Nematostella vectensis, a member of cnidarians, the ancient sister group of bilaterians. We found that β-catenin signalling is crucial for the early induction of the embryonic nervous system. β-Catenin activity at the blastopore induces specific neurogenic genes required for development of the oral nervous system. β-Catenin signalling induces also Bmp signalling, which, at later larval stages, becomes indispensible for the maintenance and asymmetric patterning of the oral nervous system along the primary and secondary (directive) axes. We hypothesize that the consecutive and functionally linked involvement of β-catenin and Bmp signalling in the formation of the cnidarian oral nervous system reflects an ancestral mechanism that evolved before the cnidarian/bilaterian split.Animal evolution is closely linked to the emergence of the nervous system. At present it is unknown how the basic mechanisms of neural induction and formation of central nervous systems evolved. We addressed this question in  The bilaterian central nervous system is thought to have evolved from a cnidarian-like ancestor, but the mechanisms of neural induction in cnidarians are largely unknown. Here the authors study the cnidarian Nematostella vectensis and show that β-catenin signalling is crucial for the early induction of its embryonic nervous system, suggesting evolutionary roots for this pathway. The emergence of nerve cells is one of the key novelties in animal evolutionThe general view holds that the bilaterian CNS can be traced back through evolution to a nerve net in a cnidarian-like ancestor345678565568910111213Xenopus and Drosophila, which showed that an inhibition of Bmp signalling is sufficient to induce the neuroectoderm, it was proposed that the primary function of Bmp signalling in bilaterians is the inhibition of the neural fate, and accordingly, the default state of the ectoderm would be the neural fate1516192223In bilaterians, several secreted factors have been demonstrated to be crucial for the early segregation of the embryonic ectoderm into a neurogenic and a non-neurogenic tissue during neural induction. Bmp signalling is the critical factor for the early development of the CNS and for its anterior–posterior (AP) and dorsal–ventral (DV) patterning. Bmp signalling is suppressed by antagonists such as Chordin and Noggin on the anterior–dorsal side in chordates1617N. vectensis. Many cnidarian polyps develop a conspicuous ring-like nerve plexus around their mouth8101031Wnt/β-catenin signalling has multiple and even opposite functions in neural development of deuterostomes, depending on the developmental stage27N. vectensis is the salt-and-pepper-like expression of neural marker messenger RNAs (mRNAs) such as NvAchaete-scute homolog A (NvAshA), NvElav1 and Rfamide in blastula epithelium613N. vectensis embryos. A whole-genome microarray analysis on NvAshA-overexpressing embryos has identified NvElav1, NvRfamide and genes involved neurotransmission as NvAshA targetsNvElav1 is required at least ectodermal development of neuropeptides-expressing neuronsNvAshA suggests that the early embryonic epithelium of N. vectensis has the potential to generate various neuronal cell types that form the larval nerve netNvAshB, which is also involved in NvRfamide neuropeptide gene expressionNvAshC, NvSoxC, NvMusashi and NvRfamide that are expressed dominantly or exclusively around the blastopore6121314The first sign for neurogenesis in N. vectensis blastulae343637N. vectensis reflects the evolutionary emergence of these major signalling axes in the evolution of the nervous system.Our study revealed that β-catenin signalling is essential for early neurogenesis during the development of the oral nervous system that starts at the blastula/gastrula transition. Wnt/β-catenin signalling is known to be active at the blastoporal side and defines the primary oral–aboral axis in Cnidaria/Bilateria ancestor41The cnidarian nervous system is rich in neuropeptides811N. vectensis that belongs to anthozoans, the most basal class in cnidarians  and GLWamidergic (GLWa+) neuronal subpopulations correspond to 10% of all neurons at the late planula stage -PCR and whole-mount in situ hybridization (WISH) data demonstrate that several neurogenic genes were strongly upregulated during early embryonic development  formed a synexpression group at the prospective and established blastopore site together with the previously described NvSoxB2a and NvAshB12NvSoxB2a was considered to be an unclassified NvSox1  axis  axis .NvSoxB2a, NvAshB and NvArp3) and other early neurogenic and neural marker genes , NvElav1 and NvRfamide) as well as the late oral neurogenic genes  using morpholino (MO) antisense oligonucleotides against the early blastoporal neurogenic genes  and endodermal (NvElav1+) nervous systems. As NvSoxB2a and NvAshB are required for the expression of many early and late oral neuronal genes, they might mainly be involved in the commitment of embryonic cells that gives rise to neural precursor cells, whereas NvArp3 might have a selective function to specify certain neuronal cell types such as Elav1+ neurons.We further analysed the neurogenic activity of the early oral TFs by monitoring the mature RFa neurons  and 7. Ia larvae , which ila stage . These rNvBrachyury (NvBra), a target of canonical Wnt/β-catenin signalling in cnidariansNvTcf expression is maintained (35NvRfamide) along the oral–aboral (primary) body axis51To identify signals required for expression of the early oral neurogenic genes, we searched for factors turned on in the blastula at the site of presumptive blastopore region. RT-PCR and WISH analyses indicated that intained 50. Wnt/βNvSoxB2a, NvAshB and NvArp3, as well as a β-catenin target gene NvBra at the blastula stage  into fertilized eggs. The augmentation of β-catenin signalling induced increased expression of the neurogenic genes la stage . A more la stage  and 9. Tla stage . Under tla stage . In blasla stage  suppressd NvArp3 . These dNvAshA, NvSoxB2c, NvElav1 and NvRfamide that show lateral expression patterns at blastula. Most of these genes except for NvElav1 were not upregulated under augmented β-catenin signalling  also induced strong upregulation of other neural genes that show a later oral expression at the gastrula stage . All neula stage . The iCR neurons . Thus, gN. vectensis. The asymmetric expression of NvBmp2/4, NvBmp5–8 and their antagonist NvChordin became visible around the blastoporal lip only after the gastrula stage  to globally elevate the Bmp pathway. hBmp2 treatment induced the phosphorylation of NvSmad1 in a dose-dependent manner , whereas no significant effect was observed on the expression level of the early oral TFs , our findings may suggest that β-catenin protein that is expressed uniformly in early blastomeres has a role to predispose the blastula epithelium to become neurogenic, as it has been suggested by previous studies on vertebrates29575859Another intriguing finding revealed in this study is the β-catenin dependency of expression of the early lateral neural genes Xenopus and DrosophilaWhen we tested the function of Bmp signalling on the early induction of oral neuronal genes, we could not detect any inhibitory Bmp effect, which was unexpected since Bmp/Chordin signalling is frequently considered as the primary and general CNS inducer15+ neurons6162+ neurons at the planula stageN. vectensis? When we analysed Bmp signalling at the planula stage, we discovered that RFa+ and GLWa+ neurons as well as early and late oral neurogenic TFs  exhibited indeed a distinct Bmp sensitivity. Only for NvAshB this Bmp dependency was weak, and it was absent for NvSoxB2a. Therefore, Bmp signalling is an important factor in the development of the oral nervous system. However, this Bmp regulation is complex. When Bmp signalling was either suppressed or over-activated at gastrula and planula stages, neurogenic TFs and GLWamide neuropeptide became downregulated under both conditions. This is reminiscent to the blastoporal regulation of NvSoxB1 and NvNk2.1 that are likewise downregulated in NvChordin and NvBmp2/4 morphantsNvBmp2/4, NvBmp5–8 and NvChordin genes is dependent on β-catenin signalling, the Wnt/β-catenin regulation of neural patterning might be mediated, at least in part, by the Bmp activity. This point is of interest for future studies.Treatment of gastrulae and planula larvae with inhibitors of GSK3  triggered β-catenin activation and the expansion of oral markers including RFaNvArp6-expressing cells and GLWa+ neurons form a distinct asymmetric pattern along the directive axis, which is Bmp dependent. This asymmetric suppression of Bmp is reminiscent to an evolutionarily conserved Bmp function in the mediolateral patterning of the trunk nervous system in bilateriansNvArp6 but suppresses other oral neuronal TFs) did not result in more and symmetric development of GLWa+ neurons. Therefore, GLWa+ neurons depend on additional neurogenic TFs, which are similarly restricted to the oral site.A striking function of Bmp signalling at the planula stage is the asymmetric induction of neurons along the (secondary) directive axis. SoxB1 and Nk2.1) are exclusively expressed in the oral nervous system in N. vectensis12Our data provide a new view on the evolution of the nervous system, and we propose that the cnidarian oral nervous system exhibits some important features that are essential for the evolution of the bilaterian CNS . (i) The41Most recently the genome of comb jellies (ctenophores), another pre-bilaterian clade with a primitive nervous system, was published65656769N. vectensis give an insight how a first step in the evolution of nervous system centralization may have been accomplished. The cnidarian nervous system is also an intriguing example, how basic positional information, which is organized in bilaterians in a Cartesian coordinate systemTo understand the emergence of the bilaterian CNS from simple neural clusters, deeper insights into the anatomical and molecular features of the cnidarian nervous systems are very helpful. Although this knowledge is still far from being complete, our data on the development of the oral nervous system in N. vectensis were cultured at a salinity one-third of normal seawater . Adult males and females were separately cultured at 18 °C in the dark and fed two to three times per week. On 2 days after the feeding, the culture boxes were washed. For spawning the release of eggs and sperms, the culture boxes were incubated at 26 °C under the light for at least 14 h. After fertilization, embryos were cultured at 20 °C.NvSoxB2a, NvAshB, NvArp3 and NvArp6 genes were subcloned with partial 5′ untranslated region (UTR) and the complete open-reading frame into pGEM-T vectors and sequenced. The insert was cut using EcoRI and inserted into the pCS2 plasmid.Gene-specific primers were then designed to recover both 3′ and 5′ rapid amplification of complementary DNA (cDNA) ends (RACE) fragments using the GeneRacer kit (Invitrogen) with annealing temperatures between 55 and 65 °C for 1st and nested PCR. Gene-specific primer sequences are listed in For semi-quantitative RT-PCR, the level of amplified product was compared in the exponential range of amplification. qRT-PCR was performed on the Bio-Rad DNAEngine equipped with Chromo4 real-time PCR detector (Bio-Rad) using Absolute QPCR SYBR Green mix (Thermo Scientific). The primer sequences are listed in −1. As a control, 1 μg ml−1 bovine serum albumin (BSA) was applied. hBmp2 protein was added at one-two-cell-stage embryos and cDNAs were prepared at the blastula (12–14 h.p.f.), gastrula (30 h.p.f.) or planula (74 