Neurite extension across regions of low cell-substratum adhesivity: implications for the guidepost hypothesis of axonal pathfinding

According to the adhesive "guidepost" hypothesis, pioneer axons follow pathways marked by specific nonadjacent cells (guidepost cells). The hypothesis implies that high adhesivity between extending axons and guidepost cells facilitates axon extension across low-adhesivity tissues or spaces between guidepost cells. This study investigates the ability of a high-adhesivity substratum to promote axonal extension across a low-adhesivity substratum in vitro. Dissociated chick embryo dorsal root ganglion neurons are cultured on a substratum consisting of areas of high-adhesivity substratum-bound laminin (i.e., model adhesive guideposts) separated by a low-adhesivity agarose substratum. Increasing the cell-substratum adhesivity of these guideposts results in an increase in the percentage of neurites spanning a given width of the low-adhesivity substratum. Filopodial processes at the tips of neurites can extend over the low-adhesivity substratum. Apparently, filopodial contact with high-adhesivity guideposts enables neurites to extend across intervening low-adhesivity substrata. The maximum width of low-adhesivity substratum discontinuities spanned by some neurites in vitro is comparable to the distance between some putative guidepost cells in insects. Consistent with the adhesive guidepost hypothesis, these findings demonstrate neurite extension on a substratum of discontinuous cell-substratum adhesivity.     

Effects of soluble laminin on organelle transport and neurite growth in cultured mouse dorsal root ganglion neurons: difference between primary neurites and branches

Laminin, an extracellular matrix molecule, is known to promote neurite growth. In the present study, the effects of soluble laminin on organelle transport and their relation to neurite growth were investigated in cultured dissociated mouse dorsal root ganglion (DRG) neurons. Laminin added into the extracellular medium was deposited on the surface of DRG neurons. DRG neurons incubated with soluble laminin exhibited branched, long, and thin neurites. Time-lapse study demonstrated that many small-diameter branches were newly formed after the addition of laminin. Thus, the growths of large-diameter primary neuritis, arising from cell bodies and branches extended from growth cones of primary neuritis, were analyzed separately. Laminin decreased the growth rate of primary neurites but increased that of branches. In primary neurites, acute addition of laminin rapidly decreased organelle movement in the neurite shaft and growth cone, accompanied by slowing of the growth cone advance. Branching of primary neurites occurred in response to laminin in some growth cones. In these growth cones, organelles protruded into nascent branches. In branches, soluble laminin increased organelle movement in the growth cone and the distal portion of the shaft. These results suggest that laminin inhibits the elongation of primary neurites but promotes branching and elongation of branches, all of which seem to be closely related to organelle transport.     

Segmental independence and age dependence of neurite outgrowth from embryonic chick sensory neurons

Targets in limb regions of the chick embryo are further removed from the dorsal root ganglia that innervate them compared with thoracic ganglion-to-target distances. It has been inferred that axons grow into the limb regions two to three times faster than into nonlimb regions. We tested whether the differences were due to intrinsic properties of the neurons located at different segmental levels. Dorsal root ganglia (DRG) were isolated from the forelimb, trunk, and hind limb regions of stage 25–30 embryos. Neurite outgrowth was measured in dissociated cell culture and in cultures of DRG explants. Although there was considerable variability in the amount of neurite outgrowth, there were no substantive differences in the amount or the rate of outgrowth comparing brachial, thoracic, or lumbosacral neurons. The amount of neurite outgrowth in dissociated cell cultures increased with the stage of development. Overall, our data suggest that DRG neurons express a basal amount of outgrowth, which is initially independent of target-derived neurotrophic influences; the magnitude of this intrinsic growth potential increases with stage of development; and the neurons of the DRG are not intrinsically specified to grow neurites at rates that are matched to the distance they are required to grow to make contact with their peripheral targets in vivo. We present a speculative model based on Poisson statistics, which attempts to account for the variability in the amount of neurite outgrowth from dissociated neurons. © 1995 John Wiley & Sons, Inc.     

