The developing heart primordium strongly expresses N-cadherin. In order to investigate the role of this adhesion molecule in heart morphogenesis, chicken embryos were cultured at stages 5-12, and injected with anti-N-cadherin antibodies that can specifically block the activity of this cadherin. In the injected embryos, the epimyocardial layers, which develop bilaterally from the splanchnic mesoderm, do not fuse to form a single cardiac tube. Each of the unfused layers becomes fragmented into epithelioid clusters. At the cellular level, large intercellular gaps are observed in the antibody-treated myocardial layers. These disorganized myocardial layers beat to some extent, suggesting that their differentiation is not blocked; however, their contraction is not coordinated. Morphogenesis of other tissues, not only N-cadherin-negative but also N-cadherin-positive tissues, such as the neural tube and notochord, proceeded normally even in the presence of anti-N-cadherin antibodies. These results suggest that N-cadherin is indispensable for heart formation (but not for morphogenesis of the other tissues) at the developmental stages examined. For the latter processes, expression of other cadherin subtypes presumably compensate for the loss of N-cadherin activity (Nakagawa, 1997).

During early heart development the expression pattern of N-cadherin, a calcium-dependent cell adhesion molecule, suggests its involvement in morphoregulation and the stabilization of cardiomyocyte differentiation. N-cadherin's adhesive activity is dependent upon its interaction with the intracellular catenins. An association with alpha-catenin and beta-catenin also is believed to be involved in cell signaling. This study details the expression patterns of alpha-catenin, beta-catenin, and gamma-catenin, during definition of the cardiac cell population as distinct compartments in the anterior regions of the chick embryo between stages 5 and 9. The restriction of N-cadherin/catenin localization at stage 5+ from a uniform pattern in vivo, to specific cell clusters that demarcate areas where mesoderm separation is initiated, suggests that the N-cadherin/catenin complex is involved in boundary formation and in the subsequent cell sorting. The latter two processes lead to the specification and formation of the somatic and cardiac splanchnic mesoderm. N-cadherin colocalizes with alpha- and beta-catenin at the cell membrane before and during the time that its expression becomes restricted to the lateral mesoderm and continues cephalocaudal into stage 8. These proteins continue to colocalize in the myocardium of the tubular heart. Plakoglobin is not expressed in this region during stages 6-8, but is detected in the myocardium later at stage 13. The observed in vivo expression patterns of alpha-catenin, beta-catenin, and plakoglobin suggest that these proteins are directly linked with the developmental regulation of cell junctions, as cardiac cells become stably committed and phenotypically differentiated to eventually form a mature myocardium. The localization of N-CAM also was analyzed during these stages to determine whether the N-cadherin-catenin localization is unique or whether other cell adhesion molecules have similar expression patterns. The results indicate that the unique pattern of N-cadherin expression is not shared with N-CAM. Perturbation of N-cadherin using a function perturbing N-cadherin antibody (NCD-2) inhibits normal early heart development and myogenesis in a cephalocaudal, stage-dependent manner. A model is proposed whereby myocardial cell compartmentalization also defines the endocardial population. The presence of beta-catenin suggests that a similar signaling pathway involving Wnt (wingless)-mediated events may function in myocardial cell compartmentalization during early vertebrate heart development, as in Drosophila contractile vessel development (Linask, 1997).

Activation of PKC by PMA promotes the separation of chicken cardiomyocytes from one another in culture. Immunofluorescence staining for N-cadherin indicates that PMA, but not its inactive isoform 4 alpha PMA, induces the separation of cardiomyocytes and of co-cultured fibroblasts at intercellular junctional regions. The PMA-induced separation of cardiomyocytes and of co-cultured fibroblasts is inhibited by the PKC inhibitor, H-7. Immunoblot analysis further demonstrates that both PKC iota and PKC lambda are expressed in the cardiomyocyte cultures. While PKC lambda is localized to the cell-cell contact areas between cardiomyocytes, PKC iota is only detectable in the perinuclear cytoplasm of the co-cultured fibroblasts. The present findings suggest the involvement of PKC in regulating N-cadherin-mediated adherens junction formation in chicken cardiomyocytes. The differential distribution of PKC lambda and PKC iota in the cardiomyocytes and in the co-cultured fibroblasts suggests that different PKC isozymes are involved in regulating the assembly of intercellular junctions in these two cell types (Wu, 1996).

