TCF/LEF regulation of the topologically associated domain ADI promotes mESCs to exit the pluripotent ground state.

Mouse embryonic stem cells (mESCs) can be maintained in vitro in defined N2B27 medium supplemented with two chemical inhibitors for GSK3 and MEK (2i) and the cytokine leukemia inhibitory factor (LIF), which act synergistically to promote self-renewal and pluripotency. Here, we find that genetic deletion of the four genes encoding the TCF/LEF transcription factors confers mESCs with the ability to self-renew in N2B27 medium alone. TCF/LEF quadruple knockout (qKO) mESCs display dysregulation of several genes, including Aire, Dnmt3l, and IcosL, located adjacent to each other within a topologically associated domain (TAD). Aire, Dnmt3l, and IcosL appear to be regulated by TCF/LEF in a ß-catenin independent manner. Moreover, downregulation of Aire and Dnmt3l in wild-type mESCs mimics the loss of TCF/LEF and increases mESC survival in the absence of 2iL. Hence, this study identifies TCF/LEF effectors that mediate exit from the pluripotent state.

Delayed maturation of thymic epithelium in mice with specific deletion of ß-catenin gene in FoxN1 positive cells.

Wnt signalling pathways have been reported to be involved in thymus development but their precise role in the development of both thymic epithelium (TE) and thymocytes is controversial. Herein, we examined embryonic, postnatal and adult thymi of mice with a specific deletion of ß-catenin gene in FoxN1+ thymic epithelial cells (TECs). Together with a high postnatal mouse mortality, the analysis showed severe thymic hypocellularity, largely due an important reduction in numbers of developing thymocytes, and delayed, partially blocked maturation of mutant TECs. Affected TECs included largely cortical (c) TEC subsets, such as immature MTS20+ TECs, Ly51+ cTECs and a remarkable, rare Ly51+MTS20+MHCIIhi cell subpopulation previously reported to contain thymic epithelial progenitor cells (TEPCs) (Ulyanchenko et al., Cell Rep 14:2819-2832, 2016). In addition, altered postnatal organization of mutant thymic medulla failed to organize a unique, central epithelial area. This delayed maturation of TE cell components correlated with low transcript production of some molecules reported to be masters for TEC maturation, such as EphB2, EphB3 and RANK. Changes in the thymic lymphoid component became particularly evident after birth, when molecules expressed by TECs and involved in early T-cell maturation, such as CCL25, CXCL12 and Dll4, exhibited minimal values. This represented a partial blockade of the progression of DN to DP cells and reduced proportions of this last thymocyte subset. At 1 month, in correlation with a significant increase in transcript production, the DP cell percentage increased in correlation with a significant fall in the number of mature TCRαßhi thymocytes and peripheral T lymphocytes.

Canonical Wnt Signaling Pathway on Polarity Formation of Utricle Hair Cells.

As part of the inner ear, the vestibular system is responsible for sense of balance, which consists of three semicircular canals, the utricle, and the saccule. Increasing evidence has indicated that the noncanonical Wnt/PCP signaling pathway plays a significant role in the development of the polarity of the inner ear. However, the role of canonical Wnt signaling in the polarity of the vestibule is still not completely clear. In this study, we found that canonical Wnt pathway-related genes are expressed in the early stage of development of the utricle and change dynamically. We conditionally knocked out ß-catenin, a canonical Wnt signaling core protein, and found that the cilia orientation of hair cells was disordered with reduced number of hair cells in the utricle. Moreover, regulating the canonical Wnt pathway (Licl and IWP2) in vitro also affected hair cell polarity and indicated that Axin2 may be important in this process. In conclusion, our results not only confirm that the regulation of canonical Wnt signaling affects the number of hair cells in the utricle but also provide evidence for its role in polarity development.

Endogenous Wnt/ß-catenin signaling in Müller cells protects retinal ganglion cells from excitotoxic damage.

