Forkhead box G1 (FOXG1) is a member of the Fox transcription factor family involved in regulation of many cancers. However, the role of FOXG1 in hepatocellular carcinogenesisis largely unclear. The present study aimed at examining the biological function and underlying mechanism of FOXG1 on hepatocellular carcinoma (HCC) tumor metastasis as well as its clinical significance.Levels of FOXG1 were determined by immunohistochemical and real-time PCR analysis in HCC cell lines and human HCC samples. The effect of FOXG1 on cancer cell invasion and metastasis was investigated in vitro and in vivo in either FOXG1-silenced or overexpressing human HCC cell lines. Immunoprecipitation and chromatin immunoprecipitation assays were performed to investigate the interaction of FOXG1, β-catenin, TCF4 and the effect on Wnt target-gene promoters.In human HCC, the level of FOXG1 progressively increased from surrounding non tumorous livers to HCC, reaching the highest levels in metastatic HCC. Furthermore, expression levels of FOXG1 directly correlated with cancer cell epithelial-mesenchymal transition (EMT) phenotype. In FOXG1-overexpressing cells, FOXG1 promotes the stabilization and nuclear accumulation of β-catenin by directly binding to β-catenin and it associates with the lymphoid enhancer factor/T cell factor proteins (LEF/TCFs) on Wnt responsive enhancers (WREs) in chromatin.The results show that FOXG1 plays a key role in mediating cancer cell metastasis through the Wnt/β-catenin pathway in HCC cells and predicts HCC prognosis after surgery. Targeting FOXG1 may provide a new approach for therapeutic treatment in the future. Worldwide, HCC is still a malignant tumor with high incidence and mortality, remaining a great threat to human health. According to the 2018 cancer statistics report, the five-year relative survival rate for HCC patients is only 18%. However, the mortality rate of males consistently sustain higher and continue to rise at a rate of 1.6% per year . BesidesEMT is a normal physiological process that converts epithelial cells into mesenchymal cells. However, the process is frequently exploited by cancer cells to migrate to surrounding and distant regions. Characteristics of EMT observed in the tumor progressions include the decreased intercellular adhesion, loss of epithelial markers (e.g. E-cadherin), acquisition of mesenchymal markers (including vimentin and N-cadherin), etc. . These EP < 0.0001) [A key role in the molecular pathway that promotes EMT is the Wnt/β-catenin signaling pathway . When th 0.0001) . Further 0.0001) . Hence, FOX transcription factor family plays an important role in the Wnt pathway , 14. BesIn our current study, we aimed to explore the possibility of FOXG1 as a key component of the Wnt signaling pathway regulating the nuclear accumulation of β-catenin, determine the biological interaction of FOXG1/β-catenin/TCF4 on HCC EMT and lesions metastasis, and further clarify their clinical significance. These findings suggest that FOXG1 may be a valuable prognostic factor and potential therapeutic target for HCC.2 at 37 °C.HCC cell lines, including HepG2, Huh7, Hep3B, PLC/PRF5y, HCCLM3, MHCC97L, and MHCC97H, were obtained from the Guangdong Provincial Key Laboratory of Liver Disease and grown in Dulbecco’s modified Eagle medium . LO2 normal human liver cells were maintained in RPMI-1640 medium . All medium was supplemented with 10% fetal bovine serum , 2 mM L-glutamine, 100 units/ml penicillin, and 100 μg/ml streptomycin in an atmosphere of 5% COA total of 105 paraffin-embedded, archived HCC samples that had been histopathologically and clinically diagnosed as HCC at the Sun Yat-sen University Cancer Center from 1995 to 2011 were used in the current study. Ten fresh HCC tissue samples were obtained from patients undergoing HCC resection at the Department of Hepatobiliary Surgery of the Third Affiliated Hospital of Sun Yat-sen University. Prior patient consent and approval of the Institutional Research Ethics Committee of the Third Affiliated Hospital were obtained for the use of the clinical materials described above for research purposes. The clinicopathologic characteristics of the 105 patients are summarized in Additional file Cells were lysed in co-IP buffer . Lysates were centrifuged and cleared by incubation with 25 μl of Protein A/G gel for 1 h at 4 °C. The pre-cleared supernatant was subjected to IP using first antibodies at 4 °C overnight. Then, the protein complexes were collected by incubation with 30 μl Protein A/G gel for 1 h at 4 °C. The collected protein complexes were washed six times with co-IP buffer and analyzed by western blotting.6 cells were prepared with the ChIP assay kit  according to the manufacturer’s instructions. DNA complexes were immunoprecipitated using the anti-FOXG1  or anti-β-catenin  or anti-TCF4  antibodies and then eluted by incubation for 30 min at 37 °C in 100 μl of 10 mM DTT. After centrifugation, the supernatant was diluted 50× with ChIP buffer. The resulting precipitated DNA samples were further analyzed by qPCR. The primers are listed in section “RNA extraction, reverse transcription, and qPCR” in Additional file For the ChIP assay, 5 × 10HCC cells, cultured on cover slips, were transfected with pcDNA3.1-FOXG1 or FOXG1 shRNA and the respective controls pcDNA-vector or nonsense siRNA control reagents for 48 h. Cells were washed, fixed in 4% paraformaldehyde for 15 min, and then rinsed in PBS three times for 5 min each, and blocked with 5% BSA in PBS for 30 min at 25 °C. Primary antibody was added and incubated for 1 h at 37 °C and then detected using FITC-conjugated secondary antibody. Images were captured using a fluorescence microscope.t-test; P < 0.05 was considered significant.We used 2-μm sections of formalin-fixed paraffin-embedded HCC tissue samples for IHC staining. All sections were treated routinely with the EnVision system . The primary antibodies were anti-FOXG1 , anti-TCF4 , and anti-β-catenin . IHC staining results were evaluated in a double-blind manner by two experienced pathologists and are presented as the average score. The results of IHC staining were scored on a scale of 0–12. The value of the staining intensity and the percentage of positive cells were graded. It was presented as the average score for the extent of IHC staining. Scores for different groups of tissues were compared using Student’s t-test was used to determine statistical differences. The chi-square test was used to analyze the relationship between FOXG1 expression and clinical pathological characteristics. Survival curves were plotted using the Kaplan–Meier method and compared by log-rank test. P < 0.05 was considered significant.All values are presented as mean ± standard deviation (SD). Significant differences were determined using SPSS 16.0 software . Student’s Other experimental procedures are available in Additional file b and c). Furthermore, FOXG1 expression was higher in HCC with vascular invasion  and cirrhotic liver tissue (CIRR), with the highest level in HCC tissues (HCC) compared with healthy liver tissue (HEAL)  or -uncoated (for migration) Transwell assay confirmed that overexpression of FOXG1 increased the invasiveness and migration of the PLC/PRF5yand Huh7 HCC cell lines Fig. c and d. An orthotopic xenograft tumor model was performed to further explore the ability of FOXG1 to promote metastasis and invasion in HCC cells in vivo. The Huh7/FOXG1 cells and their corresponding vector control cells were injected in the tail vein of nude mice. Strikingly, mice subjected to Huh7/FOXG1 tumors cells displayed prominent lung metastasis, whereas a much lower level of metastasis was found in mice transplanted with control Huh7/vector cells Fig. a and b. It is yet to be proven whether FOXG1 is merely a physical marker of EMT or it also contributes to the traits of the progressive phenotype. Therefore, we performed FOXG1 knockdown experiment using a retroviral-based approach in Huh7 and PLC/PRF5y cells. After confirming that 70–80% of FOXG1 were knocked down successfully, we found the elevation of E-cadherin expression in epithelial markers, and the decrease of vimentin and fibronectin levels in mesenchymal markers by Western blotting Fig. a and ImmWe used the TOPFlash reporter to ascertain whether FOXG1 affected the transcriptional activity of β-catenin-TCF/LEF. As we expected, TOP/FOP flash assays revealed that overexpression of FOXG1 significantly increased the transcriptional activation of TCF/LEF in Huh7 and PLC/PRF5y cells, whereas knockdown of FOXG1 attenuated the transcriptional activation of TCF/LEF /WREs in chromatin. The results showed that overexpression of FOXG1 in 293 T cells increased the activity of the LEF-1 promoter Fig. d. And thIn addition, the association of β-catenin or FOXG1 with the LEF-1 promoter diminished upon the depletion of TCF4 Fig. . By usinP < 0.05)  and β-catenin   Fig. b. The ex15) Fig. . In geneIn the present study, we provide evidence for a novel mechanistic link between FOXG1 and the oncogenic Wnt/β-catenin signaling pathway in HCC. We found that FOXG1 was significantly up-regulated in HCC and was positively correlated with poor prognosis. Overexpression of FOXG1 promotes the metastasis and invasion of HCC in vitro or in vivo, while the down-regulation of Foxg1 inhibits these occurrences. Furthermore, we demonstrated that FOXG1 could activate the Wnt/β-catenin pathway by directly stimulating β-catenin nuclear translocation through binding β-catenin and TCF4. It implies that FOXG1 plays a role as an oncogene in HCC and may be an important target for clinical intervention.FOXG1, as a member of FOX gene superfamily, has been revealed to play an important role in embryogenesis and cancers. Other members of this superfamily, such as FOXKs , FOXM1 , FOXQ1 , FOXP3 , FOXF2 , FOXO1 , FOXR2 , FOXA2  and FOXLAberrant activation of the Wnt/β-catenin signaling pathway is commonly found in many types of human cancers and is thought to promote tumor progression . MultiplAdditionally, JASPAR database analysis showed that the FOXG1 promoter contains a NF-κB and MET-specific binding site. In HCC, close to 80% of patients develop tumors against a background of chronic liver inflammation. However, little is known about the specific molecular mechanisms underlying this process. As confirmed the activation of NF-κβ mediates liver oncogenes in mice, the lymphotoxin is over-expressed in a liver-specific manner . Mice inIn summary, the present study demonstrated that FOXG1 promotes tumor growth in HCC. The up-regulation of FOXG1 dramatically promoted EMT of HCC cells by inducing nuclear shuttle of β-catenin and consequently retains it in the nucleus. In the nucleus, FOXG1/β-catenin complex binds to TCF4 and enhances its transcriptional activity. These findings reveal a novel mechanism of hyperactivation of the Wnt/β-catenin signaling pathway in HCC, suggesting that FOXG1 might activate that pathway through multiple mechanisms by inhibiting β-catenin degradation, accumulating nuclear β-catenin levels and increasing TCF4 transcriptional activity. More broadly, FOXG1 may serve as a potential therapeutic target for HCC.Additional file 1: Table S1. Clinicopathological Characteristics of Clinical Samples and Expression of FOXG1 in HCC. Table S2. Correlation between FOXG1 Expression and Clinico-pathologic. (DOCX 24 kb)Additional file 2. Supplemental material and methods. (DOCX 23 kb)


