Regulation of embryonic cell adhesion by the cadherin cytoplasmic domain.

Differential adhesion between embryonic cells has been proposed to be mediated by a family of closely related glycoproteins called the cadherins. The cadherins mediate adhesion in part through an interaction between the cadherin cytoplasmic domain and intracellular proteins, called the catenins. To determine whether these interactions could regulate cadherin function in embryos, a form of N-cadherin was generated that lacks an extracellular domain. Expression of this mutant in Xenopus embryos causes a dramatic inhibition of cell adhesion. Analysis of the mutant phenotype shows that at least two regions of the N-cadherin cytoplasmic domain can inhibit adhesion and that the mutant cadherin can inhibit catenin binding to E-cadherin. These results suggest that cadherin-mediated adhesion can be regulated by cytoplasmic interactions and that this regulation may contribute to morphogenesis when emerging tissues coexpress several cadherin types.     

Adhesion molecules during somitogenesis in the avian embryo

In avian embryos, somites constitute the morphological unit of the metameric pattern. Somites are epithelia formed from a mesenchyme, the segmental plate, and are subsequently reorganized into dermatome, myotome, and sclerotome. In this study, we used somitogenesis as a basis to examine tissue remodeling during early vertebrate morphogenesis. Particular emphasis was put on the distribution and possible complementary roles of adhesion-promoting molecules, neural cell adhesion molecule (N-CAM), N-cadherin, fibronectin, and laminin. Both segmental plate and somitic cells exhibited in vitro calcium-dependent and calcium-independent systems of cell aggregation that could be inhibited respectively by anti-N-cadherin and anti-N-CAM antibodies. In vivo, the spatio-temporal expression of N-cadherin was closely associated with both the formation and local disruption of the somites. In contrast, changes in the prevalence of N-CAM did not strictly accompany the remodeling of the somitic epithelium into dermamyotome and sclerotome. It was also observed that fibronectin and laminin were reorganized secondarily in the extracellular spaces after CAM-mediated contacts were modulated. In an in vitro culture system of somites, N-cadherin was lost on individual cells released from somite explants and was reexpressed when these cells reached confluence and established intercellular contacts. In an assay of tissue dissociation in vitro, antibodies to N-cadherin or medium devoid of calcium strongly and reversibly dissociated explants of segmental plates and somites. Antibodies to N-CAM exhibited a smaller disrupting effect only on segmental plate explants. In contrast, antibodies to fibronectin and laminin did not perturb the cohesion of cells within the explants. These results emphasize the possible role of cell surface modulation of CAMs during the formation and remodeling of some transient embryonic epithelia. It is suggested that N-cadherin plays a major role in the control of tissue remodeling, a process in which N-CAM is also involved but to a lesser extent. The substratum adhesion molecules, fibronectin and laminin, do not appear to play a primary role in the regulation of these processes but may participate in cell positioning and in the stabilization of the epithelial structures.     

N-cadherin is required for neural crest remodeling of the cardiac outflow tract

Cardiac neural crest cells undergo extensive cell rearrangements during the formation of the aorticopulmonary septum in the outflow tract. However, the morphogenetic mechanisms involved in this fundamental process remain poorly understood. To determine the function of the Ca2+-dependent cell adhesion molecule, N-cadherin, in murine neural crest, we applied the Cre/loxP system and created mouse embryos genetically mosaic for N-cadherin. Specifically, deletion of N-cadherin in neural crest cells led to embryonic lethality with distinct cardiovascular defects. Neural crest cell migration and homing to the cardiac outflow tract niche were unaffected by loss of N-cadherin. However, N-cadherin-deficient neural crest cells were unable to undergo the normal morphogenetic changes associated with outflow tract remodeling, resulting in persistent truncus arteriosus in the majority of mutant embryos. Other mutant embryos initiated aorticopulmonary septum formation; however, the neural crest cells were unable to elongate and align properly along the midline and remained rounded with limited contact with their neighbors. Interestingly, rotation of the outflow tract was incomplete in these mutants suggesting that alignment of the channels is dependent on N-cadherin-generated cytoskeletal forces. A second cardiac phenotype was observed where loss of N-cadherin in the epicardium led to disruption of heterotypic cell interactions between the epicardium and myocardium resulting in a thinned ventricular myocardium. Thus, we conclude that in addition to its role in myocardial cell adhesion, N-cadherin is required for neural crest cell rearrangements critical for patterning of the cardiac outflow tract and in the maintenance of epicardial-myocardial cell interactions.     