The myocardial wall of the vertebrate heart changes from a simple epithelium to a trabeculated structure during embryogenesis. This process occurs when epithelioid cardiomyocytes migrate toward the endocardium, which is coincident with up-regulation of the cell adhesion molecule, N-cadherin. To study the role of N-cadherin expressed at the trabeculation stage, a replication-defective retrovirus expressing a dominant negative mutant of N-cadherin (DeltaN-cadherin) was engineered. Control viruses were designed to express either beta-galactosidase or a full-length N-cadherin. Viruses were introduced into epithelioid presumptive myocytes at the time they initiated the epithelial-mesenchymal transformation. Individual cells infected with control viruses generate daughter myocytes, which migrate toward endocardium as a tight cluster, thereby generating a clone that forms a single or at most two trabeculae. In contrast, myocytes expressing DeltaN-cadherin are sparsely distributed within the myocardium and fail to form the ridge-shaped clone. Thus, in addition to its known roles in myocyte epithelialization and intercalated disc formation, N-cadherin appears to play a role in homotypic interactions between nonepithelial migratory myocytes during trabecular formation of the embryonic heart (Ong, 1998).

Cell-cell adhesion mediated by some members of the cadherin family is essential for embryonic survival. The N-cadherin-null embryo dies during mid-gestation, with multiple developmental defects. N-cadherin-null embryos expressing cadherins using muscle-specific promoters, alpha- or beta-myosin heavy chain, are partially rescued. Somewhat surprisingly, either N-cadherin or E-cadherin is effective in rescuing the embryos. The rescued embryos exhibit an increased number of somites, branchial arches and the presence of forelimb buds; however, in contrast, brain development is severely impaired. In rescued animals, the aberrant yolk sac morphology seen in N-cadherin-null embryos is corrected, demonstrating that this phenotype is secondary to the cardiac defect. Dye injection studies and analysis of chimeric animals that have both wild-type and N-cadherin-null cells support the conclusion that obstruction of the cardiac outflow tract represents a major defect that is likely to be the primary cause of pericardial swelling seen in null embryos. Although rescued embryos are more developed than null embryos, they are smaller than wild-type embryos, even though the integrity of the cardiovascular system appears normal. The smaller size of rescued embryos may be due, at least in part, to increased apoptosis observed in tissues not rescued by transgene expression, indicating that N-cadherin-mediated cell adhesion provides an essential survival signal for embryonic cells. These data provide in vivo evidence that cadherin adhesion is essential for cell survival and for normal heart development. These data also show that E-cadherin can functionally substitute for N-cadherin during cardiogenesis, suggesting a critical role for cadherin-mediated cell-cell adhesion, but not cadherin family member-specific signaling, at the looping stage of heart development (Luo, 2001).

To date most of the studies involving the maintenance of ovarian cell viability have focused on the endocrine, paracrine, and autocrine factors that inhibit these cells from undergoing programmed cell death or apoptosis. Recently, studies have demonstrated that cell contact also prevents ovarian cells from dying via an apoptotic mechanism. N-cadherin homophilic binding (1) is part of the mechanism through which cell contact maintains cell viability, (2) results in the activation (i.e. tyrosine phosphorylation) of the fibroblast growth factor (FGF) receptor, and (3) prevents a sustained elevation in intracellular free calcium ([Ca2+]i) which triggers apoptosis. These studies also revealed that hepatocyte growth factor (HGF), also known as scatter factor (SF), disrupts cell contact, which leads to a sustained increase in [Ca2+]i levels and ultimately, to cell death. Based on these studies, a putative mechanism is presented that relates the cellular and molecular mechanism through which basic FGF, N-cadherin, and HGF/SF interact to regulate [Ca2+]i levels and ultimately ovarian cell survival (Peluso, 1997).

