Regulation Of Canonical Wnt Signaling Research Articles

High-Temperature Requirement A1 (Htra1) - A Novel Regulator of Canonical Wnt Signaling

Different cancer types as well as many other diseases are caused by aberrant activation of the canonical Wnt signal transduction pathway, and it is especially implicated in the development and progression of colorectal cancer (CRC). The main effector protein of the canonical Wnt signaling cascade is β-catenin, which binds to the T- cell factor/lymphoid enhancer factor (TCF/LEF) and triggers the activation of Wnt target genes. Here, we identify the serine protease High-Temperature Requirement A1 (HTRA1) as a novel component of the canonical Wnt pathway. We show that the HTRA1 protein inhibits the Wnt/β-catenin signaling, in both paracrine and autocrine manners, and affects the expression of several Wnt target genes. Moreover, HTRA1 forms a complex with β-catenin and reduces the proliferation rates of cells. Taken together, our findings indicate that HTRA1 functions as a novel suppressor of the canonical Wnt signaling pathway.

Coupling optogenetics and light-sheet microscopy, a method to study Wnt signaling during embryogenesis

Optogenetics allows precise, fast and reversible intervention in biological processes. Light-sheet microscopy allows observation of the full course of Drosophila embryonic development from egg to larva. Bringing the two approaches together allows unparalleled precision into the temporal regulation of signaling pathways and cellular processes in vivo. To develop this method, we investigated the regulation of canonical Wnt signaling during anterior-posterior patterning of the Drosophila embryonic epidermis. Cryptochrome 2 (CRY2) from Arabidopsis Thaliana was fused to mCherry fluorescent protein and Drosophila β–catenin to form an easy to visualize optogenetic switch. Blue light illumination caused oligomerization of the fusion protein and inhibited downstream Wnt signaling in vitro and in vivo. Temporal inactivation of β–catenin confirmed that Wnt signaling is required not only for Drosophila pattern formation, but also for maintenance later in development. We anticipate that this method will be easily extendable to other developmental signaling pathways and many other experimental systems.

Cinnamaldehyde induces cell apoptosis mediated by a novel circular RNA hsa_circ_0043256 in non-small cell lung cancer

Cinnamaldehyde (CA), the primary chemical component of the Chinese traditional herb Cinnamomum cassia, is an effective cytotoxic agent against various human cancers. Our previous study indicated that CA could trigger apoptosis in three kinds of non-small cell lung cancer (NSCLC) cells. However, CA mechanism of action in NSCLC has not been unveiled completely. Herein, we showed that a novel circular RNA hsa_circ_0043256 was upregulated in NSCLC cells in response to CA treatment, as detected by microarray and real-time PCR. Hsa_circ_0043256 could inhibit cell proliferation and induce apoptosis, while hsa_circ_0043256 knock-down could promote cell proliferation and restrain apoptosis induced by CA. Bioinformatics analysis predicted that hsa_circ_0043256 could work as a miR-1252 sponge, which could in turn directly target a vital negative regulator of Canonical Wnt signaling, Itchy E3 ubiquitin protein ligase (ITCH), as validated by dual-luciferase assay. Western blot results further confirmed that hsa_circ_0043256 could upregulate ITCH expression, whereas miR-1252 could partially abolish this effect. Interestingly, hsa_circ_0043256 knock-down could weaken Wnt/β-catenin pathway inhibition induced by CA. Finally, we discovered that CA induced apoptosis and meanwhile upregulated hsa_circ_0043256 expression in vivo. Immunohistochemical analysis revealed that ITCH expression was positively association with hsa_circ_0043256 levels. Above all, we characterized a new mechanism mediated by hsa_circ_0043256/miR-1252/ITCH axis in CA function against NSCLC, providing a novel insight into lung cancer therapy.

Directed differentiation and long-term maintenance of epicardial cells derived from human pluripotent stem cells under fully defined conditions.