Zebrafish neural tube morphogenesis requires Scribble-dependent oriented cell divisions

How control of subcellular events in single cells determines morphogenesis on the scale of the tissue is largely unresolved. The stereotyped cross-midline mitoses of progenitors in the zebrafish neural keel provide a unique experimental paradigm for defining the role and control of single-cell orientation for tissue-level morphogenesis in vivo. We show here that the coordinated orientation of individual progenitor cell division in the neural keel is the cellular determinant required for morphogenesis into a neural tube epithelium with a single straight lumen. We find that Scribble is required for oriented cell division and that its function in this process is independent of canonical apicobasal and planar polarity pathways. We identify a role for Scribble in controlling clustering of α-catenin foci in dividing progenitors. Loss of either Scrib or N-cadherin results in abnormally oriented mitoses, reduced cross-midline cell divisions, and similar neural tube defects. We propose that Scribble-dependent nascent cell-cell adhesion clusters between neuroepithelial progenitors contribute to define orientation of their cell division. Finally, our data demonstrate that while oriented mitoses of individual cells determine neural tube architecture, the tissue can in turn feed back on its constituent cells to define their polarization and cell division orientation to ensure robust tissue morphogenesis.     

A role for N-cadherin in mesodermal morphogenesis during gastrulation

Cell adhesion molecules mediate numerous developmental processes necessary for the segregation and organization of tissues. Here we show that the zebrafish biber (bib) mutant encodes a dominant allele at the N-cadherin locus. When knocked down with antisense oligonucleotides, bib mutants phenocopy parachute (pac) null alleles, demonstrating that bib is a gain-of-function mutation. The mutant phenotype disrupts normal cell-cell contacts throughout the mesoderm as well as the ectoderm. During gastrulation stages, cells of the mesodermal germ layer converge slowly; during segmentation stages, the borders between paraxial and axial tissues are irregular and somite borders do not form; later, myotomes are fused. During neurulation, the neural tube is disorganized. Although weaker, all traits present in bib mutants were found in pac mutants. When the distribution of N-cadherin mRNA was analyzed to distinguish mesodermal from neuroectodermal expression, we found that N-cadherin is strongly expressed in the yolk cell and hypoblast in the early gastrula, just preceding the appearance of the bib mesodermal defects. Only later is N-cadherin expressed in the anlage of the CNS, where it is found as a radial gradient in the forming neural plate. Hence, besides a well-established role in neural and somite morphogenesis, N-cadherin is essential for morphogenesis of the mesodermal germ layer during gastrulation.     

Functional role for stable microtubules in lens fiber cell elongation

The process of tissue morphogenesis, especially for tissues reliant on the establishment of a specific cytoarchitecture for their functionality, depends a balanced interplay between cytoskeletal elements and their interactions with cell adhesion molecules. The microtubule cytoskeleton, which has many roles in the cell, is a determinant of directional cell migration, a process that underlies many aspects of development. We investigated the role of microtubules in development of the lens, a tissue where cell elongation underlies morphogenesis. Our studies with the microtubule depolymerizing agent nocodazole revealed an essential function for the acetylated population of stable microtubules in the elongation of lens fiber cells, which was linked to their regulation of the activation state of myosin. Suppressing myosin activation with the inhibitor blebbistatin could attenuate the loss of acetylated microtubules by nocodazole and rescue the effect of this microtubule depolymerization agent on both fiber cell elongation and lens integrity. Our results also suggest that acetylated microtubules impact lens morphogenesis through their interaction with N-cadherin junctions, with which they specifically associate in the region where lens fiber cell elongate. Disruption of the stable microtubule network increased N-cadherin junctional organization along lateral borders of differentiating lens fiber cells, which was prevented by suppression of myosin activity. These results reveal a role for the stable microtubule population in lens fiber cell elongation, acting in tandem with N-cadherin cell-cell junctions and the actomyosin network, giving insight into the cooperative role these systems play in tissue morphogenesis.     