Purpose: To analyze whether activation of endogenous wingless (Wnt)/ß-catenin signaling in Müller cells is involved in protection of retinal ganglion cells (RGCs) following excitotoxic damage. Methods: Transgenic mice with a tamoxifen-dependent ß-catenin deficiency in Müller cells were injected with N-methyl-D-aspartate (NMDA) into the vitreous cavity of one eye to induce excitotoxic damage of the RGCs, while the contralateral eye received PBS only. Retinal damage was quantified by counting the total number of RGC axons in cross sections of optic nerves and measuring the thickness of the retinal layers on meridional sections. Then, terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay was performed to identify apoptotic cells in retinas of both genotypes. Western blot analyses to assess the level of retinal ß-catenin and real-time RT-PCR to quantify the retinal expression of neuroprotective factors were performed. Results: Following NMDA injection of wild-type mice, a statistically significant increase in retinal ß-catenin protein levels was observed compared to PBS-injected controls, an effect that was blocked in mice with a Müller cell-specific ß-catenin deficiency. Furthermore, in mice with a ß-catenin deficiency in Müller cells, NMDA injection led to a statistically significant decrease in RGC axons as well as a substantial increase in TUNEL-positive cells in the RGC layer compared to the NMDA-treated controls. Moreover, in the retinas of the control mice a NMDA-mediated statistically significant induction of leukemia inhibitory factor (Lif) mRNA was detected, an effect that was substantially reduced in mice with a ß-catenin deficiency in Müller cells. Conclusions: Endogenous Wnt/ß-catenin signaling in Müller cells protects RGCs against excitotoxic damage, an effect that is most likely mediated via the induction of neuroprotective factors, such as Lif.

Aptamer/Peptide-Functionalized Genome-Editing System for Effective Immune Restoration through Reversal of PD-L1-Mediated Cancer Immunosuppression.

Effective reversal of tumor immunosuppression is of critical importance in cancer therapy. A multifunctional delivery vector that can effectively deliver CRISPR-Cas9 plasmid for ß-catenin knockout to reverse tumor immunosuppression is constructed. The multi-functionalized delivery vector is decorated with aptamer-conjugated hyaluronic acid and peptide-conjugated hyaluronic acid to combine the tumor cell/nuclear targeting function of AS1411 with the cell penetrating/nuclear translocation function of TAT-NLS. Due to the significantly enhanced plasmid enrichment in malignant cell nuclei, the genome editing system can induce effective ß-catenin knockout and suppress Wnt/ß-catenin pathway, resulting in notably downregulated proteins involved in tumor progression and immunosuppression. Programmed death-ligand 1 (PD-L1) downregulation in edited tumor cells not only releases the PD-1/PD-L1 brake to improve the cancer killing capability of CD8+ T cells, but also enhances antitumor immune responses of immune cells. This provides a facile strategy to reverse tumor immunosuppression and to restore immunosurveillance and activate anti-tumor immunity.

Hormonal and spatial control of SUMOylation in the human and mouse adrenal cortex.

SUMOylation is a highly conserved and dynamic post-translational mechanism primarily affecting nuclear programs for adapting organisms to stressful challenges. Alteration of SUMOylation cycles leads to severe developmental and homeostatic defects and malignancy, but signals coordinating SUMOylation are still unidentified. The adrenal cortex is a zonated endocrine gland that controls body homeostasis and stress response. Here, we show that in human and in mouse adrenals, SUMOylation follows a decreasing centripetal gradient that mirrors cortical differentiation flow and delimits highly and weakly SUMOylated steroidogenic compartments, overlapping glomerulosa, and fasciculata zones. Activation of PKA signaling by acute hormonal treatment, mouse genetic engineering, or in Carney complex results in repression of small ubiquitin-like modifier (SUMO) conjugation in the inner cortex by coordinating expression of SUMO pathway inducers and repressors. Conversely, genetic activation of canonical wingless-related integration site signaling maintains high SUMOylation potential in the outer neoplastic cortex. Thus, SUMOylation is tightly regulated by signaling pathways that orchestrate adrenal zonation and diseases.-Dumontet, T., Sahut-Barnola, I., Dufour, D., Lefrançois-Martinez, A.-M., Berthon, A., Montanier, N., Ragazzon, B., Djari, C., Pointud, J.-C., Roucher-Boulez, F., Batisse-Lignier, M., Tauveron, I., Bertherat, J., Val, P., Martinez, A. Hormonal and spatial control of SUMOylation in the human and mouse adrenal cortex.