Here, we describe how to efficiently direct human pluripotent stem cells (hPSCs) differentiation into self-renewing epicardial cells in a completely defined, xeno-free system by temporal modulation of regulators of canonical Wnt signaling. Appropriate differentiation-stage-specific application of Gsk3 inhibitor, Wnt inhibitor, and Gsk3 inhibitor (GiWiGi) is sufficient to produce cells expressing epicardial markers and exhibiting epicardial phenotypes with a high yield and purity from multiple hPSC lines in 16 d. Characterization of differentiated cells is performed via flow cytometry and immunostaining to assess quantitative expression and localization of epicardial cell-specific proteins. In vitro differentiation into fibroblasts and smooth muscle cells (SMCs) is also described. In addition, culture in the presence of transforming growth factor (TGF)-β inhibitors allows long-term expansion of hPSC-derived epicardial cells (for at least 25 population doublings). Functional human epicardial cells differentiated via this protocol may constitute a potential cell source for heart disease modeling, drug screening, and cell-based therapeutic applications.

Extracellular LDLR repeats modulate Wnt signaling activity by promoting LRP6 receptor endocytosis mediated by the Itch E3 ubiquitin ligase.

The LOW-density lipoprotein related protein 6 (LRP6) receptor is an important effector of canonical Wnt signaling, a developmental pathway, whose dysregulation has been implicated in various diseases including cancer. The membrane proximal low-density lipoprotein (LDL) receptor repeats in LRP6 exhibit homology to ligand binding repeats in the LDL receptor (LDLR), but lack known function. We generated single amino acid substitutions of LRP6-LDLR repeat residues, which are highly conserved in the human LDLR and mutated in patients with Familial Hypercholesteremia (FH). These substitutions negatively impacted LRP6 internalization and activation of Wnt signaling. By mass spectrometry, we observed that the Itch E3 ubiquitin ligase associated with and ubiquitinated wild type LRP6 but not the LDLR repeat mutants. These findings establish the involvement of LRP6-LDLR repeats in the regulation of canonical Wnt signaling.

Coupling optogenetics and light-sheet microscopy to study signal transduction in vivo

Optogenetics allows precise, fast and reversible intervention in biological processes. Coupled with light-sheet microscopy, this approach allows unparalleled insight into the regulation of signaling pathways and cellular processes from a spatial and temporal perspective in vivo. To develop this method, we investigated the spatial and temporal regulation of canonical Wnt signaling during anterior-posterior patterning of the Drosophila embryonic epidermis. Cryptochrome C (Cry2) from Arabidopsis Thaliana was fused to mCherry fluorescent protein and Drosophila β-catenin to form an easy to visualize optogenetic switch. Blue light illumination caused reversible oligomerization of the fusion protein and inhibited downstream Wnt signaling in vitro and in vivo. Temporal inactivation of β-catenin showed that Wnt signaling is required not only for Drosophila patterning formation, but also for maintenance throughout development. By applying light-sheet microscopy to activate the pathway and then record subsequent behavior in toto, we show precise spatial regulation of Wnt signaling in vivo. We anticipate that this method will be easily extendable to other developmental signaling pathways and many other experimental systems.

Increased Runx2 expression associated with enhanced Wnt signaling in PDLLA internal fixation for fracture treatment.

Poly-D-L lactide (PDLLA) biodegradable implants to heal fractures are widely applied in orthopedic surgeries. However, whether the process of fracture healing is regulated differently when PDLLA is used compared with traditional metal materials remains unclear. Runt-related transcription factor 2 (Runx2) and canonical Wnt signaling are essential and may interact reciprocally in the regulation of osteogenesis during bone repair. In the present study, a rat femoral open osteotomy model was used to compare the curative efficacy of a PDLLA rod and Kirschner wire under intramedullary fixation for fracture treatment. The dynamic expression of Runx2 and key components of the canonical Wnt signaling in callus tissue during fracture healing was also investigated. The results of the current study indicate that at weeks 4 and 6 following fixation, the callus bone structural parameters of microCT were significantly improved by PDLLA rod compared to that of Kirschner wire. In addition, at weeks 4 and 6 after fixation, the protein and mRNA expression of Runx2 and the positive regulators of canonical Wnt signaling, such as Wnts and β-catenin, were significantly increased. However, the protein and mRNA expression levels of the negative regulators of canonical Wnt signaling, such as glycogen synthase kinase-3β, were significantly decreased in callus tissue when treated with PDLLA rod compared with Kirschner wire. Collectively, these data indicate that compared to the traditional metal material, using PDLLA internal fixation for fracture treatment may further improve bone formation, which is associated with the increased expression of Runx2 and the enhancement of canonical Wnt signaling.

