The Wnt/β-catenin signaling pathway is involved in the normal development of thyroid gland, but its disregulation provokes the appearance of several types of cancers, including papillary thyroid carcinomas (PTC) which are the most common thyroid tumours. The follow-up of PTC patients is based on the monitoring of serum thyroglobulin levels which is regulated by the thyroid transcription factor 1 (TTF-1): a tissue-specific transcription factor essential for the differentiation of the thyroid. We investigated whether the Wnt/β-catenin pathway might regulate TTF-1 expression in a human PTC model and examined the molecular mechanisms underlying this regulation. Immunofluorescence analysis, real time RT-PCR and Western blot studies revealed that TTF-1 as well as the major Wnt pathway components are co-expressed in TPC-1 cells and human PTC tumours. Knocking-down the Wnt/β-catenin components by siRNAs inhibited both TTF-1 transcript and protein expression, while mimicking the activation of Wnt signaling by lithium chloride induced TTF-1 gene and protein expression. Functional promoter studies and ChIP analysis showed that the Wnt/β-catenin pathway exerts its effect by means of the binding of β-catenin to TCF/LEF transcription factors on the level of an active TCF/LEF response element at  in TTF-1 promoter. In conclusion, we demonstrated that the Wnt/β-catenin pathway is a direct and forward driver of the TTF-1 expression. The localization of TCF-4 and TTF-1 in the same area of PTC tissues might be of clinical relevance, and justifies further examination of these factors in the papillary thyroid cancers follow-up. Papillary thyroid carcinoma (PTC) is the most common thyroid cancer representing about 80% of all thyroid cancers TTF-1 mRNA is detected in papillary carcinomas (PTC) but not in anaplastic carcinomas; therefore TTF-1 is considered as a marker to distinguish between these two types of thyroid neoplasms The transcriptional factor TTF-1  is commonly expressed in the thyroid gland, lung and central nervous system c-myc, cyclin D1, matrix metalloproteases, CD44 and homeodomain-containing genes The Wnt signaling pathway is a complex network of proteins described to be involved in the control of thyrocyte proliferation The role of Wnt/β-catenin signaling in TTF-1 regulation remains unclear. The aim of this study is to investigate whether the Wnt/β-catenin pathway could regulate TTF-1 expression in a papillary thyroid carcinoma model and to examine the mechanism(s) involved in this regulation. We show herein that Wnt/β-catenin pathway is a direct driver of TTF-1 expression.We investigated by real-time PCR the relative expression in the TPC-1 cell line of TTF-1 as well as the major components of the Wnt/β-catenin pathway , the LRP6 co-receptor, Dishevelled (Dsh1-2-3), β-catenin and the transcription factors: LEF-1, TCF-1, TCF-3, TCF-4. Most of these genes were less expressed 300-fold to 3-fold than RPL13A, except for Dsh3 (1.2-fold higher than RPL13A) and β-catenin (4.8-fold higher than RPL13A) (Immunocytochemistry (ICC) experiments were performed to determine the presence and the subcellular localization of Wnt/β-catenin signaling pathway and of TTF-1 protein in the human TPC-1 cells. As depicted in Altogether, our results clearly showed that the major components of Wnt/β-catenin signaling pathway as well as TTF-1 are expressed in TPC-1 cells. Thus, we aimed to explore the potential regulation of TTF-1 expression by Wnt/β-catenin pathway.TTF-1 mRNA . TTF-1 promoter activity was decreased by 80% when LRP6Dn was transfected, and by 95% with the DEPXDn, β-cateninDn and TCFDn . These results demonstrate that the Wnt/β-catenin signaling pathway is crucial for the basal expression of the TTF-1 gene.To assess if the effect of Wnt/β-catenin is exerted on the level of TTF-1 promoter, we transfected TPC-1 cells with a luciferase reporter plasmid baring 2.11 kbp of TTF-1 promoter, with plasmids expressing dominant negative forms of either LRP6 (LRP6Dn), Dsh (DEPXDn), β-catenin (β-cateninDn) or TCF (TCFDn). The efficiency of the knockdown of Wnt/β-catenin pathway was tested on TOP-Flash-Luc construct that contains four copies of TCF/LEF response element. TOP-Flash-Luc transcriptional activity was strongly inhibited when the dominant negative forms of Wnt components were overexpressed (up to 95% inhibition) , for 10 min, 24 h and 48 h. We observed a 10-fold increase in the expression of TTF-1 at 24 h when cells were incubated with 5 to 20 mM LiCl  functional deletion of TTF-1 promoter, (ii) ChIP analysis of the recruitment of β-catenin on the level of TTF-1 promoter.First, the effect of LiCl on the regulation of TTF-1 transcription was investigated by transfecting TPC-1 cells with a reporter plasmid coupled to different fragments of the TTF-1 promoter  revealed by Genomatix software the presence of two putative binding sites for TCF/LEF  or downstream (TCF-4) of the β-catenin. We found a downregulation of TTF-1 mRNA and protein. The loss-of-function experiments revealed a strong inhibition of the transcriptional TTF-1 activity with dominant negative LRP6, Dsh, β-catenin and TCF expression vectors. Taking together, these findings confirm that the molecular partners of Wnt/β-catenin are responsible for the TTF-1 regulation by post-transcriptional effects influencing the TTF-1 mRNA and protein expression and that they are required for the basal expression of the TTF-1 promoter.The first one consists of knocking-down the molecular compounds of Wnt/β-catenin upstream  or tumour regression .From the medical point of view, inactivation of GSK-3β by LiCl alone The study was approved by the Ethical Committee of the Institut Gustave Roussy. Patients gave written informed consent. No industry gave support for this study.2.The human TPC-1 cell line was kindly provided by Dr. C. Dupuy . The cell line was grown in Dulbecco's Modified Eagle Medium (DMEM)  supplemented with 10% fetal bovine serum (FBS), 100 units/ml penicillin and 100 µg/ml streptomycin (Invitrogen). Cells were incubated at 37°C in a humidified atmosphere containing 5% CO4 cells per well. The protocol used in this study was as previously described TPC-1 cells were plated in 6-well plates with coverslide at a density of 2×10Human thyroid tissues embedded in paraffin blocks were obtained from archive files of Institut Gustave-Roussy, pathology department. They included four primary papillary carcinoma and three apparently normal thyroid tissues taken at distance of benign adenoma. An immunohistochemical study was done on freshly cut 4-µm serial sections with anti-TTF1 (Upstate) and anti-TCF4 (Millipore). Immune complexes were visualized using HRP anti-Rabbit  and DAB chromogen solution . Nuclei were lightly counterstained with hematoxylin and sections were mounted with Histomount .Firefly luciferase reporter plasmids containing various regions of the TTF-1 promoter  and the control empty vector (pSV0AL-A(delta)5′) were used as previously described 5 TPC-1 cells were seeded in 6-well plates. Transfections were performed in serum-free OPTI-MEM using a 50 nM siRNAs and 6 µl Lipofectamine. Cells were incubated with transfectants for 48 h.To knockdown some of the Wnt/β-catenin pathway molecules  transient transfections were carried out using Lipofectamine 2000 transfection reagent (Invitrogen). Briefly, 24 h before transfection, 3×10For promoter studies, transient transfection of plasmids was carried out using the jetPEI™ reagent  according to the manufacturer's instructions. We used 3 µg of TTF-1-luc plasmids or 0.5 µg of TOP-Flash-luc or FOP-Flash-luc. Twenty four hours after transfection, cells were exposed to LiCl (10 mM) for 24 hours. After harvesting, cell extracts were assayed for luciferase activity  and luminescence was measured in a Lumat LB 9507 luminometer . The luciferase activity was normalized to the protein levels .The same protocol was used for transient transfections with dominant-negative expression vectors. TPC-1 cells were cotransfected with 3 µg of TTF-1-luc-2.11 plasmid and 1 µg of dominant-negative expression vectors. Results were expressed as percent of transcriptional activity: [luciferase activity elicited by each sample×100]/luciferase activity elicited by the TOP-Flash-Luc plasmid alone or TTF-1 luc-2.11 alone].4cells/well and incubated with different LiCl concentrations  for 10 min, 24 h and 48 h. qRT-PCR and Western Blot were performed after harvesting the cells.To determine the effect of LiCl (an inhibitor of GSK-3β) on TTF-1 expression, kinetic studies were performed on TPC-1. Cells were plated in 6-well plates at 3×10Total RNA was extracted from TPC-1 cells using RNeasy mini-kit . First-strand cDNA was generated with M-MLV RT buffer pack .in −ΔΔCt method Primers used for Real-time PCR (qRT-PCR) are described Total protein extracts were obtained using M-PER reagent (Pierce) with protease inhibitors cocktail . Proteins were titrated by the BCA method using the BCA protein assay . Cell extracts were separated on 10% SDS-PAGE gel. Proteins were transferred using the iBlot™ Dry Blotting System (Invitrogen) on pre-cut nitrocellulose membranes, then blocked with 0.2% casein  in PBS with 0.1% Tween-20. The membranes were incubated overnight at 4°C with the primary antibody specific for TTF-1 , β-catenin  or β-actin  used as internal control. Blots were washed and incubated with anti-rabbit antibody conjugated to alkaline phosphatase  for 1H at room temperature, and subsequently washed and revealed using CDP Star chemoluminescence reagent (Perkin Elmer).6cells/plate) and treated or not with LiCl for 1 h and 4 h then fixed with 1% formaldehyde added to the medium for 10 min, scraped, and collected by centrifugation. Cells were resuspended in 300 µl of lysis buffer  (PIPES pH 8.0), 85 mM KCl, 0.5% NP-40) with a cocktail of protease inhibitors (Roche). Cells were pelleted by centrifugation and suspended in 300 µl of 1% SDS, 10 mM EDTA, and 50 mM Tris-HCl (pH 8.0) containing protease inhibitors. After incubation on ice for 10 min, they were sonicated six times for 30 seconds using Bioruptor (Diagenode). Lysates were then cleared by centrifugation and the concentration of DNA was determined. Equal amounts of DNA were diluted ten times in dilution-buffer , 167 mM NaCl). The chromatin solution was precleared for one hour at 4°C on Protein G-Agarose/Salmon Sperm DNA beads from Upstate. After brief centrifugation and removal of the beads, DNA was incubated overnight at 4°C on a rotating wheel with 1 µg of either anti-β-catenin, 2206, Sigma) or non-relevant antibody . Immune complexes were collected on Protein G-Agarose/Salmon Sperm DNA beads from Upstate. Beads were washed sequentially in TSE , with 150 mM NaCl, TSE with 500 mM NaCl, buffer A , and twice with Tris-EDTA and then eluted with 200 µl 1% SDS and 0.1 M NaHCO3. Cross-links were reversed by heating at 65°C for 4 h after adding NaCl to 200 mM final concentration. After treatment with Proteinase K (50 µg/ml) for 1 h at 37°C, DNA was purified using Geneclean Turbo kit (Q-Biogene). Next, real-time PCR analysis of inputs and immunoprecipitated DNA samples were performed using the following primers: P1F: CCCTCTTCCTCCTAAGCAGTTTC, P1R: TTAAAGTGGTGGCGTAAATGGC. P2F CCCAACTCTTCCTTTTACTCAG, P2R: GTCACCCAGCTTCTTTCTCTC. Data are compared to input and normalized to anti-nucleolin C23 and expressed as 2−ΔΔCt to determine the assay site IP-fold enrichment. The results are given as relative fold in site occupancy compared with cells cultured without LiCl.TPC-1 cells were plated in 10 cm plates (3×10U test was used to compare the mean of Wnt compounds mRNA relative levels after transfection with siRNAs and the mean of transcriptional activity after transfection with plasmids. For all other studies, means of treatment groups were compared with one-way ANOVA. When the ANOVA showed that there were significant differences between the groups, Dunnett's test or Bonferroni's test was used to identify the sources of these differences. p≤0.05 was considered statistically significant.Data are represented as mean ± SD. Mann-Whitney


