A novel Axin2 knock‐in mouse model for visualization and lineage tracing of WNT/CTNNB1 responsive cells

Wnt signal transduction controls tissue morphogenesis, maintenance and regeneration in all multicellular animals. In mammals, the WNT/CTNNB1 (Wnt/β‐catenin) pathway controls cell proliferation and cell fate decisions before and after birth. It plays a critical role at multiple stages of embryonic development, but also governs stem cell maintenance and homeostasis in adult tissues. However, it remains challenging to monitor endogenous WNT/CTNNB1 signaling dynamics in vivo. Here, we report the generation and characterization of a new knock‐in mouse strain that doubles as a fluorescent reporter and lineage tracing driver for WNT/CTNNB1 responsive cells. We introduced a multi‐cistronic targeting cassette at the 3′ end of the universal WNT/CTNNB1 target gene Axin2. The resulting knock‐in allele expresses a bright fluorescent reporter (3xNLS‐SGFP2) and a doxycycline‐inducible driver for lineage tracing (rtTA3). We show that the Axin2^{P2A‐rtTA3‐T2A‐3xNLS‐SGFP2} strain labels WNT/CTNNB1 responsive cells at multiple anatomical sites during different stages of embryonic and postnatal development. It faithfully reports the subtle and dynamic changes in physiological WNT/CTNNB1 signaling activity that occur in vivo. We expect this mouse strain to be a useful resource for biologists who want to track and trace the location and developmental fate of WNT/CTNNB1 responsive stem cells in different contexts.     

E‐cadherin can limit the transforming properties of activating β‐catenin mutations

Wnt pathway deregulation is a common characteristic of many cancers. Only colorectal cancer predominantly harbours mutations in APC, whereas other cancer types (hepatocellular carcinoma, solid pseudopapillary tumours of the pancreas) have activating mutations in β‐catenin (CTNNB1). We have compared the dynamics and the potency of β‐catenin mutations in vivo. Within the murine small intestine (SI), an activating mutation of β‐catenin took much longer to achieve Wnt deregulation and acquire a crypt‐progenitor cell (CPC) phenotype than Apc or Gsk3 loss. Within the colon, a single activating mutation of β‐catenin was unable to drive Wnt deregulation or induce the CPC phenotype. This ability of β‐catenin mutation to differentially transform the SI versus the colon correlated with higher expression of E‐cadherin and a higher number of E‐cadherin:β‐catenin complexes at the membrane. Reduction in E‐cadherin synergised with an activating mutation of β‐catenin resulting in a rapid CPC phenotype within the SI and colon. Thus, there is a threshold of β‐catenin that is required to drive transformation, and E‐cadherin can act as a buffer to sequester mutated β‐catenin.     

miR‐150‐5p suppresses the stem cell‐like characteristics of glioma cells by targeting the Wnt/β‐catenin signaling pathway

Glioma is the most common brain tumor malignancy with high mortality and poor prognosis. Emerging evidence suggests that cancer stem cells are the key culprit in the development of cancer. MicroRNAs have been reported to be dysregulated in many cancers, while the mechanism underlying miR‐150‐5p in glioma progression and proportion of stem cells is unclear. The expression levels of miR‐150‐5p and catenin beta 1 (CTNNB1, which encodes β‐catenin) were measured by quantitative real‐time polymerase chain reaction (qRT‐PCR) and western blot. The expression levels of downstream genes of the Wnt/β‐catenin pathway and stem cell markers were detected by qRT‐PCR. Tumorigenesis was investigated by cell viability, colony formation, and tumor growth in vitro and in vivo. The interaction between miR‐150‐5p and β‐catenin was explored via bioinformatics analysis and luciferase activity assay. We found that miR‐150‐5p was downregulated in glioma and its overexpression inhibited cell proliferation, colony formation, and tumor growth. Moreover, miR‐150‐5p directly suppressed CTNNB1 and negatively regulated the abundances of downstream genes of the Wnt/β‐catenin pathway and stem cell markers. Furthermore, miR‐150‐5p expression was decreased and β‐catenin level was enhanced in CD133+ glioma stem cells. Knockdown of miR‐150‐5p contributed to CD133? cells with stem cell‐like phenotype, whereas overexpression of miR‐150‐5p suppressed CD133+ glioma stem cell‐like characteristics. In conclusion, miR‐150‐5p inhibited the progression of glioma by controlling stem cell‐like characteristics via regulating the Wnt/β‐catenin pathway, providing a novel target for glioma treatment.     