h.p.f.) stages. For WISH experiments, hBmp-treated blastulae and gastrulae (12 h.p.f. (blastula) and 24 h.p.f. (gastrula) were fixed. For experiments using the GSK3β inhibitors to enhance β-catenin signalling, embryos were treated with ALP, BIO or 1-AZA (Sigma) at the concentrations indicated. cDNAs were prepared at the blastula and early gastrula stages (12–18 h.p.f.) for the qPCR analyses. For immunostaining of neuropeptides and mOrange protein, embryos were treated with GSK3β inhibitors from one- and two-cell stage to blastula stage. After being washed, the embryos were cultured in Nematostella medium until early (72 h.p.f.) or late (98 h.p.f.) planula stages. To pharmacologically inhibit β-catenin signalling, embryos were treated with 10 μM or 50 μM iCRT14 (Sigma), a Tcf/β-catenin inhibitor, for 14 h (blastula), 3 days (early planula) or 4 days (late planula).Embryos were treated with recombinant hBmp2 (a gift from Dr Wölfl) at concentrations from 0.1 to 1 μg ml−1 rhodamine- or Alxa488-dextran (Invitrogen). Embryos were then rinsed with N. vectensis medium and incubated until desired stages.pCS2 constructs, pCS2-MT-β-Eng (DN-β-catenin) (a gift from Dr McCrea), pCS2-NvBmp2/4 and DN-GSK3β, were linearized using appropriate restriction enzymes. Capped mRNAs were synthesized using the SP6 mMessage mMachine mRNA synthesis kit (Ambion). Capped mRNAs or MO antisense oligonucleotide for control (5′- CCTCTTACCTCAGTTACAATTTATA ), neurogenic TFs NvSoxB2a , NvAshB , NvArp3 , NvArp6 , NvBmp2/4 (5′- GTAAGAAACAGCGTAAGAGAAGCAT -3′)The translation-blocking MOs were tested using the TnT SP6 quick coupled transcription/translation system (Promega). pCS2 plasmids (250–500 ng) containing MO-target sites (5′ UTR and first ATG) were incubated with control or gene-specific MOs (50 μM) at 30 °C for 90 min. The biotinylated proteins synthesized were detected by western blotting using horseradish peroxidase-conjugated streptavidin .In situ hybridization was performed as described previouslyN. vectensis embryos were solubilized with 2 × SDS–polyacrylamide gel electrophoresis sample buffer and immediately boiled at 90 °C for 5 min. After being sonicated, proteins separated by SDS–polyacrylamide gel electrophoresis were transferred to a PVDF (polyvinylidene difluoride) membrane (Millipore). The membranes were blocked with 5% BSA in buffer consisting of 50 mM Tris-HCl, pH 8.0, 2 mM CaCl2, 80 mM NaCl and 0.2% Nonidet P-40, incubated with the blocking solution containing anti-phospho-Smad1/5/8 antibody  (1:200 dilution), anti-Smad1/5/8 (N-18) antibody (Santa Cruz Biotechnology) (1:300 dilution) or anti-β-tubulin (2-28-33) antibody (Sigma)  followed by incubation with horseradish peroxidase-conjugated secondary antibodies (Jackson ImmunoResearch)  in blocking solution. Immunoreactive proteins were detected with an ECL immunoblotting detection reagent (Amersham Pharmacia Biotech). Full images of western blots are shown in −1 BSA+5% normal goat serum in PBS), incubated with anti-FMRFamide antibody (1:600 dilution) (Sigma) or anti-GLWamide antibody (1:400 dilution) (a gift from Dr Koizumi) in the blocking solution containing 0.1% Triton X-100, and then with Alexa488- or Alexa568-conjugated anti-Rabbit antibody (1:800 dilution) (Invitrogen). Specimens were imaged using a Nikon ECLIPSE 80i fluorescent microscope equipped with DIGITAL SIGH DSU1 (Nikon). For confocal microscopy, a fluorescent microscope ECLIPSE Ti (Nikon) equipped with an A1R MP confocal scanner (Nikon) was used.The NvElav1::mOrange transgenic embryos were fixed with 4% paraformaldehyde/PBT for 1 h at room temperature and stained using anti-DsRed antibody (1:300 dilution)(Clontech), as was previously reportedH.W. performed most of the experiments. A.K. and M.F. performed some WISH analyses. H.W., K.A., S.Ö., T.F. and T.W.H. designed the project. H.W. and T.W.H. wrote the manuscript.How to cite this article: Watanabe, H. et al. Sequential actions of β-catenin and Bmp pattern the oral nerve net in Nematostella vectensis. Nat. Commun. 5:5536 doi: 10.1038/ncomms6536 (2014).Supplementary Figures 1-15 and Supplementary Table 1


Predictive computational modelling in biomedical research offers the potential to integrate diverse data, uncover biological mechanisms that are not easily accessible through experimental methods and expose gaps in knowledge requiring further research. Recent developments in computing and diagnostic technologies have initiated the advancement of computational models in terms of complexity and specificity. Consequently, computational modelling can increasingly be utilised as enabling and complementing modality in the clinic—with medical decisions and interventions being personalised. Myocardial infarction and heart failure are amongst the leading causes of death globally despite optimal modern treatment. The development of novel MI therapies is challenging and may be greatly facilitated through predictive modelling. Here, we review the advances in patient-specific modelling of cardiac mechanics, distinguishing specificity in cardiac geometry, myofibre architecture and mechanical tissue properties. Thereafter, the focus narrows to the mechanics of the infarcted heart and treatment of myocardial infarction with particular attention on intramyocardial biomaterial delivery. Cardiovascular diseases are the single leading cause of death worldwide, accounting for 30 % of all human mortality . DespiteComputational models can provide a unique framework for assessing efficacy of therapy approaches with relatively low resources: Therapeutic parameters can be easily modified and assessed in multiple concurrent in silico experiments, and computational sensitivity studies are easily conducted to optimise treatment efficacy. Advancing research and technologies have sparked a great deal of interest in integrating FE models into the clinical environment. This is becoming more achievable each year, making it likely that computational models will serve as the first line of the screening for future therapies in the years to come .Reliable computational models can also provide a richer source of information for clinical decision support and treatment planning. Patient- and subject-specific computational modelling has been increasing at an exponential rate Fig. a, and soThis review aims at contextualising the advances and challenges of patient-specific computational modelling with particular focus on cardiac and infarct mechanics and the translation of therapeutic concepts, based on intramyocardial biomaterial injections, for the myocardial infarction (MI) and infarct-induced heart failure (HF). The developments in subject- and patient-specific modelling are detailed with focus on cardiac geometries, myofibre architecture and the constitutive properties of cardiac tissue. Thereafter, the focus narrows on computational modelling of infarct mechanics and therapies for MI, in particular intramyocardial biomaterial injection.Anatomical simplification needs to balance model accuracy and computational demands. Until recently, this motivated the use of simplified left ventricular (LV) geometries introduced by various groups –9 as theThe first three-dimensional patient-specific computational geometry of a heart was introduced by Okajima et al.  to studyTo date, patient-specific cardiac geometries have been introduced as representative cases as proof of concept that a given computational approach can be applied in a patient-specific framework. Extending the concept to computational studies with a large number of patient-specific geometries could generate statistically meaningful results for a patient population. An alternative approach is to utilise a statistically averaged geometry that is representative of a patient population .The myofibre orientation has a critical influence on cardiac mechanics and electrophysiology. However, the description of myofibre orientation is a highly intricate and sophisticated task, which has been the subject of substantial historical disagreement –18. The post mortem data set. In this case, the derived fibre orientations are projected (or mapped) onto other subject- or patient-specific geometries obtained from computed tomography (CT) or magnetic resonance imaging (MRI) . Ma. Ma95]. For more viscous injectable materials or when the mechanical effects of the injectate at a microstructural level are being investigated –102, modResidual stress in the cardiac wall due to material injections has only recently been considered. Using a patient-specific LV FE model based on MRI data of a patient with HF, ischaemic cardiomyopathy and hypertension, Lee et al.  revealedLee et al.  investigAdvances in patient-specific computational cardiac mechanics over the last decade have been significant in almost every aspect. The quality of imaging and segmentation techniques coupled with increasing computational resources have allowed for unprecedented growth. Geometrically realistic multi-physics models are becoming the new standard of computational cardiology. As structural and functional data of the heart become more easily available, the calibration and validation of these models becomes more reliable.Patient-specific models hold promise for personalising diagnosis, treatment planning and therapy design. The example of SVR emphasises that interventions based on accurate patient-specific information have clear advantages over treatments that are not personalised. Large-cohort patient-specific computational studies, simulating treatments in silico, will also be able to unlock novel and statistically meaningful findings for entire patient populations—something that cannot be achieved with a small number of computational models. The ability to re-use computational models, perturb parameters and perform sensitivity studies will not only provide an unprecedented wealth of information in the aid of therapy design but can also accelerate the translation of therapy approaches into the clinical setting.Despite these advantages, there has yet to be a single high-resolution patient-specific computational cardiac model, constructed and calibrated using data from a single patient. Accounting for patient-specific myofibre structure and calibrating material laws using comprehensive in vivo data are still largely lacking in computational models investigating cardiac mechanics.While recent computational research on biomaterial injection MI therapies has made substantial progress, more work is needed to further elucidate the mechanisms underlying the benefits observed. The local and global changes in myocardial tissue structure after MI, including necrosis, fibrosis and scar formation, and the representation in computational models are one area that need to receive increased attention. Pending availability of experimental data, advanced numerical methods to model growth –109 and Realistic predictive patient-specific computational models require comprehensive in vivo data for calibration and validation. In the context of cardiac diseases and therapies, current in vivo imaging technologies are not yet advanced enough to provide such patient-specific data as part of the clinical diagnostic modalities. Until cutting-edge modalities such as in vivo cardiac DTMRI become more available in the clinic routine, the pursuit of fully subject-specific computational modelling remains limited to preclinical research, where a richer resource of in vivo and ex vivo data can be utilised. Subject-specific computational modelling can, however, offer great potential to complement experimental research and can play a crucial role in advancing biomaterial injection therapies for MI.


