Prmt5 (Protein arginine methyltransferase 5) encodes a protein arginine methyltransferase which has been demonstrated to play important roles in germ cell development during embryonic stages. In the present study, we found that Prmt5 was also abundantly expressed in male germ cells after birth. Inactivation of this gene by crossing with Stra8-Cre transgenic mice resulted in germ cell loss during spermatogenesis. Further study revealed that the germ cell development was grossly normal before P10. However, most of the germ cells in Prmt5Δ/f; Stra8-Cre mice were blocked at meiotic stage. The expression of meiosis associated genes was reduced in Prmt5Δ/f; Stra8-Cre testes compared to control testes at P10. γH2AX was detected in sex body of control germ cells at P12, whereas multiple foci were observed in Prmt5-deficient germ cells. Further study revealed that H4R3me2s was virtually absent in germ cells after Prmt5 inactivation. The results of this study indicate that Prmt5 also plays important roles in germ cell development during spermatogenesis.During germ cell development, epigenetic modifications undergo extensive remodeling. Abnormal epigenetic modifications usually result in germ cell loss and reproductive defect.  Epigenetic modification is one of the important mechanisms regulating the gene expression, which is involved in a number of biological processes. Epigenetic processes include DNA methylation, histone modifications, and chromatin remodeling235Prmt5 belongs to the PRMT family and is responsible for the formation of symmetric dimethylarginine (SMDA) in arginine-rich protein motifsPrmt5 is essential for maintaining the pluripotency of mouse embryonic stem cells (ES). Deletion of Prmt5 results in the down-regulation of pluripotency transcription factors and causes embryonic lethality before implantationPrmt5 is not required to maintain pluripotency in human ES cellsPrmt5 is also expressed in primordial germ cells (PGCs) and directs histone arginine methylation in mouse germ cellsPrmt5 in PGCs using Blimp1-Cre resulted in germ cells death before E12.517Prmt5 plays essential roles in PGCs survival.Post-translational histone modifications include methylation, acetylation, phosphorylation, ubiquitylation and sumoylation. Methylation is one of the most prevalent histone modifications monitored by histone methyltransferases11Prmt5 was also abundantly expressed in germ cells of adult testis, suggesting that histone methylation probably also plays roles in spermatogenesis. To investigate the functions of Prmt5 in later stage of germ cell development, it was specifically inactivated in male germ cells by crossing Prmt5flox mice with Stra8-Cre transgenic mice. We found that the germ cells were gradually lost after day 12 and very few germ cells were survived in adult testes. The results of this study indicate that Prmt5 is required for male germ cell survival during spermatogenesis.In this study, we found that Prmt5 is abundantly expressed in the germ cells during embryonic stages, and inactivation of Prmt5 in PGCs results in germ cell loss in both male and female gonads1617Prmt5 in testes after birth was examined by immunofluorescence. As shown in Prmt5 protein was localized in the nucleus of Dazl-posotive germ cells at P1 . It was continually expressed in germ cells at P7  and P10 , whereas the signal was mainly detected in cytoplasm at these stages. Interestingly, Prmt5 protein was translocated from cytoplasm to the nucleus of germ cells at P12 . In adult testes, Prmt5 protein was abundantly expressed in the nucleus of spermatocytes . The dynamics of nuclear-cytoplasm translocation of PRMT5 in germ cells postnatally was illustrated with the schematic diagram , whereas no PRMT5 was detected in the germ cells from Prmt5Δ/f; Stra8-Cre mice  and only Leydig cells  were labeled with anti-PRMT5 antibody. The results of immunofluorescence also showed that Prmt5 protein was absent in the germ cells of Prmt5Δ/f; Stra8-Cre testes at P7  positive, whereas multiple foci were noted  were significantly reduced in testes from Prmt5Δ/f; Stra8-Cre males. By contrast, the expression of germ cell specific genes, Dazl and Vasa, were not decreased in Prmt5-deficient germ cells. Interestingly, the mRNA level of Plzf was significantly increased in Prmt5Δ/f; Stra8-Cre testes. It has been reported that the expression of transposable elements in germ cells was repressed by PRMT5 during embryonic stagesIAP-LTR and L1-ORF2 was not increased in Prmt5Δ/f; Stra8-Cre testes  at P10. Robust signal of H4R3me2s was detected in germ cells of control testes , whereas no signal was observed in the germ cells of Prmt5Δ/f; Stra8-Cre testes  at this stage.As a protein arginine methyltransferase, PRMT5 is responsible for the symmetrical dimethylation of H4R3 in PGCs, and H4R3me2s is virtually absent in PGCs after Dnmt3L in mouse model up-regulates the transcription of LINE and IAP retrotransposons in spermatogonia and spermatocytes. The mutant mice display meiotic failure with widespread non-homologous chromosome synapsis and progressive loss of germ cells by the mid-pachytene stage2122Suv39h1 and Suv39h2 causes defects in meiosis in male germ cellsPrmd9 (PR domain-containing 9) is an H3K4 trimethyltransferase that is specifically expressed in early meiotic germ cells both in the testis and ovary. Analysis of Prdm9-null mice indicates that it is involved in germ cell meiosis by regulating synapsis formation and recombination of homologous chromosomes during meiotic prophaseEhmt2 (euchromatic histone-lysine N-methyltransferase 2) in germ cells causes the arrest of meiosis at the early pachytene stage in both testis and ovary, indicating that H3K9 mono- and dimethylation is also essential for early meiotic progressionThe functions of epigenetic modification in germ cell development have been reported previously. DNA methylation plays critical roles in retrotransposon silencing and germ cell development, deletion of Prmt5 encodes a protein arginine methyltransferase which catalyzes the symmetric dimethylarginine (SMDA) in both glycine and arginine-rich protein motifs. Previous studies have demonstrated that Prmt5 is expressed in PGCs during embryonic stages. Specifically deletion of this gene in PGCs by crossing with Blimp1-Cre causes germ cell loss before sex determination. Kim et al. found that Prmt5 functions in PGCs development by repressing transposable elements and that inactivation of Prmt5 results in the up-regulation of IAP and LINE1Prmt5 is required for germ cell survival by enabling the correct splicing of primary RNA transcripts that function in the DNA damage response pathwayPrmt5 was continually expressed in the germ cells of adult testis. Deletion of this gene after birth caused massive germ cell loss, and only a few germ cells were noted in adult testes. These results indicate that Prmt5 is also required for postnatal germ cell survival in testis which is consistent with the functions during embryonic stages17Stra8-Cre is reported to be activated in germ cells of testis at approximately 3 days after birthPrmt5 were virtually absent in germ cells at P7. However, the development of germ cells was grossly normal in Prmt5Δ/f; Stra8-Cre testes at P10, Stra8-positive and Scp3-positive germ cells were observed in both control and Prmt5-deficient testes, indicating that the initiation of meiosis is not affected in Prmt5-deficient germ cells. The defect of germ cell development was first noted at P12 in Prmt5Δ/f; Stra8-Cre testes. In control mice, the number of germ cells was significantly increased at P12, and Stra8-positive and Scp3-positive germ cells were noted in the lumen of most seminiferous tubules. By contrast, most of the germ cells were still localized at the peripheral region of seminiferous tubules in Prmt5-deficient testes at this stage. A small number of Stra8-positive and Scp3-positive germ cells were detected in Prmt5-deficient testes. However, multiple foci of γH2AX were noted in Prmt5-deficient germ cells. In control germ cell, γH2AX was only detected in sex bodies. These results suggest that the DNA double strain breaks during meiosis are not properly repaired in Prmt5-deficient germ cells. Immunofluorescence results also showed that Prmt5 protein was mainly expressed in spermatocytes, not in germ cells at other developmental stages. Based on these results, we speculate that Prmt5 is probably not involved in the development of germ cells before meiosis, but it is required for the process of meiosis. The death of Prmt5-deficient germ cells is probably due to the defects of meiosis. However, we could not exclude the possibility that the blockage of meiosis is a deleterious effect after Prmt5 inactivation.In this study, we found that Prmt5 inactivation, whereas H3R2me2s was not changed in Prmt5-deficient germ cells, suggesting that PRMT5 is responsible for H4R3me2s in germ cell during later developmental stages. The functions of Prmt5 in germ cell development are probably mediated by H4R3me2s which are consistent with other histone modifiers2425Prmt5 is involved in germ cell development mediated by histone methylation or indirectly catalyzed the methylation of other proteins is unclear which need further investigation.Previous studies found that both H4R3me2s and H3R2me2s are catalyzed by PRMT516Prmt5 is required for germ survival during embryonic stage by repressing the transcription of retrotransposonIAP and LINE1 was not increased in Prmt5-deficient germ cells, suggesting that Prmt5 is required for germ cell survival at different developmental stages but the underlying mechanisms are different.It has been reported that Prmt5 was also expressed in male germ cells after birth which also plays important roles in spermatogenesis. Deletion of this gene results in aberrant spermatogenesis and most of germ cells die in adult testes. The results suggest that Prmt5 is probably required for germ cell meiosis and the death of Prmt5-deficient germ cells is likely due to the defects of meiosis. However, we could not exclude that the defect of meiosis is a deleterious effects after Prmt5 inactivation.Taken together, in the present study, we found that Prmt5flox mice strain was obtained from the European Conditional Mouse Mutagenesis Program Wtsi)Prmt5Δ/+mice were obtained by crossing with ZP3-Cre mice. Prmt5Δ/f; Stra8-Cre mice were obtained by crossing Prmt5Δ/+; Stra8-Cre males with Prmt5flox/flox females. Genotyping was performed by PCR as described previously using DNA isolated from tail tips28All animal works were carried out in accordance with the protocols approved by the Institutional Animal Care and Use Committee at the Institute of Zoology, Chinese Academy of Sciences (CAS). All the mice were maintained in a C57BL/6;129/SvEv mixed background. Prmt5Δ/f; Stra8-Cre and control mice immediately after euthanasia, fixed in 4% paraformaldehyde for up to 24 hrs, stored in 70% ethanol, and embedded in paraffin. Five-micrometer-thick sections were cut and mounted on glass slides. After deparaffinization, sections were processed for immunohistochemistry and immunofluorescence analysis.Testes were dissected from IHC analysis of tissues from at least three mice for each genotype was performed using a Vectastain ABC (avidin–biotin–peroxidase) kit  as recommended and using antibodies to MVH , PRMT5 . The IHC procedure was performed as described previouslyAfter rehydration and antigen retrieval, the 5 μm sections were incubated with 5% donkey serum in 0.3% triton X-100 for 1 hr. Then the sections were incubated with the primary antibodies for 1.5 hrs, and the corresponding TRITC-conjugated donkey anti-rabbit IgG  and FITC-conjugated donkey anti-mouse IgG  for 1.5 hrs at room temperature. The following dilutions of primary antibodies were used: PRMT5 , DAZL , STRA8 , SCP3 , γH2AX , DMC1 , H3R2me2s , H4R3me2s . After three times wash in PBS, the nuclei was stained with DAPI. The sections were examined with confocal laser scanning microscope .Gapdh was used as an endogenous control. All gene expression was quantified relative to Gapdh expression. The relative concentration of the candidate gene expression was calculated using the formula 2−ΔΔCT as described in the SYBR Green user manual. Primers used for real-time PCR were listed in The testes were dissected at P10, and stored at −80 °C after snap frozen with liquid nitrogen. Total RNA was extracted using a Qiagen RNeasy kit in accordance with the manufacturer’s instructions. Two micrograms of total RNA was used to synthesize first-strand cDNA. To quantify gene expression, real-time SYBR Green assay was performed with the isolated RNA. In Situ Cell Death Detection Kit, Fluorescein , as recommended. The images were captured by Nikon DS-Ri1 CCD camera.TUNEL assays were conducted with the Prmt5-deficient gonads at each time point were used for immunostaining. The quantitative results are presented as mean ± SEM. The data were evaluated for statistical differences using student T-test. P-value < 0.05 was considered as significant.Experiments were repeated at least three times. Three to five control or How to cite this article: Wang, Y. et al. Prmt5 is required for germ cell survival during spermatogenesis in mice. Sci. Rep.5, 11031; doi: 10.1038/srep11031 (2015).


