[E-cadherin as a novel surface marker of spermatogonial stem cells].

Spermatogenesis is a fundamental biological process that ensures gee transmission from one generation to another trough gametes. This process relies on a rare population of testicular cells, called spermatogonial stem cells (SSCs), that self-renew throughout adult male life and differentiate into mature gametes. Despite the longstanding research of SSCs, their biological properties remain largely unknown which is partly due to very limited availability of these cells. Here we show that cell adhesion protein E-cadherin is a highly specific surface marker of mouse SSCs, which can be successfully used for their enrichment.

Role of beta-catenin in the developing cortical and hippocampal neuroepithelium.

beta-Catenin plays a pivotal role in Wnt signaling during embryogenesis and is a component of adherens junctions. Since targeted disruption of the beta-catenin gene is lethal at gastrulation we have used a D6-Cre mouse line for conditional inactivation of beta-catenin in the mouse cerebral cortex and hippocampus after embryonic day (E) 10.5. In D6-Cre floxed beta-catenin mice, hippocampal CA1-CA2 fields are disrupted in similar manner as in Wnt-3a and LEF-1 mutants. The cortex of D6-Cre floxed beta-catenin mutants is strongly affected which contrasts with the normal cortex observed in Wnt-3a and LEF-1 mutants. Severe abnormalities in the organization of the neuroepithelium are observed that include disrupted interkinetic nuclear migration, loss of adherens junctions, impaired radial migration of neurons toward superficial layers and decreased cell proliferation after E15.5. At newborn stage, a premature disassembly of the radial glial scaffold and increased numbers of astrocytes are found in the cortex.

Expression patterns of Wnt genes in mouse gut development.

Wnt signaling regulates cell fate decisions and cell proliferation during development and in adult tissues in both invertebrates and vertebrates. Here we describe the identification of Wnt genes, Wnt2a, 4, 5a, 5b, 6 and 11, expressed in mouse embryonic gut development. Each of these genes exhibits a characteristic and regional-specific expression pattern along the anterior-posterior axis of the digestive tube between embryonic day (E) 12.5 and 16.5 of embryonic development. The expression of Wnt5a is confined to the mesenchymal compartment, while expression of Wnt4 is found both in the intestinal epithelium and the mesenteric anlage. Wnt11 is expressed in the epithelium of esophagus and colon, but also in mesenchymal cells of the stomach. Wnt5b and Wnt6 exhibit restricted expression in the epithelium of the esophagus. A characteristic regionalized expression pattern is observed in the developing stomach. Wnt5a is expressed in the mesenchymal layer of the prospective gland region but becomes restricted to the tip of the gland region by E14.5. Wnt11 is highly expressed at the gastro-esophageal junctions, while Wnt4 is found in the epithelium lining the pyloric region of the stomach but not in the epithelium of the prospective gland region.

Differential adhesion leads to segregation and exclusion of N-cadherin-deficient cells in chimeric embryos.

Cadherin-mediated cell-cell interactions are thought to be critical in controlling cell sorting during embryogenesis. Here, we report that chimeric embryos generated with N-cadherin-deficient (N-cadherin(-/-)) embryonic stem cells develop further than embryos completely lacking N-cadherin only when the myocardium consists of N-cadherin-positive cells. Initially, the N-cadherin-negative and -positive cells mix together to form chimeric tissues; however, by embryonic day 9.5, the N-cadherin(-/-) cells segregate from the wild-type cells forming distinct aggregates. The chimeric embryos have large aggregates of N-cadherin(-/-) myocardial cells in the heart lumen, indicating that the cells are unable to maintain cell-cell contacts with N-cadherin-positive myocytes. This sorting-out phenomenon also is apparent in somites, neural tube, and developing brain where N-cadherin(-/-) cells form distinct lumenal structures. These studies demonstrate that N-cadherin-mediated adhesion is critical for maintaining cell-cell interactions in tissues undergoing active cellular rearrangements and increased mechanical stress associated with morphogenesis.

