Prognostic Significance of Ror2 and Wnt5a Expression in Medulloblastoma

Medulloblastoma (MB) is a clinically and biologically heterogeneous group of tumors, and currently classified into four molecular subgroups (Wnt, Shh, Group 3 and Group 4). Intracellular signaling of the Wnt pathway has been divided into two classes: the “canonical” and the “non‐canonical” signaling pathway. The canonical signaling pathway is a well‐established, β‐catenin‐dependent signaling pathway in MB. In contrast, very little research about the non‐canonical WNT signaling pathway in MB exists. In order to identify the roles of Wnt‐5a and Ror2, two non‐canonical WNT pathway‐related genes, we studied 76 cases of MB with immunohistochemistry and quantitative real‐time PCR and correlated the results with clinicopathological and other molecular parameters and prognosis. Wnt5a and Ror2 were immunopositive in 20 (29.4%) and 35 (51.5%) of 68 cases, respectively. There were positive associations among protein expressions of Wnt5a, Ror2 and β‐catenin. Ror2 mRNA levels were well correlated with immunoexpression. Ror2 mRNA expression was significantly associated with CTNNB1 mutation. High Ror2 mRNA expression was an independent favorable prognostic factor. In conclusion, our study demonstrates the first attempt to identify Wnt5a and Ror2 as additional mechanisms contributing to dysregulation of the non‐canonical WNT signaling pathway in MB. Ror2 may play a role as an oncosuppressor in MB.     

An ATF2‐based luciferase reporter to monitor non‐canonical Wnt signaling in xenopus embryos

Non‐canonical/planar cell polarity (PCP) Wnt signaling plays important roles in embryonic development and tissue homeostasis, and is implicated in human disease. Monitoring Wnt/PCP signaling relies mostly on semi‐quantitative bioassays or biochemical analysis. Here we describe a luciferase reporter assay based on an ATF2 response element, which faithfully monitors non‐canonical Wnt signaling in Xenopus embryos. The assay is simple, quantitative, and robust. It can be used to detect non‐canonical Wnt signaling changes following gain and loss of function of pathway components, including Wnt, Frizzled, Ror2, Disheveled, Rac1, MKK7, and JNK. Wnt/PCP signaling has recently been implicated in left‐right asymmetry and our reporter assay suggests that in gastrula embryos there is a right‐ward bias in Wnt/PCP signaling. We also mapped Wnt/PCP signaling in the early Xenopus embryo and find that it peaks in the dorso‐vegetal region, paralleling Wnt/β‐catenin signaling. Developmental Dynamics, 2011. © 2010 Wiley‐Liss, Inc.     

Loss of adenomatous polyposis coli (apc) results in an expanded ciliary marginal zone in the zebrafish eye

The distal region of neural retina (ciliary marginal zone [CMZ]) contains stem cells that produce non‐neural and neuronal progenitors. We provide a detailed gene expression analysis of the eyes of apc mutant zebrafish where the Wnt/β‐catenin pathway is constitutively active. Wnt/β‐catenin signaling leads to an expansion of the CMZ accompanied by a central shift of the retinal identity gene sox2 and the proneural gene atoh7. This suggests an important role for peripheral Wnt/β‐catenin signaling in regulating the expression and localization of neurogenic genes in the central retina. Retinal identity genes rx1 and vsx2, as well as meis1 and pax6a act upstream of Wnt/β‐catenin pathway activation. Peripheral cells that likely contain stem cells can be identified by the expression of follistatin, otx1, and axin2 and the lack of expression of myca and cyclinD1. Our results introduce the zebrafish apc mutation as a new model to study signaling pathways regulating the CMZ. Developmental Dynamics 239:2066–2077, 2010. © 2010 Wiley‐Liss, Inc.     

Twist1 contributes to cranial bone initiation and dermal condensation by maintaining wnt signaling responsiveness

Background: Specification of cranial bone and dermal fibroblast progenitors in the supraorbital arch mesenchyme is Wnt/β‐catenin signaling‐dependent. The mechanism underlying how these cells interpret instructive signaling cues and differentiate into these two lineages is unclear. Twist1 is a target of the Wnt/β‐catenin signaling pathway and is expressed in cranial bone and dermal lineages. Results: Here, we show that onset of Twist1 expression in the mouse cranial mesenchyme is dependent on ectodermal Wnts and mesenchymal β‐catenin activity. Conditional deletion of Twist1 in the supraorbital arch mesenchyme leads to cranial bone agenesis and hypoplastic dermis, as well as craniofacial malformation of eyes and palate. Twist1 is preferentially required for cranial bone lineage commitment by maintaining Wnt responsiveness. In the conditional absence of Twist1, the cranial dermis fails to condense and expand apically leading to extensive cranial dermal hypoplasia with few and undifferentiated hair follicles. Conclusions: Thus, Twist1, a target of canonical Wnt/β‐catenin signaling, also functions to maintain Wnt responsiveness and is a key effector for cranial bone fate selection and dermal condensation. Developmental Dynamics 245:144–156, 2016. © 2015 Wiley Periodicals, Inc.     