Identification of a novel intermediate filament-linked N-cadherin/gamma-catenin complex involved in the establishment of the cytoarchitecture of differentiated lens fiber cells

Tissue morphogenesis and maintenance of complex tissue architecture requires a variety of cell-cell junctions. Typically, cells adhere to one another through cadherin junctions, both adherens and desmosomal junctions, strengthened by association with cytoskeletal networks during development. Both β- and γ-catenins are reported to link classical cadherins to the actin cytoskeleton, but only γ-catenin binds to the desmosomal cadherins, which links them to intermediate filaments through its association with desmoplakin. Here we provide the first biochemical evidence that, in vivo, γ-catenin also mediates interactions between classical cadherins and the intermediate filament cytoskeleton, linked through desmoplakin. In the developing lens, which has no desmosomes, we discovered that vimentin became linked to N-cadherin complexes in a differentiation-state specific manner. This newly identified junctional complex was tissue specific but not unique to the lens. To determine whether in this junction N-cadherin was linked to vimentin through γ-catenin or β-catenin we developed an innovative “double” immunoprecipitation technique. This approach made possible, for the first time, the separation of N-cadherin/γ-catenin from N-cadherin/β-catenin complexes and the identification of multiple members of each of these isolated protein complexes. The study revealed that vimentin was associated exclusively with N-cadherin/γ-catenin junctions. Assembly of this novel class of cadherin junctions was coincident with establishment of the unique cytoarchitecture of lens fiber cells. In addition, γ-catenin had a distinctive localization to the vertices of these hexagonally shaped differentiating lens fiber cells, a region devoid of actin; while β-catenin co-localized with actin at lateral cell interfaces. We believe this novel vimentin-linked N-cadherin/γ-catenin junction provides the tensile strength necessary to establish and maintain structural integrity in tissues that lack desmosomes.     

Adhesive subdivisions intrinsic to the epithelial somites.

Developing somites express two subtypes of classic cadherin adhesion receptors, N-cadherin and cadherin-11 (cad11). To investigate the role of these adhesion molecules in somite morphogenesis, we analyzed the somites of mice whose N-cadherin and cad11 genes were disrupted. The epithelial somites of N-cadherin null mutant mice were fragmented as reported, whereas those of cad11(-/-) mice showed no structural anomaly. In mice double homozygous for N-cadherin and cad11 mutation, however, somites were further fragmented into smaller clusters than in the N-cadherin-deficient mice, suggesting that these two cadherins cooperate in the maintenance of epithelial somites. Despite the disorganization of epithelial structures, dorsoventral polarity markers were expressed in their correct patterns in all of these mutant somites. Uncx4.1, whose expression is localized only in the caudal region of each somite, was also expressed in a normal pattern in the mutant somites. However, the staining for Uncx4.1 revealed that, in the N-cadherin mutants, each somite tended to be cleaved at the border between the Uncx4. 1-positive and -negative regions and that the cleaved subunits maintained the clustered state, often exhibiting epithelioid morphology. This separation of the rostral and caudal regions was observed as soon as the epithelial somites had been formed. In the N-cadherin/cad11 double-homozygous mutants, this tendency was also observed, although each half of the somite further disintegrated into randomly arranged cell clusters. These results suggest that cells of the rostral and caudal regions of each epithelial somite have an activity to aggregate independently or separate from one another and that one role of N-cadherin and cad11 is to connect the two halves into a single unit.     

