N-cadherin and neural development

XmN-cadherin (Xenopus maternally expressed neural cadherin) cDNA consistes of 3690 bp and encodes 922 amino acid residues. XmN-cadherin preserves five extracellular cadherin motifs, a single transmembrane domain, and a cytoplasmic domain, and is closely related by its sequence to R- and N-cadherin. In the adult frog, XmN-cadherin mRNA is detected strongly in ovary, testis, brain, eye, and kidney, and weakly in stomach and intestine. In the egg, the mRNA occurs as a maternal mRNA at a relatively high level; its level becomes very low by the neurula stage, then increases steadily thereafter. Dissection experiments with 8-cell stage and neurula stage embryos reveal that the maternally inherited mRNA is relatively uniformly distributed within the embryo. In sharp contrast, whole mount in situ hybridization reveals that the zygotically expressed mRNA occurs almost exclusively in neural tissues such as brain, the anterior part of the spinal cord, and the optic and otic vesicles. Thus, XmN-cadherin appears to have at least three functions: it probably contributes in early embryos to cell-type non-specific cell adhesion, but in post-neurula embryos may be responsible for the development and/or maintenance of anterior neural tissues, and may be used in adult frog for the development and/or maintenance of neural, endodermal and reproductive organs (Tashiro, 1996).

Neural (N)- and epithelial (E)-cadherin are localized to synaptic complexes in mutually exclusive distributions. In cerebellum, N-cadherin is frequently found associated with synapses, some of which are perforated; in hippocampus, N- and E-cadherin-containing synapses are found aligned along dendritic shafts within the stratum lucidum of CA3. It is proposed that the cadherins function as primary adhesive moieties between pre- and postsynaptic membranes in the synaptic complex. According to this model, once neurites have been guided to the vicinity of their cognate targets, it is the differential distribution of cadherins along the axonal and dendritic plasma membranes, and ultimately cadherin self-association, that "locks in" nascent synaptic connections (Fannon, 1996).

Cell adhesion molecule (CAM) expression is highly regulated during nervous system development to control cell migration, neurite outgrowth, fasciculation, and synaptogenesis. Using electrical stimulation of mouse dorsal root ganglion (DRG) neurons in cell culture, this work shows that N-cadherin expression is regulated by neuronal firing, and that expression of different CAMs is regulated by distinct patterns of neural impulses. N-cadherin is down-regulated by 0.1 or 1 Hz electrical stimulation, but NCAM mRNA and protein levels are not altered by stimulation. L1 is down-regulated by 0.1 Hz stimulation, but not by 0.3 Hz, 1 Hz, or pulsed stimulation. N-cadherin expression is lowered with faster kinetics than L1 (1 vs. 5 days), and L1 mRNA returns to higher levels after terminating the stimulus. The RSLE splice variant of L1 is not regulated by action potential stimulation, and activity-dependent influences on L1 expression are blocked by target-derived influences. The results are consistent with changes in firing pattern accompanying DRG development and suggest that functional activity can influence distinct developmental processes by regulating the relative abundance of different CAMs (Itoh, 1997).

In the vertebrate brain, neurons grouped in parallel laminae receive distinct sets of synaptic inputs. In the avian optic tectum, arbors and synapses of most retinal axons are confined to 3 of 15 laminae. The adhesion molecule N-cadherin and cell surface glycoconjugates recognized by a plant lectin are selectively associated with these "retinorecipient" laminae. The lectin and a monoclonal antibody to N-cadherin perturb laminar selectivity in distinct fashions. In contrast, neurotrophins increase the complexity of retinal arbors without affecting their laminar distribution. Thus, cell surface molecules and soluble trophic factors may collaborate to shape lamina-specific arbors in the brain, with the former predominantly affecting their position and the latter their size (Inoue, 1997).

The cell-cell adhesion molecule N-cadherin strongly promotes neurite outgrowth in cultured retinal neurons. To test whether cadherins regulate process outgrowth in retinal neurons in vivo, cadherin function was blocked in single cells by expression of a dominant negative N-cadherin mutant. When cadherin function is inhibited, axon and dendrite outgrowth are severely impaired, particularly in retinal ganglion cells. Laminar migration and cell type specification, by contrast, appear unaffected. Expression of the catenin-binding domain of N-cadherin, which blocks cadherin-mediated adhesion in early embryos, does not affect axon outgrowth, suggesting that outgrowth and adhesion are mediated by distinct regions of the cytoplasmic domain. These findings indicate that cadherins play an essential role in the initiation and extension of axons from retinal ganglion cells in vivo (Riehl, 1996).