Mice lacking the orphan receptor ror1 have distinct skeletal abnormalities and are growth retarded

Ror1 is a member of the Ror‐family receptor tyrosine kinases. Ror1 is broadly expressed in various tissues and organs during mouse embryonic development. However, so far little is known about its function. The closely related family member Ror2 was shown to play a crucial role in skeletogenesis and has been shown to act as a co‐receptor for Wnt5a mediating non‐canonical Wnt‐signaling. Previously, it has been shown that during embryonic development Ror1 acts in part redundantly with Ror2 in the skeletal and cardiovascular systems. In this study, we report that loss of the orphan receptor Ror1 results in a variety of phenotypic defects within the skeletal and urogenital systems and that Ror1 mutant mice display a postnatal growth retardation phenotype. Developmental Dynamics 239:2266–2277, 2010. © 2010 Wiley‐Liss, Inc.     

Blocking Dishevelled signaling in the noncanonical Wnt pathway in sea urchins disrupts endoderm formation and spiculogenesis,but not secondary mesoderm formation

Dishevelled (Dsh) is a phosphoprotein key to beta‐catenin dependent (canonical) and beta‐catenin independent (noncanonical) Wnt signaling. Whereas canonical Wnt signaling has been intensively studied in sea urchin development, little is known about other Wnt pathways. To examine roles of these beta‐catenin independent pathways in embryogenesis, we used Dsh‐DEP, a deletion construct blocking planar cell polarity (PCP) and Wnt/Ca²⁺ signaling. Embryos overexpressing Dsh‐DEP failed to gastrulate or undergo skeletogenesis, but produced pigment cells. Although early mesodermal gene expression was largely unperturbed, embryos exhibited reduced expression of genes regulating endoderm specification and differentiation. Overexpressing activated beta‐catenin failed to rescue Dsh‐DEP embryos, indicating that Dsh‐DEP blocks endoderm formation downstream of initial canonical Wnt signaling. Because Dsh‐DEP‐like constructs block PCP signaling in other metazoans, and disrupting RhoA or Fz 5/8 in echinoids blocks subsets of the Dsh‐DEP phenotypes, our data suggest that noncanonical Wnt signaling is crucial for sea urchin endoderm formation and skeletogenesis. Developmental Dynamics 238:1649–1665, 2009. © 2009 Wiley‐Liss, Inc.     

Beta‐catenin signaling in hepatic development and progenitors: Which way does the WNT blow?

The Wnt/β‐catenin pathway is an evolutionarily conserved signaling cascade that plays key roles in development and adult tissue homeostasis and is aberrantly activated in many tumors. Over a decade of work in mouse, chick, xenopus, and zebrafish models has uncovered multiple functions of this pathway in hepatic pathophysiology. Specifically, beta‐catenin, the central component of the canonical Wnt pathway, is implicated in the regulation of liver regeneration, development, and carcinogenesis. Wnt‐independent activation of beta‐catenin by receptor tyrosine kinases has also been observed in the liver. In liver development across various species, through regulation of cell proliferation, differentiation, and maturation, beta‐catenin directs foregut endoderm specification, hepatic specification of the foregut, and hepatic morphogenesis. Its role has also been defined in adult hepatic progenitors or oval cells especially in their expansion and differentiation. Thus, beta‐catenin undergoes tight temporal regulation to exhibit pleiotropic effects during hepatic development and in hepatic progenitor biology. Developmental Dynamics 240:486–500, 2011. © 2010 Wiley‐Liss, Inc.     

Essential role of Wnt5a‐Ror1/Ror2 signaling in metanephric mesenchyme and ureteric bud formation

Spatiotemporally regulated interaction between the metanephric mesenchyme (MM) and Wolffian duct (WD) is essential for the induction of a single ureteric bud (UB). The MM then interacts with the tip of the UB to induce outgrowth and branching of the UB, which in turn promotes growth of the adjacent MM. The Ror family receptor tyrosine kinases, Ror1 and Ror2, have been shown to act as receptors for Wnt5a to mediate noncanonical Wnt signaling. Previous studies have shown that Ror2‐mutant mice exhibit ectopic formation of the UB, due to abnormal juxtaposition of the MM to the WD. We show here that both Ror1 and Ror2 are expressed in the mesenchyme between the MM and WD during UB formation. Although Ror1‐mutant mice show no apparent defects in UB formation, Ror1;Ror2‐double‐mutant mice exhibit either defects in UB outgrowth and branching morphogenesis, associated with the loss of the MM from the UB domain, or ectopic formation of the UB. We also show genetic interactions between Ror1 and Wnt5a during UB formation. These findings suggest that Wnt5a‐Ror1/Ror2 signaling regulates cooperatively the formation of the MM at the proper position to ensure normal development of the UB.     