Dalbergia odorifera has been traditionally used as a medicine to treat many diseases. However, the role of 2,4,5-trimethoxyldalbergiquinol (TMDQ) isolated and extracted from D. odorifera in osteoblast function and the underlying molecular mechanisms remain poorly understood. The aim of this study was to investigate the effects and possible underlying mechanisms of TMDQ on osteoblastic differentiation of primary cultures of mouse osteoblasts as an in vitro assay system. TMDQ stimulated osteoblastic differentiation, as assessed by the alkaline phosphatase (ALP) activity, ALP staining, mineralized nodule formation, and the levels of mRNAs encoding the bone differentiation markers, including ALP, bone sialoprotein (BSP), osteopontin, and osteocalcin. TMDQ upregulated the expression of Bmp2 and Bmp4 genes, and increased the protein level of phospho-Smad1/5/8. Furthermore, TMDQ treatment showed the increased mRNA expression of Wnt ligands, phosphorylation of GSK3, and the expression of β-catenin protein. The TMDQ-induced osteogenic effects were abolished by Wnt inhibitor, Dickkopf-1 (DKK1), and bone morphogenetic protein (BMP) antagonist, noggin. TMDQ-induced runt-related transcription factor 2 (Runx2) expression was attenuatted by noggin and DKK1. These data suggest that TMDQ acts through the activation of BMP, Wnt/β-catenin, and Runx2 signaling to promote osteoblast differentiation, and we demonstrate that TMDQ could be a potential agent for the treatment of bone loss-associated diseases such as osteoporosis. The aim of this work was to investigate the effects and its underlying signal mechanism of TMDQ on osteoblastic differentiation in primary culture of mouse calvarial osteoblasts as an in vitro assay system.Although we isolated and identified novel compound 2,4,5-trimethoxyldalbergiquinol (TMDQ) compound from the heartwood of To determine the cytotoxic potential of TMDQ, its effect on viability of the osteoblasts was evaluated. Up to a concentration of 10 μM, no cytotoxic effects could be observed using 3--2,5-diphenyltetrazolium bromide (MTT) assay . To inveTo understand the signal pathways involved in the regulation of cell differentiation by TMDQ, mitogen-activated protein kinase (MAPK), BMPs, and Wnt/β-catenin pathways were analyzed via reverse transcriptase-PCR (RT-PCR), real-time PCR, western blot analysis, and immunofluorescence. As shown in 13 we examined the effect of TMDQ on Bmp expression and BMP signaling. We found that TMDQ enhanced Bmp2 and Bmp4 mRNA expression as well as the downstream signaling molecules of BMP, and the phosphorylation of Smad1/5/8 in the osteoblasts  and nuclear β-catenin (N) in the osteoblasts were increased by treatment with TMDQ , and BMP antagonist, noggin, for 1 h before treatment with TMDQ. Results showed that noggin pretreatment drastically inhibited TMDQ-mediated ALP staining, ALP activity, and mineralized nodule formation, whereas DKK1 partially attenuated , which is a key transcription factor that has an essential role in osteoblastic differentiation.14 The expression of Runx2 was significantly increased by TMDQ after 48 h incubation , and increased mineralized nodule formation in the osteoblasts. These data suggest that the early and late osteoblastic differentiation, and the maturation were stimulated by TMDQ.The formation of new bone involves a complex series of events including the proliferation and differentiation of osteoblasts, and eventually the formation of a mineralized extracellular matrix. Thus, ALP is a phenotypic marker for the early differentiations of osteoblasts, whereas the mineralization of extracellular matrix by Alizarin red staining is a biological marker for terminal differentiation.β superfamily and have pivotal roles in the regulation of bone formation and remodeling.21 BMP signaling is initiated by binding to receptor, propagated by the phosphorylation of Smad1/5/8 complex, and finally translocated into the nucleus to regulate the transcription of target genes.13 Several natural or chemical compounds have been reported to induce osteoblastic differentiation by the induction of BMP signaling, such as daidzein,22 osthole,23 and betulinic acid.24 Our data showed that the treatment of osteoblasts with TMDQ enhanced the Bmp2 and Bmp4 expression as well as the phosphorylation of Smad1/5/8, which is the central molecule in BMP signaling. In addition, the BMP antagonist, noggin, blocked TMDQ-mediated ALP activity and mineralized nodule formation, indicating that BMP production is required in TMDQ-mediated osteoblastic differentiation. MAP kinases signaling  also represents as an alternative and non-canonical pathway for BMP2 signal transduction to promote osteogenic factors including Runx2.26 However, TMDQ did not affect MAP kinases signaling in the osteoblasts. These data suggest that TMDQ promotes osteoblastic differentiation through the BMP/Smad pathway but not the MAPKs pathway.BMPs form a unique group of proteins within the transforming growth factor β-catenin signaling has critical roles in the bone formation.27In vitro and in vivo studies showed that the activation of Wnt/β-catenin signaling promotes osteoblastic differentiation and mineralization, whereas it inhibits apoptosis in osteoblasts and osteocyte.29 The Wnt ligands bind to Frizzled and LRP5/6 receptors, and induce the stabilization of cytoplasmic β-catenin by inhibiting GSK3β.30 Consequently, the β-catenin was accumulated in the cytoplasm and translocated into the nucleus to regulate gene expression.32 In the present study, we demonstrated that TMDQ significantly increased the expression of canonical Wnt ligands and stabilized the β-catenin by inactivating GSK3β, allowing the stabilization and nuclear translocation of β-catenin. Moreover, the TMDQ-induced osteogenic effects were abolished by Wnt inhibitor, DKK1. These results suggest that TMDQ promotes osteoblastic differentiation and mineralization through the activation of the BMP and Wnt/β-catenin signaling pathways.Wnt/33 BMP2-mediated Runx2 expression has an important role in the osteoblastic differentiation.35 In the present study, the protein level of Runx2 was significantly increased in the TMDQ-treated osteoblasts. In addition, the pharmacological and genomic downregulation for Runx2 attenuated TMDQ-mediated osteoblastic differentiation. It was reported that Wnt/β-catenin signaling increases BMP2 expression and vice versa,37 as well as osteogenic genes including Runx2 integrate the BMP2 and Wnt/β-catenin signaling pathways in the regulation of osteoblastic differentiation.41 Based on the above results and the upregulation of Runx2 by TMDQ, Runx2 is activated by the BMP- and Wnt/β-catenin-dependent signaling pathways in the osteoblasts, suggesting functional cross talk between BMP2 and Wnt/β-catenin siganaling.Runx2 is the main transcription factor required for the activation of osteoblastic differentiation and is crucial for the regulation of genes responsible for the production of bone-specific proteins.β-catenin signaling pathway to activate Runx2 in primary cultures of mouse calvarial osteoblasts. These findings suggest that TMDQ may be useful in inducing osteogenesis and facilitating bone mineralization for bone diseases such as osteoporosis.In summary, the present study is the first report that TMDQ promotes osteoblastic differentiation through the BMP and Wnt/D. odorifera (1.65 kg) were extracted with EtOH by untrasonics for 1 h. After concentrated in vacuo, the EtOH extract (200 g) was suspended in H2O and partitioned with EtOAc to give EtOAc  and aqueous fractions. Fraction DOE was chromatographed over a silica gel column, eluting with acetone in n-hexane  and washing with MeOH to provide seven subfractions (DOE1–6). Subfraction DOE3 was separated by a silica gel column chromatography, eluting with n-hexane-CH2Cl2 (1 : 2) to give five subfractions (DOE31–5). Subfraction DOE34 was further separated by a silica gel column chromatography, eluting with n-hexane-EtOAc (4 : 1) to give 2,4,5-trimethoxydalbergiquinol (10 mg). The structure of this compound was elucidated through comparison of the 1H- and 13C-NMR spectral data with those of the literature.42 4,5-Trimethoxydalbergiquinol: yellow amorphous powder. 1H-NMR  δ 5.15 , 4.87 , 6.26 , 5.04 , 6.70 , 6.63 , 7.11–7.24 , 3.69 , 3.82 . 