Wnt7b regulates placental development in mice.

Secreted Wnt proteins regulate many developmental processes in multicellular organisms. We have generated a targeted mutation in the mouse Wnt7b gene. Homozygous Wnt7b mutant mice die at midgestation stages as a result of placental abnormalities. Wnt7b expression in the chorion is required for fusion of the chorion and allantois during placental development. The alpha4 integrin protein, required for chorioallantoic fusion, is not expressed by cells in the mutant chorion. Wnt7b also is required for normal organization of cells in the chorionic plate. Thus, Wnt7b signaling is central to the early stages of placental development in mammals.     

Wnt5b regulates mesenchymal cell aggregation and chondrocyte differentiation through the planar cell polarity pathway

Although genetic evidence has demonstrated a role for Wnt5b during cartilage and limb development, little is known about the mechanisms underlying Wnt5b-regulated chondrocyte differentiation. We observed that Wnt5b inhibited chondrocyte hypertrophy and expression of type X collagen. In addition, Wnt5b regulated the overall size of chondrogenic cultures, suggesting that Wnt5b regulates other processes involved in cartilage development. We therefore investigated the signaling pathways by which Wnt5b influences differentiation. Wnt5b activated known calcium-dependent signaling pathways and JNK, a component of the planar cell polarity pathway. Since the planar cell polarity pathway regulates process such as cell migration and cell aggregation that are involved in limb development, we assayed for effects of Wnt5b on these processes. We observed a marked increase chondroprogenitor cell migration with Wnt5b expression. This effect was blocked by inhibition of JNK, but not by inhibition of other Wnt5b-responsive factors. Expression of Wnt5b also disrupted the cellular aggregation associated with mesenchymal condensation. Decreased aggregation was associated with reduced cadherin expression as well as increased cadherin receptor turnover. This increase in cadherin receptor turnover was associated with an increase in Src-dependent beta-catenin phosphorylation downstream of Wnt5b. Our data demonstrate that not only does Wnt5b inhibit chondrocyte hypertrophy, but document a novel role for Wnt5b in modulating cellular migration through the JNK-dependent and cell adhesion through an activation of Src and subsequent cadherin receptor turnover.     

Wnt signaling regulates the proliferation potential and lineage commitment of human umbilical cord derived mesenchymal stem cells

Relatively less is known about the interactions that tightly regulate the mesenchymal stem cells (MSCs) to maintain their pluripotency. Recent studies reports that Wnt proteins might play an important role in governing the MSC cell fate. In this study, we tested the hypothesis that Wnt proteins differentially regulate in vitro differentiation of human umbilical cord derived MSCs. Stromal cells from human umbilical cord (hUCMSCs) were isolated and treated with Wnt inhibitor/activator. FACS analysis of hUCMSCs for CD29, CD90, CD73, CD44, CD45 marker expression and gene expression of Wnt target genes and lineage specific genes were performed after Lithium Chloride (LiCl) and Quercetin treatment for 6 days. The cultured primary hUCMSCs demonstrated elevated MSC surface marker expression with clonogenic properties and differentiation potentials towards osteogenic, adipogenic and chondrogenic lineages. Downregulation in the expression of Wnt with Quercetin treatment was noted. LiCl treatment increased cellular proliferation but did not influence differentiation suggesting that the cells retain pluripotency whereas Quercetin treatment downregulated stemness markers, Wnt target gene expression and promoted osteogenesis as demonstrated by FACS analysis, calcium estimation and gene expression studies. Shift of differentiation potential after the inhibition of Wnt signaling by Quercetin was evident from the gene expression data and elevated calcium production, driving MSCs towards probable osteogenic lineage. The findings in particular are likely to open an interesting avenue of biomedical research, summarizing the impact of Wnt signaling on lineage commitment of MSCs.