Inactivation of the beta-catenin gene by Wnt1-Cre-mediated deletion results in dramatic brain malformation and failure of craniofacial development.

beta-Catenin is a central component of both the cadherin-catenin cell adhesion complex and the Wnt signaling pathway. We have investigated the role of beta-catenin during brain morphogenesis, by specifically inactivating the beta-catenin gene in the region of Wnt1 expression. To achieve this, mice with a conditional ('floxed') allele of beta-catenin with required exons flanked by loxP recombination sequences were intercrossed with transgenic mice that expressed Cre recombinase under control of Wnt1 regulatory sequences. beta-Catenin gene deletion resulted in dramatic brain malformation and failure of craniofacial development. Absence of part of the midbrain and all of the cerebellum is reminiscent of the conventional Wnt1 knockout (Wnt1(-/-)), suggesting that Wnt1 acts through beta-catenin in controlling midbrain-hindbrain development. The craniofacial phenotype, not observed in embryos that lack Wnt1, indicates a role for beta-catenin in the fate of neural crest cells. Analysis of neural tube explants shows that (beta-catenin is efficiently deleted in migrating neural crest cell precursors. This, together with an increased apoptosis in cells migrating to the cranial ganglia and in areas of prechondrogenic condensations, suggests that removal of beta-catenin affects neural crest cell survival and/or differentiation. Our results demonstrate the pivotal role of beta-catenin in morphogenetic processes during brain and craniofacial development.

Pontin52 and reptin52 function as antagonistic regulators of beta-catenin signalling activity.

In Wnt-stimulated cells, beta-catenin becomes stabilized in the cytoplasm, enters the nucleus and interacts with HMG box transcription factors of the lymphoid-enhancing factor-1 (LEF-1)/T-cell factor (TCF) family, thereby stimulating the transcription of specific target genes. We recently identified Pontin52 as a nuclear protein interacting with beta-catenin and the TATA-box binding protein (TBP), suggesting its involvement in regulating beta-catenin-mediated transactivation. Here, we report the identification of Reptin52 as an interacting partner of Pontin52. Highly homologous to Pontin52, Reptin52 likewise binds beta-catenin and TBP. Using reporter gene assays, we show that the two proteins antagonistically influence the transactivation potential of the beta-catenin-TCF complex. Furthermore, we demonstrate the evolutionary conservation of this mechanism in Drosophila. dpontin and dreptin are essential genes that act antagonistically in the control of Wingless signalling in vivo. These results indicate that the opposite action of Pontin52 and Reptin52 on beta-catenin-mediated transactivation constitutes an additional mechanism for the control of the canonical Wingless/Wnt pathway.

Wnt/(beta)-catenin signaling regulates the expression of the homeobox gene Cdx1 in embryonic intestine.

During mammalian development, the Cdx1 homeobox gene exhibits an early period of expression when the embryonic body axis is established, and a later period where expression is restricted to the embryonic intestinal endoderm. Cdx1 expression is maintained throughout adulthood in the proliferative cell compartment of the continuously renewed intestinal epithelium, the crypts. In this study, we provide evidence in vitro and in vivo that Cdx1 is a direct transcriptional target of the Wnt/(beta)-catenin signaling pathway. Upon Wnt stimulation, expression of Cdx1 can be induced in mouse embryonic stem (ES) cells as well as in undifferentiated rat embryonic endoderm. Tcf4-deficient mouse embryos show abrogation of Cdx1 protein in the small intestinal epithelium, making Tcf4 the likely candidate to transduce Wnt signal in this part of gut. The promoter region of the Cdx1 gene contains several Tcf-binding motifs, and these bind Tcf/Lef1/(beta)-catenin complexes and mediate (beta)-catenin-dependent transactivation. The transcriptional regulation of the homeobox gene Cdx1 in the intestinal epithelium by Wnt/(beta)-catenin signaling underlines the importance of this signaling pathway in mammalian endoderm development.

The p300/CBP acetyltransferases function as transcriptional coactivators of beta-catenin in vertebrates.

Wnt growth factors regulate a variety of developmental processes by altering specific gene expression patterns. In vertebrates beta-catenin acts as transcriptional activator, which is needed to overcome target gene repression by Groucho/TLE proteins, and to permit promoter activation as the final consequence of Wnt signaling. However, the molecular mechanisms of transcriptional activation by beta-catenin are only poorly understood. Here we demonstrate that the closely related acetyltransferases p300 and CBP potentiate beta-catenin-mediated activation of the siamois promoter, a known Wnt target. beta-catenin and p300 also synergize to stimulate a synthetic reporter gene construct, whereas activation of the cyclin D1 promoter by beta-catenin is refractory to p300 stimulation. Axis formation and activation of the beta-catenin target genes siamois and Xnr-3 in Xenopus embryos are sensitive to the E1A oncoprotein, a known inhibitor of p300/CBP. The C-terminus of beta-catenin interacts directly with a region overlapping the CH-3 domain of p300. p300 could participate in alleviating promoter repression imposed by chromatin structure and in recruiting the basal transcription machinery to promoters of particular Wnt target genes.