13C-NMR  111.16 (C-2), 141.98 (C-3), 48.66 (C-4), 115.86 (C-5), 144.76 (C-6), 150.00 (C-7), 99.8 (C-8), 153.1 (C-9), 124.9 (C-10), 144.2 (C-1'), 129.0 , 129.5 , 126.9 (C-4'), 56.7 (6-OCH3), 56.9 (7-OCH3), 57.6 (9-OCH3).Dried heartwoods of α-minimum essential medium without L-ascorbic acid supplemented with 10% fetal bovine serum, penicillin (100 U/ml), and streptomycin (100 μg/ml) at 37 °C in a humidified atmosphere of 5% CO2 and 95% air. The cells were detached and reseeded at ~70–80% confluence, and then the cells (passage 1) were used for the experiments reported here. To test the effects of TMDQ on osteoblast differentiation, osteoblastic differentiation was induced by changing OS medium containing 50 μg/ml L-ascorbic acid and 10 mM β-glycerophosphate when the cells are ~80% confluent. The recombinant noggin and DKK1 were purchased from Invitrogen  and PeproTech , respectively. The medium was replaced every 2 days during the incubation period. TMDQ was dissolved in 100% DMSO and then diluted it (1 : 1000) directly into the medium. Final concentration of 0.1% DMSO was used as the vehicle control. For siRNA transfection, cells were transfected using Lipofectamine RNAiMAX according to the manufacturer's specification (Invitrogen).Primary osteoblasts were isolated from calvariae of 1-day-old ICR mice after dissected aseptically and treated with 0.2% collagenase-dispase enzyme solution . Cells (passage 0) were collected by centrifuge after repeated digestions and cultured in Cell toxicity was measured by an MTT assay to detect NADH-dependent dehydrogenase activity. Fifty microliters of MTT solution (5 mg/ml) in 1 × phosphate-buffered saline (PBS) were directly added to the cells, which was then incubated for 2 h to allow MTT to metabolize to formazan. Absorbance was measured at a wavelength of 540 nm using an ELISA reader .Mp-NPP in 0.1 M glycine-NaOH (pH 10.3) at 37 °C for 30 min. The reaction was stopped by adding 0.25 N NaOH. The absorbance was measured at 405 nm using an ELISA reader (Beckman Coulter).ALP activity was measured by spectrophotometry. Cells were homogenized in 0.5 ml distilled water with a sonicator, and centrifuged. The aliquots of cell homogenate were incubated with 15 mCells were washed with 1 × PBS and then fixed in 4% formaldehyde for 20 min at room temperature. The cells were rinsed with distilled water, and permeabilized by 0.1% Triton X-100. Cells were incubated at 37 °C for 30 min in the Naphthol-AS-BL alkaline solution mixture (Sigma-Aldrich).After 14 days of culture, cells were fixed in 70% ice-cold ethanol for 1 h and rinsed with distilled water. Cells were stained with 40 mM Alizarin Red S (pH 4.2) for 10 min with gentle agitation. The level of Alizarin red S staining was observed under light microscopy. Stains were eluted with 100% DMSO to quantify the amount of Alizarin red staining and measured at 590 nm.15 primers with AccuPower RT PreMix . Next, the generated cDNAs were amplified with AccuPower PCR PreMix . The sequences of primers are listed in CT−ΔΔ formula was used, where –ΔΔCT= experimental sample— control sample.The total RNA of cells was extracted with Trizol reagent  according to the manufacturer's instructions. One microgram quantity of RNAs isolated from each sample was reverse-transcribed with oligo (dT)μg) resolved on SDS-polyacrylamide gel electrophoresis were transferred to a polyvinylidene fluoride membrane , and the membrane was blocked with 1 × TBS containing 0.05% Tween-20 and 5% skim milk or 2% BSA for 1 h at room temperature. After blocking, the membranes were incubated overnight at 4 °C with the respective primary antibodies as follows: β-catenin , p-ERK1/2 , ERK1/2 , p-p38 , p38 , p-JNK , JNK , GSK3β , p-GSK3β , Runx2 , p-Smad1/5/8 , Smad1/5/8 , β−actin , Lamin B . The membranes were washed with 1 × PBS and incubated with diluted horseradish peroxidase-conjugated secondary antibodies  for 1 h at room temperature. After three washes, the membranes were detected using the enhanced chemiluminescence kit (Millipore).Cells were washed twice with ice-cold PBS, and lysed in 20 mM Tris-HCl buffer (pH 7.4) containing a protease inhibitor mixture . Protein concentration was determined by Bradford reagent . Equal amounts of lysates  antibodies for overnight at room temperature, washed three times, and incubated with Alexa-488-conjugated secondary antibodies  for 2 h at room temperature. The cells was stained with DAPI (Sigma-Aldrich) and washed three times, mounted on glass slides, and viewed on confocal microscopy .Cells were grown on glass coverslips and incubated with TMDQ for 48 h. Cells were fixed in 10% formalin for 15 min at room temperature. After washing three times in 1 × PBS, the cells were permeabilized with 0.2% Triton X-100 in 1 × PBS for 20 min, washed three times in 1 × PBS, and then blocked with 5% BSA in 1 × PBS for 1 h at room temperature. After then, the cells were incubated with anti-p-Smad1/5/8  and anti- P<0.05 was considered to be statistically significant.The data were analyzed using the GraphPad Prism version 5 program . Data are presented as mean±S.E.M. Satistical significance was performed on the data using one-way analysis of variance and the differences were assessed by the Dunnett's test. A value of


We analyzed the protein distribution of two cadherin-associated molecules, plakoglobin and β-catenin, during the different stages of tooth development and tooth replacement in zebrafish. Plakoglobin was detected at the plasma membrane already at the onset of tooth development in the epithelial cells of the tooth. This pattern remained unaltered during further tooth development. The mesenchymal cells only showed plakoglobin from cytodifferentiation onwards. Plakoglobin 1a morpholino-injected embryos showed normal tooth development with proper initiation and differentiation. Although plakoglobin is clearly present during normal odontogenesis, the loss of plakoglobin 1a does not influence tooth development. β-catenin was found at the cell borders of all cells of the successional lamina but also in the nuclei of surrounding mesenchymal cells. Only membranous, not nuclear, β-catenin, was found during morphogenesis stage. However, during cytodifferentiation stage, both nuclear and membrane-bound β-catenin was detected in the layers of the enamel organ as well as in the differentiating odontoblasts. Nuclear β-catenin is an indication of an activated Wnt pathway, therefore suggesting a possible role for Wnt signalling during zebrafish tooth development and replacement. During the various phases of development of the vertebrate body, it is of major importance that cells are capable of communicating with each other and of rearranging themselves. Adhesion molecules play a very important role during the morphogenetic processes that underlie development –4.There are different types of cell junctions. Adherens junctions (AJs) are an important type of intercellular junction, consisting of the cadherin-catenin complex. Cadherins represent an important family of cell adhesion molecules, and include classical cadherins and desmosomal cadherins. Classical cadherins are connected to the actin cytoskeleton through β-catenin. At the cell membrane the cadherins cluster laterally to achieve strong adhesive forces. By connecting to cadherins at the neighbouring cell membrane, cells are held closely together –7. DesmoThe cadherin-associated molecules plakoglobin and β-catenin are close homologues. Their sequence is partially similar and they compete for the same binding site at the C-terminal region of classical cadherins . While pApart from functioning in cell adhesion, β-catenin is also known to play a role in the Wnt pathway by binding to the LEF/TCF binding site in the nucleus –15. GiveTooth development has been used for many years as a paradigm for investigating organ development. Not only do teeth result from multiple, reciprocal interactions between two tissue layers, the epithelium and underlying mesenchyme, but these two tissue layers undergo extensive morphogenetic movements. Tooth formation starts with the establishment of an epithelial thickening, called placode. As the epithelium and underlying mesenchyme pass through different stages of morphogenesis, both their cells need to rearrange before they can differentiate and deposit tooth-specific matrices –20. How 1, superscript indicating the generation number). soon followed by the adjacent teeth in the same row . At around 80 hours post-fertilization (hpf) the first developing tooth attaches to the branchial arch, thereby becoming functional. At the same time, a replacement tooth  starts to develop from the base of the crypt surrounding the functional tooth [We have set out for a study aiming at elucidating the role of cell adhesion molecules during development and replacement of teeth. To this end, we focus on the zebrafish since this species, contrary to mammalian species, replaces its teeth throughout life. Zebrafish lack jaw teeth in the oral cavity, but have pharyngeal teeth located on the fifth branchial arch. The full zebrafish dentition consists of 11 teeth on each side, divided into three rows, called ventral (V), mediodorsal (MD) and dorsal (D) rows. The first tooth starts to develop at two days post-fertilization (dpf) in position 4 of the ventral row . β-catenin is also expressed at the plasma membrane of the cells constituting the epithelial crypts surrounding the functional tooth .In this study we show the distribution of two closely related cell adhesion molecules during the development and replacement of zebrafish teeth. β-catenin and plakoglobin are expressed by the same cell layers, but β-catenin has a broader distribution. Plakoglobin expression is restricted to the epithelial-derived part of the tooth during initiation and morphogenesis stage but expands to the dental papilla from cytodifferentiation onwards. β-catenin is also expressed both in the epithelial enamel organ and in the mesenchymal-derived dental papilla. However, unlike plakoglobin, β-catenin is also found in the nuclei of cells of the enamel organ and of differentiating odontoblasts. The analysis of plakoglobin morphant embryos revealed no abnormalities as the first three teeth developed normally.The scarce data available on plakoglobin distribution in teeth are limited to the mouse model, where its expression parallels that observed here in zebrafish. In both species, plakoglobin is expressed in the epithelial part of the developing tooth. In mice, an asymmetrical distribution has been observed, the plakoglobin protein signal being weaker on the medial side of the tooth , 29. WhiThe distribution of β-catenin during tooth development has been studied previously in mice and in humans , 31–33. In previous studies, we have shown that both first-generation or later-generation zebrafish teeth form without a detectable down-regulation of E-cadherin , 34, refSince plakoglobin showed such a strong and specific expression during zebrafish tooth development, plakoglobin morpholino-injected zebrafish were