Dispersion of neural crest cells and their ultimate regroupment into peripheral ganglia are associated with precisely coordinated regulations both in time and space of the expression and function of cell adhesion receptors. In particular, the disappearance of N-cadherin from the cell surface at the onset of migration and its reexpression during cell aggregation suggest that, during migration, N-cadherin expression is repressed in neural crest cells. Although migrating neural crest cells move as a dense population, each individual does not establish extensive and permanent intercellular contacts with its neighbors. However, cells synthesize and express mature N-cadherin molecules at levels comparable to those found in cells that exhibit stable intercellular contacts, but in contrast to them, the bulk of N-cadherin molecules is not connected with the cytoskeleton. Agents that block a broad spectrum of serine-threonine kinases or that selectively affect protein kinases C, inhibitors of protein tyrosine kinases, and inhibitors of phosphatases all restore tight cell-cell associations among neural crest cells, accompanied by a slight increase in the overall cellular content of N-cadherin and its accumulation to the regions of intercellular contacts. The effect of the kinase and phosphatase blockers is inhibitable by agents known to affect protein synthesis and exportation, indicating that the restored cell-cell contacts are mediated chiefly by an intracellular pool of N-cadherin molecules recruited to the membrane. N-cadherin molecules are constitutively phosphorylated in migrating neural crest cells, but their levels and states of phosphorylation are apparently not modified in the presence of kinase and phosphatase inhibitors. These observations therefore suggest that N-cadherin-mediated cell-cell interactions are not stable in neural crest cells migrating in vitro, and that they are under the control of a complex cascade of intracellular signals involving kinases and phosphatases and are probably elicited by surface receptors (Monier-Gavelle, 1995).

During the emergence of neural crest cells from the neural tube, the expression of cadherins changes dynamically. In the chicken embryo, the early neural tube expresses two cadherins, N-cadherin and cadherin-6B (cad6B), in the dorsal-most region where neural crest cells are generated. However, the expression of these two cadherins is downregulated in the neural crest cells migrating from the neural tube; instead they begin expressing cadherin-7 (cad7). As an attempt to investigate the role of these changes in cadherin expression, various cadherin constructs, including N-cadherin, cad7, and a dominant negative N-cadherin (cN390), were overexpressed in neural crest-generating cells. This was achieved by injecting adenoviral expression vectors encoding these molecules into the lumen of the closing neural tube of chicken embryos at stage 14. In neural tubes injected with the viruses, efficient infection was observed at the neural crest-forming area, resulting in the ectopic cadherin expression seen also in migrating neural crest cells. Notably, the distribution of neural crest cells with the ectopic cadherins changes depending on which constructs are expressed. Many crest cells fail to escape from the neural tube when N-cadherin or cad7 is overexpressed. Moreover, none of the cells with these ectopic cadherins migrate along the dorsolateral (melanocyte) pathway. When these samples are stained for Mitf, an early melanocyte marker, positive cells are found accumulated within the neural tube, suggesting that the failure of their migration is not due to differentiation defects. In contrast to these phenomena, cells expressing non-functional cadherins exhibit a normal migration pattern. Thus, the overexpression of a neuroepithelial cadherin (N-cadherin) and a crest cadherin (cad7) results in the same blocking effect on neural crest segregation from neuroepithelial cells, especially for melanocyte precursors. These findings suggest that the regulation of cadherin expression or its activity at the neural crest-forming area plays a critical role in neural crest emigration from the neural tube (Nakagawa,1998).

The cell adhesion molecule N-cadherin is ubiquitously expressed in the early neuroepithelium, with strongest expression in the ependymal lining. The function of N-cadherin was blocked during early chicken brain development by injecting antibodies against N-cadherin into the tectal ventricle of embryos at 4-5 d of incubation [embryonic day 4 (E4)-E5]. N-cadherin blockage results in massive morphological changes in restricted brain regions. At approximately E6, these changes consist of invaginations of pieces of the ependymal lining and the formation of neuroepithelial rosettes. The rosettes are composed of central fragments of ependymal lining, surrounded by an inner ventricular layer and an outer mantle layer. Radial glia processes are radially arranged around the ependymal centers of the rosettes. The normal layering of the neural tissue is thus preserved, but its coherent epithelial structure is disrupted. The observed morphological changes are restricted to specific brain regions such as the tectum and the dorsal thalamus, whereas the ventral thalamus and the pretectum are almost undisturbed. At E10-E11, analysis of late effects of N-cadherin blockage reveals that in the dorsal thalamus, gray matter is fragmented and disorganized; in the tectum, additional layers have formed at the ventricular surface. Together, these results indicate that N-cadherin function is required for the maintenance of a coherent sheet of neuroepithelium in specific brain regions. Disruption of this sheet results in an abnormal morphogenesis of brain gray matter (Ganzler-Odenthal, 1998).