EWSR1-ETS gene fusions resulting in oncogenic transcription factors, currently, no targeted therapy could be implemented. It seems even more difficult to develop a targeted therapeutic strategy in osteosarcoma which is characterized by high complexity and heterogeneity in genomic alterations. Nevertheless, the common point between these different bone tumors is their ability to deregulate bone homeostasis and remodeling and divert them to their benefit. Therefore, targeting different actors of the bone tumor microenvironment has been hypothesized to develop new therapeutic strategies. In this context, it is well known that the Wnt/β-catenin signaling pathway plays a key role in cancer development, including osteosarcoma and Ewing sarcoma as well as in bone remodeling. Moreover, recent studies highlight the implication of the Wnt/β-catenin pathway in angiogenesis and immuno-surveillance, two key mechanisms involved in metastatic dissemination. This review focuses on the role played by this signaling pathway in the development of primary bone tumors and the modulation of their specific microenvironment.Osteosarcoma and Ewing sarcoma are the most common malignant primary bone tumors mainly occurring in children, adolescents and young adults. Current standard therapy includes multidrug chemotherapy and/or radiation specifically for Ewing sarcoma, associated with tumor resection. However, patient survival has not evolved for the past decade and remains closely related to the response of tumor cells to chemotherapy, reaching around 75% at 5 years for patients with localized forms of osteosarcoma or Ewing sarcoma but less than 30% in metastatic diseases and patients resistant to initial chemotherapy. Despite Ewing sarcoma being characterized by specific  Osteosarcoma and Ewing sarcoma are the most common primary bone tumors mainly occurring in children, adolescents and young adults. Current standard therapy includes multidrug chemotherapy and/or radiation for Ewing sarcoma, associated with tumor resection. However, the 5-year survival rates have not been improved during the last decades reaching 70–80% for patients with localized forms, but less than 30% in metastatic diseases and patients resistant to initial chemotherapy ,2,3,4,5.EWSR1 and FLI1 genes in 90% of cases, or by the fusion of EWSR1 with other transcription factors of the E26 Transformation-Specific (ETS) gene family in 10% of cases [Osteosarcoma that mainly occurs at the ends of long bones is not associated with any clinical signs except severe pain or spontaneous fracture. Osteosarcoma does not express specific oncogenic markers but exhibits a large number and variety of genetic alterations. Ewing sarcoma is a tumor composed of small undifferentiated round cells that can appear mainly in bones but also in soft tissues in 15% of cases. In contrast to osteosarcoma, Ewing sarcoma is characterized by a chromosomal translocation between the of cases ,8.Despite progress in understanding the biology of osteosarcoma and Ewing sarcoma, no targeted therapy could be currently implemented to improve patient survival. Nevertheless, the common point between these two pediatric bone tumors is their ability to deregulate bone homeostasis and remodeling to divert them to their benefit. Therefore, new therapeutic research is moving towards targeting different actors of the bone microenvironment.It is well established that the bone tumor microenvironment (TME) plays a major role during tumor initiation, progression, and metastatic processes. This very complex and dynamic environment includes different cell types such as osteoblasts, osteoclasts, stromal cells, mesenchymal stem cells, immune or endothelial cells and non-cellular components such as the extracellular matrix compounds . These cThe crucial role played by the TME on sarcoma development has been recently highlighted in several studies. As an example, a modification of the transcriptome has been observed in cell lines established from synovial sarcomas compared to initial tumors or directly engrafted tumors in mice, demonstrating the impact of TME on tumor behavior, particularly through interactions with the immune system and stromal cells . The bonOne of the main hallmarks of osteosarcoma and Ewing sarcoma is their ability to disrupt bone remodeling in favor of an exacerbated osteoclast-mediated bone resorption, leading to the release of pro-tumoral factors initially embedded in the bone matrix ,20,21. IHuman Mesenchymal Stem Cells (hMSCs) play also a major role in the communication between tumor cells and their microenvironment, as highlighted by recent studies, including increased proliferation and migration of tumor cells. On one hand, bone marrow MSCs promote migration and invasion through direct secretion of several cytokines such as C-X-C motif chemokine ligand 12 (CXCL12) that binds to C-X-C motif chemokine receptor 4 (CXCR4) or CXCR7 on osteosarcoma associated with lower overall patient survival ,27,28. I+ M2-polarized macrophages is crucial for the inhibition of osteosarcoma progression [Bone sarcoma is able to modulate the recruitment, expansion and differentiation of immune cells to establish an immunosuppressive microenvironment, allowing tumor development and metastatic dissemination . Regardigression . In contgression ,40. Moregression . These rgression .Besides cellular components, the physical properties of the TME can influence the behavior of cancer cells. Indeed, mechanical (composition of the extracellular matrix) and chemical stress (hypoxia) increase the motility and capacity of sarcoma cells to metastasize, through increased expression of procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2 (PLOD2), a collagen cross-linker . Hypoxic+/CD4+ infiltrating T lymphocytes, leading to a better response to chemotherapy and overall patient survival [+ iNOS+, inducible nitric oxide synthase), as compared to metastatic diseases [Several studies have highlighted the importance of analyzing TME in order to predict patient outcomes. For example, Volchenboum et al. have demonstrated the importance of studying stromal cells in contact with the Ewing sarcoma, to predict patients at high risk of relapse, thanks to a gene set enrichment analysis conducted on human Ewing sarcomas samples by the Children’s Oncology Group (COG) . A stronsurvival . In ostediseases . In addihttps://clinicaltrials.gov/ct2/show/NCT00470223) has been developed to evaluate the combination of zoledronate with current chemotherapy and surgery in osteosarcoma patients. Unfortunately, the results do not recommend the use of zoledronate as no improvement in event-free survival was observed, zoledronate showing a trend to increase the risk of failure in osteosarcoma patients [https://clinicaltrials.gov/ct2/show/NCT02301039) and Nivolumab, NCT02304458 ).Osteoclasts and immune cells are the main potential therapeutic targets for osteosarcoma and Ewing sarcoma. Based on preclinical data, the clinical trial OS2006 , or to a co-receptor lipoprotein receptor-related protein (LRP)-5 or LRP-6, receptor tyrosine-kinase-like orphan receptor (ROR), related to tyrosine kinase (Ryk), to activate cell signaling. In the absence of Wnt ligands, the Wnt/β-catenin pathway is inactive, β-catenin is sequestered by a protein complex for degradation. This complex is composed of scaffold proteins, dishevelled (Dvl), adenomatous polyposis coli (APC) and Axin1/2 and Wilms tumor gene on X chromosome (WTX) protein , and twoAXIN2, LEF1, cMYC, CCND1, BIRC5  to an Fzd receptor and its co-receptor LRP5/6 activates the canonical Wnt pathway, inducing the recruitment of Dvl on Fzd receptor and its activation. CK1α and GSK3β kinases then phosphorylate the LRP5/6 co-receptor, allowing the recruitment of Axin on LRP5/6 and the inactivation of the β-catenin degradation complex which is trapped to the membrane. Neosynthesized β-catenin is no longer degraded and accumulates in the cytoplasm , before Numerous molecules are implicated in the regulation of Wnt ligands and the canonical Wnt pathway activation. These include secreted extracellular inhibitors: dickkopf (DKK), secreted-Fzd-related proteins (SFRPs), Wnt inhibitory factor 1 (WIF1), sclerostin (SOST), cerberus, insulin-like growth factor binding protein 4 (IGFBP4). DKK and SOST proteins are competitive receptor and co-receptor antagonists that prevent signaling activation by Wnt ligands. The WIF1, SFRPs  and cerberus proteins are able to bind to Wnt ligands preventing their interaction with receptors and co-receptors ,66.The canonical Wnt signaling pathway is involved in many cancers, notably in gastrointestinal cancers , hematopoietic cancers , breast cancer, melanoma, and brain tumors such as glioblastoma ,68. Its The most recent researches on Wnt/β-catenin implication in osteosarcoma deal with metastatic dissemination and recurrence that are associated with poor prognosis for patients. Thus, following a brief description of the implication of the canonical Wnt/β-catenin signaling pathway in osteosarcoma development, this part will focus on these recent studies.Canonical Wnt/β-catenin signaling pathway has been involved in the development of osteosarcoma but current knowledge remains unclear because of the high complexity of this pathway. Many studies support an aberrant activation of the canonical Wnt signaling pathway in osteosarcoma cells which seems to play a crucial role in tumorigenicity as well as metastatic dissemination ,78. As aA key issue of osteosarcoma metastasis is the epithelial-mesenchymal transition (EMT)-like multi-step process, based on a transformation from an epithelial to a mesenchymal phenotype, promoting the invasive capacities of cancer cells. EMT is characterized by repression of key components of the intercellular junctions such as E-cadherin associated with an increase in mesenchymal markers including N-cadherin, vimentin and fibronectin. This modulation of gene expression is regulated by different transcription factors such as Snail-1, Snail-2, ZEB-1, ZEB-2 or Twist . DespiteEMT can also promote tumor cells to develop stem cell characteristics and consequently actively participate in the cancer stem cell (CSC) phenotype acquisition . CSCs aa. These New insights about the role of long non-coding RNA (lncRNA) and micro RNA (miR) in osteosarcoma progression have also recently emerged. On one hand, the overexpression of a number of miR has been demonstrated in osteosarcoma cells leading to increased osteosarcoma growth and metastasis. Most of these miRs downregulate antagonists of the Wnt/β-catenin signaling pathway. As an example, miR-552-5p is overexpressed in osteosarcoma tissues and cell lines compared to healthy tissue and osteoblasts respectively. Knockdown of this miR decreases osteosarcoma proliferation and metastasis by directly inhibiting WIF1 . On the In addition to β-catenin, other modulators of the canonical Wnt signaling pathway, in particular, Wnt antagonists, are affected during osteosarcoma primary and metastatic development, but have already been described in several reviews and will not be developed here ,108.The involvement of the Wnt signaling pathway in Ewing sarcoma is less described than in osteosarcoma and seems to be controversial. First, no recurrent mutations in the Wnt/β-catenin signaling pathway have been reported in Ewing sarcoma but the activation of this pathway has been demonstrated in aggressive sarcoma, the bone microenvironment being the source of Wnt ligands. Moreover, this activation can be potentiated through the R-spondin (RSPO)/leucine rich repeat containing G protein-coupled receptor 5 (LGR5)/β-catenin axis. Indeed, LGR5 is highly expressed in aggressive Ewing sarcoma and CSCs and the binding of RSPO-2 on LGR5 was able to potentiate Wnt3a-induced canonical Wnt signaling. However, it seems that an important variability in Wnt responsiveness exists among Ewing sarcoma cells based on the differential expression of LGR5 and the availability of Wnt and RSPO ligands in the tumor microenvironment . The proThe analysis of the secretome of Wnt3a-activated Ewing sarcoma cells has revealed an increased secretion of proteins implicated in the composition and the structure of the extracellular matrix. In details, Wnt3a-activated cells present an upregulation of the signaling pathways involved in the communication between the tumor and its microenvironment, such as integrin linked kinase, focal adhesion kinase, cadherin angiogenesis, TGFβ and matrix metalloproteinase pathways .Finally, the effects of WNT974, a Porcupine protein inhibitor involved in the palmitoylation of Wnt ligands, an indispensable process for Wnt secretion has been investigated in Ewing sarcoma. The WNT974-mediated inhibition of endogenous Wnt ligands secreted by tumor cells decreases the migration of Ewing sarcoma cells in vitro, downregulating the expression of genes involved in cell migration. In addition, WNT974 appears to reduce the development of metastatic disease in vivo in