Neurite outgrowth on immobilized axonin-1 is mediated by a heterophilic interaction with L1(G4)

Axonin-1 is an axon-associated cell adhesion molecule with dualistic expression, one form being glycophosphatidylinositol-anchored to the axonal membrane, the other secreted from axons in a soluble form. When presented as a substratum for neuronal cultures it strongly promotes neurite outgrowth from chicken embryonic dorsal root ganglia neurons. In this study, the axon-associated cell adhesion molecule G4, which is identical with Ng-CAM and 8D9, and homologous or closely related to L1 of the mouse and NILE of the rat, was investigated with respect to a receptor function for axonin-1. Using fluorescent microspheres with covalently coupled axonin-1 or L1(G4) at their surface we showed that these proteins bind to each other. Within the sensitivity of this microsphere assay, no interaction of axonin-1 with itself could be detected. Axonin-1-coated microspheres also bound to the neurites of cultured dorsal root ganglia neurons. This interaction was exclusively mediated by L1(G4), as indicated by complete binding suppression by monovalent anti-L1(G4) antibodies. The interaction between neuritic L1(G4) and immobilized axonin-1 was found to mediate the promotion of neurite growth on axonin-1, as evidenced by the virtually complete arrest of neurite outgrowth in the presence of anti-L1(G4) antibodies. Convincing evidence has recently been presented that neurite growth on L1(8D9) is mediated by the homophilic binding of neuritic L1(G4) (1989. Neuron. 2: 1597-1603). Thus, both L1(G4)- and axonin-1-expressing axons may serve as "substrate pathways" for the guidance of following axons expressing L1(G4) into their target area. Conceivably, differences in the concentration of axonin-1 and L1(G4), and/or modulatory influences on their specific binding parameters in leading pathways and following axons could represent elements in the control of axonal pathway selection.     

Neurite outgrowth on a step gradient of chondroitin sulfate proteoglycan (CS-PG).

Sulfated proteoglycans (PGs) may play a significant role in the regulation of neurite outgrowth. They are present in axon-free regions of the developing nervous system and repel elongating neurites in a concentration-dependent manner in vitro. The addition of growth-promoting molecules, such as laminin, can modify the inhibitory effect of PGs on neurite outgrowth (Snow, Steindler, and Silver, 1990b). Substrata containing a high-PG/low-laminin ratio completely inhibit neurite outgrowth, while normal, unimpeded outgrowth is observed on low-PG/high-laminin substrata. Therefore, different patterns of neurite outgrowth may result from regulation of the ratio of growth-promoting molecules to growth-inhibiting molecules. Using video microscopy, embryonic chicken dorsal root ganglia neurons (DRG), chicken retinal ganglia neurons (RGC), and rat forebrain neurons (FB) were analyzed as they extended processes from a substratum consisting of laminin alone onto a step gradient of increasing concentrations of chondroitin sulfate proteoglycan (CS-PG) bound to laminin. In contrast to neurite outgrowth inhibition that occurs at the border of a single stripe of high concentration of CS-PG (Snow et al., 1990b and this study), growth cones grew onto and up CS-PG presented in a step-wise graded distribution. Although the behavior of the different cell types was unique, a common behavior of each cell type was a decrease in the rate of neurite outgrowth with increasing CS-PG concentration. These data suggest that appropriate concentrations of growth-promoting molecules combined with growth-inhibiting molecules may regulate the direction and possibly the timing of neurite outgrowth in vivo. The different responses of different neuronal types suggest that the presence of sulfated PG may have varying effects on different aspects of neuronal development.     

The role of endogenous heparan sulfate proteoglycan in adhesion and neurite outgrowth from dorsal root ganglia

Some phases of dorsal root ganglion (DRG) substratum attachment and growth cone morphology are mediated through endogenous cell surface heparan sulfate proteoglycan. The adhesive behavior of intact embryonic chicken DRG (spinal sensory ganglia) is examined on substrata coated with fibronectin, fibronectin treated with antibody to the cell-binding site (anti-CBS), and the heparan sulfate-binding protein platelet factor four. DRG attach to fibronectin, anti-CBS-treated fibronectin, and platelet factor four. The ganglia extend an extensive halo of unfasciculated neurites on fibronectin and produce fasciculated neurite outgrowth on platelet factor four and anti-CBS antibody-treated FN. Treatment with heparinase, but not chondroitinase, abolishes adhesion to fibronectin and platelet factor four. Growth cones of DRG on fibronectin have well-spread lamellae and microspikes. On platelet factor four, and anti-CBS-treated FN, growth cones exhibit microspikes only. Isolated Schwann cells adhere equally well to fibronectin and platelet factor four, spreading more rapidly on fibronectin. Isolated DRG neurons adhere equally well on both substrata, but only 10% of the neurons extend long neurites on platelet factor four. The majority of the isolated neurons on platelet factor four exhibit persistent microspike production resembling that of the early stages of normal neurite extension. Endogenous heparan sulfate proteoglycan supports the adhesion of whole DRG, isolated DRG neurons, and Schwann cells, as well as extensive microspike activity by DRG neurons, one important part of growth cone activity.     