The procession of round spermatids through stages VII and VIII of the rat spermatogenic cycle is critically dependent on testosterone (T). When intratesticular T levels are reduced, round spermatids appear to slough from the seminiferous epithelium, resulting in the disappearance of elongated spermatids. It has been hypothesized that T-dependent cell adhesion molecules are involved in Sertoli cell-round spermatid interactions. This study examined the hormonal regulation of one candidate cell adhesion molecule, N-cadherin, in vitro and its participation in Sertoli cell-round spermatid adhesion in coculture. Sertoli cells were treated with FSH and T, alone or in combination. Together FSH and T significantly increase the cellular content of N-cadherin, whereas FSH or T alone have no effect. Round spermatids cultered in the presence of FSH, T, or FSH plus T show an increase in round spermatid density with increasing T doses in the presence of FSH, whereas FSH and T alone produce no effect. T also increases the N-cadherin content of the cocultures in a dose-dependent manner in the presence of FSH. Addition of an N-cadherin antiserum to the Sertoli cell-round spermatid coculture in the presence of FSH and T significantly reduces round spermatid density. It is concluded that both the production of N-cadherin by Sertoli cells and the binding of round spermatids to Sertoli cells are stimulated in a synergistic manner by T and FSH. The immunoneutralization data suggest the active involvement of N-cadherin in round spermatid-Sertoli cell adhesion in vitro. N-Cadherin may be one of the factors that subserve the androgen-dependent process of round to elongated spermatid maturation (Perryman, 1996).

During embryonic development, cell migration and cell differentiation are associated with dynamic modulations both in time and space of the repertoire and function of adhesion receptors, but the nature of the mechanisms responsible for their coordinated occurrence remains to be elucidated. Thus, migrating neural crest cells adhere to fibronectin in an integrin-dependent manner while maintaining reduced N-cadherin-mediated intercellular contacts. In these cells, the control of N-cadherin may rely directly on the activity of integrins involved in the process of cell motion. Prevention of neural crest cell migration using RGD peptides or antibodies to fibronectin and to beta1 and beta3 integrins causes rapid N-cadherin-mediated cell clustering. Restoration of stable intercellular contacts results essentially from the recruitment of an intracellular pool of N-cadherin molecules that accumulate into adherens junctions in tight association with the cytoskeleton and not from the redistribution of a preexisting pool of surface N-cadherin molecules. Agents that cause elevation of intracellular Ca2+ after entry across the plasma membrane are potent inhibitors of cell aggregation and reduce the N-cadherin- mediated junctions in the cells. Elevated serine/threonine phosphorylation of catenins associated with N-cadherin accompany the restoration of intercellular contacts. These results indicate that in migrating neural crest cells, beta1 and beta3 integrins are at the origin of a cascade of signaling events which involve transmembrane Ca2+ fluxes, followed by activation of phosphatases and kinases, and which ultimately control the surface distribution and activity of N-cadherin. Such a direct coupling between adhesion receptors by means of intracellular signals may be significant for the coordinated interplay between cell-cell and cell-substratum adhesion that occurs during embryonic development, in wound healing, and during tumor invasion and metastasis (Monier-Gavelle, 1997).