a murine subcutaneous xenograft model and to delay the time of first lung metastasis establishment. WNT974 inhibits Wnt signaling pathways  and cell migration and dissemination, therefore demonstrated the role of the Wnt signaling pathway during Ewing sarcoma metastatic spread .As described in the first part of the review, the bone TME is a complex and dynamic environment composed of both cellular and non-cellular components. This TME which includes bone, vascular and immune “niches” plays a key role in initiation, primary development and metastasis of osteosarcoma and Ewing sarcoma . This paBone remodeling results from a balance between osteoclasts-mediated bone resorption and osteoblasts-promoted bone formation. Osteocytes, trapped in the bone matrix, also contribute to the regulation of osteoblast and osteoclast functions during bone remodeling. In this context, the Wnt/β-catenin signaling pathway is able to modulate both osteoblast and osteoclast differentiation and activity. Indeed, osteoblasts and their precursors, osteocytes and osteoclasts are able to secrete various Wnt ligands which participate in the control of bone remodeling ,117,118.The early-stage development of osteosarcoma and Ewing sarcoma initiates a dysregulation of the bone remodeling in favor of an exacerbated bone resorption, leading to the release of pro-tumorigenic factors initially trapped into the bone tissue b. NevertAn important step in the metastatic process of bone sarcoma is the degradation of the extracellular matrix related to upregulation of proteolytic enzymes such as MMPs. Several studies have highlighted that the invasive capacities of osteosarcoma cells are correlated with MMPs expression, particularly MMP-2 and MMP-9 . MoreoveAngiogenesis is a complex biological process that leads to new blood vessel development and plays a key role in primary tumor progression and metastatic potential of several cancers including bone sarcoma. Stages of angiogenesis include the proliferation and migration of endothelial cells to form a vessel sprout and then the recruitment of pericytes or smooth muscle cells to produce a mature and functional blood vessel . This prVEGFA gene was detected in osteosarcoma and significantly associated with high microvessels density and low patient disease-free survival [Osteosarcoma patients with high VEGF expression levels exhibit both lower disease-free survival and lower overall survival . Similarsurvival . Severalsurvival ,138,139.survival ,141.VEGF gene promoter. Moreover, defects in APC, resulting in constitutively active β-catenin, induce the over-expression of VEGF [Regarding the role of the Wnt/β-catenin signaling pathway in angiogenesis, transcriptional regulation of VEGF by the β-catenin/TCF complex has been demonstrated, involving TCF binding sites in the  of VEGF ,143. Fur of VEGF . First,  of VEGF ,146. Sec of VEGF . All theOnly a few studies deal with the implication of the canonical Wnt/β-catenin signaling pathway in bone sarcoma-associated angiogenesis. Nevertheless, a study has described that naked cuticle homolog 2 (NKD2), a negative regulator of Wnt signaling, downregulates the expression of genes involved in the vasculature development of osteosarcoma , suggestIt is well recognized that hypoxia is an important regulator of angiogenesis during tumor development . A majorBesides its regulation of angiogenesis, hypoxia has been directly correlated to bone sarcoma progression, metastasis and patient prognosis. Thus, hypoxia enhances the expression and the transcriptional function of the fusion protein EWS/FLI1 in a HIF-1α dependent-manner in Ewing sarcoma cells and consequently increases their invasiveness , HIF-1α The implication of the Wnt/β-catenin signaling pathway in hypoxia has mainly been reported in carcinoma. As an example, hypoxia promotes β-catenin nuclear localization related to a more invasive phenotype in colorectal adenocarcinoma cells . MoreoveThe immune microenvironment of bone sarcomas is highly heterogeneous composed of effectors of the innate immunity such as natural killer (NK) cells, dendritic cells (DC) and tumor-associated macrophages (TAMs) and cytotoxic T cells which drive adaptive immunity ,35. Neve+ macrophages and survival [Regarding innate immunity, bone sarcomas are mainly invaded by TAMs which have been associated with a good prognosis in osteosarcoma patients. However, as described above, it remains difficult to conclude about the role of M2-polarized macrophages in osteosarcoma progression, probably because of the difficulty to clearly identify the different TAMs subtypes in tumors and by the fact that, in contrast to the binary M1/M2 subtype, TAMs correspond to several distinct populations that often share features of both subtypes . In Ewinsurvival ,40. The survival  , correspond to the second most important immune cells in osteosarcoma ,166. TILAnother important point is that cross regulations between TAMs and TILs play a key role in osteosarcoma development. Thus, M2-polarized macrophages promote the suppression of TILs in osteosarcoma and, conversely, anti-PD1 therapy targeting TILs is able to stimulate a transition from M2 macrophages to M1 macrophages, leading to a decrease in lung metastases ,178.Finally, it seems important to note that, besides TAMs and TILs, the Wnt/β-catenin pathway is able to modulate the proliferation, recruitment, and/or functions of the different effectors of the immune system and to regulate multiple aspects of the immune cells/tumor crosstalk. As an example, Wnt ligands secreted by tumor cells can activate the Wnt/β-catenin signaling pathway in DC, leading to up regulated interleukin-10 and indoleamine 2,3-dioxygenase 1 secretion. This results in an increase in regulatory T cells inside the tumor and consequently an inhibition of TILs cytotoxic properties .The canonical Wnt/β-catenin signaling pathway plays a crucial role during the different steps of bone sarcoma growth and the metastatic process, although its involvement has been more studied in osteosarcoma than in Ewing sarcoma. This pathway seems to promote bone sarcoma development through both a direct effect on bone sarcoma cells and an indirect modulation of the bone TME cell activities. Thus, aberrant activation of the canonical Wnt signaling pathway in osteosarcoma cells seems to be highly involved in cell proliferation, induction of an EMT-like process, acquisition of stem cells properties of osteosarcoma CSCs as well as metastatic dissemination. The Wnt/β-catenin pathway activation does not affect the proliferation of Ewing sarcoma cells but appears to play a role in the cellular motility of these cells. The Wnt/β-catenin pathway also participates to the hijacking of the bone TME by the bone sarcoma cells, promoting modulation of the ECM to allow the tumor cells invasiveness and inducing immune tolerance and angiogenesis in the TME. This results in an increase in metastatic dissemination.The crucial role of the canonical Wnt/β-catenin signaling pathway is not restricted to bone sarcoma. That is why different therapeutic strategies targeting the Wnt/β-catenin signaling pathway in cancers have been developed. Corresponding clinical trials have been recently described in details in two reviews ,174. BriThe complexity of the Wnt signaling offers multiple targets to reduce β-catenin activation in tumor cells. However, better knowledge about the role of Wnt/β-catenin in each tumor cell type, in particular, osteosarcoma and Ewing sarcoma, is necessary to safely target this pathway. It is also necessary to focus on crosstalks between the Wnt/β-catenin and other signaling pathways during bone sarcoma growth and metastatic processes.


Amyotrophic lateral sclerosis (ALS) represents a devastating, progressive, heterogeneous, and the most common motor neuron (MN) disease. To date, no cure has been available for the condition. Studies with transgenic mice have yielded significant results that help us understand the underlying mechanisms of ALS. Nonetheless, none of more than 30 large clinical trials over the past 20 years proved successful, which led some researchers to challenge the validity of the preclinical models.Human-induced pluripotent cells (iPSCs) were established by introducing Sendai virus into fibroblast cells. We established TDP-43 HES by inserting CAG-TDP43 (G298S) cassette or the CAG-EGFP cassette into PPP1R12C-locus of human embryonic stem cells  by TALEN-mediated homologous recombination. iPSCs or HESC were differentiated to motor neurons and non-motor neuron as control. Relevant biomarkers were detected in different differentiated stages. TDP-43 aggregates, neurofilament, and mitochondria analyses were performed.TDP43 G298S mutation and two sporadic ALS patients, we showed that both sporadic and familial ALS were characterized by TDP-43 aggregates in the surviving MN. Significantly higher neurofilament (NF) inclusion was also found in ALS MN compared with wild-type (WT) GM15 controls (P < 0.05). The neurite mitochondria density was significantly lower in ALS MN than that in the control MNs. Transgenesis of TDP-43 G298S into AAVS locus in human embryonic stem cells reproduced phenotype of patient-derived G289S MN. By challenging MNs with a proteasome inhibitor, we found that MNs were more vulnerable to MG132, with some accompanying phenotype changes, such as TDP43 translocation, NF inclusion, mitochondria distribution impairment, and activation of caspase3.In this study, using iPSCs-derived human MN from an ALS patient with a Our results suggested that changes in TDP43 protein, NF inclusion, and distribution impairment of mitochondria are common early pathology both in familial and sporadic ALS. These findings will help us gain insight into the pathogenesis of the condition and screen relevant drugs for the disease.The online version of this article (10.1186/s13287-018-1048-1) contains supplementary material, which is available to authorized users. C9orf72, SOD1, TARDBP, FUS, and ANG. TARDBP gene mutations account for about 4% of familial ALS. The protein encoded by TAR DNA-binding protein 43 (TDP-43) was a major component of the histopathological hallmark of degenerating neurons in most sporadic and familial ALS cases [Amyotrophic lateral sclerosis (ALS) is a devastating, progressive, heterogeneous, and the most common motor neuron (MN) disease . IncidenLS cases .Work with transgenic mice has led to important advances in the understanding of mechanisms of ALS. Since 20- to 40-fold of mutant protein is needed to produce a phenotype in mice, artificial phenotypes might result . GeneticThe fibroblast information of GM15, G298S TDP43, and two sporadic was provided in Additional file http://www.jax.org). Two months after the development of tumors, mice were perfused with 4% paraformaldehyde and teratomas were HE-stained.Human ESCs and iPSCs were subcutaneously injected into the back of mice with severe combined immunodeficient (SCID) mice , non-essential amino acids, and N2 supplement in the presence of 2 μM SB431542, 3 μM CHIR99021 and 300 nM LDN193189 . At day 8, for MN induction, the neuroepithelia were treated with 0.1 μM retinoic acid (RA) and 0.5 μM purmorphamine (both from Stemgent) for 7 days. 0.5 μM cyclopamine was added in place of purmorphamine to generate non-MNs. At day 14, both the rosettes-like MN and non-MN progenitors were isolated with a pipette and expanded as floating clusters in suspension in the same respective medium but without SB431542, LDN193189, and CHIR99021 for an additional 7 days. The immature MN and non-MN clusters at day 21 were dissociated into individual cells or plated neurospheres onto laminin substrate in the presence of 0.1 μM compound E, a notch inhibitor to block cell proliferation for 10 days to produce synchronized post-mitotic MN and non-MN populations. The MNs or non-MNs were assayed from day 1 to day 10 after day 21 plating.Human iPSCs or ESCs were first differentiated to neuroepithelia for 7 days in a neural medium consisting of Dulbecco’s modified Eagle’s medium/Ham’s F-12 medium  for 30 min before incubation with TDP43 antibody (1:200) at 4 °C overnight. After rinsing six times, the cultures were incubated with the Ultra