Component Of Wnt Signaling Pathway Research Articles

Downregulation of the Canonical WNT Signaling Pathway by TGFβ1 Inhibits Photoreceptor Differentiation of Adult Human Müller Glia with Stem Cell Characteristics.

Müller glia are responsible for the retina regeneration observed in zebrafish. Although the human retina harbors Müller glia with stem cell characteristics, there is no evidence that they regenerate the retina after disease or injury. Transforming growth factor-β (TGFβ) and Wnt signaling regulate retinal neurogenesis and inflammation, but their roles in the neural differentiation of human Müller stem cells (hMSC) are not known. We examined hMSC lines in vitro for the expression of various Wnt signaling components and for their modulation by TGFβ1, as well as the effect of this cytokine on the photoreceptor differentiation of these cells. Culture of hMSC with a combination of factors that induce photoreceptor differentiation of hMSC (FGF2, taurine, retinoic acid, and insulin-like growth factor type1; FTRI), markedly upregulated the expression of components of the canonical Wnt signaling pathway, including WNT2B, DKK1, and active β-CATENIN. Although FTRI did not modify mRNA expression of WNT5B, a component of the noncanonical/planar cell polarity Wnt pathway, it upregulated its secretion. Furthermore, TGFβ1 not only decreased WNT2B expression, but also inhibited FTRI-induced photoreceptor differentiation of hMSC, as determined by expression of the photoreceptor markers NR2E3, RHODOPSIN, and RECOVERIN. Inhibition of TGFβ1 signaling by an ALK5 inhibitor prevented TGFβ1-induced changes in the expression of the two Wnt ligands examined. More importantly, inhibition of the canonical WNT signaling by XAV-939 prevented FTRI-induced photoreceptor differentiation. These observations suggest that TGFβ may play a key role in preventing neural differentiation of hMSC and may constitute a potential target for induction of endogenous regeneration of the human retina.

Abstract 1042: Suppression of colorectal cancer cell proliferation by resveratrol involves upregulation of the non-canonical ligand Wnt9a

Abstract Our previous studies showed that the suppression of colorectal cancer (CRC) cell proliferation by resveratrol (RV) was accompanied by a disproportionate increase of the non-canonical Wnt signaling pathway (N-CAN) components relative to the canonical Wnt signaling pathway (CAN) components. Wnt signaling, which consists of CAN and N-CAN is an intricate ensemble of signaling components involved with various cellular processes. The CAN utilizes β-catenin and stimulates cell proliferation whereas the N-CAN is independent of β-catenin and is instrumental in cell differentiation. These opposing mechanisms act in concert to maintain homeostasis in healthy tissues which is dysregulated in cancer. These observations have led us to propose a novel hypothesis that suppression of CRC proliferation may be mediated by the activation of the non-canonical Wnt pathway by nutritional supplements (NNS) such as RV. As far as we know, no known studies have determined the effects of RV on the non-canonical pathways in human CRC. The aim of this study was to determine whether suppression of CRC cell proliferation by RV involves non-canonical Wnt pathway components. CRC cells derived from surgical specimens (n = 5) were treated with RV (5ug/ml) for 72h. Cell proliferation was determined using MTS assay and gene expression was performed using commercially available quantitative RT-PCR Wnt pathway gene expression arrays. RV significantly increased N-CAN ligand Wnt A and Wnt 11(p<0.01) and CAN ligand Wnt 10A (p<0.05) by 18, 9 and 4-folds, respectively. RV treatment resulted in a significant decrease of CRC cell proliferation (56% inhibition; p<0.01) compared to untreated cells. This inhibition was partially reversed by IWP-2, a Wnt ligand production inhibitor. Conditioned media from CRC cells treated with RV significantly suppressed CRC cell proliferation (p<0.05) compared to cells treated with conditioned media from untreated cells and that from cells treated with conditioned media from RV+IWP treated cells. Proliferation was not different in cells treated with conditioned media from untreated cells, IWP-2 or RV+IWP-2-treated cells. Wnt 9A antibody partially reversed the RV-mediated suppression of CRC cell proliferation relative to cells treated with IgG controls. To the best of our knowledge this is the first known study to show the involvement of a non-canonical Wnt pathway ligand, Wnt9A, in the suppression of CRC proliferation mediated by a NNS, resveratrol. This study suggests the presence of a novel mechanism for the suppression of CRC proliferation which entails activation of the non-canonical pathway. Given the excellent tolerance profile of NNS such as RV, the capacity of such agents to activate the non-canonical pathway to inhibit tumor proliferation, offers the opportunity to investigate the effects of chronic administration of these agents on tumor growth and progression in patients. Citation Format: Irshad Ali, Bani M. Fagla, Donald P. Braun. Suppression of colorectal cancer cell proliferation by resveratrol involves upregulation of the non-canonical ligand Wnt9a. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1042. doi:10.1158/1538-7445.AM2015-1042