N-cadherin function is required for differentiation-dependent cytoskeletal reorganization in lens cells in vitro

Members of the cadherin family of cell adhesion molecules participate in calcium-dependent cell-cell adhesions that are necessary for the cell sorting events that regulate early developmental processes. Although individual cadherin molecules have been shown to participate in tissue histogenesis, the regulation of function of these receptors in cell differentiation has been more difficult to identify. We have determined that N-cadherin linkage to the cytoskeleton is correlated with lens cell differentiation in vivo. Through the use of a chick embryo lens culture system that mimics differentiation in vivo, we have determined that N-cadherin linkage to the cytoskeleton is altered and lens differentiation is blocked by function-blocking antibodies to N-cadherin. In the presence of the N-cadherin function-blocking antibody, NCD-2, both N-cadherin and filamentous actin are prevented from organizing at the cortical membranes. This correlates with an inhibition of lens morphogenesis and differentiation. These results are paralleled by changes in the expression of the molecular components of the cadherin-catenin complex and their linkage to the actin cytoskeleton. In the presence of NCD-2, expression of N-cadherin, alpha-catenin, and beta-catenin is inhibited and their association with the cytoskeleton blocked. Overall cadherin expression, however, remains unchanged as demonstrated by studies with a pan-cadherin antibody. This is accompanied by an increase in expression of the cadherin cytoskeletal protein plakoglobin. Although the cells have tried to compensate for the loss of N-cadherin by up-regulation of another cadherin(s) and plakoglobin, this is unable to compensate for N-cadherin function. The data strongly suggest that N-cadherin and its associated cytoskeleton play an important role in the differentiation process that leads to the formation of the crystalline lens.     

The cleavage of N-cadherin is essential for chondrocyte differentiation

The aggregation of chondroprogenitor mesenchymal cells into precartilage condensation represents one of the earliest events in chondrogenesis. N-cadherin is a key cell adhesion molecule implicated in chondrogenic differentiation. Recently, ADAM10-mediated cleavage of N-cadherin has been reported to play an important role in cell adhesion, migration, development and signaling. However, the significance of N-cadherin cleavage in chondrocyte differentiation has not been determined. In the present study, we found that the protein turnover of N-cadherin is accelerated during the early phase of chondrogenic differentiation in ATDC5 cells. Therefore, we generated the subclones of ATDC5 cells overexpressing wild-type N-cadherin, and two types of subclones overexpressing a cleavage-defective N-cadherin mutant, and examined the response of these cells to insulin stimulation. The ATDC5 cells overexpressing cleavage-defective mutants severely prevented the formation of cartilage aggregates, proteoglycan production and the induction of chondrocyte marker gene expression, such as type II collagen, aggrecan and type X collagen. These results suggested that the cleavage of N-cadherin is essential for chondrocyte differentiation.     

The Mechanical Role of Microtubules in Tissue Remodeling

During morphogenesis, tissues undergo extensive remodeling to get their final shape. Such precise sculpting requires the application of forces generated within cells by the cytoskeleton and transmission of these forces through adhesion molecules within and between neighboring cells. Within individual cells, microtubules together with actomyosin filaments and intermediate filaments form the composite cytoskeleton that controls cell mechanics during tissue rearrangements. While studies have established the importance of actin-based mechanical forces that are coupled via intercellular junctions, relatively little is known about the contribution of other cytoskeletal components such as microtubules to cell mechanics during morphogenesis. In this review the focus is on recent findings, highlighting the direct mechanical role of microtubules beyond its well-established role in trafficking and signaling during tissue formation.     

Tissue and age-specificity of post-translational modifications of N-cadherin during chick embryo development

Our previous studies indicated that regulation of N-cadherin expression differs spatially and temporally among tissues of the eye, possibly reflecting the distinct roles it has in the development and maintenance of eye tissues. To understand this regulation of N-cadherin expression and its function in different tissues during embryonic development, we investigated the post-translational modifications of N-cadherin and its association with the cytoskeleton. We show that N-cadherin is a sulfated and phosphorylated protein. The phosphorylation of N-cadherin occurs in an age- and tissue-specific pattern during development in the neural retina, brain, lens and heart. The extent of sulfation of N-cadherin is also age-dependent, and both sulfated and unsulfated pools of N-cadherin exist in the same tissue as indicated by two-dimensional electrophoresis. The degree of association of N-cadherin with the cytoskeleton differs from one tissue to another, as well as within a single tissue at different stages of development. A positive correlation was found between the extent, developmental timing, and tissue specificity of N-cadherin phosphorylation and the degree of N-cadherin association with the cytoskeleton. Our results suggest the existence of a microheterogeneous population of N-cadherin molecules, within which posttranslational modification of N-cadherin may affect its association with the cytoskeleton and its expression and function during development.     

