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)     

Differential contributions of Ng-CAM and N-CAM to cell adhesion in different neural regions

Individual neurons can express both the neural cell adhesion molecule (N-CAM) and the neuron-glia cell adhesion molecule (Ng-CAM) at their cell surfaces. To determine how the functions of the two molecules may be differentially controlled, we have used specific antibodies to each cell adhesion molecule (CAM) to perturb its function, first in brain membrane vesicle aggregation and then in tissue culture assays testing the fasciculation of neurite outgrowths from cultured dorsal root ganglia, the migration of granule cells in cerebellar explants, and the formation of histological layers in the developing retina. Our strategy was initially to delineate further the binding mechanisms for each CAM. Antibodies to Ng-CAM and N-CAM each inhibited brain membrane vesicle aggregation but the binding mechanisms of the two CAMs differed. As expected from the known homophilic binding mechanism of N-CAM, anti-N- CAM-coated vesicles did not co-aggregate with uncoated vesicles. Anti- Ng-CAM-coated vesicles readily co-aggregated with uncoated vesicles in accord with a postulated heterophilic binding mechanism. It was also shown that N-CAM was not a ligand for Ng-CAM. In contrast to assays with brain membrane vesicles, cellular systems can reveal functional differences for each CAM reflecting its relative amount (prevalence modulation) and location (polarity modulation). Consistent with this, each of the three cellular processes examined in vitro was preferentially inhibited only by anti-N-CAM or by anti-Ng-CAM antibodies. Both neurite fasciculation and the migration of cerebellar granule cells were preferentially inhibited by anti-Ng-CAM antibodies. Anti-N-CAM antibodies inhibited the formation of histological layers in the retina. The data on perturbation by antibodies were correlated with the relative levels of expression of Ng-CAM and N-CAM in each of these different neural regions. Quantitative immunoblotting experiments indicated that the relative Ng-CAM/N-CAM ratios in comparable extracts of brain, dorsal root ganglia, and retina were respectively 0.32, 0.81, and 0.04. During culture of dorsal root ganglia in the presence of nerve growth factor, the Ng-CAM/N-CAM ratio rose to 4.95 in neurite outgrowths and 1.99 in the ganglion proper, reflecting both polarity and prevalence modulation. These results suggest that the relative ability of anti-Ng-CAM and anti-N-CAM antibodies to inhibit cell-cell interactions in different neural tissues is strongly correlated with the local Ng-CAM/N-CAM ratio.(ABSTRACT TRUNCATED AT 400 WORDS)     

IKAP/Elp1 is required in vivo for neurogenesis and neuronal survival, but not for neural crest migration

Familial Dysautonomia (FD; Hereditary Sensory Autonomic Neuropathy; HSAN III) manifests from a failure in development of the peripheral sensory and autonomic nervous systems. The disease results from a point mutation in the IKBKAP gene, which encodes the IKAP protein, whose function is still unresolved in the developing nervous system. Since the neurons most severely depleted in the disease derive from the neural crest, and in light of data identifying a role for IKAP in cell motility and migration, it has been suggested that FD results from a disruption in neural crest migration. To determine the function of IKAP during development of the nervous system, we (1) first determined the spatial-temporal pattern of IKAP expression in the developing peripheral nervous system, from the onset of neural crest migration through the period of programmed cell death in the dorsal root ganglia, and (2) using RNAi, reduced expression of IKBKAP mRNA in the neural crest lineage throughout the process of dorsal root ganglia (DRG) development in chick embryos in ovo. Here we demonstrate that IKAP is not expressed by neural crest cells and instead is expressed as neurons differentiate both in the CNS and PNS, thus the devastation of the PNS in FD could not be due to disruptions in neural crest motility or migration. In addition, we show that alterations in the levels of IKAP, through both gain and loss of function studies, perturbs neuronal polarity, neuronal differentiation and survival. Thus IKAP plays pleiotropic roles in both the peripheral and central nervous systems.     

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.     