Wnt/β-catenin signaling of cartilage canal and osteochondral junction chondrocytes and full thickness cartilage in early equine osteochondrosis.

The objective of this study was to elucidate gene and protein expression of Wnt signaling molecules in chondrocytes of foals having early osteochondrosis (OC) versus normal controls. The hypothesis was that increased expression of components of Wnt signaling pathway in osteochondral junction (OCJ) and cartilage canal (CC) chondrocytes would be found in early OC when compared to controls. Paraffin-embedded osteochondral samples (7 OC, 8 normal) and cDNA from whole cartilage (7 OC, 10 normal) and chondrocytes surrounding cartilage canals and osteochondral junctions captured with laser capture microdissection (4 OC, 6 normal) were obtained from femoropatellar joints of 17 immature horses. Equine-specific Wnt signaling molecule mRNA expression levels were evaluated by two-step real-time qPCR. Spatial tissue protein expression of β-catenin, Wnt-11, Wnt-4, and Dkk-1 was determined by immunohistochemistry. There was significantly decreased Wnt-11 and increased β-catenin, Wnt-5b, Dkk-1, Lrp6, Wif-1, Axin1, and SC-PEP gene expression in early OC cartilage canal chondrocytes compared to controls. There was also significantly increased β-catenin gene expression in early OC osteochondral junction chondrocytes compared to controls. Based on this study, abundant gene expression differences in OC chondrocytes surrounding cartilage canals suggest pathways associated with catabolism and inhibition of chondrocyte maturation are targeted in early OC pathogenesis.

Identification ofin vivoDNA-binding mechanisms of Pax6 and reconstruction of Pax6-dependent gene regulatory networks during forebrain and lens development

The transcription factor Pax6 is comprised of the paired domain (PD) and homeodomain (HD). In the developing forebrain, Pax6 is expressed in ventricular zone precursor cells and in specific subpopulations of neurons; absence of Pax6 results in disrupted cell proliferation and cell fate specification. Pax6 also regulates the entire lens developmental program. To reconstruct Pax6-dependent gene regulatory networks (GRNs), ChIP-seq studies were performed using forebrain and lens chromatin from mice. A total of 3514 (forebrain) and 3723 (lens) Pax6-containing peaks were identified, with ∼70% of them found in both tissues and thereafter called ‘common’ peaks. Analysis of Pax6-bound peaks identified motifs that closely resemble Pax6-PD, Pax6-PD/HD and Pax6-HD established binding sequences. Mapping of H3K4me1, H3K4me3, H3K27ac, H3K27me3 and RNA polymerase II revealed distinct types of tissue-specific enhancers bound by Pax6. Pax6 directly regulates cortical neurogenesis through activation (e.g. Dmrta1 and Ngn2) and repression (e.g. Ascl1, Fezf2, and Gsx2) of transcription factors. In lens, Pax6 directly regulates cell cycle exit via components of FGF (Fgfr2, Prox1 and Ccnd1) and Wnt (Dkk3, Wnt7a, Lrp6, Bcl9l, and Ccnd1) signaling pathways. Collectively, these studies provide genome-wide analysis of Pax6-dependent GRNs in lens and forebrain and establish novel roles of Pax6 in organogenesis.

