Altered expression of neuronal cell adhesion molecules induced by nerve injury and repair

Peripheral nerve injury results in short-term and long-term changes in both neurons and glia. In the present study, immunohistological and immunoblot analyses were used to examine the expression of the neural cell adhesion molecule (N-CAM) and the neuron-glia cell adhesion molecule (Ng-CAM) within different parts of a functionally linked neuromuscular system extending from skeletal muscle to the spinal cord after peripheral nerve injury. Histological samples were taken from 3 to 150 d after crushing or transecting the sciatic nerve in adult chickens and mice. In unperturbed tissues, both N-CAM and Ng-CAM were found on nonmyelinated axons, and to a lesser extent on Schwann cells and myelinated axons. Only N-CAM was found on muscles. After denervation, the following changes were observed: The amount of N-CAM in muscle fibers increased transiently on the surface and in the cytoplasm, and in interstitial spaces between fibers. Restoration of normal N-CAM levels in muscle was dependent on reinnervation; in a chronically denervated state, N-CAM levels remained high. After crushing or cutting the nerve, the amount of both CAMs increased in the area surrounding the lesion, and the predominant form of N-CAM changed from a discrete Mr 140,000 component to the polydisperse high molecular weight embryonic form. Anti-N-CAM antibodies stained neurites, Schwann cells, and the perineurium of the regenerating sciatic nerve. Anti-Ng-CAM antibodies labeled neurites, Schwann cells and the endoneurial tubes in the distal stump. Changes in CAM distribution were observed in dorsal root ganglia and in the spinal cord only after the nerve was cut. The fibers within affected dorsal root ganglia were more intensely labeled for both CAMs, and the motor neurons in the ventral horn of the spinal cord of the affected segments were stained more intensely in a ring pattern by anti-N-CAM and anti-Ng-CAM than their counterparts on the side contralateral to the lesion. Taken together with the previous studies (Rieger, F., M. Grumet, and G. M. Edelman, J. Cell Biol. 101:285-293), these data suggest that local signals between neurons and glia may regulate CAM expression in the spinal cord and nerve during regeneration, and that activity may regulate N-CAM expression in muscle. Correlations of the present observations are made here with established events of nerve degeneration and suggest a number of roles for the CAMs in regenerative events.(ABSTRACT TRUNCATED AT 400 WORDS)     

The neuronal chondroitin sulfate proteoglycan neurocan binds to the neural cell adhesion molecules Ng-CAM/L1/NILE and N-CAM, and inhibits neuronal adhesion and neurite outgrowth

We have previously shown that aggregation of microbeads coated with N- CAM and Ng-CAM is inhibited by incubation with soluble neurocan, a chondroitin sulfate proteoglycan of brain, suggesting that neurocan binds to these cell adhesion molecules (Grumet, M., A. Flaccus, and R. U. Margolis. 1993. J. Cell Biol. 120:815). To investigate these interactions more directly, we have tested binding of soluble 125I- neurocan to microwells coated with different glycoproteins. Neurocan bound at high levels to Ng-CAM and N-CAM, but little or no binding was detected to myelin-associated glycoprotein, EGF receptor, fibronectin, laminin, and collagen IV. The binding to Ng-CAM and N-CAM was saturable and in each case Scatchard plots indicated a high affinity binding site with a dissociation constant of approximately 1 nM. Binding was significantly reduced after treatment of neurocan with chondroitinase, and free chondroitin sulfate inhibited binding of neurocan to Ng-CAM and N-CAM. These results indicate a role for chondroitin sulfate in this process, although the core glycoprotein also has binding activity. The COOH-terminal half of neurocan was shown to have binding properties essentially identical to those of the full-length proteoglycan. To study the potential biological functions of neurocan, its effects on neuronal adhesion and neurite growth were analyzed. When neurons were incubated on dishes coated with different combinations of neurocan and Ng-CAM, neuronal adhesion and neurite extension were inhibited. Experiments using anti-Ng-CAM antibodies as a substrate also indicate that neurocan has a direct inhibitory effect on neuronal adhesion and neurite growth. Immunoperoxidase staining of tissue sections showed that neurocan, Ng-CAM, and N-CAM are all present at highest concentration in the molecular layer and fiber tracts of developing cerebellum. The overlapping localization in vivo, the molecular binding studies, and the striking effects on neuronal adhesion and neurite growth support the view that neurocan may modulate neuronal adhesion and neurite growth during development by binding to neural cell adhesion molecules.     