Rhabdomyosarcoma is the most common soft tissue sarcoma of childhood and adolescence, accounting for approximately 7% of childhood cancers. Current therapies include nonspecific cytotoxic chemotherapy regimens, radiation therapy, and surgery; however, these multimodality strategies are unsuccessful in the majority of patients with high-risk disease. It is generally believed that these tumors represent arrested or aberrant skeletal muscle development, and, accordingly, developmental signaling pathways critical to myogenesis such as Notch, WNT, and Hedgehog may represent new therapeutic targets. In this paper, we summarize the current preclinical studies linking these embryonic pathways to rhabdomyosarcoma tumorigenesis and provide support for the investigation of targeted therapies in this embryonic cancer. Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma of childhood and adolescence, accounting for approximately 7% of all pediatric cancers , 2. TumoThe current treatment strategies for RMS include chemotherapy, radiation, and surgery. Over the last 50 years, the survival rates for patients with low- and intermediate-risk disease have improved significantly due to risk stratification, combination chemotherapy regimens, and cooperative clinical trials . The IRSRMS is believed to arise from skeletal muscle precursors that fail to undergo appropriate terminal differentiation. Although the precise cell of origin is unclear, skeletal muscle precursors are obvious suspects because they already possess “stemness” characteristics—features that are shared with cancer cells—such as self-renewal, high rates of proliferation, resistance to senescence, and reversal of quiescence. In addition to these stem cell properties, skeletal muscle precursors share stemness markers with RMS cancer cells including PAX3, PAX7, MET, and myogenic regulatory factors , 12. In Myogenesis is a complex and carefully timed process that results in discrete muscle groups that are fully functional even shortly after birth. In mammals, skeletal myogenesis begins during embryonic life and proceeds through fetal, neonatal, and adult developmental stages . There aEmbryonic signaling pathways including Notch, WNT, and Hedgehog play a critical role in the transition of muscle precursors from self-renewal to differentiation fates during both muscle development and postnatal muscle regeneration. During muscle development and postnatal muscle regeneration, mononucleated progenitor cells undergo amplification and asymmetric differentiation along a myogenic lineage to form multinucleated myotubes and ultimately myofibers, which are the terminally differentiated unit of skeletal muscle. In both fetal and postnatal development, there remains a small population of mononuclear muscle stem cells  or satellite cells  that remain undifferentiated and retain the ability to proliferate and differentiate in response to growth signals or tissue damage. In normal myogenesis, the Notch, WNT, and Hedgehog signaling pathways have been shown to regulate the progression of muscle stem cells towards lineage-committed progenitors, and, therefore, these embryonic signaling pathways function to preserve and expand this subpopulation of muscle stem cells. Given the critical role of these pathways in myogenesis, aberrant expression, activation, or regulation of these pathways may be involved in RMS tumorigenesis and may present novel therapeutic targets in the treatment of RMS. Specifically, RMS may exploit the function of these pathways in order to allow for persistent stem cell maintenance, self-renewal, and tumor initiation . The Notch signaling pathway is an evolutionarily conserved signaling pathway that mediates many tissue progenitor cell fate decisions including the determination between self-renewal and differentiation. This pathway includes multiple transmembrane receptors and ligands that function through direct cell surface contact. In mammals, there are four Notch receptors (Notch1–4) and five Notch ligands . Signaling is initiated when neighboring cells come into direct contact resulting in ligand-receptor binding. Activation of the receptor by ligand binding is followed by a two-step proteolytic cleavage of the cytoplasmic portion of the receptor and release of the cleaved active Notch receptor . ICN then translocates to the nucleus where it binds to the transcription factor RBP-J in cooperation with the coactivator Mastermind, resulting in a large transcriptional activation complex that promotes the transcription of target genes . In muscNotch signaling maintains muscle progenitors during both embryogenesis and postnatal development by suppressing myogenic differentiation. Studies in the 1990s demonstrated that overexpression of ICN in murine myoblasts inhibits their differentiation , with suIn addition to inhibition of differentiation, Notch signaling promotes the renewal of stem cells during embryogenesis and satellite cells during postnatal myogenesis. Over-expression of activated Notch (ICN) in myoblasts results in proliferation of satellite cells and inhibition of differentiation into fusion-competent myoblasts . In agreβ-catenin, the noncanonical planar cell polarity (PCP), and the Wnt/Ca2+ pathways [β-catenin pathway is the best understood. In the absence of Wnt, the pathway is inactive, allowing for the phosphorylation of β-catenin by glycogen synthase kinase 3 (GSK3) and casein kinase 1 (CK1). These two kinases along with scaffolding proteins Axin and adenomatous polyposis coli gene product (APC) form the destruction complex. Once β-catenin is phosphorylated, it is recognized and targeted for proteasomal degradation by β-Trcp, an E3 ubiquitin ligase [β-catenin, which accumulates in the cytoplasm and translocate to the nucleus where it forms complexes with TCF/LEF and activates Wnt target genes  following injury did not alter myogenesis; however, activating the Wnt pathway late (four days) following injury increased myogenic differentiation [β-catenin expression [ In adult myogenesis, the role of the Wnt pathway and ntiation . Furtherntiation . In CD45ntiation . This myntiation –39. Intentiation . Finallypression .β-catenin increased proliferation of Pax7+ satellite cells and inhibited their differentiation in both an in vitro and an in vivo setting [β-catenin in myogenic differentiation.In contrast,  setting , 43. Fur setting . Finally setting . Future The Hedgehog signaling pathway has been rightly described as an enigmatic developmental pathway in metazoans, due in part to an incomplete understanding of how the pathway constituent proteins interact . CurrentDrosophila melanogaster, Hedgehog signaling is mediated by the transcription factor Cubitus interruptus (Ci). In contrast, the mammalian ortholog to Ci is comprised of the three GLI family members GLI1, GLI2, and GLI3. Under homeostatic conditions, cells with the potential to respond to Hedgehog signaling  express the cell surface receptor Patched1 (PTCH1), which constitutively inhibits the membrane protein Smoothened (SMO) [In ed (SMO) . When a ed (SMO) . SMO loced (SMO) .Similar to the Notch and Wnt pathways, the Hedgehog pathway has been shown to play a crucial role in the regulation of early myogenesis in vertebrates. Hedgehog ligand diffuses from cells within the notochord and ventral neural tube into the developing somites where Hedgehog signaling is required to maintain MYF5 expression in the dorsomedial lip of the dermomyotome . During in vivo. Furthermore, the addition of Shh to C2C12 myoblasts in vitro led to an increase in proliferation [ Although postnatal skeletal muscle is largely quiescent, the satellite cell has a regenerative capacity akin to highly proliferative stem cell populations such as the hematopoietic stem cell. Recent data suggests that Hedgehog signaling plays a unique role in adult myogenesis because inhibition of Shh via cyclopamine impaired the activation of Myf5 and MyoD in skeletal muscle progenitors and led to a reduction in the number of activated satellite cells and myoblasts following injury feration . In addiferation . This grferation . Althougβ. Briefly, it has been demonstrated in epithelium that loss of Shh in the hair follicle leads to an increase in GSK3β, suggesting that activated Hedgehog signaling can, given the appropriate cellular context, potentiate Wnt signaling activation via β-catenin stabilization [The ability of Notch signaling to maintain muscle stem cells in both fetal and adult muscle likely requires the input of the Wnt and Hedgehog signaling pathways important in tissue differentiation and stem cell biology. Brack et al. demonstrated that coordinated transition from Notch activation to WNT activation is required to promote myogenic lineage progression from satellite cells to fusion-competent myoblasts . They adlization . These s Traditionally the study of RMS tumorigenesis has focused on genes and proteins that have been found to be dysregulated in human, and later murine models of RMS cell lines and tissues. Thus, there has been intense focus, rightly so, on the classic tumor suppressors Rb and p53, classic oncogenes such as Ras and Myc, a variety of receptor tyrosine kinases such as IGF1R, cMET, and of course the aRMS-specific chromosomal translocations. As such, newer treatment agents that can target these moieties, such as the anti-IGF1R monoclonals and small-molecule RTK inhibitors, have made their way through preclinical studies and are being tested in human clinical trials. However, recently there have been developed classes of drugs that can inhibit the Notch, Wnt, and Hedgehog pathways, and this has likely spurred a renewed interest in understanding the biology of these pathways in RMS. The first study linking the Notch