Systemic administration of lithium improves distracted bone regeneration in rats.

Lithium, popular in psychology field, has been recognized as an activator component of the canonical Wnt signaling pathway. The effect of lithium on osteogenesis or on the human fracture risk has been widely reported. However, little is known on its role in distraction osteogenesis to date. In this study, the effect of systematic administrated lithium on distraction osteogenesis in a rat model was investigated. The osteotomy was performed on the right tibia in 40 adult male Sprague-Dawley rats. Then they were randomly assigned into two equal groups (n = 20/group), which underwent Lithium or saline treatment through gastric gavage until the day they were killed. One week after the osteotomy, the tibias were distracted for 14 days (rate 0.6 mm/day). Following 8 weeks consolidation period, the distracted tibias in both groups were harvested and examined by X-ray plain radiography, histology, dual-energy X-ray absorptiometry, Micro-CT, and biomechanical tests. The results showed that lithium group possessed higher bone mineral density, more mature new bone tissue, and better regenerated bone mass continuity in the distraction gaps without any local or systemic adverse effects was encountered. This study suggested lithium could increase bony callus ossification volume and accelerate distracted tissue mineralization to facilitate bone regeneration in distraction gap.

Transposon mutagenesis identifies genetic drivers of Braf(V600E) melanoma.

Although nearly half of human melanomas harbor oncogenic BRAF(V600E) mutations, the genetic events that cooperate with these mutations to drive melanogenesis are still largely unknown. Here we show that Sleeping Beauty (SB) transposon-mediated mutagenesis drives melanoma progression in Braf(V600E) mutant mice and identify 1,232 recurrently mutated candidate cancer genes (CCGs) from 70 SB-driven melanomas. CCGs are enriched in Wnt, PI3K, MAPK and netrin signaling pathway components and are more highly connected to one another than predicted by chance, indicating that SB targets cooperative genetic networks in melanoma. Human orthologs of >500 CCGs are enriched for mutations in human melanoma or showed statistically significant clinical associations between RNA abundance and survival of patients with metastatic melanoma. We also functionally validate CEP350 as a new tumor-suppressor gene in human melanoma. SB mutagenesis has thus helped to catalog the cooperative molecular mechanisms driving BRAF(V600E) melanoma and discover new genes with potential clinical importance in human melanoma.

Mutations in DVL1 Cause an Osteosclerotic Form of Robinow Syndrome

Robinow syndrome (RS) is a phenotypically and genetically heterogeneous condition that can be caused by mutations in genes encoding components of the non-canonical Wnt signaling pathway. In contrast, germline mutations that act to increase canonical Wnt signaling lead to distinctive osteosclerotic phenotypes. Here, we identified de novo frameshift mutations in DVL1, a mediator of both canonical and non-canonical Wnt signaling, as the cause of RS-OS, an RS subtype involving osteosclerosis, in three unrelated individuals. The mutations all delete the DVL1 C terminus and replace it, in each instance, with a novel, highly basic sequence. We showed the presence of mutant transcript in fibroblasts from one individual with RS-OS and demonstrated unimpaired protein stability with transfected GFP-tagged constructs bearing a frameshift mutation. Invitro TOPFlash assays, in apparent contradiction to the osteosclerotic phenotype, revealed that the mutant allele was less active than the wild-type allele in the canonical Wnt signaling pathway. However, when the mutant and wild-type alleles were co-expressed, canonical Wnt activity was 2-fold higher than that in the wild-type construct alone. This work establishes that DVL1 mutations cause a specific RS subtype, RS-OS, and that the osteosclerosis associated with this subtype might be the result of an interaction between the wild-type and mutant alleles and thus lead to elevated canonical Wnt signaling.