N-cadherin expression during development is regulated by several mechanisms, including mRNA expression, cytokine modulation, and proteolytically mediated turnover, yielding the NCAD90 protein. N-cadherin is the target of endogenous kinase and phosphatase action in the retina; modulation of different classes of these enzymes can result in either stimulation or inhibition of NCAD90 production. These results provide a mechanistic explanation for observations that cadherin function is downregulated following expression of exogenously introduced viral tyrosine kinases and provide a function for the tyrosine phosphatases recently found in association with cadherins. The results indicate that N-cadherin expression during retinal development is possibly regulated in part by modulation of its phosphorylation state, the balance of which may determine whether N-cadherin remains stably expressed or is targeted for proteolytically mediated turnover to produce NCAD90 (Lee, 1997).

Cadherins are homophilic adhesion molecules that, together with their intracellular binding partners the catenins, mediate adhesion and signaling at a variety of intercellular junctions. This study shows that neural (N)-cadherin and beta-catenin, an intracellular binding partner for the classic cadherins, are present in axons and dendrites before synapse formation and then cluster at developing synapses between hippocampal neurons. N-cadherin is expressed initially at all synaptic sites but rapidly becomes restricted to a subpopulation of excitatory synaptic sites. Sites of GABAergic, inhibitory synapses in mature cultures therefore lack N-cadherin but are associated with clusters of beta-catenin, implying that they contain a different classic cadherin. These findings indicate that N-cadherin adhesion may stabilize early synapses that can then be remodeled to express a different cadherin and that cadherins systematically differentiate between functionally (excitatory and inhibitory) and spatially distinct synaptic sites on single neurons. These results suggest that differential cadherin expression may orchestrate the point-to-point specificity displayed by developing synapses (Benson, 1998).

Cell adhesion is critical to the establishment of proper connections in the nervous system. Some receptor-type protein tyrosine phosphatases (RPTPs) have adhesion molecule-like extracellular segments with intracellular tyrosine phosphatase domains that may transduce signals in response to adhesion. PTPmu is an RPTP that mediates cell aggregation and is expressed at high levels in the nervous system. PTPmu promotes neurite outgrowth of retinal ganglion cells when used as a culture substrate. In addition, PTPmu is found in a complex with N-cadherin in retinal cells. To determine the physiological significance of the association between PTPmu and N-cadherin, the expression level and enzymatic activity of PTPmu were perturbed in retinal explant cultures. Downregulation of PTPmu expression through antisense techniques results in a significant decrease in neurite outgrowth on an N-cadherin substrate, whereas there is no effect on laminin or L1-dependent neurite outgrowth. The overexpression of a catalytically inactive form of PTPmu significantly decreases neurite outgrowth on N-cadherin. These data indicate that PTPmu specifically regulates signals required for neurites to extend on an N-cadherin substrate, implicating reversible tyrosine phosphorylation in the control of N-cadherin function. Together, these results suggest that PTPmu plays a dual role in the regulation of neurite outgrowth (Burden-Gulley, 1999).

In ventricular cells of the mouse telencephalon, differential expression of cadherin cell adhesion molecules defines neighboring regions; R-cadherin delineates the future cerebral cortex, while cadherin-6 delineates the lateral ganglionic eminence. By using cell labelling analyses in the whole embryo culture system, it has been demonstrated that the interface between R-cadherin and cadherin-6 expression is a boundary for cell lineage restriction at embryonic day 10.5. Interestingly, when a group of cells with exogenous cadherin-6 were generated to straddle the cortico-striatal boundary by electroporation at embryonic day 11.0, ectopic cadherin-6-expressing cortical cells were sorted into the striatal compartment, and the reverse was the trend for ectopic R-cadherin-expressing striatal cells. Although cadherin-6 gene knockout mice engineered in this study showed no obvious phenotype in telencephalic compartmentalization, the preferential sorting of ectopic cadherin-6-expressing cells was abolished in this mutant background. Thus, the differential expression pattern of cadherins in the embryonic telencephalon is responsible for maintaining the cortico-striatal compartment boundary (Inoue, 2001).

The complex, yet highly ordered and predictable, structure of the neural retina is one of the most conserved features of the vertebrate central nervous system. In all vertebrate classes, retinal neurons are organized into laminae with each neuronal class adopting specific morphologies and patterns of connectivity. Using genetic analyses in zebrafish, it has been demonstrated that N-cadherin (Ncad) has several distinct and crucial functions during the establishment of retinal organization. Although the location of cell division is disorganized in embryos with reduced or no Ncad function, different classes of retinal neurons are generated. However, these neurons fail to organize into correct laminae, most probably owing to compromised adhesion between retinal cells. In addition, amacrine cells exhibit exuberant and misdirected outgrowth of neurites that contributes to severe disorganization of the inner plexiform layer. Retinal ganglion cells also exhibit defects in process outgrowth, with axons exhibiting fasciculation defects and adopting incorrect ipsilateral trajectories. At least some of these defects are likely to be due to a failure to maintain compartment boundaries between eye, optic nerve and brain. Although in vitro studies have implicated Fgf receptors in modulating the axon outgrowth promoting properties of Ncad, most aspects of the Ncad mutant phenotype are not phenocopied by treatments that block Fgf receptor function (Masai, 2003).

