Wnt2 Regulates Progenitor Proliferation in the Developing Ventral Midbrain

Wnts are secreted, lipidated proteins that regulate multiple aspects of brain development, including dopaminergic neuron development. In this study, we perform the first purification and signaling analysis of Wnt2 and define the function of Wnt2 in ventral midbrain precursor cultures, as well as in Wnt2-null mice in vivo. We found that purified Wnt2 induces the phosphorylation of both Lrp5/6 and Dvl-2/3, and activates β-catenin in SN4741 dopaminergic cells. Moreover, purified Wnt2 increases progenitor proliferation, and the number of dopaminergic neurons in ventral midbrain precursor cultures. In agreement with these findings, analysis of the ventral midbrain of developing Wnt2-null mice revealed a decrease in progenitor proliferation and neurogenesis that lead to a decrease in the number of postmitotic precursors and dopaminergic neurons. Collectively, our observations identify Wnt2 as a novel regulator of dopaminergic progenitors and dopaminergic neuron development.     

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 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.     

Wnt-3a utilizes a novel low dose and rapid pathway that does not require casein kinase 1-mediated phosphorylation of Dvl to activate beta-catenin

The current view of canonical Wnt signalling is that following Wnt binding to its receptors (Frizzled-Lrp5/6), dishevelled (Dvl) becomes hyperphosphorylated, and the signal is transduced to the APC-GSK3beta-axin-beta-catenin multiprotein complex, which subsequently dissociates. As a result beta-catenin is not phosphorylated, escapes proteosomal degradation and activates its target genes after translocation to the nucleus. Here, we analyzed the importance of the Wnt-3a-induced phosphorylation and shift in electrophoretic migration of Dvl (PS-Dvl) for the activation of beta-catenin. Analysis of Wnt-3a time- and dose-responses in a dopaminergic cell line showed that beta-catenin is activated rapidly (within minutes) and at a low dose of Wnt-3a (1 ng/ml). Surprisingly, PS-Dvl appeared only after 30 min and at greater doses (> or =20 ng/ml) of Wnt-3a. Moreover, we found that a casein kinase 1 inhibitor (D4476) or siRNA for casein kinase 1 delta/epsilon (CK1delta/epsilon) blocked the Wnt-3a-induced PS-Dvl. Interestingly, CK1 inhibition or siRNA for CK1delta/epsilon did not ablate the activation of beta-catenin by Wnt-3a, indicating that there is a PS-Dvl-independent path to activate beta-catenin. The increase in beta-catenin activation by Wnt-3a (PS-Dvl-dependent or -independent) were blocked by Dickkopf1 (Dkk1), suggesting that the effect of Wnt-3a is in both cases mediated by Lrp5/6 receptors. Thus, our results show that Wnt-3a rapidly induce a partial activation of beta-catenin in the absence of PS-Dvl at low doses, while at high doses induce a full activation of beta-catenin in a PS-Dvl-dependent manner.     

Wnt-5a is involved in TGF-beta3-stimulated chondrogenic differentiation of chick wing bud mesenchymal cells

In this study we investigated whether signalling by TGF-beta3 and Wnt-5a cross-talk during chondrogenic differentiation of chick wing mesenchyme. Using differential display polymerase chain reaction screening, we found the expression of Wnt-5a to be significantly increased during transforming growth factor-beta3 (TGF-beta3)-induced precartilage condensation in mesenchyme micromass cultures. Transfection of cells with a Wnt-5a expression construct promoted precartilage condensation and chondrogenesis in micromass cultures, similar to that observed when chondrogenic-competent cells were exposed to TGF-beta3. Overexpression of Wnt-5a or treatment with TGF-beta3 stimulated the activation of protein kinase C-alpha (PKC-alpha) and p38 mitogen-activated protein kinase (MAPK), both positive regulators of chondrogenic differentiation. Inactivation of PKC-alpha and p38 MAPK by specific inhibitors abrogated chondrogenesis stimulated by both TGF-beta3 and Wnt-5a. Similarly, partial reduction in TGF-beta3-induced Wnt-5a expression by small interfering RNA resulted in decreased activities of PKC-alpha and p38 MAPK, and abolished the chondro-stimulatory effect of TGF-beta3. Collectively, these findings indicate that Wnt-5a, a non-canonical Wnt, can mediate the chondro-stimulatory effect of TGF-beta3 through upregulation of PKC-alpha and p38MAPK signaling.     