Recent progresses in stem cell field, in particular in the generation of induced Pluripotent Stem Cells (iPSCs) has made possible for the first time obtaining substantial amounts of human cells to recapitulate in vitro some of the key pathogenetic processes underlying MNDs. In the present review, recently published studies involving the use of iPSCs to unravel aspects of ALS and SMA pathogenesis are discussed with an overview of their implications in the process of finding a cure for these still orphan disorders.Motor neuron diseases (MNDs) are neuromuscular disorders affecting rather exclusively upper motor neurons (UMNs) and/or lower motor neurons (LMNs). The clinical phenotype is characterized by muscular weakness and atrophy leading to paralysis and almost invariably death due to respiratory failure. Adult MNDs include sporadic and familial amyotrophic lateral sclerosis (sALS-fALS), while the most common infantile MND is represented by spinal muscular atrophy (SMA). No effective treatment is ccurrently available for MNDs, as for the vast majority of neurodegenerative disorders, and cures are limited to supportive care and symptom relief. The lack of a deep understanding of MND pathogenesis accounts for the difficulties in finding a cure, together with the scarcity of reliable  Concerning their epidemiology, the prevalence of MNDs accounts for about 5–7 cases in every 100,000 people, with an annual incidence of approximately two new cases per 100,000 people [ type 1) .The biological substrate of MNDs is responsible for the extremely disabling clinical phenotype, characterized by progressive weakness with muscle wasting, eventually leading to paralysis and death, mostly secondary to respiratory insufficiency . No effeMNDs are usually differentiated relating to the subset of motor neurons that are mainly involved in the disease course. Upper motor neurons are especially affected in primary lateral sclerosis (PLS) and hereditary spastic paraplegias (HSP). On the other hand, SMA, progressive muscular atrophy (PMA), SBMA and hereditary motor neuropathies (HMNs) involve mainly lower motor neurons.C9ORF72 are found in 40% of cases, followed by mutations of superoxide dismutase-1 (SOD1) (20% of cases), TARDBP (4% of cases), FUS (4% of cases) and other genes [ALS is the most common adult form of MND, characterized by the simultaneous degeneration of UMNs and LMNs. ALS can be divided into a sporadic form (sALS), representing 90%–95% of all cases, and a familial form (fALS), accounting for the remaining 5%–10% of cases . The spoThe group of SMAs comprises a series of LMN disorders characterized by extreme heterogeneity in both clinical presentation and genetic condition.SMN) gene mapped in 5q11.2–q13.3 [SMN are localized on chromosome 5q13: the telomeric SMN1 gene and its inverted centromeric homologue SMN2. SMN2 only differs from SMN1 for five base pair changes, of which a C to T substitution at +6 of exon 7 (c.840C > T) is the only nucleotide change in the coding region [pre-mRNA of SMN2 that excludes exon 7 from the majority of SMN2 transcripts. The result is the production of 10%–50% of the full-length functional protein and 50%–90% of a truncated, non-functional and unstable transcript (SMNΔ7) [SMN2, which correlates to the severity of the disease. The exact functions of the SMN protein, as much as the reasons accounting for the disruption of MNs in SMA, are yet to be fully disclosed.The most prevalent forms of SMAs go under the name of “Proximal Spinal Muscular Atrophy”, often referred to simply as “SMA”, and are caused by genetic mutations on chromosome 5q (hence the term “5q-SMA”). SMA is the most common MND during childhood: with an incidence of 1:6000–1:10,000 live births and a carrier frequency of 1:40–1:60, it is the leading genetic cause of infantile mortality ,12. SMA .2–q13.3 . In the g region . This is (SMNΔ7) . All indSMN1 and present with early denervation weakness, but different clinical symptoms than those stated above, including joint contractures (infantile SMA with arthrogryposis—XL-SMA), distal rather than proximal weakness , diaphragmatic paralysis (SMA with respiratory distress 1—SMARD1), and pontocerebellar degeneration (SMA with pontocerebellar hypoplasia—SMA-PCH) [Further, less common forms of SMAs recognize defects in genes other than SMA-PCH) ,18.The inaccessibility to the cell type of interest majorly involved in MNDs and the lack of established models for the elucidation of pathogenetic mechanisms underlying such disorders represent a fundamental obstacle for progresses to be made in the field of therapies development. In order to resolve these issues and make durable discoveries, new strategies should be taken into account. In this respect, stem cell technology may represent a valuable solution.in vitro and give rise to teratomas in vivo. They can be identified with stem cell markers and resemble embryonic stem cells (ESCs) both in morphology and behaviour, but they are not burdened by ethical concerns [in vitro models that recapitulate mechanisms responsible for the establishment of pathologies [in vitro models obtained from iPSC-derived differentiated cells, like iPSC-MNs, exhibiting the affected genotype peculiar to the disease. So far, the great potential of iPSCs led to the generation of patient-specific cells for several neurodegenerative disorders, including Alzheimer’s Disease [Induced pluripotent stem cells (iPSCs) are originated from patients’ differentiated cells (oftentimes fibroblasts) through the use of reprogramming factors, first identified by Yamanaka in 2006 . IPSCs aconcerns . Indeed,hologies . Obtainehologies . Further Disease , Hunting Disease , Parkins Disease  and Amyo Disease . These cSOD1) [i.e., the role of the oxidative damage). The identification of C9ORF72 repeat expansion as the major factor responsible for ALS onset in the familial forms has focused attention on the causative role of alterations in RNA metabolism [TARDBP and FUS genes (encoding DNA/RNA binding proteins) in ALS development [Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease with adult onset. Symptoms reflect the dysfunction and death of motor neurons (MNs) and are characterized by muscular weakness and atrophy progressively leading to paralysis . Upper aSOD1) , researctabolism ,34,35, aelopment .in vitro some of these pathogenetic hypotheses and to model and investigate early disease mechanisms. Eggan’s group pioneered the field in 2008, when they investigated the potential of human MNs derived from embryonic stem cells (ESCs) to provide key data on ALS pathogenesis [SOD1 mutated mice. After 10 days in culture, a significant decrease in MN number could be observed in a time-dependent manner, thus suggesting a strong implication of non-cell autonomous mechanisms in ALS onset. Moreover, they tested if the same toxic glial effect could be detected with human interneurons and they found that the glia-induced toxicity is rather specific for MNs: interneurons treated for 20 days with SOD1G93A glia-conditioned medium appeared fully preserved. Complementary, MNs co-cultured with SOD1G93A mouse embryonic fibroblasts remained unaffected, thus indicating that the toxic effect is specifically related to the presence of astrocytes. Oligonucleotide arrays were exploited to identify genes differentially expressed in mutant glia. After these analyses, Di Giorgio and colleagues focused their investigation on the role of prostaglandin D2, which resulted responsible for a significant decrease of MN survival in culture. This work proved the advantages to use human cells for in vitro disease studies and provided the basis for further investigations on ALS pathogenesis.The establishment of human cell platforms has allowed for the first time to test ogenesis . ESC-derSOD1 in human MNs [SOD1 iPSC lines presented a further reduced survival when co-cultured with SOD1 glia, but this effect was much more amplified in the latter case implying the presence of a strong cell-autonomous component in ALS pathology. SOD1 MNs presented also an unhealthy morphology with shorter processes and reduced soma, thus summarizing the changes observed in the human pathology. Importantly, ZFN-mediated gene correction of SOD1 mutation resulted in the rescue of both altered morphology and reduced lifespan. Data from RNA-seq of SOD1 MNs highlighted a strong down-regulation of genes related to mitochondria functions and protein translation. Further analyses showed impairment in mitochondria motility in addition to an enrichment of mitochondria number located in neuronal processes. A substrate of ER stress has been found in healthy human MNs, which could be related to the cell size; this result is in line with the well-known early degeneration of largest alpha MNs in ALS. A combination of oxidative and ER stress and the up-regulation of the unfolded protein response (UPR) have been shown and could be additive causative mechanisms of neuronal toxicity. Moreover, the electrical activity of MNs could be involved in the ER stress, a result which nicely correlates with the report of the intrinsic hyperexcitability displayed by iPSCs-derived MNs from ALS patients [C9ORF72 and SOD1 lines led to the discovery of common pathways downstream of these mutations related to enhanced oxidative stress response and decreased mitochondria activity.The same research group proceeded beyond these results with a recent study aiming to elucidate pathways which are impaired by the expression of mutated uman MNs . Kiskinipatients . FinallyC9ORF72 iPSC lines were obtained by Sareen et al. to investigate the pathological processes underlying the most common genetic form of ALS [C9ORF72 was due to either gain of function or loss of function or both mechanisms. To address this question, iPSCs lines were developed from different C9ORF72 hexanucleotide expansion carriers affected by ALS and/or frontotemporal lobar degeneration (FTLD). Southern blot analyses were performed on derived MNs assessing the expansion and also highlighting differences among patients, which in some cases could be reflected in more severe forms of the disease. FISH analyses detected the presence of RNA foci within C9ORF72-ALS MNs , in line with previous data from patients’ tissues [C9ORF72 and TDP43 ALS forms. This work provided data in the direction of a “gain of function” mechanism linked to C9ORF72 hexanucleotide expansion. It has been shown that the mutated allele is usually transcribed and its downregulation using antisense oligonucleotides did not affect cell viability, but resulted in the correction of cell transcriptional profile. The specific pathogenetic role of the involvement of different RNA binding proteins interacting with RNA foci needs further investigations and could represent a common causative mechanism shared by different forms of ALS. Indeed, the aberrant cytoplasmatic aggregation of TDP43 represents a rather common pathological hallmark both in familial and sporadic ALS. Processes leading from the cytoplasmic aggregation to the selective MN loss appear to be exquisitely human and not easily detectable in animal models, where overexpression of TDP43 is ubiquitously provoked. Indeed, the majority of cell platforms and animal models considered in ALS pathogenesis studies relied on the overexpression of TDP43 in nonhuman or nonneuronal cells. Reason underlying the selective vulnerability of MNs to the disease process could be misled as well as the investigation of key molecular events that cause the human disease [et al. generated iPSCs from a patient affected by ALS carrying the TDP43 M337V mutation and used them as a tool to investigate TDP43 pathology in human neuronal cells [m of ALS . They in tissues . Observe tissues  and its  disease . The estal cells . No diffet al. proceeded beyond these data to elucidate the role of TDP43 in physiological conditions and its impairment in ALS related disease [Alami et al. could investigate the causative role of mutant SOD1 in impairing neurofilament (NF) turnover within MNs [D90ASOD1 mutation, thus obtaining isogenic controls. A modified