p120cas (CAS) is a tyrosine kinase substrate whose phosphorylation has been implicated in cell transformation by Src and in ligand-induced signaling through the EGF, PDGF, and CSF-1 receptors. More recently, CAS has been shown to associate with E-cadherin and its cofactors (catenins), molecules that are involved in cell adhesion. Although both CAS and beta-catenin contain armadillo repeat domains (Arm domains), the amino acid identity between these proteins in this region is only 22%. It is not yet clear whether CAS will emulate other catenins by associating with other members of the cadherin family. In addition to binding E-cadherin, wild-type CAS associates with N-cadherin and P-cadherin. Transient transfection of cloned CAS isoforms into MDCK epithelial cells indicates that CAS1 and CAS2 isoforms are equally capable of binding to E-cadherin even though these cells preferentially express CAS2 isoforms. CAS also colocalizes with N-cadherin in NIH3T3 cells; analysis of CAS mutants in vivo indicates that the CAS-N-cadherin interaction requires an intact CAS Arm domain. The data suggest that CAS-cadherin interactions in general are dictated by the conserved armadillo repeats and are not heavily influenced by sequences added outside the Arm domain by alternative splicing. Interestingly, overexpression of CAS in NIH3T3 cells induce a striking morphological phenotype characterized by the presence of long dendrite-like processes. This branching phenotype is specific for CAS, since (1) overexpression of the structurally similar beta-catenin has little effect on cell morphology, and (2) the branching is abolished by deletions in the CAS Arm domain. These data indicate that CAS, like other catenins, is a cofactor for multiple members of the cadherin family. However, the dramatically distinct phenotype exhibited by fibroblasts overexpressing CAS, versus beta-catenin, support recent data suggesting that these catenins have fundamentally different and possibly opposing roles in cadherin complexes (Reynolds, 1996).

N-cadherin (CDH2) is a member of the cadherin family of Ca2(+)-dependent cell-cell adhesion molecules. To investigate mechanisms controlling CDH2 transcription, a genomic DNA sequence containing 2.8 kb of 5' flanking region and the first two exons of chicken CDH2 was isolated and analyzed. Sequence analysis of the promoter region of CDH2 reveals no CCATT or TATA boxes, but showed a high overall GC content, high CpG dinucleotide content, and several consensus Sp1 and Ap2 binding sequences. When fused to the cat reporter gene in transient transfection experiments, the sequence from positions -3231 to -118 (relative to the translation start site) directs high-level expression in CDH2-expressing chicken primary retinal cells and mouse N2A cells, but is much less active in chicken embryonic fibroblast cells and mouse 3T3 cells which do not express CDH2. Similarly, this promoter fragment directs variable, but neuronal-specific, expression of reporter genes in adult transgenic mice, but fails to produce the correct pattern of expression in other tissues, implying that additional sequences further upstream and/or within introns of CDH2 may play important roles in the transcriptional control (Li, 1997).

The cadherins are a family of cell-cell adhesion molecules that mediate Ca2+-dependent homophilic interactions between cells and transduce signals by interacting with cytoplasmic proteins. In the hippocampus, immunostaining combined with confocal microscopy has revealed that both neural- (N-) and epithelial- (E-) cadherin are present at synaptic sites, implying a role in synaptic function. Pretreatment of hippocampal slices with antibodies (Abs) raised against the extracellular domain of either N-cad or E-cad had no effect on basal synaptic properties but significantly reduces long-term potentiation (LTP). Infusion of antagonistic peptides containing the His-Ala-Val (HAV) consensus sequence for cadherin dimerization also attenuate LTP induction without affecting previously established LTP. Because the intense synaptic stimulation associated with LTP induction might transiently deplete extracellular Ca2+ and hence potentially destabilize cadherin-cadherin interactions, an examination was carried out to determine if slices could be protected from inhibition by N-cad Abs or HAV peptides by raising the extracellular Ca2+ concentration. Indeed, high extracellular Ca2+ prevents the block of LTP by these agents. Taken together, these results indicate that cadherins are involved in synaptic plasticity, and the stability of cadherin-cadherin bonds may be regulated by synaptic stimulation (Tang, 1998).

It is an open question whether new synapses form during hippocampal long-term potentiation (LTP). Late-phase LTP (L-LTP) is associated with a significant increase in numbers of synaptic puncta identified by synaptophysin and N-cadherin, an adhesion protein involved in synapse formation during development. During potentiation, protein levels of N-cadherin are significantly elevated and N-cadherin dimerization is enhanced. The increases in synaptic number and N-cadherin levels are dependent on cAMP-dependent protein kinase (PKA) and protein synthesis, both of which are also required for L-LTP. Blocking N-cadherin adhesion prevents the induction of L-LTP, but not the early-phase of LTP (E-LTP). These data suggest that N-cadherin is synthesized during the induction of L-LTP and recruited to newly forming synapses. N-cadherin may play a critical role in L-LTP by holding nascent pre- and post-synaptic membranes in apposition, enabling incipient synapses to acquire function and contribute to potentiation (Bozdagi, 2000).