Stromelysin-1 and mesothelin are differentially regulated by Wnt-5a and Wnt-1 in C57mg mouse mammary epithelial cells

Background  

The Wnt signal transduction pathway is important in a wide variety of developmental processes as well as in the genesis of human cancer. Vertebrate Wnt pathways can be functionally separated into two classes, the canonical Wnt/beta-catenin pathway and the non-canonical Wnt/Ca²⁺ pathway. Supporting differences in Wnt signaling, gain of function of Wnt-1 in C57mg mouse mammary epithelial cells leads to their morphological transformation while loss of function of Wnt-5a leads to the same transformation. Many downstream target genes of the Wnt/beta-catenin pathway have been identified. In contrast, little is known about the Wnt/Ca²⁺ pathway and whether it regulates gene expression.     

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.     

Analysis of the expression and function of Wnt-5a and Wnt-5b in developing and regenerating axolotl (Ambystoma mexicanum) limbs

Urodele amphibians are unique adult vertebrates because they are able to regenerate body parts after amputation. Studies of urodele limb regeneration, the key model system for vertebrate regeneration, have led to an understanding of the origin of blastema cells and the importance of positional interactions between blastema cells in the control of growth and pattern formation. Progress is now being made in the identification of the signaling pathways that regulate dedifferentiation, blastema morphogenesis, growth and pattern formation. Members of the Wnt family of secreted proteins are expressed in developing and regenerating limbs, and have the potential to control growth, pattern formation and differentiation. We have studied the expression of two non-canonical Wnt genes, Wnt-5a and Wnt-5b . We report that they are expressed in equivalent patterns during limb development and limb regeneration in the axolotl ( Ambystoma mexicanum ), and during limb development in other tetrapods, implying conservation of function. Our analysis of the effects of ectopic Wnt-5a expression is consistent with the hypothesis that canonical Wnt signaling functions during the early stages of regeneration to control the dedifferentiation of stump cells giving rise to the regeneration-competent cells of the blastema.     

Wnt5a controls neurite development in olfactory bulb interneurons

Neurons born in the postnatal SVZ (subventricular zone) must migrate a great distance before becoming mature interneurons of the OB (olfactory bulb). During migration immature OB neurons maintain an immature morphology until they reach their destination. While the morphological development of these cells must be tightly regulated, the cellular pathways responsible are still largely unknown. Our results show that the non-canonical Wnt pathway induced by Wnt5a is important for the morphological development of OB interneurons both in vitro and in vivo. Additionally, we demonstrate that non-canonical Wnt signalling works in opposition to canonical Wnt signalling in neural precursors from the SVZ in vitro. This represents a novel role for Wnt5a in the development of OB interneurons and suggests that canonical and non-canonical Wnt pathways dynamically oppose each other in the regulation of dendrite maturation.     

Both Canonical and Non-Canonical Wnt Signaling Independently Promote Stem Cell Growth in Mammospheres

The characterization of mammary stem cells, and signals that regulate their behavior, is of central importance in understanding developmental changes in the mammary gland and possibly for targeting stem-like cells in breast cancer. The canonical Wnt/β-catenin pathway is a signaling mechanism associated with maintenance of self-renewing stem cells in many tissues, including mammary epithelium, and can be oncogenic when deregulated. Wnt1 and Wnt3a are examples of ligands that activate the canonical pathway. Other Wnt ligands, such as Wnt5a, typically signal via non-canonical, β-catenin-independent, pathways that in some cases can antagonize canonical signaling. Since the role of non-canonical Wnt signaling in stem cell regulation is not well characterized, we set out to investigate this using mammosphere formation assays that reflect and quantify stem cell properties. Ex vivo mammosphere cultures were established from both wild-type and Wnt1 transgenic mice and were analyzed in response to manipulation of both canonical and non-canonical Wnt signaling. An increased level of mammosphere formation was observed in cultures derived from MMTV-Wnt1 versus wild-type animals, and this was blocked by treatment with Dkk1, a selective inhibitor of canonical Wnt signaling. Consistent with this, we found that a single dose of recombinant Wnt3a was sufficient to increase mammosphere formation in wild-type cultures. Surprisingly, we found that Wnt5a also increased mammosphere formation in these assays. We confirmed that this was not caused by an increase in canonical Wnt/β-catenin signaling but was instead mediated by non-canonical Wnt signals requiring the receptor tyrosine kinase Ror2 and activity of the Jun N-terminal kinase, JNK. We conclude that both canonical and non-canonical Wnt signals have positive effects promoting stem cell activity in mammosphere assays and that they do so via independent signaling mechanisms.     