MiR-577 inhibits glioblastoma tumor growth via the Wnt signaling pathway.

microRNAs (miRNAs) are commonly altered in glioblastoma. Publicly available algorithms suggest the Wnt pathway is a potential target of miR-577 and the Wnt pathway is commonly altered in glioblastoma. Glioblastoma has not been previously evaluated for miR-577 expression. Glioblastoma tumors and cell lines were evaluated for their expression of miR-577. Cell lines were transfected with miR-577, miR-577-mutant, or control mimics to evaluate the effect of miR-577 expression on cell proliferation in vitro and in an animal model. Wnt pathway markers were also evaluated for their association with miR-577 expression. miR-577 expression was decreased in 33 of 40 (82.5%) glioblastoma tumors and 5 of 6 glioblastoma cell lines. miR-577 expression correlated negatively with cell growth and cell viability. miR-577 down-regulation was associated with increased expression of the Wnt signaling pathway genes lipoprotein receptor-related protein (LRP) 6 (LRP6) and β-catenin. Western blot analysis confirmed decreased expression of the Wnt signaling pathway genes Axin2, c-myc, and cyclin D1 in miR-577 transfected cells. miR-577 expression is down-regulated in glioblastoma. miR-577 directly targets Wnt signaling pathway components LRP6 and β-catenin. miR-577 suppresses glioblastoma multiforme (GBM) growth by regulating the Wnt signaling pathway.

MiRNA-320 in the human follicular fluid is associated with embryo quality in vivo and affects mouse embryonic development in vitro.

Previous work from our laboratory demonstrated the existence of miRNAs in human follicular fluid. In the current study, we have sought to identify miRNAs that might affect oocyte/embryo quality in patients undergoing intracytoplasmic sperm injection and to investigate their roles in in vitro fertilization outcomes in mouse oocytes. 53 samples were classified as Group 1 (high quality) if the day-3 embryos had seven and more cells or as Group 2 (low quality) if the embryos had six and fewer cells. TaqMan Human microRNAs cards and qRT-PCR were performed to verify differently expressed miRNAs. The function of the corresponding miRNA was investigated in mouse oocytes by injecting them with miRNA-inhibitor oligonucleotides. We found that hsa-miR-320a and hsa-miR-197 had significantly higher expression levels in the Group 1 follicular fluids than in Group 2 (p = 0.0073 and p = 0.008, respectively). Knockdown of mmu-miR-320 in mouse oocytes strongly decreased the proportions of MII oocytes that developed into two-cell and blastocyst stage embryos (p = 0.0048 and p = 0.0069, respectively). Wnt signaling pathway components had abnormal expression level in miR-320 inhibitor-injected oocytes. This study provides the first evidence that miRNAs in human follicular fluid are indicative of and can influence embryo quality.

Baicalin can attenuate the inhibitory effects of mifepristone on Wnt pathway during peri-implantation period in mice

Wnts are considered as important factors in uterus developmental process and embryo implantation. Baicalin has been demonstrated to possess tocolytic properties. In order to investigate the effect of baicalin on the Wnt signaling pathway during the peri-implantation, pregnant Kuming mice were randomly divided into four groups: control group, baicalin group administered with 40mg/kg BW of baicalin through an intragastric gavage on day 2 to 7 of the pregnancy (Pd2–Pd7), mifepristone group treated with 4mg/kg BW of mifepristone, an abortifacient agent, via subcutaneous administration on Pd4, and baicalin+mifepristone group treated with their combination. The concentrations of the implantation-related steroid hormones (progesterone and estradiol) in the blood serum were measured with RIA. The gene and protein expression levels of the important molecules of the Wnt pathway (Wnt4, LRP6, Dkk1 and β-catenin) in the endometrium were detected with RT-PCR and western blot, respectively. The results showed that baicalin decreased (P<0.05) the estradiol levels on Pd4–Pd8 and increased (P<0.05) the progesterone levels on Pd3–Pd8. Mifepristone increased (P<0.05) the estradiol levels on Pd5–Pd8 and decreased (P<0.05) the progesterone levels on Pd6–Pd8. Compared with the control group, baicalin increased the gene and protein expression levels of Wnt4, LRP6 and β-catenin (P<0.05) and decreased the gene and protein expression levels of Dkk1 (P<0.05) during the middle-to-late stage of the experiment in mice uterine tissue. Baicalin alleviated the mifepristone-induced increase or decrease in the serum levels of progesterone and estradiol, and the gene or protein expression levels of Wnt4, LRP6 and β-catenin. The tocolytic properties tocolysis of baicalin may be realized through regulating the levels of estrogen/progesterone and the important components of canonical Wnt signaling pathway during the embryo implantation process intervened with the subcutaneous administration of mifepristone in the mice.