The segmental pattern of neural-crest-derived sympathetic ganglia arises as a direct result of signals that restrict neural crest cell migratory streams through rostral somite halves. The spatiotemporal pattern of chick sympathetic ganglia formation is a two-phase process. Neural crest cells migrate laterally to the dorsal aorta, then surprisingly spread out in the longitudinal direction, before sorting into discrete ganglia. This study investigated the function of two families of molecules that are thought to regulate cell sorting and aggregation. By blocking Eph/ephrins or N-cadherin function, changes were measured in neural crest cell migratory behaviors that lead to alterations in sympathetic ganglia formation using a sagittal slice explant culture and 3D confocal time-lapse imaging. The results demonstrate that local inhibitory interactions within inter-ganglionic regions, mediated by Eph/ephrins, and adhesive cell-cell contacts at ganglia sites, mediated by N-cadherin, coordinate to sculpt discrete sympathetic ganglia (Kasemeier-Kulesa, 2006).

During neural crest ontogeny, an epithelial to mesenchymal transition is necessary for cell emigration from the dorsal neural tube. This process is likely to involve a network of gene activities, which remain largely unexplored. N-cadherin inhibits the onset of crest delamination both by a cell adhesion-dependent mechanism and by repressing canonical Wnt signaling found to be necessary for crest delamination by acting downstream of BMP4. Furthermore, N-cadherin protein, but not mRNA, is normally downregulated along the dorsal tube in association with the onset of crest delamination, and this process is triggered by BMP4. BMP4 stimulates cleavage of N-cadherin into a soluble cytoplasmic fragment via an ADAM10-dependent mechanism. Intriguingly, when overexpressed, the cytoplasmic N-cadherin fragment translocates into the nucleus, stimulates cyclin D1 transcription and crest delamination, while enhancing transcription of β-catenin. CTF2 also rescues the mesenchymal phenotype of crest cells in ADAM10-inhibited neural primordia. Hence, by promoting its cleavage, BMP4 converts N-cadherin inhibition into an activity that is likely to participate, along with canonical Wnt signaling, in the stimulation of neural crest emigration (Shoval, 2007).

Receptor tyrosine kinases of the EGFR family exert their various effects on cellular function through the formation of different dimeric receptor complexes. To investigate the functional impact of EGFR-HER2 heterodimers on migration of glial tumour cells, different HER2 constructs, including a constitutively active (HER2VE) and a dominant-negative (HER2VEKA) receptor, were stably transfected in the EGFR-overexpressing human glioma cell line LN18. Interference of EGFR activation through HER2VEKA inhibited cellular migration, whereas EGFR activation through HER2VE increased migration. These results were corroborated by inhibition of EGFR-HER2 signalling with tyrosine kinase inhibitors, because only the blocking of both receptors in HER2VE-cells with the bi-specific inhibitor AEE788 downregulated migration to levels comparable with those in HER2VEKA cells. The non-migratory phenotype was mediated through upregulation of N-cadherin and its recruitment to the cell membrane in HER2VEKA cells; downregulation of N-cadherin by RNAi restored migration in HER2VEKA cells and N-cadherin was also downregulated in migrating HER2VE-cells. Downregulation of N-cadherin levels in the plasma membrane was accompanied by a direct interaction of the EGFR-HER2 and N-cadherin-beta-catenin complexes, leading to tyrosine phosphorylation of beta-catenin. These results indicate that HER2 affects glial-cell migration by modulating EGFR-HER2 signal transduction, and that this effect is mediated by N-cadherin (Rappl, 2008).

Xenopus Cadherin-11 (Xcad-11) is expressed when cranial neural crest cells (CNC) acquire motility. However, its function in stimulating cell migration is poorly understood. This study demonstrates that Xcad-11 initiates filopodia and lamellipodia formation, which is essential for CNC to populate pharyngeal pouches. The cytoplasmic tail of Xcad-11 was identified as both necessary and sufficient for proper CNC migration as long as it was linked to the plasma membrane. These results showing that guanine nucleotide exchange factor (GEF)-Trio binds to Xcad-11 and can functionally substitute for it like constitutively active forms of RhoA, Rac, and cdc42 unravel a novel cadherin function (Kashef, 2009).