     

Wnt5a regulates Shh and Fgf10 signaling during lung development

The role of WNT signaling and its interactions with other morphogenetic pathways were investigated during lung development. Previously, we showed that targeted disruption of Wnt5a results in over-branching of the epithelium and thickening of the interstitium in embryonic lungs. In this study, we generated and characterized transgenic mice with lung-specific over-expression of Wnt5a from the SpC promoter. Over-expression of Wnt5a interfered with normal epithelial-mesenchymal interactions resulting in reduced epithelial branching and dilated distal airways. During early lung development, over-expression of Wnt5a in the epithelium resulted in increased Fgf10 in the mesenchyme and decreased Shh in the epithelium. Both levels and distribution of SHH receptor, Ptc were reduced in SpC-Wnt5a transgenic lungs and were reciprocally correlated to changes of Fgf10 in the mesenchyme, suggesting that SHH signaling is decreased by over-expression of Wnt5a. Cultured mesenchyme-free epithelial explants from SpC-Wnt5a transgenic lungs responded abnormally to recombinant FGF10 supplied uniformly in the Matrigel with dilated branch tips that mimic the in vivo phenotype. In contrast, chemotaxis of transgenic epithelial explants towards a directional FGF10 source was inhibited. These suggest that over-expression of Wnt5a disrupts epithelial-response to FGF10. In conclusion, Wnt5a regulates SHH and FGF10 signaling during lung development.     

LDL receptor-related protein LRP6 regulates proliferation and survival through the Wnt cascade in vascular smooth muscle cells

Initial studies have established expression of low-density lipoprotein (LDL) receptor-related protein 6 (LRP6) in vascular smooth muscle cells (VSMCs). We hypothesized that LRP6 is a critical mediator governing the regulation of the canonical Wnt/beta-catenin/T cell factor 4 (Tcf-4) cascade in the vasculature. This hypothesis was based on our previous work demonstrating a role for the beta-catenin/Tcf-4 pathway in vascular remodeling as well as work in other cell systems establishing a role for LRP family members in the Wnt cascade. In line with our hypothesis, LRP6 upregulation significantly increased Wnt-1-induced Tcf activation. Moreover, a dominant interfering LRP6 mutant lacking the carboxyl intracellular domain (LRP6DeltaC) abolished Tcf activity. LRP6-induced stimulation of Tcf was blocked in VSMCs harboring constitutive expression of a dominant negative Tcf-4 transgene lacking the beta-catenin binding domain, suggesting that LRP6-induced activation of Tcf was mediated through a beta-catenin-dependent signal. Expression of the dominant interfering LRP6DeltaC transgene was sufficient to abolish the Wnt-induced survival as well as cyclin D1 activity and cell cycle progression. In conclusion, these findings provide the first evidence of a role for an LDL receptor-related protein in the regulation of VSMC proliferation and survival through the evolutionary conserved Wnt signaling cascade.     

Bmp7 regulates branching morphogenesis of the lacrimal gland by promoting mesenchymal proliferation and condensation

The lacrimal gland provides an excellent model with which to study the epithelial-mesenchymal interactions that are crucial to the process of branching morphogenesis. In the current study, we show that bone morphogenetic protein 7 (Bmp7) is expressed with a complex pattern in the developing gland and has an important role in regulating branching. In loss-of-function analyses, we find that Bmp7-null mice have distinctive reductions in lacrimal gland branch number, and that inhibition of Bmp activity in gland explant cultures has a very similar consequence. Consistent with this, exposure of whole-gland explants to recombinant Bmp7 results in increased branch number. In determining which cells of the gland respond directly to Bmp7, we have tested isolated mesenchyme and epithelium. We find that, as expected, Bmp4 can suppress bud extension in isolated epithelium stimulated by Fgf10, but interestingly, Bmp7 has no discernible effect. Bmp7 does, however, stimulate a distinct response in mesenchymal cells. This manifests as a promotion of cell division and formation of aggregates, and upregulation of cadherin adhesion molecules, the junctional protein connexin 43 and of alpha-smooth muscle actin. These data suggest that in this branching system, mesenchyme is the primary target of Bmp7 and that formation of mesenchymal condensations characteristic of signaling centers may be enhanced by Bmp7. Based on the activity of Bmp7 in promoting branching, we also propose a model suggesting that a discrete region of Bmp7-expressing head mesenchyme may be crucial in determining the location of the exorbital lobe of the gland.     