Binding between the neural cell adhesion molecules axonin-1 and Nr- CAM/Bravo is involved in neuron-glia interaction

Neural cell adhesion molecules of the immunoglobulin superfamily mediate cellular interactions via homophilic binding to identical molecules and heterophilic binding to other family members or structurally unrelated cell-surface glycoproteins. Here we report on an interaction between axonin-1 and Nr-CAM/Bravo. In search for novel ligands of axonin-1, fluorescent polystyrene microspheres conjugated with axonin-1 were found to bind to peripheral glial cells from dorsal root ganglia. By antibody blockage experiments an axonin-1 receptor on the glial cells was identified as Nr-CAM. The specificity of the interaction was confirmed with binding studies using purified axonin-1 and Nr-CAM. In cultures of dissociated dorsal root ganglia antibodies against axonin-1 and Nr-CAM perturbed the formation of contacts between neurites and peripheral glial cells. Together, these results implicate a binding between axonin-1 of the neuritic and Nr-CAM of the glial cell membrane in the early phase of axon ensheathment in the peripheral nervous system.     

Studies of adhesion molecules mediating interactions between cells of peripheral nervous system indicate a major role for L1 in mediating sensory neuron growth on Schwann cells in culture

The involvement of the adhesion molecules L1, N-CAM, and J1 in adhesion and neurite outgrowth in the peripheral nervous system was investigated. We prepared Schwann cells and fibroblasts (from sciatic nerves) and neurons (from dorsal root ganglia) from 1-d mice. These cells were allowed to interact with each other in a short-term adhesion assay. We also measured outgrowth of dorsal root ganglion neurons on Schwann cell and fibroblast monolayers. Schwann cells (which express L1, N-CAM, and J1) adhered most strongly to dorsal root ganglion neurons by an L1-dependent mechanism and less by N-CAM and J1. Schwann cell-Schwann cell adhesion was mediated by L1 and N-CAM, but not J1. Adhesion of fibroblasts (which express N-CAM, but not L1 or J1) to neurons or Schwann cells was mediated by L1 and N-CAM and not J1. However, inhibition by L1 and N-CAM antibodies was found to be less pronounced with fibroblasts than with Schwann cells. N-CAM was also strongly involved in fibroblast-fibroblast adhesion. Neurite outgrowth was most extensive on Schwann cells and less on fibroblasts. A difference in extent of neurite elongation was seen between small- (10-20 microns) and large- (20-35 microns) diameter neurons, with the larger neurons tending to exhibit longer neurites. Fab fragments of polyclonal L1, N-CAM, and J1 antibodies exerted slightly different inhibitory effects on neurite outgrowth, depending on whether the neurites were derived from small or large neurons. L1 antibodies interfered most strikingly with neurite outgrowth on Schwann cells (inhibition of 88% for small and 76% for large neurons), while no inhibition was detectable on fibroblasts. Similarly, although to a smaller extent than L1, N-CAM appeared to be involved in neurite outgrowth on Schwann cells and not on fibroblasts. Antibodies to J1 only showed a very small effect on neurite outgrowth of large neurons on Schwann cells. These observations show for the first time that identified adhesion molecules are potent mediators of glia-dependent neurite formation and attribute to L1 a predominant role in neurite outgrowth on Schwann cells which may be instrumental in regeneration.     