studied. Until 96 hpf, there was no detectable difference between control-injected and morphant larvae. Both morphant and control zebrafish developed teeth at the correct positions. Hence, reduction of plakoglobin did not alter the possibility to initiate and further develop teeth. Furthermore, the morphant teeth were also capable of differentiating, as enameloid was formed. This indicates that plakoglobin is not necessary for the differentiation of the inner dental epithelium or the odontoblasts . ToPlakoglobin KO mice die because of severe defects in the developing heart due to the lack of desmosomes. The intestinal epithelium and hair follicle development of these plakoglobin null embryos was not altered and AJs were unaffected demonstrating that epithelial-derived tissues can develop normally without plakoglobin , 41. Celichabod is not useful either as their embryos are deficient for maternal, but not zygotically expressed β-catenin-2 [Next to its important function in cell adhesion, β-catenin is also known to be a key mediator of Wnt signalling. The presence of nuclear β-catenin is considered a valid proxy for active Wnt signalling . Severalatenin-2 .While β-catenin and plakoglobin share at least some functions, plakoglobin was long thought not to be involved in Wnt signalling. Only later it was discovered that plakoglobin can act on its own in the Wnt signalling pathway by binding to LEF/TCF in the nucleus , 54, 55.Summarizing, we were able to show the distribution of β-catenin and plakoglobin during the different stages of zebrafish tooth development. These cell adhesion-related molecules were expressed in both the epithelial as well as the mesenchymal component of the developing tooth. Unlike plakoglobin, β-catenin signal could be detected in numerous nuclei suggesting that Wnt signalling is active during initiation but repressed during morphogenesis stage. Although plakoglobin is strongly expressed in developing teeth, the loss of plakoglobin does not seem to affect tooth development as plakoglobin morphants do not show any tooth defect. Neither β-catenin nor plakoglobin is down-regulated during initiation of a new tooth placode. How and which cell adhesion molecules are regulated to obtain the fine balance between reduced adhesion and proper development, needs to be further examined.Wild-type (wt) zebrafish were raised in a 14h light / 10h dark light regime at the standard temperature of 28.5°C . EmbryosSince euthanasia is not considered an animal experiment, and since developing zebrafish should be regarded as protected animals under the new legislation of animal welfare (EU 2010/63/EU) only from 120 hpf onwards , an authPlakoglobin morpholino-injected, control morpholino-injected and wild-type zebrafish were obtained from the lab of Dr. M. Grealy . One- to 2-cell stage embryos have been injected with 5 ng of a morpholino constructed to prevent translation of plakoglobin-1a mRNA or with a 5 base pair (bp) mismatch morpholino as control . The batPlakoglobin MO-injected embryos and larvae were fixed overnight at 4°C in 4% paraformaldehyde (PFA) in phosphate buffered saline (PBS) and embedded in epon according to standard procedures. Semithin (1 μm) sections were made using a diamond knife. Next, the epon sections were stained with toluidine blue  for detailed histological analysis.Wild-type embryos and larvae were fixed overnight at 4°C in 4% PFA in PBS, depigmented, dehydrated in an increasing methanol (MeOH) series and stored in 100% MeOH at -20°C for whole-mount analysis.The fifth branchial arches dissected from adult zebrafish were fixed overnight at 4°C in 4% PFA in PBS, decalcified in Morse’s solution  for several days at 4°C, embedded in paraffin according to standard procedures and sectioned at 5 μm with a Prosan Microm HM360 microtome.2), embryos and larvae were processed for whole mount immunohistochemistry as described previously [To generate data on first-generation teeth and the first replacement tooth , the sections were blocked with 1% Bovine serum albumin and 1% sheep serum in 1xPBS for 2h at RT and next incubated overnight at 4°C with the primary antibody. We used anti-plakoglobin clone 15  or anti-β-catenin C7207 . The latter was used by different authors  to i to i2O2


Canonical Wnt signals, transduced by stabilized β-catenin, play similar roles across animals in maintaining stem cell pluripotency, regulating cell differentiation, and instructing normal embryonic development. Dysregulated Wnt/β-catenin signaling causes diseases and birth defects, and a variety of regulatory processes control this pathway to ensure its proper function and integration with other signaling systems. We previously identified GTP-binding protein 2 (Gtpbp2) as a novel regulator of BMP signaling, however further exploration revealed that Gtpbp2 can also affect Wnt signaling, which is a novel finding reported here.Xenopus embryos causes severe axial defects and reduces expression of Spemann-Mangold organizer genes. Gtpbp2 knockdown blocks responses to ectopic Wnt8 ligand, such as organizer gene induction in ectodermal tissue explants and induction of secondary axes in whole embryos. However, organizer gene induction by ectopic Nodal2 is unaffected by Gtpbp2 knockdown. Epistasis tests, conducted by activating Wnt signal transduction at sequential points in the canonical pathway, demonstrate that Gtpbp2 is required downstream of Dishevelled and Gsk3β but upstream of β-catenin, which is similar to the previously reported effects of Axin1 overexpression in Xenopus embryos. Focusing on Axin in Xenopus embryos, we find that knockdown of Gtpbp2 elevates endogenous or exogenous Axin protein levels. Furthermore, Gtpbp2 fusion proteins co-localize with Dishevelled and co-immunoprecipitate with Axin and Gsk3b.Knockdown of Gtpbp2 in Xenopus embryos. Our data suggest a model in which Gtpbp2 suppresses the accumulation of Axin protein, a rate-limiting component of the β-catenin destruction complex, such that Axin protein levels negatively correlate with Gtpbp2 levels. This model is supported by the similarity of our Gtpbp2-Wnt epistasis results and previously reported effects of Axin overexpression, the physical interactions of Gtpbp2 with Axin, and the correlation between elevated Axin protein levels and lost Wnt responsiveness upon Gtpbp2 knockdown. A wide variety of cancer-causing Wnt pathway mutations require low Axin levels, so development of Gtpbp2 inhibitors may provide a new therapeutic strategy to elevate Axin and suppress aberrant β-catenin signaling in cancer and other Wnt-related diseases.We conclude that Gtpbp2 is required for canonical Wnt/β-catenin signaling in The online version of this article (doi:10.1186/s12964-016-0138-x) contains supplementary material, which is available to authorized users. Wnt signaling regulates various cell behaviors including proliferation, differentiation, polarization and migration, and is required to generate embryonic polarity as well as maintain normal tissue homeostasis –4. InappCentral to the mechanism of canonical Wnt signaling is the stabilization of a cytoplasmic pool of β-catenin protein, which results in its subsequent nuclear translocation and induction of target genes. In the absence of Wnt ligand, cytoplasmic β-catenin is phosphorylated by glycogen synthase kinase-3 β (Gsk3b) as part of the multisubunit destruction complex that also includes the Adenomatous Polyposis Coli (Apc) and Axin proteins. Phosphorylated β-catenin is recognized by the F-box protein, β-transducin repeat-containing protein (β-TrCP), part of an E3 ubiquitin ligase that catalyzes β-catenin ubiquitination and proteasomal degradation . Upon WnXenopus embryos triggered by overexpression of Wnt ligand or a kinase-dead form of Gsk3b (dnGsk3b) [Relative to other Wnt pathway components, Axin protein is maintained at low endogenous levels and imparts a rate-limiting step in β-catenin targeting by the destruction complex , 15. Oveaxin2 expression is itself transactivated, allowing for initial promotion and subsequent negative feedback of signaling [Although the precise regulation of Axin turnover is not yet fully understood, several aspects of its degradation and regulation have recently been uncovered. In the presence of Wnt ligand, Axin protein is targeted for degradation while ignaling , 17. Morignaling –20.Xenopus embryos. Furthermore, we demonstrate that Gtpbp2 is required for transduction of Wnt signaling and negatively correlates with Axin protein levels. Our findings reveal a new member involved in the regulation of proteins that transduce canonical Wnt signals, and illuminate Gtpbp2 as a potential drug target for diseases involving disregulated Wnt signaling.In this study we have examined the requirements for GTP binding protein 2 (Gtpbp2), a largely uncharacterized protein that is distantly related to the Eef1a (Ef1α) family of large GTPases , 22, in Xenopus embryos, and it is expressed in a tissue specific manner [Xenopus embryos that could not be explained by defects in BMP signaling. The phenotypes of morpholino-injected embryos (morphants) included reduced head structures when translation-blocking morpholinos were targeted to dorsal blastomeres , chordin, and goosecoid  was also severely reduced, while the expression of another mesodermal T-box gene, zygotic vegt, was not affected in those morphants  in pluripotent Xenopus blastula ectodermal explants   and either control or Gtpbp2-specific morpholinos, and animal caps were excised and scored for the induction of nodal3.1 or siamois. Wnt8 failed to induce either gene upon Gtpbp2 knockdown  Fig. . The orgnodal3.1 , siamois[siamois ) or indi chordin , 29) actnos Fig. , and