Wnt5a-mediated non-canonical Wnt signalling regulates human endothelial cell proliferation and migration

Cell to cell interaction is one of the key processes effecting angiogenesis and endothelial cell function. Wnt signalling is mediated through cell-cell interaction and is involved in many developmental processes and cellular functions. In this study, we investigated the possible function of Wnt5a and the non-canonical Wnt pathway in human endothelial cells. We found that Wnt5a-mediated non-canonical Wnt signalling regulated endothelial cell proliferation. Blocking this pathway using antibody, siRNA or a down-stream inhibitor led to suppression of endothelial cell proliferation, migration, and monolayer wound closure. We also found that the mRNA level of Wnt5a is up-regulated when endothelial cells are treated with a cocktail of inflammatory cytokines. Our findings suggest non-canonical Wnt signalling plays a role in regulating endothelial cell growth and possibly in angiogenesis.     

Wnt7b stimulates embryonic lung growth by coordinately increasing the replication of epithelium and mesenchyme

The effects of Wnt7b on lung development were examined using a conditional Wnt7b-null mouse. Wnt7b-null lungs are markedly hypoplastic, yet display largely normal patterning and cell differentiation. In contrast to findings in prior hypomorphic Wnt7b models, we find decreased replication of both developing epithelium and mesenchyme, without abnormalities of vascular smooth muscle development. We further demonstrate that Wnt7b signals to neighboring cells to activate both autocrine and paracrine canonical Wnt signaling cascades. In contrast to results from hypomorphic models, we show that Wnt7b modulates several important signaling pathways in the lung. Together, these cascades result in the coordinated proliferation of adjacent epithelial and mesenchymal cells to stimulate organ growth with few alterations in differentiation and patterning.     

Secreted frizzled-related protein 4 regulates two Wnt7a signaling pathways and inhibits proliferation in endometrial cancer cells

In the endometrium, hormonal effects on epithelial cells are often elicited through stromal hormone receptors via unknown paracrine mechanisms. Several lines of evidence support the hypothesis that Wnts participate in stromal-epithelial cell communication. Wnt7a is expressed in the luminal epithelium, whereas the extracellular modulator of Wnt signaling, secreted frizzled-related protein 4 (SFRP4), is localized to the stroma. Studies have reported that SFRP4 expression is significantly decreased in endometrial carcinoma and that both SFRP4 and Wnt7a genes are differentially regulated in response to estrogenic stimuli. Aberrant Wnt7a signaling irrevocably causes organ defects and infertility and contributes to the onset of disease. However, specific frizzled receptors (Fzd) that bind Wnt7a and the particular signal transduction pathway each Wnt7a-Fzd pair activates have not been identified. Additionally, the function of SFRP4 in the endometrium has not been addressed. We show here that Wnt7a coimmunoprecipitates with Fzd5, Fzd10, and SFRP4 in Ishikawa cells. Wnt7a binding to Fzd5 was shown to activate beta-catenin/canonical Wnt signaling and increase cellular proliferation. Conversely, Wnt7a signaling mediated by Fzd10 induced a noncanonical c-Jun NH2-terminal kinase-responsive pathway. SFRP4 suppresses activation of Wnt7a signaling in both an autocrine and paracrine manner. Stable overexpression of SFRP4 and treatment with recombinant SFRP4 protein inhibited endometrial cancer cell growth in vitro. These findings support a mechanism by which the nature of the Wnt7a signal in the endometrium is dependent on the Fzd repertoire of the cell and can be regulated by SFRP4.     

