Canonical Wnt Activity Research Articles

A dual function for canonical Wnt/β-catenin signaling in the developing mammalian cochlea

The canonical Wnt/β-catenin signaling pathway is known to play crucial roles in organogenesis by regulating both proliferation and differentiation. In the inner ear, this pathway has been shown to regulate the size of the otic placode from which the cochlea will arise; however, direct activity of canonical Wnt signaling as well as its function during cochlear mechanosensory hair cell development had yet to be identified. Using TCF/Lef:H2B-GFP reporter mice and transfection of an independent TCF/Lef reporter construct, we describe the pattern of canonical Wnt activity in the developing mouse cochlea. We show that prior to terminal mitosis, canonical Wnt activity is high in early prosensory cells from which hair cells and support cells will differentiate, and activity becomes reduced as development progresses. Using an in vitro model we demonstrate that Wnt/β-catenin signaling regulates both proliferation and hair cell differentiation within the developing cochlear duct. Inhibition of Wnt/β-catenin signaling blocks proliferation during early mitotic phases of development and inhibits hair cell formation in the differentiating organ of Corti. Conversely, activation increases the number of hair cells that differentiate and induces proliferation in prosensory cells, causing an expansion of the Sox2-positive prosensory domain. We further demonstrate that the induced proliferation of Sox2-positive cells may be mediated by the cell cycle regulator cyclin D1. Lastly, we provide evidence that the mitotic Sox2-positive cells are competent to differentiate into hair cells. Combined, our data suggest that Wnt/β-catenin signaling has a dual function in cochlear development, regulating both proliferation and hair cell differentiation.

Noncanonical Wnt5a Is Induced by HTLV-1 bZIP Factor, and Supports Proliferation and Migration of Adult T-Cell Leukemia Cell.

Abstract 2405Adult T-cell leukemia (ATL) is a fatal malignancy of CD4-positive T-cells caused by human T-cell leukemia virus type1 (HTLV-1). HTLV-1 bZIP factor (HBZ) is encoded in the minus strand of HTLV-1 provirus, and is consistently expressed in HTLV-1-infected cells. It has been shown that HBZ promotes proliferation of T-cells, and HBZ-expressing transgenic mice develop T-cell lymphomas and systemic inflammations, suggesting that HBZ has the critical roles in pathogenesis of HTLV-1. However, the molecular mechanisms of HBZ-induced oncogenesis have not been clarified yet. The Wnt signaling is a highly conserved cellular signaling pathway in eukaryotes and the Wnt ligands have been found to trigger multiple pathways of Wnt signaling. The most well-studied cascade is the Wnt/β-catenin pathway (also known as the canonical Wnt pathway), which is β-catenin-dependent and mainly controls cell differentiation, proliferation, and apoptosis. Aberrant activation of the canonical Wnt pathway has been linked to many cancers. In contrast, it is known that the noncanonical Wnt pathway antagonizes the canonical pathway, while a representative noncanonical Wnt ligand, Wnt5a, is suggested to have both oncogenic and tumor suppressive properties in several malignancies. The significance of those Wnt pathways in HTLV-1-associated oncogenesis is still obscure. In this study, we found that HBZ markedly suppressed the canonical Wnt/β-catenin pathway. As a mechanism of HBZ-mediated Wnt suppression, we found that HBZ targets LEF1 and TCF1, the key transcription factors of the pathway, and impairs its DNA-binding ability. In contrast, HTLV-1 Tax protein interacted with Dvl and DAPLE, which are the regulators of the canonical Wnt signaling, and activated the pathway. Indeed, the canonical Wnt activation is not observed in ATL cells, and enforced activation of β-catenin in ATL cells inhibited cellular proliferation, implying that activation of the canonical Wnt pathway might have suppressive effect on ATL cells. On the other hand, HBZ induces the transcription of the noncanonical Wnt5a by enhancing its promoter activity. In addition, Wnt5a transcription level in ATL cells is significantly higher than in HTLV-1-uninifected control cells. Knockdown of Wnt5a impairs both proliferation and migration of ATL cells, suggesting that HBZ-induced Wnt5a supports development and progression of ATL. Our results implicate novel roles of HBZ in ATL leukemogenesis through dysregulation of both the canonical and noncanonical Wnt pathways. Disclosures:No relevant conflicts of interest to declare.