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 pattern of BM88 in the developing nervous system of the chick and mouse embryo

We isolated a chick homologue of BM88 (cBM88), a cell-intrinsic nervous system-specific protein and examined the expression of BM88 mRNA and protein in the developing brain, spinal cord and peripheral nervous system of the chick embryo by in situ hybridization and immunohistochemistry. cBM88 is widely expressed in the developing central nervous system, both in the ventricular and mantle zones where precursor and differentiated cells lie, respectively. In the spinal cord, particularly strong cBM88 expression is detected ventrally in the motor neuron area. cBM88 is also expressed in the dorsal root ganglia and sympathetic ganglia. In the early neural tube, cBM88 is first detected at HH stage 15 and its expression increases with embryonic age. At early stages, cBM88 expression is weaker in the ventricular zone (VZ) and higher in the mantle zone. At later stages, when gliogenesis persists instead of neurogenesis, BM88 expression is abolished in the VZ and cBM88 is restricted in the neuron-containing mantle zone of the neural tube. Association of cBM88 expression with cells of the neuronal lineage in the chick spinal cord was demonstrated using a combination of markers characteristic of neuronal or glial precursors, as well as markers of differentiated neuronal, oligodendroglial and astroglial cells. In addition to the spinal cord, cBM88 is expressed in the HH stage 45 (embryonic day 19) brain, including the telencephalon, diencephalon, mesencephalon, optic tectum and cerebellum. BM88 is also widely expressed in the mouse embryonic CNS and PNS, in both nestin-positive neuroepithelial cells and post-mitotic betaIII-tubulin positive neurons.     

IKAP/hELP1 downregulation in neuroblastoma cells causes enhanced cell adhesion mediated by contactin overexpression

A splicing mutation in the IKBKAP gene encoding the IKAP/hELP1 (IKAP) protein was found to be the major cause of Familial Dysautonomia (FD). This mutation affects both the normal development and survival of sensory and sympathetic neurons of the peripheral nervous system (PNS). To understand the FD phenotype it is important to study the specific role played by IKAP in developing and mature PNS neurons. We used the neuroblastoma SHSY5Y cell line, originated from neural crest adrenal tumor and simulated the FD phenotype by reducing IKAP expression with retroviral constructs. We observed that IKAP-downregulated cells formed cell clusters compared to control cells under regular culture conditions. We examined the ability of these cells to differentiate into mature neurons in the presence of laminin, an essential extracellular matrix for developing PNS neurons. We found that the cells showed reduced attachment to laminin, morphological changes and increased cell-to-cell adhesion resulting in cell aggregates. We identified Contactin as the adhesion molecule responsible for this phenotype. We show that Contactin expression is related to IKAP expression, suggesting that IKAP regulates Contactin levels for appropriate cell-cell adhesion that could modulate neuronal growth of PNS neurons during development.Key words: Familial Dysautonomia, IKAP/hELP1, neuronal differentiation, laminin, contactin, peripheral nervous system     

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.     

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.     

IKAP/hELP1 down-regulation in neuroblastoma cells causes enhanced cell adhesion mediated by contactin overexpression

A splicing mutation in the IKBKAP gene encoding the IKAP/hELP1 (IKAP) protein was found to be the major cause of Familial Dysautonomia (FD). This mutation affects both the normal development and survival of sensory and sympathetic neurons of the peripheral nervous system (PNS). To understand the FD phenotype it is important to study the specific role played by IKAP in developing and mature PNS neurons. We used the neuroblastoma SHSY5Y cell line, originated from neural crest adrenal tumor, and simulated the FD phenotype by reducing IKAP expression with retroviral constructs. We observed that IKAP – down - regulated cells formed cell clusters compared to control cells under regular culture conditions. We examined the ability of these cells to differentiate into mature neurons in the presence of laminin, an essential extracellular matrix for developing PNS neurons. We found that the cells showed reduced attachment to laminin, morphological changes and increased cell-to-cell adhesion resulting in cell aggregates. We identified Contactin as the adhesion molecule responsible for this phenotype. We show that Contactin expression is related to IKAP expression, suggesting that IKAP regulates Contactin levels for appropriate cell-cell adhesion that could modulate neuronal growth of PNS neurons during development.     

Synemin Isoforms in Astroglial and Neuronal Cells from Human Central Nervous System

The intermediate filament (IF) synemin gene encodes three IF proteins (H 180, M 150, L 41 kDa) with overlapping distributions. Synemin M was present early with vimentin and nestin. Synemin H was found later in the nervous system and mesodermic derivatives concomitantly with angiogenesis and the migration of neural crest cells. Synemin L appeared later in neurons. A series of in vitro cell cultures were done to identify the linkage between synemin isoforms and specific cell types of the central nervous system (CNS). The neurons and glia from the brains of humans and rats were cultured and double immunostaining done with antibodies against the H/M or L synemin isoforms and neural cell types (βIII-tubulin or NeuN) or astrocyte intermediate filaments (GFAP or vimentin). In neurons of the CNS, synemin H/M were co-expressed with GFAP, vimentin or nestin in glial cells, whereas synemin L was found in neurons.     