signaling pathway to RMS investigated the influence of Hes1, a Notch target gene, on reversibility of cellular quiescence. In this work, Sang et al. demonstrated that HES1 mRNA expression was increased in 21 primary RMS tumors and three RMS cell lines (one eRMS and two aRMS) . They thγ-secretase inhibitors and dominant negative MAML1 coactivator construct, decreases RMS cell line mobility and invasiveness in vitro as measured by wound healing assays and Matrigel/Transwell invasion assays, respectively. A subsequent study by Roma et al. evaluated the contribution of the Notch signaling pathway to RMS mobility and invasiveness . Using qγ-secretase inhibitors in vitro resulted in decreased proliferation of eRMS cell lines, and reduction of the Notch target gene Hey1 resulted in an increase in early differentiation. In murine eRMS xenograft models, treatment with either Notch1 shRNA or γ-secretase inhibitors inhibited tumor growth, providing preclinical evidence of a role for Notch pathway inhibition in eRMS. The role of Notch signaling in RMS stem cell maintenance has not been specifically addressed, although it appears that Notch upregulation via constitutively active ICN1 enhances the formation of “rhabdospheres” (unpublished data), a recently described source of eRMS cancer stem cells that exhibits upregulated stem cell genes and increased tumorigenicity [ Recently we have investigated the role of the Notch-Hey1 pathway inhibition in eRMS . Inhibitgenicity . Furtherβ-catenin pathway was first identified as protumorigenic when an inactivating mutation in APC was described in human colon cancer [β-catenin activity and target gene expression [β-catenin) causes cell cycle arrest in leukemia [β-catenin pathway necessarily meant that it was tumorigenic, the role of Wnt pathway members such as GSK3 in other signaling pathways and the cell or tissue context of the Wnt pathway signals suggest that the role of Wnt signaling in tumorigenesis is more complex.The Wnt/n cancer . Since tpression . On the leukemia , decreasleukemia , and apoleukemia . Furtherleukemia . Thus, w−/−/cfos−/− mice and human eRMS cells (RD cells) [β-catenin activity when compared to normal muscle myoblasts. Reactivating the Wnt pathway in eRMS cells induced MyoD expression and promoted differentiation. This work suggests that inducing the Wnt pathway could be a potential therapeutic approach for treating eRMS. Also in a small set of samples from patients' tumors, aRMS, eRMS, and sclerosing RMS (sRMS) all show cytoplasmic localization of β-catenin, suggesting that a downregulation of the Wnt pathway is a common feature of RMS tumors; however this downregulation is not due to a mutation in β-catenin [β-catenin and only 15% of RMS contained any cells staining positive for nuclear β-catenin [Currently, very little is known about the role of Wnt in RMS. In eRMS, the Wnt pathway was investigated using cells derived from eRMS tumors formed in p53D cells) . These c-catenin . This wa-catenin .β-catenin, appeared to preferentially inhibit cell proliferation and induce apoptosis when compared with eRMS cells [In aRMS, GSK3 inhibitors, which result in the activation of MS cells . FurtherMS cells . More woIn conclusion, downregulation of the Wnt pathway appears to play an important role in RMS tumorigenesis. Thus, activating the Wnt pathway in RMS tumors may be a promising future strategy for the treatment of both aRMS and eRMS.PTCH1 (Gorlin syndrome) are susceptible to multiple malignancies such as basal cell carcinoma (BCC), medulloblastoma, and to a lesser extent RMS [PTCH1 mutations in sporadic human RMS is less clear. Recent studies in human tumors that searched for mutations in either PTCH1 or SMO have yielded conflicting results. For example, one study found that only one out of 14 RMS samples sequences demonstrated loss of heterozygosity (LOH) at the PTCH1 locus and there were no point mutations or deletions found in the RMS samples tested [PTCH1), suggesting that it may play a role in sporadic RMS [PTCH1 locus [ Clinical evidence supporting a role for Hedgehog signaling in RMS etiology is well established. Individuals with germline mutations in tent RMS . In paratent RMS ; howevertent RMS . The rols tested . In contadic RMS . In addiH1 locus .+/−Ptch1 mutant mice [in vivo [+/−Ptch1 mouse tumors depend on Smo for RMS initiation, the SMO inhibitor cyclopamine does not appear to suppress tumor growth in vivo [in vitro and in vivo. However, Hedgehog signaling was still implicated in these same RMS cell lines because they responded to a novel GLI antagonist GANT 61 (Gli Antagonist 61) [Because eRMS is one of the three major tumor types that develop in ant mice  and the [in vivo , it was  in vivo . Studiesnist 61) . In addinist 61) . In thesnist 61) .GLI1 gene did not lead to increased protein expression [In contrast to eRMS, the Hedgehog pathway is not known to play a significant role in aRMS. In fact, recent studies on human tissue samples demonstrated not only that aRMS tumors had reduced levels of GLI1 transcript but that aRMS samples that harbored the 12q13-15 amplicon containing the pression .γ-secretase inhibitors, which block the proteolytic cleavage and subsequent activation of the Notch receptors [As discussed above, defects in the embryonic signaling pathways that control muscle development may underlie some of the aberrant proliferation and arrested differentiation in RMS. Developmental programs responsible for controlling self-renewal and differentiation in muscle progenitor cells, including Notch, WNT, and Hedgehog, may contribute to RMS tumorigenesis and are therefore attractive pathways for therapeutic targeting. Currently, several drugs targeting the Notch, WNT, or Hedgehog pathways are in pediatric  and/or seceptors . While teceptors . More dieceptors .β-catenin-TCF interaction [β regulates growth and hormone production in NETs; however, it was ineffective at inhibiting NET growth [in vitro model of osteoporosis [Regarding the Wnt pathway, most drugs being developed as cancer therapeutics are aimed at inhibiting the pathway , 81 sinceraction , 83. Receraction . Undersceraction , and eveeraction , 87. LitT growth . In receT growth , 90. WAYoporosis .Although cyclopamine does not appear to be effective for Ptch1-mutant RMS, it is possible that pharmacologic blockade of the Hedgehog pathway with other drugs will be a useful approach for treating these tumors. For example, other drugs that inhibit SMO have recently been developed including SANT-1, SANT-2, and GDC-0449 , 93 and In summary, there is enough evidence supporting a link between the Notch, Wnt, and Hedgehog embryonic pathways and RMS tumorigenesis to continue evaluating the blockade of these pathways in preclinical studies. But the optimism of these observations must be balanced by the feasibility of targeting these pathways in children. Therapies designed to target aberrant stem cell pathways involved in tumor development may also unwittingly inhibit normal, essential stem cell pathways required for normal growth and development into adulthood, a defining characteristic of the pediatric population.Embryonic cancers such as RMS are believed to result at least in part from defective developmental processes. As such, the genes and signaling pathways responsible for normal regulation of myogenic proliferation and differentiation remain vulnerable for transformation and may act as potential RMS oncogenes. It has been hypothesized that RMS may arise from muscle stem cells or, alternatively, differentiated muscle cells may undergo mutations that stimulate dedifferentiation and reactivate self-renewal. Regardless of the precise cell of origin, mutations in embryonic signaling pathways may function as primary events in tumorigenesis, or secondary, cooperating mutations. An improved understanding of the roles of embryonic signaling pathways in RMS tumorigenesis will allow for the identification of critical targets and promote the development of rational therapies.The Notch, WNT, and Hedgehog pathways are regulators of myogenic lineage determination and maturation, and, accordingly, RMS may arise from disordered regulation of these normal developmental pathways. In this paper, we have summarized the known role of these signaling pathways during myogenesis and the current data supporting a link to rhabdomyosarcomagenesis. In summary, these embryonic signaling pathways are responsible for controlling the balance between undifferentiated muscle progenitor cells and nonproliferative differentiated myotubes. By favoring self-renewal versus a differentiated phenotype, muscle stem cells are at an increased risk for accumulating mutations necessary for oncogenesis; this is more likely to occur in cells that self-renew and do not exit the cell cycle or undergo apoptosis.Rational drug targets are being identified and employed in RMS clinical trials . However, progress has been slow for a variety of reasons. First, despite being the most common soft tissue sarcoma in childhood, RMS is still a rare disease. Therefore, there are relatively few patients available to enroll on early phase trials, and there are an increasing number of novel therapies with promising preclinical data competing for patient accrual. Second, many patients who are eligible for phase I studies do not choose to participate due to a history of heavy pretreatment and a general sense of “hopelessness” associated with “toxicity trials.” One strategy to overcome both of these obstacles is to open phase I trials of targeted therapeutics to patients who achieve remission but remain at high risk for disease relapse. This approach of investigating novel therapeutics as “maintenance therapy” would increase the number of patients available for enrollment in phase I trials and would also likely result in a higher accrual rate. A population of patients in remission would be healthier than patients with relapse, and they might be more likely to perceive hope with novel, single-agent therapies aimed at maintaining a first remission as opposed to inducing a second remission. In support of this approach, the most recent European Cooperative Sarcoma Group trial, CWS-96, demonstrated an improved survival for patients with metastatic soft tissue sarcomas treated with oral maintenance therapies compared to high-dose chemotherapy with a five-year overall survival of 52% versus 15%, respectively, . While tBased on the data presented in this paper, we conclude that the Notch, Wnt, and Hedgehog embryonic signaling pathways are important in RMS for the transformation of precursor cells, initiation of tumors, and maintenance of self-renewal properties. Given the persistently dismal cure rates for patients with high-risk RMS, an improved understanding of the role of these pathways in RMS may provide an opportunity to pharmacologically target these embryonic pathways and improve survival of patients with RMS.