Initial appearance and regional distribution of the neuron-glia cell adhesion molecule in the chick embryo

This study represents a global survey of the times of the first appearance of the neuron-glia cell adhesion molecule (Ng-CAM) in various regions and on particular cells of the chick embryonic nervous system. Ng-CAM, originally characterized by means of an in vitro binding assay between glial cells and brain membrane vesicles, first appears in development at the surface of early postmitotic neurons. By 3 d in the chick embryo, the first neurons detected by antibodies to Ng-CAM are located in the ventral neural tube; these precursors of motor neurons emit well-stained fibers to the periphery. To identify locations of appearance of Ng-CAM in the peripheral nervous system (PNS), we used a monoclonal antibody called NC-1 that is specific for neural crest cells in early embryos to show the presence of numerous crest cells in the neuritic outgrowth from the neural tube; neither these crest cells nor those in ganglion rudiments bound anti-Ng-CAM antibodies. The earliest neurons in the PNS stained by anti-Ng-CAM appeared by 4 d of development in the cranial ganglia. At later stages and progressively, all the neurons and neurities of the PNS were found to contain Ng-CAM both in vitro and in vivo. Many central nervous system (CNS) neurons also showed Ng-CAM at these later stages, but in the CNS, the molecule was mostly associated with neuronal processes (mainly axons) rather than with cell bodies; this regional distribution at the neuronal cell surface is an example of polarity modulation. In contrast to the neural cell adhesion molecule and the liver cell adhesion molecule, both of which are found very early in derivatives of more than one germ layer, Ng-CAM is expressed only on neurons of the CNS and the PNS during the later epoch of development concerned with neural histogenesis. Ng-CAM is thus a specific differentiation product of neuroectoderm. Ng-CAM was found on developing neurons at approximately the same time that neurofilaments first appear, times at which glial cells are still undergoing differentiation from neuroepithelial precursors. The present findings and those of previous studies suggest that together the neural cell adhesion molecule and Ng-CAM mediate specific cellular interactions during the formation of neuronal networks by means of modulation events that govern their prevalence and polarity on neuronal cell surfaces.     

Heterotypic binding between neuronal membrane vesicles and glial cells is mediated by a specific cell adhesion molecule

By means of a multistage quantitative assay, we have identified a new kind of cell adhesion molecule (CAM) on neuronal cells of the chick embryo that is involved in their adhesion to glial cells. The assay used to identify the binding component (which we name neuron-glia CAM or Ng-CAM) was designed to distinguish between homotypic binding (e.g., neuron to neuron) and heterotypic binding (e.g., neuron to glia). This distinction was essential because a single neuron might simultaneously carry different CAMs separately mediating each of these interactions. The adhesion of neuronal cells to glial cells in vitro was previously found to be inhibited by Fab' fragments prepared from antisera against neuronal membranes but not by Fab' fragments against N-CAM, the neural cell adhesion molecule. This suggested that neuron-glia adhesion is mediated by specific cell surface molecules different from previously isolated CAMs . To verify that this was the case, neuronal membrane vesicles were labeled internally with 6-carboxyfluorescein and externally with 125I-labeled antibodies to N-CAM to block their homotypic binding. Labeled vesicles bound to glial cells but not to fibroblasts during a 30-min incubation period. The specific binding of the neuronal vesicles to glial cells was measured by fluorescence microscopy and gamma spectroscopy of the 125I label. Binding increased with increasing concentrations of both glial cells and neuronal vesicles. Fab' fragments prepared from anti-neuronal membrane sera that inhibited binding between neurons and glial cells were also found to inhibit neuronal vesicle binding to glial cells. The inhibitory activity of the Fab' fragments was depleted by preincubation with neuronal cells but not with glial cells. Trypsin treatment of neuronal membrane vesicles released material that neutralized Fab' fragment inhibition; after chromatography, neutralizing activity was enriched 50- fold. This fraction was injected into mice to produce monoclonal antibodies; an antibody was obtained that interacted with neurons, inhibited binding of neuronal membrane vesicles to glial cells, and recognized an Mr = 135,000 band in immunoblots of embryonic chick brain membranes. These results suggest that this molecule is present on the surfaces of neurons and that it directly or indirectly mediates adhesion between neurons and glial cells. Because the monoclonal antibody as well as the original polyspecific antibodies that were active in the assay did not bind to glial cells, we infer that neuron- glial interaction is heterophilic, i.e., it occurs between Ng-CAM on neurons and an as yet unidentified CAM present on glial cells.     