Inactive Wnt/β‐catenin pathway in conventional high‐grade osteosarcoma

Osteosarcoma is the most common malignant bone tumour, with a peak incidence in children and young adolescents, suggesting a role of rapid bone growth in its pathogenesis. The Wnt/β‐catenin pathway plays a crucial role in skeletal development and is indispensable for osteoblasts' lineage determination. Previous studies suggesting an oncogenic role for the Wnt/β‐catenin pathway in osteosarcoma were based on cytoplasmic staining of β‐catenin or the detection of one component of this pathway. However, those approaches are inappropriate to address whether the Wnt/β‐catenin pathway is functionally active. Therefore, in this study, we examined nuclear β‐catenin expression in 52 human osteosarcoma biopsies, 15 osteoblastomas (benign bone tumours), and four human osteosarcoma cell lines by immunohistochemistry. Furthermore, we modulated Wnt/β‐catenin pathway activity using a GIN (GSK3β inhibitor) and evaluated its effect on cell growth and osteogenic differentiation. Absence of nuclear β‐catenin staining was found in 90% of the biopsies and all osteosarcoma cell lines, whereas strong nuclear β‐catenin staining was observed in all osteoblastomas. Wnt‐luciferase activity was comparable to the negative control in all osteosarcoma cell lines. GIN stimulated the Wnt/β‐catenin pathway, as shown by translocation of β‐catenin into the nucleus and increased Wnt‐luciferase activity as well as mRNA expression of AXIN2, a specific downstream target gene. Stimulation of the Wnt/β‐catenin pathway by GIN significantly reduced cell proliferation in the cell lines MG‐63 and U‐2‐OS and enhanced differentiation in the cell lines HOS and SJSA‐1, as shown by an increase in alkaline phosphatase (ALP) activity and mineralization. In contrast with the oncogenic role of the Wnt/β‐catenin pathway in osteosarcoma as previous studies suggested, here we demonstrate that this pathway is inactivated in osteosarcoma. Moreover, activation of the Wnt/β‐catenin pathway inhibits cell proliferation or promotes osteogenic differentiation in osteosarcoma cell lines. Our data suggest that loss of Wnt/β‐catenin pathway activity, which is required for osteoblast differentiation, may contribute to osteosarcoma development. Copyright © 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Wnt/β‐Catenin Signaling Pathway Upregulates c‐Myc Expression to Promote Cell Proliferation of P19 Teratocarcinoma Cells

Aberrant activation of the Wnt/β‐catenin signaling pathway is a common event in human tumor progression. Wnt signaling has also been implicated in maintaining a variety of adult and embryonic stem cells by imposing a restraint to differentiation. To understand the function and mechanism of Wnt/β‐catenin signaling on the pathogenesis of teratocarcinoma, we used the mouse teratocarcinoma P19 cell line as a model in vitro. Gsk3β specific inhibitor (SB216763) was used to activate Wnt/β‐catenin signaling. All trans‐retinoic acid (RA) was used to induce P19 cell differentiation. At different culture times, gene expression was examined by immunofluorescence staining, quantitative real‐time PCR, and Western‐blotting; BrdU incorporation assays were performed to measure P19 cell proliferation. Small interference RNA technology was used to downregulate c‐myc expression. The results showed that SB216763 induced the nuclear translocation of β‐catenin, upregulated the expression of c‐myc and pluripotency related genes, oct4, sox2 and nanog, and blocked cell differentiation induced by all trans‐RA. The proliferation of P19 cells was significantly enhanced by SB216763, as well as c‐myc overexpression. C‐myc downregulation inhibited P19 cell proliferation caused by activation of Wnt/β‐catenin signaling and induced P19 cell differentiation. In conclusion, activation of the Wnt/β‐catenin pathway could promote the proliferation and inhibit the differentiation of mouse teratocarcinoma cells by upregulation of c‐myc expression. Anat Rec, 2012. © 2012 Wiley Periodicals, Inc.     

Critical role of Frizzled1 in age‐related alterations of Wnt/β‐catenin signal in myogenic cells during differentiation

Activation of Wnt/β‐catenin signal in muscle satellite cells (mSCs) of aged mice during myogenic differentiation has been appreciated as an important age‐related feature of the skeletal muscles, resulting in impairment of their regenerative ability following muscle injury. However, it remains elusive about molecules involved in this age‐related alteration of Wnt/β‐catenin signal in myogenic cells. To clarify this issue, we carried out expression analyses of Wnt receptor genes using real‐time RT‐PCR in mSCs isolated from the skeletal muscles of young and aged mice. Here, we show that expression of Frizzled1 (Fzd1) was detected at high levels in mSCs of aged mice. Higher expression levels of Fzd1 were also detected in mSC‐derived myogenic cells from aged mice and associated with activation of Wnt/β‐catenin signal during their myogenic differentiation in vitro. We also provide evidence that suppressed expression of Fzd1 in myogenic cells from aged mice results in a significant increase in myogenic differentiation, and its forced expression in those from young mice results in its drastic inhibition. These findings indicate the critical role of Fzd1 in altered myogenic differentiation associated with aging.     