differentiation protocol was applied to originate synchronized mature cells avoiding the generation of later-born MNs or glial cells.Neurofilament aggregation is a well-known pathological hallmark of ALS. Thanks to iPSC-based technology, Chen thin MNs . To bypaIndeed, challenges that need to be overcome when producing iPSC-derived neurons involve both the generation of immature cells and significant differences in the time rate of differentiation among iPSC colonies. These issues could lead to the production of heterogonous populations in terms of neural differentiated phenotype. The optimization of differentiation protocols has allowed generating neural cell populations that are synchronized regarding their growth in culture, in order to properly observe and investigate all the phases of their development .in vivo when patients present phenotypically asymptomatic or with very mild symptoms. This effect was due to the presence of mutated SOD1, as demonstrated by gain of function and loss of function experiments. Further analyses revealed that binding of SOD1 to 3′ UTR of NF-L mRNA could cause the alteration of NF structure, making them prone to aggregate. These events were selectively present in ALS-MNs and not in control cells or non-MNs. Moreover, human ALS MNs presented in vitro lower/normal levels of SOD1 (and increased amount of NF) compared to control MNs, strongly contrasting data from animal models where mutant SOD1 levels are much higher.NF subunits in ALS-MNs appeared to be unbalanced and developed a tendency to aggregate leading to neurite degeneration, an effect that probably interests the early phases of the disease in vitro has provided crucial data for this purpose  were able to reduce MN degeneration and increase lifespan in a murine model of mutant SOD1 ALS [Overall, these results highlight the importance of validating data obtained from animal models in human cells and using them as a precious tool to elucidate mechanisms which are peculiar of human physiology and pathology. The identification of the mechanisms underlying the development of ALS is crucial for the discovery of new therapeutic compounds. Indeed, the elucidation of the role of misregulated neurofilament turnover , granula purpose . The optentified . FinallySOD1 ALS . ALS astSMN1 gene. RT-PCR analysis also detected a few truncated transcripts lacking exon 7, along with the full-length SMN transcripts, witnessing the maintenance of functional SMN2 gene and its alternative splicing. Since lower alpha MNs are the main target of the pathological processes of SMA, iPSCs from SMA and WT fibroblasts were directed towards a motor neuronal and glial fate, in order to disclose in which phase of maturation the pathogenetic events occur. If in the early stages of the differentiation protocol a robust production of MNs was described and no significant difference was documented between the SMA- and WT-derived MNs, interestingly, around week 10 of the protocol, the SMA-derived MNs appeared to be decreased both from a quantitative and a qualitative point of view, as they were fewer and smaller compared to the WT-derived MNs. It was then speculated that the SMA phenotype hampers MN ontogenesis at later developmental time periods, either by inhibiting the generation of new cells or increasing the degeneration of those already matured. This was the first study to report that human iPSCs can be adopted as a reliable model of a genetically inherited disease, such as SMA, and serve as a powerful tool for a better understanding of its etiopathogenesis.Ebert and Svendsen were the first authors to demonstrate that human iPSCs can be employed to recapitulate the specific pathology that underlies SMA . Until tin vitro [Sareen and Svendsen’s group carried on the experiment for a more precise definition of the mechanisms that are responsible for the post-developmental damage that leads to cell death, as observed in vitro . Sareen in vitro . This wain vitro . Taken tSMN2 sequence-specific oligodeoxynucleotides. The correction of SMA iPSCs consisted in the exchange of a T to C at position +6 of exon 7, thus converting SMN2 into SMN1 through the inclusion of exon 7 in SMN2 transcripts. Subsequently, the model required the differentiation of SMA fibroblasts-derived iPSCs, both treated and untreated, into MNs, the cell-type majorly involved in SMA, in order to evaluate the effects of SMN1 deficiency on these cells. At week 8 of the multistage differentiation protocol, based on the administration of Retinoic Acid and Sonic Hedgehog, they observed specific disease effects of the SMN1 defect on untreated SMA iPSCs-derived MNs compared to the cultures treated with oligodeoxynucleotides. The statistically significant alterations included an overall reduction in cell soma size, axonal elongation and in the ability to form neuromuscular junctions, resulting in a decreased survival of MNs derived from untreated iPSCs. This experiment well demonstrates that a SMA model reproducing at least some aspects of the disease can be generated, and that MN dysfunction in SMA is the result of multiple neuropathological events likely occurring at a late differentiative state. Genetic correction of SMN2 via oligodeoxynucleotides proved to rescue the cellular damage to the defective SMN1 gene and to significantly increase the number of detectable gems, nuclear aggregates of SMN protein which correlate with the rate of translation of the full-length protein.Another contribution to the knowledge on the pathogenic mechanisms of SMA was given by Corti and colleagues . Using aSMN1 deficiency.Given the recent hypothesis on the role of RNA and splicing abnormalities as a primary determinant of selective MN death in SMA ,53, the SOD1 mutated models of ALS [Following the hypothesis that a cell population other than MNs may be implied in the activation of the apoptotic cascade, the attention was then focused on glial cells. Indeed, astrocytes had already proved to contribute to MN dysfunction in s of ALS ,55 and ts of ALS . In ordes of ALS , the moss of ALS . They deOverall, the possibility to isolate cell types that are specifically damaged in the disease process, like MNs and glia cells in SMA, makes iPSCs an unprecedented tool to disclose pathogenetic mechanisms that have been inaccessible so far . MoreoveSOD1 which characterizes mutant SOD1 mice may influence observed molecular phenotypes [SMN2 gene, which plays a crucial role in determining clinical phenotypes [in vitro studies. On the other hand, the development of iPSC-based technology has allowed to generate human pluripotent cells in abundance to perform any in vitro study bypassing ethical constraints [i.e., fibroblasts or astrocytes) into relevant cell types  [The pathogenesis of several MNDs is still obscure under multiple aspects, thus hampering the development of potential therapies . Experimenotypes . With reenotypes . As a coenotypes ,61, but straints . Furtherstraints . Anotherstraints . It is cstraints . Recentlneurons) . These rThe development and optimization of iPSC-based platforms has allowed elucidating disease-specific mechanisms, which are exquisitely human . Further


Cervical cancer continues to threaten women’s health worldwide, and the incidence of cervical adenocarcinoma (AD) is rising in the developed countries. Previously, we showed that glucose-regulated protein 58 (Grp58) served as an independent factor predictive of poor prognosis of patients with cervical AD. However, the molecular mechanism underlying the involvement of Grp58 in cervical carcinogenesis is currently unknown.DNA microarray and enrichment analysis were used to identify the pathways disrupted by knockdown of Grp58 expression.Among the pathway identified, the WNT signaling pathway was one of those that were significantly associated with knockdown of Grp58 expression in HeLa cells. Our experiments showed that β-catenin, a critical effector of WNT signaling, was stabilized thereby accumulated in stable Grp58 knockdown cells. Membrane localization of β-catenin was observed in Grp58 knockdown, but not control cells. Using a transwell assay, we found that accumulated β-catenin induced by Grp58 knockdown or lithium chloride treatment inhibited the migration ability of HeLa cells. Furthermore, an inverse expression pattern of Grp58 and β-catenin was observed in cervical tissues.Our results demonstrate that β-catenin stability is negatively regulated by Grp58 in HeLa cells. Overexpression of Grp58 may be responsible for the loss of or decrease in membranous β-catenin expression in cervical AD.The online version of this article (doi:10.1186/1471-2407-14-555) contains supplementary material, which is available to authorized users. Cervical cancer is the third leading cause of cancer-related mortality among women worldwide, althougPathway enrichment analysis of a set of differentially expressed genes upon Grp58 knockdown was performed using the GeneGo MetaCore analysis tool . Genes displaying differential expression, by comparing stable control and Grp58 knockdown cells, greater than 1.2 fold were uploaded. A pathway map with a false discovery rate of <0.01 was considered significant.The human cervical cancer cell line, HeLa, was obtained from the American Type Culture Collection , and cultured as recommended. Stable Grp58 knockdown cells were established as described earlier. For MG1Total RNA was extracted from cells using TRIzol. The first cDNA strand was synthesized using the superscript III kit for RT-PCR . qRT-PCR was performed using SYBR Green, as described previously. The priWestern blot analysis was performed as described previously. Anti-Gr4 in 100 μl) were seeded in the upper chamber  in triplicate. Lower chambers were supplemented with 20% fetal bovine serum in medium. Traversed cells were stained with crystal violet after 24 h incubation.Cells were trypsinized and re-suspended using serum-free medium. Equal amounts of cells  for 10 min, blocked with 1% bovine serum albumin for 30 min, and stained with the indicated primary antibody for 3 h at RT. After washing three times with PBST, slides were incubated with secondary antibody for 2 h at RT. Fluorescence images were acquired using confocal microscopy . Grp58 and β-catenin primary antibodies were the same as those used for Western blotting. The secondary antibodies employed were Alexa Fluor 488 goat-anti-mouse and 568 goat-anti-rabbit antibody .Formalin-fixed and paraffin-embedded tissues were examined using IHC, according to previously described procedures. The GrpData from a total of 109 cervical carcinoma patients subjected to primary definitive surgery between 2000 and 2008 at Chang Gung Memorial Hospital  were retrieved from the hospital database, and the histological types confirmed by pathologists. Thirty-four patients with cervical AD, classified as stage I to IIB according to the International Federation of Gynecology and Obstetrics (FIGO) staging system, were enrolled under the protocol approved by the Institutional Review Board (IRB: 95-1241B); all patients provided informed consent. The personal rights of the patients were preserved.P values < 0.05 were considered significant. SPSS statistical software was used for statistical analyses.One-way analysis of variance (ANOVA) was used to compare means of more than two groups. Mann–Whitney test was applied to compare the means of two independent groups. Previously, we used an Affymetrix microarray to identify the genes disrupted upon knockdown of Grp58 expression. Differeβ-Catenin is a crucial molecule of the WNT signaling pathway. The β-catenin-dependent canonical pathway is the Wnt signaling pathway that has been extensively characterized to date. β-catenin transduces Wnt-activated signals to the nucleus and serves as a transcriptional co-activator in T cell factor/lymphoid enhancer-binding factor (TCF/LEF)-dependent gene transcription. SeveralIn general, β-catenin mainly localizes to the cytoplasmic membrane as a component of adherens junctions. Subcellular localization of β-catenin in stable HeLa cells was determined using IF staining, with images visualized using confocal microscopy. As shown in Figure To explore the impact of stable accumulated β-catenin on cell migration, the GSK3 inhibitor, LiCl, was used to suppress β-catenin protein degradation. LiCl enhanced β-catenin protein levels in a dose-dependent manner . Conversely, the Grp58 level was significantly higher in tumors (186.2 ± 60) than adjacent normal tissues . In paired samples , while β-catenin was significantly downregulated in AD . Four my tumors. SET comy tumors. Downregy tumors. AccordiPatients with cervical AD are generally considered to have a poorer prognosis than those with SCC. HoweverSupplementary methods and figures.(DOCX 23 KB)Additional file 1:Additional file 2: Figure S1: Verification of the expression of WNT signaling pathway-related genes. (TIFF 12 MB)Additional file 3: Figure S2: Invasion and proliferation abilities of stable cells. (TIFF 7 MB)Additional file 4: Figure S3: Migration abilities of Grp58 knockdown Caski and C33A cells. (TIFF 6 MB)