The role of Wnt/β‐catenin signaling in proliferation and regeneration of the developing basilar papilla and lateral line

Canonical Wnt/β‐catenin signaling has been implicated in multiple developmental events including the regulation of proliferation, cell fate, and differentiation. In the inner ear, Wnt/β‐catenin signaling is required from the earliest stages of otic placode specification through the formation of the mature cochlea. Within the avian inner ear, the basilar papilla (BP), many Wnt pathway components are expressed throughout development. Here, using reporter constructs for Wnt/β‐catenin signaling, we show that this pathway is active throughout the BP (E6‐E14) in both hair cells (HCs) and supporting cells. To characterize the role of Wnt/β‐catenin activity in developing HCs, we performed gain‐ and loss‐of‐function experiments in vitro and in vivo in the chick BP and zebrafish lateral line systems, respectively. Pharmacological inhibition of Wnt signaling in the BP and lateral line neuromasts during the periods of proliferation and HC differentiation resulted in reduced proliferation and decreased HC formation. Conversely, pharmacological activation of this pathway significantly increased the number of HCs in the lateral line and BP. Results demonstrated that this increase was the result of up‐regulated cell proliferation within the Sox2‐positive cells of the prosensory domains. Furthermore, Wnt/β‐catenin activation resulted in enhanced HC regeneration in the zebrafish lateral line following aminoglycoside‐induced HC loss. Combined, our data suggest that Wnt/β‐catenin signaling specifies the number of cells within the prosensory domain and subsequently the number of HCs. This ability to induce proliferation suggests that the modulation of Wnt/β‐catenin signaling could play an important role in therapeutic HC regeneration. © 2013 Wiley Periodicals, Inc. Develop Neurobiol 74: 438–456, 2014     

A new gain‐of‐function mouse line to study the role of Wnt3a in development and disease

Wnt/β‐catenin signals are important regulators of embryonic and adult stem cell self‐renewal and differentiation and play causative roles in tumorigenesis. Purified recombinant Wnt3a protein, or Wnt3a‐conditioned culture medium, has been widely used to study canonical Wnt signaling in vitro or ex vivo. To study the role of Wnt3a in embryogenesis and cancer models, we developed a Cre recombinase activatable Rosa26^Wnt3a allele, in which a Wnt3a cDNA was inserted into the Rosa26 locus to allow for conditional, spatiotemporally defined expression of Wnt3a ligand for gain‐of‐function (GOF) studies in mice. To validate this reagent, we ectopically overexpressed Wnt3a in early embryonic progenitors using the T‐Cre transgene. This resulted in up‐regulated expression of a β‐catenin/Tcf‐Lef reporter and of the universal Wnt/β‐catenin pathway target genes, Axin2 and Sp5. Importantly, T‐Cre; Rosa26^Wnt3a mutants have expanded presomitic mesoderm (PSM) and compromised somitogenesis and closely resemble previously studied T‐Cre; Ctnnb1^ex3 (β‐catenin^GOF) mutants. These data indicate that the exogenously expressed Wnt3a stimulates the Wnt/β‐catenin signaling pathway, as expected. The Rosa26^Wnt3a mouse line should prove to be an invaluable tool to study the function of Wnt3a in vivo.     