A dual function for canonical Wnt/β-catenin signaling in the developing mammalian cochlea

The canonical Wnt/β-catenin signaling pathway is known to play crucial roles in organogenesis by regulating both proliferation and differentiation. In the inner ear, this pathway has been shown to regulate the size of the otic placode from which the cochlea will arise; however, direct activity of canonical Wnt signaling as well as its function during cochlear mechanosensory hair cell development had yet to be identified. Using TCF/Lef:H2B-GFP reporter mice and transfection of an independent TCF/Lef reporter construct, we describe the pattern of canonical Wnt activity in the developing mouse cochlea. We show that prior to terminal mitosis, canonical Wnt activity is high in early prosensory cells from which hair cells and support cells will differentiate, and activity becomes reduced as development progresses. Using an in vitro model we demonstrate that Wnt/β-catenin signaling regulates both proliferation and hair cell differentiation within the developing cochlear duct. Inhibition of Wnt/β-catenin signaling blocks proliferation during early mitotic phases of development and inhibits hair cell formation in the differentiating organ of Corti. Conversely, activation increases the number of hair cells that differentiate and induces proliferation in prosensory cells, causing an expansion of the Sox2-positive prosensory domain. We further demonstrate that the induced proliferation of Sox2-positive cells may be mediated by the cell cycle regulator cyclin D1. Lastly, we provide evidence that the mitotic Sox2-positive cells are competent to differentiate into hair cells. Combined, our data suggest that Wnt/β-catenin signaling has a dual function in cochlear development, regulating both proliferation and hair cell differentiation.

HTLV-1 bZIP factor dysregulates the Wnt pathways to support proliferation and migration of adult T-cell leukemia cells

Human T-cell leukemia virus type 1 (HTLV-1) is the causative agent of adult T-cell leukemia (ATL). HTLV-1 bZIP factor (HBZ), the viral gene transcribed from the antisense strand, is consistently expressed in ATL cells and promotes their proliferation. In this study, we found that a Wnt pathway-related protein, disheveled-associating protein with a high frequency of leucine residues (DAPLE), interacts with both HTLV-1 Tax and HBZ. In the presence of DAPLE, Tax activated canonical Wnt signaling. Conversely, HBZ markedly suppressed canonical Wnt activation induced by either Tax/DAPLE or β-catenin. As a mechanism of HBZ-mediated Wnt suppression, we found that HBZ targets lymphoid enhancer-binding factor 1, one of the key transcription factors of the pathway, and impairs its DNA-binding ability. We also observed that the canonical Wnt pathway was not activated in HTLV-1-infected cells, whereas the representative of noncanonical Wnt ligand, Wnt5a, which antagonizes canonical Wnt signaling, was overexpressed. HBZ was able to induce Wnt5a transcription by enhancing its promoter activity through the TGF-β pathway. Importantly, knocking down of Wnt5a in ATL cells repressed cellular proliferation and migration. Our results implicate novel roles of HBZ in ATL leukemogenesis through dysregulation of both the canonical and noncanonical Wnt pathways.

Reactive oxygen species and Wnt signalling crosstalk patterns mouse extraembryonic endoderm

Primitive endoderm formation from the inner cell mass is one of the earliest known cell fate decisions made in the mouse embryo. The mechanisms involved in orchestrating this process are not fully understood and are difficult to study in vivo. The F9 teratocarcinoma cell line is an in vitro model used to circumvent many technical problems surrounding the study of extraembryonic endoderm differentiation. F9 cells treated with retinoic acid differentiate to primitive endoderm and this is accompanied by the activation of canonical Wnt–β-catenin signalling. Reactive oxygen species can modulate this signalling pathway [1], but whether they are sufficient to induce extraembryonic endoderm in vitro is not known. In the present study, a sustained increase in ROS levels was found in retinoic acid-treated F9 cells. An increase in Tcf-Lef transcriptional activity, a read out of Wnt–β-catenin signalling, was also seen in response to exogenous H2O2. Analysis from immunoblots, immunocytochemistry and real time PCR revealed the presence of markers of differentiation and a reduction in the expression of a marker of proliferation, confirming that H2O2-treated F9 cells developed into primitive endoderm. In contrast, exposing retinoic acid-treated cells to antioxidants impeded differentiation. Real time PCR was also used to identify candidates responsible for the observed elevation in ROS production. Results indicated that the NADPH oxidase 1, 2, 3 and 4 and Duox2 genes were RA responsive. Furthermore, the NADPH oxidase inhibitor, diphenyleneiodonium chloride was shown to attenuate primitive endoderm formation. Together, these results shed new light on how early mouse embryogenesis might be influenced by the crosstalk involving ROS and the Wnt–β-catenin signalling pathway.

