P120-catenin regulates WNT signaling and EMT in the mouse embryo.
ABSTRACT: Epithelial-to-mesenchymal transitions (EMTs) require a complete reorganization of cadherin-based cell-cell junctions. p120-catenin binds to the cytoplasmic juxtamembrane domain of classical cadherins and regulates their stability, suggesting that p120-catenin may play an important role in EMTs. Here, we describe the role of p120-catenin in mouse gastrulation, an EMT that can be imaged at cellular resolution and is accessible to genetic manipulation. Mouse embryos that lack all p120-catenin, or that lack p120-catenin in the embryo proper, survive to midgestation. However, mutants have specific defects in gastrulation, including a high rate of p53-dependent cell death, a bifurcation of the posterior axis, and defects in the migration of mesoderm; all are associated with abnormalities in the primitive streak, the site of the EMT. In embryonic day 7.5 (E7.5) mutants, the domain of expression of the streak marker Brachyury (T) expands more than 3-fold, from a narrow strip of posterior cells to encompass more than one-quarter of the embryo. After E7.5, the enlarged T+ domain splits in 2, separated by a mass of mesoderm cells. Brachyury is a direct target of canonical WNT signaling, and the domain of WNT response in p120-catenin mutant embryos, like the T domain, is first expanded, and then split, and high levels of nuclear ?-catenin levels are present in the cells of the posterior embryo that are exposed to high levels of WNT ligand. The data suggest that p120-catenin stabilizes the membrane association of ?-catenin, thereby preventing accumulation of nuclear ?-catenin and excessive activation of the WNT pathway during EMT.
Project description:MACF1 (microtubule actin cross-linking factor 1) is a multidomain protein that can associate with microfilaments and microtubules. We found that MACF1 was highly expressed in neuronal tissues and the foregut of embryonic day 8.5 (E8.5) embryos and the head fold and primitive streak of E7.5 embryos. MACF1(-/-) mice died at the gastrulation stage and displayed developmental retardation at E7.5 with defects in the formation of the primitive streak, node, and mesoderm. This phenotype was similar to Wnt-3(-/-) and LRP5/6 double-knockout embryos. In the absence of Wnt, MACF1 associated with a complex that contained Axin, beta-catenin, GSK3beta, and APC. Upon Wnt stimulation, MACF1 appeared to be involved in the translocation and subsequent binding of the Axin complex to LRP6 at the cell membrane. Reduction of MACF1 with small interfering RNA decreased the amount of beta-catenin in the nucleus, and led to an inhibition of Wnt-induced TCF/beta-catenin-dependent transcriptional activation. Similar results were obtained with a dominant-negative MACF1 construct that contained the Axin-binding region. Reduction of MACF1 in Wnt-1-expressing P19 cells resulted in decreased T (Brachyury) gene expression, a DNA-binding transcription factor that is a direct target of Wnt/beta-catenin signaling and required for mesoderm formation. These results suggest a new role of MACF1 in the Wnt signaling pathway.
Project description:Activation of Wnt/?-catenin signaling is crucial for the differentiation of pluripotent stem cells, namely the epiblast, embryonic stem, and embryonal carcinoma cells, into mesendoderm. However, downstream events of Wnt/?-catenin signaling that control the formation of mesendoderm are still unclear. In the present study, we used mouse P19 embryonal carcinoma cells as a model, and identified a homeodomain protein Nkx1-2 as a key regulator of mesendoderm formation. In the mouse embryo, Nkx1-2 was expressed in the primitive streak, in which the nascent mesendoderm emerges. In P19 cells, the expression of Nkx1-2 was activated by Wnt/?-catenin signaling independently of Brachyury, an evolutionary conserved early mesendoderm gene. In contrast, the expression of Nkx1-2 was both necessary and sufficient for the activation of Brachyury. Nkx1-2 acted as a transcriptional repressor to mediate the action of Wnt/?-catenin signaling to activate the Brachyury expression. We found Tcf3 as a potential target of gene repression by Nkx1-2, and the down-regulation of Tcf3 was partly required for effective activation of Brachyury by Wnt/?-catenin signaling. These results suggest that Nkx1-2 is a critical component of the gene regulatory network that operates downstream of Wnt/?-catenin signaling to regulate the formation of mesendoderm.
