiple tissues in partially overlapping, gradient-like patterns, some of which correlate with known patterns of Wnt activity. Mouse Nkd1 can block Wnt1-mediated, but not beta-catenin-mediated, activation of a Wnt-dependent reporter construct in mammalian cell culture. Misexpression of mouse nkd1 in Drosophila antagonizes Wg function. The data suggest that the vertebrate Nkd-related proteins, similar to their fly counterpart, may act as inducible antagonists of Wnt signals.     

Naked1 antagonizes Wnt signaling by preventing nuclear accumulation of β-catenin

Cyto-nuclear shuttling of β-catenin is at the epicenter of the canonical Wnt pathway and mutations in genes that result in excessive nuclear accumulation of β-catenin are the driving force behind the initiation of many cancers. Recently, Naked Cuticle homolog 1 (Nkd1) has been identified as a Wnt-induced intracellular negative regulator of canonical Wnt signaling. The current model suggests that Nkd1 acts between Disheveled (Dvl) and β-catenin. Here, we employ the zebrafish embryo to characterize the cellular and biochemical role of Nkd1 in vivo. We demonstrate that Nkd1 binds to β-catenin and prevents its nuclear accumulation. We also show that this interaction is conserved in mammalian cultured cells. Further, we demonstrate that Nkd1 function is dependent on its interaction with the cell membrane. Given the conserved nature of Nkd1, our results shed light on the negative feedback regulation of Wnt signaling through the Nkd1-mediated negative control of nuclear accumulation of β-catenin.     

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.     

Wnt/beta-catenin 通路在成骨细胞中的作用

Wnt信号通路包括经典通路和非经典通路两种,其中Wnt经典通路又称为Wnt/β-catenin通路,其在成骨细胞的分化、增殖过程中发挥这重要的作用。Wnt信号通路实现过程中有多种因子参与,包括Wnt蛋白、β-catenin、蛋白激酶GSK-3β以及APC蛋白等多种。Wnt蛋白家族是由19种Wnt蛋白组成的,主要分为经典Wnt蛋白和非经典Wnt蛋白,其本质是一系列高度保守的分泌性糖蛋白,并且不同的Wnt蛋白对成骨细胞发挥着不同的作用,其中经典Wnt蛋白通过经典Wnt信号作用于成骨细胞对成骨细胞的增殖、分化有着重要的影响。本综述通过对Wnt经典信号通路过程中的多种因子与成骨细胞分化、增殖的关系进行分析总结,了解Wnt/β-catenin通路对成骨细胞的作用。     

Activation of non-canonical Wnt/JNK pathway by Wnt3a is associated with differentiation fate determination of human bone marrow stromal (mesenchymal) stem cells

The canonical Wnt signaling pathway can determine human bone marrow stromal (mesenchymal) stem cell (hMSC) differentiation fate into osteoblast or adipocyte lineages. However, its downstream targets in MSC are not well characterized. Thus, using DNA microarrays, we compared global gene expression patterns induced by Wnt3a treatment in two hMSC lines: hMSC-LRP5^T253 and hMSC-LRP5^T244 cells carrying known mutations of Wnt co-receptor LRP5 (T253I or T244M) that either enhances or represses canonical Wnt signaling, respectively. Wnt3a treatment of hMSC activated not only canonical Wnt signaling, but also the non-canonical Wnt/JNK pathway through upregulation of several non-canonical Wnt components e.g. naked cuticle 1 homolog (NKD1) and WNT11. Activation of the non-canonical Wnt/JNK pathway by anisomycin enhanced osteoblast differentiation whereas its inhibition by SP600125 enhanced adipocyte differentiation of hMSC. In conclusion, canonical and non-canonical Wnt signaling cooperate in determining MSC differentiation fate.     

Dickkopf-1 antagonizes Wnt signaling independent of beta-catenin in human mesothelioma

Dickkopf-1 (Dkk-1) is a secreted protein that acts as a potent inhibitor of the Wnt signal transduction pathway. It is thought that the antagonistic effect of Dkk-1 is specific to the canonical (Wnt/beta-catenin) pathway. In this study, we demonstrate that restoration of Dkk-1 expression suppresses cell growth and induces apoptotic cell death in beta-catenin-deficient mesothelioma cell lines H28 and MS-1. Furthermore, we found that a small-molecule inhibitor of JNK inhibited the apoptosis induced by Dkk-1 overexpression in these cells. Together, our data suggest that Dkk-1 may be able to antagonize Wnt signaling and exert its tumor suppressive effects through beta-catenin-independent non-canonical pathways (i.e., the Wnt/JNK pathway).     