Selective Activation of p120ctn-Kaiso Signaling to Unlock Contact Inhibition of ARPE-19 Cells without Epithelial-Mesenchymal Transition

Contact-inhibition ubiquitously exists in non-transformed cells and explains the poor regenerative capacity of in vivo human retinal pigment epithelial cells (RPE) during aging, injury and diseases. RPE injury or degeneration may unlock mitotic block mediated by contact inhibition but may also promote epithelial-mesenchymal transition (EMT) contributing to retinal blindness. Herein, we confirmed that EMT ensued in post-confluent ARPE-19 cells when contact inhibition was disrupted with EGTA followed by addition of EGF and FGF-2 because of activation of canonical Wnt and Smad/ZEB signaling. In contrast, knockdown of p120-catenin (p120) unlocked such mitotic block by activating p120/Kaiso, but not activating canonical Wnt and Smad/ZEB signaling, thus avoiding EMT. Nuclear BrdU labeling was correlated with nuclear release of Kaiso through p120 nuclear translocation, which was associated with activation of RhoA-ROCK signaling, destabilization of microtubules. Prolonged p120 siRNA knockdown followed by withdrawal further expanded RPE into more compact monolayers with a normal phenotype and a higher density. This new strategy based on selective activation of p120/Kaiso but not Wnt/β-catenin signaling obviates the need of using single cells and the risk of EMT, and may be deployed to engineer surgical grafts containing RPE and other tissues.

β-Catenin–SOX2 signaling regulates the fate of developing airway epithelium

Wnt–β-catenin signaling regulates cell fate during organ development and postnatal tissue maintenance, but its contribution to specification of distinct lung epithelial lineages is still unclear. To address this question, we used a Cre recombinase (Cre)-LoxP approach to activate canonical Wnt signaling ectopically in developing lung endoderm. We found that persistent activation of canonical Wnt signaling within distal lung endoderm was permissive for normal development of alveolar epithelium, yet led to the loss of developing bronchiolar epithelium and ectasis of distal conducting airways. Activation of canonical Wnt led to ectopic expression of a lymphoid-enhancing factor and a T-cell factor (LEF and TCF, respectively) and absence of SRY (sex-determining region Y)-box 2 (SOX2) and tumor protein p63 (p63) expression in proximal derivatives. Conditional loss of SOX2 in airways phenocopied epithelial differentiation defects observed with ectopic activation of canonical Wnt. Our data suggest that Wnt negatively regulates a SOX2-dependent signaling program required for developmental progression of the bronchiolar lineage.

β-Catenin–SOX2 signaling regulates the fate of developing airway epithelium

Wnt-β-catenin signaling regulates cell fate during organ development and postnatal tissue maintenance, but its contribution to specification of distinct lung epithelial lineages is still unclear. To address this question, we used a Cre recombinase (Cre)-LoxP approach to activate canonical Wnt signaling ectopically in developing lung endoderm. We found that persistent activation of canonical Wnt signaling within distal lung endoderm was permissive for normal development of alveolar epithelium, yet led to the loss of developing bronchiolar epithelium and ectasis of distal conducting airways. Activation of canonical Wnt led to ectopic expression of a lymphoid-enhancing factor and a T-cell factor (LEF and TCF, respectively) and absence of SRY (sex-determining region Y)-box 2 (SOX2) and tumor protein p63 (p63) expression in proximal derivatives. Conditional loss of SOX2 in airways phenocopied epithelial differentiation defects observed with ectopic activation of canonical Wnt. Our data suggest that Wnt negatively regulates a SOX2-dependent signaling program required for developmental progression of the bronchiolar lineage.