Small gold-conjugated secondary antibody overnight at 4 °C. The samples were then washed and post-fixed in 2% glutaraldehyde in 0.1 M PB and then subjected to regular embedding and EM processing. In order to increase the contrast of citrate, some thin sections were stained with uranyl acetate. For better visualization of immune-gold particles some sections were not stained. The samples were examined under a Philips 120 electron microscope.Cultures were fixed in 2.0% paraformaldehyde in 0.1 M cacodylate buffer (pH 7.4), 2.5% glutaraldehyde for 30 min for regular EM. Cultures were fixed in 4% paraformaldehyde in 0.1 M cacodylate buffer (pH 7.4), 0.1% glutaraldehyde for immuno-EM. The fixed cultures were rinsed with PBS, and residual aldehyde was inactivated by incubation for 10 min in 0.1% NaBH4 in 0.1 M PB. They were then permeabilized in 0.2% Triton-X-100 and incubated in AURION Blocking Solution  for detection.Cells were lysed using a buffer consisting of 62.5 mMTris-HCl at pH 6.8, 20% glycerol, 2% SDS, 2 mM DTT, 100 μM PMSF, and protease inhibitor cocktail. Lysates were resolved by SDS-PAGE, and Western blotting was carried out using horseradish peroxidase-conjugated IgG as a secondary antibody and the ECL system . Then, the cells were fixed in 4% PFA made from culture medium. The length of entire neuritis stained with NF 200 antibody was measured. Images were analyzed using Image J (NIH) software to determine the number of mitochondria within the neurite. One hundred fifty neurons were randomly selected for analysis. The number of the mitochondria on a 50-μm segment from cell body was counted, and the sum of the mitochondria was divided by neuritis length.2 in a sealed observation chamber during image acquisition.For labeling of NF-inclusion, 0.5 μg/ml doxycycline was added into the MN medium to induce NFL-GFP fusion protein expression 24 h before assay. MN mitochondria were then labeled for 15 min at 37 °C with 50 nM MitoTracker Orange CM-H2TMRos . MNs were washed and then replaced in complemented medium. Cells were observed at least 1 h after the washout of MitoTracker. Time-lapse recordings of movement of mitochondrial and NF inclusions were acquired every 5 s for 10 min by using a Nikon Biostation IM, in which the cells were maintained at 37 °C, in 5% COhttp://www.addgene.org) was replaced by CAG-MCS cassette by SpeIand MIuIdouble digestion. To elicit the expression of G298S TDP43, the cDNA was inserted into the SalIand MIuIsites of donor plasmid. For the expression of Tet-On 3G, Tet-On 3G sequence was inserted into the MCS by XhoIand AscIdigestion. In order to construct Tet-inducible expression systems, pTRE3G-SV40-polyA cassette  was inserted into the SpeIsite of donor plasmid. For expression NFL-EGFP, the sequence was inserted into the SalIand MIuIsites of the inducible donor plasmid. TALENs pairs targeting the AAVS1 locus was designed as previously described [http://www.addgene.org/), TALEN repeat arrays were constructed using standard restriction digestion and ligation reactions. TALEN activity was assayed via surveyor nuclease . Human iPSCs or ESCs were cultured in 1 μM Rho Kinase (ROCK)-inhibitor  24 h prior to electroporation. Cells (1 × 107) were electroporated with 40 μg of donor plasmids and 5 μg of each TALEN-encoding plasmid . Cells were subsequently plated onto MEF feeder layers in the MEF-conditioned medium supplemented with ROCK inhibitor for the first 24 h. Individual colonies were selected and expanded after puromycin selection (1 μg/ml) for 10 to 14 days. The correct cell clones were identified by PCR. The following primers are used: Forward: 5′ACCAACGCCGACGGTATCAG3′; Reverse: 5′CAGACCCTTGCCCTGGTGGT3′.We successfully induced constitutive and inducible expression of transgenes in human ESCs and iPSCs by using TALEN-mediated homologous recombination. CAG-GFP cassette of the donor plasmid AAV-CAGGS-EGFP , TDP43-1, TDP43-2, SPOR-1, and SPOR-2 iPSCs were established by introducing Sendai virus into fibroblast cells . All iPS-43 Fig. .Fig. 1iPTDP43-1, TDP43-2, SPOR-1, and SPOR-2 iPSCs can differentiate into OLIG2+cells, HB9+/TuJ1+, and CHAT+ motorneurons with high efficiency  motor neurons . Meanwhile, we used non-MNs as controls to confirm that the pathology is specific for MNs.We generated spinal cord motor neurons (MNs) by using a chemical protocol described in the “Materials and methods” section Fig. . The GM1ncy Fig. . Seven dons Fig. . There wSOD1 mutant familial ALS patients, and is supposed to impair the cell through multiple mechanisms [TDP-43 G298S MNs, TDP-43 was found in both the nucleus and the cytoplasm . The protein aggregates might damage the protein clearance system of the cell. So we quantified the TDP43 protein level in MNs. Our results showed that there existed no significant difference in TDP43 protein level between ALS MNs and control MNs at this early stage    Fig. . When cu05) Fig. . The siznts Fig. . At the 05) Fig. , d.Fig. In order to directly observe the neurofilament inclusion, we inserted pTRE3G-NFL-EGFP cassette to the PPP1R12C locus by using TALEN-mediated homologous recombination in ALS iPSCs to induce the expression of NFL-EGFP fusion protein Fig. . At the It was reported that NF serves as a docking site for the mitochondria and regulates their spatial distribution along axons . Since nSince NF-inclusion affected mitochondria movement and distribution, we determined the density of the mitochondria on a 50-μm segment from cell body of MNs. The results exhibited that the neurite mitochondria density was significantly lower in ALS MNs than in the control MNs, but the distribution of mitochondria of non-MN was not affected Fig. , f.TDP-43 G298S MNs and controls, but was caused by the toxicity of mutant TDP-43, we inserted the mutant cDNA into PPP1R12C-locus of human embryonic stem cells (H9) by TALEN-mediated homologous recombination  technology to integrate the same mutation into a safe harbor gene locus  in the genome of human embryonic stem cells (H9). Until now, it is believed that nuclear depletion, cytoplasmic accumulation of insoluble TDP-43, or both of them may contribute to the onset and progression of ALS. However, no definite conclusion has been reached [TDP-43 caused abnormal MN phenotype by gaining toxicity of TDP-43.Individual variations and even differences among individual stem cell clones pose great obstacles to the establishment of a disease model by using human stem cells. To address this barrier and establish cause-effect relationships between G298S  reached . But ourTDP-43 and sporadic MNs were more vulnerable to perturbation. This vulnerability was specific for MNs, with some accompanying phenotype changes, such as NF inclusion, mitochondria distribution impairment, and activation of CASP3.To confirm the phenotype obtained from the stem cell model and obtain prominent phenotypes, we further challenged MNs with a proteasome inhibitor, MG132. Our data suggested that G298S In summary, we established an enriched MN platform which can model the hallmark pathology of ALS. We found that TDP43 protein pathology appeared early in ALS spinal MNs, which is consistent with other studies. For the first time, our data proved that NF inclusion and impairment of mitochondria distribution are early and common pathology in both family and sporadic ALS. The pathology subjects MNs to the huge risk of degeneration triggered by environmental factors. These findings will help us gain insight into pathogenesis of the condition and screen relevant drugs for the disease.Additional file 1:Table S1. (DOCX 13 kb)Additional file 2:Table S2. (DOCX 13 kb)Additional file 3:Video S1. NF-LGFP40X_7_ch1.AVI. NF inclusions in NF-L-GFP merge MN neurites in light microscope (× 40). (AVI 15305 kb)Additional file 4:Video S2. NF-LGFP40X_7_ch2.AVI. NF inclusions in NF-L-GFP merge MN neurites in green channel. (AVI 15305 kb)Additional file 5:Video S3. NF-LGFP40X_7_ch3.AVI. Mitochondrial movement in NF-L-GFP merge MN neurites in red channel. (AVI 15305 kb)Additional file 6:Video S4. NF-LGFP40X_7_ch4.AVI. Some mitochondria moved into, rather out of NF inclusions. (AVI 45903 kb)


T2/b-Cateninfl(Ex3)/fl(Ex3), cACT) or knocked out . cACT discs had promoted expression of Wnt-targets and -ligands, brachyury, extracellular matrix production and 34% greater compressive stiffness than WT (b-Cateninfl(Ex3)/fl(Ex3)) discs, but 50% less tensile stiffness. By contrast, knockout reversed the cACT phenotype: less protein expression of b-catenin in the nucleus pulposus, less expression of brachyury, heightened expression of extracellular matrix breakdown and 46% less compressive stiffness than wild-type  discs. These data suggest that intervertebral disc degeneration is associated with loss of Wnt signaling and that the concomitant increase in b-catenin is a regenerative response, potentially offering a therapeutic approach to degeneration.B-Catenin, transcription factor of Wnt signaling, is promoted in patients with intervertebral disc (IVD) degeneration, but Wnt signaling decreases with aging. We hypothesize that IVD degeneration is associated with decreased Wnt signaling despite more b-Catenin. Chronic compression of tail IVDs of young-adult and aged Wnt-reporter (TOPGAL) animals initiated an age-related cascade of degenerative-like changes, which included reduced Wnt ligand expression and Wnt signaling in nucleus pulposus cells, despite elevation of b-Catenin protein and gene expression. To determine the effect of upregulated and downregulated Wnt signaling in adult discs, b-Catenin in the nucleus pulposus was stabilized (Shh-CreEr IVD degeneration is a multi-factorial disease with mechanical loads, age and genetics each contributing to its induction. Clarifying the mechanism(s) involved in the initiation of disc degeneration will ultimately offer strategies to treat or possibly prevent this disease.Intervertebral disc (IVD) degeneration is a major etiological factor of low back pain, which carries an estimated economic burden of at least $50 billion in treatment to U.S. and is the number one cause of job disability worldwide4. Whereas moderate mechanical forces may protect the IVD from degradation8, excessive mechanical loads shift the balance of extracellular homeostasis toward catabolism. Compressive mechanical loading of murine tail IVDs is a well characterized model that leads to extensive cellular and matrix degradation11. However, studies of IVD degeneration using mechanical loading have focused on describing the consequences to structure and matrix constituents, but have provided little insight into the possible underlying mechanism(s). Moreover, most tail compression studies involve young, growing rodents, which may not model adult intervertebral discs12.Persistent, abnormal mechanical loading may instigate back pain and increase the propensity for functional failure of the IVDs. For instance, frequent bending and twisting engender IVD wedging, prolapse, annular fissures, and shifting of the nucleus pulposus (NP)13. In short, the demarcation between the NP and annulus fibrosus becomes ambiguous by the end of the second decade of life because of collagen fibril growth in diameter and number14. Dramatic dehydration and loss of glycosaminoglycans of the IVD occurs by middle-age and is concentrated to the NP15. But how aging influences cellular signaling in response to altered, harmful mechanical loading is unclear.Aging is another factor that provokes IVD degeneration16 and is putatively involved in the catabolism of IVDs. In brief, if negative regulators secreted frizzled-related proteins (sFRP) and SOST/DKK do not bind to Wnt and LRP5 or 6, respectively, Wnt ligands bind to Low-density lipoprotein receptor-related protein 5/6 (LRP5/6) receptors and Frizzled co-receptors. Once activated, the tail end of the complex inhibits glycogen synthase kinase (GSK) and allows the stabilization of b-Catenin, which may then translocate to the nucleus, interact with T-Cell factor/lymphoid enhancer factor (TCF/LEF) and initiate gene transcription. Both patients with IVD degeneration and canines with an age-related propensity for degeneration have up-regulated levels of b-Catenin18. In vitro, Wnt signaling and greater