Acute Wnt pathway activation positively regulates leptin gene expression in mature adipocytes

Genome-wide association studies (GWAS) have revealed the implication of several Wnt signaling pathway components, including its effector transcription factor 7-like 2 (TCF7L2) in diabetes and other metabolic disorders. As TCF7L2 is expressed in adipocytes, we investigated its expression and function in rodent fat tissue and mature adipocytes. We found that TCF7L2 mRNA expression in C57BL/6 mouse epididymal fat tissue was up-regulated by feeding but down-regulated by intraperitoneal insulin injection. In high-fat diet (HFD) fed mice, db/db mice and Zucker (fa/fa) rats, epididymal fat TCF7L2 mRNA levels were lower than the corresponding controls. Treating rat adipocytes with 100nM insulin repressed TCF7L2 mRNA and protein levels, associated with the repression of leptin mRNA level. The treatment with 1nM insulin, however, stimulated TCF7L2 and leptin mRNA levels. This stimulation could be attenuated by iCRT14, an inhibitor of β-catenin/TCF-responsive transcription. Wnt3a stimulated leptin mRNA level, which was also blocked by iCRT14 co-treatment. Utilizing the leptin-expressing cell line HTR8 as a tool, we defined an evolutionarily conserved CREB binding motif that mediated Wnt3a activation. Although Wnt activation is known to repress the differentiation of 3T3-L1 cells towards mature adipocytes, short-term Wnt3a treatment of differentiated 3T3-L1 cells stimulated leptin mRNA levels. Thus, wnt pathway plays a dual function in adipocytes, including the well-known repressive effect on adipogenesis and the stimulation of leptin production in mature adipocytes in response to nutritional status.

Association between polymorphisms in AXIN1 gene and atrial septal defect

Context: AXIN1 is a central component of Wnt signalling pathway which is essential for embryonic development.Objective: To investigate whether polymorphisms of AXIN1 contribute to ASD susceptibility.Materials and methods: Three tag SNPs (rs12921862, rs370681 and rs1805105) in AXIN1 were genotyped in 208 ASD patients and 302 healthy controls using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) in a Chinese population.Results: Significantly increased ASD risk was observed to be associated with the A allele of rs12921862 (p < 0.0001, OR = 3.096, 95% CI = 2.037–4.717). Increased ASD risk was observed to be associated with rs370681 in a codominant (p = 0.043, OR = 1.52, 95% CI = 1.04–2.22) and overdominant model (p = 0.016, OR = 1.57, 95% CI = 1.08–2.27).Conclusion: rs12921862 and rs370681 may contribute to ASD susceptibility.

De novo mutations in beta-catenin (CTNNB1) appear to be a frequent cause of intellectual disability: expanding the mutational and clinical spectrum.

Recently, de novo heterozygous loss-of-function mutations in beta-catenin (CTNNB1) were described for the first time in four individuals with intellectual disability (ID), microcephaly, limited speech and (progressive) spasticity, and functional consequences of CTNNB1 deficiency were characterized in a mouse model. Beta-catenin is a key downstream component of the canonical Wnt signaling pathway. Somatic gain-of-function mutations have already been found in various tumor types, whereas germline loss-of-function mutations in animal models have been shown to influence neuronal development and maturation. We report on 16 additional individuals from 15 families in whom we newly identified de novo loss-of-function CTNNB1 mutations (six nonsense, five frameshift, one missense, two splice mutation, and one whole gene deletion). All patients have ID, motor delay and speech impairment (both mostly severe) and abnormal muscle tone (truncal hypotonia and distal hypertonia/spasticity). The craniofacial phenotype comprised microcephaly (typically -2 to -4 SD) in 12 of 16 and some overlapping facial features in all individuals (broad nasal tip, small alae nasi, long and/or flat philtrum, thin upper lip vermillion). With this detailed phenotypic characterization of 16 additional individuals, we expand and further establish the clinical and mutational spectrum of inactivating CTNNB1 mutations and thereby clinically delineate this new CTNNB1 haploinsufficiency syndrome.