Expression of Eph receptors and ephrins is differentially regulated by E-cadherin.

E-cadherin is the main cell adhesion molecule of early embryonic and adult epithelial cells. Downregulation of E-cadherin is associated with epithelial-mesenchymal transition during embryonic mesoderm formation and tumor progression. To identify genes whose expression is affected by the loss of E-cadherin, we compared mRNA expression patterns between wild-type and E-cadherin null mutant embryonic stem (ES) cells. We found that expression of several Eph receptors and ephrins is dependent on E-cadherin. Rescue of E-cadherin null ES cells with E-cadherin cDNA restores the wild-type expression pattern of Eph family members. Rescue of E-cadherin null ES cells with N-cadherin cDNA does not restore the wild-type expression pattern, indicating that the regulation of differential expression of Eph family members is specific to E-cadherin. Constitutive ectopic expression of E-cadherin in non-epithelial NIH3T3 cells results in the production of the EphA2 receptor. In epithelial cells, E-cadherin is required for EphA2 receptor localization at cell-cell contacts; in the absence of functional E-cadherin, EphA2 localizes to the perinuclear region. Our results indicate that E-cadherin may be directly or indirectly required for the membrane localization of Eph receptors and their membrane-bound ligands.

Brachyury is a target gene of the Wnt/beta-catenin signaling pathway.

To identify target genes of the Wnt/beta-catenin signaling pathway in early mouse embryonic development we have established a co-culture system consisting of NIH3T3 fibroblasts expressing different Wnts as feeder layer cells and embryonic stem (ES) cells expressing a green fluorescent protein (GFP) reporter gene transcriptionally regulated by the TCF/beta-catenin complex. ES cells specifically respond to Wnt signal as monitored by GFP expression. In GFP-positive ES cells we observe expression of Brachyury. Two TCF binding sites located in a 500 bp Brachyury promoter fragment bind the LEF-1/beta-catenin complex and respond specifically to beta-catenin-dependent transactivation. From these results we conclude that Brachyury is a target gene for Wnt/beta-catenin signaling.

Casein kinase II phosphorylation of E-cadherin increases E-cadherin/beta-catenin interaction and strengthens cell-cell adhesion.

Beta-catenin, a member of the Armadillo repeat protein family, binds directly to the cytoplasmic domain of E-cadherin, linking it via alpha-catenin to the actin cytoskeleton. A 30-amino acid region within the cytoplasmic domain of E-cadherin, conserved among all classical cadherins, has been shown to be essential for beta-catenin binding. This region harbors several putative casein kinase II (CKII) and glycogen synthase kinase-3beta (GSK-3beta) phosphorylation sites and is highly phosphorylated. Here we report that in vitro this region is indeed phosphorylated by CKII and GSK-3beta, which results in an increased binding of beta-catenin to E-cadherin. Additionally, in mouse NIH3T3 fibroblasts expression of E-cadherin with mutations in putative CKII sites resulted in reduced cell-cell contacts. Thus, phosphorylation of the E-cadherin cytoplasmic domain by CKII and GSK-3beta appears to modulate the affinity between beta-catenin and E-cadherin, ultimately modifying the strength of cell-cell adhesion.

Curbing the nuclear activities of beta-catenin. Control over Wnt target gene expression.

Wnt molecules control numerous developmental processes by altering specific gene expression patterns, and deregulation of Wnt signaling can lead to cancer. Many Wnt factors employ beta-catenin as a nuclear effector. Upon Wnt stimulation, beta-catenin heterodimerizes with T-cell factor (TCF) DNA-binding proteins to form a transcriptional activator complex. As the activating subunit of this complex, beta-catenin performs dual tasks: it alleviates repression of target gene promoters and subsequently it activates them. Beta-catenin orchestrates these effects by recruiting chromatin modifying cofactors and contacting components of the basal transcription machinery. Although beta-catenin and TCFs are universal activators in Wnt signaling, their target genes display distinct temporal and spatial expression patterns. Apparently, post-translational modifications modulate the interactions between TCFs and beta-catenin or DNA, and certain transcription factors can sequester beta-catenin from TCFs while others synergize with beta-catenin-TCF complexes in a promoter-specific manner. These mechanisms provide points of intersection with other signaling pathways, and contribute to the complexity and specificity of Wnt target gene regulation.

Mutations affecting transmembrane segment interactions impair adhesiveness of E-cadherin.