Sequential expression and differential function of multiple adhesion molecules during the formation of cerebellar cortical layers

We have correlated the times of appearance of the neural cell adhesion molecule (N-CAM), the neuron-glia cell adhesion molecule (Ng-CAM), and the extracellular matrix protein, cytotactin, during the development of the chicken cerebellar cortex, and have shown that these molecules make different functional contributions to granule cell migration. Immunofluorescent staining showed distinct spatiotemporal expression sequences for each adhesion molecule. N-CAM was present at all times in all layers. However, the large cytoplasmic domain polypeptide of N-CAM was always absent from the external granular layer and was enriched in the molecular layer as development proceeded. Ng-CAM began to be expressed in the premigratory granule cells just before migration and later disappeared from cell bodies but remained on parallel fibers. Cytotactin, which is synthesized by glia and not by neurons, appeared first in a speckled pattern within the external granular layer and later appeared in a continuous pattern along the Bergmann glia; it was also enriched in the molecular layer. After we established their order of appearance, we tested the separate functions of these adhesion molecules in granule cell migration by adding specific antibodies against each molecule to cerebellar explant cultures that had been labeled with tritiated thymidine and then measuring the differential distribution of labeled cells in the forming layers. Anti-N-CAM showed marginal effects. In contrast, anti-Ng-CAM arrested most cells in the external granular layer, while anti-cytotactin arrested most cells in the molecular layer. Time course analyses combined with sequential addition of different antibodies in different orders showed that anti-Ng-CAM had a major effect in the early period (first 36 h in culture) and a lesser effect in the second part of the culture period, while anti-cytotactin had essentially no effect at the earlier time but had major effects at a later period (18-72 h in culture). The two major stages of cerebellar granule cell migration thus appear to be differentially affected by distinct adhesion molecules of different cellular origins, binding mechanisms, and overall distributions. The results indicated that local cell surface modulation of adhesion molecules of different specificities at defined stages and sites is essential to the formation of cerebellar cortical layers.     

Nerve growth factor enhances expression of neuron-glia cell adhesion molecule in PC12 cells

The neuron-glia cell adhesion molecule (Ng-CAM) has been identified in mammalian brain tissue and PC12 pheochromocytoma cells as Mr 200,000 and Mr 230,000 species, respectively. When PC12 cells were treated with nerve growth factor (NGF), the amount of Ng-CAM at the cell surface was increased approximately threefold, whereas the amount of the neural cell adhesion molecule (N-CAM) remained unchanged. An NGF-inducible large external glycoprotein (NILE) has been previously identified by its enhanced expression in NGF-treated PC12 cells. Ng-CAM and NILE are similar in molecular weight, expression during development, and responsiveness to NGF in PC12 cells, suggesting that the two molecules are related. In addition, antibodies to Ng-CAM and NILE cross-reacted and the molecules had similar peptide maps after limited proteolysis. Moreover, antibodies to Ng-CAM inhibited fasciculation of neurites, a functional property shared with NILE. The results show that cell adhesion molecules can respond selectively to growth factors and suggest that NILE is, in fact, mammalian Ng-CAM.     

Neuron-glia cell adhesion molecule interacts with neurons and astroglia via different binding mechanisms