Learning impairments and molecular changes in the brain caused by ß-catenin loss.

Intellectual disability (ID), defined as IQ<70, occurs in 2.5% of individuals. Elucidating the underlying molecular mechanisms is essential for developing therapeutic strategies. Several of the identified genes that link to ID in humans are predicted to cause malfunction of ß-catenin pathways, including mutations in CTNNB1 (ß-catenin) itself. To identify pathological changes caused by ß-catenin loss in the brain, we have generated a new ß-catenin conditional knockout mouse (ß-cat cKO) with targeted depletion of ß-catenin in forebrain neurons during the period of major synaptogenesis, a critical window for brain development and function. Compared with control littermates, ß-cat cKO mice display severe cognitive impairments. We tested for changes in two ß-catenin pathways essential for normal brain function, cadherin-based synaptic adhesion complexes and canonical Wnt (Wingless-related integration site) signal transduction. Relative to control littermates, ß-cat cKOs exhibit reduced levels of key synaptic adhesion and scaffold binding partners of ß-catenin, including N-cadherin, α-N-catenin, p120ctn and S-SCAM/Magi2. Unexpectedly, the expression levels of several canonical Wnt target genes were not altered in ß-cat cKOs. This lack of change led us to find that ß-catenin loss leads to upregulation of γ-catenin (plakoglobin), a partial functional homolog, whose neural-specific role is poorly defined. We show that γ-catenin interacts with several ß-catenin binding partners in neurons but is not able to fully substitute for ß-catenin loss, likely due to differences in the N-and C-termini between the catenins. Our findings identify severe learning impairments, upregulation of γ-catenin and reductions in synaptic adhesion and scaffold proteins as major consequences of ß-catenin loss.

Conditional Haploinsufficiency of ß-Catenin Aggravates Neuronal Damage in a Paraquat-Based Mouse Model of Parkinson Disease.

The canonical Wnt pathway is critical for both the development and adulthood survival and homeostatic maintenance of the midbrain dopaminergic (DA) neurons. Expanding evidence has demonstrated that genetic factors associated with familial Parkinson disease (PD) deregulate this important pathway, suggesting that a disturbed canonical Wnt pathway is likely involved in PD pathogenesis; yet, the specific role of this pathway in sporadic PD remains unclear. In this study, we aimed to determine the effects of specific inhibition of the canonical pathway by hemizygous knockout of ß-catenin, the obligatory component of the canonical Wnt pathway, on paraquat (PQ)-induced DA neuronal degeneration in the substantia nigra in vivo. We found that while hemizygous conditional knockout of ß-catenin in DA neurons did not cause any significant TH+ neuronal loss in the substantia nigra at basal level, it triggered elevated oxidative stress at basal level and further enhanced PQ-induced oxidative damage and loss of TH+ neurons in the substantia nigra and axonal termini in the striatum that manifested as exacerbated motor deficits. These data support the notion that reduced Wnt/ß-catenin signaling in sporadic PD likely contributes to DA neuronal loss through an enhanced oxidative stress-response pathway.

Myeloid ß-Catenin Deficiency Exacerbates Atherosclerosis in Low-Density Lipoprotein Receptor-Deficient Mice.