Efficiency is defined as the use of resources in such a way as to maximize the production of goods and services. Improving efficiency has been the focus of management in many industries; however, it has not been until recently that incorporating efficiency models into healthcare has occurred. In particular, the study and development of improvement projects aimed at enhancing efficiency in GI have been growing rapidly in recent years. This focus on improving efficiency in GI has been spurred by the dramatic rise in the demand for endoscopic procedures as well as the rising number of insured patients requiring GI care coupled at the same time with limited resources in terms of staffing and space in endoscopy centers. This paper will critically review the history of efficiency in endoscopy centers, first by looking at other healthcare industries that have extensively studied and improved efficiency in their fields, examine a number of proposed efficiency metrics and benchmarks in endoscopy centers, and finally discuss opportunities where endoscopy centers could improve their efficiency.  Efficiency has many varying definitions and historically has been employed in economic models and businesses. At its core efficiency is defined as the use of resources in such a way as to maximize the production of goods and services . While tIn the last several years there has been a dramatic surge in the request for GI specialty care and in particular endoscopic services –4. Such In the field of healthcare efficiency, anesthesia has been the clear leader for the last four decades. Specifically, there has been a tremendous amount of research which is focused on improving efficiency in ambulatory surgery centers and hospital operating rooms (OR) and is an area from which the GI field can learn. From this expansive field of literature, three key steps emerge on how to improve efficiency within a healthcare setting. The first step is the establishment and use of metrics so that improvement work can be measured and monitored. Clearly defined metrics have been established by the Association of Anesthesia Clinical Directors which have been utilized and validated in several studies . The sec metric is any type of measurement used to gauge some quantifiable component of an organization's performance [ benchmark is a standard or point of reference in measuring or judging the current value or success of an organization in order to determine its success or overall performance in comparison to the performance of other similar organizations [Along the same lines as lessons learned in anesthesia, in order to begin enhancing efficiency within an endoscopy center, one of the first steps is to clearly define the metrics that will be used to initially assess the organization and to eventually measure its success. Aformance . Moreoveizations . Metricsizations , 40.In an attempt to study efficiency within endoscopy centers, a few authors have established endoscopy center metrics ad hoc . These mWhile much research has focused on identifying and examining a laundry list of metrics, some studies have taken the approach of identifying and studying a few key metrics. For example, several studies have examined only one or two metrics such as procedure duration, recovery room time, and/or elapsed time between endoscopic procedures  in an effort to more easily study efficiency , 42–44. Once an endoscopy center has determined which set of metrics to utilize and measure, it is critical to have benchmarks. Initially, one may not need benchmarks as internal metrics can be used to gauge the success or failure of implementing process or technology changes. However, benchmarks serve to help one guide their organization in accordance with accepted industry standards and best practices and to further build their practice. Multiple industries such as the automobile, airline, and banking sectors have clearly defined benchmarks for which one measures success and operational/productivity improvements. Yet, as with metrics, few available and published benchmarks are known for endoscopy centers. Scant data is available for some measurements that are used in research, but again it is unclear whether these measurements represent the “optimal” assessment. Furthermore, no clear consensus has been reached on them. Additionally, of the few published benchmarks, there is considerable heterogeneity in their numbers with a multitude of confounding factors such as type of sedation utilized for each procedure, procedure type, and type of endoscopy center where the data was collected  . Much ofWith an emphasis on cost containment and improving efficiency in healthcare two methods, (time and motion studies and discrete event simulation modeling), have been successfully advocated and performed in order to attain these goals. While using these two methods have been incredibly successful in a number of other healthcare areas, their use in endoscopy centers has been less evident. However, limited GI research has illustrated some promising results with using these two methods with respect to two areas examining several simultaneous changes to an endoscopy center versus studying one focused change.With regard to the first area, two groups have employed time and motion studies and simulation modeling to fully examine a number of factors that impact endoscopy center efficiency , 24. IniIn the second area, smaller studies have modeled the effect on efficiency by examining one specific change to an endoscopy center rather than multiple, simultaneous ones. Some studies have focused on altering staffing specifically focusing on the endoscopist , 42, 44 In assessing efficiency within endoscopy centers there are several potential areas where process improvements can be implemented. Process improvement methods have been demonstrated to remove inefficiencies within endoscopy centers. For example, Schembre and colleagues utilized manufacturing efficiency tools from the Toyota Corporation  to improve flow within their endoscopy center. After extensively studying patient and staff travel patterns, resource utilization, and equipment standardization, they then implemented several changes based on their observations. Consequently, they were successful in reducing patient waiting times while increasing procedure volume . ThroughA crucial factor within an endoscopy center is the staff of it and how to best utilize staffing resources. Work has been conducted in this arena and can serve as a useful resource for endoscopy centers. With respect to nursing, some GI societies have proposed nursing staff models, but while being helpful it should be noted that no data exist to support or refute these recommendations . LikewisAnother proposed endoscopist model is having nonphysicians (nurse practitioners/physician assistants) perform endoscopy. Numerous data have shown that nonphysicians can safely perform endoscopic procedures with similar quality to physicians –54. ExpaLastly, how the entire team of providers is organized is a critical decision. Many advocate a “Pod layout” whereby a team is assigned to one physician/room for the day. This team is responsible for all aspects of patient care from preparation through recovery , 35. ThiWithin ambulatory surgery centers several studies have demonstrated that shortening procedure time does not improve efficiency, but rather other factors such as scheduling and operational improvements are more instrumental. One proposed method for scheduling procedures focuses on utilizing open access endoscopy . Such a method can improve patient access to both clinic and endoscopy, but some limitations may exist. For example, some studies suggest that open access yields more inappropriate indications for procedures with a reported 10–28% prevalence of incorrectly scheduled procedures . A seconEqually important to scheduling factors is minimizing delays within the endoscopy center. There are two types of endoscopy center delays: patient and procedure related. There are a number of interventions the endoscopy center can employ to minimize patient related delays. These include ensuring that patient instructions are clear, providing excellent directions to the endoscopy center, performing an advance call to patients to review preparatory instructions/medications, and ensuring an efficient check-in process once the patient arrives at the endoscopy center. Along these lines, a number of modalities have been shown to improve patient adherence to pre-endoscopy instructions and reduce patient related delays; such modalities include multimedia, interactive computer programs, and education classes . On the While reducing endoscopic procedure time does not appear to increase efficiency of an endoscopy center , other fOne area that has been used as a marker for improving efficiency is reducing endoscopy room turnover time. Using procedure volume per hour as a marker for efficiency, Zamir and Rex demonstrated a clear inverse relationship between this marker and room turnover time . Yet, thLastly, reducing recovery room time can help increase efficiency. With regard to recovery room time, Grossman et al. modeled an ambulatory surgery center and demonstrated that recovery time was the main bottleneck. In fact, a 50% reduction in recovery time increased the number of patients per room per day and shortened the overall length of stay of patients . HoweverImproving efficiency in endoscopy centers has been an increasingly important topic. Overall, there are three key steps to consider when beginning the journey of improving endoscopy center efficiency. The first step in improving efficiency within an endoscopy center is to determine which metrics would be the most relevant and feasible to measure within one's organization. At the same time, it is crucial to remember that metrics help identify where to improve but do not directly cause improvements to take place. Also, while no accepted or standardized benchmarks have been adopted for endoscopy centers, there is some limited data available for which one can compare their data measurements. Secondly, an initial assessment of potential areas where inefficiencies may exist in an endoscopy center should be conducted with particular focuses on patient flow, staffing, facility, and equipment. Along these lines, there is evidence to suggest that time and motion studies coupled with simulation modeling can aid endoscopy centers in better understanding their operations and how potential changes can affect the efficiency of their center. Finally, a number of areas exist where endoscopy centers can focus in order to begin improvement work in efficiency. Examining endoscopist models, minimizing patient and procedure related delays, utilizing block scheduling, reducing room turnover time through clear communication and role definition, and considering the types of sedation administered are factors that have been demonstrated to impact efficiency and procedure volume in endoscopy centers. Lastly, one has to tailor their proposed innovations to be in alignment with the goals of their organization and patient population.