The neuron-glia cell adhesion molecule (Ng-CAM) is present in the central nervous system on postmitotic neurons and in the periphery on neurons and Schwann cells. It has been implicated in binding between neurons and between neurons and glia. To understand the molecular mechanisms of Ng-CAM binding, we analyzed the aggregation of chick Ng-CAM either immobilized on 0.5-micron beads (Covaspheres) or reconstituted into liposomes. The results were correlated with the binding of these particles to different types of cells as well as with cell-cell binding itself. Both Ng-CAM-Covaspheres and Ng-CAM liposomes individually self-aggregated, and antibodies against Ng-CAM strongly inhibited their aggregation; the rate of aggregation increased approximately with the square of the concentration of the beads or the liposomes. Much higher rates of aggregation were observed when the ratio of Ng-CAM to lipid in the liposome was increased. Radioiodinated Ng-CAM on Covaspheres and in liposomes bound both to neurons and to glial cells and in each case antibodies against Ng-CAM inhibited 50-90% of the binding. Control preparations of fibroblasts and meningeal cells did not exhibit significant binding. Adhesion between neurons and glia within and across species (chick and mouse) was explored in cellular assays after defining markers for each cell type, and optimal conditions of shear, temperature, and cell density. As previously noted using chick cells (Grumet, M., S. Hoffman, C.-M. Chuong, and G. M. Edelman. 1984 Proc. Natl. Acad. Sci. USA. 81:7989-7993), anti-Ng-CAM antibodies inhibited neuron-neuron and neuron-glia binding. In cross-species adhesion assays, binding of chick neurons to mouse astroglia and binding of mouse neurons to chick astroglia were both inhibited by anti-Ng-CAM antibodies. To identify whether the cellular ligands for Ng-CAM differed for neuron-neuron and neuron-glia binding, cells were preincubated with specific antibodies, the antibodies were removed by washing, and Ng-CAM-Covasphere binding was measured. Preincubation of neurons with anti-Ng-CAM antibodies inhibited Ng-CAM-Covasphere binding but similar preincubation of astroglial cells did not inhibit binding. In contrast, preincubation of astroglia with anti-astroglial cell antibodies inhibited binding to these cells but preincubation of neurons with these antibodies had no effect. Together with the data on Covaspheres and liposome aggregation, these findings suggested that Ng-CAM-Covaspheres bound to Ng-CAM on neurons but bound to different molecules on astroglia.(ABSTRACT TRUNCATED AT 400 WORDS)     

Functional characterization of chondroitin sulfate proteoglycans of brain: interactions with neurons and neural cell adhesion molecules

Ng-CAM and N-CAM are cell adhesion molecules (CAMs), and each CAM can bind homophilically as demonstrated by the ability of CAM-coated beads (Covaspheres) to self-aggregate. We have found that the extent of aggregation of Covaspheres coated with either Ng-CAM or N-CAM was strongly inhibited by the intact 1D1 and 3F8 chondroitin sulfate proteoglycans of rat brain, and by the core glycoproteins resulting from chondroitinase treatment of the proteoglycans. Much higher concentrations of rat chondrosarcoma chondroitin sulfate proteoglycan (aggrecan) core proteins had no significant effect in these assays. The 1D1 and 3F8 proteoglycans also inhibited binding of neurons to Ng-CAM when mixtures of these proteins were adsorbed to polystyrene dishes. Direct binding of neurons to the proteoglycan core glycoproteins from brain but not from chondrosarcoma was demonstrated using an assay in which cell-substrate contact was initiated by centrifugation, and neuronal binding to the 1D1 proteoglycans was specifically inhibited by the 1D1 monoclonal antibody. Different forms of the 1D1 proteoglycan have been identified in developing and adult brain. The early postnatal form (neurocan) was found to bind neurons more effectively than the adult proteoglycan, which represents the C-terminal half of the larger neurocan core protein. Our results therefore indicate that certain brain proteoglycans can bind to neurons, and that Ng-CAM and N-CAM may be heterophilic ligands for neurocan and the 3F8 proteoglycan. The ability of these brain proteoglycans to inhibit adhesion of cells to CAMs may be one mechanism to modulate cell adhesion and migration in the nervous system.     

Evidence for the Binding of Ng-CAM to Laminin

Ng-CAM is a cell adhesion molecule mediating neuron-glia and neuron-neuron adhesion via different binding mechanisms. While its binding can be homophilic as demonstrated by the self-aggregation of Ng-CAM coated beads (Covaspheres), Ng-CAM has also been shown to bind to glia by a heterophilic mechanism. In the present study, we found that the extent of Ng-CAM Covasphere aggregation was strongly diminished in the presence of the extracellular matrix glycoprotein laminin. When proteolytic fragments of laminin were tested, the P1' fragment (obtained from the short arms by pepsin treatment) was found to inhibit aggregation of Ng-CAM-Covaspheres while the elastase fragments E3 and E8 (from the long arm) were ineffective. To provide other means of analyzing interactions between laminin and Ng-CAM, the two proteins were covalently linked to differently fluorescing Covaspheres and tested for coaggregation. Laminin-Covaspheres coaggregated with Ng-CAM-Covaspheres, and this binding was inhibited both by anti-Ng-CAM and by anti-laminin antibodies. Covaspheres coated with other proteins including BSA and fibronectin did not coaggregate with Ng-CAM-Covaspheres. Moreover, using a solid phase binding assay, we found that ¹²⁵I-labeled Ng-CAM bound to laminin and to Ng-CAM but not to fibronectin. The results suggest that regions in the short arms of laminin can bind to Ng-CAM. To test whether Ng-CAM present on neurons could be involved in binding to laminin, adhesion of neurons to substrates coated with various proteins was tested in the presence of specific antibodies. Anti-Ng-CAM Fab' fragments inhibited neuronal binding to laminin but not binding to fibronectin. The combined results open the possibility that Ng-CAM on the surface of neurons may mediate binding to laminin in vivo, and that interactions with laminin can modulate homophilic Ng-CAM binding.     