Vertebrate cranial sensory ganglia have a dual origin from the neural crest and ectodermal placodes. In the largest of these, the trigeminal ganglion, Slit1-Robo2 signaling is essential for proper ganglion assembly. This study demonstrates a crucial role for the cell adhesion molecule N-cadherin and its interaction with Slit1-Robo2 during gangliogenesis in vivo. A common feature of chick trigeminal and epibranchial ganglia is the expression of N-cadherin and Robo2 on placodal neurons and Slit1 on neural crest cells. Interestingly, N-cadherin localizes to intercellular adherens junctions between placodal neurons during ganglion assembly. Depletion of N-cadherin causes loss of proper ganglion coalescence, similar to that observed after loss of Robo2, suggesting that the two pathways might intersect. Consistent with this possibility, blocking or augmenting Slit-Robo signaling modulates N-cadherin protein expression on the placodal cell surface concomitant with alteration in placodal adhesion. Lack of an apparent change in total N-cadherin mRNA or protein levels suggests post-translational regulation. Co-expression of N-cadherin with dominant-negative Robo abrogates the Robo2 loss-of-function phenotype of dispersed ganglia, whereas loss of N-cadherin reverses the aberrant aggregation induced by increased Slit-Robo expression. This study suggests a novel mechanism whereby N-cadherin acts in concert with Slit-Robo signaling in mediating the placodal cell adhesion required for proper gangliogenesis (Shiau, 2009).

The development of neural crest cells involves an epithelial-mesenchymal transition (EMT) associated with the restriction of cadherin 6B expression to the pre-migratory neural crest cells (PMNCCs), as well as a loss of N-cadherin expression. Cadherin 6B, which is highly expressed in PMNCCs, persists in early migrating neural crest cells and is required for their emigration from the neural tube. Cadherin 6B-expressing PMNCCs exhibit a general loss of epithelial junctional polarity and acquire motile properties before their delamination from the neuroepithelium. Cadherin 6B selectively induces the de-epithelialization of PMNCCs, which is mediated by stimulation of BMP signaling, whereas N-cadherin inhibits de-epithelialization and BMP signaling. As BMP signaling also induces cadherin 6B expression and represses N-cadherin, cadherin-regulated BMP signaling may create two opposing feedback loops. Thus, the overall EMT of neural crest cells occurs via two distinct steps: a cadherin 6B and BMP signaling-mediated de-epithelialization, and a subsequent delamination through the basement membrane (Par, 2010).

Ovo1 links Wnt signaling with N-cadherin localization during neural crest migration

A fundamental issue in cell biology is how migratory cell behaviors are controlled by dynamically regulated cell adhesion. Vertebrate neural crest (NC) cells rapidly alter cadherin expression and localization at the cell surface during migration. Secreted Wnts induce some of these changes in NC adhesion and also promote specification of NC-derived pigment cells. This study shows that the zebrafish transcription factor Ovo1 is a Wnt target gene that controls migration of pigment precursors by regulating the intracellular movements of N-cadherin (Ncad). Ovo1 genetically interacts with Ncad and its depletion causes Ncad to accumulate inside cells. Ovo1-deficient embryos strongly upregulate factors involved in intracellular trafficking, including several rab GTPases, known to modulate cellular localization of cadherins. Surprisingly, NC cells express high levels of many of these rab genes in the early embryo, chemical inhibitors of Rab functions rescue NC development in Ovo1-deficient embryos and overexpression of a Rab-interacting protein leads to similar defects in NC migration. These results suggest that Ovo proteins link Wnt signaling to intracellular trafficking pathways that localize Ncad in NC cells and allow them to migrate. Similar processes probably occur in other cell types in which Wnt signaling promotes migration (Piloto, 2010).

PDGF controls contact inhibition of locomotion by regulating N-cadherin during neural crest migration

A fundamental property of neural crest (NC) migration is Contact inhibition of locomotion (CIL), a process by which cells change their direction of migration upon cell contact. CIL has been proven to be essential for NC migration in amphibian and zebrafish by controlling cell polarity in a cell contact dependent manner. Cell contact during CIL requires the participation of the cell adhesion molecule N-cadherin (see Drosophila CadN), which starts to be expressed by NC cells as a consequence of the switch between E- and N-cadherins during epithelial to mesenchymal transition (EMT). However, the mechanism that controls the upregulation of N-cadherin remains unknown. This study shows that PDGFRα (see Drosophila Pvr) and its ligand PDGF-A (see Drosophila Pvf1) are co-expressed in migrating cranial NC. Inhibition of PDGF-A/PDGFRα blocks NC migration by inhibiting N-cadherin and, consequently impairing CIL. Moreover, PI3K/AKT (see Drosophila Akt) was found to be a downstream effector of the PDGFRα cellular response during CIL. These results lead to a proposal that PDGF-A/PDGFRα signalling is a tissue-autonomous regulator of CIL by controlling N-cadherin upregulation during EMT. Finally, it was shown that once NC have undergone EMT, the same PDGF-A/PDGFRα works as NC chemoattractant guiding their directional migration (Bahm, 2017).