The Wnt/β-catenin signaling pathway plays a central role in the development and regulation of the central nervous system and several genes belonging to the cascade have been genetically associated with ASDs. In the present paper, we review basic information regarding the role of Wnt/β-catenin signaling in excitatory/inhibitory balance  through the regulation of pre- and postsynaptic compartments. Furthermore, we integrate information supporting the role of the glycogen synthase kinase 3β (GSK3β) in the onset/development of ASDs through direct modulation of Wnt/β-catenin signaling. Finally, given GSK3β activity as key modulator of synaptic plasticity, we explore the potential of this kinase as a therapeutic target for ASD. Hundreds of genes have been associated with autism spectrum disorders (ASDs) and the interaction of weak and TSC1 and TSC2), Rett syndrome (MECP2), Fragile X syndrome , and Cowden syndrome (PTEN), but, altogether, these disorders do not account for more than 10% of cases [ de novo occurring SNVs and CNVs have a larger contribution to the onset of ASD [ de novo CNVs are significantly enriched in individuals affected with the disorder and it is estimated that 8% of cases that carry these variants are likely to be pathogenic [ de novo SNVs in affected individuals are disruptive or frameshift mutations that generate nonconserved amino acid changes such as premature stop codons or alternative splice sites ultimately affecting the normal biological function of the resulting protein [ de novo variants affect ASD susceptibility in 10–15% of probands [ de novo mutations are part of an interconnected network of chromatin remodeling, synaptic plasticity, and Wnt/β-catenin signaling genes [Autism spectrum disorders (ASDs) are highly heterogeneous, pervasive developmental disorders characterized by impaired social communication skills, repetitive behaviors, and a restricted range of interests . The widof cases . In the of cases , 6. Whilof cases . Rare vathogenic . On the  protein . Overallprobands . Neverthprobands , 13. In β-catenin signaling components and/or genes coding for molecules that modulate its functional activity [+2, and the canonical Wnt/β-catenin signaling pathway [β-catenin signaling is the most well understood cascade and it starts via binding of the Wnt ligand to cell membrane receptors Frizzled (FZD), belonging to the 7-transmembrane domains family of proteins and to members of the low density lipoprotein receptor related proteins 5 and 6 (LRP5/6), which act as coreceptors [β-catenin from the degradation complex consisting of Axin and adenomatous polyposis coli (APC) scaffolds [β (GSK3β) [β-catenin accumulates in the cytosol and translocates to the nucleus where it interacts with T-cell factor/lymphoid enhancing factor (TCF/LEF) transcription factors to activate transcription of target genes [β sequential phosphorylation of β-catenin [β-transducing repeat-containing protein (β-TrCP) and subsequent proteasome degradation [Through the analysis of biochemical and pharmacological data, animal models of the disease, and genetic association studies, we predicted earlier that the onset/development of ASDs might involve the additive effect of genetic variants within Wnt/activity , and sucactivity , 17, 18. pathway . Wnt/β-ceceptors . Wnt bincaffolds , and the (GSK3β) . As a neet genes . Convers-catenin  targetinradation .β-catenin degradation complex and thus modulates the action of Wnt signaling [β-catenin signaling [ CDH5, CDH8, CDH9, CDH10, CDH13, CDH15, PCDH10, PCDH19, and PCDHb4 [β-catenin in cell-cell adhesion complexes. More recently, the chromo-helicase domain protein 8 (CHD8) [β-catenin through direct binding [ de novo genetic variants in the β-catenin (CTNNB1) gene itself have been implicated in severe intellectual disability [β-catenin pathway. Furthermore, we trace functional defects to GSK3β activity and explore its pharmacological regulation as a potential therapeutic target for ASD, particularly in relation to synaptic plasticity.It is interesting to note that the tumor suppressor complex formed by TSC1 and TSC2 interacts with the ignaling , 26. Othignaling , the hepignaling , 29, whiignaling , and sevd PCDHb4 , some of8 (CHD8) , 32, whi binding , and DYR binding  have bee binding , which i binding . Finallysability . TherefoNLGN3) mutant mice, and the models for Rett, Fragile X, and Angelman syndromes . In hum signaling downstream of Wnt ligands [At the presynaptic region, canonical Wnt signaling has a major role in clustering and recycling of synaptic vesicles (SVs). Conditioned media containing Wnt7a, and to a lesser extent Wnt3a, were found to enhance SVs recycling in primary cultures of rat hippocampal neurons . Similar ligands . Interes ligands . Moreove ligands , which pβ-catenin signaling also seems to trigger neurotransmitter release and SV trafficking by modulating the function of SVs-associated phosphoproteins, including membrane-trafficking proteins such as synapsin and synaptotagmin. While all three members of the synapsin (SYN) gene family (SYN1-3) [ SYN2 is predicted as a Wnt/β-catenin target gene [Wnt/(SYN1-3)  have bee(SYN1-3) –60, it h(SYN1-3)  and phos(SYN1-3)  of Syn1 get gene  and is uget gene . Finallyget gene .β-catenin adhesion complexes have an essential function during the recruitment and clustering of SVs to synapses [β-catenin results in the mislocalization of SVs along the axon, while clustering of active zone proteins like Bassoon is unchanged [β-catenin weakens cadherin-catenin interactions [β-catenin phosphorylation and strengthens cadherin mediated adhesion [β-catenin dissociation from cell adhesion complexes that have been genetically linked with ASD is the MET receptor tyrosine kinase [β-catenin and promotes its dissociation from cadherins [β-catenin signaling in synaptic structure stability and function through modulating cell adhesion, vesicle exocytosis, and clustering well beyond β-catenin functioning solely as a TCF/LEF transcriptional coactivator.Other mechanisms modulating the activity of the presynaptic terminal involve the function of cell adhesion proteins, most notably trans-synaptic cadherin interactions. It is widely accepted that cadherin-synapses –69. Indenchanged . Tyrosinractions . Interesractions , activatadhesion . Among oe kinase , which padherins , thus li2+ related pathways. Canonical and noncanonical Wnt pathways have been extensively related to Ca2+ homeostasis and signaling [2+ influxes in neurons. It is accepted that activation of L-type voltage sensitive Ca2+ channels (L-VSCCs) or NMDA receptors allows the entrance of Ca2+ which in turn activate CAMKII triggering actin cytoskeleton reorganization to regulate dendritic growth [β-catenin dependent dendritic growth upon Ca2+ influx [Experience driven plasticity is highly dependent on proper synaptic transmission and is mainly modulated by Caignaling , 74, 75.ignaling , Wnt5a [ignaling  have allc growth . In thisc growth  activatec growth . CAMKIV c growth  and addi+ influx , 80.2+ influx is an event preceding the establishment of long-term synaptic potentiation (LTP) that allows the recruitment of AMPARs at the postsynaptic terminal, which in turn enhances long lasting excitatory transmission [NLGN1) protein increasing its surface expression [β-catenin signaling impairs LTP and conversely its activation facilitates it [ TSC2 missense mutations fail to inhibit the Wnt pathway [FMR1), and also for the disrupted in schizophrenia 1 (DISC1) genes, exhibit diminished capacity to establish LTP [ DISC1 genetic variants, directly impact Wnt/β-catenin signaling function (see below) [β-catenin pathway plays a central role in Ca2+ homeostasis at postsynaptic terminals, which is commonly disrupted in ASD. In addition, abnormal establishment of LTP, phenomenon in which the signaling cascade plays an important role, has profound effects in activity driven plasticity affecting efficient synaptic transmission and disrupting the E/I balance.Activation of CAMKII and other kinases through NMDAR-mediated Casmission . Additiopression . Notablytates it , and bottates it  and that pathway , it is llish LTP , 85. Beslish LTP –88, comme below) . Altogetβ-catenin pathway specifically through the modulation of GSK3β could improve core ASD symptomatology and open a therapeutic window for the treatment of ASD through the fine-tuning of synaptic plasticity.LTP is the most well understood paradigm of activity driven plasticity and is considered to be one of the synaptic mechanisms underlying learning and memory . In turnα and GSK3β, have been described in mammals and are involved in multiple cellular processes including glycogen metabolism, gene transcription, microtubule stability, and apoptosis [β is as a convergence point of major prevalent neurological disorders, including Alzheimer's disease, schizophrenia, and bipolar disorder [ DISC1 gene has an essential role in modulating brain structure and function and when mutated leads to neuropsychiatric behavior. DISC1 inhibits GSK3β activity by direct physical interaction resulting in reduced β-catenin phosphorylation and activation of Wnt/β-catenin signaling cascade [β-catenin signaling activity [β has been documented in animal models of FXS [β displays similar social preference abnormalities as FMR1 KO mice [GSK3 is an evolutionary conserved serine/threonine kinase highly abundant in the brain. Two homologous isoforms, GSK3poptosis . GSK3β idisorder –95, and  cascade  and commactivity . Regardis of FXS –99. For β in mice forebrain has anxiolytic and prosocial effects [β leads to reduced spine density where persistent spines are lost and newly formed spines are unstable [β-catenin [β has been shown to increase internalization of NMDA and AMPA receptors, effect that is mainly observed for NR2B containing receptors [β impairs the establishment of LTP [β in a Ca2+ dependent mechanism [β-catenin signaling through hyperactivation of GSK3β might help to explain transmission anomalies as it is observed in ASD.Mouse models for Fragile X, Phellan-McDermid, and Angelman syndromes, as well as for Tuberous Sclerosis, all present an abnormal number of dendritic spines that suggest a dysregulation in synaptic turnover –102. In  effects  and its  effects . Interesunstable . These s-catenin . Furthereceptors . Converst of LTP  and highechanism . Given t In utero exposure to anticonvulsive medication is known to cause neurodevelopmental abnormalities [β [β, VPA induces the stabilization of β-catenin and the activation of Wnt target genes, though the exact mechanism of GSK3β is not currently understood. Indeed, in utero exposure to VPA increases the incidence of autism in the offspring [Due to its high heterogeneity, genetic factors cannot be held accountable for the entire spectrum of autism suggesting a role for environmental factors in the onset of ASD.malities . The mosities [β  and of hities [β  activitiffspring , 113 andffspring , 114. Cuffspring . This VPffspring .