Wnt-4 activates the canonical beta-catenin-mediated Wnt pathway and binds Frizzled-6 CRD: functional implications of Wnt/beta-catenin activity in kidney epithelial cells

The Wnt signaling pathway is central to the development of all animals and to cancer progression, yet largely unknown are the pairings of secreted Wnt ligands to their respective Frizzled transmembrane receptors or, in many cases, the relative contributions of canonical (beta-catenin/LEF/TCF) versus noncanonical Wnt signals. Specifically, in the kidney where Wnt-4 is essential for the mesenchymal to epithelial transition that generates the tissue's collecting tubules, the corresponding Frizzled receptor(s) and downstream signaling mechanism(s) are unclear. In this report, we addressed these issues using Madin-Darby Canine Kidney (MDCK) cells, which are competent to form tubules in vitro. Employing established reporter constructs of canonical Wnt/beta-catenin pathway activity, we have determined that MDCK cells are highly responsive to Wnt-4, -1, and -3A, but not to Wnt-5A and control conditions, precisely reflecting functional findings from Wnt-4 null kidney mesenchyme ex vivo rescue studies. We have confirmed that Wnt-4's canonical signaling activity in MDCK cells is mediated by downstream effectors of the Wnt/beta-catenin pathway using beta-Engrailed and dnTCF-4 constructs that suppress this pathway. We have further found that MDCK cells express the Frizzled-6 receptor and that Wnt-4 forms a biochemical complex with the Frizzled-6 CRD. Since Frizzled-6 did not appear to transduce Wnt-4's canonical signal, data supported recently by Golan et al., there presumably exists another as yet unknown Frizzled receptor(s) mediating Wnt-4 activation of beta-catenin/LEF/TCF. Finally, we report that canonical Wnt/beta-catenin signals cells help maintain cell growth and survival in MDCK cells but do not contribute to standard HGF-induced (nonphysiologic) tubule formation. Our results in combination with work from Xenopus laevis (not shown) lead us to believe that Wnt-4 binds both canonical and noncanonical Frizzled receptors, thereby activating Wnt signaling pathways that may each contribute to kidney tubulogenesis.     

Wnt5a regulates ventral midbrain morphogenesis and the development of A9-A10 dopaminergic cells in vivo

Wnt5a is a morphogen that activates the Wnt/planar cell polarity (PCP) pathway and serves multiple functions during development. PCP signaling controls the orientation of cells within an epithelial plane as well as convergent extension (CE) movements. Wnt5a was previously reported to promote differentiation of A9-10 dopaminergic (DA) precursors in vitro. However, the signaling mechanism in DA cells and the function of Wnt5a during midbrain development in vivo remains unclear. We hereby report that Wnt5a activated the GTPase Rac1 in DA cells and that Rac1 inhibitors blocked the Wnt5a-induced DA neuron differentiation of ventral midbrain (VM) precursor cultures, linking Wnt5a-induced differentiation with a known effector of Wnt/PCP signaling. In vivo, Wnt5a was expressed throughout the VM at embryonic day (E)9.5, and was restricted to the VM floor and basal plate by E11.5-E13.5. Analysis of Wnt5a-/- mice revealed a transient increase in progenitor proliferation at E11.5, and a precociously induced NR4A2+ (Nurr1) precursor pool at E12.5. The excess NR4A2+ precursors remained undifferentiated until E14.5, when a transient 25% increase in DA neurons was detected. Wnt5a-/- mice also displayed a defect in (mid)brain morphogenesis, including an impairment in midbrain elongation and a rounded ventricular cavity. Interestingly, these alterations affected mostly cells in the DA lineage. The ventral Sonic hedgehog-expressing domain was broadened and flattened, a typical CE phenotype, and the domains occupied by Ngn2+ DA progenitors, NR4A2+ DA precursors and TH+ DA neurons were rostrocaudally reduced and laterally expanded. In summary, we hereby describe a Wnt5a regulation of Wnt/PCP signaling in the DA lineage and provide evidence for multiple functions of Wnt5a in the VM in vivo, including the regulation of VM morphogenesis, DA progenitor cell division, and differentiation of NR4A2+ DA precursors.     