Chemical biology reveals CARF as a positive regulator of canonical Wnt signaling by promoting TCF/\u03b2-catenin transcriptional activity

Wnt/β-catenin signaling regulates multiple biological processes and aberration of this pathway is frequently observed in human cancers. Previously, we uncovered NC043 as a small-molecule inhibitor of Wnt/β-catenin signaling. Here, we identified CARF as the cellular target of NC043. We found that NC043 binds directly to CARF through forming a covalent bond with the Cys-516 residue of CARF. Further study revealed that CARF interacts with Dvl, which potentiates the Dvl–c-Jun–β-catenin–TCF transcriptional complex and thus promotes Wnt signaling activation. NC043 could disrupt the interaction between CARF and Dvl, thereby impairing Wnt signal transduction. In line with this, knockdown of CARF in zebrafish leads to impairment of embryonic development, hematopoietic stem cell generation and caudal fin regeneration. Collectively, we identified CARF as the cellular target of NC043 and revealed CARF as a positive regulator of Wnt/β-catenin signal transduction.

Role of Androgen Dependent TFPI-Regulating Protein (ADTRP) in Vascular Development and Function

We used in vitro and in vivo models to characterize the physiological role of the novel protein encoded by C6ORF105. This gene’s expression is androgen-responsive, and the encoded protein is predicted to be palmitoylated and membrane multi-spanning. Previously we showed that C6ORF105 expression co-regulates with tissue factor pathway inhibitor (TFPI)in human endothelial cells (EC); hence we named this protein "androgen-dependent TFPI-regulating protein" (ADTRP). Using in vitro cell-based TOP-Flash reporter assay we identified ADTRP as a negative regulator of canonical Wnt signaling in human cells. Overexpressing ADTRP in HEK293T cells inhibited the activity of beta-catenin/TCF-dependent transcriptional reporter, while silencing ADTRP increased the expression of Wnt target genes LEF-1, AXIN-2, IL-8 and DKK-2 in EA.hy926 EC line and HUVEC. Addition of LiCl showed that the effect of ADTRP was upstream of GSK3, therefore we focused the investigations on the Wnt signalosome proteins. ADTRP expression in HEK293T cells led to decreased phosphorylation of Wnt co-receptor LRP6, suggesting that ADTRP can affect this critical membrane-located event of Wnt signaling. Furthermore, ADTRP expression in reporter cells transfected with a constitutively phosphorylated form of LRP6 (LRP6DN mutant) inhibited Wnt3a- induced signaling, which suggests that ADTRP can interfere with events downstream of LRP6 phosphorylation, such as Axin-2 binding. Altogether, these data indicate that the Wnt signaling inhibitory activity of ADTRP takes place at the plasma membrane level. Site directed mutagenesis of the predicted palmitoylation site Cys61 showed that Wnt inhibitory effects of ADTRP require palmitoyl-mediated anchoring, highlighting the importance of proper membrane location of ADTRP for Wnt pathway inhibition. In vivo morpholino-based knockdown of adtrp in zebrafish embryos produced aberrant angiogenesis, defective branching and ruptured vessels, hemorrhage spots, pericardial edema and slow heart-beat, all reminiscent of defects caused by activation of canonical Wnt signaling. Indeed, adtrp knock down increased Wnt mediated lef-1 and pax-2a as well as mmp2 and mmp9 mRNA expression. Co-injection of ADTRP mRNA partially recovered the adtrp morpholino- induced morphologic abnormalities. Also, knock down of adtrp in a Wnt reporter zebrafish showed increased expression of ectopic Wnt signaling. Furthermore, our recently established Adtrp-/- mice also display some typical Wnt-mediated vascular defects, including: (i) abnormal patterning, increased capillary tortuosity, abnormal branching and increased density of the capillary network; (ii) dilated vessels, especially venules and veins; (iii) increased leakeage of permeability tracers (Evans blue and fluorescent dextran) without evident changes in endothelial junctions; (iv) hemorrhage spots in the skin, meningeal layers, heart, bladder and kidneys; (v) intravascular and interstitial fibrin deposition in the lung, liver and kidney. ADTRP deficiency decreased plasma TFPI antigen by ~2-times. Furthermore, TFPI antigen and anticoagulant activity in lung extracts and isolated lung EC were similarly decreased, which confirms our previous in vitro data. We aslo noticed increased tail bleeding time (>500 sec vs. 200 sec in WT littermates) and blood volume loss, which likely was caused by increased dilation of the tail vein. Gene expression analysis of whole organs showed upregulation of Wnt target genes involved in vascular contractility (Nos3), and extracellular matrix remodeling (Mmp2). Similarly, skin fibroblasts and lung EC isolated from Adtrp-/- mice showed increased expression of Wnt target genes (Lef-1, Cyclin D, Dkk2, c-Myc), which indicates constitutive activation of canonical Wnt signaling. In conclusion, we used genetic animal models and cell culture systems to show for the first time that the novel protein ADTRP plays major roles in vascular development and function. Lack of, or low levels of ADTRP associate with activation of coagulation and vascular development defects, which may be due, at least in part, to intrinsic high levels of ectopic canonical Wnt signaling. DisclosuresNo relevant conflicts of interest to declare.