Evidence for the Binding of Ng-CAM to Laminin

Ng-CAM is a cell adhesion molecule mediating neuron-glia and neuron-neuron adhesion via different binding mechanisms. While its binding can be homophilic as demonstrated by the self-aggregation of Ng-CAM coated beads (Covaspheres), Ng-CAM has also been shown to bind to glia by a heterophilic mechanism. In the present study, we found that the extent of Ng-CAM Covasphere aggregation was strongly diminished in the presence of the extracellular matrix glycoprotein laminin. When proteolytic fragments of laminin were tested, the P1′ fragment (obtained from the short arms by pepsin treatment) was found to inhibit aggregation of Ng-CAM-Covaspheres while the elastase fragments E3 and E8 (from the long arm) were ineffective. To provide other means of analyzing interactions between laminin and Ng-CAM, the two proteins were covalently linked to differently fluorescing Covaspheres and tested for coaggregation. Laminin-Covaspheres coaggregated with Ng-CAM-Covaspheres, and this binding was inhibited both by anti-Ng-CAM and by anti-laminin antibodies. Covaspheres coated with other proteins including BSA and fibronectin did not coaggregate with Ng-CAM-Covaspheres. Moreover, using a solid phase binding assay, we found that ¹²⁵I-labeled Ng-CAM bound to laminin and to Ng-CAM but not to fibronectin. The results suggest that regions in the short arms of laminin can bind to Ng-CAM. To test whether Ng-CAM present on neurons could be involved in binding to laminin, adhesion of neurons to substrates coated with various proteins was tested in the presence of specific antibodies. Anti-Ng-CAM Fab' fragments inhibited neuronal binding to laminin but not binding to fibronectin. The combined results open the possibility that Ng-CAM on the surface of neurons may mediate binding to laminin in vivo, and that interactions with laminin can modulate homophilic Ng-CAM binding.     

Interactions of the chondroitin sulfate proteoglycan phosphacan, the extracellular domain of a receptor-type protein tyrosine phosphatase, with neurons, glia, and neural cell adhesion molecules

Phosphacan is a chondroitin sulfate proteoglycan produced by glial cells in the central nervous system, and represents the extracellular domain of a receptor-type protein tyrosine phosphatase (RPTP zeta/beta). We previously demonstrated that soluble phosphacan inhibited the aggregation of microbeads coated with N-CAM or Ng-CAM, and have now found that soluble 125I-phosphacan bound reversibly to these neural cell adhesion molecules, but not to a number of other cell surface and extracellular matrix proteins. The binding was saturable, and Scatchard plots indicated a single high affinity binding site with a Kd of approximately 0.1 nM. Binding was reduced by approximately 15% after chondroitinase treatment, and free chondroitin sulfate was only moderately inhibitory, indicating that the phosphacan core glycoprotein accounts for most of the binding activity. Immunocytochemical studies of embryonic rat spinal phosphacan, Ng-CAM, and N-CAM have overlapping distributions. When dissociated neurons were incubated on dishes coated with combinations of phosphacan and Ng-CAM, neuronal adhesion and neurite growth were inhibited. 125I-phosphacan bound to neurons, and the binding was inhibited by antibodies against Ng-CAM and N-CAM, suggesting that these CAMs are major receptors for phosphacan on neurons. C6 glioma cells, which express phosphacan, adhered to dishes coated with Ng-CAM, and low concentrations of phosphacan inhibited adhesion to Ng-CAM but not to laminin and fibronectin. Our studies suggest that by binding to neural cell adhesion molecules, and possibly also by competing for ligands of the transmembrane phosphatase, phosphacan may play a major role in modulating neuronal and glial adhesion, neurite growth, and signal transduction during the development of the central nervous system.     