Project description:Wnt signaling pathways have fundamental roles in animal development and tumor progression. Here, employing Xenopus embryos and mammalian cell lines, we report that the degradation machinery of the canonical Wnt pathway modulates p120-catenin protein stability through mechanisms shared with those regulating ?-catenin. For example, in common with ?-catenin, exogenous expression of destruction complex components, such as GSK3? and axin, promotes degradation of p120-catenin. Again in parallel with ?-catenin, reduction of canonical Wnt signals upon depletion of LRP5 and LRP6 results in p120-catenin degradation. At the primary sequence level, we resolved conserved GSK3? phosphorylation sites in the amino-terminal region of p120-catenin present exclusively in isoform-1. Point-mutagenesis of these residues inhibited the association of destruction complex components, such as those involved in ubiquitylation, resulting in stabilization of p120-catenin. Functionally, in line with predictions, p120 stabilization increased its signaling activity in the context of the p120-Kaiso pathway. Importantly, we found that two additional p120-catenin family members, ARVCF-catenin and ?-catenin, associate with axin and are degraded in its presence. Thus, as supported using gain- and loss-of-function approaches in embryo and cell line systems, canonical Wnt signals appear poised to have an impact upon a breadth of catenin biology in vertebrate development and, possibly, human cancers.
Project description: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/?-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.
Project description:To explore the loss of Dullurd function in mouse embryo development, we have performed the Agilent microarray analysis between a Dullard-heterozygous and a Dullard-homozygous E7.5 embryo. Our findings show that Dullard does not act in concert with BMP4 and Smad1/5/8, whereas loss of Dullard is associated with a reduced level of WNT/Î²-catenin signaling activity, accompanied by elevated expression of Wnt3 and WNT antagonists including Dkk1, Sfrp1 and Sfrp5 in mouse germ cell formation. E7.5 mouse embryos were collected. One Dullard-heterozygous embryo and one Dullard-homozygous embryo were used for the analysis.
Project description:The Wnt pathways contribute to many processes in cancer and development, with ?-catenin being a key canonical component. p120-catenin, which is structurally similar to ?-catenin, regulates the expression of certain Wnt target genes, relieving repression conferred by the POZ- and zinc-finger-domain-containing transcription factor Kaiso. We have identified the kinase Dyrk1A as a component of the p120-catenin-Kaiso trajectory of the Wnt pathway. Using rescue and other approaches in Xenopus laevis embryos and mammalian cells, we found that Dyrk1A positively and selectively modulates p120-catenin protein levels, thus having an impact on p120-catenin and Kaiso (and canonical Wnt) gene targets such as siamois and wnt11. The Dyrk1A gene resides within the Down's syndrome critical region, which is amplified in Down's syndrome. A consensus Dyrk phosphorylation site in p120-catenin was identified, with a mutant mimicking phosphorylation exhibiting the predicted enhanced capacity to promote endogenous Wnt-11 and Siamois expression, and gastrulation defects. In summary, we report the biochemical and functional relationship of Dyrk1A with the p120-catenin-Kaiso signaling trajectory, with a linkage to canonical Wnt target genes. Conceivably, this work might also prove relevant to understanding the contribution of Dyrk1A dosage imbalance in Down's syndrome.
Project description:Epithelial to mesenchymal transitions (EMTs) are thought to be essential to generate diversity of tissues during early fetal development, but these events are essentially impossible to study at the molecular level in vivo in humans. The first EMT event that has been described morphologically in human development occurs just prior to generation of the primitive streak. Because human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) are thought to most closely resemble cells found in epiblast-stage embryos prior to formation of the primitive streak, we sought to determine whether this first human EMT could be modeled in vitro with pluripotent stem cells. The data presented here suggest that generating embryoid bodies from hESCs or hiPSCs drives a procession of EMT events that can be observed within 24-48 hours after EB generation. These structures possess the typical hallmarks of developmental EMTs, and portions also display evidence of primitive streak and mesendoderm. We identify PTK7 as a novel marker of this EMT population, which can also be used to purify these cells for subsequent analyses and identification of novel markers of human development. Gene expression analysis indicated an upregulation of EMT markers and ECM proteins in the PTK7+ population. We also find that cells that undergo this developmental EMT retain developmental plasticity as sorting, dissociation and re-plating reestablishes an epithelial phenotype.