Growing evidence suggests the Wnt family of secreted glycoproteins and their associated signaling pathways, linked to development, are recapitulated during wound repair and regeneration events. However, the role of the Wnt pathway in such settings remains unclear. In the current study, we treated mouse fibroblasts with 250 ng/mL of recombinant Wnt3a for 72 hours and examined its affect on cell morphology and function. Wnt3a induced a spindle-like morphology in fibroblasts characterized by the increased formation of stress fibres. Wnt3a decreased the proliferation of fibroblasts, but significantly increased cell migration as well as fibroblast-mediated contraction of a collagen lattice. Wnt3a significantly increased the expression of TGF-β and its associated signaling through SMAD2. Consistent with this, we observed significantly increased smooth muscle α-actin expression and incorporation of this contractile protein into stress fibres following Wnt3a treatment. Knockdown of β-catenin using siRNA reversed the Wnt3a-induced smooth muscle α-actin expression, suggesting these changes were dependent on canonical Wnt signaling through β-catenin. Neutralization of TGF-β with a blocking antibody significantly inhibited the Wnt3a-induced smooth muscle α-actin expression, indicating these changes were dependent on the increased TGF-β signaling. Collectively, this data strongly suggests Wnt3a promotes the formation of a myofibroblast-like phenotype in cultured fibroblasts, in part, by upregulating TGF-β signaling through SMAD2 in a β-catenin-dependent mechanism. As myofibroblasts are critical regulators of wound healing responses, these findings may have important implications for our understanding of normal and aberrant injury and repair events. Wound healing is a complex and dynamic process involving the interplay of many cellular and non-cellular components, typically culminating in the replacement of injured tissue with a fibrotic scar The Wnt signaling pathway is best recognized for its critical role in development of multi-cellular organisms In the current study, we examined the affect of Wnt3a, a canonical Wnt ligand, on fibroblast morphology and function. Our data suggests Wnt3a stimulates a spindle-like morphology in murine fibroblasts characterized by the increased expression of smooth muscle α-actin-positive stress fibres. These changes appear to be mediated, at least in part, by Wnt3a upregulating the expression of transforming growth factor (TGF)-β signaling through SMAD2 in a β-catenin-dependent manner. Collectively, this data suggests Wnt3a promotes the formation of a myofibroblast-like phenotype in cultured fibroblasts. As myofibroblasts are critical regulators of a wound healing response, these findings suggest a central role for Wnt signaling in regulating normal and aberrant injury and repair events.2 and used for experiments between passages 6–12. Recombinant murine Wnt3a  was added to cells at a concentration of 250 ng/mL for 72 hours prior to performing functional studies or collecting cells for analysis. Morphological changes were photographed with a Nikon 50i series upright microscope equipped with a digital camera. Confirmation of Wnt pathway activation was performed using a TOPFlash Reporter assay, as well as qPCR gene expression of axin2, as we have previously described Mouse embryonic fibroblasts  were cultured in DMEM containing 10% FBS and 100 U/mL penicillin/streptomycin. Cells were maintained in a humidified incubator at 37°C with 5% COin vitro scratch wound assay, as previously described Cell proliferation was measured by MTS assay (Promega) 72 hours after Wnt3a treatment. Migration was measured using the 5 cells/mL. The collagen/cell suspension was vortexed, and 1 mL per well was added to the BSA-coated dishes and the solution was allowed to polymerize for 45 minutes at 37°C. Fresh growth media was added to the solidified collagen gels and plates were returned to the incubator. Collagen gel contraction was monitored over a period of 24 hours and the surface area of contracted gels was measured using Image-Pro Plus software .Twelve-well culture dishes were coated with 1% bovine serum albumin (BSA) and incubated for 1 hour at 37°C to create a non-stick surface that prevents gels from attaching to the dishes. Prior to performing contraction assays, fibroblasts were treated for 72 hours with Wnt3a. Cells were then trypsinized, counted and seeded into a 0.5 mg/mL Type I collagen solution  in growth media at a concentration of 1×104P2O7, 50 mM NaF, 50 mM NaCl, 5 mM EDTA, 5 mM EGTA, 2 mM Na3VO4, and 1 mM phenylmethylsulfonyl fluoride, with 0.1% Triton X-100 and 10 µg/mL leupeptin) followed by centrifugation at high speed (14000 × g at 4°C for 10 minutes) to recover proteins. The protein concentration of samples was measured by a Bradford protein assay. Equal amounts of protein from each sample were separated with sodium dodecylsufate-polyacrylamide gel electrophoresis  and transferred to a nitrocellulose membrane. Membranes were blocked for 1 hour in 5% milk/TBS Tween 20 and incubated overnight at 4°C with primary antibody in 2.5% milk/TBST. Following 3 washes in TBST, secondary antibody (Santa Cruz biotechnology) at a concentration of 1∶2000 in 2.5% milk/TBST was added for 1 hour at room temperature. Antibody binding was visualized with the enhanced chemiluminescence detection system (Thermo Fischer Scientific). Images were captured with a Chemigenius2 system  and band intensities were calculated with GeneTools software (Syngene).Cell lysates were collected in lysis buffer (10 mM HEPES (pH 7.4), 50 mM NaAntibodies used were as follows: β-catenin , TGF-β , p-SMAD2 , SMAD2/3 , smooth muscle α-actin .Cells were fixed for 20 minutes in 3.7% formaldehyde, permeabilized with 0.1% triton X-100 for 20 minutes, blocked for 30 minutes with 1% BSA in PBS and incubated overnight at 4°C with the indicated primary antibody at a concentration of 1∶100 in 1% BSA. Following primary antibody, cells were washed with PBS and incubated with anti-mouse Alexa-fluor488 conjugated secondary antibody (Invitrogen) at a concentration of 1∶200 in 1% BSA for 1 hour at room temperature in the dark. To visualize f-actin, permeabilized cells were stained for 20 minutes with phalloidin conjugated to Alexa-fluor594 (Invitrogen). Cells were coverslipped with VectaShield mounting medium containing DAPI (Vector Laboratories) and images were captured using a Leica AOBS SP2 confocal microscope as we have previously described Cells were seeded into 24-well culture dishes and siRNA transfection was performed using Oligofectamine (Invitrogen) as per the manufacturer's instructions. Control siRNA or mouse β-catenin siRNA  were added to the cells at a concentration of 60 pmol/well for 24 hours prior to treating with Wnt3a. Cell lysates were harvested for Western blotting after 72 hours of Wnt3a treatment.t-test. For all analyses, P<0.05 was considered statistically significant.Results are represented as the mean±standard deviation. Significant differences in treatment groups were determined using the unpaired Student's Mouse fibroblasts were treated for 24 hours with 250 ng/mL Wnt3a (or the vehicle control) to determine if recombinant Wnt3a induces nuclear accumulation of β-catenin. Immunohistochemistry demonstrated a strong nuclear signal for β-catenin in Wnt3a-treated fibroblasts while control cells remained negative for nuclear β-catenin , suggestWnt3a induced a marked change in fibroblast morphology after 72 hours of treatment. The Wnt-treated cells appeared spindle-shaped and organized into parallel sheets as visualized by light microscopy . Consistin vitro scratch wound assay (Cell proliferation was observed to be significantly decreased after 72 hours Wnt3a treatment (Morphologically and functionally, our data suggests Wnt3a stimulates a myofibroblast-like phenotype in cultured fibroblasts. We next examined whether Wnt3a alters the TGF-β signaling axis in these cells. Western blot demonstrated expression of TGF-β to be upregulated after 72 hours Wnt3a treatment, and densitometry showed this change to be significant . ConsistTo determine if the altered cell phenotype induced by Wnt3a was dependent on β-catenin, we used siRNA to knock down β-catenin expression prior to treating cells with Wnt3a. Western blot showed siRNA significantly decreased β-catenin expression in both vehicle- and Wnt3a-treated cells . Knock dTo better characterize the role of TGF-β signaling in Wnt3a-treated fibroblasts, a time course experiment was performed over 72 hours. Representative Western blots are shown in In this study, we have shown that Wnt3a alters the phenotype of mouse fibroblasts. Structurally, Wnt3a induced a spindle-shaped morphology characterized by increased expression and incorporation of smooth muscle α-actin into stress fibres. Functionally, Wnt3a inhibited fibroblast proliferation, but increased cell migration and contraction. These changes were mediated, at least in part, by Wnt3a-induced TGF-β expression and signaling through SMAD2 in a β-catenin-dependent mechanism. Collectively, this data suggests Wnt3a stimulates the formation of a myofibroblast-like phenotype in cultured fibroblasts.et al showed overexpression of the Frizzled 1 receptor in combination with Wnt3a treatment stimulated the expression of myofibroblast markers in cardiac fibroblasts, changes that occurred in a β-catenin-independent pathway Our data are consistent with recent studies suggestive of an interaction between Wnt/β-catenin and TGF-β/SMAD signaling in controlling gene transcription and cell phenotype in vivo, the Wnt signaling pathway may be shown as a critical regulator of the wound healing response. More work will be needed to determine how manipulating the Wnt pathway alters injury and repair events in vivo.Growing evidence points to an active role for Wnt signaling in normal wound repair and in a number of human diseases. Increased canonical Wnt signaling has been observed during cutaneous wound repair 2+ pathway Wnt proteins are believed to signal through three distinct pathways, of which the canonical Wnt/β-catenin cascade is the best understood. The other pathways include the noncanonical planar cell polarity pathway and the Wnt/CaIn summary, we provide data on a novel role for Wnt3a in stimulating myofibroblast differentiation in cultured fibroblasts. Our data suggest Wnt3a treatment promotes a contractile and migratory fibroblast phenotype that is characterized by increased expression of smooth muscle α-actin. These changes appear to be mediated by increased expression of TGF-β and signaling through SMAD2 in a β-catenin-dependent manner. As myofibroblasts play a central role in normal and aberrant injury and repair events, this data suggests Wnt3a may be critically involved in the wound healing response.


Similarly to the Wnt protein palmitoyltransferase, porcupine (PPN) is essential to the activation of the Wnt/β-catenin signaling pathway. However, little is known about the role of PPN activity in human gastric cancer, one of the most common causes of cancer-related mortality. Real-time quantitative PCR was used to detect the expression levels of PPN in paired gastric cancer tissues. Cell proliferation, migration and invasion assays were performed following treatment using a newly developed small molecule PPN inhibitor  in the gastric cancer MKN28 cell line. Expression of downstream target genes and transcriptional activity of the Wnt/β-catenin signaling pathway were examined following IWP-2 treatment in MKN28. We identified that PPN was overexpressed in human gastric cancer tissue samples and cell lines. Following treatment of the gastric cancer cell line MKN28 with IWP-2, we detected that IWP-2 decreased MKN28 cell proliferation, migration and invasion, and elevated caspase 3/7 activity. Further analysis demonstrated that IWP-2 downregulated the transcriptional activity of the Wnt/β-catenin signaling pathway and downregulated the expression levels of downstream Wnt/β-catenin target genes in MKN28 cells. As current Wnt pathway-targeting strategies used for anticancer therapy have mainly focused on Wnt-receiving cells, our data shed light on the potential use of Wnt palmitoyltransferase PPN inhibitors to abrogate Wnt production in Wnt-producing cells, thus providing a potential therapeutic option for gastric cancer. The Wnt signaling pathway is important in embryonic development as well as the initiation and progression of a number of types of human cancer –3. The WDrosophila segment polarity gene, porcupine (PPN) encodes a transmembrane protein located in the endoplasmic reticulum which is necessary for the normal processing of Wingless protein . Tissue samples were stored at −80°C prior to analysis.−ΔCt value demonstrates the relative PPN expression  in cancer cell lines and normal tissues. The 2−ΔΔCt value demonstrates the fold change of the relative PPN expression  in cancer tissues normalized to adjacent normal tissues . The concentration of RNA was examined by Nanodrop 1000 . Reverse transcription and real-time PCR were performed as previously reported . The seq tissues  and 2−ΔΔ2. Wnt palmitoyltransferase inhibitor (IWP-2) was purchased from Sigma Aldrich  and 30 μM was used to treat the cells.Human gastric cancer cell lines MKN28 were obtained from the China Center for Type Culture Collection . The cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum. Cells were cultured at 37°C in a humid incubator with 5% COThe detailed western blot procedures were performed as previously reported . The priCellTiter 96 Aqueous Non-Radioactive Cell Proliferation Assay kit  was used as previously reported .The detailed procedures for colony formation and soft agar assays were performed as previously reported .Dual-Glo Luciferase Assay System (Promega Corporation) was used as previously reported . The ratμl of 5×103) were incubated with an equal volume of the assay reagent at room temperature. After 1.5 h, luminescence was measured to calculate caspase-3/7 activity.Caspase-Glo 3/7 Assay (Promega Corporation) was used for measuring caspase-3/7 activity according to the manufacturer’s instructions. Treated cells  without or with 5 mg/ml matrigel (Sigma Aldrich) were used for migration and invasion assays, respectively. The experiments were repeated three times independently.Haptotaxis chambers . The differences in the relative expression levels of genes between two unpaired and paired groups were analyzed by the Student’s t-test and the Wilcoxon matched pairs test, respectively. For independently repeated experiments, mean values ± SD (error bars) are represented in PPN was overexpressed in 62.5% (10/16) of gastric cancer tissue samples compared with adjacent normal tissue samples  specific for PPN activity ,11. Foll P<0.05; . In addi P<0.05; .In cell migration and invasion assays, MKN28 cells demonstrated almost 50% reduction in migration  and invaDue to the correlation of proliferation, migration and invasion of MKN28 cells with the activity of the Wnt/β-catenin signaling pathway , we examAlthough PPN has been demonstrated to be important in the processing and distribution of Wnt proteins –9, littlWe previously demonstrated that the proliferation of MKN28 cells correlated with the activity of the Wnt/β-catenin signaling pathway in a Wnt ligand-dependent manner , suggestAs downstream target genes of the Wnt/β-catenin signaling pathway, CCND1 and C-MYC encode regulators of cancer cell proliferation, migration and invasion ,23,24. TIn summary, we demonstrated that PPN expression levels are upregulated in gastric cancer tissues. We also revealed that IWP-2, a palmitoyltransferase inhibitor specific for PPN, inhibited cell proliferation, migration and invasion, as well as inducing apoptosis in gastric cancer cells. Further analysis showed that activity of the Wnt/β-catenin signaling pathway was also downregulated by IWP-2 in these gastric cancer cells. Our data indicate that PPN activity may be critical for cell proliferation and activation of the Wnt/β-catenin signaling pathway in gastric cancer. These data also provide evidence for the viability of targeting the Wnt/β-catenin signaling pathway in Wnt-producing cells of gastric cancer and shed light on the potential use of PPN inhibitors as a therapeutic strategy for the treatment of gastric cancer.