β-catenin signaling is central through the regulation of GSK3β. VPA mice exhibit elevated NMDA receptor levels and enhanced LTP [ WNT1 and WNT2 genes further enhancing Wnt/β-catenin signaling [β-catenin signaling by enhancing GSK3β expression in the prefrontal cortex or the hippocampal region of VPA mice [β-catenin, neurotrophin, and LTP signaling, the same pathways which also appear enriched in the transcriptome of lithium [β-catenin signaling by inhibiting GSK3β [β-catenin signaling, most of the data comes from in vitro cell cultures exposed to VPA and not from in vivo studies using mice prenatally exposed to the drug. In this context, it is interesting to note that chronic VPA treatment in mice has been shown to correct dendritic spine deficits and to improve novel object recognition [β-catenin pathway is still unknown. In this context, it is interesting to note that ASD could result from a transient gain of function of the Wnt/β-catenin pathway during embryonic development and a subsequent decline after birth.While several molecular mechanisms regarding the onset of ASDs in VPA mice have been reported, the activation of Wnt/nced LTP  and inhinced LTP , suggestignaling . In thisVPA mice , improveVPA mice , 121. In lithium , which mng GSK3β . Nonetheognition ; thus, tβ phosphorylation (phosphor-Ser9 and phosphor-Ser21) [β rescues LTP and hippocampal neurogenesis defects in FMR1 knockout mice and improves cognitive tasks [β inhibition similarly rescues dendritic spines deficit observed in FXS mice suggesting that inhibition of this kinase and thus activation the Wnt/β-catenin play a role in reactivating synaptic plasticity and these effects might play an important role in the behavioral and learning improvements observed.Lithium has been widely used to manage mood disorders, such as bipolar disorders, and it is not uncommon for ASD children to feature symptoms within this spectrum such as euphoria, mania, or paranoia . Few stur-Ser21) . Interesve tasks , 103. Fuβ phosphorylation selectively in FMR1 knockout mice [Antagonists for metabotropic glutamate receptor (mGluRs) are up to date the most successful pharmacological modulators improving ASD symptomatology probably through regulation of abnormal mRNA translation at synapses . In thisout mice , effect out mice , furtherout mice .β-catenin signaling plays a central role. As described in this review, several lines of evidence indicate that Wnt signaling regulation of serine/threonine kinase GSK3β has profound effects in activity dependent synaptic plasticity and thus in the regulation of the E/I balance. Through dissecting Wnt/GSK3β activity and pharmacology in cells and animal models of ASDs, it seems plausible that there may be differential effects driven by Wnt/β-catenin signaling activity during the initial patterning of brain structures and later on when these structures have been established. Overall, the therapeutic value of GSK3β modulation that seems to rescue synaptic plasticity events that could be disrupted in ASD brains warrants further basic and clinical investigation.ASD displays a high genetic heterogeneity that results in a wide range of abnormal phenotypes and settling a unified paradigm that accounts for the gain or loss of function of genetically associated genes has been an elusive task. Currently, most elements associated with ASDs converge in signaling pathways important for synaptic plasticity, where Wnt/


Xenopus forebrain. Depletion of Fezf2 induces apoptosis in postmitotic neural progenitors, with concomitant reduction in forebrain size and neuronal differentiation. Mechanistically, we found that Fezf2 stimulates neuronal differentiation by promoting Wnt/β-catenin signalling in the developing forebrain. In addition, we show that Fezf2 promotes activation of Wnt/β-catenin signalling by repressing the expression of two negative regulators of Wnt signalling, namely lhx2 and lhx9. Our findings suggest that Fezf2 plays an essential role in controlling when and where neuronal differentiation occurs within the developing forebrain and that it does so by promoting local Wnt/β-catenin signalling via a double-repressor model.Brain regionalisation, neuronal subtype diversification and circuit connectivity are crucial events in the establishment of higher cognitive functions. Here we report the requirement for the transcriptional repressor Fezf2 for proper differentiation of neural progenitor cells during the development of the  The vertebrate forebrain, which carries out higher neural functions, is a highly organised and complex structure derived from the anteriormost region of the neural plate. Although the extent of elaboration and the size of the various subdomains of the anterior central nervous system vary between species, the molecular mechanisms that generate the brain, including the patterning of the different forebrain subdomains and subsequent neuronal differentiation within each compartment, are highly conserved amongst vertebrates. Therefore, studies on the early development of the forebrain in zebrafish, frog, chick and mouse embryos have shed light on the conserved developmental programmes that contribute to the formation and development of the vertebrate brain .Forebrain development comprises three distinct stages. The first stage is neural induction during gastrulation, which defines both the position and identity of the anterior neuroectoderm. This stage is quickly followed by a second, patterning stage, whereby the anterior neuroectoderm is regionalised into the various forebrain subdomains by means of both transcriptional regulation and signal transduction. The third and final phase of forebrain development, which lasts until adulthood, is associated with regionalised growth of the various forebrain subdomains and concomitant specification, migration and differentiation of the various neuronal subtypes that make up the adult brain .Wnt/β-catenin signalling has been reported to play an essential role during the patterning and differentiation stages of the forebrain. During the patterning stage, low or absent Wnt/β-catenin signalling in the anterior region of the forebrain is required for telencephalic specification, whereas high Wnt/β-catenin signalling, together with BMP, FGF and Shh signalling, is important for diencephalic specification . Later, Fezf2, which is also known as fezl/Earmuff, too few, ZNF312 and Zfp312, is a highly conserved gene that encodes a zinc finger transcriptional repressor, which is expressed in the forebrain (Fezf2 homologues have been identified and studied in Drosophila (Fezf2 orthologues encode transcription factors characterised by six DNA-binding C2H2-type zinc fingers and an Engrailed homology 1 (Eh1) repressor motif that interacts with Transducin-like enhancer of split (TLE)-type transcriptional co-repressors , reaching a maximum at the mid-neurula stage (stage 15), at which time its expression decreased slightly and plateaued thereafter . This pattern was very similar to that obtained from whole-exome deep sequencing  . At the tailbud stage (stage 28), fezf2 was expressed in the telencephalon, ventral diencephalon and the eye vesicle . At early tadpole stages (stage 35), the expression of fezf2 remained restricted to the forebrain and eye vesicle .We isolated elopment . Subseququencing . We thenfezf2, we first performed a series of knockdown experiments using an antisense morpholino oligonucleotide (MO) targeting the exon 3-intron 3 splice junction of the pre-mRNA . The knockdown efficiency of this MO was validated using RT-PCR and qPCR . Embryos injected with control MO exhibited normal forebrain development, whereas fezf2 MO caused significant disruption in the development of the forebrain, as revealed by diminution in the expression of the rostral forebrain-specific marker arx   embryos did not exhibit altered expression of the forebrain markers arx, otx2 and pax6 .To dissect the function of rker arx A as wellig. S4A) . Notablyfezf2, we injected the fezf2 MO into one cell of 2-cell stage embryos, and then we assessed the effect of this perturbation on specific cell populations in the injected side of the forebrain versus the control side at stage 30. No significant change was observed in the number of Sox3+ neural progenitor cells in the fezf2 MO-injected side versus non-injected side  . No reduction in either Sox3+ or MyT1+ cells was observed in embryos injected with a control MO . Therefore, fezf2 is required for neuronal differentiation, but is not essential for the maintenance of neural progenitor cell populations.To characterise the cell types in the forebrain that were affected by loss of ted side B-D (Wangted side . However1 (MyT1) E-G (Bell1 (MyT1) . We furtfezf2 is required for cell survival during differentiation. To address this, we performed TUNEL assays on control versus knockdown sides of the embryos. These experiments revealed that injection of fezf2 MO, but not the control MO, caused a 3-fold increase in the number of apoptotic cells in the knockdown side versus the control side of the forebrain . Taken together, we conclude that fezf2 controls the transition from neuronal progenitors to differentiated neurons, but is not required for the early forebrain patterning events, nor for the maintenance of neural progenitor cells prior to neuronal differentiation.One possibility for the reduction in the number of differentiated neurons in the forebrain area is that fezf2 mRNA are significantly dorsoanteriorised, resembling LiCl-treated embryos . Together, these phenotypic and signalling changes suggested that fezf2 overexpression might lead to hyperactivation of Wnt/β-catenin signalling.We next investigated the mechanism(s) by which Fezf2 acts during development. It was first noted during the functional screen  that emb embryos  or those embryos   (fezf2 led to a robust increase in the expression level of these two Wnt-responsive genes in early embryos (fezf2 exhibited quantitatively higher levels of expression of goosecoid (gsc) and chordin (chd), two additional Wnt/β-catenin-responsive genes . By contrast, expression of the ventral markers vent1 .To confirm whether is (sia) . Indeed, embryos C,D. In ave genes , and ledrs vent1  and bmp4t1 , consistent with previous findings . In addition, injection of fezf2 mRNA into one of the two ventral blastomeres