Distribution of laminin in the developing peripheral nervous system of the chick

During axonal elongation in the developing peripheral nervous system, the temporal and spatial distribution of adhesive molecules in extracellular matrices and on neighboring cell surfaces may provide "choices" of pathways for growth cone migration. The extracellular matrix glycoprotein laminin appears in early embryos and mediates neuronal adhesion and neurite extension in vitro. In this study, we have examined the distribution of laminin at early periods of peripheral nervous system development. The distribution of laminin, demonstrated by immunostaining frozen sections of chick embryos, was compared to the distribution of fibronectin and of early peripheral neurites as revealed with an antibody to a neurofilament-associated protein. Laminin is present in the neural tube basement membrane, in early ganglia, and in developing dorsal and ventral roots, where the laminin staining pattern parallels that of neurofilaments. In early ganglia and nerve roots, laminin immunostaining defines loose "meshworks" rather than basement membranes, which seem to form slightly later in these structures. In contrast, fibronectin is absent in neural tube basement membrane, ganglia, and nerve roots, although it is present along neural crest migratory pathways and in intersomitic spaces. Our observations of laminin distribution are consistent with the possibility that laminin provides an adhesive surface for neurite extension at some stages of early peripheral nervous system development.     

Axonal growth in response to experimentally applied mechanical tension

A machine was constructed, called a Cell Puller, that allows the steady advance or withdrawal of a microelectrode at very slow speeds—up to 170 μm/hr. Specially prepared microelectrodes held in the Cell Puller were placed in cultures of dissociated chick sensory ganglion neurons in such a way that growth cones attached to their tips. Movements of the microelectrodes, at speeds up to about 100 μm/hr, then resulted in the elongation of the neurites for up to 24 hr and for increases in length up to 960 μm; more rapid towing failed to cause extensive neurite elongation. Estimates of neurite diameter before and after “towing” indicated that a net increase in neurite volume had occurred. Furthermore, long neurites could be produced by towing from previously rounded neuronal cell bodies confined to small adhesive “islands” on a nonadhesive substratum. Neurites produced by microelectrode towing had a normal appearance, showed rapid saltatory movements of internal organelles and were capable of resuming growth on the substratum. Electron microscopy of bundles of neurites produced in this way from explanted dorsal root ganglia showed an ultrastructure typical of cultured neurites, with abundant longitudinally aligned microtubules and neurofilaments. These experiments demonstrate that neurites can grow in response to mechanical tension under tissue culture conditions. It is proposed that they do so also in normal development, the tension in this case being supplied initially by the locomotory activity of the growth cones and subsequently by the morphogenetic movements of the surrounding tissues.     

Choroid plexus ependymal cells enhance neurite outgrowth from dorsal root ganglion neurons in vitro

The epithelial cells of the choroid plexus are a continuation of the ventricular ependymal cells and are regarded as modified ependymal cells. The present study was carried out to determine the influence of choroid plexus ependymal cells (CPECs) on axonal growth in vitro. Choroid plexuses were dissected from the fourth ventricle of postnatal day-1–10 mice, mechanically dissociated, and plated in fibronectin-coated culture dishes. CPECs had spread into monolayers with few endothelial cells in 3-week cultures. Some macrophages were scattered on the monolayer of CPECs. Dorsal root ganglia (DRG) were excised from mouse fetuses of 14-day gestation, dissociated with trypsin and cocultured on the CPEC monolayers. For comparison, dissociated DRG neurons were cocultured on astrocyte monolayers or cultured on laminin-coated plates. After 4.5 h culturing, the cultures were fixed and immunohistochemically double-stained for neurites and CPECs using antibodies against β-tubulin III and S-100 β, respectively. It was demonstrated that neurons extended many long neurites with elaborate branching on the surface of S-100-stained CPECs. In contrast, DRG neurons cultured on the astrocytes and on the laminin-coated plates had much shorter primary neurites with fewer branches than those cultured on the CPECs. The total length of neurites including primary neurites and their branches, of a single DRG neuron was 285 ± 14, 395 ± 15 and 565 ± 12 μm on the laminin-coated plates, on astrocytes and on CPECs, respectively. Scanning electron microscopy revealed extension of neurites with well-developed growth cones on the ependymal cells. These results suggest that CPECs have a great capacity to promote neurite outgrowth from DRG neurons in vitro.     

Adult mouse dorsal root ganglia neurons in cell culture.