Activation of the pathway at the cerebellar ventricular zone led to a reduction in the number of cells expressing the glial lineage markers Sox9 and GFAP and the interneuron marker Pax2, but had no consistent effect on either proliferation or apoptosis. Our findings suggest that activation of the Wnt/β-catenin pathway in the cerebellar ventricular zone causes a shift in the cell types produced, most likely due to disruption of normal differentiation. Thus, we propose that regulation of Wnt/β-catenin signalling levels are required for normal development of cells arising from the cerebellar ventricular zone during late embryogenesis.Development of the cerebellum proceeds under the precise spatio-temporal control of several key developmental signalling pathways, including the Wnt/β-catenin pathway. We recently reported the activity of Wnt/β-catenin signalling in the perinatal cerebellar ventricular zone (VZ), a germinal centre in the developing cerebellum that gives rise to GABAergic and glial cells. In order to investigate the normal function of Wnt/β-catenin signalling in the VZ and the cell lineages it gives rise to, we used a combination of  The adult cerebellum contains a variety of types of neurons and glia, arranged in a highly characteristic laminar structure. At the centre of the cerebellum sits the white matter (WM), consisting of axonal tracts surrounding three clusters of deep cerebellar nuclei (DCN). Outside the WM sits the internal granule layer (IGL), densely populated by glutamatergic granule cells (GCs), γ-aminobutyric acid (GABA)ergic interneurons and protoplasmic astrocytes. Above the IGL sits the Purkinje cell (PC) layer (PCL), which contains the cell bodies of PCs arranged in a characteristic monolayer and interspersed with Bergmann glia (BG). PCs, GCs and Bergmann glia all extend processes into the cell-sparse molecular layer (ML), which contains a further population of interneurons 10.5 in the mouse. Cells arise from two distinct germinal centres - the ventricular zone (VZ) - which lines the dorsal aspect of the fourth ventricle, and the upper rhombic lip (URL) – a transient structure at the caudal limit of the cerebellum It is likely that many aspects of this developmental programme are under the control of intercellular signalling pathways. Indeed, both sonic hedgehog (Shh) and fibroblast growth factor (FGF) signalling are known to play important roles in cerebellum development Further support for a developmental function of the Wnt/β-catenin signalling pathway has been revealed from investigations into the cerebellar malignancy medulloblastoma. Activating mutations in multiple components of the pathway have been identified in tumour samples in vivo. Activation of Wnt/β-catenin signalling led to a marked decrease in the production of both Sox9+ and Pax2+ cell lineages – key markers of glia and interneurons respectively. This decrease did not correlate with consistent changes in proliferation or cell death and we propose a potential role for Wnt/β-catenin signalling in regulating differentiation at the cerebellar ventricular zone.We recently reported a highly specific spatio-temporal pattern of Wnt/β-catenin signalling pathway activity in the developing cerebellum lacZ reporter under the control of a β-catenin/T cell factor (TCF) responsive element and is widely used as an in vivo reporter of Wnt/-catenin activity LacZ protein product β-galactosidase  alongside expression of Pax2, which marks VZ-derived interneurons and their precursors At perinatal stages, Wnt/β-catenin signalling is active in the cerebellar ventricular zone and in cells migrating radially from it ex vivo slice culture assay. E18.5 BAT-gal+ cerebellum slices were cultured in the presence of the GSK3β inhibitors BIO in vivoTo evaluate a potential role for Wnt/β-catenin signalling in regulating aspects of cerebellum development, we first tested the effect of activating the pathway on development of the cerebellum in an Axin2 using qRT-PCR on RNA extracted from individual whole slices. Expression of Axin2 (relative to EIF4A2) was significantly increased in BIO and CHIR treated slices compared to DMSO-treated controls  and CHIR . CombineWe next investigated whether increased Wnt/β-catenin signalling affected the other cell type born in the cerebellar VZ during this developmental window, the interneuron lineage, by examining expression of the transcription factor Pax2. Pax2 is expressed in lineage restricted interneuron progenitors that arise from multipotent VZ or WM progenitors We next sought to determine the effects of constitutive Wnt/β-catenin pathway activation on the developing PC population. While these GABAergic neurons are born at the VZ and become post mitotic by E13.5, morphological maturation of the individual PCs and their development into a monolayer proceeds until the early postnatal period. Immunohistochemistry for the PC marker Calbindin One possible explanation for the decreased numbers of Sox9 and Pax2 expressing cells in cerebellum slices treated with BIO or CHIR could be reduced proliferation of progenitors. To test this possibility, we added BrdU to the cultures two hours prior to fixation and determined the proportions of BrdU labelled cells in the same defined regions of the cultured slices . With thAn alternative possible explanation for the reduction in Pax2 and Sox9 expressing cells is an increase in apoptosis. To test this possibility, we examined cultured slices for expression of the apoptotic marker Caspase3 in vivo. We used a strain of mice harbouring the 580sApc conditional allele of ApcloxApc. In these mice, exon 14 of Apc is flanked by loxP sites and cre-mediated deletion leads to loss of functional Apc protein, thereby promoting constitutive Wnt/β-catenin activity Apc specifically in the VZ, we used in utero electroporation to deliver a pCAG-Cre-IRES2-EGFP (Cre-GFP) plasmid lox/loxApc embryos , suggesting that loss of Apc did not affect cell numbers at this stage of development.We next sought to determine whether these effects of activating Wnt/β-catenin signalling during cerebellum development could also be observed  embryos , the ear plasmid . In embr plasmid , indicat embryos . In bothApclox allele led to activation of Wnt/β-catenin signalling, we examined electroporated lox/loxApc embryos for the ectopic localisation of β-catenin to the nucleus (lox/loxApc embryos contained nuclear β-catenin (lox/+Apc embryos (data not shown). Quantitation of randomly chosen fields in ten electroporated lox/loxApc embryos revealed nuclear localised β-catenin in 82% (±6.2%) of GFP+ cells, indicative of active Wnt/β-catenin activity.To determine whether cre-mediated inactivation of the  nucleus . At E14.-catenin . No nuclin vivo, we first examined expression of the VZ derived lineage markers Sox9 and Pax2 at E18.5. We used double immunofluorescence to determine the proportion of electroporated cells and their progeny (i.e. GFP+ cells) that expressed Sox9  of GFP+ cells in lox/+Apc controls  in lox/+Apc control embryos  compared to that in lox/+Apc controls (31.1%±4.2%) , a marker of mitotic cells . We foun1%±4.2%) . Therefoex vivo slice culture and in vivo genetic manipulation via in utero electroporation.Following up on our previous finding that Wnt/β-catenin signalling is active at the perinatal VZ Axin2  causes increased self-renewal and an expansion of the NSC population While at first these observations appear inconsistent and the different model systems employed make comparison difficult, our data suggest that activation of the Wnt/β-catenin pathway elicits a differential response depending on the developmental window during which activation takes place and the length of time allowed for an effect to become manifest. This conclusion is supported by recent findings from Pei et al. While a precise developmental function for the Wnt/β-catenin pathway throughout the key stages of cerebellum development awaits further clarification, taken together our findings support the conclusion that activation of the Wnt/β-catenin pathway at the VZ during late embryonic development leads to a decrease in the output of Sox9, GFAP and Pax2 expressing cells from the VZ and early WM, most likely as a result of altered differentiation.The licence authorising this work was approved by the University of Edinburgh's Ethical Review Committee on 22nd September 2008 (application number PL35-08) and by the Home Office on 6th November 2008. Animal husbandry was in accordance with the UK Animals (Scientific Procedures) Act 1986 regulations. To minimise animal suffering, pregnant dams were culled by cervical dislocation under terminal anaesthesia according to the Code of Practice for Humane Killing of Animals under Schedule 1 to the Animals (Scientific Procedures) Act 1986 issued by the Home Office.loxApc allele were maintained on a CBA/C57Bl/6J genetic background. Genotyping was carried out as described previously The day on which the vaginal plug was detected was designated E0.5 and the day of birth as P0. BAT-gal mice were maintained as hemizygotes on a C57BL/6J background and were genotyped as described previously Ex vivo organotypic slice culture procedures generally followed those of Anderson et al. 2 for one hour before media was replaced with serum-free neurobasal medium (Gibco) supplemented with 2% B-27 (Sigma), glucose, L-glutamine and Penicillin-Streptomycin. Small molecule GSK3β inhibitors were dissolved in DMSO and added to pre-warmed Neurobasal medium+supplements at a final concentration of 20 µM for BIO (Sigma) and 50 µM for CHIR (Cambridge Biosciences). This was added to the slice cultures after they were allowed to equilibrate to the Neurobasal medium+supplements and then cultured for 24 hours. 2 hours prior to the end point BrdU was added to the medium at a final concentration of 10 mg/ml.2 delivered through a facemask. Uterine horns were surgically exposed and approximately 3 µl of purified pCAG-Cre-IRES2-EGFP plasmid DNA 2O with 2 mg/ml Fast Green (Sigma) was injected into the fourth ventricle to each embryo using pulled glass capillaries inserted through the roof plate into the ventricle. A PV820 Picrospritzer (WPI) was used to deliver short bursts of plasmid solution until the Fast Green dye was visible filling the ventricle. Forceps-type electrodes  were positioned around the midbrain-hindbrain boundary of embryos outside the uterine wall (anode oriented to the right) and a CUY21 electroporator (Nepa-gene) was used to deliver five pulses with the following parameters: intensity = 45 V, pulse length = 50 ms, pulse interval = 950 ms. At the end of the procedure uterine horns were repositioned in the abdomen and the smooth muscle lining was closed with grade 5 sutures and the skin was closed with 5 mm wound staples. Brains were dissected at defined stages during embryonic development and those with successful electroporations were identified by epifluorescence microscopy for GFP expression within the cerebellum.E13.5 timed pregnant females were anaesthetised by inhalation with isoflurane and maintained in anaesthesia with Oin vivo analysis, heads were collected from E14.5 embryos and brains were dissected from E18.5 embryos. For all histological analysis, including cultured cerebellum slices, tissue was immersion fixed in 4% paraformaldehyde (PFA) in PBS overnight at 4°C. Tissue was then cryoprotected in 30% sucrose/PBS overnight before embedding in 30% sucrose/OCT (1∶1) and snap freezing. Sections were cut on a cryostat at a thickness of 12 µm for ex vivo slices and 14 µm for in vivo tissue.For Immunohistochemistry and immunofluorescence were performed according to standard