Lateral clustering of E-cadherin molecules is required for the adhesive properties of this cell-cell adhesion molecule. Both the extracellular domain and the cytoplasmic region of E-cadherin were previously reported to contribute to lateral clustering, but little is known about a role of the transmembrane domain in this respect. Following our previous findings indicating self-assembly of artificial transmembrane segments based on leucine residues, we asked whether the leucine-rich transmembrane segment of E-cadherin participates in lateral clustering. Here, we demonstrate that its transmembrane domain self-assembles as analyzed using the ToxR reporter system. Certain point mutations within the transmembrane domain markedly reduced self-assembly. To study whether the same point mutations also affect E-cadherin-mediated adhesion in vivo, wild-type and mutant E-cadherin cDNAs were transfected into Ltk(-) cells. Indeed, cell aggregation assays revealed significantly reduced adhesiveness when mutations had been introduced which disrupted transmembrane segment interaction. In control experiments, cell-surface expression, interaction with catenins and the cytoskeleton as well as trypsin-resistance of the protein were unaffected. These data suggest that interactions between the transmembrane segments are important for the lateral association of E-cadherin molecules required for cell-cell adhesion.

Beta-catenin is a major tyrosine-phosphorylated protein during mouse oocyte maturation and preimplantation development.

During mouse preimplantation development, the components of the E-cadherin-catenin complex are derived from both maternal and zygotic gene activity and the adhesion complex is increasingly accumulated and stored in a nonfunctional form, ready to be used for compaction and the formation of the trophectoderm cell layer (Ohsugi et al., Dev. Dyn. 206:391-402, 1996). Here, we show that beta-catenin is a major tyrosine-phosphorylated protein in oocytes and early cleavage-stage embryos and that the relative amount of phosphorylated beta-catenin is greatly reduced during the morula-blastocyst transition. Peptide-specific antibodies indicate that beta-catenin undergoes conformational changes and/or that the carboxy-terminal region of beta-catenin is blocked during preimplantation development. Moreover, the availability of a carboxy-terminal epitope seems to depend on the tyrosine phosphorylation state of beta-catenin and becomes unmasked when oocytes are treated with the tyrosine kinase inhibitor genistein. Our results suggest that tyrosine phosphorylation of beta-catenin represents a molecular mechanism to keep the accumulating E-cadherin adhesion complex in a nonfunctional form. Dev Dyn 1999;216:168-176.

Functional characterization of multiple transactivating elements in beta-catenin, some of which interact with the TATA-binding protein in vitro.

beta-Catenin, a member of the family of Armadillo repeat proteins, plays a dual role in cadherin-mediated cell adhesion and in signaling by Wnt growth factors. Upon Wnt stimulation beta-catenin undergoes nuclear translocation and serves as transcriptional coactivator of T cell factor DNA-binding proteins. Previously the transactivation potential of different portions of beta-catenin has been demonstrated, but the precise location of transactivating elements has not been established. Also, the mechanism of transactivation by beta-catenin and the molecular basis for functional differences between beta-catenin and the closely related proteins Armadillo and Plakoglobin are poorly understood. Here we have used a yeast system for the detailed characterization of the transactivation properties of beta-catenin. We show that its transactivation domains possess a modular structure, consist of multiple subelements that cover broad regions at its N and C termini, and extend considerably into the Armadillo repeat region. Compared with beta-catenin the N termini of Plakoglobin and Armadillo have different transactivation capacities that may explain their distinct signaling properties. Furthermore, transactivating elements of beta-catenin interact specifically and directly with the TATA-binding protein in vitro providing further evidence that a major function of beta-catenin during Wnt signaling is to recruit the basal transcription machinery to promoter regions of Wnt target genes.

Cadherins and tissue formation: integrating adhesion and signaling.

Cadherins and other cell-substrate and cell-cell adhesion molecules play an essential role during development. Through their cytoplasmic interaction with the cytoskeleton, cell adhesion molecules physically link cells with the extracellular matrix and/or with each other. These interactions create architectural and structural entities that enable the tissues in the embryo to restrain the physical forces encountered during development. Regulated cell adhesion is also often the driving force of morphogenetic movements. This review goes beyond the adhesive aspect of cadherins, focusing on their roles as signaling molecules in development. We discuss how cadherins, through their effects on cell proliferation, cell death, cell polarization, and differentiation, play a role in the formation of tissues and organs in the developing embryo.

The C-terminal transactivation domain of beta-catenin is necessary and sufficient for signaling by the LEF-1/beta-catenin complex in Xenopus laevis.