OBJECTIVE: The Wnt/ß-catenin signaling is an ancient and evolutionarily conserved pathway that regulates essential aspects of cell differentiation, proliferation, migration and polarity. Canonical Wnt/ß-catenin signaling has also been implicated in the pathogenesis of atherosclerosis. Macrophage is one of the major cell types involved in the initiation and progression of atherosclerosis, but the role of macrophage ß-catenin in atherosclerosis remains elusive. This study aims to investigate the impact of ß-catenin expression on macrophage functions and atherosclerosis development. APPROACH AND RESULTS: To investigate the role of macrophage canonical Wnt/ß-catenin signaling in atherogenesis, we generated ß-cateninΔmyeLDLR-/- mice (low-density lipoprotein receptor-deficient mice with myeloid-specific ß-catenin deficiency). As expected, deletion of ß-catenin decreased macrophage adhesion and migration properties in vitro. However, deficiency of ß-catenin significantly increased atherosclerotic lesion areas in the aortic root of LDLR-/- (low-density lipoprotein receptor-deficient) mice without affecting the plasma lipid levels and atherosclerotic plaque composition. Mechanistic studies revealed that ß-catenin can regulate activation of STAT (signal transducer and activator of transcription) pathway in macrophages, and ablation of ß-catenin resulted in STAT3 downregulation and STAT1 activation, leading to elevated macrophage inflammatory responses and increased atherosclerosis. CONCLUSIONS: This study demonstrates a critical role of myeloid ß-catenin expression in atherosclerosis by modulating macrophage inflammatory responses.

Fibroblast-Specific ß-Catenin Signaling Dictates the Outcome of AKI.

AKI is a devastating condition with high morbidity and mortality. The pathologic features of AKI are characterized by tubular injury, inflammation, and vascular impairment. Whether fibroblasts in the renal interstitium have a role in the pathogenesis of AKI is unknown. In this study, we investigated the role of fibroblast-specific ß-catenin signaling in dictating the outcome of AKI, using conditional knockout mice in which ß-catenin was specifically ablated in fibroblasts (Gli1-ß-cat-/-). After ischemia-reperfusion injury (IRI), Gli1-ß-cat-/- mice had lower serum creatinine levels and less morphologic injury than Gli1-ß-cat+/+ littermate controls. Moreover, we detected fewer apoptotic cells, as well as decreased cytochrome C release; reduced expression of Bax, FasL, and p53; and increased phosphorylation of Akt, in the Gli1-ß-cat-/- kidneys. Gli1-ß-cat-/- kidneys also exhibited upregulated expression of proliferating cell nuclear antigen and Ki-67, which are markers of cell proliferation. Furthermore, Gli1-ß-cat-/- kidneys displayed suppressed NF-κB signaling and cytokine expression and reduced infiltration of inflammatory cells. Notably, loss of ß-catenin in fibroblasts induced renal expression of hepatocyte growth factor (HGF) and augmented the tyrosine phosphorylation of c-met receptor after IRI. In vitro, treatment with Wnt ligands or ectopic expression of active ß-catenin inhibited HGF mRNA and protein expression and repressed HGF promoter activity. Collectively, these results suggest that fibroblast-specific ß-catenin signaling can control tubular injury and repair in AKI by modulating HGF expression. Our studies uncover a previously unrecognized role for interstitial fibroblasts in the pathogenesis of AKI.

ß-Catenin in stromal progenitors controls medullary stromal development.

The renal stroma is a population of matrix-producing fibroblast cells that serves as a structural framework for the kidney parenchyma. The stroma also regulates branching morphogenesis and nephrogenesis. In the mature kidney, the stroma forms at least three distinct cell populations: the capsular, cortical, and medullary stroma. These distinct stromal populations have important functions in kidney development, maintenance of kidney function, and disease progression. However, the development, differentiation, and maintenance of the distinct stroma populations are not well defined. Using a mouse model with ß-catenin deficiency in the stroma cell population, we demonstrate that ß-catenin is not involved in the formation of the stromal progenitors nor in the formation of the cortical stroma population. In contrast, ß-catenin does control the differentiation of stromal progenitors to form the medullary stroma. In the absence of stromal ß-catenin, there is a marked reduction of medullary stromal markers. As kidney development continues, the maldifferentiated stromal cells locate deeper within the kidney tissue and are eliminated by the activation of an intrinsic apoptotic program. This leads to significant reductions in the medullary stroma population and the lack of medulla formation. Taken together, our results indicate that stromal ß-catenin is essential for kidney development by regulating medulla formation through the differentiation of medullary stromal cells.