Knockdown of mediator subunit Med19 suppresses bladder cancer cell proliferation and migration by downregulating Wnt/β‐catenin signalling pathway

Mediator complex subunit 19 (Med19), a RNA polymerase II‐embedded coactivator, is reported to be involved in bladder cancer (BCa) progression, but its functional contribution to this process is poorly understood. Here, we investigate the effects of Med19 on malignant behaviours of BCa, as well as to elucidate the possible mechanisms. Med19 expression in 15 BCa tissues was significantly higher than adjacent paired normal tissues using real‐time PCR and Western blot analysis. Immunohistochemical staining of 167 paraffin‐embedded BCa tissues was performed, and the results showed that high Med19 protein level was positively correlated with clinical stages and histopathological grade. Med19 was knocked down in BCa cells using short‐hairpin RNA. Functional assays showed that knocking‐down of Med19 can suppress cell proliferation and migration in T24, UM‐UC3 cells and 5637 in vitro, and inhibited BCa tumour growth in vivo. TOP/FOPflash reporter assay revealed that Med19 knockdown decreased the activity of Wnt/β‐catenin pathway, and the target genes of Wnt/β‐catenin pathway were down‐regulated, including Wnt2, β‐catenin, Cyclin‐D1 and MMP‐9. However, protein levels of Gsk3β and E‐cadherin were elevated. Our data suggest that Med19 expression correlates with aggressive characteristics of BCa and Med19 knockdown suppresses the proliferation and migration of BCa cells through down‐regulating the Wnt/β‐catenin pathway, thereby highlighting Med19 as a potential therapeutic target for BCa treatment.     

CpG site DNA methylation of the CCAAT/enhancer‐binding protein,alpha promoter in chicken lines divergently selected for fatness

CCAAT/enhancer‐binding protein (C/EBP), alpha (CEBPA) is a master regulator of adipogenesis and, together with peroxisome proliferator‐activated receptor gamma (PPARG), plays a critical role in adipocyte differentiation. Previous studies have demonstrated that CEBPA is regulated by DNA methylation and involved in the osteogenesis and adipogenesis of mouse C3H10T1/2 and bone marrow stromal cells. However, it is unclear whether CEBPA is regulated by DNA methylation in adipose tissues. Thus, the objectives of the present study were to investigate CpG site methylation in a 357‐bp CEBPA promoter region and to assess the correlation between promoter CpG site methylation and CEBPA gene expression in the abdominal adipose tissues of Northeast Agricultural University broiler lines divergently selected for abdominal fat content. The results showed that the methylation percentage of the analyzed CEBPA promoter region was significantly higher in lean broilers than in fat broilers at 2 weeks (80.3% vs. 43.4%, P < 0.0001), 3 weeks (95.4% vs. 74.0%, P < 0.0001) and 7 weeks of age (82.6% vs. 57.2%, P < 0.0001). Real‐time quantitative RT‐PCR analysis showed that CEBPA expression was significantly higher in the fat vs. the lean line at 2 weeks of age (P = 0.0013) but not at 3 or 7 weeks of age. The correlation analysis showed that only at 2 weeks of age was the methylation percentage negatively correlated with CEBPA expression (Pearson's r = ?0.8312, P = 0.0029). Of all seven tested CpGs, only two, the CpGs at ?1494 and ?1478 bp, displayed a significantly negative correlation with CEBPA mRNA expression. These results suggest that the CEBPA is methylated in adipose tissue and may regulate chicken early adipose development.     

Aberrant DNA methylation of WNT pathway genes in the development and progression of CIMP-negative colorectal cancer

The WNT signaling pathway has an essential role in colorectal carcinogenesis and progression, which involves a cascade of genetic and epigenetic changes. We aimed to analyze DNA methylation affecting the WNT pathway genes in colorectal carcinogenesis in promoter and gene body regions using whole methylome analysis in 9 colorectal cancer, 15 adenoma, and 6 normal tumor adjacent tissue (NAT) samples by methyl capture sequencing. Functional methylation was confirmed on 5-aza-2′-deoxycytidine-treated colorectal cancer cell line datasets. In parallel with the DNA methylation analysis, mutations of WNT pathway genes (APC, β-catenin/CTNNB1) were analyzed by 454 sequencing on GS Junior platform. Most differentially methylated CpG sites were localized in gene body regions (95% of WNT pathway genes). In the promoter regions, 33 of the 160 analyzed WNT pathway genes were differentially methylated in colorectal cancer vs. normal, including hypermethylated AXIN2, CHP1, PRICKLE1, SFRP1, SFRP2, SOX17, and hypomethylated CACYBP, CTNNB1, MYC; 44 genes in adenoma vs. NAT; and 41 genes in colorectal cancer vs. adenoma comparisons. Hypermethylation of AXIN2, DKK1, VANGL1, and WNT5A gene promoters was higher, while those of SOX17, PRICKLE1, DAAM2, and MYC was lower in colon carcinoma compared to adenoma. Inverse correlation between expression and methylation was confirmed in 23 genes, including APC, CHP1, PRICKLE1, PSEN1, and SFRP1. Differential methylation affected both canonical and noncanonical WNT pathway genes in colorectal normal-adenoma-carcinoma sequence. Aberrant DNA methylation appears already in adenomas as an early event of colorectal carcinogenesis.     