Expression of cell-adhesion molecules in embryonic induction. I. Morphogenesis of nestling feathers

The potential relationship of cell adhesion to embryonic induction during feather formation was examined by immunohistochemical analysis of the spatiotemporal distribution of three cell-adhesion molecules (CAMs), neural CAM (N-CAM), liver CAM (L-CAM), and neuron-glia CAM (Ng-CAM), and of substrate molecules (laminin and fibronectin) in embryonic chicken skin. The N-CAM found at sites of embryonic induction in the feather was found to be similar to brain N-CAM as judged by immuno-cross-reactivity, migratory position in PAGE, and the presence of embryonic to adult conversion. In contrast to the N-CAM found in the brain, however, only one polypeptide of Mr 140,000 was seen. N-CAM-positive dermal condensations were distributed periodically under L-CAM-positive feather placodes at those sites where basement membranes are known to be disrupted. After initiation of induction, L-CAM-positive placode cells became transiently N-CAM-positive. N-CAM was asymmetrically concentrated in the dorsal region of the feather bud, while fibronectin was concentrated in the ventral region. During feather follicle formation, N-CAM was expressed in the dermal papilla and was closely apposed to the L-CAM-positive papillar ectoderm, while the dermal papilla showed no evidence of laminin or fibronectin. The collar epithelium was both N-CAM- and L-CAM-positive. During the formation of the feather filament, N-CAM appeared periodically and asymmetrically on basilar cells located in the valleys between adjacent barb ridges. In contrast to the two primary CAMs, Ng-CAM was found only on nerves supplying the feather and the skin. These studies indicate that at each site of induction during feather morphogenesis, a general pattern is repeated in which an epithelial structure linked by L-CAM is confronted with periodically propagating condensations of cells linked by N-CAM.     

N-CAM at the vertebrate neuromuscular junction

We have detected the neural cell adhesion molecule, N-CAM, at nerve-muscle contacts in the developing and adult mouse diaphragm. Whereas we found N-CAM staining with fluorescent antibodies consistently to overlap with the pattern of alpha-bungarotoxin staining at nerve-muscle contacts both during development and in the adult, we observed N-CAM staining on the surfaces of developing myofibers and at much lower levels on adult myofibers. Consistent with its function, N-CAM was also detected on axons and axon terminals. Immunoblotting experiments with anti-N-CAM antibodies on detergent extracts of embryonic (E) diaphragm muscle revealed a polydisperse polysialylated N-CAM polypeptide, which in the adult (A) was converted to a discrete form of Mr 140,000; this change, called E-to-A conversion, was previously found to occur in different neural tissues at different rates. The Mr 140,000 component was not recognized by monoclonal antibody anti-N-CAM No. 5, which specifically recognizes antigenic determinants associated with N-linked oligosaccharide determinants on N-CAM from neural tissue. The relative concentration of the Mr 140,000 component prepared from diaphragm muscle increased during fetal development and then decreased sharply to reach adult values. Nevertheless, expression of N-CAM in muscle could be induced after denervation: one week after the sciatic nerve was severed, the relative amount of N-CAM increased dramatically as detected by immunoblots of extracts of whole muscle. Immunofluorescent staining confirmed that there was an increase in N-CAM, both in the cell and at the cell surface; at the same time, however, staining at the motor endplate was diminished. Our findings indicate that, in muscle, in addition to chemical modulation, cell-surface modulation of N-CAM occurs both in amount and distribution during embryogenesis and in response to denervation.     

Neuronal cell adhesion molecules and cytotactin are colocalized at the node of Ranvier