Role of nectin in formation of E-cadherin-based adherens junctions in keratinocytes: analysis with the N-cadherin dominant negative mutant

E-cadherin is a Ca(2+)-dependent cell-cell adhesion molecule at adherens junctions (AJs) of epithelial cells. A fragment of N-cadherin lacking its extracellular region serves as a dominant negative mutant (DN) and inhibits cell-cell adhesion activity of E-cadherin, but its mode of action remains to be elucidated. Nectin is a Ca(2+)-independent immunoglobulin-like cell-cell adhesion molecule at AJs and is associated with E-cadherin through their respective peripheral membrane proteins, afadin and catenins, which connect nectin and cadherin to the actin cytoskeleton, respectively. We showed here that overexpression of nectin capable of binding afadin, but not a mutant incapable of binding afadin, reduced the inhibitory effect of N-cadherin DN on the cell-cell adhesion activity of E-cadherin in keratinocytes. Overexpressed nectin recruited N-cadherin DN to the nectin-based cell-cell adhesion sites in an afadin-dependent manner. Moreover, overexpression of nectin enhanced the E-cadherin-based cell-cell adhesion activity. These results suggest that N-cadherin DN competitively inhibits the association of the endogenous nectin-afadin system with the endogenous E-cadherin-catenin system and thereby reduces the cell-cell adhesion activity of E-cadherin. Thus, nectin plays a role in the formation of E-cadherin-based AJs in keratinocytes.     

Remodeling Tissue Interfaces and the Thermodynamics of Zipping during Dorsal Closure in Drosophila

Dorsal closure during Drosophila embryogenesis is an important model system for investigating the biomechanics of morphogenesis. During closure, two flanks of lateral epidermis (with actomyosin-rich purse strings near each leading edge) close an eye-shaped opening that is filled with amnioserosa. At each canthus (corner of the eye) a zipping process remodels the tissue interfaces between the leading edges of the lateral epidermis and the amnioserosa. We investigated zipping dynamics and found that apposing leading edge cells come together at their apical ends and then square off basally to form a lateral junction. Meanwhile, the purse strings act as contractile elastic rods bent toward the embryo interior near each canthus. We propose that a canthus-localized force contributes to both bending the ends of the purse strings and the formation of lateral junctions. We developed a thermodynamic model for zipping based on three-dimensional remodeling of the tissue interfaces and the reaction dynamics of adhesion molecules in junctions and elsewhere, which we applied to zipping during unperturbed wild-type closure and to laser or genetically perturbed closure. We identified two processes that can contribute to the zipping mechanism, consistent with experiments, distinguished by whether amnioserosa dynamics do or do not augment canthus adhesion dynamics.     

N-cadherin cell-cell adhesion complexes are regulated by fibronectin matrix assembly