Wnt 3a promotes proliferation and suppresses osteogenic differentiation of adult human mesenchymal stem cells

Multipotential adult mesenchymal stem cells (MSCs) are able to differentiate along several known lineages, and lineage commitment is tightly regulated through specific cellular mediators and interactions. Recent observations of a low/high bone-mass phenotype in patients expressing a loss-/gain-of-function mutation in LRP5, a coreceptor of the Wnt family of signaling molecules, suggest the importance of Wnt signaling in bone formation, possibly involving MSCs. To analyze the role of Wnt signaling in mesenchymal osteogenesis, we have profiled the expression of WNTs and their receptors, FRIZZLEDs (FZDs), and several secreted Wnt inhibitors, such as SFRPs, and examined the effect of Wnt 3a, as a representative canonical Wnt member, during MSC osteogenesis in vitro. WNT11, FZD6, SFRP2, and SFRP3 are upregulated during MSC osteogenesis, while WNT9A and FZD7 are downregulated. MSCs also respond to exogenous Wnt 3a, based on increased beta-catenin nuclearization and activation of a Wnt-responsive promoter, and the magnitude of this response depends on the MSC differentiation state. Wnt 3a exposure inhibits MSC osteogenic differentiation, with decreased matrix mineralization and reduced alkaline phosphatase mRNA and activity. Wnt 3a treatment of fully osteogenically differentiated MSCs also suppresses osteoblastic marker gene expression. The Wnt 3a effect is accompanied by increased cell number, resulting from both increased proliferation and decreased apoptosis, particularly during expansion of undifferentiated MSCs. The osteo-suppressive effects of Wnt 3a are fully reversible, i.e., treatment prior to osteogenic induction does not compromise subsequent MSC osteogenesis. The results also showed that sFRP3 treatment attenuates some of the observed Wnt 3a effects on MSCs, and that inhibition of canonical Wnt signaling using a dominant negative TCF1 enhances MSC osteogenesis. Interestingly, expression of Wnt 5a, a non-canonical Wnt member, appeared to promote osteogenesis. Taken together, these findings suggest that canonical Wnt signaling functions in maintaining an undifferentiated, proliferating progenitor MSC population, whereas non-canonical Wnts facilitate osteogenic differentiation. Release from canonical Wnt regulation is a prerequisite for MSC differentiation. Thus, loss-/gain-of-function mutations of LRP5 would perturb Wnt signaling and depress/promote bone formation by affecting the progenitor cell pool. Elucidating Wnt regulation of MSC differentiation is important for their potential application in tissue regeneration.     

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.     

Zebrafish colgate/hdac1 functions in the non-canonical Wnt pathway during axial extension and in Wnt-independent branchiomotor neuron migration

Vertebrate gastrulation involves the coordinated movements of populations of cells. These movements include cellular rearrangements in which cells polarize along their medio-lateral axes leading to cell intercalations that result in elongation of the body axis. Molecular analysis of this process has implicated the non-canonical Wnt/Frizzled signaling pathway that is similar to the planar cell polarity pathway (PCP) in Drosophila. Here we describe a zebrafish mutant, colgate (col), which displays defects in the extension of the body axis and the migration of branchiomotor neurons. Activation of the non-canonical Wnt/PCP pathway in these mutant embryos by overexpressing DeltaNdishevelled, rho kinase2 and van gogh-like protein 2 (vangl2) rescues the extension defects suggesting that col acts as a positive regulator of the non-canonical Wnt/PCP pathway. Further, we show that col normally regulates the caudal migration of nVII facial hindbrain branchiomotor neurons and that the mutant phenotype can be rescued by misexpression of vangl2 independent of the Wnt/PCP pathway. We cloned the col locus and found that it encodes histone deacetylase1 (hdac1). Our previous results and studies by others have implicated hdac1 in repressing the canonical Wnt pathway. Here, we demonstrate novel roles for zebrafish hdac1 in activating non-canonical Wnt/PCP signaling underlying axial extension and in promoting Wnt-independent caudal migration of a subset of hindbrain branchiomotor neurons.     