Increase in Proliferation and Differentiation of Neural Progenitor Cells Isolated from Postnatal and Adult Mice Brain by Wnt-3a and Wnt-5a

Wnt signaling is implicated in the control of cell growth and differentiation during CNS development. These findings are based on studies of mouse and chick models. However, the action of Wnt signaling, at the cellular level, is poorly understood. In this study, we investigated the roles of Wnt-3a and Wnt-5a on differentiation and proliferation of postnatal neural progenitor cells (NPCs) in mice.NPCs were isolated from the subventricular zone (SVZ) of PN-1 and adult ICR mice. Plasmids containing active Wnt-3a or Wnt-5a were transfected to NPCs; their effects on the formation of neurospheres and differentiation into neuronal cells were then determined. Transfection of Wnt-3a and Wnt-5a plasmids promoted regeneration of neurospheres and differentiation into Map2-positive cells, and decreased differentiation into GFAP-positive cells. The conditioned media obtained from Wnt-3a or Wnt-5a transfected NPCs showed similar effects on differentiation of NPCs with cDNA transfection, although the magnitude of stimulatory effect was less than that by plasmid transfection. Wnt-3a and Wnt-5a transfection did not affect Brdu incorporation of neuronal or glial progenitors in differentiation media. Wnt-3a and Wnt-5a plasmid transfection and the treatment of Wnt-3a and Wnt-5a conditioned media increased $β −cateninlevelsinNPCs. Wnt−3ahadagreatereffecton β $-catenin levels than Wnt-5a. The PKC inhibitor completely blocked the Wnt-5a effect on neuronal differentiation in NPCs. These findings suggest that Wnt-3a and Wnt-5a each have distinct effects on the proliferation and differentiation of NPCs in postnatal mice.     

Wnt-5a increases NO and modulates NMDA receptor in rat hippocampal neurons

Wnt signaling has a crucial role in synaptic function at the central nervous system. Here we evaluate whether Wnts affect nitric oxide (NO) generation in hippocampal neurons. We found that non-canonical Wnt-5a triggers NO production; however, Wnt-3a a canonical ligand did not exert the same effect. Co-administration of Wnt-5a with the soluble Frizzled related protein-2 (sFRP-2) a Wnt antagonist blocked the NO production. Wnt-5a activates the non-canonical Wnt/Ca²⁺ signaling through a mechanism that depends on Ca²⁺ release from Ryanodine-sensitive internal stores. The increase in NO levels evoked by Wnt-5a promotes the insertion of the GluN2B subunit of the NMDA receptor (NMDAR) into the neuronal cell surface. To the best of our knowledge, this is the first time that Wnt-5a signaling is related to NO production, which in turn increases NMDARs trafficking to the cell surface.     

Phasic modulation of Wnt signaling enhances cardiac differentiation in human pluripotent stem cells by recapitulating developmental ontogeny