Activation/Proliferation-associated Protein 2 (Caprin-2) Positively Regulates CDK14/Cyclin Y-mediated Lipoprotein Receptor-related Protein 5 and 6 (LRP5/6) Constitutive Phosphorylation

Low-density lipoprotein receptor-related proteins 5 and 6 (LRP5/6) are co-receptors for Wnt ligands. Upon ligand binding, LRP5/6 undergo glycogen synthase kinase 3 (GSK3)/casein kinase I (CKI)-mediated phosphorylation at multiple PPP(S/T)P motifs in the intracellular domain, which is essential for canonical Wnt signal transduction. On the other hand, in the Wnt-off state, the mitosis-specific CDK14-Cyclin Y kinase complex phosphorylates Ser-1490 of LRP5/6 at G2/M, thereby priming the receptor for Wnt-induced phosphorylation. However, it remains unclear how CDK14/Cyclin Y is recruited to LRP5/6 and whether there are other cofactors involved in this process. Previously, we identified Caprin-2 as a positive regulator of canonical Wnt signaling by promoting GSK3-depedent LRP5/6 phosphorylation upon Wnt stimulation. Here we uncovered that Caprin-2 positively regulates constitutive LRP5/6 Ser-1490 phosphorylation by complexing with CDK14/Cyclin Y. Caprin-2-mediated LRP5/6 phosphorylation is cell cycle-dependent in a pattern similar to that of CDK14/Cyclin Y-dependent LRP5/6 phosphorylation. Moreover, knockdown of Caprin-2 disrupts not only the interaction between CDK14 and Cyclin Y but also the interaction between CDK14/Cyclin Y and LRP6. Overall, our findings revealed an unrecognized role of Caprin-2 in facilitating LRP5/6 constitutive phosphorylation at G2/M through forming a quaternary complex with CDK14, Cyclin Y, and LRP5/6.

LMO2 attenuates tumor growth by targeting the Wnt signaling pathway in breast and colorectal cancer.