Tanshinone IIA pretreatment renders free flaps against hypoxic injury through activating Wnt signaling and upregulating stem cell-related biomarkers.

Partial or total flap necrosis after flap transplantation is sometimes clinically encountered in reconstructive surgery, often as a result of a period of hypoxia that exceeds the tolerance of the flap tissue. In this study, we determine whether tanshinone IIA (TSA) pretreatment can protect flap tissue against hypoxic injury and improve its viability. Primary epithelial cells isolated from the dorsal skin of mice were pretreated with TSA for two weeks. Cell counting kit-8 and Trypan Blue assays were carried out to examine the proliferation of TSA-pretreated cells after exposure to cobalt chloride. Then, Polymerase chain reaction and Western blot analysis were used to determine the expression of β-catenin, GSK-3β, SOX2, and OCT4 in TSA-treated cells. In vivo, after mice were pretreated with TSA for two weeks, a reproducible ischemic flap model was implemented, and the area of surviving tissue in the transplanted flaps was measured. Immunohistochemistry was also conducted to examine the related biomarkers mentioned above. Results show that epidermal cells, pretreated with TSA, showed enhanced resistance to hypoxia. Activation of the Wnt signaling pathway in TSA-pretreated cells was characterized by the upregulation of β-catenin and the downregulation of GSK-3β. The expression of SOX2 and OCT4 controlled by Wnt signaling were also found higher in TSA pretreated epithelial cells. In the reproducible ischaemic flap model, pretreatment with TSA enhanced resistance to hypoxia and increased the area of surviving tissue in transplanted flaps. The expression of Wnt signaling pathway components, stem-cell related biomarkers, and CD34, which are involved in the regeneration of blood vessels, was also upregulated in TSA-pretreated flap tissue. The results show that TSA pretreatment protects free flaps against hypoxic injury and increases the area of surviving tissue by activating Wnt signaling and upregulating stem cell-related biomarkers.

The cellular story of dishevelleds.

Dishevelled (DVL) proteins, three of which have been identified in humans, are highly conserved components of canonical and noncanonical Wnt signaling pathways. These multifunctional proteins, originally discovered in the fruit fly, through their different domains mediate complex signal transduction: DIX (dishevelled, axin) and PDZ (postsynaptic density 95, discs large, zonula occludens-1) domains serve for canonical beta-catenin signaling, while PDZ and DEP (dishevelled, Egl-10, pleckstrin) domains serve for non-canonical signaling. In canonical or beta-catenin signaling, DVL forms large molecular supercomplexes at the plasma membrane consisting of Wnt-Fz-LRP5/6-DVL-AXIN. This promotes the disassembly of the beta-catenin destruction machinery, beta-catenin accumulation, and consequent activation of Wnt signaling. Therefore, DVLs are considered to be key regulators that rescue cytoplasmic beta-catenin from degradation. The potential medical importance of DVLs is in both human degenerative disease and cancer. The overexpression of DVL has been shown to potentiate the activation of Wnt signaling and it is now apparent that up-regulation of DVLs is involved in several types of cancer.

Tanshinone IIA pretreatment protects free flaps against hypoxic injury by upregulating stem cell-related biomarkers in epithelial skin cells.