Non-canonical Wnt signaling through Wnt5a/b and a novel Wnt11 gene, Wnt11b, regulates cell migration during avian gastrulation

Knowledge of the molecular mechanisms regulating cell ingression, epithelial–mesenchymal transition and migration movements during amniote gastrulation is steadily improving. In the frog and fish embryo, Wnt5 and Wnt11 ligands are expressed around the blastopore and play an important role in regulating cell movements associated with gastrulation. In the chicken embryo, although Wnt5a and Wnt5b are expressed in the primitive streak, the known Wnt11 gene is expressed in paraxial and intermediate mesoderm, and in differentiated myocardial cells, but not in the streak. Here, we identify a previously uncharacterized chicken Wnt11 gene, Wnt11b, that is orthologous to the frog Wnt11 and zebrafish Wnt11 (silberblick) genes. Chicken Wnt11b is expressed in the primitive streak in a pattern similar to chicken Wnt5a and Wnt5b. When non-canonical Wnt signaling is blocked using a Dishevelled dominant-negative protein, gastrulation movements are inhibited and cells accumulate in the primitive streak. Furthermore, disruption of non-canonical Wnt signaling by overexpression of full-length or dominant-negative Wnt11b or Wnt5a constructions abrogates normal cell migration through the primitive streak. We conclude that non-canonical Wnt signaling, mediated in part by Wnt11b, is important for regulation of gastrulation cell movements in the avian embryo.     

Wnt5a and Wnt5b exhibit distinct activities in coordinating chondrocyte proliferation and differentiation

Proper longitudinal growth of long bones relies on the regulation of specific spatial patterns of chondrocyte proliferation and differentiation. We have studied the roles of two members of the Wnt family, Wnt5a and Wnt5b in long bone development. We show that Wnt5a is required for longitudinal skeletal outgrowth and that both Wnt5a and Wnt5b regulate the transition between different chondrocyte zones independently of the Indian hedgehog (Ihh)/parathyroid hormone-related peptide (PTHrP) negative feedback loop. We find that important cell cycle regulators such as cyclin D1 and p130, a member of the retinoblastoma family, exhibit complimentary expression patterns that correlate with the distinct proliferation and differentiation states of chondrocyte zones. Furthermore, we show that Wnt5a and Wnt5b appear to coordinate chondrocyte proliferation and differentiation by differentially regulating cyclin D1 and p130 expression, as well as chondrocyte-specific Col2a1 expression. Our data indicate that Wnt5a and Wnt5b control the pace of transitions between different chondrocyte zones.     

Dok5 regulates proliferation and differentiation of osteoblast via canonical Wnt/β-catenin signaling

Objective:In bone tissue engineering, the use of osteoblastic seed cells has been widely adopted to mediate the osteogenic differentiation so as to prompt bone regeneration and repair. It is hypothesized that Dok5 can regulate the proliferation and differentiation of osteoblasts. In this study, the role of Dok5 in osteoblast proliferation and differentiation was investigated.Methods:A lentiviral vector to silence Dok5 was transferred to C3H10, 293T and C2C12 cells. CCK-8 assay was used to detect the cell proliferation. Cells were stained by ALP and AR-S staining. Western blot and RT-PCR were used to detect the expression levels of related factors.Results:Dok5 expression level was gradually up-regulated during the osteoblast differentiation. Dok5 silencing down-regulated the expression levels of osteogenic biosignatures OPN, OCN, and Runx2 and suppressed the osteogenesis. Additionally, the osteoblast proliferation and canonical Wnt/β-catenin signaling were suppressed upon Dok5 knockdown, β-catenin expression level was significantly down-regulated in the knockdown group, while the expression levels of GSK3-β and Axin, negative regulators in the Wnt signaling pathway, were up-regulated. Furthermore, overexpression of Dok5 promoted the proliferation and osteogenesis and activated the canonical Wnt/β-catenin signaling pathway.Conclusion:Dok5 may regulate the osteogenic proliferation and differentiation via the canonical Wnt/β-catenin signaling pathway.     