knoXenopus Gtpbp2 protein co-immunoprecipitated with tagged components of the β-catenin destruction complex, Gsk3b and Axin, in human cells  resulted in a more general cytoplasmic localization  that lacks Gsk3b target residues and thus avoids targeting by the destruction complex , and dominant-negative kinase-dead Gsk3b (dnGsk3b)  Fig.  a-c. Howcat Fig. , consistgtpbp2 mRNA . Plasmids for gtpbp2 were previously described [wnt8) [nodal2) [myc-dsh) [, Xg134 pCS2MT-Gsk3 (myc-gsk3) [, Xg137 pCS2-dnGsk3 (dngsk3) [, XE49 pCS2MT-ptβ − catenin (ptbcat) [gfp-dvl2) [, pCS2-Axin MtFu1 [myc-axin). Synthetic mRNA was generated in vitro from linearized plasmids using AmpliCap™ SP6 High Yield Message Maker Kit (Cellscript). Reporter assays were conducted using the thymidine kinase promoter-Renilla luciferase  and M50 Super 8x TOPFlash plasmids [Xenopus Axin 1066, 1:1000) [Gtpbp2 and control morpholinos were as previously described , and a mescribed , and remd [wnt8) , pCS2-xn[nodal2) , pCS2MT-myc-dsh) , Xg134 pyc-gsk3) , Xg137 p(dngsk3) , XE49 pC(ptbcat) , XE43 pC 1:1000) , or purcXenopus laevis husbandry, microinjection and animal cap dissections were done as described previously [Xenopus laevis were obtained as a non-specific wild type strain from Xenopus Express and Nasco Inc., and maintained in cycled colony.eviously . For theodc transcripts, and statistical analyses were conducted using R. Whole mount in situ RNA hybridization (WISH) was performed as described [Embryos or explants were treated as indicated and cDNA was generated as previously described . Real-tiescribed .Xenopus embryos or explants were lysed in NP40 lysis buffer and extracted with an equal volume of 1,1,2-trichlorotrifluoro-ethane (Sigma), and then separated using SDS-page. Detection of western blots was conducted with IR secondary antibodies (Alexa 680 and IRdye800) scanned on a Licor Odyssey Classic Imager. Levels of ectopic myc-Axin were normalized to levels of co-injected HA-mCherry.Hek293t cells were co-transfected using linear PEI (1 mg/ml) with plasmids encoding HA-Gtpbp2, Myc, Myc-Gsk3b, and/or Myc-Axin. Cells were incubated overnight and lysed in NP40 lysis buffer . Cell lysates were incubated with mouse anti-Myc (9E10) antibody overnight, purified using anti-IgG magnetic beads (NEB) according to the manufacturers instructions. Immunoprecipitations and whole cell lysates (1:10 of IP volumes) were blotted with mouse anti-myc (1:100) and rabbit anti-HA antibodies (1:500). For western blotting Xenopus embryos were injected into the animal pole with mcherry-gtpbp2 mRNA (500 pg) into both cells, with one cell co-injected with gfp-dvl2 (500 pg), followed by imaging at late blastula (stage 9). Embryos were first examined live, then gently fixed in 1 % paraformaldehyde in PBS for 15 min, washed 3x in PBS, and costained with 2 mg/ml 4,6-diamino-2-phenylindole (DAPI). Embryos were then imaged on using a 10x objective on a Zeiss fluorescence microscope (Motorized Axio Imager Z1) with an ApoTome analysis unit.Two-cell stage Xenopus embryos with 100 pg Super TopFlash together with 50 pg pRL-TK reporter plasmids, and 10 pg of xwnt8 mRNA, and 30 ng of gtpbp2 M1, M2, or control morpholino as indicated. Animal caps were cut at mid-blastula stage 8 and cultured until mid-gastrula stage 11, at which point cell extracts were prepared and analyzed using Dual-Glo Luciferase Assay Reporter Assays (Promega).Reporter assays were conducted by injecting CK1, Casein-kinase 1; Dsh, Dishevelled; frzb, frizzled-b; gsc, goosecoid; Gsk3b, glycogen synthase kinase-3 β; Gtpbp2, GTP-binding protein 2; β-TrCP, beta-transducin repeat-containing protein; odc, ornithine decarboxylase


Objective. To investigate the effects of Toll-like receptors in stem cell osteogenesis. Methods. Bone marrow mesenchymal stem cells (BMSCs) were divided into the blank group, the TLR-3 activated group, and the TLR-4 activated group. After 10 days' osteogenic-promoting culture, expression of type I collagen and osteocalcin was determined by Western blot. Osteoblasts (OBs) were also divided into three groups mentioned above. Alkaline phosphatase (ALP) and alizarin red staining were performed after 10 days' ossification-inducing culture. The expression of β-catenin was investigated by Western blot. Results. Both the TLR-3 and TLR-4 activated groups had increased expression of type I collagen and osteocalcin; the effect of TLR-4 was stronger. The intensity of alizarin red and ALP staining was strongest in the TLR-3 activated group and weakest in the TLR-4 activated group. Activation of TLR-4 decreased the expression of β-catenin, whilst activation of TLR-3 did not affect the expression of β-catenin. Discussion. This study suggested that both TLR-3 and -4 promoted differentiation of BMSCs to OBs. TLR-3 had an inducing effect on the ossification of OBs to osteocytes, whilst the effect of TLR-4 was the opposite because of its inhibitory effect on the Wnt signaling pathway. It is widely accepted that stem cells, as seed cells, in conjunction with bone tissue engineering scaffolding, are beneficial to bone healing due to their ability to differentiate to osteoblasts (OBs). Following ossification, OBs transform to osteocytes, which then form primary bone tissue. This is not, however, the final stage of bone formation. The physiological function of bone tissue does not develop until reconstruction of the bone tissue is completed by both OBs and osteoclasts (OCs). The whole process of the differentiation of stem cells to mature bone tissue is defined as osteogenesis of stem cells, which is regulated by various cellular factors, such as bone morphogenetic protein-2 (BMP-2) and fibroblast growth factor (FGF) , 2. Tollβ and induces chronic inflammation. Consequently, it is reasonable to propose that the effects of TLR-3 and TLR-4 are most likely also different in the osteogenesis of stem cells.Toll-like receptors (TLRs) are important types of pattern-recognition receptors (PRRs) and are type I transmembrane glycoproteins. They are widely expressed in most human and other animal cells and tissues and not only initiate the primary immune response but also play an important role in the general immune system and the regulation of other bodily functions including proliferation of stem cells . In addiβ-catenin is the downstream signal molecule of Wnt pathway. Cytoplasmic β-catenin is accumulated as a result of the activation of Wnt signaling pathway and then transferred into cell nucleus. Nuclear β-catenin is able to upregulate the expression of RUX-2, which is crucial in the osteogenesis of stem cells, by integrating with T cell factor/lymphoid enhancer factor (TCF/LEF).The Wnt signaling pathway may be an important mechanism in the processes of osteogenesis of stem cells by regulating the OB function in gene transcription level , 12. ActThis study aims to determine the differing effects of TLR-3 and TLR-4 in the differentiation of BMSCs to OBs and the ossification of OBs to osteocytes. Another purpose is to investigate the effect of Wnt signaling pathway in the process of osteogenesis of stem cells.n = 5) as described  , 10 ng/mL. Cells in the TLR-4 activated group were treated with lipopolysaccharide (LPS) , 10 ng/mL, the activator of TLR-4. Cells in the blank group were not treated. All treatment groups were cultured in osteoblastogenic-promoting differentiating medium . On day 10 of cell culture, the protein expression of type I collagen and osteocalcin of cells was detected by Western blot as previously described [Following isolation from rats , the TLR-4 activated group (n = 12 wells), and the bank group (n = 12 wells) and then treated with the same method mentioned above. Then, all cells in these groups were induced with ossification-inducing medium  for another 10 days. After that, ALP staining and alizarin red staining were then performed to cells in these groups.In order to demonstrate the effects of different TLR subtypes in ossification of OBs to osteocytes, alkaline phosphatase (ALP) and alizarin red staining were performed as following protocols. In the first 10 days, BMSCs isolated from rats were induced to OBs with osteoblastogenic-promoting differentiating medium mentioned above. OBs induced from BMSCs were divided into TLR-3 activated group  and the TLR-4 activated group  compared to the blank group. Moreover, cells in the TLR-4 activated group expressed higher levels of type I collagen than cells in the TLR-3 activated group . Similarly, the expression of osteocalcin was higher in the TLR-3 activated group  and the TLR-4 activated group  than that in the blank group. Between TLR-4 activated group and TLR-3 activated group, expression of osteocalcin was higher in the former one . Data of quantitative analysis of the western blot images was in Results of flow cytometry showed that the surface markers of BMSCs were positive . The celt = 4.017, P = 0.015 < 0.05) and even weaker in TLR-3 activated group . The result of ALP staining , whilst the intensity of ALP in the blank group was stronger than that in the TLR-4 activated group . Data of quantitative analysis was in The intensity of alizarin red staining  in the bstaining  by immunβ-catenin was mentioned  and the blank group . However, there was no difference of the expression of β-catenin between TLR-3 activated group and the blank group . Data of quantitative analysis was in When expression of total and nuclear entioned , it was β, IL-6, and IL-8 are secreted [α, a typical proinflammatory factor, augments the recruitment and differentiation of stem cells and as a consequence promotes bone healing [α knockout mice compared to the wild types [This study clearly demonstrated