Nectin-2 and N-cadherin interact through extracellular domains and induce apical accumulation of F-actin in apical constriction of Xenopus neural tube morphogenesis

Neural tube formation is one of the most dynamic morphogenetic processes of vertebrate development. However, the molecules regulating its initiation are mostly unknown. This study demonstrates that nectin-2, an immunoglobulin-like cell adhesion molecule, is involved in the neurulation of Xenopus embryos in cooperation with N-cadherin. First, it was found that, at the beginning of neurulation, nectin-2 is strongly expressed in the superficial cells of neuroepithelium. The knockdown of nectin-2 impairs neural fold formation by attenuating F-actin accumulation and apical constriction, a cell-shape change that is required for neural tube folding. Conversely, the overexpression of nectin-2 in non-neural ectoderm induced ectopic apical constrictions with accumulated F-actin. However, experiments with domain-deleted nectin-2 revealed that the intracellular afadin-binding motif, which links nectin-2 and F-actin, is not required for the generation of the ectopic apical constriction. Furthermore, it was found that nectin-2 physically interacts with N-cadherin through extracellular domains, and they cooperatively enhance apical constriction by driving the accumulation of F-actin at the apical cell surface. Interestingly, the accumulation of N-cadherin at the apical surface of neuroepithelium is dependent on the presence of nectin-2, but that of nectin-2 is not affected by depletion of N-cadherin. A novel mechanism of neural tube morphogenesis is proposed, regulated by the two types of cell adhesion molecules (Morita, 2010).

Cortical neural precursors inhibit their own differentiation via N-cadherin maintenance of beta-catenin signaling

Little is known about the architecture of cellular microenvironments that support stem and precursor cells during tissue development. Although adult stem cell niches are organized by specialized supporting cells, in the developing cerebral cortex, neural stem/precursor cells reside in a neurogenic niche lacking distinct supporting cells. This study finds that neural precursors themselves comprise the niche and regulate their own development. Precursor-precursor contact regulates beta-catenin signaling and cell fate. In vivo knockdown of N-cadherin reduces beta-catenin signaling, migration from the niche, and neuronal differentiation in vivo. N-cadherin engagement activates beta-catenin signaling via Akt, suggesting a mechanism through which cells in tissues can regulate their development. These results suggest that neural precursor cell interactions can generate a self-supportive niche to regulate their own number (Zhang, 2010).

Whether Akt might link N-cadherin to β-catenin activation in cortical precursors was investigated . It was found that function-blocking antibodies to N-cadherin or shRNA to N-cadherin led to a significant reduction in phosphorylated (active) Akt in primary cortical precursors. To test the link between Akt activation and phosphorylation of β-catenin at Ser552, Akt was inhibited in neural precursors using triciribine (API-2), a small molecule Akt pathway inhibitor. Triciribine treatment of primary cortical precursors reduced the fraction of cells expressing β-catenin Ser552 in a dose-dependent fashion. Finally, expression of a dominant-negative (kinase-dead) Akt also reduced both baseline β-catenin signaling in high-density primary cortical precursor cultures as well as Wnt-stimulated β-catenin signaling. To confirm whether Akt functions downstream of N-cadherin to mediate β-catenin signaling, myristoylated (active) Akt was coexpressed along with shRNA to N-cadherin and β-catenin signaling was measured by TOP-flash reporters. It was found that that myrAkt rescued β-catenin signaling following N-cadherin knockdown. It was also found that myrAkt alone could increase β-catenin signaling, a finding consistent with the idea that this pathway may exist in parallel with the canonical Wnt signaling pathway. Together, these observations suggest that N-cadherin engagement leads to phosphorylation of Akt and subsequent Akt-mediated phosphoryation and activation of β-catenin (Zhang, 2010).

N-cadherin and muscle development

Limb muscle formation involves invasion of the limb bud mesoderm by myogenic precursor cells from the dermomyotomes at limb bud level. Directed cell migration, homing, and differentiation of myogenic cells are controlled by the stationary cells of the limb bud mesoderm. At the level of the extracellular matrix, the molecular basis of migration control has been suggested to be exerted by the distribution of hyaluronan. N-cadherin-mediated interactions play a role at cell-membrane level in myoblast distribution and differentiation. N-cadherin is strongly expressed by myogenic cells in the chick limb bud and more moderately expressed by stationary mesodermal cells in the myogenic zones and progress zone. After in vivo injection of antibodies and Fab-fragments against the homophilic binding site of N-cadherin into the wing bud mesoderm, aggregates of myoblasts are found predominantly in the dorsal myogenic zone 36 hr after injection, apparently due to immobilization. In the same position, areas of myf-5-positive cells are also observed. In injected limb buds, Pax-3-positive cells are less evenly distributed than in uninjected limbs. They are found to spread up to the epidermis and also form loosely arranged aggregates. After prolonged reincubation periods, injected limbs show ectopic myoblasts that are rich in desmin and areas of strongly desmin-expressing myoblasts within muscle blastemas. These effects are not observed after application of antibodies against other parts of the N-cadherin molecule. It is concluded that N-cadherin is involved in myoblast migration in the limb buds via homophilic interactions and that it plays a role in signal transduction during myogenesis (Brand-Saberi, 1996).