The proto-oncogene LIM-domain only 2 (lmo2) was traditionally considered to be a pivotal transcriptional regulator in hematopoiesis and leukemia. Recently, the cytosolic localization of LMO2 was revealed in multiple epithelial tissues and a variety of solid tumors. However, the function of LMO2 in these epithelia and solid tumors remains largely unclear. The Wnt signaling pathway is a crucial determinant of development, and abnormalities in several key segments of this pathway contribute to oncogenesis. The current study demonstrated that LMO2 participates in the regulation of canonical Wnt signaling in the cytoplasm by binding to Dishevelled-1/2 (DVL-1/2) proteins. These interactions occurred at the PDZ domain of Dishevelled, and LMO2 subsequently attenuated the activation of the key factor β-catenin in the canonical Wnt signaling pathway. Meanwhile, significantly decreased expression of LMO2 was detected in breast and colorectal cancers, and the downregulation of LMO2 in these cells increased cell proliferation and reduced apoptosis. Taken together, the data in this study revealed a novel crosstalk between LMO2 and the Wnt signaling pathway during tumorigenesis and suggested that LMO2 might be a tumor suppressor in certain solid tumors, in contrast to its traditional oncogenic role in the hematopoietic system.

Kaiso overexpression promotes intestinal inflammation and potentiates intestinal tumorigenesis in ApcMin/+ mice

Constitutive Wnt/β-catenin signaling is a key contributor to colorectal cancer (CRC). Although inactivation of the tumor suppressor adenomatous polyposis coli (APC) is recognized as an early event in CRC development, it is the accumulation of multiple subsequent oncogenic insults facilitates malignant transformation. One potential contributor to colorectal carcinogenesis is the POZ-ZF transcription factor Kaiso, whose depletion extends lifespan and delays polyp onset in the widely used ApcMin/+ mouse model of intestinal cancer. These findings suggested that Kaiso potentiates intestinal tumorigenesis, but this was paradoxical as Kaiso was previously implicated as a negative regulator of Wnt/β-catenin signaling. To resolve Kaiso's role in intestinal tumorigenesis and canonical Wnt signaling, we generated a transgenic mouse model (KaisoTg/+) expressing an intestinal-specific myc-tagged Kaiso transgene. We then mated KaisoTg/+ and ApcMin/+ mice to generate KaisoTg/+:ApcMin/+ mice for further characterization. KaisoTg/+:ApcMin/+ mice exhibited reduced lifespan and increased polyp multiplicity compared to ApcMin/+ mice. Consistent with this murine phenotype, we found increased Kaiso expression in human CRC tissue, supporting a role for Kaiso in human CRC. Interestingly, Wnt target gene expression was increased in KaisoTg/+:ApcMin/+ mice, suggesting that Kaiso's function as a negative regulator of canonical Wnt signaling, as seen in Xenopus, is not maintained in this context. Notably, KaisoTg/+:ApcMin/+ mice exhibited increased inflammation and activation of NFκB signaling compared to their ApcMin/+ counterparts. This phenotype was consistent with our previous report that KaisoTg/+ mice exhibit chronic intestinal inflammation. Together our findings highlight a role for Kaiso in promoting Wnt signaling, inflammation and tumorigenesis in the mammalian intestine.

WISP1 aggravates osteoarthritis by modulation of TGF-beta signaling and positive regulation of canonical Wnt signaling

WISP1 aggravates osteoarthritis by modulation of TGF-beta signaling and positive regulation of canonical Wnt signaling