The role of protein synthesis in hippocampal L-LTP and the identity of the proteins synthesized have remained elusive. In Aplysia, the cAMP-dependent PKA pathway, acting through the transcription factor CREB, stimulates the formation of new synaptic contacts during the transition from short-term to long-term memory storage. L-LTP of Schaffer collateral-CA1 synapses was induced by bath application of the membrane-permeable, cAMP analog Sp-cyclic adenosine 3',5'-monophosphorothioate (Sp-cAMPS) to hippocampal slices for 15 min, which results in a persistent potentiation of the area CA1 field excitatory postsynaptic potential (EPSP). There are striking similarities of the mammalian results to those obtained in Aplysia in that the increase in numbers of labeled synapses in response to Sp-cAMPS-induced L-LTP is blocked by inhibitors of PKA and protein synthesis. The finding that Sp-cAMPS stimulates a protein synthesis-dependent rise in levels of N-cadherin suggests that N-cadherin synthesis is a critical target of the cAMP-dependent PKA signaling cascade required for L-LTP. Like long-term facilitation in Aplysia, cAMP-dependent PKA signaling in hippocampal neurons activates the transcription factor CREB, which in turn regulates gene expression during L-LTP. It is not yet known if CREB regulates N-cadherin expression. Nevertheless, it is likely that the source of N-cadherin, as it progressively accumulates at synaptic sites during L-LTP, is from both new N-cadherin protein synthesis and from a locally recruitable pool of surface N-cadherin, one which is not tethered via the catenins to the actin cytoskeleton and therefore highly mobile (Bozdagi, 2000).

Strong depolarization of cultured hippocampal neurons changes the conformation of synaptic N-cadherin to the lateral strand dimer configuration, which, in turn, confers a pronounced resistance of N-cadherin to proteolytic degradation (Tanaka, 2000) -- indices of strong adhesive activity. The fact that N-cadherin bonds can display such a dynamic modulation of adhesive strength by the functional status of the very synapses at which they are localized (Tanaka, 2000 ), and perhaps as well by a variety of signaling pathways, suggests an ongoing and reversible modulation of adhesive strength. The date presented here support this view in that the strand dimer is detected in the control slices and is slightly enhanced in the potentiated ones, raising the possibility that under conditions of increasing synaptic efficacy, such as during LTP, synapses are stabilized by increased adhesive force during the initial phases of long-term memory encoding, followed subsequently by diminished adhesive force to promote synapse disassembly as the information encoded by such new circuitry is then transferred to other brain areas. It is likely that a broad array of adhesion molecules that utilize different adhesive mechanisms will be found to act coordinately to modify temporally and mechanistically distinct aspects of synaptic plasticity, which underscores the dynamic relationship between the signaling function of synapses and the adhesive elements that build, maintain, and modify the structural scaffolding for signaling (Bozdagi, 2000).

Cadherin-mediated interactions are integral to synapse formation and potentiation. N-cadherin is required for memory formation and regulation of a subset of underlying biochemical processes. N-cadherin antagonistic peptide containing the His-Ala-Val motif (HAV-N) transiently disrupted hippocampal N-cadherin dimerization and impaired the formation of long-term contextual fear memory while sparing short-term memory, retrieval, and extinction. HAV-N impaired the learning-induced phosphorylation of a distinctive, cytoskeletally associated fraction of hippocampal Erk-1/2 and altered the distribution of IQGAP1, a scaffold protein linking cadherin-mediated cell adhesion to the cytoskeleton. This effect was accompanied by reduction of N-cadherin/IQGAP1/Erk-2 interactions. Similarly, in primary neuronal cultures, HAV-N prevented NMDA-induced dendritic Erk-1/2 phosphorylation and caused relocation of IQGAP1 from dendritic spines into the shafts. The data suggest that the newly identified role of hippocampal N-cadherin in memory consolidation may be mediated, at least in part, by cytoskeletal IQGAP1/Erk signaling (Schrick, 2007).