Immunocytochemical methods were used to show that Ng-CAM (the neuron-glia cell adhesion molecule), N-CAM (the neural cell adhesion molecule), and the extracellular matrix protein cytotactin are highly concentrated at nodes of Ranvier of the adult chicken and mouse. In contrast, unmyelinated axonal fibers were uniformly stained by specific antibodies to both CAMs but not by antibodies to cytotactin. Ultrastructural immunogold techniques indicated that both N-CAM and Ng-CAM were enriched in the nodal axoplasm and axolemma of myelinated fibers as well as within the nodal regions of the myelinating Schwann cell. At embryonic day 14, before myelination had occurred, small-caliber fibers of chick embryos showed periodic coincident accumulations of the two CAMs but not of cytotactin, with faint labeling in the axonal regions between accumulations. Cytotactin was found on Schwann cells and in connective tissue. By embryonic day 18, nodal accumulations of CAMs were first observed in a few medium- and large-caliber fibers. Immunoblot analyses indicated that embryonic to adult conversion of N-CAM and a progressive decrease in the amount of Ng-CAM and N-CAM occurred while nodes were forming. Sciatic nerves of mouse mutants with defects in cell interactions showed abnormalities in the distribution patterns and amount of Ng-CAM, N-CAM, and cytotactin that were consistent with the known morphological nodal disorders. In trembler (+/Tr), intense staining for both CAMs appeared all along the fibers and the amounts of N-CAM in the sciatic nerve were found to be increased. In mice with motor endplate disease (med/med), Ng-CAM and N-CAM, but not cytotactin, were localized in the widened nodes. Both trembler and med/med Schwann cells stained intensely for cytotactin, in contrast to normal Schwann cells which stained only slightly. All of these findings are consistent with the hypothesis that surface modulation of neuronal CAMs mediated by signals shared between neurons and glia may be necessary for establishing and maintaining the nodes of Ranvier.     

Functional analysis of posttranslational cleavage products of the neuron-glia cell adhesion molecule, Ng-CAM

Neuron-glia cell adhesion molecule (Ng-CAM) mediates cell adhesion between neurons homophilically and between neurons and glia heterophilically; it also promotes neurite outgrowth. In the chick brain, Ng-CAM is detected as glycoproteins of 190 and 210 kD (Ng- CAM200) with posttranslational cleavage products of 135 kD (F135, which contains most of the extracellular region) and 80 kD (F80, which includes the transmembrane and the cytoplasmic domains). To examine the functions of each of these components, we have expressed Ng-CAM200, F135, and F80 in murine L cells, and F135 and F80 as GST fusion proteins in the pGEX vector in bacteria. Appropriately transfected L cells expressed each of these proteins on their surfaces; F135 was also found in the media of cells transfected with Ng-CAM200 and F135. In addition to binding homophilically, cells transfected with Ng-CAM200 and F135 bound heterophilically to untransfected L cells, suggesting that there is a ligand for Ng-CAM on fibroblasts that may be related to the glial ligand. Detailed studies using the transfected cells and the fusion proteins indicated that both the homophilic and the heterophilic binding activities of Ng-CAM are localized in the F135 fragment of the molecule. The results also indicated that proteolytic cleavage of Ng- CAM200 is not required either for its expression on the cell surface or for cell adhesion and that there is an "anchor" for F135 on L cells (and presumably on neurons). In contrast to the cell binding results, the F80 but not the F135 fusion protein enhanced the outgrowth of neurites from dorsal root ganglion cells; this activity was associated with the FnIII repeats of F80. The observations that a protein corresponding to F135 contains the cell aggregation sites whereas one corresponding to the F80 has the ability to promote neurite outgrowth suggest that proteolytic cleavage may be an important event in regulating these Ng-CAM activities during embryonic development and neural regeneration.     

Analysis of high PSA N-CAM expression during mammalian spinal cord and peripheral nervous system development.

Using a monoclonal antibody that recognizes specifically a high polysialylated form of N-CAM (high PSA N-CAM), the temporal and spatial expression of this molecule was studied in developing spinal cord and neural crest derivatives of mouse truncal region. Temporal expression was analyzed on immunoblots of spinal cord and dorsal root ganglia (DRGs) extracts microdissected at different developmental stages. Analysis of the ratio of high PSA N-CAM to total N-CAM indicated that sialylation and desialylation are independently regulated from the expression of polypeptide chains of N-CAM. Motoneurons, dorsal root ganglia cells and commissural neurons present a homogeneous distribution of high PSA N-CAMs on both their cell bodies and their neurites. Sialylation of N-CAM can occur in neurons after their aggregation in peripheral ganglia as demonstrated for dorsal root ganglia at E12. Furthermore, peripheral ganglia express different levels of high PSA N-CAM. With in vitro models using mouse neural crest cells, we found that expression of high PSA N-CAM was restricted to cells presenting an early neuronal phenotype, suggesting a common regulation for the expression of high PSA N-CAM molecules, neurofilament proteins and sodium channels. Using perturbation experiments with endoneuraminidase, we confirmed that high PSA N-CAM molecules are involved in fasciculation and neuritic growth when neurons derived from neural crest grow on collagen substrata. However, we demonstrated that these two parameters do not appear to depend on high PSA N-CAM molecules when cells were grown on a fibronectin substratum, indicating the existence of a hierarchy among adhesion molecules.     