that both TLR-3 and TLR-4 affected the osteogenesis of BMSCs. However, these two TLR subtypes played different roles in different stages of osteogenesis of BMSCs. The differentiation of BMSCs to OBs was promoted by the activation of both TLR-3 and TLR-4. Type I collagen and osteocalcin were detected to illuminate the effect of TLR-3 and TLR-4. Type I collagen, the primary composition of bone matrix and osteocalcin, is secreted by OBs. Secretion of type I collagen climaxes between day 10 and day 14, following the formation of OBs in preparation for the ossification to osteocytes . Osteocasecreted . Indeed ld types , 22. TheDuring the ossification stage, activation of TLR-3 and TLR-4 showed opposite effects. ALP and alizarin red staining were performed to illuminate the effect of ossification. Mature OBs release a large number of matrix vesicles, each of which contains a large amount of calcium and is helpful in mineralization , 24. Aliβ-catenin in the cytoplasm, the downstream signaling molecule of the Wnt signaling pathway, is vital for complete transduction of the Wnt signaling pathway [β-catenin results in abnormal bone metabolism and dysfunction of bone [β-catenin in OBs was investigated in the TLR-4 activated group and also in the TLR-3 activated group. The results in current study support the inhibitory effect of TLR-4 in the Wnt signaling pathway, thereby possibly explaining the inhibitory effect of TLR-4 in the ossification of OBs to osteocytes, observed in the present study. However, activation of TLR-3 did not affect the expression of β-catenin and the Wnt signaling pathway. Therefore, TLR-3 shows differential effect in ossification of OBs compared to TLR-4.Given that activation of both TLR-3 and TLR-4 had the same effect on the promotion of the differentiation of stem cells to OBs, it was interesting to observe that TLR-3 and TLR-4 had differing effects on the ossification of OBs to osteocytes. It became clear that it was necessary to investigate the downstream mechanisms of TLR-3 and TLR-4, as alterations in these downstream mechanisms may provide insight into the differing effects of TLR-3 and TLR-4 in stem cell osteogenesis. The Wnt signaling pathway promotes bone formation and inhibits bone absorption and is one of the most important signaling pathways of bone metabolism . Existen pathway . Lack of of bone . It has  of bone . Consequ


Influence of WNT proteins on the formation of blood vessel, taking place both under healthy and pathological conditions, is also considered. Participation of WNT proteins, β-catenin, and inhibitors and inducers of WNT signaling in the process of endometrial angiogenesis is largely unknown. Thus, confirmation of their local and systemic participation in the process of endometrial angiogenesis may in the long term help to establish new diagnostic and therapeutic approaches in conditions associated with the pathology of the female reproductive system.WNT proteins are involved in embryonic development, sex determination, stem cell recruitment, angiogenesis, and cancer. They take part in morphological changes in the endometrium during development, regulate processes of endometrial proliferation and differentiation. This review presents current knowledge about implication of WNT proteins and  WNT are proteins involved in physiological and pathological processes such as embryonic development, sex determination, malignant transformation, endothelial cell differentiation, and angiogenesis , 2. Angiβ-catenin in physiological endometrial functions as well as their involvement in uterine carcinogenesis. We also present current knowledge about influence of WNT proteins on the formation of blood vessel, both under healthy and pathological conditions. The proposed hypothesis about participation of WNT proteins in the regulation of the formation of microvessels might provide a conceptual framework for the design of future experiments.In this review, we present data about implication of WNT proteins and Wingless gene is responsible for segmentation during embryogenesis and legs formation during transformation of Drosophila melanogaster. Homologous gene int-1, detected in mammals, becomes activated upon cell integration of the MMTV virus (mouse mammary tumor virus), which causes mammary tumors in mice. Comparison of the amino acid sequences of the proteins encoded by these two genes showed their high homology, while combination of the abbreviations of gene names gives the name for the whole gene family, WNT (WNT = Wg + INT). To date, the largest number (19) of WNT genes was found in mice and humans. Their existence was also confirmed in the nematode Caenorhabditis elegans, zebrafish (Danio rerio), amphibians of the Xenopus genus, and chicken . Based on their capability to induce the malignant transformation in cell lines derived from murine mammary gland epithelial cells (C57MG), WNT genes were divided into two groups. The first group includes genes encoding cysteine-rich, secretory glycoproteins with oncogenic characteristics: WNT1, WNT3, WNT3A, WNT7, WNT8, and WNT8B, while the other encodes proteins lacking the properties to induce malignant transformation: WNT2, WNT4, WNT5A, WNT5B, WNT6, WNT7B, and WNT11 [β-catenin signaling pathway is activated by WNT proteins joined with the complex of Frizzled (FZD)/low-density-lipoprotein receptor-related protein (LRP) on the cell surface. Activated cytoplasmic protein called Dishevelled (DSH) inhibits the activity of a protein APC/GSK3β complex (Axin/Adenomatous Polyposis Coli/Glycogen Synthase Kinase 3β) responsible for the degradation of β-catenin. After stimulation with WNT, cytoplasmic β-catenin is translocated to the nucleus, where it activates transcription factors: T cell factor (TCF) and lymphoid enhancer factor (LEF). These transcription factors change the expression level of target genes encoding proteins implicated in cell proliferation and survival , cellular migration (CD44), cell adhesion (CDH1), digestion of extracellular matrix (MMP7), and many others [β-catenin binds to α-catenin and cytoplasmic domain of E-cadherin, one of the methods of inhibiting cell signal induced by WNT is to increase the level of E-cadherin. In mammals, the activation of the WNT/β-catenin pathway leads to the enhanced recruitment of stem cells and amplification of their pluripotency.WNT proteins activate the canonical  and noncanonical  cell signaling pathways. Canonical signaling pathway known also as WNT/β-catenin-independent pathway can activate FZD receptor family. The initiation of signal cascades results in the release of calcium ions, activation of protein kinase C (PKC) and calcium-calmodulin dependent protein kinase II (CAMKII). It has been established that the WNT/Ca2+ signal transduction pathway antagonizes the action of WNT/β-catenin signaling [The other group of WNT which act through ignaling  and may ignaling . In the WNT-mediated signaling pathways can be modulated through secreted Frizzled-related proteins (sFRP) and Dickkopf (DKK) proteins. sFRP proteins bind directly to WNT proteins, while DKK proteins block LRP5/6 coreceptors. In both cases, WNT ability for signal transduction is blocked . WNT4, WNT5A, and WNT7A genes as demonstrated in mouse [ WNT4, WNT5A, and WNT7A genes expression was presented also in developed uterus in humans [Developmental changes of the endometrium are mainly associated with the expression ofin mouse –8 and piin mouse , 10. Hown humans , 12, shen humans , horse [n humans , and pign humans , 16.Wnt4 gene is expressed in the primordial gonads of mouse embryos [ Wnt4 gene is expressed in stromal cells of the forming endometrium [ Wnt4 gene, sex reversion, partial atrophy of the Müllerian ducts, masculinization, and morphological and functional changes of the gonads were described [ Wnt4 gene in mouse causes ectopic expression of Leydig cells markers  [ embryos  and Wnt4 embryos . During ometrium . In miceescribed . Moreoveogenase) . Increasogenase) . WNT5A gene has been documented by finding that Wnt5a knockout mice have no reproductive organs and live no longer than 24 hours [ Wnt5a gene was observed in mice gonadal ridges [ in situ hybridization [The key role of the24 hours . Constitl ridges . Wnt5a pdization , 22, 23. Wnt7a genes expression have deformed wall of the uterus and undeveloped ovaries [Female mice lacking ovaries . Moreove ovaries . WNT4 gene expression is higher in endometrial stroma in comparison to its expression in epithelial cells [2) into ovariectomized mice upregulated, while administration of progesterone (P4) had no effect on, Wnt4 gene expression in stromal cells of endometrium [ Wnt4 was presented by Wnt5a during the luteal phase of the estrous cycle in mice [ Wnt5a  was observed only in stromal cells just before and soon after estrus occurrence [4 and antagonist of P4 receptor at the same time (RU 486) increased endometrial WNT5A mRNA level at day 12 of pregnancy [In physiological condition , WNT4 geal cells . Injectiometrium . Similar in mice  but exprcurrence . Howevercurrence . Treatmeregnancy . WNT7A mRNA levels in the female endometrial tissue were higher in the proliferative phase in comparison to secretory phase of the menstrual cycle. However, other authors did not find correlation of the WNT7A gene expression with phases of the menstrual cycle with the use of real-time PCR, digoxigenin-labeled cRNA probes, and in situ hybridization technique [ WNT7A expression is stimulated by estradiol [ WNT7A gene expression was presented in in vitro culture of luminal epithelial cells of human endometrium [ WNT7A gene expression may be essential for the transdifferentiation of endometrium during its transition to the