Cardiomyocytes (CMs) derived from human pluripotent stem cells (hPSCs) offer immense value in studying cardiovascular regenerative medicine. However, intrinsic biases and differential responsiveness of hPSCs towards cardiac differentiation pose significant technical and logistic hurdles that hamper human cardiomyocyte studies. Tandem modulation of canonical and non-canonical Wnt signaling pathways may play a crucial role in cardiac development that can efficiently generate cardiomyocytes from pluripotent stem cells. Our Wnt signaling expression profiles revealed that phasic modulation of canonical/non-canonical axis enabled orderly recapitulation of cardiac developmental ontogeny. Moreover, evaluation of 8 hPSC lines showed marked commitment towards cardiac-mesoderm during the early phase of differentiation, with elevated levels of canonical Wnts (Wnt3 and 3a) and Mesp1. Whereas continued activation of canonical Wnts was counterproductive, its discrete inhibition during the later phase of cardiac differentiation was accompanied by significant up-regulation of non-canonical Wnt expression (Wnt5a and 11) and enhanced Nkx2.5⁺ (up to 98%) populations. These Nkx2.5⁺ populations transited to contracting cardiac troponin T-positive CMs with up to 80% efficiency. Our results suggest that timely modulation of Wnt pathways would transcend intrinsic differentiation biases of hPSCs to consistently generate functional CMs that could facilitate their scalable production for meaningful clinical translation towards personalized regenerative medicine.     

Noncanonical Wnt-4 signaling enhances bone regeneration of mesenchymal stem cells in craniofacial defects through activation of p38 MAPK

Mesenchymal stem cells (MSCs) are multipotent cells that can be differentiated into osteoblasts and provide an excellent cell source for bone regeneration and repair. Recently, the canonical Wnt/beta-catenin signaling pathway has been found to play a critical role in skeletal development and osteogenesis, implying that Wnts can be utilized to improve de novo bone formation mediated by MSCs. However, it is unknown whether noncanonical Wnt signaling regulates osteogenic differentiation. Here, we find that Wnt-4 enhanced in vitro osteogenic differentiation of MSCs isolated from human adult craniofacial tissues and promoted bone formation in vivo. Whereas Wnt-4 did not stabilize beta-catenin, it activated p38 MAPK in a novel noncanonical signaling pathway. The activation of p38 was dependent on Axin and was required for the enhancement of MSC differentiation by Wnt-4. Moreover, using two different models of craniofacial bone injury, we found that MSCs genetically engineered to express Wnt-4 enhanced osteogenesis and improved the repair of craniofacial defects in vivo. Taken together, our results reveal that noncanonical Wnt signaling could also play a role in osteogenic differentiation. Wnt-4 may have a potential use in improving bone regeneration and repair of craniofacial defects.     

Wnt signal transduction pathways

The Wnt signaling pathway is an ancient and evolutionarily conserved pathway that regulates crucial aspects of cell fate determination, cell migration, cell polarity, neural patterning and organogenesis during embryonic development. The Wnts are secreted glycoproteins and comprise a large family of nineteen proteins in humans hinting to a daunting complexity of signaling regulation, function and biological output. To date major signaling branches downstream of the Fz receptor have been identified including a canonical or Wnt/β-catenin dependent pathway and the non-canonical or β-catenin-independent pathway which can be further divided into the Planar Cell Polarity and the Wnt/Ca²⁺ pathways, and these branches are being actively dissected at the molecular and biochemical levels. In this review, we will summarize the most recent advances in our understanding of these Wnt signaling pathways and the role of these pathways in regulating key events during embryonic patterning and morphogenesis.Key words: Wnt, frizzled, dishevelled, canonical, non-canonical, β-catenin, Planar Cell Polarity     

Dkk1 regulates ventral midbrain dopaminergic differentiation and morphogenesis

Dickkopf1 (Dkk1) is a Wnt/β-catenin inhibitor that participates in many processes during embryonic development. One of its roles during embryogenesis is to induce head formation, since Dkk1-null mice lack head structures anterior to midbrain. The Wnt/β-catenin pathway is also known to regulate different aspects of ventral midbrain (VM) dopaminergic (DA) neuron development and, in vitro, Dkk1-mediated inhibition of the Wnt/β-catenin pathway improves the DA differentiation in mouse embryonic stem cells (mESC). However, the in vivo function of Dkk1 on the development of midbrain DA neurons remains to be elucidated. Here we examined Dkk1(+/-) embryos and found that Dkk1 is required for the differentiation of DA precursors/neuroblasts into DA neurons at E13.5. This deficit persisted until E17.5, when a defect in the number and distribution of VM DA neurons was detected. Furthermore, analysis of the few Dkk1(-/-) embryos that survived until E17.5 revealed a more severe loss of midbrain DA neurons and morphogenesis defects. Our results thus show that Dkk1 is required for midbrain DA differentiation and morphogenesis.