b-Catenin in rodent IVD cells may trigger cellular senescence, apoptosis and biomarkers of matrix breakdown19. Further, brachyury T is a transcription factor for notochordal cells and requires TCF/LEF signaling20. Notochordal cell fate is driven by Wnt signaling activity21 and, T expression decreases in canines with an age-related propensity for intervertebral disc degeneration22, which is consistent with an age-related loss of Wnt signaling24. In mice, constitutive activation of b-Catenin in the cells of the annulus fibrosus leads to IVD degradation by increasing biomarkers of matrix breakdown17. By contrast, Wnt signaling decreases with aging, with concentrated decreases in the NP23. Disrupting Wnt signaling and b-Catenin during development deteriorates the entire IVD25. Therefore, the aim here was to clarify the early changes in canonical Wnt signaling and b-catenin during IVD degeneration and the role of altering b-catenin in the NP and not the annulus fibrosus.Transcription factor b-Catenin (bCAT) is regulated by the canonical Wnt pathwayin vivo model of murine intervertebral disc degeneration and of the exclusive regulation of Wnt/b-Catenin signaling in the NP, a hallmark location for the beginning of IVD degeneration. We hypothesize that IVD degeneration is associated with reduced Wnt signaling despite the upregulation of b-Catenin, which can be a regenerative response. Here, we apply in vivo chronic loads to adult tail IVD of transgenic mice designed to report Wnt activity. Next, b-Catenin was constitutively activated (cACT) in Sonic hedgehog (Shh)-expressing cells of the NP that resist degradation to demonstrate the in vivo consequences of greater b-Catenin, which appear in degenerated IVDs. Further, transcription factor b-Catenin was suppressed in Shh-expressing cells and consequences noted after 1 or 3 months. Lastly, the tail intervertebral discs of b-Catenin gain-of-function mice were subjected to static compression to determine the effect of regulating b-Catenin in a model of disc degeneration to offer insight to potential therapeutics and caveats.The approach applied here is the first known instance of the determination of Wnt signaling/b-Catenin in an 23. We previously demonstrated that aging-related changes of tail IVD from 5 to 12 months (mo) of age model human aging: increased stiffness and collagen content, and loss of proteoglycan content23. Therefore, IVDs of Wnt-reporter TOPGAL mice aged 5 and 12 mo old were instrumented at vertebrae CC7 and CC9  expression  declined with compression  or not compressed (Sham). LacZ staining in control IVDs showed blue staining in the NP, which was representative of Wnt signaling and loading decreased Wnt signaling in 5 mo and 12 mo IVDs, but was severely ablated in 12 mo IVD IVD Fig. . 5 mo anIVD Fig. . Sham anIVD Fig.  and protIVD Fig. . The effIVD Fig. , potentiIn tissues other than the NP, active Wnt signaling appears in the growth plate of vertebrae from young-adult and middle-aged mice Fig. . Punctur32. Biomechanical changes to each disc level were determined and the level with greatest change in biomechanical properties was used as the deciding factor for further analysis. To mimic the accumulation of b-Catenin in the NP during tail compression and determine its contribution, we crossed b-Cateninfl(Ex3)/fl(Ex3) (WT) with Shh-CreERT2 mice to yield b-Cateninfl(Ex3)/fl(Ex3)/Shh-CreERT2mice (bCAT cACT). bCAT cACT and WT mice were dosed daily with tamoxifen (5 d) and their IVDs were harvested 3 weeks later  IVDs was determined by crossing Ai9(RCL-tdT) mice with Shh-CreERT2 (Shh-CreERT2/tdT) and injecting them with tamoxifen for two days. tdT served as controls and showed no expression. Shh was expressed in the cells of the NP and cartilage endplate of tail IVDs  of the whole IVD (NP and annulus fibrosus) corroborated the Cre-mediated excision of exon3 by the reduced expression of full-length b-Catenin mRNA expression Fig. , but quawild-type b-Catenin prior to loading /fl(Ex3)) discs and increased b-Catenin expression in cACT IVDs  mice were crossed with Shh-CreERT2 to yield b-Cateninfl/fl/Shh-CreERT2 mice (bCAT cKO). bCAT cKO and WT mice were injected daily with tamoxifen for 5 days, and IVDs harvested 3 weeks later for gene expression, mechanics and histology  complex is a mechanotransductive pathway proposed to be involved in b-Catenin-driven transcription in the cell nucleus41, they persist in human IVDs with aging and degeneration42 and their loss may contribute to degeneration. Corroborative of other studies43, tail compression suppresses the expression of notochordal cell marker T but we newly demonstrated that T expression can be modulated by endogenous regulation of b-Catenin in nucleus pulposus cells. In both the lumbar and tail IVDs, stabilization of b-Catenin in the cells of the nucleus pulposus increased the number of vacuolated cells, reminiscent of healthy, immature notochordal NP cells44. In relation to osterix, modulation of b-Catenin differentially influenced osterix and T expression, i.e., loss of b-Catenin downregulated T expression and upregulated osterix expression and vice versa. Osterix is a critical transcription factor in stem cell differentiation, where loss of osterix45 or loss of b-catenin47 diverts differentiation from osteoblasts to chondrocytes, but is needed for hypertrophic differentiation48. The role of osterix during intervertebral disc degeneration is not well understood but our findings suggest a relationship where its regulation may contribute to the vastly different expression profiles of notochordal and chondrocytic-like cells22. Nucleus pulposus-specific genetic manipulation of the Wnt/b-Catenin pathway regulated the biomechanics of the IVD, potentially by mediating the differentiation and/or proliferation of resident progenitors in the nucleus pulposus.Concomitantly, notochordal cell expression (T) and osterix were differentially regulated by Wnt signaling in the NP. Notochordal cells of the NP are closely associated with a healthy IVD49 that was regulated in our studies by compression, Shh-driven b-Catenin-deficiency and b-Catenin stabilization; (ii) Deletion of b-Catenin protein expression in the cKO animal studies was regulated in the NP; and (iii) Suppression and upregulation of b-Catenin expression in the NP was highly correlated to the mechanics of the entire IVD. Further, b-Catenin expression of the whole IVD and of the NP were similarly correlated to the compressive stiffness of the cKO lumbar IVDS. Nonetheless, this limitation must be kept in mind and detailed interpretations must be applied cautiously. (2) There was a lack of biomechanical change in the tails of cKO IVDs and we suggest that a prolonged suppression of Wnt signaling may be necessary to alter the functional impact. While lumbar IVD are more comparable to human IVD than tails31 and differ from tail IVD in mechanics, metabolism and change differently with aging32, we studied tail IVDs because they have the advantage of their ease of access for manipulation. Our data suggest that b-catenin levels may have been normalized in tail IVDs after a short period of tamoxifen. Lumbar IVDs demonstrated a similar recovery effect, but over a longer period (3 mo). (3) There were a few disparities in the effect of tail compression between the WT mice and TOPGAL mice, e.g., downregulation of aggrecan and b-catenin gene expression in WT IVDs versus no effect in the 5 mo IVDs in TOPGAL mice. The applied force was the same between the mouse strains, but the size of the WT IVD was smaller and therefore, the applied stress was larger in and more harmful to the WT discs than TOPGAL discs.There were a few limitations in the study. (1) All of the gene expression in the study was conducted in whole intervertebral discs which diluted the alterations of the NP, but many outcomes were determined that were specific to the NP. For instance: (i) Brachyury T is a NP specific marker in mice and humansIn conclusion, early intervertebral disc degeneration by tail compression reduced Wnt signaling, but concomitantly increased b-Catenin with minimal Wnt activation. The mechanism was unclear, but it may have included disruption of the LINC complex, which may limit translocation of b-Catenin into the cell nucleus. Upregulation of b-Catenin and downregulation of Wnt activity in the nucleus pulposus was modeled by stabilization and deletion of b-Catenin in Shh-expressing cells, respectively. Deficiency of b-Catenin reduced the compressive stiffness of the intervertebral disc. By contrast, stabilized b-Catenin increased the compressive stiffness of the intervertebral disc and prevented compression-induced loss of aggrecan expression, albeit with some consequences. Overall, stimulating Wnt/b-Catenin signaling may be a therapeutic approach to intervertebral disc degeneration but will have to be approached with caution.6, contained the LacZ transgene under the control of the TCF/LEF promotor27, and were used to assay Wnt activity. Shh-CreERT2 mice (B6.129S6-Shhtm2(cre/ERT2)Cjt/J, stock # 005623) and b-Cateninfl/fl mice  were purchased from The Jackson laboratory. To suppress b-Catenin in nucleus pulposi, b-Cateninfl/fl  mice were crossed with Shh-CreERT2 to yield b-Cateninfl/fl/Shh-CreERT2 mice . Shh is expressed in the NP of embryonic and postnatal IVDs50, is required for the formation of the notochord sheath and patterning of the NP51, and is accepted as a marker of NP cells up to 19 months of age52. To induce activation of b-Catenin in nucleus pulposi, b-Cateninfl(Ex3)/fl(Ex3)  were crossed with Shh-CreERT2 to yield Shh-CreERT2/b-Cateninfl(Ex3)/fl(Ex3) mice . Dr. Roberta Faccio generously donated the β-Cateninfl(Ex3)/fl(Ex3) mice53. In order to report expression of Shh in adult intervertebral discs, Ai9(RCL-tdT) mice were crossed with Shh-CreERT2 to generate the experimental Shh-CreERT2/tdT mice. Shh-CreERT2/tdT mice were injected with tamoxifen for two days and the IVDs harvested on the third day. tdT mice (No Cre) served as controls. Three mice were used to determine the targeting efficiency in the lumbar and 2 in the tail. The experimental design is listed in Table Female TOPGAL  transgenic mice  were inbred to bear experimental mice that were aged to 5 (n = 19) or 12 months (n = 16). The homozygous TOPGAL mice on a CD-1 background were genotyped as previously published-lacZ34Ef10 and degeneration12. Tail puncture is another model of severe IVD degeneration54 and was applied to 5 mo and 12 mo animals to determine the effect.Once the C7 and C9 vertebra were identified with preoperative radiographs Fig. , 23-G ne6. Prior to mechanical testing, the bone-disc-bone segments were excised, zygapophysial joints and superficial tissue were removed, and spinal units were hydrated in 1X PBS for 18 h at 4 °C. Superior and inferior vertebra were gripped by microvises and, once secured, the sample was immersed in 1X PBS. A materials testing system  applied a load range of −1.2 Newtons (N) to 0.6 N for twenty compression-tension cycles at a frequency of 0.5 Hz, respectively.Controlled mechanical tests were performed on L6-S1 and CC6-CC7 motion segments as previously described6. Briefly, the compressive and tensile loading curves were isolated and a 6th order polynomial was fit to the 20th loading and unloading tension-compression cycle. The minimum derivative of the curve represented the neutral zone stiffness and the derivative measured at 80% of the maximum load magnitude in the compressive and tensile portion of the curve constituted the compressive and tensile stiffness, respectively.The trilinear fit model determined the compressive, tensile and neutral zone stiffness of the motion segment55 on coccygeal (CC7-CC12) motion segments. Motion segments from TOPGAL mice were freshly harvested, fixed in 4% paraformaldehyde for 1 h, incubated in Xgal  for 48 h, fixed overnight, decalcified in Immunocal for two days and embedded in paraffin using routine methods. These, coronal sections (10 µm) were used to visualize galactosidase cells and serial sections were counter stained with eosin or Safranin-O. All other intervertebral discs from other mice were not incubated with Xgal or fixed overnight. Sections for