Coordinate regulation of N-glycosylation gene DPAGT1, canonical Wnt signaling and E-cadherin adhesion

The metabolic pathway of protein N-glycosylation influences intercellular adhesion by affecting the composition and cytoskeletal association of E-cadherin protein complexes, or adherens junctions (AJs). In sparse cells, E-cadherin is modified extensively with complex N-glycans and forms nascent AJs, while in dense cultures, hypoglycosylated E-cadherin drives the assembly of mature AJs with increased levels of γ- and α-catenins. N-glycosylation of E-cadherin is controlled by the DPAGT1 gene, a key regulator of the N-glycosylation pathway. DPAGT1 is a target of the canonical Wnt signaling pathway, with both β- and γ-catenins binding to Tcf at its promoter. We now report that DPAGT1 senses cell density through canonical Wnt signaling. In dense cells, depletion of β-catenin from the DPAGT1 promoter correlated with downregulation of its cellular abundance, while loss of nuclear γ-catenin reflected its greater recruitment to AJs. DPAGT1 itself affected canonical Wnt signaling, with forced changes in its expression resulting in corresponding changes in transcriptionally active β-catenin and canonical Wnt activity. Remarkably, a 2.4-fold increase in the DPAGT1 mRNA level resulted in increased N-glycosylation and reduced membrane localization of E-cadherin, coincident with dramatic changes in cell morphology. Lastly, we present evidence that N-glycosylation status of E-cadherin controls its antagonism of canonical Wnt signaling. Transfection of hypoglycosylated E-cadherin mutant, V13, but not fully N-glycosylated E-cadherin, into sparse cells inhibited canonical Wnt activity by depleting nuclear β- and γ-catenins. Collectively, our studies show that cells coordinate DPAGT1 expression and protein N-glycosylation with canonical Wnt signaling and E-cadherin adhesion via positive and negative feedback mechanisms.

A Switch from Canonical to Noncanonical Wnt Signaling Mediates Drug Resistance in Colon Cancer Cells

Butyrate, a fermentation product of fiber in the colon, acts as a histone deacetylase inhibitor (HDACi) and induces apoptosis in colon cancer (CC) cells in vitro. We have reported that the apoptotic effects of butyrate are dependent upon the hyperactivation of the Wnt/beta-catenin pathway. However, prolonged exposure of CC cells to increasing concentrations of butyrate results in the acquisition of resistance to the Wnt/beta-catenin- and apoptosis-inducing effects of this agent, as well as cross-resistance to structurally different HDACis. Here we report that one mechanism whereby HDACi resistance arises is through the increase of beta-catenin-independent (noncanonical) Wnt signaling. Compared to HDACi-sensitive HCT-116 CC cells, HDACi-resistant HCT-R cells exhibit higher levels of AKT/PKB cell survival signaling, which is in part induced by WNT5A and its receptor ROR2. The induction of AKT signaling by HDACis is also detected in other CC cell lines, albeit to a lesser extent than in the drug-resistant HCT-R cells. The observations suggested that the apoptotic effect of butyrate and other HDACis in CC cells can be augmented by inhibitors of pAKT. In agreement with the hypothesis, the combination of MK2206, a pAKT inhibitor, and a HDACi (butyrate or LBH589) induced higher apoptosis in CC cells compared to each agent alone. The exposure to both agents also re-sensitized the HCT-R cells to apoptosis. Finally, the concept of simultaneously inducing canonical Wnt activity and suppressing AKT signaling was translated into a combination of diet-derived agents. Diet-derived pAKT inhibitors (caffeic acid phethyl ester, sulforaphane, dilallyl trisulfide) suppressed the butyrate-induced levels of pAKT, and increased the apoptotic effects of butyrate in both drug-sensitive and drug-resistant CC cells.Our findings can be translated into (a) CC therapy employing combinations of synthetic HDACis and inhibitors of pAKT, as well as (b) CC prevention based upon diets that result in sufficient amounts of butyrate and pAKT inhibitors.