Beta-catenin is a multifunctional protein involved in cell adhesion and communication. In response to signaling by Wnt growth factors, beta-catenin associates with nuclear TCF factors to activate target genes. A transactivation domain identified at the C-terminus of beta-catenin can stimulate expression of artificial reporter genes. However, the mechanism of target gene activation by TCF/beta-catenin complexes and the physiological relevance of the beta-catenin transactivation domain still remain unclear. Here we asked whether the beta-catenin transactivation domain can generate a Wnt-response in a complex biological system, namely axis formation during Xenopus laevis embryogenesis. We show that a chimeric transcription factor consisting of beta-catenin fused to the DNA-binding domain of LEF-1 induces a complete secondary dorsoanterior axis when expressed in Xenopus. A LEF-1-beta-catenin fusion lacking the C-terminal transactivation domain is impaired in signaling while fusion of just the beta-catenin transactivator to the DNA-binding domain of LEF-1 is sufficient for axis-induction. The latter fusion molecule is blocked by dominant negative LEF-1 but not by excess cadherin indicating that all events parallel or upstream of the transactivation step mediated by beta-catenin are dispensable for Wnt-signaling. Moreover, beta-catenin can be replaced by a heterologous transactivator. Apparently, the ultimate function of beta-catenin in Wnt signaling is to recruit the basal transcription machinery to promoter regions of specific target genes.

E-cadherin binding prevents beta-catenin nuclear localization and beta-catenin/LEF-1-mediated transactivation.

Beta-catenin is a multifunctional protein found in three cell compartments: the plasma membrane, the cytoplasm and the nucleus. The cell has developed elaborate ways of regulating the level and localization of beta-catenin to assure its specific function in each compartment. One aspect of this regulation is inherent in the structural organization of beta-catenin itself; most of its protein-interacting motifs overlap so that interaction with one partner can block binding of another at the same time. Using recombinant proteins, we found that E-cadherin and lymphocyte-enhancer factor-1 (LEF-1) form mutually exclusive complexes with beta-catenin; the association of beta-catenin with LEF-1 was competed out by the E-cadherin cytoplasmic domain. Similarly, LEF-1 and adenomatous polyposis coli (APC) formed separate, mutually exclusive complexes with beta-catenin. In Wnt-1-transfected C57MG cells, free beta-catenin accumulated and was able to associate with LEF-1. The absence of E-cadherin in E-cadherin-/- embryonic stem (ES) cells also led to an accumulation of free beta-catenin and its association with LEF-1, thereby mimicking Wnt signaling. beta-catenin/LEF-1-mediated transactivation in these cells was antagonized by transient expression of wild-type E-cadherin, but not of E-cadherin lacking the beta-catenin binding site. The potent ability of E-cadherin to recruit beta-catenin to the cell membrane and prevent its nuclear localization and transactivation was also demonstrated using SW480 colon carcinoma cells.

Nuclear endpoint of Wnt signaling: neoplastic transformation induced by transactivating lymphoid-enhancing factor 1.

The interaction between beta-catenin and LEF-1/TCF transcription factors plays a pivotal role in the Wnt-1 signaling pathway. The level of beta-catenin is regulated by partner proteins, including glycogen synthase kinase-3beta (GSK-3beta) and the adenomatous polyposis coli (APC) tumor suppressor protein. Genetic defects in APC are responsible for a heritable predisposition to colon cancer. APC protein and GSK-3beta bind beta-catenin, retain it in the cytoplasm, and facilitate the proteolytic degradation of beta-catenin. Abrogation of this negative regulation allows beta-catenin to translocate to the nucleus and to form a transcriptional activator complex with the DNA-binding protein lymphoid-enhancing factor 1 (LEF-1). This complex is thought to be involved in tumorigenesis. Here we show that covalent linkage of LEF-1 to beta-catenin and to transcriptional activation domains derived from the estrogen receptor or the herpes simplex virus protein VP16 generates transcriptional regulators that induce oncogenic transformation of chicken embryo fibroblasts. The chimeras between LEF-1 and beta-catenin or VP16 are constitutively active, whereas fusions of LEF-1 to the estrogen receptor are regulatable by estrogen. These experiments document the oncogenicity of transactivating LEF-1 and show that the transactivation domain normally provided by beta-catenin can be replaced by heterologous activation domains. These results suggest that the transactivating function of the LEF-1/beta-catenin complex is critical for tumorigenesis and that this complex transforms cells by activating specific LEF-1 target genes.