Wnt/β‐catenin signaling in the mouse embryonic cranial mesenchyme is required to sustain the emerging differentiated meningeal layers

Cranial neural crest cells (CNCCs) give rise to cranial mesenchyme (CM) that differentiates into the forebrain meningeal progenitors in the basolateral and apical regions of the head. This occurs in close proximity to the other CNCC‐CM‐derivatives, such as calvarial bone and dermal progenitors. We found active Wnt signaling transduction in the forebrain meningeal progenitors in basolateral and apical populations and in the non‐meningeal CM preceding meningeal differentiation. Here, we dissect the source of Wnt ligand secretion and requirement of Wnt/β‐catenin signaling for the lineage selection and early differentiation of the forebrain meninges. We find persistent canonical Wnt/β‐catenin signal transduction in the meningeal progenitors in the absence of Wnt ligand secretion in the CM or surface ectoderm, suggesting additional sources of Wnts. Conditional mutants for Wntless and β‐catenin in the CM showed that Wnt ligand secretion and Wnt/β‐catenin signaling were dispensable for specification and proliferation of early meningeal progenitors. In the absence of β‐catenin in the CM, we found diminished laminin matrix and meningeal hypoplasia, indicating a structural and trophic role of mesenchymal β‐catenin signaling. This study shows that β‐catenin signaling is required in the CM for maintenance and organization of the differentiated meningeal layers in the basolateral and apical populations of embryonic meninges.     

Wnt antagonist SFRP3 inhibits the differentiation of mouse hepatic progenitor cells

Wnt/β‐catenin pathway plays an important role in regulating embryonic development. Hepatocytes differentiate from endoderm during development. Hepatic progenitor cells (HPCs) have been isolated from fetal liver and extrahepatic tissues. Most current studies in liver development and hepatic differentiation have been focused on Wnts, β‐catenin, and their receptors. Here, we sought to determine the role of Wnt antagonists in regulating hepatic differentiation of fetal liver‐derived HPCs. Using mouse liver tissues derived from embryonic day E12.5 to postnatal day (PD) 28, we found that 13 of the 19 Wnt genes and almost all of Wnt receptors/co‐receptors were expressed in most stages. However, Wnt antagonists SFRP2, SFRP3, and Dkk2 were only detected in the early stages. We established and characterized the reversible stable HPCs derived from E14.5 mouse fetal liver (HP14.5). HP14.5 cells were shown to express high levels of early liver progenitor cell markers, but low levels or none of late liver markers. HP14.5 cells were shown to differentiate into mature hepatocytes upon dexamethasone (Dex) stimulation. Dex‐induced late marker expression and albumin promoter activity in HP14.5 cells were inhibited by exogenous expression of SFRP3. Furthermore, Dex‐induced glycogen synthesis of PAS‐positive HP14.5 cells was significantly inhibited by SFRP3. Therefore, our results have demonstrated that the expression of Wnt antagonists decreases as hepatic differentiation progresses, suggesting that a balanced Wnt signaling may be critical during mouse liver development and hepatic differentiation. J. Cell. Biochem. 108: 295–303, 2009. © 2009 Wiley‐Liss, Inc.     