immunohistochemistry were deparaffinized and stained with total β-catenin antibody  . For frozen sections  tissues were fixed in 4% paraformaldehyde, decalcified in 14% EDTA for 3 days, infiltrated with 30% sucrose overnight, embedded in OCT, sectioned coronally (10 µm), and stained with DAPI.Beta-galactosidase staining for Wnt activity, Safranin-O, and IHC was performed as previously described23. Images of sections were thresholded using ImageJ 1.40 g . The ratio of stained area-to-total area of the nucleus pulposus was normalized to the internal control and used as an outcome. One section was analyzed per IVD. The internal control IVDs were CC10–12 and were not subjected to static compression .Wnt activity (LacZ expression) quantification within the nucleus pulposus was carried out as previously describedion Fig. . HistoloIpo8 (Mm01255158_m1) and then normalized to the control intervertebral disc (2−∆∆CT) or to the average WT value. Brachyury  expression was used as Wnt signaling indicator because it requires TCF/LEF signaling20 and notochordal fate is driven by Wnt signaling26.For instrumented animals, tail intervertebral discs between CC7-CC9 and CC10–12 and, for genetic mouse models, tail intervertebral discs CC10–12 and lumbar discs L3-L5 were separated from all other tissues and snap frozen in liquid nitrogen. Then samples were pulverized in a Mikro Dismembrator  and suspended in TRIZOL (Ambion) until further processing. Total RNA extraction was performed using a standard kit . RNA concentration was quantified . First strand cDNA was synthesized  from 500 ng of total RNA for Taqman (Life Technologies) probes Aggrecan (Mm00545794_m1), AdamtS5 (Mm00478620_m1), b-Catenin , Col1a (00801666_g1), Col2a (Mm001309565_m1), MMP13 (Mm00439491_m1), DKK1 (Mm00438422_m1), CCND1 (Mm00432359_m1), Brachyury (T) (Mm00436877_m1), osterix (Mm04209856), Axin2 (MM00443610), Sun1 (Mm00659179_m1), Sun2 (Mm01299500_m1), Nesp1 (Hs00326979_m1), Nesp2 (Hs00794881_m1) and Wnt ligands . The relative gene expression in each loaded intervertebral disc was represented by normalizing to Paired Student’s t-tests compared control to loaded intervertebral discs. Unpaired Student’s t-tests compared intervertebral discs of 5 mo to 12 mo animals or WT to genetic KO animals. A linear regression correlated the relative expression (cACT/WT or cKO/WT) of b-Catenin to Adamts5, aggrecan or compressive stiffness of both WT and cACT/cKO IVDs. Another linear regression correlated the fraction of cells positive for b-Catenin in the NP to the stiffness of both WT and cACT/cKO IVDs. Statistical computations were completed using SPSS (IBM SPSS Statistics 21) and significance was set at p < 0.05.Data may be made available upon request.Supplementary Information


Here we extend our previous work on cluster false positive rates, which used one‐sided tests, to two‐sided tests. Briefly, we found that parametric methods perform worse for two‐sided t‐tests, and that nonparametric methods perform equally well for one‐sided and two‐sided tests.One‐sided  In our findings, it appears that modest inaccuracies in the null cluster size distribution corresponding to FWE 0.05 (see Figure On this last assumption, the control of FWE depends on the accuracy of the cluster size distribution in its tail. For example, it is of little consequence if the true cluster size FWE t‐test is only based on the assumption of exchangeability between subjects, and therefore performs equally well for two one‐sided tests at In contrast, the nonparametric permutation test for a two‐sample


The number of patients diagnosed with chronic bile duct disease is increasing and in most cases these diseases result in chronic ductular scarring, necessitating liver transplantation. The formation of ductular scaring affects liver function; however, scar-generating portal fibroblasts also provide important instructive signals to promote the proliferation and differentiation of biliary epithelial cells. Therefore, understanding whether we can reduce scar formation while maintaining a pro-regenerative microenvironment will be essential in developing treatments for biliary disease. Here, we describe how regenerating biliary epithelial cells express Wnt-Planar Cell Polarity signalling components following bile duct injury and promote the formation of ductular scars by upregulating pro-fibrogenic cytokines and positively regulating collagen-deposition. Inhibiting the production of Wnt-ligands reduces the amount of scar formed around the bile duct, without reducing the development of the pro-regenerative microenvironment required for ductular regeneration, demonstrating that scarring and regeneration can be uncoupled in adult biliary disease and regeneration. In fibrotic biliary disease, portal fibroblasts promote both biliary scarring and bile duct regeneration. Here, the authors report that the non-canonical Wnt-PCP signalling promotes bile duct scarring in mice, and inhibition of Wnt-ligands reduces the scarring without impairing regeneration. Chronic damage to the bile duct typifies biliary disease and results in the proliferation of biliary epithelial cells (BECs) and the formation of a regenerative microenvironment populated by immune cells and fibroblasts2. These cells provide both instructive signals to BECs4 and maintain tissue integrity by depositing scar tissue7, thereby promoting bile duct regeneration. It remains unclear whether BECs communicate to the microenvironment to maintain it appropriately and regulate the amount of extracellular matrix (ECM) that is deposited by surrounding cells.Introduction: Biliary diseases, also known as cholangiopathies, account for approximately one-third of all adult liver disease and 70% of childhood liver disease10, and previous studies have concluded that BEC proliferation is β-catenin dependent12. Recent advances in both animal models13 and single-cell RNA sequencing however, have challenged the central role for Wnt-β-catenin signalling in BEC proliferation and have suggested that the role of Wnt in these processes could be more complex than previously thought15. In addition to stabilising β-catenin, Wnt ligands integrate into a much larger signalling network, thereby activating a number of Wnt-dependent signalling cascades17. These, so-called, non-canonical Wnt signalling pathways are highly conserved from Drosophila through to vertebrates and utilise a broad range of cell surface receptors to activate diverse downstream processes20. Wnt-Planar Cell Polarity (Wnt-PCP) signalling represents one of these non-canonical pathways and is required for a number of morphogenic processes in the embryo;22 moreover, Wnt-PCP signalling has been implicated in the pathogenesis of a number of adult diseases and cancers25. Whether Wnt-PCP signalling plays a role in bile duct disease and regeneration has not been determined.In a number of tissues, epithelial regeneration relies on the activation of Wnt-β-catenin signalling by locally produced Wnt ligands26, are upregulated in biliary injury. In this context, therapeutic inhibition of the Wnt signalling pathway, through the prevention of Wnt-ligand secretion, reduces the level of fibrosis deposited around proliferating BECs, without affecting BEC number. We then go on to demonstrate that Wnt ligands regulate this process through Planar Cell Polarity receptors that activate the JNK/c-JUN signalling pathway specifically in BECs. In turn, this Wnt-PCP signal promotes BEC crosstalk with portal fibroblasts and regulates the capacity of fibroblasts to synthesise collagen and form scar tissue. This study demonstrates how non-canonical Wnt signalling functions to regulate adult tissue scarring by integrating a number of cell types and offers a novel therapeutic target to treat biliary diseases in patients.Here, we demonstrate that Wnt ligands associated with non-canonical Wnt signalling, particularly Wnt5a27 however, the role that Wnt signalling plays in this process remains controversial, with conflicting reports describing variable roles for Wnt-β-catenin28. Using tissue from patients with primary sclerosing cholangitis (PSC), a progressive human biliary disease in which BECs proliferate29 and also two mouse models of BEC proliferation  we sought to determine whether the Wnt-β-catenin pathway is activated in BECs. To do this, we assessed Axin2 mRNA expression and the nuclear translocation of β-catenin in BECs. We failed to see that β-catenin translocates into the nucleus of BECs nor did we see the expected increase in Axin2 expression in any of these contexts  is significantly increased, and while c-JUN is expressed broadly within BECs, c-JUN phosphorylation (phospho-c-JUNS73) is increased in PSC patients compared with those without disease  and connective tissue growth factor (Ctgf), known c-JUN target genes39, which are associated with biliary scarring41, are also transcriptionally upregulated . Inhibition of Porcupine results in the global suppression of Wnt-ligand secretion and renders Wnt ligands unable to bind to the CRD domain of Frizzled. Our experimental strategy is summarised in Fig. Previous studies have found that loss of Wnt-ligand secretion following Wnt-less (Wls) deletion alters biliary regeneration and fibrosisCtgf and Mmp7  and fibroblasts (stained for Desmin) remained the same between Porcupine-i and control mice Fig. , the amo45, including the archetypal Wnt-PCP ligand, Wnt5a46, and indeed, in CD45+/CD11b+ macrophages isolated from the livers of mice with biliary injury, we found that macrophages expressed WNT5A. Moreover, in macrophages from mice treated with porcupine-i, 50% of cells retained WNT5A in their ER compared with 32% in vehicle-treated animals  are a source of multiple Wnt ligandsury Fig. . In thes 47 Fig. . Moreove 47 Fig. , suggestWnt5aflox, Supplementary Fig. Ctgf and Mmp7 when myeloid Wnt5a was deleted compared with ducts isolated from mice in which Wnt5a was intact  are viable, reach adulthood and have been used to determine the role of Vangl2 in a number of adult contexts53. In isolated bile ducts from Vangl2S464N mice given either DDC or TAA injury 54.The Vangl2 can be deleted in the BEC lineage, we isolated bile ducts from Vangl2flox/flox mice or wild-type littermates and grew these as organoids . Following Vangl2 loss, Vangl2ΔTM organoids show reduced levels of nuclear phospho-c-JUNS73 . In K-19CreERT::Vangl2flox mice, following injury, we failed to detect phospho-JNKT183/Y185 or phospho-c-JUNS73 in Vangl2ΔTM BECs  are retained in vivo in the bile duct following injury  pathway, is necessary for establishing any number of axes within the embryo, and through its control of cell shape, migration and planar cell movement is absolutely required for normal organogenesis63. The role of Wnt ligands in establishing these body plans likely predates their role in activating β-catenin-dependent transcription, yet whether Wnt-PCP signalling plays a role in adult homoeostasis and regeneration remains largely mysterious, and work unpicking the role of Wnt-PCP in adult disease and tissue regeneration remains in its infancy.Wnt ligands do however, act through a number of highly conserved developmental pathways27. The role of Wnt signalling in the context of BEC proliferation and bile duct regeneration has been contested, with a number of early papers demonstrating activation of the canonical, Wnt-β-catenin-dependent pathway in these cells28. Whether Wnt-β-catenin is as important as these early papers suggested remains contentious, as we and others have failed to find nuclear translocation of β-catenin or activation of canonical Wnt-β-catenin receptors in BECs during bile duct regeneration35. Furthermore, recent studies using single-cell RNA (scRNA) sequencing have failed to find an obvious canonical Wnt signature in BECs15, and deletion of the Wnt-potentiating R-Spondin (RSPO)-LGR system in BECs had little effect on their proliferation14. These data are not unequivocal, as recent work has indicated that RSPO can function independently of LGR proteins64. Though if this is the case in BECs they would stand out as unusual as the majority of Wnt-responsive cells in the adult have been shown to require the RSPO-LGR