protocols. Antigen retrieval was achieved by microwaving sections on medium power in 10 mM citrate buffer (pH6.0) for five minutes followed by 15 minutes at low power. Primary antibodies were rabbit anti-β-galactosidase , mouse anti-β-galactosidase (DSHB 1∶100), mouse anti-PCNA , mouse anti-Pax2 , goat anti-GFP , rabbit anti-GFP , rabbit anti-Sox9 , mouse anti-β-catenin , mouse anti-BrdU , mouse anti-Caspase3  and mouse anti-Calbindin . All primary antibodies were detected using goat anti-rabbit IgG, goat anti-mouse IgG or donkey anti-goat IgG secondary antibodies conjugated to Alexa fluor 568 or 488 dyes  and sections were blocked using serum from the secondary host species. Appropriate controls were used in all cases by incubating sections with all but the primary antibodies. No staining was observed under these conditions. Nuclear counterstaining was performed with TOPRO-3 .A Leica brightfield microscope connected to a Leica DFC360Fx camera was used to capture images of fluorescently labelled sections. Confocal microscopy for high resolution image analysis was carried out in the IMPACT Imaging Facility at the University of Edinburgh using an inverted Zeiss LSM510 confocal microscope.Counting frames were placed in four defined regions of each section analysed : three a2O. Reactions were set up in a 96 well white PCR plate (Bio-Rad) using a QIAgility automated pipetter (Qiagen). H2O and –RT negative controls were included for each gene tested. Each reaction was begun with a 10 minute 95°C denaturation step followed by 40 cycles of: 15 seconds at 95°C, 60 seconds at 60°C. Fluorescence was monitored at each cycle and then a melt curve analysis was performed.RNA extraction for reverse transcriptase PCR (RT-PCR) was performed on individual snap frozen cerebellum slices using an RNeasy Microkit (Qiagen) following the manufacturer's instructions. On-column DNA digestion was performed in all cases during extraction to eliminate genomic DNA contamination. cDNA was synthesised from 50 ng RNA  using a Sensiscript reverse transcription kit (Qiagen). The reactions were prepared with 50 ng sample RNA added to 1× RT-Buffer, 10 µM random hexamer primers (Promega), 0.5 mM dNTPs (Invitrogen), 10 u RNAse inhibitor (Promega), 1 µl Sensiscript reverse transcriptase and made up to 20 µl with nuclease free water. Identical reactions were set up in tandem excluding reverse transcriptase (−RT) to serve as negative controls for possible genomic DNA carryover. Reactions were carried out at 37°C for 60 minutes and stored at −20°C. Quantitative RT-PCR (qRT-PCR) was performed using a Precision qPCR SYBRgreen detection kit (PrimerDesign) and commercially synthesised primer pairs (PrimerDesign) with an Opticon DNA Engine (MJ Research) following the manufacturer's instructions. All primers used had annealing temperatures of ∼58°C and an amplicon size of 90–120 bp. Reactions were prepared in 20 µl with Precision qPCR mastermix (PrimerDesign), 300 nM custom primer pairs, 0.5 µl cDNA and made up to 20 µl with nuclease free HPLUS (BioGazelle). This analysis determined EIF4A2 as the optimal reference gene. Relative amounts of transcript for each gene analysed were normalised to EIF4A2 in each case and expressed as a ratio. For every gene analysed, samples were run in triplicate using cDNA collected from three individual slices from each sample group.For each gene analysed a set of six two-fold serial dilutions of control cDNA  was tested in order to generate a standard curve of arbitrarily assigned transcript units. Quantitation was carried out by comparing the relative amounts of gene-specific template cDNA to a housekeeping reference gene. The appropriate reference gene for analysis was identified using a GeNorm reference gene selection kit (PrimerDesign). This involved a test of six common reference genes by qPCR with a sample of the test cDNA followed by statistical analysis using qBase


To test whether membrane fluidity and its changes are important for the sensitivity of cells to the action of perifosine (OPP), a new anticancer drug targeting cell membrane and not DNA.Influence of OPP on the membrane structure of OPP-resistant MCF7, and OPP-sensitive MT3 breast cancer cell lines, as well as of mouse fibroblasts (L929) cell lines, and model cells (liposomes) was investigated by electron paramagnetic resonance, using spin labeled derivative of OPP (P5) and 5-doxylpalmitoyl methylester ) as spin probes.P ≤ 0.05, t test). In cells, the differences were observed only by P5 and not by MeFASL. Average order parameter effS decreased for about 12% in MCF7 and L929 and only for 8% in OPP-sensitive MT3 cells, showing that there was no correlation between membrane fluidity changes and sensitivity of cells to OPP. The only correlation we found was between OPP sensitivity and the cell growth rate. In liposomes, both spin probes were sensitive to the action of OPP. effS decreased with increasing concentration of OPP. For MeFASL a significant decrease was observed at 4 mol% OPP, while for P5 it was observed at 8 mol%.OPP increased membrane fluidity of all cell lines at concentrations higher than 50 µM (on the level of Influence of OPP on plasma membrane fluidity of breast cancer cells is not the determining factor in the sensitivity of cells to OPP. Perifosine (OPP) belongs to the group of alkylphospholipids (APL), a new class of anticancer agents, targeting directly cell membrane and not DNA. They show a selective apoptotic response in tumor cells, sparing normal cells. The mechanism of APLs action is not yet completely understood. It is known that due to their amphiphilic nature, APLs are easily incorporated into cell membranes in substantial amounts and then spread further into intracellular membrane compartments, where they accumulate and interfere with a wide variety of key enzymes ,2. At loEarly research focusing on the immune stimulating activity of APLs demonstrated that miltefosine (hexadecylphosphatidylcholine) and other lipids of this class are able to activate T-cells and macrophages to express and release chemokines GM-CSF , IFgammaIn this study, EPR with spin probes was used to investigate the influence of OPP on the membrane structure of OPP-resistant MCF7 and OPP-sensitive MT3 breast cancer cells. Results were compared to those obtained on fast growing mouse fibroblasts (L929) and model cell membranes (liposomes).The spin probes 1,1-dimethylpiperidin-1-IUm-4-yl octadecyl phosphate , spin labeled perifosine  and 5-doxylpalmitoyl-methylester ) were synthetized at the Faculty of Pharmacy, University of Ljubljana, Slovenia and used to monitor the membrane properties close to water-lipid interface .The cell lines studied were experimental human breast cancer estrogen receptor positive (ER+) MCF7, estrogen receptor negative (ER-) MT3  and mouse fibroblasts L929 .)  and cholesterol  were used.For liposome preparation, the phospholipids: 1-Palmitoyl-2-oleoyl-phosphatidylcholine (POPC), 1-Palmitoyl-2-oleoyl-phosphatidylethanolamine (POPE), 1-Palmitoyl-2-oleoyl-phosphatidylserine  or P5 spin probe and different concentrations of OPP  by a thin lipid film method described in detail elsewhere . Mixing TM-I (Gibco) supplemented with fetal calf serum and penicillin- streptomycin in same concentrations. Approximately 106 cells were seeded into a 75 cm2 flask at 37°C, 90% humidity, and 5% CO2. On the day of the measurement cells were trypsinized at confluence state using Trypsin-EDTA 0.02% in PBS (PAA). On the day of the measurement, the cells were trypsinised in the plateau phase. The cell suspension was centrifuged at cca 400∙g and the pellet  was resuspended in medium without serum (3 mL). The cell viability was checked by Trypan Blue Stain 0.4% (Gibco) and the number of cells in a sample was calculated after counting the cells using Bürker -Türck's plate.Human breast cancer cell lines (ER-) MT3 were cultured in RPMI-1640 medium  supplemented with 4 μmol/mL L-glutamine , penicillin-streptomycin with 10 000 units penicillin and 10 mg streptomycin per mL , and heat-inactivated 10% fetal calf serum , while (ER+) MCF7 and mouse fibroblasts L929 were cultured in DMEM media – GlutaMAXLabeling the cells with P5 or MeFASL (3 µmol) was performed by a thin lipid film method . BrieflyEPR spectra were acquired at 20°C .max and 2Amin) were measured as shown in eff was calculated:From the spectra, maximal and minimal hyperfine splitting : xxA = 0.63 mT; yyA = 0.58 mT; and Azz = 3.36 mT , rotational correlation time c(τ), the line-width broadening (W), and the polarity correction of the magnetic tensors g and A . Order parameter describes the orientational order of the phospholipids’ acyl chains in the membrane regions and varies from S = 1 for perfectly ordered membrane to S = 0 for isotropic distribution; rotational correlation time (cτ) describes the rate of nitroxide group motion, which is influenced by the acyl chain motion of the surrounding lipids; polarity correction factors gp and Ap arise from the polarity of the spin probe nitroxide group environment; and the line-width broadening (W) appears due to the paramagnetic impurities, spin-spin interactions, unresolved hydrogen super-hyperfine interactions, etc. Besides, a fraction (weight) of each spectral component has to be determined, which describes the relative amount of the spin probes with particular motional mode. Since partition of MeFASL was found to be approximately equal between different membrane regions of phospholipid/cholesterol vesicles . For more precise description of membrane characteristics, a computer simulation of EPR spectra is necessary, which takes into account that the experimental EPR spectrum is a superposition of several spectral components reflecting different modes of restricted rotational motion of spin probes in different environments of the membrane ,24. Eachvesicles , we assuvesicles . Therefohttp://www.ijs.si/ijs/dept/epr/).To obtain the best fit of the calculated EPR spectrum to the experimental one, the multi-run hybrid evolutionary optimization algorithm was used , togetheCritical micellar concentration (CMC) of OPP and P5 was determined with Krüss processor tensiometer, as described in more details elsewhere . CMC valCell viability determined using Trypan Blue Stain depended on the OPP concentration added . At 50 µBefore measuring cell viability and performing further EPR studies, the rate of cell growth was evaluated for all cell lines . After hModel cells – liposomes. EPR spectra of MeFASL and P5 in giant liposomes changed with different concentrations of OPP  spin probe was slightly more sensitive to the perturbation of the liposome membrane by OPP than P5. With MeFASL the difference in spectra line-shape was detected already at 4 mol% of OPP, while 8 mol% of OPP was needed to see any spectral change with P5. Distribution of MeFASL and P5 in different membrane local environments was well visible from the differences in the EPR spectra line-shape between the two probes  was highest in the most ordered regions (R1), indicating that these regions occupy the largest area of the membrane. OPP decreased order parameter S of R1 regions as detected with both spin probes, indicating higher fluidity of the most ordered domains  spin probe  and P5 in the membrane of different cell lines showed the spectra typical for restricted spin probe motion (effS) of P5 were calculated directly from the spectra according to the equation 1 , while the opposite effect was found for breast cancer cell lines; for OPP-sensitive MT3 eff =S 0.68 ± 0.01 and for OPP-resistant MCF7 eff =S 0.70 ± 0.01.e motion . Similaruation 1 . At roomuation 1 . HoweverAp in