Mechanism of skin morphogenesis. I. Analyses with antibodies to adhesion molecules tenascin, N-CAM, and integrin.

To understand cell interactions during induction of skin appendages, we studied the roles of adhesion molecules N-CAM, tenascin, integrin, and fibronectin during feather development. Tenascin appeared in a periodic pattern on epithelia and was so far the earliest molecule detected in placodes. Three placode domains were identified: the anterior was positive for tenascin, the distal positive for N-CAM, and the posterior lacking both. Integrin appeared in dermal-epidermal junctions of placodes. In feather buds, sagittal sections revealed a transient anterior-posterior asymmetry with tenascin and N-CAM enriched in the anterior mesoderm. Tangential sections revealed a lateral-medial asymmetry with tenascin distributed in a ring shape and N-CAM in an "X" shape. Integrin was diffusely distributed within buds. Later tenascin and N-CAM were enriched in dermal papilla, the inducer of skin appendages. Perturbation of embryonic skin explant cultures with antibodies showed that anti-integrin beta 1 and anti-fibronectin blocked epithelial-mesenchymal interaction, anti-N-CAM caused uneven segregation of mesenchymal condensation, and anti-tenascin inhibited feather bud elongation. Dose-response curves showed gradual effects by these antibodies. The results indicated that these adhesion molecules are independently regulated and each contributes in different phases during morphogenesis of skin appendages.     

Cell adhesion molecules NgCAM and axonin-1 form heterodimers in the neuronal membrane and cooperate in neurite outgrowth promotion

The axonal surface glycoproteins neuronglia cell adhesion molecule (NgCAM) and axonin-1 promote cell-cell adhesion, neurite outgrowth and fasciculation, and are involved in growth cone guidance. A direct binding between NgCAM and axonin-1 has been demonstrated using isolated molecules conjugated to the surface of fluorescent microspheres. By expressing NgCAM and axonin-1 in myeloma cells and performing cell aggregation assays, we found that NgCAM and axonin-1 cannot bind when present on the surface of different cells. In contrast, the cocapping of axonin-1 upon antibody-induced capping of NgCAM on the surface of CV- 1 cells coexpressing NgCAM and axonin-1 and the selective chemical cross-linking of the two molecules in low density cultures of dorsal root ganglia neurons indicated a specific and direct binding of axonin- 1 and Ng-CAM in the plane of the same membrane. Suppression of the axonin-1 translation by antisense oligonucleotides prevented neurite outgrowth in dissociated dorsal root ganglia neurons cultured on an NgCAM substratum, indicating that neurite outgrowth on NgCAM substratum requires axonin-1. Based on these and previous results, which implicated NgCAM as the neuronal receptor involved in neurite outgrowth on NgCAM substratum, we concluded that neurite outgrowth on an NgCAM substratum depends on two essential interactions of growth cone NgCAM: a trans-interaction with substratum NgCAM and a cis-interaction with axonin-1 residing in the same growth cone membrane.     

Isolation of mouse N-CAM-related cDNA: detection and cloning using monoclonal antibodies.

Clones coding for the mouse neural cell adhesion molecule (N-CAM) were isolated from a cDNA library prepared in the expression vector lambda gt 11 from mRNA extracted from a mouse neuroblastoma cell line. This library was screened with two anti-N-CAM monoclonal antibodies directed against different sites on the molecule and with rabbit anti-N-CAM serum. Two clones were identified with the first monoclonal antibody, three with the second one, none reacted with both. The relevance of these cDNA clones to N-CAM was confirmed by several observations. First, cDNA sequences detected with one monoclonal antibody cross-hybridized with those identified by the other antibody. Second, the different fusion proteins all bound the rabbit serum in addition to one monoclonal antibody. Finally, the probes hybridized to discrete mRNA species of sufficient lengths to code for the very large N-CAM polypeptides in RNA preparations from N-CAM-expressing, but not from N-CAM-negative cells. An additional mRNA species not seen in embryonic brain was expressed in adult mouse brain. Genomic blot experiments indicated that sequences corresponding to one of our probes are present only a few times in the mouse genome.