rheostat65. Several papers have now suggested that Notch and Yap signalling, not canonical Wnt signalling, are the favoured routes to promote BEC proliferation66. Our data presented here are in agreement with this observation; however, whereas other groups fail to see Axin2 transcripts in BECs15, we find infrequent transcripts that do not increase with injury, and therefore we conclude that canonical Wnt-β-catenin signalling is not upregulated in patients with biliary disease or animal models.The bile duct, like other epithelial structures in the liver, has a remarkable regenerative capability, and following injury, biliary epithelial cells (BECs) begin to proliferate and form new tubular networks, ultimately restoring flow though the duct and maintaining the function of the liver28, and from our data in this paper and those data from other groups it appears that in the context of bile duct injury and repair at least that myeloid-derived Wnts play an important role in regulating this process43. Importantly, others have shown that BEC proliferation is dependent on Wnt ligands, but that this proliferation in BECs is β-catenin independent, suggesting that non-canonical Wnt signalling pathways, including but not limited to Wnt-PCP, regulate cholangiocyte proliferation13. In this study, we repeatedly fail to see a reduction in cholangiocyte number when we inhibit Wnt-PCP signalling, as would be expected based on previous data13. We cannot exclude, however, the possibility that there are compensatory pro-proliferative mechanisms that take place following pharmacological inhibition of Wnt-ligand production, nor can we exclude the possibility that BEC proliferation is driven through a non-canonical or atypical Wnt pathway that does not depend on Vangl2, such as Wnt-STOP or Wnt-YAP/TAZ signalling68.There is little doubt that Wnt ligands are expressed during biliary regeneration15. Inhibition of Wnt ligands, either genetically or using a therapeutic Wnt inhibitor, significantly decreases the amount of biliary scarring that occurs following injury, leading us to demonstrate that Wnt-PCP signalling is upstream of fibrogenic cytokine production. Surprisingly, heterozygous loss of Vangl2, through the Vangl2S464N mutation, is sufficient to reduce biliary scarring, suggesting that this system requires tight regulation. Emerging data from other chronically diseased tissues indicate that Wnt-PCP signalling can play a role in the establishment and maintenance of scarring and deposition of the extracellular matrix70; here we show that Wnt-PCP is also involved in the formation of biliary scars in the adult liver.In this study, we do, however, find a consistent and reproducible suppression of bile duct fibrosis when Wnt-PCP signalling is inhibited in BECs. In this model, the binding of Wnt ligands, through Wnt-PCP receptor activation, including Ptk7 and Vangl2, results in the phosphorylation of Jnk, which promotes c-Jun-dependent transcription. These observations are concordant with scRNA data in which the AP-1 transcription factor network is enriched in BECs66. Deleting these pro-fibrogenic cells or inhibiting their activation/proliferation would therefore have significant consequences for the regenerating biliary epithelium and could ultimately prevent ductular regrowth following injury. Inhibition of Wnt signalling or loss of Wnt-PCP receptors on proliferating BECs does not affect the formation of the cellular regenerative microenvironment, with pro-regenerative fibroblast numbers and the numbers of BECs remaining constant following inhibition. Our data demonstrate that it is possible to pharmacologically uncouple regeneration from scar fomation in the diseased bile duct and suggest that inhibition of Wnt-ligand production should be considered as a therapy in chronic biliary diseases.Adult tissue repair, by its very nature, requires fine balance between epithelial regeneration and scarring. While fibroblasts in the regenerative microenvironment are the primary cells that make scar tissue, we and others have previously shown that the fibroblast microenvironment in biliary regeneration provides a number of signals that BECs require to maintain both their proliferation and lineage specification2. Animal experiments were approved by the University of Edinburgh Animal Welfare Ethical Review Board (AWERB).All mice were maintained in 12-h light/dark cycles and had access to food and water ad libitum in accordance with UK Home Office Regulations. All experiments were performed under UK Home Office licence PPL: 70/8150 held by Dr Luke Boulter; mice were euthanised by an escalating dose of CO2O). Mice were dosed for 48 h prior to the administration of DDC to ensure inhibited Wnt-ligand production prior to injury. Effectiveness of LGK974 was confirmed through reduction of GS staining adjacent to central veins and is known to be Wnt-ligand dependent. Finally, to generate livers that overexpressed β-cateninNF90, we used a hydrodynamic model in which 20 μg of pT3-EF1aH N90-beta-catenin ) was co-injected with 6 μg of PT2/C-Luc//PGK-SB13 ). These plasmids were delivered in 10% w/v physiological saline in <7 s.For the DDC mouse model, 6–8-week-old, male CD1 mice were fed 0.1% 5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) in their diet for up to 14 days and provided with normal drinking water. Mice that drop >20% of their bodyweight were given DDC food softened with water. For the TAA model, mice were given 400 mg/L thioacetamide in sweetened water for up to 12 weeks. Thioacetamide water was present throughout the model and mice would access this ad libitum. In studies where mice received the Porcupine inhibitor, LGK974 experimental mice were dosed with 5 mg/kg LGK974, twice daily via oral gavage  were maintained on a C3H background, and throughout these studies, wild-type littermates served as a control. Krt19CreERT:R26RtdTomato:Vangl2flox/flox mice were generated by crossing the Krt19CreERT mouse line initially with the tdTomato Ai14 line from Jax labs. Mice heterozygous for the K-19CreERT knock-in and homozygous for tdTomato were then crossed with Vangl2flox mice, which were generated and kindly provided by Prof Deborah Henderson, Newcastle. In this study, all Krt19CreERT:R26RtdTomato:Vangl2flox/flox mice were positive for tdTomato and CreERT and were then homozygous, heterozygous or wild type for the Vangl2flox allele. Induction of Cre was achieved through repeat administration of tamoxifen (three times for 1 week) at a concentration of 4 mg per mouse for each injection. Tamoxifen was diluted in 10% molecular-grade ethanol: 90% corn oil. LysMCre:Wnt5aflox/flox mice were generated by crossing the LysMCre line kindly provided by Dr Steven Jenkins, University of Edinburgh, with Wnt5aflox/flox mice purchased from Jax labs. Mice were maintained as Cre positive and homozygous, heterozygous or wild type for the floxed allele. All genetically engineered mice were genotyped by Transnetyx Inc.Vangl2Five-micrometre unstained sections of formalin-fixed paraffin-embedded explant fibrotic human liver from the deep right lobe of native explants from patients with primary sclerosing cholangitis undergoing allograft liver transplantation were received. Explant livers were received fresh on ice and fixed immediately by the installation of 4% v/v neutral buffered formalin  via the hepatic veins as part of the standard specimen pathway. Human tissue was obtained by approved application to the Lothian NRS Human Annotated Bioresource that is authorised to provide unconsented anonymised tissue under ethical approval number 15/ES/0094 from the East of Scotland Research Ethics Service REC 1. Informed consent was waived due to the historical nature of these samples.2O and quantified using a Nanodrop (Thermofisher). In all, 1 µg of total RNA was treated with gDNA wipe-out buffer (Qiagen) and reverse transcribed using the Quantitect reverse transcription kit (Qiagen) as per the manufacturer’s instructions. For quantitative PCR ~20 ng of total cDNA was used per reaction and was run using FastStart SYBR Green reagent (Roche), cycle conditions were defined by the manufacturer’s protocol. All qRT-PCR reactions were run using a Lightcycler 480-II. All primer sets used in this study were purchased from Qiagen  and the manufacturer’s protocol was followed. RNA was suspended in RNAse/DNAse-free H2O2, washed, and then native streptavidin and biotin were blocked by using a commercial avidin and biotin blocking kit. Following this, sections were blocked with a universal protein block. Tissue sections were incubated overnight with primary antibodies diluted in antibody diluent. Following washing, biotin-conjugated secondary antibodies were incubated with tissue sections as per Table 2O2 and Avidin/Biotin blocking stage. DAB-stained sections were imaged on an Olympus Dotslide, Hamamatzu Nanozoomer or an Olympus BX53 upright microscope. Immunofluorescence was imaged using a Nikon A1R confocal microscope.Following euthanasia, livers were flushed through with 20 ml of physiological saline. Dissected liver lobes were then fixed overnight in 10% neutral buffered formalin in PBS. Following fixation, tissue was dehydrated in 70% ethanol and processed to wax using a tissue processor. Four-micrometre tissue sections were cut and rehydrated through graduated alcohols. Tissue sections were antigen retrieved as per Table RNAscope was performed on formalin-fixed liver tissue. Four-micrometre sections were used throughout, and all RNAScope performed in this study was done by Aquila Histoplex, Edinburgh, with the exception of Axin2, which was performed at the core histology facility, The Beatson Institute, Glasgow.3 pieces and digested in DMEM/F-12 media containing Collagenase-IV (Roche) and DNASe-I (Roche). Following digestion and dissociation, bile ducts become obvious as parenchyma is digested away. Bile ducts are strained through a 70-µm filter and extensively washed in PBS to remove any residual cells. Bile ducts are then used for downstream applications.To isolate bile ducts from both uninjured and injured livers, dissected liver was chopped into 5-mmLiver organoids were derived from isolated bile ducts. Briefly, isolated bile ducts were resuspended in 100% GFR Matrigel. Within 24 h, bile ducts form closed structures, and within 48 h budding can be seen from the duct. Following expansion, these ducts were removed from Matrigel by incubating with ice-cold Versene. Organoids were dissociated with pipetting and then re-plated in fresh 100% Matrigel. This process was repeated to expand organoids. The growth medium used in this study consisted of a base media of DMEM/F-12 supplemented with Glutamax, Penicillin/Streptomycin, Fungizone and HEPES. Just prior to feeding, the base media was supplemented with HGF, EGF, FGF10, Gastrin, Nicotinamide, N-Acetylcystine, B-27, Forskolin, Y-27632 (ROCK inhibitor), A83-01 (TGF-β inhibitor) and Chir99021 (GSK3β inhibitor).flox/flox) versus control organoids (Vangl2WT/WT) Cre was expressed from the CMV promoter, in vitro, by infection of organoid structures with Lv-CMV-Cre  at an MOI of 5. For infection media was supplemented with 1/100 of Transdux reagent. For assays in which signalling was monitored in response to genotype, growth media was withdrawn from organoid cultures, and organoids were cultured in base media containing B-27, Forskolin, Nicotinamide and N-Acetylcystine alone for 48 h, at which point organoids were lysed for analysis.To evaluate the effects of Vangl2 knockout on bile duct organoids (Vangl2l-thyronine), 0.025 μg/ml EGF and 4 μg/ml Forskolin, and were pH adjusted with 3.4 N NaOH.The non-immortalised human cholangiocyte line (NHC-3 cells) was provided by Dr Jesus Banales . Cells were grown in DMEM/F-12 supplemented with 5% FCS, 1× MEM non-essential amino acids, 1× Lipid mixture , 1× MEM vitamin solution, 1× Penicillin/Streptomycin, 25 mg Soybean Trypsin Inhibitor, 1× Insulin/Transferrin/Selenium, 13.4 μg/ml Bovine Pituitary Extract, 3.93 μg/ml Dexamethasone, 3.4 μg/ml T3  were plated at 1 × 10g. The remaining cells were then pelleted and red cells lysed using Red Cell Lysis buffer (Gibco). Following lysis, cells were blocked in PBS containing BSA and