Activation of nuclear ß-catenin/c-Myc axis promotes oxidative stress injury in streptozotocin-induced diabetic cardiomyopathy.

Myocardial oxidative stress injury plays a crucial role in the pathogenesis of diabetic cardiomyopathy (DCM). Wnt/ß-catenin signaling has been reported to involve in various heart diseases. However, the underlying mechanism associated with ß-catenin in DCM remains elusive. This study intended to explore the effect of ß-catenin on oxidative damage of DCM by establishing streptozotocin (STZ)-induced diabetic mouse model and hydrogen peroxide (H2O2)-treated myocardial cell model. Cardiac oxidative stress in DCM was detected by measurements of lipid peroxidation and anti-oxidative enzyme activities as well as DHE staining. Nuclear ß-catenin activity and oxidative damage degree were measured by western blotting, qPCR, MTT assay and TUNEL staining. Cardiac function and morphology were evaluated by echocardiography and histopathology. Under diabetic oxidative stress or H2O2 stimulation, nuclear ß-catenin accumulation upregulated downstream c-Myc and further facilitated DNA damage and p53-mediated apoptosis as well as cell viability reduction, followed by phenotypic changes of cardiac dysfunction, interstitial fibrosis deposition and myocardial atrophy. Conversely, through directly inhibiting nuclear ß-catenin/c-Myc axis, not only did siRNA knockdown of ß-catenin or c-Myc attenuate cell injury in H2O2-stimulated cardiomyocytes, but also diabetic cardiac-specific ß-catenin-knockout mice displayed the same prevention of heart injury as insulin-treated diabetic mice. The present study demonstrated that activated nuclear ß-catenin/c-Myc axis was responsible for oxidative cardiac impairment of DCM. Therefore, repressing functional nuclear ß-catenin may provide a hopeful therapeutic strategy for DCM.

Mice lacking liver-specific ß-catenin develop steatohepatitis and fibrosis after iron overload.

BACKGROUND & AIMS: Iron overload disorders such as hereditary hemochromatosis and iron loading anemias are a common cause of morbidity from liver diseases and increase risk of hepatic fibrosis and hepatocellular carcinoma (HCC). Treatment options for iron-induced damage are limited, partly because there is lack of animal models of human disease. Therefore, we investigated the effect of iron overload in liver-specific ß-catenin knockout mice (KO), which are susceptible to injury, fibrosis and tumorigenesis following chemical carcinogen exposure. METHODS: Iron overload diet was administered to KO and littermate control (CON) mice for various times. To ameliorate an oxidant-mediated component of tissue injury, N-Acetyl-L-(+)-cysteine (NAC) was added to drinking water of mice on iron overload diet. RESULTS: KO on iron diet (KO +Fe) exhibited remarkable inflammation, followed by steatosis, oxidative stress, fibrosis, regenerating nodules and occurrence of occasional HCC. Increased injury in KO +Fe was associated with activated protein kinase B (AKT), ERK, and NF-κB, along with reappearance of ß-catenin and target gene Cyp2e1, which promoted lipid peroxidation and hepatic damage. Addition of NAC to drinking water protected KO +Fe from hepatic steatosis, injury and fibrosis, and prevented activation of AKT, ERK, NF-κB and reappearance of ß-catenin. CONCLUSIONS: The absence of hepatic ß-catenin predisposes mice to hepatic injury and fibrosis following iron overload, which was reminiscent of hemochromatosis and associated with enhanced steatohepatitis and fibrosis. Disease progression was notably alleviated by antioxidant therapy, which supports its chemopreventive role in the management of chronic iron overload disorders. LAY SUMMARY: Lack of animal models for iron overload disorders makes it hard to study the disease process for improving therapies. Feeding high iron diet to mice that lack the ß-catenin gene in liver cells led to increased inflammation followed by fat accumulation, cell death and wound healing that mimicked human disease. Administration of an antioxidant prevented hepatic injury in this model.

Inhibition of Smooth Muscle ß-Catenin Hinders Neointima Formation After Vascular Injury.