the presence of OPP in the two most ordered regions for all cell lines, indicating that OPP decreases permeability of the cell membrane for polar molecules.Similarly as in the spectral analysis of liposomes, in cell membranes good fit of experimental spectra was obtained with three spectral components. The main contribution to the decrease in the effective order parameter, measured directly from the spectra, in the presence of OPP was the decrease in the fraction of the region with the highest order parameter (R1) and the increase in the fraction of the region with intermediate order parameter (R2) . The mosThe results showed that our hypothesis that sensitivity of breast cancer cells to OPP was connected with cell membrane fluidity was not confirmed, but a possible correlation between the rate of cell growth and sensitivity of cells to OPP was indicated.3) and its analogues (Ap) of less ordered regions as detected by MeFASL than P5 as a spin probes. This indicates greater influence of OPP in the part of the membrane where nitroxide group of MeFASL was distributed. MeFASL is highly lipophilic, with partition coefficient between membrane and solution log P = 5.12 . It distThe effect of OPP on model membranes is expected as it is known that APLs insert progressively from the aqueous medium into lipid monolayers as monomers below the critical micellar concentration (CMC), and as the combination of monomers and micelles above the CMC . Like otIn the second part of our investigation, we determined the influence of OPP on two breast cancer cell lines and mouse fibroblasts. The influence of OPP on cell membrane fluidity was less pronounced than in model membranes (higher amount of OPP was needed to observe the difference). This indicated that different metabolic processes in the cells accelerated the transport of OPP into the cell interior or prevented OPP uptake by the cells.It is interesting to note that in contrast to the significant influence of OPP on cell membrane fluidity observed with spin probe P5, no influence was observed with MeFASL10,3). The possible reason could be different locations of MeFASL and P5 in the membrane, which is evident from the line-shape of the EPR spectra. In the model membranes, MeFASL is distributed equally in all membrane regions and is rapidly equilibrated between both layers of the membrane . We supp,3. The pWhen we compared viability and membrane fluidity of cells in the presence of OPP, the most sensitive to OPP was the mouse fibroblast cell line L929, which is also the most rapidly growing. This is in accordance with the studies that measured the correlation between the degree of proliferative activity of cells and the degree of apoptosis . Not onl50) was observed at lower concentrations (below 20 µM)   than wasAccording to our knowledge, there are only few data about the influence of APLs on cell membrane fluidity showing similar membrane fluidity increase as observed in this study, but using other APLs and other cell lines -30. HighSo far we proved that plasma membrane fluidity of cells and fluidity changes caused by OPP were not the determining factors in sensitivity of cells to OPP. In the future, our investigations should be extended to more cell lines with different growing rates and sensitivity to OPP. Beside membrane fluidity changes, the uptake and accumulation of OPP by different cell lines should be followed and possible transport mechanisms should be further investigated. Only an extensive investigation of cell-OPP interaction using different methods and approaches will make this promising anticancer drug more reliable for cancer treatment.


Epiblast stem cells (EpiSCs) are primed pluripotent stem cells and can be derived from postimplantation mouse embryos. We now show that the absence of canonical Wnt/β-catenin signaling is essential for maintenance of the undifferentiated state in mouse EpiSCs and in the epiblast of mouse embryos. Attenuation of Wnt signaling with the small-molecule inhibitor XAV939 or deletion of the β-catenin gene blocked spontaneous differentiation of EpiSCs toward mesoderm and enhanced the expression of pluripotency factor genes, allowing propagation of EpiSCs as a homogeneous population. EpiSCs were efficiently established and propagated from single epiblast cells in the presence of both XAV939 and the Rho kinase (ROCK) inhibitor Y27632. Cell transplantation revealed that EpiSCs were able to contribute to primordial germ cells and descendants of all three germ layers in a host embryo, suggesting that they maintained pluripotency, even after prolonged culture with XAV939. Such an improvement in the homogeneity of pluripotency achieved with the use of a Wnt inhibitor should prove advantageous for manipulation of primed pluripotent stem cells. The canonical Wnt/β-catenin signaling pathway plays pivotal roles not only in early embryogenesis but also in stem cell homeostasis and tumorigenesis Cdh1) were able to contribute to embryos after blastocyst injection if they had been treated with the Rho kinase (ROCK) inhibitor Y27632 Mouse epiblast stem cells (EpiSCs), which are derived from the epiblast at embryonic day (E) 5.5 to E7.5, exhibit features of pluripotency and require Nodal-Activin and fibroblast growth factor (Fgf) signaling for maintenance of this characteristic. They are therefore thought to be more closely related to human ESCs than to mouse ESCs in this regard We have now investigated the role of canonical Wnt signaling in mouse EpiSCs with the use of small-molecule inhibitors and deletion of the β-catenin gene. Consistent with our previous observations in human ESCs lacZ, β-cateninfl/fl, and UBC-CreER mouse strains (The Jackson Laboratory) were maintained on the B6/129 hybrid background. Mouse embryos were collected at E6.5, with noon of the day on which the vaginal plug was detected being designated E0.5. Mice harboring the UBC-CreER transgene were crossed with β-cateninfl/fl mice. Both UBC-CreER and β-cateninfl/fl alleles were identified by PCR analysis as described previously ROSA26-lacZ, or β-cateninfl/fl mice with a modified version of a method described previously All EpiSC lines used in the present study were newly established from E6.5 epiblasts of ICR, ROSA26-Total RNA was extracted and purified from EpiSCs with the use of Sepasol-RNA I Super G (Nakarai) and was subjected to RT with the use of a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). Real-time PCR analysis was performed with an ABI Prism 7500 Real-time PCR system and PowerSYBR Green PCR Master Mix (Applied Biosystems). The abundance of each mRNA was normalized by the amount of 18S rRNA and calculated by the standard curve method. Details of PCR primers are available on request.2O2 for 10 minutes at room temperature, exposed to 5% BSA in PBS, and then incubated consecutively with primary antibodies and biotinylated secondary antibodies (Jackson Immunoresearch). They were then treated with ABC staining solution (Vector) and immersed in 3,3‘-diaminobenzidine solution (Vector) for detection of immunoreactivity. Primary antibodies included those to Oct3/4, to E-cadherin, and to PCNA (Santa Cruz Biotechnology); those to β-catenin (BD Biosciences); those to Sox2 ; and those to Nanog (Cosmo Bio).Lysates of EpiSCs were prepared and subjected to SDS-polyacrylamide gel electrophoresis followed by immunoblot analysis as described previously lacZ embryos were treated with Y27632 (10 µM) for 1 hour and then dissociated into single cells with trypsin-EDTA. ICR embryos at E6.5 and EpiSCs were handled with manipulators (Narishige) under an inverted microscope (Zeiss). EpiSCs (10 to 20 cells) were injected between the posterior epiblast and visceral endoderm layers of E6.5 embryos in DMEM supplemented with 10% fetal bovine serum. The injected embryos were then allowed to develop in whole-embryo culture. Cells expressing lacZ (those derived from EpiSCs) were visualized by fixation of embryos with 1% paraformaldehyde and 0.2% glutaraldehyde in PBS for 10 minutes followed by staining with 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside .Cell transplantation was performed as described previously 2 at 37°C in DMEM supplemented with 75% rat serum either in four-well plates (for inhibitor experiments) or with rotation in 15-ml tubes (for cell transplantation experiments).Whole-embryo culture was performed as previously described Foxd3 probe plasmid was newly generated by cloning a 1410-bp fragment of the coding region of mouse Foxd3 amplified by PCR into pBS (Stratagene).EpiSCs and embryos were fixed overnight at 4°C with 4% paraformaldehyde in PBS, dehydrated with a graded series of methanol solutions, and stored at –20°C prior to analysis. In situ hybridization was performed as described previously Brachyury (T), Mixl1, and Nanog expression as well as in the repression of Sox2 and Foxd3 expression, indicative of the onset of mesoderm formation , Sox2, and Foxd3 in the entire epiblast . In contrast, inhibition of Wnt signaling with XAV939 improved the morphology of EpiSC colonies and extinguished differentiated cells usually found in EpiSC colonies cultured with standard medium . Given that the expression profiles of EpiSCs harboring a transgene for green fluorescent protein (GFP) under the control of the Oct4 regulatory region were found to differ between cell subpopulations positive or negative for GFP expression T expression and the partial loss of Oct4 expression within control single EpiSC colonies, indicating that EpiSCs constitute a heterogeneous population as a result of spontaneous differentiation along the mesoderm and endoderm lineages during culture in the presence of Activin and Fgf2  and which also harbored a transgene (UBC-CreER) for a tamoxifen-activatable form of Cre recombinase. The cells were rendered null for β-catenin (β-cateninΔ/Δ) by treatment with tamoxifen. Two independent cell lines (designated #3 and #4) were established, one of which (#3) was subjected to further characterization  were established with XAV939, treated with Y27632, and transplanted into the space between the epiblast and visceral endoderm of E6.5 embryos, which were then cultured for 2 days to allow development to the four- to six-somite stage. The embryos (n = 18) were then stained with the β-galactosidase substrate X-gal and examined for the distribution of EpiSC-derived cells. EpiSCs were found to contribute preferentially to the extraembryonic mesoderm including the allantoic bud (n = 17/18), yolk sac (n = 10/18), and amnion (n = 7/18) (n = 11/18), mesoderm (n = 7/18), and ectoderm (n = 3/18) (n = 3/8 embryos)  . Some ce = 3/18) . Teratomembryos) , indicatWnt3 is expressed in the posterior epiblast for induction of the PS, where mesoderm cells are generated We have shown that the canonical Wnt/β-catenin signaling pathway attenuates the undifferentiated state of EpiSCs and the epiblast of postimplantation mouse embryos. Importantly, expression of PS-mesoderm markers, which has been considered characteristic of EpiSCs, was found to be heterogeneous in these cells and to be extinguished by inhibition of Wnt signaling. These data indicate that the expression of PS-mesoderm markers is a consequence of spontaneous differentiation rather than a general hallmark of the primed pluripotent state. In mouse embryos, Esrrb is a pivotal target of β-catenin/Tcf3 as well as of Nanog in regulation of the naïve pluripotent state Esrrb in EpiSCs Nanog, Fgf5, and Fgf8, all of which are downstream target genes of Nodal signaling β-cateninΔ/Δ cells even in the presence of Activin and Fgf2. The nuclear translocation of Smad2/3 did not differ between β-cateninfl/fl