Effects of Wnt/β-catenin signalling on proliferation and differentiation of apical papilla stem cells

Objectives: The Wnt signalling pathway has been shown to play an important role in tooth development, however its effects with stem cells from the apical papilla (SCAP) have remained unclear. The purpose of this study was to determine effects of Wnt/β‐catenin on proliferation and differentiation of SCAP in vitro. Materials and methods: SCAP were obtained, identified and cultured. Cell proliferation, alkaline phosphatase (ALP) activity, mRNA expression of mineralization‐related genes and mineralized nodule formation were measured in presence or absence of various concentrations of lithium chloride. Results: MTT assay and flow cytometry demonstrated that Wnt/β‐catenin activity could promote proliferation of SCAP. Real‐time PCR analysis found that Wnt/β‐catenin strongly upregulated expression of dentine sialophosphoprotein, osteocalcin and ALP in SCAP after incubation with mineralization induction medium, while ALP and alizarin red staining indicated that Wnt/β‐catenin enhanced ALP activity and formation of mineralized nodules. Conclusion: Our results suggest that canonical Wnt/β‐catenin signalling promotes proliferation and odonto/osteogenic differentiation of SCAP.     

Genistein and daidzein repress adipogenic differentiation of human adipose tissue-derived mesenchymal stem cells via Wnt/β-catenin signalling or lipolysis

Objectives: One aspect of the effects of isoflavones against fat deposition might be at least associated with the mechanism by which Wnt/β‐catenin signalling inhibits adipocyte differentiation. However, it remains completely unknown as to whether isoflavones might influence Wnt signalling during commitment of pluripotent mesenchymal stem cells (MSCs) to adipose lineages. In the present study, we have investigated the mechanisms underlying effects of genistein and daidzein, the major soy isoflavones, on anti‐adipogenic Wnt/β‐catenin signalling. Materials and methods: Adipose tissue‐derived (AD) MSCs were exposed continuously to genistein and daidzein (0.01–100 μm ) during adipogenic differentiation (21 days). An oestrogen antagonist, ICI 182,780, was used to determine whether or not the isoflavones activated Wnt signalling via oestrogen receptors (ERs). Results: Genistein and daidzein suppressed adipogenic differentiation of AD‐MSCs in a dose‐dependent manner and inhibited expression of adipogenic markers, PPARγ, SREBP‐1c and Glut 4, from mid‐phase differentiation. Microarrays showed that anti‐adipogenic effects of genistein were principally attributable to activation of Wnt signalling via ERs‐dependent pathway, such as Erk/JNK signalling and LEF/TCF4 co‐activators. These findings were supported by evidence that the effects of genistein were offset by ICI182,780. Unlike genistein, daidzein inhibited adipogenesis through stimulation of lipolysis, with for example, PKA‐mediated hormone sensitive lipase. This is consistent with the increase in glycerol released from AD‐MSCs. In conclusion, understanding that different sets of mechanisms of the two isoflavones on adipogenesis will help the design of novel strategies to prevent observed current epidemic levels of obesity, using isoflavones.     

Wnt/β‐catenin signalling induces MLL to create epigenetic changes in salivary gland tumours

We show that activation of Wnt/β‐catenin and attenuation of Bmp signals, by combined gain‐ and loss‐of‐function mutations of β‐catenin and Bmpr1a, respectively, results in rapidly growing, aggressive squamous cell carcinomas (SCC) in the salivary glands of mice. Tumours contain transplantable and hyperproliferative tumour propagating cells, which can be enriched by fluorescence activated cell sorting (FACS). Single mutations stimulate stem cells, but tumours are not formed. We show that β‐catenin, CBP and Mll promote self‐renewal and H3K4 tri‐methylation in tumour propagating cells. Blocking β‐catenin–CBP interaction with the small molecule ICG‐001 and small‐interfering RNAs against β‐catenin, CBP or Mll abrogate hyperproliferation and H3K4 tri‐methylation, and induce differentiation of cultured tumour propagating cells into acini‐like structures. ICG‐001 decreases H3K4me3 at promoters of stem cell‐associated genes in vitro and reduces tumour growth in vivo. Remarkably, high Wnt/β‐catenin and low Bmp signalling also characterize human salivary gland SCC and head and neck SCC in general. Our work defines mechanisms by which β‐catenin signals remodel chromatin and control induction and maintenance of tumour propagating cells. Further, it supports new strategies for the therapy of solid tumours.