OBJECTIVE: Smooth muscle cells (SMCs) contribute to neointima formation after vascular injury. Although ß-catenin expression is induced after injury, whether its function is essential in SMCs for neointimal growth is unknown. Moreover, although inhibitors of ß-catenin have been developed, their effects on SMC growth have not been tested. We assessed the requirement for SMC ß-catenin in short-term vascular homeostasis and in response to arterial injury and investigated the effects of ß-catenin inhibitors on vascular SMC growth. APPROACH AND RESULTS: We used an inducible, conditional genetic deletion of ß-catenin in SMCs of adult mice. Uninjured arteries from adult mice lacking SMC ß-catenin were indistinguishable from controls in terms of structure and SMC marker gene expression. After carotid artery ligation, however, vessels from mice lacking SMC ß-catenin developed smaller neointimas, with lower neointimal cell proliferation and increased apoptosis. SMCs lacking ß-catenin showed decreased mRNA expression of Mmp2, Mmp9, Sphk1, and S1pr1 (genes that promote neointima formation), higher levels of Jag1 and Gja1 (genes that inhibit neointima formation), decreased Mmp2 protein expression and secretion, and reduced cell invasion in vitro. Moreover, ß-catenin inhibitors PKF118-310 and ICG-001 limited growth of mouse and human vascular SMCs in a dose-dependent manner. CONCLUSIONS: SMC ß-catenin is dispensable for maintenance of the structure and state of differentiation of uninjured adult arteries, but is required for neointima formation after vascular injury. Pharmacological ß-catenin inhibitors hinder growth of human vascular SMCs. Thus, inhibiting ß-catenin has potential as a therapy to limit SMC accumulation and vascular obstruction.

90K Glycoprotein Promotes Degradation of Mutant ß-Catenin Lacking the ISGylation or Phosphorylation Sites in the N-terminus.

ß-Catenin is a major transducer of the Wnt signaling pathway, which is aberrantly expressed in colorectal and other cancers. Previously, we showed that ß-catenin is downregulated by the 90K glycoprotein via ISGylation-dependent degradation. However, the further mechanisms of ß-catenin degradation by 90K-mediated ISGylation pathway were not investigated. This study aimed to identify the ß-catenin domain responsible for the action of 90K and to compare the mechanism of 90K on ß-catenin degradation with phosphorylation-dependent ubiquitinational degradation of ß-catenin. The deletion mutants of ß-catenin lacking N- or C-terminal domain or mutating the N-terminal lysine or nonlysine residue were employed to delineate the characteristics of ß-catenin degradation by 90K-mediated ISGylation pathway. 90K induced Herc5 and ISG15 expression and reduced ß-catenin levels in HeLa and CSC221 cells. The N-terminus of ß-catenin is required for 90K-induced ß-catenin degradation, but the N-terminus of ß-catenin is not essential for interaction with Herc5. However, substituting lysine residues in the N-terminus of ß-catenin with arginine or deleting serine or threonine residue containing domains from the N-terminus does not affect 90K-induced ß-catenin degradation, indicating that the N-terminal 86 amino acids of ß-catenin are crucial for 90K-mediated ISGylation/degradation of ß-catenin in which the responsible lysine or nonlysine residues were not identified. Our present results highlight the action of 90K on promoting degradation of mutant ß-catenin lacking the phosphorylation sites in the N-terminus. It provides further insights into the discrete pathway downregulating the stabilized ß-catenin via acquiring mutations at the serine/threonine residues in the N-terminus.

Efficient CRISPR/Cas9-Mediated Versatile, Predictable, and Donor-Free Gene Knockout in Human Pluripotent Stem Cells.