Cells with the potential to form skeletal muscle are present in the chick embryo prior to gastrulation. Muscle differentiation begins after gastrulation within the somites. The role of cadherin-mediated adhesion in the commitment and differentiation of skeletal muscle precursor cells was examined by analyzing the expression of cell-cell adhesion molecules in cultures of epiblast, segmental plate, and somite cells and by determining the effects of adhesion-perturbing antibodies on the accumulation of MyoD and sarcomeric myosin. Cultured primitive streak stage epiblast cells downregulate E-cadherin and upregulate N-cadherin. This switch in cadherin expression also occurs in vivo as epiblast cells enter the primitive streak. Although MyoD protein is present in cells with N- or E-cadherin, only cells with N-cadherin differentiate into skeletal muscle. In contrast to the primitive streak stage epiblast cells, prestreak epiblast cells maintain the expression of E-cadherin in vitro. While the majority of prestreak cells contain MyoD, only a few synthesize myosin. Treatment of primitive streak stage epiblast cells with function-perturbing antibodies to N-cadherin results in an inhibition of myosin accumulation and a decrease in the percentage of cells with MyoD. Segmental plate and somite cells are similar to primitive streak stage epiblast cells in that most differentiate into skeletal muscle when cultured in serum-free medium. While function-perturbing antibodies to N-cadherin inhibit the accumulation of myosin in these mesoderm cells, the number of MyoD positive cells is unaffected in somite cultures and only partially reduced in segmental plate cultures. These results suggest that N-cadherin-mediated cell-cell adhesion is involved in both the commitment of muscle precursors and their terminal differentiation (George-Weinstein 1997).

The cell-cell adhesion molecule N-cadherin, with its associated catenins, is expressed by differentiating skeletal muscle and its precursors. Although N-cadherin's role in later events of skeletal myogenesis (such as adhesion during myoblast fusion) is well established, less is known about its role in earlier events, such as commitment and differentiation. N-cadherin- mediated adhesion enhances skeletal muscle differentiation in three-dimensional cell aggregates. The cadherin-negative BHK fibroblastlike cell line was transfected with N-cadherin. Expression of exogenous N-cadherin upregulates endogenous beta-catenin and induces strong cell-cell adhesion. When BHK cells are cultured as three-dimensional aggregates, N-cadherin enhances withdrawal from the cell cycle and stimulates differentiation into skeletal muscle as measured by increased expression of sarcomeric myosin and the 12/101 antigen. In contrast, N-cadherin does not stimulate differentiation of BHK cells in monolayer cultures. The effect of N-cadherin is not unique since E-cadherin also increases the level of sarcomeric myosin in BHK aggregates. However, a nonfunctional mutant N-cadherin that increases the level of beta-catenin fails to promote skeletal muscle differentiation, suggesting an adhesion-competent cadherin is required. These results suggest that cadherin-mediated cell-cell interactions during embryogenesis can dramatically influence skeletal myogenesis (Redfield, 1997).

Myoblast fusion is essential to muscle tissue development yet remains poorly understood. N-cadherin, like other cell surface adhesion molecules, has been implicated by others in muscle formation based on its pattern of expression and on inhibition of myoblast aggregation and fusion by antibodies or peptide mimics. Mice rendered homozygous null for N-cadherin reveal the general importance of the molecule in early development, but do not test a role in skeletal myogenesis, since the embryos die before muscle formation. To test genetically the proposed role of N-cadherin in myoblast fusion, N-cadherin null primary myoblasts were obtained in culture. Fusion of myoblasts expressing or lacking N-cadherin is found to be equivalent, both in vitro by intracistronic complementation of lacZ and in vivo by injection into the muscles of adult mice. An essential role for N-cadherin in mediating the effects of basic fibroblast growth factor is also excluded. These methods for obtaining genetically homozygous null somatic cells from adult tissues should have broad applications. Here, they demonstrate clearly that the putative fusion molecule, N-cadherin, is not essential for myoblast fusion (Charlton, 1997).