at the 4-cell stage induced axis duplication with complete head in more than 75% of embryos , as is often observed following ectopic activation of Wnt/β-catenin signalling   or nlk1 mRNA, a direct inhibitor of Wnt/β-catenin signalling   . Thus, ffezf2 on Wnt/β-catenin signalling, we performed in vivo luciferase assays using a Wnt-responsive construct, TOPFlash  control . These experiments confirmed that fezf2 overexpression activates Wnt/β-catenin signalling in early embryos.In order to observe a more direct effect of TOPFlash . Co-inje control E, whereaFezf2 negatively regulates Wnt/β-catenin signalling in mouse embryonic stem cells (mESCs) by repressing the expression of Wnt ligands .A previous investigation has suggested that  ligands . We teststage 15 . The expfezf2 expression in the forebrain correlates with active Wnt/β-catenin signalling. We isolated ∼3 kb of the fezf2 proximal promoter region and used it to drive the expression of Katushka in transgenic embryos (fezf2-Katushka cassette using our recently developed pTransgenesis system to generate the transgenic embryos (fezf2 promoter activity in the same embryos (fezf2) in the telencephalic and diencephalic areas, although a broader fezf2 expression was observed in the eye, which might reflect the much longer half-life of Katushka relative to dEGFP  at stage 32 in the forebrain  into an immortalised mouse C17.2 neural stem cell line, together with either empty vector (pCS2) or the Wnt-inhibitory truncated ΔTcf3 construct (pCS107-ΔN51-Tcf3)  , followe51-Tcf3) . Transfen+ axons Ia,c,J. Hy vector Ia,d,J. To effect Ib,J. Thein vivo, we electroporated a construct containing CMV promoter-driven fezf2 (pCS107 backbone) with or without the Wnt-inhibitory construct (ΔN51-Tcf3) into the third ventricle of stage 26 X. laevis embryos, and allowed them to develop until stage 31 for analysis. Electroporation of pCS107-fezf2 significantly increased the number of differentiated primary neurons (MyT1+) in the forebrain area, as found in previous studies , a gene involved in neuronal differentiation, is inducible by Wnt/β-catenin signalling and is fezf2 responsive . Misexpression of fezf2 mRNA induced ngn1 expression; however, this induction was attenuated when fezf2 mRNA was co-injected with ΔN51-tcf3 mRNA. Injection of either lacZ mRNA (control) or ΔN51-tcf3 mRNA alone had no effect on ngn1 expression in the animal cap explants  , together with a reversed pattern of Smad1/5/8 and Smad2/3 phosphorylation  , and thied (Eve) A. Injectfezf2-expressing regions affects forebrain development. We inserted a cassette comprising a neural-specific β-tubulin promoter driving tauGFP (NβT-tauGFP) into the p1 site of the pTransgenesis system to assess differentiated neural tissue in transgenic embryos . However, the antimorphic VP16-Fezf2 transgenic embryos displayed a significant reduction in arx staining, NβT-tauGFP marked neural tissue, and decreased eye size , similar to transgenic embryos expressing the Wnt-antagonising ΔN51-Tcf3 and GSK3βS9A constructs, which had reduced Wnt activity in fezf2-expressing regions . By contrast, transgenic embryos expressing the Wnt-agonising ΔN90-β-catenin construct demonstrated expansion of arx staining, excessive growth of differentiated neural tissue, and enlarged eyes, suggesting that elevated Wnt activity promotes the growth of neural tissue within the forebrain . These results confirmed that the antimorphic Fezf2 acts as a negative regulator of Wnt signalling, and that proper Wnt signalling in fezf2-expressing areas is crucial for normal forebrain development in vivo.We next investigated whether endogenously augmented Wnt/β-catenin signalling within the  embryos . The 3.5promoter C  possess the Eh1-interacting WD domain (in vivo co-immunoprecipitation assay performed with gastrula stage (10.5) embryos, Fezf2 interacted with all three TLEs that possess the Eh1-interacting WD domain  that has a point mutation in the DNA-binding zinc finger domain but an intact Eh1 domain   . Thus, ofezf2 activates Wnt/β-catenin signalling within the forebrain, we noted previous reports suggesting that Fezf2 binds to the promoter region of lhx2  . A high ChIP enrichment was detected around the −12 kb region , whereas no ChIP enrichments were found in the other two regions tested .To investigate the regulatory mechanism by which  of lhx2 . Furtherorebrain . We therig. S7A) , 2010. Wlhx9 as the available sequence data for this region in the Xenopus tropicalis genome is incomplete. Instead, we employed an alternate strategy to determine whether Fezf2 directly influences the transcriptional activity of lhx9. Antimorphic VP16-Fezf2, if activated, should be able to trigger the expression of Fezf2 direct target genes, even in the absence of protein synthesis. Hence, we made a VP16-Fezf2 construct fused to the 3′-end of human glucocorticoid receptor (hGR) (termed p3hGR-VP16-Fezf2), which can be activated by the addition of dexamethasone (DEX) . Furthermore, we were able to show that the potent protein synthesis inhibitor cycloheximide (CHX) . Importantly, however, treatment of animals caps overexpressing p3hGR-VP16-Fezf2 with both CHX and DEX led to 2-fold and 6-fold increases in the expression levels of lhx2 and lhx9, respectively  . Reassurde (CHX)  had no eectively B,C. Thuslhx2 and/or lhx9 act downstream of fezf2 during forebrain development. Both lhx2 and lhx9 are expressed in the anterior neural ectoderm . Animal cap explants neuralised by chd and aged to stage 20 expressed significant levels of both lhx2 and lhx9 compared with control lacZ-injected embryos, indicating that these explants recapitulate anterior neuroectoderm . However, the expression of both lhx2 and lhx9 was inhibited by co-expressing fezf2 in chd-neuralised animal cap explants .We next assessed whether explants D,E, sugglhx2 and lhx9  and examined whether the activity of fezf2 could be rescued by simultaneously knocking down both lhx2 and lhx9. We found that, whereas most embryos injected with fezf2 MO displayed reduced expression of arx, embryos injected with fezf2 and lhx2/lhx9 MOs showed partial rescue in the expression of arx at stage 28 . Furthermore, no significant changes in arx expression were observed in embryos injected with lhx2/lhx9 MOs alone   . Hence, lhx2 and lhx9 act as an intermediary in the ability of Fezf2 to activate ngn1 expression in the forebrain. Whereas fezf2 morphant embryos were almost devoid of ngn1 expression in the forebrain . We also found that, although fezf2 overexpression in chd-neuralised explants induced the expression of the Wnt-responsive gene xnr3  experiments using the pTransgenesis recombination system and associated cloning primers are provided in supplementary Materials and Methods and Table S2.Details of lacZ mRNA co-injected as a tracer in some cases . For further details see supplementary Materials and Methods.Microinjection of mRNA was performed as described previously, with X. tropicalis genes were supplied by Gene Tools. Typically, 10 ng MO was injected per X. tropicalis embryo at the 1- to 2-cell stage. Further details, including MO sequences, are provided in the supplementary Materials and Methods.MOs designed against Xenopus embryos followed by electric pulses. Electroporated embryos were harvested at stage 30 for analysis.Electroporation was performed as described . Brieflyin situ hybridisation were prepared by T7 RNA polymerase-mediated transcription (Roche). X-Gal staining and in situ hybridisation were carried out as previously described .Antisense digoxigenin-labelled RNA probes for whole-mount Xenopus embryos were cryosectioned for immunofluorescence . Mouse c17.2 cells were grown in Lab-TEK II chambered slides (NUNC) and fixed with MEMFA. Details of c17.2 cell culture and transfection are provided in the supplementary Materials and Methods. Primary antibodies were: anti-Sox3  (Fixed  1:1000) , anti-My 1:1000)  and mousfezf2 or control lacZ mRNA. Injected Xenopus embryos were collected at stage 10.5 and analysed with the DLR system (Promega). For further details see supplementary Materials and Methods.Briefly, 50 pg pTK-Renilla and 100 pg M50 TOPFlash  or M51 FOPFlash   were co-XbaI-NotI restriction sites  were excised at stage 8 and allowed to develop until stage 12. A final concentration of 5 µg/ml CHX with or without 2 µM DEX in ethanol was used. Carrier alone (0.05% ethanol) was used as control. Animal cap explants were collected 2 h post treatment (The DEX-inducible VP16-Fezf2 construct was made by fusing the VP16-Fezf2 protein to the 3′-end of human glucocorticoid receptor (hGR) using on sites . 500 pg reatment .X. laevis embryos was determined by western blot analysis as described in the supplementary Materials and Methods.The phosphorylation status of signalling molecules in gastrula stage X. tropicalis embryos were injected with 50 pg FLAG-tagged fezf2 mRNA, harvested at stage 15, crosslinked with 3.7% formaldehyde for 15 min and stored at −80°C until use. Approximately 300 embryos were used for each sample. Fezf2-binding fragments were enriched using anti-FLAG M2 antibody (Sigma) as described  was performed using a modification of published methods . Brieflyescribed . DNA regn is the number of individual embryos from at least three independent fertilisations and injections. For qPCR analyses, collected animal cap explants from individual experiments were pooled for RNA extraction, and all data were from at least three independent experiments (n=3), unless otherwise indicated. Statistical analysis was performed using GraphPad Prism software with either two-tailed unpaired Student's t-test (for two samples) or two-tailed unpaired one-way ANOVA (for multiple samples) and s.e.m. was calculated.For Sox3, MyT1 and TUNEL assays, positive cells were counted on two consecutive sections in the corresponding brain area for determination of the mean ; n is th