Fibronectin is a principal component of the extracellular matrix. Soluble fibronectin molecules are assembled into the extracellular matrix as insoluble, fibrillar strands via a cell-dependent process. In turn, the interaction of cells with the extracellular matrix form of fibronectin stimulates cell functions critical for tissue repair. Cross-talk between cell-cell and cell-extracellular matrix adhesion complexes is essential for the organization of cells into complex, functional tissue during embryonic development and tissue remodeling. Here, we demonstrate that fibronectin matrix assembly affects the organization, composition, and function of N-cadherin-based adherens junctions. Using fibronectin-null mouse embryonic myofibroblasts, we identified a novel quaternary complex composed of N-cadherin, β-catenin, tensin, and actin that exists in the absence of a fibronectin matrix. In the absence of fibronectin, homophilic N-cadherin ligation recruited both tensin and α5β1 integrins into nascent cell-cell adhesions. Initiation of fibronectin matrix assembly disrupted the association of tensin and actin with N-cadherin, released α5β1 integrins and tensin from cell-cell contacts, stimulated N-cadherin reorganization into thin cellular protrusions, and decreased N-cadherin adhesion. Fibronectin matrix assembly has been shown to recruit α5β1 integrins and tensin into fibrillar adhesions. Taken together, these studies suggest that tensin serves as a common cytoskeletal link for integrin- and cadherin-based adhesions and that the translocation of α5β1 integrins from cell-cell contacts into fibrillar adhesions during fibronectin matrix assembly is a novel mechanism by which cell-cell and cell-matrix adhesions are coordinated.     

Gene replacement reveals a specific role for E-cadherin in the formation of a functional trophectoderm

During mammalian embryogenesis the trophectoderm represents the first epithelial structure formed. The cell adhesion molecule E-cadherin is ultimately necessary for the transition from compacted morula to the formation of the blastocyst to ensure correct establishment of adhesion junctions in the trophectoderm. Here, we analyzed to what extent E-cadherin confers unique adhesion and signaling properties in trophectoderm formation in vivo. Using a gene replacement approach, we introduced N-cadherin cDNA into the E-cadherin genomic locus. We show that the expression of N-cadherin driven from the E-cadherin locus reflects the expression pattern of endogenous E-cadherin. Heterozygous mice co-expressing E- and N-cadherin are vital and show normal embryonic development. Interestingly, N-cadherin homozygous mutant embryos phenocopy E-cadherin-null mutant embryos. Upon removal of the maternal E-cadherin, we demonstrate that N-cadherin is able to provide sufficient cellular adhesion to mediate morula compaction, but is insufficient for the subsequent formation of a fully polarized functional trophectoderm. When ES cells were isolated from N-cadherin homozygous mutant embryos and teratomas were produced, these ES cells differentiated into a large variety of tissue-like structures. Importantly, different epithelial-like structures expressing N-cadherin were formed, including respiratory epithelia, squamous epithelia with signs of keratinization and secretory epithelia with goblet cells. Thus, N-cadherin can maintain epithelia in differentiating ES cells, but not during the formation of the trophectoderm. Our results point to a specific and unique function for E-cadherin during mouse preimplantation development.     

Role of N-cadherin cell adhesion molecules in the histogenesis of neural retina

We investigated the role of N-cadherin cell adhesion molecules in the histogenesis of the chicken neural retina. In the undifferentiated retina of early embryos, N-cadherin is almost evenly distributed. With differentiation, N-cadherin was gradually localized in particular cell layers. In the 8.5 to 10.5 day embryos, N-cadherin was most abundant in the optic nerve fiber layer, the plexiform layers and the outer limiting membrane. Thereafter, this molecule gradually diminished from most parts of the retina, except in the outer limiting membrane. When incubated with Fab fragments of a polyclonal antibody to N-cadherin, retinas of early embryos tended to dissociate and could not be maintained as a tissue mass. Retinas from older embryos were not dissociated by the Fab, but their morphogenesis was severely affected. We conclude that N-cadherin is essential for maintaining the overall structure of the undifferentiated retina, but during development, its role becomes restricted to maintaining more specific regions of the tissue. We also suggest that there might be additional, unidentified cadherin-like molecules in the retina.     