Slow-twitch muscle fibers of the zebrafish myotome undergo a unique set of morphogenetic cell movements. During embryogenesis, slow-twitch muscle derives from the adaxial cells, a layer of paraxial mesoderm that differentiates medially within the myotome, immediately adjacent to the notochord. Subsequently, slow-twitch muscle cells migrate through the entire myotome, coming to lie at its most lateral surface. The cellular and molecular basis for slow-twitch muscle cell migration has been examined. Slow-twitch muscle cell morphogenesis is marked by behaviors typical of cells influenced by differential cell adhesion. Dynamic and reciprocal waves of N-cadherin and M-cadherin expression within the myotome, that correlate precisely with cell migration, generate differential adhesive environments that drive slow-twitch muscle cell migration through the myotome. Removing or altering the expression of either protein within the myotome perturbs migration. These results provide a definitive example of homophilic cell adhesion shaping cellular behavior during vertebrate development (Cortés, 2003).

N-cadherin and hematopoietic cell differentiation

The cadherins, an important family of cell adhesion molecules, are known to play major roles during embryonic development and in the maintenance of solid tissue architecture. In the hematopoietic system, however, little is known of the role of this cell adhesion family. By RT-PCR, Western blot analysis and immunofluorescence staining it has been shown that N-cadherin, a classical type I cadherin mainly expressed on neuronal, endothelial and muscle cells, is expressed on the cell surface of resident bone marrow stromal cells. FACS analysis of bone marrow mononuclear cells reveals that N-cadherin is also expressed on a subpopulation of early hematopoietic progenitor cells. Triple-color FACS analysis defined a new CD34(+) CD19(+) N-cadherin(+) progenitor cell population. During further differentiation, however, N-cadherin expression is lost. Treatment of CD34(+) progenitor cells with function-perturbing N-cadherin antibodies drastically diminishes colony formation, indicating a direct involvement of N-cadherin in the differentiation program of early hematopoietic progenitors. N-cadherin can also mediate adhesive interactions within the bone marrow as demonstrated by inhibition of homotypic interactions of bone-marrow-derived cells with N-cadherin antibodies. Together, these data strongly suggest that N-cadherin is involved in the development and retention of early hematopoietic progenitors within the bone marrow microenvironment (Puch, 2001).

The activity of adult stem cells is essential to replenish mature cells constantly lost due to normal tissue turnover. By a poorly understood mechanism, stem cells are maintained through self-renewal while concomitantly producing differentiated progeny. Genetic evidence is provided for an unexpected function of the c-Myc protein in the homeostasis of hematopoietic stem cells (HSCs). Conditional elimination of c-Myc activity in the bone marrow (BM) results in severe cytopenia and accumulation of HSCs in situ. Mutant HSCs self-renew and accumulate due to their failure to initiate normal stem cell differentiation. Impaired differentiation of c-Myc-deficient HSCs is linked to their localization in the differentiation preventative BM niche environment, and correlates with up-regulation of N-cadherin and a number of adhesion receptors, suggesting that release of HSCs from the stem cell niche requires c-Myc activity. Accordingly, enforced c-Myc expression in HSCs represses N-cadherin and integrins leading to loss of self-renewal activity at the expense of differentiation. Endogenous c-Myc is differentially expressed and induced upon differentiation of long-term HSCs. Collectively, these data indicate that c-Myc controls the balance between stem cell self-renewal and differentiation, presumably by regulating the interaction between HSCs and their niche (Wilson, 2004).

The stem cell niche is defined as a subset of tissue cells and extracellular substrates that can harbor one or more stem cells controlling their self-renewal and progeny production in vivo. Retention of stem cells in the niche is thought to be accomplished by stem cell niche and stem-cell extracellular matrix (ECM)-ligand interactions. It has been shown in the Drosophila ovary that DE-cadherin-mediated anchoring of germ line and somatic stem cells to the niche is essential for their maintenance. Putative niches have also been identified in vertebrates, including the bulge region in the skin epidermis and the stem cell-bearing base of intestinal crypts. In the BM, HSCs are located at the endosteal lining of the BM cavities, and recent studies show that specialized spindle-shaped N-cadherin+ osteoblasts (SNO) are a key component of the BM stem cell niche. HSCs are thought to be anchored to SNO cells via a homotypic N-cadherin interaction (Wilson, 2004 and references therein).

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. This study analyzed the extent to which E-cadherin confers unique adhesion and signaling properties in trophectoderm formation in vivo. Using a gene replacement approach, N-cadherin cDNA was introduced into the E-cadherin genomic locus. 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, 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. These results point to a specific and unique function for E-cadherin during mouse preimplantation development (Kan, 2007).

N-cadherin and heart development

Evolutionary homologs continued: part 3/3 | back to part 1/3

Cadherin-N: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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