Project description:Mouse embryonic stem cells (mESCs) and mouse epiblast stem cells (mEpiSCs) are the pluripotent stem cells (PSCs), derived from the inner cell mass (ICM) of preimplantation embryos at embryonic day 3.5 (E3.5) and postimplantation embryos at E5.5-E7.5, respectively. Depending on their environment, PSCs can exist in the so-called naïve (ESCs) or primed (EpiSCs) states. Exposure to EpiSC or human ESC (hESC) culture condition can convert mESCs towards an EpiSC-like state. Here, we show that the undifferentiated epiblast state is however not stabilized in a sustained manner when exposing mESCs to hESC or EpiSC culture condition. Rather, prolonged exposure to EpiSC condition promotes a transition to a primitive streak- (PS-) like state via an unbiased epiblast-like intermediate. We show that the Brachyury-positive PS-like state is likely promoted by endogenous WNT signaling, highlighting a possible species difference between mouse epiblast-like stem cells and human Embryonic Stem Cells.
Project description:The establishment of the primitive streak and its derivative germ layers, mesoderm and endoderm, are prerequisite steps in the formation of many tissues. To model these developmental stages in vitro, an ES cell line was established that expresses CD4 from the foxa2 locus in addition to GFP from the brachyury locus. A GFP-Bry(+) population expressing variable levels of CD4-Foxa2 developed upon differentiation of this ES cell line. Analysis of gene-expression patterns and developmental potential revealed that the CD4-Foxa2(hi)GFP-Bry(+) population displays characteristics of the anterior primitive streak, whereas the CD4-Foxa2(lo)GFP-Bry(+) cells resemble the posterior streak. Using this model, we were able to demonstrate that Wnt and TGF-beta/nodal/activin signaling simultaneously were required for the generation of the CD4-Foxa2(+)GFP-Bry(+) population. Wnt or low levels of activin-induced a posterior primitive streak population, whereas high levels of activin resulted in an anterior streak fate. Finally, sustained activin signaling was found to stimulate endoderm commitment from the CD4-Foxa2(+)GFP-Bry(+) ES cell population. These findings demonstrate that the early developmental events involved in germ-layer induction in the embryo are recapitulated in the ES cell model and uncover insights into the signaling pathways involved in the establishment of mesoderm and endoderm.
Project description:A role for Rac1 GTPase in canonical Wnt signaling has recently been demonstrated, showing that it is required for ?-catenin translocation to the nucleus. In this study, we investigated the mechanism of Rac1 stimulation by Wnt. Upregulation of Rac1 activity by Wnt3a temporally correlated with enhanced p120-catenin binding to Rac1 and Vav2. Vav2 and Rac1 association with p120-catenin was modulated by phosphorylation of this protein, which was stimulated upon serine/threonine phosphorylation by CK1 and inhibited by tyrosine phosphorylation by Src or Fyn. Acting on these two post-translational modifications, Wnt3a induced the release of p120-catenin from E-cadherin, enabled the interaction of p120-catenin with Vav2 and Rac1, and facilitated Rac1 activation by Vav2. Given that p120-catenin depletion disrupts gastrulation in Xenopus, we analyzed p120-catenin mutants for their ability to rescue this phenotype. In contrast to the wild-type protein or other controls, p120-catenin point mutants that were deficient in the release from E-cadherin or in Vav2 or Rac1 binding failed to rescue p120-catenin depletion. Collectively, these results indicate that binding of p120-catenin to Vav2 and Rac1 is required for the activation of this GTPase upon Wnt signaling.