A method has been developed for the long-term culture of dissociated adult mouse dorsal root ganglia (DRG). Of critical importance to the success of this technique was a three-hour incubation in collagenase which softened the DRG and permitted gentle dissociation. The morphological and electrophysiological features of the dissociated adult DRG were similar to those observed in previous studies of immature (i.e., embryonic and newborn) DRG in culture and also to those of adult DRG in situ. With regard to electrophysiological work, the adult DRG neurons are superior to embryonic and newborn neurons because of their larger size and greatly increased survival in culture (no degeneration for first six days, and thereafter a relatively slow decrease). The adult neurons regenerated nerve fibers to an extent comparable to that of immature neurons. Therefore, the adult DRG cultures might be useful to study factors influencing regeneration in the adult mammalian nervous system. The adult cultures might also be useful to investigated factors influencing the aging process.     

Interactions of Schwann cells with neurites and with other Schwann cells involve the calcium-dependent adhesion molecule, N-cadherin.

During embryogenesis, Schwann cells interact with axons and other Schwann cells, as they migrate, ensheath axons, and participate in organizing peripheral nervous tissues. The experiments reported here indicate that the calcium-dependent molecule, N-cadherin, mediates adhesion of Schwann cells to neurites and to other Schwann cells. Cell cultures from chick dorsal root ganglia and sciatic nerves were maintained in media containing either 2 mM Ca++ or 0.2 mM Ca++, a concentration that inactivates calcium-dependent cadherins. When the leading lamellae of Schwann cells encountered migrating growth cones in medium with 2 mM Ca++, they usually remained extended, and the growth cones often advanced onto the Schwann cell upper surface. In the low Ca++ medium, the frequency of withdrawal of the Schwann cell lamella after contact with a growth cone was much greater, and withdrawal was the most common reaction to growth cone contact in medium with 2 mM Ca++ and anti-N-cadherin. Similarly, when motile leading margins of two Schwann cells touched in normal Ca++ medium, they often formed stable areas of contact. N-cadherin and vinculin were co-concentrated at these contact sites between Schwann cells. However, in low Ca++ medium or in the presence of anti-N-cadherin, interacting Schwann cells usually pulled away from each other in a behavior reminiscent of contact inhibition between fibroblasts. In cultures of dissociated cells in normal media, Schwann cells frequently were aligned along neurites, and ultrastructural examination showed extensive close apposition between plasma membranes of neurites and Schwann cells. When dorsal root ganglia explants were cultured with normal Ca++, Schwann cells migrated away from the explants in close association with extending neurites. All these interactions were disrupted in media with 0.2 mM Ca++. Alignment of Schwann cells along neurites was infrequent, as were extended close apposition between axonal and Schwann cell plasma membranes. Finally, migration of Schwann cells from ganglionic explants was reduced by disruption of adhesive contact with neurites. The addition of antibodies against N-cadherin to medium with normal Ca++ levels had similar effects as lowering the Ca++ concentration, but antibodies against the neuronal adhesive molecule, L1, had no effects on interactions between Schwann cells and neurites.     

Adult mouse dorsal root ganglia neurons in cell culture

A method has been developed for the long-term culture of dissociated adult mouse dorsal root ganglia (DRG). Of critical importance to the success of this technique was a three-hour incubation in collagenase which softened the DRG and permitted gentle dissociation. The morphological and electrophysiological features of the dissociated adult DRG were similar to those observed in previous studies of immature (i.e., embryonic and newborn) DRG in culture and also to those of adult DRG in situ. With regard to electrophysiological work, the adult DRG neurons are superior to embryonic and newborn neurons because of their larger size and greatly increased survival in culture (no degeneration for first six days, and thereafter a relatively slow decrease). The adult neurons regenerated nerve fibers to an extent comparable to that of immature neurons. Therefore, the adult DRG cultures might be useful to study factors influencing regeneration in the adult mammalian nervous system. The adult cultures might also be useful to investigate factors influencing the aging process.     

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.     