The roles of canonical and non-canonical Wnt signaling in human de-differentiated articular chondrocytes

Osteoarthritis is the most prevalent form of arthritis in the world and it is becoming a major public health problem. Osteoarthritic chondrocytes undergo morphological and biochemical changes that lead to de-differentiation. The involvement of signaling pathways, such as the Wnt pathway, during cartilage pathology has been reported. Wnt signaling regulates critical biological processes. Wnt signals are transduced through at least three intracellular signaling pathways including the canonical Wnt/β-catenin pathway, the Wnt/Ca2 + pathway and the Wnt/planar cell polarity pathway. We investigated the involvement of the Wnt canonical and non-canonical pathways in human articular chondrocyte de-differentiation in vitro. Human articular chondrocytes were cultured through four passages with no treatment, or with sFRP3 treatment, an inhibitor of Wnt pathways, or with DKK1 treatment, an inhibitor of the canonical pathway. Chondrocyte-secreted markers and Wnt pathway components were analyzed using western blotting and qPCR. Inhibition of the Wnt pathway showed that the canonical Wnt signaling probably is responsible for inhibition of collagen II expression, activation of metalloproteinase 13 expression and regulation of Wnt7a and c-jun expression during chondrocyte de-differentiation in vitro. Our results also suggest that expressions of eNOS, Wnt5a and cyclinE1 are regulated by non-canonical Wnt signaling.     

Ror2 modulates the canonical Wnt signaling in lung epithelial cells through cooperation with Fzd2

Background  

Wnt signaling is mediated through 1) the beta-catenin dependent canonical pathway and, 2) the beta-catenin independent pathways. Multiple receptors, including Fzds, Lrps, Ror2 and Ryk, are involved in Wnt signaling. Ror2 is a single-span transmembrane receptor-tyrosine kinase (RTK). The functions of Ror2 in mediating the non-canonical Wnt signaling have been well established. The role of Ror2 in canonical Wnt signaling is not fully understood.     

Abnormal lens morphogenesis and ectopic lens formation in the absence of beta-catenin function

beta-Catenin plays a key role in cadherin-mediated cell adhesion as well as in canonical Wnt signaling. To study the role of beta-catenin during eye development, we used conditional Cre/loxP system in mouse to inactivate beta-catenin in developing lens and retina. Inactivation of beta-catenin does not suppress lens fate, but instead results in abnormal morphogenesis of the lens. Using BAT-gal reporter mice, we show that beta-catenin-mediated Wnt signaling is notably absent from lens and neuroretina throughout eye development. The observed defect is therefore likely due to the cytoskeletal role of beta-catenin, and is accompanied by impaired epithelial cell adhesion. In contrast, inactivation of beta-catenin in the nasal ectoderm, an area with active Wnt signaling, results in formation of crystallin-positive ectopic lentoid bodies. These data suggest that, outside of the normal lens, beta-catenin functions as a coactivator of canonical Wnt signaling to suppress lens fate.     

Heparan Sulfate 6-O-endosulfatases (Sulfs) Coordinate the Wnt Signaling Pathways to Regulate Myoblast Fusion during Skeletal Muscle Regeneration

Skeletal muscle regeneration is mediated by satellite cells (SCs). Upon injury, SCs undergo self-renewal, proliferation, and differentiation into myoblasts followed by myoblast fusion to form new myofibers. We previously showed that the heparan sulfate (HS) 6-O-endosulfatases (Sulf1 and -2) repress FGF signaling to induce SC differentiation during muscle regeneration. Here, we identify a novel role of Sulfs in myoblast fusion using a skeletal muscle-specific Sulf double null (Sulf^SK-DN) mouse. Regenerating Sulf^SK-DN muscles exhibit reduced canonical Wnt signaling and elevated non-canonical Wnt signaling. In addition, we show that Sulfs are required to repress non-canonical Wnt signaling to promote myoblast fusion. Notably, skeletal muscle-relevant non-canonical Wnt ligands lack HS binding capacity, suggesting that Sulfs indirectly repress this pathway. Mechanistically, we show that Sulfs reduce the canonical Wnt-HS binding and regulate colocalization of the co-receptor LRP5 with caveolin3. Therefore, Sulfs may increase the bioavailability of canonical Wnts for Frizzled receptor and LRP5/6 interaction in lipid raft, which may in turn antagonize non-canonical Wnt signaling. Furthermore, changes in subcellular distribution of active focal adhesion kinase (FAK) are associated with the fusion defect of Sulf-deficient myoblasts and upon non-canonical Wnt treatment. Together, our findings uncover a critical role of Sulfs in myoblast fusion by promoting antagonizing canonical Wnt signaling activities against the noncanonical Wnt pathway during skeletal muscle regeneration.