Sonic hedgehog signaling regulates Gli3 processing, mesenchymal proliferation, and differentiation during mouse lung organogenesis

Lack of Sonic hedgehog (Shh) signaling, mediated by the Gli proteins, leads to severe pulmonary hypoplasia. However, the precise role of Gli genes in lung development is not well established. We show Shh signaling prevents Gli3 proteolysis to generate its repressor forms (Gli3R) in the developing murine lung. In Shh(-/-) or cyclopamine-treated wild-type (WT) lung, we found that Gli3R level is elevated, and this upregulation appears to contribute to defects in proliferation and differentiation observed in the Shh(-/-) mesenchyme, where Gli3 is normally expressed. In agreement, we found Shh(-/-);Gli3(-/-) lungs exhibit enhanced growth potential. Vasculogenesis is also enhanced; in contrast, bronchial myogenesis remains absent in Shh(-/-);Gli3(-/-) compared with Shh(-/-) lungs. Genes upregulated in Shh(-/-);Gli3(-/-) relative to Shh(-/-) lung include Wnt2 and, surprisingly, Foxf1 whose expression has been reported to be Shh-dependent. Cyclins D1, D2, and D3 antibody labelings also reveal distinct expression patterns in the normal and mutant lungs. We found significant repression of Tbx2 and Tbx3, both linked to inhibition of cellular senescence, in Shh(-/-) and partial derepression in Shh(-/-); Gli3(-/-) lungs, while Tbx4 and Tbx5 expressions are less affected in the mutants. Our findings shed light on the role of Shh signaling on Gli3 processing in lung growth and differentiation by regulating several critical genes.     

Wnt signaling in biliary development,proliferation, and fibrosis

Biliary fibrosis is an important pathological indicator of hepatobiliary damage. Cholangiocyte is the key cell type involved in this process. To reveal the pathogenesis of biliary fibrosis, it is essential to understand the normal development as well as the aberrant generation and proliferation of cholangiocytes. Numerous reports suggest that the Wnt signaling pathway is implicated in the physiological and pathological processes of cholangiocyte development and ductular reaction. In this review, we summarize the effects of Wnt pathway in cholangiocyte development from embryonic stem cells, as well as the underlying mechanisms of cholangiocyte responses to adult ductal damage. Wnt signaling pathway is regulated in a step-wise manner during each of the liver differentiation stages from embryonic stem cells to functional mature cholangiocytes. With the modulation of Wnt pathway, cholangiocytes can also be generated from adult liver progenitor cells and mature hepatocytes to repair liver damage. Non-canonical Wnt signaling is triggered in the active ductal cells during biliary fibrosis. Targeted control of the Wnt signaling may hold the great potential to reduce and/or reverse the biliary fibrogenic process.     

CFTR and Wnt/beta-catenin signaling in lung development

Background  

Cystic fibrosis transmembrane conductance regulator (CFTR) was shown previously to modify stretch induced differentiation in the lung. The mechanism for CFTR modulation of lung development was examined by in utero gene transfer of either a sense or antisense construct to alter CFTR expression levels.     

Canonical Wnt signaling regulates smooth muscle precursor development in the mouse ureter

Smooth muscle cells (SMCs) are a key component of many visceral organs, including the ureter, yet the molecular pathways that regulate their development from mesenchymal precursors are insufficiently understood. Here, we identified epithelial Wnt7b and Wnt9b as possible ligands of Fzd1-mediated β-catenin (Ctnnb1)-dependent (canonical) Wnt signaling in the adjacent undifferentiated ureteric mesenchyme. Mice with a conditional deletion of Ctnnb1 in the ureteric mesenchyme exhibited hydroureter and hydronephrosis at newborn stages due to functional obstruction of the ureter. Histological analysis revealed that the layer of undifferentiated mesenchymal cells directly adjacent to the ureteric epithelium did not undergo characteristic cell shape changes, exhibited reduced proliferation and failed to differentiate into SMCs. Molecular markers for prospective SMCs were lost, whereas markers of the outer layer of the ureteric mesenchyme fated to become adventitial fibroblasts were expanded to the inner layer. Conditional misexpression of a stabilized form of Ctnnb1 in the prospective ureteric mesenchyme resulted in the formation of a large domain of cells that exhibited histological and molecular features of prospective SMCs and differentiated along this lineage. Our analysis suggests that Wnt signals from the ureteric epithelium pattern the ureteric mesenchyme in a radial fashion by suppressing adventitial fibroblast differentiation and initiating smooth muscle precursor development in the innermost layer of mesenchymal cells.