Cranial Nerve Development Requires Co-Ordinated Shh and Canonical Wnt Signaling

Cranial nerves govern sensory and motor information exchange between the brain and tissues of the head and neck. The cranial nerves are derived from two specialized populations of cells, cranial neural crest cells and ectodermal placode cells. Defects in either cell type can result in cranial nerve developmental defects. Although several signaling pathways are known to regulate cranial nerve formation our understanding of how intercellular signaling between neural crest cells and placode cells is coordinated during cranial ganglia morphogenesis is poorly understood. Sonic Hedgehog (Shh) signaling is one key pathway that regulates multiple aspects of craniofacial development, but whether it co-ordinates cranial neural crest cell and placodal cell interactions during cranial ganglia formation remains unclear. In this study we examined a new Patched1 (Ptch1) loss-of-function mouse mutant and characterized the role of Ptch1 in regulating Shh signaling during cranial ganglia development. Ptch1Wig/ Wig mutants exhibit elevated Shh signaling in concert with disorganization of the trigeminal and facial nerves. Importantly, we discovered that enhanced Shh signaling suppressed canonical Wnt signaling in the cranial nerve region. This critically affected the survival and migration of cranial neural crest cells and the development of placodal cells as well as the integration between neural crest and placodes. Collectively, our findings highlight a novel and critical role for Shh signaling in cranial nerve development via the cross regulation of canonical Wnt signaling.

To cleave or not to cleave: the proteolytic and non‐proteolytic functions of ADAMs in neural crest induction (609.11)

The induction and subsequent development of the neural crest require highly coordinated interactions among genes and pathways. The disintegrin metalloproteinase (ADAM) family proteases have emerged as key regulators of gene and pathway interactions, mainly through their functions as proteolytic “molecular scissors”. Here we show that both the proteolytic and non‐proteolytic activities of ADAMs are important for neural crest induction. Knockdown of either ADAM13 or ADAM19 inhibits the induction of cranial neural crest (CNC) in Xenopus tropicalis. ADAM13 functions as a major regulator of canonical Wnt signaling, a signaling pathway that is essential for CNC induction. This is achieved through ADAM13‐mediated cleavage of class B ephrins, which we identified as new Wnt antagonists. By contrast, the proteolytic activity of ADAM19 seems to be dispensable for its function in CNC induction. Surprisingly, we found that ADAM19 directs CNC induction by binding and stabilizing ADAM13. These results highlight the importance of protease‐protease interaction as part of the gene interaction network in neural crest development.Grant Funding Source: Supported by the NIH

Dissecting the signaling pathways associated with the oncogenic activity of MLK3 P252H mutation

BackgroundMLK3 gene mutations were described to occur in about 20% of microsatellite unstable gastrointestinal cancers and to harbor oncogenic activity. In particular, mutation P252H, located in the kinase domain, was found to have a strong transforming potential, and to promote the growth of highly invasive tumors when subcutaneously injected in nude mice. Nevertheless, the molecular mechanism underlying the oncogenic activity of P252H mutant remained elusive.MethodsIn this work, we performed Illumina Whole Genome arrays on three biological replicas of human HEK293 cells stably transfected with the wild-type MLK3, the P252H mutation and with the empty vector (Mock) in order to identify the putative signaling pathways associated with P252H mutation.ResultsOur microarray results showed that mutant MLK3 deregulates several important colorectal cancer- associated signaling pathways such as WNT, MAPK, NOTCH, TGF-beta and p53, helping to narrow down the number of potential MLK3 targets responsible for its oncogenic effects. A more detailed analysis of the alterations affecting the WNT signaling pathway revealed a down-regulation of molecules involved in the canonical pathway, such as DVL2, LEF1, CCND1 and c-Myc, and an up-regulation of DKK, a well-known negative regulator of canonical WNT signaling, in MLK3 mutant cells. Additionally, FZD6 and FZD10 genes, known to act as negative regulators of the canonical WNT signaling cascade and as positive regulators of the planar cell polarity (PCP) pathway, a non-canonic WNT pathway, were found to be up-regulated in P252H cells.ConclusionThe results provide an overall view of the expression profile associated with mutant MLK3, and they support the functional role of mutant MLK3 by showing a deregulation of several signaling pathways known to play important roles in the development and progression of colorectal cancer. The results also suggest that mutant MLK3 may be a novel modulator of WNT signaling, and pinpoint the activation of PCP pathway as a possible mechanism underlying the invasive potential of MLK3 mutant cells.