Nuclear β-catenin localization is not sufficient for canonical Wnt signaling activation in human melanoma cell lines

In most cases, advanced stages of melanoma are practically incurable due to high metastatic potential of tumor cells. Multiple observations support the idea that aberrations in Wnt signaling pathway play a significant role in melanoma development and progression. Canonical Wnt signaling activation results in stabilization and accumulation of the major effector molecule called beta-catenin. Mutations promoting beta-catenin stabilization and, thereby, activation of canonical Wnt signaling pathway are frequently found in different cancers, but rarely observed in melanomas. Nevertheless, beta-catenin nuclear and cytoplasmic accumulation is the feature of many human melanoma cell lines and original tumors. That is why, the aim of the investigation was to elucidate the relation between beta-catenin intracellular localization and activity status of Wnt signaling pathway in human melanoma cell lines. Ten human melanoma cell lines were characterized on the basis of the following parameters: canonical Wnt ligand expression, intracellular beta-catenin localization, and activity status of canonical Wnt signaling pathway. Here, it has been demonstrated that nuclear localization of beta-catenin does not always correspond to active status canonical Wnt signaling pathway. Moreover, in the majority of cell lines with nuclear beta-catenin canonical Wnt signaling can't be activated by exogenous expression of an appropriate ligand. Human melanoma cell lines differ in activity of canonical Wnt signaling pathway as well as in mechanisms of its regulation. Therefore, the pathway-targeted potential antineoplastic therapy requires the formation of a "molecular pattern of cancer" for localization of the defect in Wnt signaling cascade in the each case.

PTK7/Otk interacts with Wnts and inhibits canonical Wnt signalling

Wnt signalling is an evolutionarily conserved pathway that directs cell-fate determination and morphogenesis during metazoan development. Wnt ligands are secreted glycoproteins that act at a distance causing a wide range of cellular responses from stem cell maintenance to cell death and cell proliferation. How Wnt ligands cause such disparate responses is not known, but one possibility is that different outcomes are due to different receptors. Here, we examine PTK7/Otk, a transmembrane receptor that controls a variety of developmental and physiological processes including the regulation of cell polarity, cell migration and invasion. PTK7/Otk co-precipitates canonical Wnt3a and Wnt8, indicating a role in Wnt signalling, but PTK7 inhibits rather than activates canonical Wnt activity in Xenopus, Drosophila and luciferase reporter assays. Loss of PTK7 function activates canonical Wnt signalling and epistasis experiments place PTK7 at the level of the Frizzled receptor. In Drosophila, Otk interacts with Wnt4 and opposes canonical Wnt signalling in embryonic patterning. We propose a model where PTK7/Otk functions in non-canonical Wnt signalling by turning off the canonical signalling branch.

T-cell factor/β-catenin activity is suppressed in two different models of autosomal dominant polycystic kidney disease

During murine kidney development, canonical WNT signaling is highly active in tubules until about embryonic days E16-E18. At this time, β-catenin transcriptional activity is progressively restricted to the nephrogenic zone. The cilial protein genes PKD1 and PKD2 are known to be mutated in autosomal dominant polycystic kidney disease (ADPKD), and previous studies proposed that these mutations could lead to a failure to suppress canonical WNT signaling activity. Several in vitro studies have found a link between cilial signaling and β-catenin regulation, suggesting that aberrant activity might contribute to the cystic phenotype. To study this, we crossed T-cell factor (TCF)/β-catenin-lacZ reporter mice with mice having Pkd1 or Pkd2 mutations and found that there was no β-galactosidase staining in cells lining the renal cysts. Thus, suppression of canonical WNT activity, defined by the TCF/β-catenin-lacZ reporter, is normal in these two different models of polycystic kidney disease. Hence, excessive β-catenin transcriptional activity may not contribute to cystogenesis in these models of ADPKD.

Tissue-specific roles of Axin2 in the inhibition and activation of Wnt signaling in the mouse embryo

Axin proteins are key negative regulators of the canonical Wnt signal transduction pathway. Although Axin2 null mice are viable, we identified an unusual ENU-induced recessive allele of Axin2, canp, that causes midgestation lethality in homozygotes. We show that the Axin2(canp) mutation is a V26D substitution in an invariant N-terminal sequence motif and that the Axin2(canp) protein is more stable than wild type. As predicted for an increased level of a negative regulator, the Axin2(canp) mutation leads to decreased Wnt signaling in most tissues, and this can account for most of the morphological phenotypes of Axin2(canp) mutants. In contrast, there is a paradoxical increase in canonical Wnt activity in the late primitive streak of all Axin2(canp) mutant embryos that is associated with the formation of an ectopic tail in some mutants. Treatment of wild-type embryos with an inhibitor of Tankyrase that stabilizes Axin proteins also causes inhibition of Wnt signaling in anterior regions of the embryo and a gain of Wnt signaling in the primitive streak. The results indicate that although increased stability of Axin2 leads to a loss of canonical Wnt signaling in most tissues, stabilized Axin2 enhances Wnt pathway activity in a specific progenitor population in the late primitive streak.