Adherens junction remodeling by the Notch pathway in Drosophila melanogaster oogenesis

Identifying genes involved in the control of adherens junction (AJ) remodeling is essential to understanding epithelial morphogenesis. During follicular epithelium development in Drosophila melanogaster, the main body follicular cells (MBFCs) are displaced toward the oocyte and become columnar. Concomitantly, the stretched cells (StCs) become squamous and flatten around the nurse cells. By monitoring the expression of epithelial cadherin and Armadillo, I have discovered that the rate of AJ disassembly between the StCs is affected in follicles with somatic clones mutant for fringe or Delta and Serrate. This results in abnormal StC flattening and delayed MBFC displacement. Additionally, accumulation of the myosin II heavy chain Zipper is delayed at the AJs that require disassembly. Together, my results demonstrate that the Notch pathway controls AJ remodeling between the StCs and that this role is crucial for the timing of MBFC displacement and StC flattening. This provides new evidence that Notch, besides playing a key role in cell differentiation, also controls cell morphogenesis.     

The Nonreceptor Protein Tyrosine Phosphatase PTP1B Binds to the Cytoplasmic Domain of N-Cadherin and Regulates the Cadherin–Actin Linkage

Cadherin-mediated adhesion depends on the association of its cytoplasmic domain with the actin-containing cytoskeleton. This interaction is mediated by a group of cytoplasmic proteins: α-and β- or γ- catenin. Phosphorylation of β-catenin on tyrosine residues plays a role in controlling this association and, therefore, cadherin function. Previous work from our laboratory suggested that a nonreceptor protein tyrosine phosphatase, bound to the cytoplasmic domain of N-cadherin, is responsible for removing tyrosine-bound phosphate residues from β-catenin, thus maintaining the cadherin–actin connection (Balsamo et al., 1996). Here we report the molecular cloning of the cadherin-associated tyrosine phosphatase and identify it as PTP1B. To definitively establish a causal relationship between the function of cadherin-bound PTP1B and cadherin-mediated adhesion, we tested the effect of expressing a catalytically inactive form of PTP1B in L cells constitutively expressing N-cadherin. We find that expression of the catalytically inactive PTP1B results in reduced cadherin-mediated adhesion. Furthermore, cadherin is uncoupled from its association with actin, and β-catenin shows increased phosphorylation on tyrosine residues when compared with parental cells or cells transfected with the wild-type PTP1B. Both the transfected wild-type and the mutant PTP1B are found associated with N-cadherin, and recombinant mutant PTP1B binds to N-cadherin in vitro, indicating that the catalytically inactive form acts as a dominant negative, displacing endogenous PTP1B, and rendering cadherin nonfunctional. Our results demonstrate a role for PTP1B in regulating cadherin-mediated cell adhesion.     

Expression of N-CAM-180 and N-cadherin during development in two South-American anuran species (Bufo arenarum and Hyla nana)

Cadherins and N-CAM are Ca++-dependent and Ca++-independent cell adhesion molecules respectively. These molecules play a key role in morphogenesis and histogenesis. We determined the spatiotemporal pattern of N-cadherin and N-CAM-180 kDa expression by immunohistochemistry during development in two South-American anuran species (Bufo arenarum, toad and Hyla nana, frog). Both N-cadherin and N-CAM were not detectable during early developmental stages. Expression of N-cadherin appeared between the inner and the outer ectoderm layers at stages 19-20. At stages 24-25, N-cadherin was expressed in the neural tube and the heart. In early tadpoles, N-cadherin expression increased along with the central nervous system (CNS) morphogenesis, and reached its maximum level at metamorphic climax stage. N-Cadherin expression was not uniformly distributed. At stage 42, olfactory placodes and retina expressed N-cadherin. Contrary to N-CAM, the strongly myelinated cranial nerves were not labeled. N-Cadherin was present in several mesoderm derivatives such as the notochord, heart and skeletal muscle. The non-neural ectoderm and the endoderm were always negative. Expression of N-CAM appeared first in the neural tube at stages 24-25 and the level of expression became uniform from pre-metamorphic to metamorphic climax tadpoles. At this latter stage, a clear N-CAM immunolabeling appeared in the nerve terminals of pharynx and heart. N-Cadherin and N-CAM were found mainly co-expressed in the CNS from early tadpole to metamorphic climax tadpole. Our results show that the expression of N-CAM and N-cadherin is evolutionary conserved. Their increased expression during late developmental stages suggests that N-CAM and N-cadherin are involved in cell contact stabilization during tissue formation.