secretory phase [ Wnt7a gene expression was completely suppressed in the surface epithelium and was undetectable in glandular epithelium and endometrial stroma after seven days of progesterone treatment [Fan and coworkers  showed technique , 27, 28.stradiol  which costradiol . The infometrium  or neonaometrium . Presencometrium , 26–28 bometrium . Moreoveβ-catenin, the mediator of Wnt/β-catenin signaling, was first isolated as an intracellular protein constituting the binding domain of E-cadherin with cell's cytoskeleton [β-catenin is essential for normal functioning of the uterus and seems to be responsible for establishing of the endometrial homeostasis [β-catenin was observed in intercellular borders of luminal and glandular epithelial cells as well as in stroma and endothelial cells [β-catenin positive staining of physiological endometrium are presented in skeleton . The avaeostasis . In humaal cells . Exampleβ-catenin (CTNNB1) mRNA level during secretory phase in human endometrium. It correlated with steroid hormone profile because progesterone but not estradiol increased CTNNB1 mRNA level in human endometrial stromal cells cultured in vitro [β-catenin increased. β-catenin was allocated from the nucleus to the cytoplasm and cell membrane during the secretory phase [ CTNNB1 gene expression in human endometrium between proliferative and secretory phases.Fujimoto and coworkers  revealedin vitro . During ry phase . Howeverry phase  showed nβ-catenin signaling, induced estradiol-mediated proliferation and hyperplasia of endometrial cells in mice [β-catenin signaling pathway increased endothelin 1 mRNA level which is target gene of β-catenin action as well as participant of endothelial cells differentiation [β-catenin signaling regulates processes of endometrial proliferation and differentiation. Specific pattern of WNT genes expression and pattern of hormonal regulations are summarized in Moreover, usage of LiCl, potential inhibitor of WNT/ in mice  and huma in mice . Activatntiation . Thus, i2 or increase P4 levels  tend to be protective [ PTEN, PIK3CA, K-ras, and CTNNBI (β-catenin) genes summarized in Endometrial cell carcinomas (ECCs) are the most common malignancy of the female genital tract in the Western world and the fourth most common one after breast, lung, and colorectal cancer in women. A constant increase of endometrial cancer has been observed in the recent years . ECCs ocotective . Type I otective . Endometotective . ESC or otective . The aveotective . Althougotective . Each su WNT4 mRNA level was lower while WNT2, WNT3, and WNT5A mRNA levels were higher in endometrial carcinoma in comparison to normal endometrium [ WNT2, WNT3, WNT4, and WNT5A genes expression was higher in normal human primary epithelial and stromal endometrial cultures compared to endometrial carcinoma cell lines, what suggest their participation in endometrial neoplasia [The expression of genes encoding WNT proteins and proteins involved in WNT signaling pathways was found to be changed also in endometrial cancer  Figure . The WNTometrium . Also WNeoplasia . WNT7A gene expression. In 63% patients of one series of endometrial carcinoma, WNT7A gene expression was absent or reduced and negatively correlated with FIGO stage, grade, lymph node metastasis, depth of myometrial invasion, lymph vascular space involvement, and peritoneal cytology [ WNT7A overexpression was found in most cases of endometrial cancer in comparison with normal endometrium and benign endometrial lesion [ WNT7A gene correlated positively with overall survival and disease-free survival of endometrial cancer [Most of the studies concentrated oncytology . In largl lesion . Howeverl cancer . WNT7A expression mediated by estradiol [ WNT7A and WNT7B genes expression was increased in endometrial carcinoma cell lines and normal endometrial tissues as compared with primary cultures of human endometrial cells [In the Ishikawa cell line model of endometrial adenocarcinoma, estrogen receptors were probably involved  in the dstradiol . Moreoveal cells . WNT10B was higher than in later stages. WNT10B proteins were mainly detected in patients with the cancer of endometrioid type, who had high graded and advanced-staged tumor without lymph node metastasis [ in vitro investigations. WNT10A gene expression was decreased in endometrial HEC1B and AN3CA cell lines while WNT10B was increased in Ishikawa cell lines [WNT10A and WNT10B proteins have been implicated in estrogen-related carcinogenesis of endometrial cancer. The amount of WNT10B protein was higher in endometrial cancer than in hyperplastic and normal endometrium as determined by Western blot technique . In earltastasis . This clll lines .β-catenin gene (CTNNB1) were found in many endometrial cancers [ CTNNB1 [β-catenin accumulation. In this case, blockage of the process of β-catenin degradation results from the lack of its phosphorylation [β-catenin was observed in endothelial cells of neovessels. Beta-catenin accumulation was observed in the nucleus of malignant changed endometrial cells [β-catenin was previously not shown in tumor vascular endothelial cells  [Vascular system is a network of arteries, capillaries, and veins for transport of gases and macromolecules. Vasculogenesis is a process ofoblasts) . Formatioblasts) . Angiogeoblasts) . Blood voblasts) . Capillaoblasts) , when enoblasts) . It is hoblasts)  and inteoblasts) . Endotheoblasts)  and/or (oblasts) . Growth oblasts) . in vitro conditions [ Wnt1-overexpressing cells accelerated differentiation of mesoderm germ layer into hematoendothelial cells via activation of canonical WNT signaling [β-catenin signaling, bovine aortic endothelial cells proliferation, and capillary stability under in vitro conditions [Participation of WNT proteins in the process of differentiation of cells of hematopoietic and endothelial cell lineage as well as vasculogenesis and angiogenesis is apparent . It has nditions . Moreoveignaling . Presencnditions . On the nditions . WNT2 gene had no impact on WNT/β-catenin signaling activation, endothelial cells proliferation [The role of the WNT2 protein in angiogenesis is less clear. Increased expression of WNT2 protein and FZD-5 receptor caused defects in the vasculature of murine placenta and changed blood flow in the mouse yolk sac , 78 as aferation , 75, or feration .WNT3A was shown to be direct, VEGF-independent, inducer of endothelial cell proliferation , 80 and β-catenin signaling [WNT5A is required for endothelial differentiation of embryonic stem cells and transformation of mouse embryonic stem cells into immature endothelial progenitor cells, taking part in healing process of damaged endothelium . Acting ignaling . Moreoveignaling . Wnt5a aignaling . Wnt7a but not VEGF promotes migration and stimulates expression of blood-brain barrier specific transporters of glucose (GLUT-1) in mouse brain endothelial cell line [ Wnt7b gene deficient mice, loss of Wnt7b gene resulted in defective smooth muscle component of the major pulmonary vessel differentiation, degradation of vessel's wall, and perinatal hemorrhage [WNT7 proteins were shown to promote normal angiogenesis in ventral regions of the CNS in mouse . Specifiell line . Moreovemorrhage . CTNNB1 gene in endothelial cells affected the formation of vasculature of head of mouse embryos, large vitelline and umbilical vessels, and the vasculature of the placenta [β-catenin absence, significant reduction in cell junctions organization and hemorrhage was observed [Inhibition of the expression ofplacenta . As a reobserved . in vivo conditions [Data provided in the proceeding chapter clearly indicate the participation of WNT proteins in the recruitment, proliferation, and migration of endothelial cells in healthy subjects and cancer patients. In the endometrium, development of the vascular network occurs simultaneously with epithelial and stromal cells expansion, expression, and influence of angiogenic factors that circulate in blood and fluctuate in menstrual cycle phase-dependent manner . Regulatnditions , this efnditions . Therefo WNT7A gene expression was upregulated during proliferative phase of the estrous cycle and downregulated in the secretory phase [β-catenin can function in the endometrium either directly on endothelial cells or indirectly through its action on endometrial cells where it promotes the expression of VEGF [We can hypothesize  that it ry phase . β-caten of VEGF  or endot of VEGF . Howeverβ-catenin, and inhibitors and inducers of WNT signaling in the process of endometrial angiogenesis is largely unknown. The main task which should be now undertaken should concentrate on defining which WNT, receptors, inhibitors, and signaling pathway are activated in the endothelial cells of the blood vessel of the endometrium. The spatiotemporal pattern of expression of elements of WNT signaling system should also be established. Differences in normal and pathological state should be also considered. Conducting this research will help to determine the angiogenic potential of the WNT family of proteins, will allow for a better understanding of the mechanism of formation of vessels in the endometrium, and will help to determine whether WNT proteins can be potential target for antiangiogenic therapy directed mainly against transcription factors as, for example, β-catenin. We are aware that our hypothesis does not present the experimentally verified information. However, we believe that our suggestion will contribute to the discussion and to some extent the development of research on the role of WNT proteins in the formation of blood vessels in the dynamically changing uterus.The participation of WNT proteins,