High-Frequency Canonical Wnt Activation in Multiple Sarcoma Subtypes Drives Proliferation through a TCF/β-Catenin Target Gene, CDC25A

Wnt canonical signaling is critical for normal development as well as homeostasis of several epithelial tissues, and constitutive activation of this pathway is commonly observed in carcinomas. We show here that 50% of human sarcomas (n = 45) and 65% of sarcoma cell lines (n = 23) of diverse histological subtypes exhibit upregulated autocrine canonical Wnt signaling. Furthermore, in Wnt autocrine cell lines, we identify alterations including overexpression or gene amplification of Wnt ligands and/or LRP5/6 coreceptors and epigenetic silencing of different cell surface Wnt antagonists. Mutations in adenomatous polyposis coli (APC) gene were observed in two nonautocrine Wnt-positive sarcoma cell lines. Finally, downregulation of the activated Wnt pathway inhibited sarcoma cell proliferation both in vitro and in vivo by a mechanism involving the downregulation of CDC25A.

The canonical Wnt signaling pathway is not involved in renal cyst development in the kidneys of inv mutant mice

Recent studies have identified several genes whose defects cause hereditary renal cystic diseases with most of the gene products located in the primary cilia. It has been suggested that primary cilia are involved in signaling pathways, defects of which result in abnormal cell proliferation and randomization of oriented cell division in the kidney leading to cyst formation. Mice with a mutation in the inv gene are a model for human nephronophthisis type 2 and develop multiple renal cysts. Inv protein (also called inversin) is located in the base of primary cilia and acts as a switch from canonical to non-canonical Wnt signaling. Here, we studied the orientation of cell division and proliferation in the kidneys of inv mutant mice, as its loss is thought to maintain activation of the canonical Wnt signaling. To establish if canonical signaling was involved in this process, we mated inv mutant with BATlacZ mice to measure canonical Wnt activity. Based on these reporter mice, nuclear localization and phosphorylation of β-catenin, and responsiveness to Wnt ligands in inv mutant cells, we found that random oriented cell division is an initial event for renal tubule expansion and precedes cell proliferation. Thus, our results do not support the hypothesis that canonical Wnt signaling causes renal cyst development in these mice.

Role of epidermal primary cilia in the homeostasis of skin and hair follicles

Skin and hair follicle morphogenesis and homeostasis require the integration of multiple signaling pathways, including Hedgehog (Hh) and Wingless (Wnt), and oriented cell divisions, all of which have been associated with primary cilia. Although studies have shown that disrupting dermal cilia causes follicular arrest and attenuated Hh signaling, little is known about the role of epidermal cilia. Here, epidermal cilia function was analyzed using conditional alleles of the ciliogenic genes Ift88 and Kif3a . At birth, epidermal cilia mutants appeared normal, but developed basaloid hyperplasia and ingrowths into the dermis of the ventrum with age. In addition, follicles in the tail were disorganized and had excess sebaceous gland lobules. Epidermal cilia mutants displayed fewer long-term label-retaining cells, suggesting altered stem cell homeostasis. Abnormal proliferation and differentiation were evident from lineage-tracing studies and showed an expansion of follicular cells into the interfollicular epidermis, as is seen during wound repair. These phenotypes were not associated with changes in canonical Wnt activity or oriented cell division. However, nuclear accumulation of the ΔNp63 transcription factor, which is involved in stratification, keratinocyte differentiation and wound repair, was increased, whereas the Hh pathway was repressed. Intriguingly, the phenotypes were not typical of those associated with loss of Hh signaling but exhibited similarities with those of mice in which ΔNp63 is overexpressed in the epidermis. Collectively, these data indicate that epidermal primary cilia may function in stress responses and epidermal homeostasis involving pathways other than those typically associated with primary cilia.