Ror2 is required for midgut elongation during mouse development

The receptor tyrosine kinase Ror2 acts as a receptor for Wnt5a to mediate noncanonical Wnt signaling, and it plays essential roles in morphogenesis. Ror2^?/? embryos exhibit phenotypes similar to, albeit generally milder than, those of Wnt5a^?/? embryos. During mouse embryogenesis, Ror2 is expressed in various organs and regions, although little is known about its expression pattern and roles in the developing gut, while Wnt5a is expressed in the developing gut, where its absence causes abnormal phenotypes. Here, we demonstrated that Ror2 was strongly and differentially expressed in the rostral and middle midgut endoderm from embryonic day (E) 10.5 through embryonic day (E) 12.5. At E11.5, Ror2^?/? embryos exhibited a shorter middle midgut with a larger diameter and more accumulation of epithelial cells in the middle midgut than control embryos, while the total cell numbers remained unaltered. These findings suggest that Ror2 plays important roles in midgut elongation by means of an epithelial convergent extension mechanism. Developmental Dynamics 239:941–953, 2010. © 2010 Wiley‐Liss, Inc.     

Wnt/β‐catenin and sonic hedgehog pathways interact in the regulation of the development of the dorsal mesenchymal protrusion

Background: The dorsal mesenchymal protrusion (DMP) is a second heart field (SHF) derived tissue involved in cardiac septation. Molecular mechanisms controlling SHF/DMP development include the Bone Morphogenetic Protein and Wnt/β‐catenin signaling pathways. Reduced expression of components in these pathways leads to inhibition of proliferation of the SHF/DMP precursor population and failure of the DMP to develop. While the Sonic Hedgehog (Shh) pathway has also been demonstrated to be critically important for SHF/DMP development and atrioventricular septation, its role in the regulation of SHF proliferation is contentious. Results: Tissue‐specific deletion of the Shh receptor Smoothened from the SHF resulted in compromised DMP formation and atrioventricular septal defects (AVSDs). Immunohistochemical analysis at critical stages of DMP development showed significant proliferation defect as well as reduction in levels of the Wnt/β‐catenin pathway‐intermediates β‐catenin, Lef1, and Axin2. To determine whether the defects seen in the conditional Smoothened knock‐out mouse could be attributed to reduced Wnt/β‐catenin signaling, LiCl, a pharmacological activator of this Wnt/β‐catenin pathway, was administered. This resulted in restoration of proliferation and partial rescue of the AVSD phenotype. Conclusions: The data presented suggest that the Wnt/β‐catenin pathway interact with the Shh pathway in the regulation of SHF/DMP‐precursor proliferation and, hence, the development of the DMP. Developmental Dynamics 245:103–113, 2016. © 2015 Wiley Periodicals, Inc.     

Activation of Wnt5a-Ror2 signaling associated with epithelial-to-mesenchymal transition of tubular epithelial cells during renal fibrosis

Activation of Wnt5a-Ror2 signaling has been shown to be associated with epithelial-to-mesenchymal transition (EMT) of epidermoid carcinoma cells via induction of matrix metalloproteinase-2 (MMP-2). Because EMT has also been implicated in the progression of tissue fibrosis, we examined the possible association of Wnt5a-Ror2 signaling with renal fibrosis. Here, we show that expression of Wnt5a and Ror2 is induced in a damaged mouse kidney after unilateral ureteral obstruction (UUO) treatment. Immunofluorescent analysis showed that Ror2 expression is clearly induced in tubular epithelial cells during renal fibrosis, and these Ror2-expressing cells also express Snail and vimentin, markers of mesenchymal cells, suggesting that Ror2 might be induced in epithelial cells undergoing EMT. We also found that MMP-2 expression is induced at Ror2-positive epithelium adjacent to significantly disrupted tubular basement membrane (TBM). Interestingly, reduced expression of MMP-2 is detected at epithelium in damaged kidneys from Ror2^+/− mice compared with those from wild-type Ror2^+/+ mice. Importantly, extents of TBM disruption are apparently reduced in damaged kidneys from Ror2^+/− mice compared with those from wild-type mice. Collectively, these findings indicate that activation of Wnt5a-Ror2 signaling in epithelial cells undergoing EMT may play an important role in disrupting TBM via MMP-2 induction during renal fibrosis.