Redox Regulation of Canonical Wnt Signaling Affects Extraembryonic Endoderm Formation

Retinoic acid (RA) induces mouse F9 cells to form primitive endoderm (PrE) and increased levels of reactive oxygen species (ROS) accompany differentiation. ROS are obligatory for differentiation and while H2O2 alone induces PrE, antioxidants attenuate the response to RA. Evidence shows that ROS can modulate the Wnt/β-catenin pathway and in this study, we show that extraembryonic endoderm formation is dependent on the redox state of nucleoredoxin (NRX). In undifferentiated F9 cells, NRX interacted with dishevelled 2 (Dvl2) and while this association was enhanced under reduced conditions, it decreased following H2O2 treatment. Depleting NRX levels caused morphological changes like those induced by RA, while increasing protein kinase A activity further induced these PrE cells to parietal endoderm. Reduced NRX levels also correlated to an increase in T-cell-factors-lymphoid enhancer factors-mediated transcription, indicative of canonical Wnt signaling. Together these results indicate that a mechanism exists whereby NRX maintains canonical Wnt signaling in the off state in F9 cells, while increased ROS levels lift these constraints. Dvl2 no longer bound to NRX is now positioned to prime the Wnt pathway(s) required for PrE formation.

Regulating Wnt signaling: a strategy to prevent neurodegeneration and induce regeneration

During the past three decades, Wingless/Int (Wnt) signaling has emerged as an essential regulator crucial for neuronal development and maintenance (Inestrosa and Arenas,2010). In addition, Wnt signaling was recently shown to be involved in the regulation of synaptic function and plasticity, which is critical for learning and memory (Oliva et al., 2013). Deregulation of Wnt signaling has been proposed as a key contributor to the pathogenesis of neurodegenerative disorders including Alzheimer’s disease (AD) and Parkinson’s disease (PD). This increasing knowledge of the specific roles of Wnt signaling cascades during different stages of life has suggested innovative therapeutic strategies for the treatment of neurodegenerative diseases. The reviews under the theme ‘WntSignalingCascadesinNeurodevelopment, Neurodegeneration and Regeneration’ published in this issue of JMCB provide an up-to-date overview of the importance of Wnt signaling in midbrain dopaminergic (mDA) development, synaptic maintenance in the hippocampus, neuroinflammatory responses, development of neurodegenerative diseases, and highlight new therapeutic approaches. The majority of dopamine-synthesizing neurones are located in the ventral midbrain. This cell population is crucial for the control and modulation of motor function, cognition, affect, motivation, and reward behaviours. Degeneration of these neurones leads to the typical extrapyramidal motor dysfunctions seen in individuals with PD. The aetiology of PD is poorly understood but work performed over the last two decades has identified a growing number of genetic defects that underlie this condition. Dr Berwick and Harvey reviewed an increasing body of evidence connecting genes implicated in PD, such as LRRK2 and PARK2, with Wnt signaling. These observations provide clues to the normal function of these proteins in healthy neurones and suggest that deregulated Wnt signaling might be a frequent pathomechanism underlying PD. The involvement of LRRK2 in Wnt signaling is of particular interest, since patients harbouring LRRK2 mutations develop PD symptoms that are indistinguishable from idiopathic PD. Dr Marchetti’s group summarized recent evidence suggesting that PD risk factors associated with increased inflammation (including aging and neurotoxin exposure) antagonize Wnt/b-catenin signaling in dopaminergic neurones and neuronal stem cells in the subventricular zone, which is important for adult neurogenesis. Wnt signaling was further suggested to participate in neuroimmune responses via crosstalk between astrocytes, microglia, neurones, and stem/ neuroprogenitor cells. Supporting evidence showed an inter-dependence of inflammation and Wnt/bcatenin signaling in MPTP-induced loss and subsequent repair of dopaminergic neurones. Pharmacological intervention targeting inflammatory responses prevented b-catenin downregulation and restored neurogenesis (L’Episcopo et al., 2012). This suggests an interaction of Wnt signaling with inflammatory pathways activated following neuronal insults with implications for the pathogenesis and treatment of PD. The review by Dr Joksimovic and Awatramani highlights the importance of b-catenin, a central component of the canonical Wnt/b-catenin signaling cascade, for the specification and differentiation of mesodiencephalic dopaminergic neurones. Wnt/b-catenin signaling was shown to be critical for the neurogenic potential of these neurones that originate from the midbrain floor plate. Conditionalb-catenin ablation in the floor plate, allowing normal early Wnt/b-catenin signaling in the midbrain, altered the function of dopaminergic progenitors, including a reduced expression of key genes such as Lmx1a, Otx2, Msx1, and Ngn2, and a decrease in postmitotic mDA neurones. Interestingly, excessive b-catenin supply to the midbrain floor plate had the opposite effect on gene expression but also resulted in fewer postmitotic mDA. This suggests that a dosage effect of b-catenin is crucial for the fine regulation of canonical Wnt signaling.