Loss-of-function studies in human pluripotent stem cells (hPSCs) require efficient methodologies for lesion of genes of interest. Here, we introduce a donor-free paired gRNA-guided CRISPR/Cas9 knockout strategy (paired-KO) for efficient and rapid gene ablation in hPSCs. Through paired-KO, we succeeded in targeting all genes of interest with high biallelic targeting efficiencies. More importantly, during paired-KO, the cleaved DNA was repaired mostly through direct end joining without insertions/deletions (precise ligation), and thus makes the lesion product predictable. The paired-KO remained highly efficient for one-step targeting of multiple genes and was also efficient for targeting of microRNA, while for long non-coding RNA over 8 kb, cleavage of a short fragment of the core promoter region was sufficient to eradicate downstream gene transcription. This work suggests that the paired-KO strategy is a simple and robust system for loss-of-function studies for both coding and non-coding genes in hPSCs.

Leukocyte Beta-Catenin Expression Is Disturbed in Systemic Lupus Erythematosus.

Wnt/ß-catenin signaling is relatively understudied in immunity and autoimmunity. ß-catenin blocks inflammatory mediators and favors tolerogenic dendritic cell (DC) phenotypes. We show here that leukocytes from lupus-prone mice and SLE patients express diminished ß-catenin transcriptional activity, particularly in myeloid cells, although other leukocytes revealed similar trends. Serum levels of DKK-1, an inhibitor under transcriptional control of Wnt/ß-catenin, were also decreased in lupus-prone mice. Surprisingly, however, preemptive deletion of ß-catenin from macrophages appears to have no effect on lupus development, even in mice with varying genetic loads for lupus. Although myeloid-specific loss of ß-catenin does not seem to be important for lupus development, the potential role of this transcription factor in other leukocytes and renal cells remain to be elucidated.

ß-catenin-mediated adhesion is required for successful preimplantation mouse embryo development.

ß-catenin (CTNNB1) is integral to cell adhesion and to the canonical Wnt signaling pathway. The effects of maternal and zygotic CTNNB1 on embryogenesis have each been separately assessed, whereas the effect of its total absence has not. As the 'traditional' conditional Ctnnb1 knockout alleles give rise to truncated CTNNB1 fragments, we designed a new knockout allele incapable of CTNNB1 production. Mouse embryos lacking intact maternal/zygotic CTNNB1 from two knockout strains were examined in detail. Preimplantation embryos are formed, yet abnormalities in their size and shape were found throughout pre- and early postimplantation development. In the absence of the zona pellucida, embryos lacking CTNNB1 undergo fission and these separated blastomeres can become small trophoblastic vesicles, which in turn induce decidual reactions. Comparing the severity of this defective adhesion phenotype in embryos bearing the null allele with those carrying the 'traditional' knockout allele suggests a hypomorphic effect of the truncated CTNNB1 protein fragment, an important observation with possible impact on previous and future studies.

Endothelial ß-Catenin Signaling Is Required for Maintaining Adult Blood-Brain Barrier Integrity and Central Nervous System Homeostasis.

BACKGROUND: The blood-brain barrier (BBB) formed by brain endothelial cells interconnected by tight junctions is essential for the homeostasis of the central nervous system. Although studies have shown the importance of various signaling molecules in BBB formation during development, little is known about the molecular basis regulating the integrity of the adult BBB. METHODS AND RESULTS: Using a mouse model with tamoxifen-inducible endothelial cell-restricted disruption of ctnnb1 (iCKO), we show here that endothelial ß-catenin signaling is essential for maintaining BBB integrity and central nervous system homeostasis in adult mice. The iCKO mice developed severe seizures accompanied by neuronal injury, multiple brain petechial hemorrhages, and central nervous system inflammation, and all had postictal death. Disruption of endothelial ß-catenin induced BBB breakdown and downregulation of the specific tight junction proteins claudin-1 and -3 in adult brain endothelial cells. The clinical relevance of the data is indicated by the observation of decreased expression of claudin-1 and nuclear ß-catenin in brain endothelial cells of hemorrhagic lesions of hemorrhagic stroke patients. CONCLUSIONS: These results demonstrate the prerequisite role of endothelial ß-catenin in maintaining the integrity of adult BBB. The results suggest that BBB dysfunction secondary to defective ß-catenin transcription activity is a key pathogenic factor in hemorrhagic stroke, seizure activity, and central nervous system inflammation.