sodium azide for 30 min on ice. Following blocking, cells were incubated in blocking buffer with CD45-PE and CD11b-PECy7 (Biolegend) antibodies for 1 h at room temperature and were then sorted using a FACS Aria II. Dead cells were excluded from sorting using DAPI.Dissected livers were minced and then digested for 2 h in DMEM/F-12 nutrient culture media containing 1% of FCS, 0.5 mg/ml Collagenase, 0.5 mg/ml Dispase and 0.1 mg/ml DNase1. Following digestion, liver tissue was mashed through a 70-μm cell strainer. Heptocytes and large cell clumps were removed from the cell mixture by slow speed centrifugation at 50 × Isolated bile ducts or organoids were lysed in RIPA buffer containing phosphatase (Thermo Fisher) and protease  inhibitors. Protein quantification was determined using Pierce BCA reagent (Pierce) and quantified using a nanodrop. The standard curve for protein quantification was derived from the BCA reagent handbook using Albumin standards provided. In all, 20 μg of total protein was loaded onto a 4–12% NuPage Bis-Tris gel (Thermo Fisher). Prior to running, proteins were reduced with NuPage LDS sample buffer (4×) and NuPage Sample Reducing Agent (10×). All gels were run using NuPage MOPS SDS Running buffer containing NuPage Antioxidant. Proteins were transferred onto PVDF membrane (Amersham) using NuPage Transfer Buffer. Following transfer, membranes were blocked in either 5% BSA (Sigma Aldrich) in TBST or 5% dried milk (Marvel) in TBST. Membranes were incubated with primary antibodies  or using one-way ANOVA to compare multiple groups, with a post hoc correction for multiple testing. Non-parametric data were analysed using a Wilcoxon–Mann–Whitney U test when comparing two groups or a Kruskall–Wallis test when comparing multiple non-parametric data. Throughout p < 0.05 was considered significant. Data are represented as mean with S.E.M. for parametric data or median with S.D. for non-parametric data.All experimental groups were analysed for normality using a D’Agostino–Pearson Omnibus test. Groups that were normally distributed were compared with either a two-tailed Student’s All animal experiments were approved by the University of Edinburgh local ethics committee and were licensed by the UK Home Office. All patient material contained in this paper was approved by the NHS Lothian Bio resource ethics committee. No prospective tissue was collected in this study.Further information on research design is available in the Supplementary InformationPeer Review FileReporting Summary


Obesity has become epidemic worldwide, which triggers several obesity-associated complications. Obesity is characterized by excess fat storage mainly in the visceral white adipose tissue (vWAT), subcutaneous WAT (sWAT), and other tissues. Myriad studies have demonstrated the crucial role of canonical Wnt/β-catenin cascade in the development of organs and physiological homeostasis, whereas recent studies show that genetic variations/mutations in the Wnt/β-catenin pathway are associated with human metabolic diseases. In this review, we highlight the regulation of updated Wnt/β-catenin signaling in obesity, especially the distinctly depot-specific roles between subcutaneous and visceral adipose tissue under high-fed diet stimulation and WAT browning process. Worldwide obesity has more than doubled since 1980; 39% of adults aged 18 years and older were overweight in 2016, and 13% were obese throughout the world . The maiWnt/β-catenin signaling controls several developmental processes in early stages of life and the homeostasis of mature tissues via regulating cell proliferation , differeWhite adipose tissues (WATs) are distributed almost in the entire body and more biologically relevant with obesity. However, brown adipose tissues (BATs) principally function in the neonatal period to combat extrauterine coldness, and recent research has shown that adult humans also possess functional BAT and its mass shows a negative correlation with adiposity and body mass index (BMI) . SeveralHere we systematically reviewed literature about Wnt/β-catenin signaling and obesity, and its precise role in the browning process of depot-specific white adipocytes, to provide information about therapeutic approaches for different subgroups of the obese population.2, which does not take fat content into account  chanisms . Increaschanisms . The funchanisms , 2013. Oeriments . However was first identified in 1982 via the discovery of the Wnt1 gene  in mice . Then thXenopus , both prXenopus , and WntXenopus , which fXenopus .Figure 1). In addition, β-catenin can bind to other transcription factors, thereby modulating many downstream biological processes including epithelial to mesenchymal transition, pluripotency, and melanocyte development  (R-spondin proteins (RSPOs), including RSPO1-4 in mammals, are secreted proteins that amplify canonical Wnt/β-catenin signaling, but not by themselves – they only sensitize cells to Wnt/β-catenin signaling. RSPOs have a similar structure consisting of one C-terminal TSR-1 domain and two N-terminal furin repeats. The two furin repeats are pivotal for potentiating Wnt/β-catenin signaling . The TSRigure 2) . ZNRF3 aigure 2) . ZNRF3/Rigure 2) .Figure 1). An initial clue to the role ZNRF3/RNF43 plays in RSPOs–LGR4/5/6 complex is that the upregulation of FZDs in cell-surface, a typical RSPOs-induced event, can be represented when ZNRF3/RNF43 lost function. This hypothesis is further supported by cell biology and biochemical experiments, in which RSPOs simultaneously combine with the extracellular regions of ZNRF3/RNF43 and/or LGR4/5/6, leading to a self-ubiquitination and endocytosis, sensitizing cells to Wnt/β-catenin signaling by increasing Wnt-FZD-LRP5/6 complex availability at the cell surface  mice have many more beige adipocytes in vWAT, and further investigation reveals the significant increase of thermogenic genes including UCP1, PGC-1α, and Cidea. These phenomena are amplified by cold stimulation or isoprenaline treatment. Our results demonstrate that ablation of Lgr4 in mice resists dietary and leptin mutant-induced obesity and its metabolic complications, and further prove the crucial role of LGR4 in obesity. Interestingly, recent data show that LGR2/TSHR . Of course, other possible mechanisms should not be excluded.Why the distinct function of Wnt/β-catenin signaling in adipose precursor cells and pre-mature/mature adipocytes is uncovered till now. As mentioned previously, when β-catenin enters the nucleus, it turns on transcription via binding to members of the TCF/LEF family of transcription factors. The critical step in the activation of Wnt/β-catenin signaling is the formation of a complex comprising β-catenin and members of the TCF/LEF family . β-catende novo adipogenesis. Adipocyte hypertrophy is a major component of the metabolic syndrome in obese individuals .Considering the possibility that the activation of Wnt/β-catenin signaling in APs promotes their proliferation whereas in pre-mature/mature adipocytes it regulates differentiation, we hypothesize that Wnt/β-catenin signaling could play an important role in depot-specific WAT expansion (including hyperplasia and hypertrophy) of HFD-induced obesity (+) adipocyte progenitors can generate a fibrogenic phenotype, which is apt to vWAT fibrosis. More importantly, the activation of PDGFRα pathway promotes PDGFRα+ CD9low cells (committed to adipogenesis) in a phenotypic shift to CD9high fibrogenic cells (originate from profibrotic cells). These observations remind us of the relationship of the PDGFRα pathway and vWAT adipocyte hypertrophy. It is possible that PDGFRα pathway activation inhibits “overproliferation” of APs by suppressing Wnt/β-catenin signaling, then triggers the hypertrophy of existing mature adipocytes. It has been hypothesized that an adipogenic signal is generated to stimulate the production of new adipocytes when mature adipocytes reach their maximal size  from hypertrophied adipocytes (Both vWAT and sWAT are composed of mature adipocytes (the fat-storing cells), adipocyte precursors (that give rise to new adipocytes), and other mesenchymal cells, which include mural, endothelial, inflammatory, and neuronal cells. In response to HFD stimulation, V-APs are first activated via Wnt/β-catenin signaling, leading to their rapid and transient burst of proliferation (“overproliferation”). Based on a previous study , the promal size . Wang alipocytes . The speIt is important to note that the manner of expansion in sWAT depot is significantly distinct from that of vWAT. The activation of Wnt/β-catenin signaling by HFD stimulation mainly promotes sWAT mature adipocytes hypertrophy, and this process is maintained for up to 12 weeks . It is pDuring the WAT expansion process, there is crosstalk among many other possible regulators with Wnt/β-catenin signaling that control the fate of sWAT and vWAT, and the mechanism deserves more study.Beige adipocytes  [or britde novo formation from adipocyte precursor cells and trans-differentiation of mature white adipocytes (-) that can be active to multilocular and UCP1+ beige adipocytes. Interestingly, a new study found that beige adipocytes of vWAT could clearly be divided into two distinct subpopulations, UCP1+ and UCP1- .It is clear that Wnt/β-catenin signaling has a crucial role in the commitment of maintenance and induction of differentiation. The study by Michael  has depi+ cells (-. In addition, it is consistent with previous observations that induced beige cells are mainly found in sWAT and are seldom detected in vWAT (In response to cold or β3-adrenergic receptor agonists or other beige adipogenic stimulations, both sWAT and vWAT adipocyte progenitors are induced to asymmetric division via some crucial signals, such as the activation of Wnt/β-catenin signaling. However, the division percentage into adipocyte progenitors and preadipocytes is different in sWAT and vWAT. Most likely, the percentage of vWAT adipocyte progenitors dividing into progenitors is far more than preadipocytes, resulting in the majority of newly emerging beige adipocytes being positive for BrdU . However+ cells  indicate in vWAT . RegardiWhat is the precise role of Wnt/β-catenin signaling in depot-specific browning and white fat expansion? Which factors interact with Wnt/β-catenin signaling underlying the different features of sWAT and vWAT? What is the mechanism by which Wnt/β-catenin signaling maintains the potency of APs and regulates the differentiation of mature adipocytes under external stimuli? If beige cells are derived from preadipocytes, the question is how Wnt/β-catenin signaling regulates the preadipocytes differentiation into beige cells. And if there are other sources for beige cells, such as trans-differentiation from mature adipocytes and activation of dormant beige adipocytes, how does Wnt/β-catenin signaling be involved in these processes, and how does it participate in and are there different roles and mechanisms in different WAT depots? Although we cannot yet definitively answer these questions, according to more and more evidences and our recent interesting discovery about the role of β-catenin in mature adipocytes, it is highly hypothesized that Wnt/β-catenin signaling is crucial for obesity development, including adipocyte hyperplasia, hypertrophy, browning, and even apoptosis, in a depot-specific manner.in vivo. As the pathogenesis, clinical features, and intervention outcomes of obesity are greatly affected by fat distribution and cellular characteristics, the study of Wnt/β-catenin signaling in WAT expansion and browning in vivo has the potential to discover novel therapeutic targets for precise clinical stratification and treatment for obesity.With the application of new methodologies and experimental models to the field of adipose tissue biology, we have the opportunity to definitively explore the WAT intrinsic mechanisms that regulate adipocyte expansion and browning JW and NC conceived the project. NC drafted the paper. JW contributed comments and advice on the manuscript. All authors were involved in editing the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer DV and handling Editor declared their shared affiliation.