Distinct Transcriptional Networks in Quiescent Myoblasts: A Role for Wnt Signaling in Reversible vs. Irreversible Arrest

Most cells in adult mammals are non-dividing: differentiated cells exit the cell cycle permanently, but stem cells exist in a state of reversible arrest called quiescence. In damaged skeletal muscle, quiescent satellite stem cells re-enter the cell cycle, proliferate and subsequently execute divergent programs to regenerate both post-mitotic myofibers and quiescent stem cells. The molecular basis for these alternative programs of arrest is poorly understood. In this study, we used an established myogenic culture model (C2C12 myoblasts) to generate cells in alternative states of arrest and investigate their global transcriptional profiles. Using cDNA microarrays, we compared G0 myoblasts with post-mitotic myotubes. Our findings define the transcriptional program of quiescent myoblasts in culture and establish that distinct gene expression profiles, especially of tumour suppressor genes and inhibitors of differentiation characterize reversible arrest, distinguishing this state from irreversibly arrested myotubes. We also reveal the existence of a tissue-specific quiescence program by comparing G0 C2C12 myoblasts to isogenic G0 fibroblasts (10T1/2). Intriguingly, in myoblasts but not fibroblasts, quiescence is associated with a signature of Wnt pathway genes. We provide evidence that different levels of signaling via the canonical Wnt pathway characterize distinct cellular states (proliferation vs. quiescence vs. differentiation). Moderate induction of Wnt signaling in quiescence is associated with critical properties such as clonogenic self-renewal. Exogenous Wnt treatment subverts the quiescence program and negatively affects clonogenicity. Finally, we identify two new quiescence-induced regulators of canonical Wnt signaling, Rgs2 and Dkk3, whose induction in G0 is required for clonogenic self-renewal. These results support the concept that active signal-mediated regulation of quiescence contributes to stem cell properties, and have implications for pathological states such as cancer and degenerative disease.

Intra-epithelial Requirement of Canonical Wnt Signaling for Tooth Morphogenesis

Multiple Wnt ligands are expressed in the developing tooth and play important and redundant functions during odontogenesis. However, the source of Wnt ligands and their targeting cells and action mechanism in tooth organogenesis remain largely elusive. Here we show that epithelial inactivation of Gpr177, the mouse Wntless (Wls) whose product regulates Wnt sorting and secretion, leads to arrest of tooth development at the early cap stage and abrogates tooth-forming capability of the dental epithelium. Gpr177 in the epithelium is necessary for the activation of canonical Wnt signaling in the dental epithelium and formation of a functional enamel knot. Epithelial deletion of Gpr177 results in defective gene expression and cellular behavior in the dental epithelium but does not alter odontogenic program in the mesenchyme. Furthermore, deletion of Axin2, a negative intracellular regulator of canonical Wnt signaling, rescues the tooth defects in mice carrying Gpr177 mutation in the dental epithelium. Together with the fact that active Wnt canonical signaling is present predominantly in the dental epithelium during tooth development, our results demonstrate that Gpr177-mediated Wnt ligands in the dental epithelium act primarily in an intra-epithelial context to regulate enamel knot formation and subsequent tooth development.