Adult skeletal muscle regeneration is a highly orchestrated process involving the activation and proliferation of satellite cells, an adult skeletal muscle stem cell. Activated satellite cells generate a transient amplifying progenitor pool of myoblasts that commit to differentiation and fuse into multinucleated myotubes. During regeneration, canonical Wnt signalling is activated and has been implicated in regulating myogenic lineage progression and terminal differentiation.Here, we have undertaken a gene expression analysis of committed satellite cell-derived myoblasts to examine their ability to respond to canonical Wnt/β-catenin signalling.in vivo promoted premature differentiation during muscle regeneration following acute injury.We found that activation of canonical Wnt signalling induces follistatin expression in myoblasts and promotes myoblast fusion in a follistatin-dependent manner. In growth conditions, canonical Wnt/β-catenin signalling prime myoblasts for myogenic differentiation by stimulating myogenin and follistatin expression. We further found that myogenin binds elements in the follistatin promoter and thus acts downstream of myogenin during differentiation. Finally, ectopic activation of canonical Wnt signalling Together, these data reveal a novel mechanism by which myogenin mediates the canonical Wnt/β-catenin-dependent activation of follistatin and induction of the myogenic differentiation process.The online version of this article (doi:10.1186/s13395-015-0038-6) contains supplementary material, which is available to authorized users. The growth, maintenance, and regeneration of skeletal muscle is attributed to the satellite cell; a mitotically quiescent cell that resides between the basal lamina and sarcolemma of the muscle fiber -3. DurinIn vivo, overexpression of Wnt3a in the tibialis anterior (TA) muscle results in an increased number of myofibers, which exhibit a dramatic reduction in the cross-sectional area and reduced regenerative efficiency. In vitro, canonical Wnt treatment of satellite cell-derived myoblasts inhibits cellular proliferation, a characteristic not observed by non-canonical Wnt treatment [Skeletal muscle regeneration is regulated through mechanisms involving cellular interactions and extracellular signalling pathways. The Wnt signalling pathway has been shown to play a critical role in regulating various developmental programs through embryonic development and in the regulation of stem cell function in adult tissues . During reatment . In addireatment ,15.To further investigate the role of canonical Wnt signalling in adult myogenesis, we analyzed the response of satellite cell-derived myoblasts to Wnt/β-catenin signalling. We show that canonical Wnt signalling is associated with committed satellite cell differentiation potential and control of follistatin expression. Investigation of the role for a Wnt-follistatin pathway leads us to discover that Wnt3a/β-catenin signalling controls the induction of myogenic differentiation via the MyoD-dependent activation of myogenin expression. These findings suggest that Wnt/β-catenin signalling controls multiple steps of adult myogenesis by promoting premature myogenic differentiation in a myogenin-dependent manner.Primary myoblasts were isolated from the hind limbs of 4- to 6-week-old wild-type C57Bl/6J mice, as previously described , and proCare of animals is in accordance with institutional guidelines as regulated by the Canadian Council of Animal Care (CCAC). Protocols were approved by Animal Research Ethics Board (AREB) at the University of Ottawa and are reviewed on an annual basis. Eight- to 12-week-old wild-type C57Bl/6J mice were used in this study. The TA muscle was injured by injection of 50 μl cardiotoxin . -6-Bromoindirubin-3′-oxime (BIO) was reconstituted in DMSO at a concentration of 5 mM  combined with saline as vehicle and injected directly into the TA muscle in a volume of 10 μl at a final concentration of 1 μM.Affymetrix MoGene 1.0 ST chipsets  were RMA normalized  using thhttp://primer3.wi.mit.edu) [Total RNA was isolated using the RNeasy Kit and subjected to on-column DNase digestion, as per manufacturer’s instructions . cDNA synthesis was performed using the Superscript III reverse transcriptase with random hexamer primers (Invitrogen). SYBR Green quantitative polymerase chain reaction (qPCR) was carried out as previously described . Transcrmit.edu) . Primer Total protein was harvested in RIPA lysis buffer fortified with protease inhibitors , and protein concentration was determined by Bradford assay . Samples (20 μg) were subjected to SDS-PAGE and electroblotted onto Immobilon-P membrane . Membranes were blocked in 5% nonfat milk in PBST, prior to sequential probing with primary antibody and HRP-conjugated secondary antibody in blocking solution. ECL  with BioMax XAR film  was used to detect target proteins. Primary antibodies used for detection are as follows: α-Pax7 , α-myogenin , α-follistatin , and α-Tubulin (Sigma). Secondary antibodies were HRP-conjugated anti-mouse and anti-rabbit .Adherent cell cultures were fixed with 4% PFA, permeabilized with 0.2% Triton-X in PBS, and incubated with primary antibodies: myogenin (M-225) from Santa Cruz , α-MyHC , and α-Laminin (Sigma). Alexa-488 and Alexa-555 conjugated secondary antibodies that matched the primary antibodies were used at 1:1,000 in PBS (Invitrogen). Nuclei were counterstained with DAPI (Sigma-Aldrich). Images were obtained using an Axioplan2 microscope , a ×20 NA 0.75 plan Apochromat  objective, and a digital Axiocam camera (Carl Zeiss). Digital images were captured using Axiovision (Carl Zeiss) and were processed with Photoshop .Protein-DNA complexes were cross-linked with 1% formaldehyde (Sigma) and sheared by sonication. Processing of samples was performed as previously described ; 2,000 μT-test. Differences were considered statistically significant at the P < 0.05 level.Three or more replicates were analyzed for each experiment presented. Data is shown as standard error of the mean (SEM), and results were assessed for statistical significance by Student’s P value <0.05); 90 genes were upregulated, while 311 genes were downregulated.Multiple Wnt signals are active during muscle regeneration . To furtTo investigate the global transcriptional effects of Wnt3a treatment, we conducted an unbiased DAVID analysis of our microarray data. Functional annotation clustering of related gene ontology (GO) terms revealed that Wnt3a stimulation strongly regulated cell cycle, cell division, and the progression of mitosis   Figure B. Furthe) Figure C,D.FigurTo validate that follistatin acts downstream of Wnt/β-catenin signalling during myogenic differentiation, we first determined the optimal concentration of siRNA against follistatin for transfection in the presence of Wnt3a to reduce follistatin expression to control levels (transfected with non-silencing Scrambled siRNAs) Figure E, left. A recent report demonstrated that myogenin expression represses genes involved in cell cycle progression, and is sufficient to induce exit from the cell cycle . Since wunpublished data). Interestingly, a recent publication from our lab identifies this element as being occupied during proliferation not by myogenin but rather by the Snail-HDAC1/2 repressive complex inhibiting myogenin from accessing this E-box motif [Previous data revealed that β-catenin directly interacts with MRFs enhancing their ability to bind E-box regulator elements and ultimately promoting muscle differentiation . More reox motif . We furtin vitro, canonical Wnt stimulation of follistatin occurs in a myogenin-dependent manner and leads to precocious myogenic differentiation.To demonstrate that follistatin acts downstream of myogenin expression, we first determined the optimal concentration of siRNA against myogenin for transfection in the presence of Wnt3a to reduce myogenin expression similar to control levels (BSA treatment transfected with non-silencing Scrambled siRNAs) (Additional file in vivo, we treated regenerating muscle with a small molecule agonist of canonical Wnt signalling (Figure in vitro data, we observed an overall increase in myogenin and follistatin expression, as well as an increase in Myh2 (Figure in vivo stimulation of β-catenin activity promotes myogenic differentiation.To investigate the role of the Wnt-follistatin pathway in muscle regeneration g Figure A. Previo2 Figure B. These in vitro observations as treatment of regenerating skeletal muscle with a canonical Wnt signalling agonist reduces its regenerative potential through initiating premature myogenic differentiation. Taken together, these observations suggest canonical Wnt signalling promotes differentiation by stimulating the expression of myogenin and follistatin in vivo.Following the direct delivery of the small molecule Wnt agonist BIO, we observed altered muscle fiber morphology Figure C, resultCanonical Wnt signalling is essential for proper myogenic development and regeneration. However, in aged skeletal muscle, excessive Wnt/β-catenin signalling results in precocious differentiation of satellite cells into the fibroblast lineage, resulting in a depletion of the satellite cell pool and inefficient muscle regeneration ,14. FurtIn situ analysis of satellite cells on single myofibers identified an increase in the percentage of satellite myogenic progenitors that express myogenin. Following Wnt3a treatment myoblasts induced to differentiate displayed enhanced fusion (Figure Interestingly, we observed that Wnt3a promoted cell cycle arrest and activation of myogenin - a core MRF transcription factor responsible for fusion and terminal differentiation Figure . In situn Figure B,C, an ein vitro [Among the genes upregulated following Wnt3a stimulation of proliferating myoblasts, the myogenic regulatory factor myogenin plays a prominent role in regulating myoblast fusion and terminal differentiation. While myogenin expression is normally upregulated during differentiation, we observed a strong increase of myogenin protein following Wnt3a stimulation (Figure in vitro , and theMyogenic differentiation is a highly orchestrated process, with cell cycle withdrawal occurring before the expression of contractile proteins or cell fusion ,42. MyogWhile its transcriptional activity is generally associated with TCF/Lef proteins, recent studies are establishing a potential TCF/Lef-independent function of β-catenin. As discussed, MyoD is able to complex with β-catenin independent of E-proteins , whose iFollistatin is a secreted glycoprotein that antagonizes various members of the TGF-β superfamily, including myostatin ,53. OrigIn the absence of exogenous canonical Wnt stimulation, myogenin is bound to the follistatin locus early during differentiation and is presumably responsible for the rapid follistatin expression observed following 24 h of myogenic differentiation Figure D. We preUpon Wnt3a stimulation, we observe myogenin binding to an E-box element not occupied during proliferation conditions (Figure Following injury-induced regeneration, we observed that treatment with a GSK3β antagonist further potentiated the differentiation effects of canonical Wnt signalling Figure B,C. SpecCanonical Wnt/β-catenin signalling promotes myogenic differentiation. The mechanism by which this occurs was previously thought to involve direct binding of β-catenin to the follistatin promoter . Our exp