BackgroundPartial or total flap necrosis after flap transplantation is sometimes encountered in reconstructive surgery, often as a result of a period of hypoxia that exceeds the tolerance of the flap tissue. The purpose of this study was to determine whether Tanshinone IIA (TSA) pretreatment can protect flap tissue against hypoxic injury and improve its viability.MethodsPrimary epithelial cells isolated from the dorsal skin of mice were pretreated with TSA for 2 weeks. Cell Counting Kit-8 and Trypan Blue assays were carried out to examine the proliferation of TSA-pretreated cells after exposure to cobalt chloride. Polymerase chain reaction and western blot analysis were used to assess the expression of β-catenin, vascular endothelial growth factor (VEGF), sex determining region Y-box 2 (SOX2), OCT4 (also known as POU domain class 5 transcription factor 1), Nanog, and glycogen synthase kinase-3 beta (GSK-3β) in TSA-treated cells. In other experiments, after mice were pretreated with TSA for 2 weeks, a reproducible ischemic flap model was implemented, and the area of surviving tissue in the transplanted flaps was measured. Immunohistochemistry was conducted to examine Wnt signaling as well as stem cell- and angiogenesis-related biomarkers in epithelial tissue in vivo.ResultsEpidermal cells, pretreated with TSA, showed enhanced resistance to hypoxia. Activation of the Wnt signaling pathway in TSA-pretreated cells was characterized by the upregulation of β-catenin and the downregulation of GSK-3β. The expression of SOX2, Nanog, and OCT4 were also higher in TSA-pretreated epithelial cells than in control cells. In the reproducible ischemic flap model, pretreatment with TSA enhanced resistance to hypoxia and increased the area of surviving tissue in transplanted flaps. The expression of Wnt signaling pathway components, stem-cell related biomarkers, and VEGF and CD34, which are involved in the regeneration of blood vessels, was also upregulated in TSA-pretreated flap tissue.ConclusionsTSA pretreatment protects free flaps against hypoxic injury and increases the area of surviving tissue by activating Wnt signaling and upregulating stem cell-related biomarkers.

Custos controls β-catenin to regulate head development during vertebrate embryogenesis.

Precise control of the canonical Wnt pathway is crucial in embryogenesis and all stages of life, and dysregulation of this pathway is implicated in many human diseases including cancers and birth defect disorders. A key aspect of canonical Wnt signaling is the cytoplasmic to nuclear translocation of β-catenin, a process that remains incompletely understood. Here we report the identification of a previously undescribed component of the canonical Wnt signaling pathway termed Custos, originally isolated as a Dishevelled-interacting protein. Custos contains casein kinase phosphorylation sites and nuclear localization sequences. In Xenopus, custos mRNA is expressed maternally and then widely throughout embryogenesis. Depletion or overexpression of Custos produced defective anterior head structures by inhibiting the formation of the Spemann-Mangold organizer. In addition, Custos expression blocked secondary axis induction by positive signaling components of the canonical Wnt pathway and inhibited β-catenin/TCF-dependent transcription. Custos binds to β-catenin in a Wnt responsive manner without affecting its stability, but rather modulates the cytoplasmic to nuclear translocation of β-catenin. This effect on nuclear import appears to be the mechanism by which Custos inhibits canonical Wnt signaling. The function of Custos is conserved as loss-of-function and gain-of-function studies in zebrafish also demonstrate a role for Custos in anterior head development. Our studies suggest a role for Custos in fine-tuning canonical Wnt signal transduction during embryogenesis, adding an additional layer of regulatory control in the Wnt-β-catenin signal transduction cascade.

CNBP modulates the transcription of Wnt signaling pathway components

Background: Cellular nucleic acid binding protein (CNBP) is a small and highly conserved protein with nucleic acid chaperone activity that binds single-stranded nucleic acids. Data collected so far suggests that CNBP is required for proper craniofacial development. Despite the advances achieved in the last decade, the identity of the molecular targets of CNBP responsible for its role in rostral head development remains elusive.Methods: In this work we used the CNBP single-stranded DNA-consensus binding sequence to find out putative CNBP target genes present in the human, mouse, chicken, Xenopus and zebrafish genomes.Results: Most of the identified genes are associated with embryonic developmental processes, being three of them (cdk14, ptk7 and tcf7l2) members of the Wnt signaling pathway. This finding, along with previous one showing that CNBP down-regulates the transcription of Wnt5, aimed our work to address the role of CNBP on the WNT signaling players and pathway regulation. Experiments carried out in zebrafish developing embryos revealed that craniofacial morphology was more adversely affected as CNBP abundance decreased. Furthermore, we observed that CNBP up-regulated in a dose-dependent fashion the transcription of cdk14, ptk7 and tcf7l2, which in turn was reflected in c-myc, ccnd1 and axin2 expression.Conclusions: Results reveal a role of CNBP in transcriptional control of components of the Wnt signaling pathway, which might explain its requirement for proper craniofacial development.