and β-cateninΔ/Δ cells (data not shown), indicating that Nodal signaling was not impaired by the loss of β-catenin. It is thus possible that β-catenin acts directly or cooperatively with Nodal signaling to regulate the expression of these genes in EpiSCs and the postimplantation epiblast. Given that XAV939 treatment conferred uniform expression of Nanog, Fgf5, and Fgf8 in EpiSCs, a Wnt-independent function of β-catenin may be required for Nodal signaling to induce the expression of these genes.Although several downstream targets of canonical Wnt/β-catenin signaling have been identified Pax6 in β-cateninΔ/Δ EpiSCs by inhibiting Fgf/MAPK signaling, even though Nanog expression was down-regulated in these cells. These results suggest that β-catenin is essential for neural induction, and that Nanog is dispensable for inhibition of neural differentiation in β-cateninΔ/Δ cells. Consistent with this notion, Nanog was recently shown not to be required for establishment and maintenance of both naïve and primed pluripotency β-cateninΔ/Δ EpiSCs is seemingly inconsistent with the fact that the neuroectoderm is formed in the anterior epiblast, which is devoid of canonical Wnt signaling. A function of β-catenin other than that in canonical Wnt signaling, such as in cell adhesion, may be required for the induction of neuroectoderm.Evidence suggests that Nanog prevents neuroectoderm differentiation and supports the pluripotent state in EpiSCs and human ESCs Sox2 and Foxd3 expression in EpiSCs and gastrulating embryos. Sox2 and Foxd3 function in the maintenance of pluripotency in epiblast cells, with corresponding mutant embryos having been shown to die after implantation as a result of a loss of epiblast cells Sox2 and Foxd3 persists in the anterior epiblast during neuroectoderm differentiation, whereas the expression of these genes in the posterior epiblast is down-regulated during PS formation (Sox2 and Foxd3. Expression of Sox2 was previously shown to be increased in the posterior region of embryos lacking β-catenin Sox2 and Foxd3We also found that the canonical Wnt/β-catenin signaling pathway negatively regulates ormation . Our resIn summary, our data show that inhibition of canonical Wnt signaling confers a uniform pluripotent ground state on EpiSCs. Given the functional and molecular differences detected among human ESC lines as well as among human iPSC lines


Though roles of β-catenin signaling during testis development have been well established, relatively little is known about its role in postnatal testicular physiology. Even less is known about its role in post-meiotic germ cell development and differentiation. Here, we report that β-catenin is highly expressed in post-meiotic germ cells and plays an important role during spermiogenesis in mice. Spermatid-specific deletion of β-catenin resulted in significantly reduced sperm count, increased germ cell apoptosis and impaired fertility. In addition, ultrastructural studies show that the loss of β-catenin in post-meiotic germ cells led to acrosomal defects, anomalous release of immature spermatids and disruption of adherens junctions between Sertoli cells and elongating spermatids . These defects are likely due to altered expression of several genes reportedly involved in Sertoli cell-germ cell adhesion and germ cell differentiation, as revealed by gene expression analysis. Taken together, our results suggest that β-catenin is an important molecular link that integrates Sertoli cell-germ cell adhesion with the signaling events essential for post-meiotic germ cell development and maturation. Since β-catenin is also highly expressed in the Sertoli cells, we propose that binding of germ cell β-catenin complex to β-catenin complex on Sertoli cell at the apical ES surface triggers a signaling cascade that regulates post-meiotic germ cell differentiation. Recent studies have shown that perturbation of β-catenin signaling in embryonic Sertoli cells results in testicular degeneration, testicular cord disruption, and Mullerian duct regression To address β-catenin's role during germ cell differentiation, we deleted β-catenin specifically in haploid spermatids. Inactivation of β-catenin in post-meiotic germ cells resulted in increased germ cell apoptosis, compromised sperm motility, acrosomal defects, abnormal chromatin compaction, and loss of Sertoli cell-germ cell adhesion at the apical ES, leading to impaired fertility. These defects may be due to altered levels of several genes associated with cell-cell signaling and cell adhesion in β-catenin-deleted germ cells. Further supporting the notion that β-catenin may be a critical regulator of Sertoli cell-germ cell adhesion were our findings that β-catenin expression was localized to the distal portion of spermatids  males and wild-type control  females revealed more than 90% of recombination of the β-catenin-flox allele in the testis (data not shown). To determine whether β-catenin deletion in post-meiotic germ cells produced any reproductive defects, we performed fertility analyses of Ctnnb1 FΔ animals. Eight-week timed mating studies revealed impaired fertility of Ctnnb1 FΔ males, as matings between Ctnnb1 FΔ males and control females not only resulted in significantly fewer litters  produced normal litter sizes and pups per litter  also produced normal litter sizes and pups per litter, suggesting that the reproductive defect did not originate from the Prm1-cre transgene .To address the role of β-catenin in post-meiotic germ cell development, we generated β-catenin conditional knockout mice by mating transgenic mice expressing xed mice , which hted mice  and S2. r litter . Similarransgene . FurtherCtnnb1 FΔ male mice. Ctnnb1 FΔ mice had significantly reduced testis weight and modestly reduced testis size compared to control mice  in Ctnnb1 FΔ testes. Light microscopic examination of sonicated testes revealed that Ctnnb1 FΔ mice possessed significantly fewer sonication-resistant spermatids than their control littermates . AO is a DNA intercalating dye that fluoresces green when bound to double-stranded DNA and red when bound to single-stranded DNA. As sperm with partially compacted chromatin will be more susceptible to acid denaturation, they will exhibit yellow to orange fluorescence (green plus red), and therefore can be readily distinguished from fully compacted chromatin with green fluorescence Ctnnb1 FΔ sperm  e testes , panel ae testes , panel de testes , panel eatocytes , panel fatocytes , panel gCtnnb1 FΔ mice prompted us to investigate whether β-catenin may also play a role in spermatid polarization. Since β-catenin and JAM-C, which is known to be critical for spermatid polarization Ctnnb1 FΔ testes when compared to control suggesting β-catenin to be one of the important constituent of spermatid polarity complex . One group of genes of particular interest was those involved in the mitogen-activated protein kinase (MAPK) pathway, which has been shown to be involved in cytoskeletal rearrangements Map2k7 (MKK7) and Mapkapk2 (MK2) were found to be upregulated, while phosphatase genes such as Dusp26 was downregulated .To further understand the mechanism by which β-catenin may regulate differentiation/maturation of post-meiotic germ cells, we performed gene expression analysis on RNA isolated from purified round spermatid as well as whole testis from 6 to 8-week old control and egulated . Anotherd Vps33a  [24], , [3k2 (MK2)  [54], [5p38 MAPK  [56], . Co-IP was carried out using Dynabeads Protein G Immunoprecipitation Kit (Invitrogen) according to manufacturer protocols. Briefly, 1.5 mg of protein lysate was precleared with 50 µl Dynabeads for 1 h. 5 µg of anti-β-catenin (Sigma) or normal rabbit IgG (Santa Cruz) were incubated with 50 µl Dynabeads for 30 min with rotation. Pre-cleared lysates were incubated with antibody-conjugated beads overnight at 4°C with rotation. Beads were collected, washed twice, and bound proteins were eluted by boiling in Laemmli sample buffer. Protein was visualized by western blot analyses using antibodies described in figure legends.Ctnnb1 FΔ mice) and whole testis (from two control and two Ctnnb1 FΔ mice) were hybridized to the Agilent 4×44 k Whole Mouse Genome Microarray according to manufacturer's protocol and scanned on the Agilent G2505B scanner. Expression levels of selected genes were further verified by qPCR analyses on RNA extracted from purified round spermatids. We have deposited the raw data at National Center for Biotechnology Information Gene Expression Omnibus (accession #GSE30773), and confirm that all details are MIAME compliant.Total RNA from purified round spermatids  and HRP-conjugated goat anti-rabbit secondary antibody . Sections were developed with DAB and counterstained with Mayer's hematoxylin. While round and elongating spermatids clearly showed β-catenin expression in control seminiferous tubules , no detectable β-catenin staining was observed in the round and elongating spermatids of Ctnnb1 FΔ tubules , suggesting that both copies of the β-catenin-flox allele has been conditionally deleted. Please note that elongating spermatids exhibited only purple hematoxylin staining in Ctnnb1 FΔ tubules (panels d-f), suggesting no β-catenin expression, while elongating spermatids in control tubules exhibited brown DAB staining (panels a-c). β-catenin staining in Sertoli cells remained unchanged in Ctnnb1 FΔ compared to control tubules . Areas in black boxes are magnified , and areas in red boxes are further magnified . Scale bar, 100 µm (panels a and d), 50 µm (panels b and e), or 25 µm (panels c and f).(TIF)Click here for additional data file.Figure S2Loss ofβ-catenin expression in Ctnnb1 FΔ elongating spermatids. Testis sections from control and Ctnnb1 FΔ mice were labeled with anti-β-catenin (1∶50) followed by AlexaFluor 488-conjugated goat anti-rabbit (1∶400). Sections were counterstained with DAPI (blue) for nuclear staining. While elongating spermatids clearly showed β-catenin expression in control seminiferous tubules , no detectable β-catenin staining was observed in the elongating spermatids of Ctnnb1 FΔ tubules . β-catenin staining in Sertoli cells remained unchanged in Ctnnb1 FΔ compared to control tubules . Areas in boxes are magnified . Scale bar, 50 µm (panels a-c and e-g) or 25 µm (panels d and h).(TIF)Click here for additional data file.Figure S3Prm1-cre males exhibit no reproductive defect. (A) Mean number of litters (n = 10) and (B) mean number of pups per litter (n = 10) obtained from eight-week timed matings of 6 to 8-week old Prm1-cre and control littermates.(TIF)Click here for additional data file.Figure S4Reduction in Ctnnb1 FΔ testis size. Testis from a control (left) and a Ctnnb1 FΔ (right) mouse, showing a modest reduction of testis size when β-catenin is conditionally deleted in haploid spermatids.(TIF)Click here for additional data file.Table S1β-catenin expression in enriched Sertoli and germ cell populations.QPCR analyses of RNA from purified Sertoli and spermatogenic cell populations pooled from four mice using primers in (DOC)Click here for additional data file.Table S2List of highly altered genes in Ctnnb1 FΔ post-meiotic germ cells. Genes listed were found to be highly altered (upregulated or downregulated) in total testis and purified round spermatids of Ctnnb1 FΔ mice compared to control mice. A complete list of altered genes in Ctnnb1 FΔ total testis and purified round spermatids microarrays is available at NCBI GEO (accession #GSE30773).(DOC)Click here for additional data file.Table S3Primers used in this study. Primer sequences were obtained from PrimerBank (DOC)Click here for additional data file.