Canonical WNT Signaling Enhances Stem Cell Expression in the Developing Heart Without a Corresponding Inhibition of Cardiogenic Differentiation

WNT signaling has been shown to influence the development of the heart. Although recent data suggested that canonical WNTs promote the emergence and expansion of cardiac progenitors in the pregastrula embryo, it has long been accepted that once gastrulation begins, canonical WNT signaling needs to be suppressed for cardiac development to proceed. Yet, this latter supposition appears to be odds with the expression of multiple canonical WNTs in the developing heart. The present study examining the effect of ectopic canonical WNT signaling on cardiogenesis in the developing frog was designed to test the hypothesis that heart formation is dependent on the inhibition of canonical WNT activity at the onset of gastrulation. Here we report that cardiac differentiation of explanted precardiac tissue from the dorsal marginal zone was not suppressed by exposure to WNT1 protein, although expression of Tbx5, Tbx20, and Nkx2.5 was selectively reduced. Pharmacological activation of WNT signaling in intact embryos using the GSK3 inhibitor SB415286 did not prevent the formation of an anatomically normal and functionally sound heart, with the only defect observed being lower levels of the cardiac transcription factor Nkx2.5. In both the explant and whole embryo studies, expression of muscle genes and proteins was unaffected by ectopic canonical WNT signaling. In contrast, canonical Wnt signaling upregulated expression of the cardiac stem cell marker c-kit and pluripotency genes Oct25 and Oct60. However, this regulatory stimulation of stem cells did not come at the expense of blocking cardiac progenitors from differentiating.

Role of epidermal primary cilia in the homeostasis of skin and hair follicles

Skin and hair follicle morphogenesis and homeostasis require the integration of multiple signaling pathways, including Hedgehog (Hh) and Wingless (Wnt), and oriented cell divisions, all of which have been associated with primary cilia. Although studies have shown that disrupting dermal cilia causes follicular arrest and attenuated Hh signaling, little is known about the role of epidermal cilia. Here, epidermal cilia function was analyzed using conditional alleles of the ciliogenic genes Ift88 and Kif3a. At birth, epidermal cilia mutants appeared normal, but developed basaloid hyperplasia and ingrowths into the dermis of the ventrum with age. In addition, follicles in the tail were disorganized and had excess sebaceous gland lobules. Epidermal cilia mutants displayed fewer long-term label-retaining cells, suggesting altered stem cell homeostasis. Abnormal proliferation and differentiation were evident from lineage-tracing studies and showed an expansion of follicular cells into the interfollicular epidermis, as is seen during wound repair. These phenotypes were not associated with changes in canonical Wnt activity or oriented cell division. However, nuclear accumulation of the ΔNp63 transcription factor, which is involved in stratification, keratinocyte differentiation and wound repair, was increased, whereas the Hh pathway was repressed. Intriguingly, the phenotypes were not typical of those associated with loss of Hh signaling but exhibited similarities with those of mice in which ΔNp63 is overexpressed in the epidermis. Collectively, these data indicate that epidermal primary cilia may function in stress responses and epidermal homeostasis involving pathways other than those typically associated with primary cilia.

Experimental myocardial infarction triggers canonical Wnt signaling and endothelial-to-mesenchymal transition

SUMMARYDespite available therapies, myocardial infarction (MI) remains a leading cause of death worldwide. Better understanding of the molecular and cellular mechanisms that regulate cardiac repair should help to improve the clinical outcome of MI patients. Using the reporter mouse line TOPGAL, we show that canonical (β-catenin-dependent) Wnt signaling is induced 4 days after experimental MI in subepicardial endothelial cells and perivascular smooth muscle actin (SMA)-positive (SMA+) cells. At 1 week after ischemic injury, a large number of canonical-Wnt-positive cells accumulated in the infarct area during granulation tissue formation. Coincidently with canonical Wnt activation, endothelial-to-mesenchymal transition (EndMT) was also triggered after MI. Using cell lineage tracing, we show that a significant portion of the canonical-Wnt-marked SMA+ mesenchymal cells is derived from endothelial cells. Canonical Wnt signaling induces mesenchymal characteristics in cultured endothelial cells, suggesting a direct role in EndMT. In conclusion, our study demonstrates that canonical Wnt activation and EndMT are molecular and cellular responses to MI and that canonical Wnt signaling activity is a characteristic property of EndMT-derived mesenchymal cells that take part in cardiac tissue repair after MI. These findings could lead to new strategies to improve the course of cardiac repair by temporal and cell-type-specific manipulation of canonical Wnt signaling.