Genome-wide microarray analysis of Atlantic cod (Gadus morhua) oocyte and embryo.

BackgroundRegulation of gene expression plays a central role in embryonic development. Early stages are controlled by gametic transcripts, which are subsequently substituted with transcripts from the genome of the zygote. Transcriptomic analyses provide an efficient approach to explore the temporal gene expression profiles in embryos and to search for the developmental regulators. We report a study of early Atlantic cod development that used a genome-wide oligonucleotide microarray to examine the composition and putative roles of polyadenylated transcripts.ResultsThe analyses were carried out in unfertilized oocytes, newly fertilized oocytes and embryos at the stages of mid-blastula transition and segmentation. Numerous genes transcribed in oocytes are involved in multiple aspects of cell maintenance and protection, including metabolism, signal perception and transduction, RNA processing, cell cycle, defense against pathogens and DNA damage. Transcripts found in unfertilized oocytes also encoded a large number of proteins implicated in cell adherence, tight junction and focal adhesion, suggesting high complexity in terms of structure and cellular interactions in embryos prior to midblastula transition (MBT). Prezygotic transcripts included multiple regulators that are most likely involved in developmental processes that take place long after fertilization, such as components of ErbB, hedgehog, notch, retinoid, TGFb, VEGF and Wnt signaling pathways, as well as transcripts involved in the development of nervous system. The major event of MBT was the activation of a large group of histones and other genes that modify chromatin structure preceding massive gene expression changes. A hallmark of events observed during segmentation was the induction of multiple transcription factors, including a large group of homeobox proteins in pace with decay of a large fraction of maternal transcripts. Microarray analyses detected a suite of master developmental regulators that control differentiation and maintenance of diverse cell lineages.ConclusionsTranscriptome profiling of the early stages in Atlantic cod revealed the presence of transcripts involved in patterning and development of tissues and organs long before activation of the zygotic genome. The switch from maternal to zygotic developmental programs is associated with large-scale modification of chromosomes.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-594) contains supplementary material, which is available to authorized users.

A Growth-Promoting Signaling Component Cyclin D1 in Neural Stem Cells Has Antiastrogliogenic Function to Execute Self-Renewal

Self-renewing proliferation of neural stem cells (NSCs) is intimately linked to the inhibition of neuronal and glial differentiation, however, their molecular linkage has been poorly understood. We have proposed a model previously explaining partly this linkage, in which fibroblast growth factor 2 (FGF2) and Wnt signals cooperate to promote NSC self-renewal via β-catenin accumulation, which leads to the promotion of proliferation by lymphoid enhancer factor (LEF)/T-cell factor (TCF)-mediated cyclin D1 expression and at the same time to the inhibition of neuronal differentiation by β-catenin-mediated potentiation of Notch signaling. To fully understand the mechanisms underlying NSC self-renewal, it needs to be clarified how these growth factor signals inhibit glial differentiation as well. Here, we demonstrate that cyclin D1, a NSC growth promoting signaling component and also a common component of FGF2 and Wnt signaling pathways, inhibits astroglial differentiation of NSCs. Interestingly, this effect of cyclin D1 is mediated even though its cell cycle progression activity is blocked. Forced downregulation of cyclin D1 enhances astrogliogenesis of NSCs in culture and in vivo. We further demonstrate that cyclin D1 binds to STAT3, a transcription factor downstream of astrogliogenic cytokines, and suppresses its transcriptional activity on the glial fibrillary acidic protein (Gfap) gene. Taken together with our previous finding, we provide a novel molecular mechanism for NSC self-renewal in which growth promoting signaling components activated by FGF2 and Wnts inhibit neuronal and glial differentiation.