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.     

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.     

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.     

NELL2 promotes motor and sensory neuron differentiation and stimulates mitogenesis in DRG in vivo

We previously identified a secreted glycoprotein, neural epidermal growth factor-like like 2 (NELL2), in a subtraction screen designed to identify molecules regulating sensory neurogenesis and differentiation in the chick dorsal root ganglion (DRG). Characterization of NELL2 expression during embryogenesis revealed that NELL2 was specifically expressed during the peak periods of both sensory and motor neuron differentiation, and within the neural crest was restricted to the sensory lineage. We now provide evidence for a function for NELL2 during neuronal development. We report here that NELL2 acts cell autonomously within CNS and PNS progenitors, in vivo, to promote their differentiation into neurons. Additionally, neuron-secreted NELL2 acts paracrinely to stimulate the mitogenesis of adjacent cells within the nascent DRG. These studies implicate dual functions for NELL2 in both the cell autonomous differentiation of neural progenitor cells while simultaneously exerting paracrine proliferative activity.     

The cfy mutation disrupts cell divisions in a stage-dependent manner in zebrafish embryos

The zebrafish curly fry (cfy) mutation leads to embryonic lethality and abnormal cell divisions starting at 12-14 h postfertilization (hpf) during neural tube formation. The mitotic defect is seen in a variety of tissues including the central nervous system (CNS). In homozygous mutant embryos, mitoses are disorganized with an increase in mitotic figures throughout the developing neural tube. One consequence of aberrant mitoses in cfy embryos is an increase in cell death. Despite this, patterning of the early CNS is relatively unperturbed with distribution of the early, primary neurons indistinguishable from that of wild-type embryos. At later stages, however, the number of neurons was dramatically decreased throughout the CNS. The effect on neurons in older cfy embryos but not young ones correlates with the time of birth of neurons: primary neurons are born before the action of the cfy gene and later neurons after. Presumably, death of neuronal progenitors that divide beginning at the neural keel stage or death of their neuronal progeny accounts for the diminution of neurons in older mutant embryos. In addition, oligodendrocytes, which also develop late in the CNS, are greatly reduced in number in cfy embryos due to an apparent decrease in oligodendrocyte precursors. Genetic mosaic analysis demonstrates that the mutant phenotype is cell-autonomous. Furthermore, there are no obvious defects in apical/basal polarity within the neuroepithelium, suggesting that the cfy gene is not critical for epithelial polarity and that polarity defects are unlikely to account for the increased mitotic figures in mutants. These results suggest that the cfy gene regulates mitosis perhaps in a stage-dependent manner in vertebrate embryos.     

Restricted neural epidermal growth factor-like like 2 (NELL2) expression during muscle and neuronal differentiation

We have identified a secreted glycoprotein, neural epidermal growth factor-like like 2 (NELL2), in a screen designed to isolate molecules regulating sensory neuron genesis and differentiation in the dorsal root ganglia (DRG). In investigating NELL2 expression during embryogenesis, we demonstrate here that NELL2 is highly regulated spatially and temporally, being only transiently expressed in discrete regions of the central (CNS) and peripheral nervous systems (PNS) and in a subset of mesoderm derived structures during their peak periods of development. In the CNS and PNS, NELL2 is maximally expressed as motor and sensory neurons differentiate. Interestingly, its expression is restricted to sublineages of the neural crest, being strongly expressed throughout the immature DRG, but excluded from sympathetic ganglia. Similarly during muscle development, NELL2 is specifically expressed by hypaxial muscle precursor cells in the differentiating somite and derivatives in the forelimbs and body wall, but not by epaxial muscle precursors. Furthermore, NELL2 is differentially regulated in the CNS and PNS; in the CNS, NELL2 is only expressed by nascent, post-mitotic neurons as they commence their differentiation, yet in the PNS, NELL2 is expressed by subsets of progenitor cells in addition to nascent neurons. Based on this restricted spatial and temporal expression pattern, functional studies are in progress to determine NELL2's role during neuronal differentiation in both the PNS and CNS.