Morphogenetic guidance cues can interact synergistically and hierarchically in steering nerve cell growth

Nerve cell growth is influenced by guiding properties of its substratum. Microfabricated cell culture substrata were used to determine whether rat dorsal root ganglia (DRG) nerve cells could detect and integrate simultaneous model adhesive and topographic guidance cues. Interference reflection microscopy demonstrated strips of surface contact under the marginal zone of growth cones on planar surfaces which were coincident with actin immunostaining at the periphery of the C-domain. Clusters of focal contacts below the growth cone C-domain delineated the track edges on adhesive gratings. Neurite extension was guided most effectively by adhesive gratings of 25-μm period where highly aligned cells were typically bipolar. Nanometric steps and differences in surface texture between the adhesive tracks was detected using atomic force microscopy (AFM). Neurites did not align to 12- to 100-μm pitch grooves which were less than 1 μm deep. The proportion of aligned neurites increased with groove depth. Maximum neurite alignment was seen when 6-μm-deep, 25-μm-wide grooves contained superimposed parallel adhesive tracks of matched pitch. Neurites aligned preferentially to adhesive tracks superimposed orthogonally over shallow grooves (1 μm deep). Primary neurites aligned increasingly to grooves with orthogonal adhesive tracks as their depth increased. These neurites frequently had highly branched terminal arbours aligned to the orthogonal adhesive tracks. We conclude that morphogenetic guidance cues can interact synergistically and hierarchically to steer nerve cell growth.     

Pure population of viable neurons from rabbit dorsal root ganglia, using gradients of Percoll

Nonneuronal cells complicate the study of neurons in vitro. A pure population of viable neurons can be obtained easily using gradients of Percoll. For each experiment, 20 dorsal root ganglia (DRG) are minced, then sequentially dissociated in collagenase and trypsin, which digest all the intercellular connections. The dissociated tissue is separated first on the basis of density, creating an interphase fraction enriched in neurons and satellite cells, which are then further separated on the basis of size. The neurons, obtained in the final pellet, number approximately 50,000 (2500 per DRG), routinely exhibit a viability of over 80% initially and are of a purity of over 90%. The viability of the neurons is confirmed by the occurrence of neurite outgrowth in culture. Thus, a pure and viable neuronal population is obtained by a simple and rapid method.     

The involvement of axonin-1/SC2 in mediating notochord-derived chemorepulsive activities for dorsal root ganglion neurites

Previous studies have suggested that the developing notochord secretes diffusible axon guidance molecules that repel dorsal root ganglion (DRG) neurites (R. Keynes et al., 1997, Neuron 18, 889-897; K. Nakamoto and T. Shiga, 1998, Dev. Biol. 202, 304-314). Neither notochord-derived chemorepellents nor their receptors on DRG neurites are, however, known. Here we investigated whether cell adhesion molecules (CAMs) of the immunoglobulin/fibronectin type III subfamily present on DRG neurites, including axonin-1/SC2, N-CAM, Ng-CAM, and Nr-CAM, are required for mediating the notochord-derived chemorepulsion. Using collagen gel cocultures of DRGs and notochord explants, we found that an antibody against axonin-1/SC2 diminished the effects of the chemorepulsive activity from the notochord, whereas antibodies against N-CAM, Ng-CAM, and Nr-CAM had no effect. We further showed that the removal of glycosylphosphatidylinositol-anchored cell surface molecules, including axonin-1/SC2, from DRG neurites diminished the effects of the notochord-derived chemorepulsive activity to an extent similar to that of treatment with the anti-axonin-1/SC2 antibody. These results suggest that axonin-1/SC2 expressed on DRG neurites may be involved in mediating the notochord-derived chemorepulsive activity.     

Promotion of neuronal cell adhesion by members of the IgLON family occurs in the absence of either support or modification of neurite outgrowth

The IgLONs are a family of glycosyl phosphatidyl inositol-linked cell adhesion molecules which are thought to modify neurite outgrowth and may play a role in cell-cell recognition. The family consists of LAMP, OBCAM, neurotrimin/CEPU-1 and neurotractin/kilon. In this paper we report the effect of recombinant LAMP, CEPU-1 and OBCAM, and transfected cell lines expressing these molecules, on the adhesion and outgrowth of dorsal root ganglion (DRG) and sympathetic neurones. CHO cells transfected with cDNA for CEPU-1 adhered to a recombinant CEPU-1-Fc substrate. However, DRG or sympathetic neurones only adhered to CEPU-1-Fc when presented on protein A. Although DRG and sympathetic neurones express IgLONs on their surface, both types of neurones exhibited differential adhesion to CEPU-1-Fc, LAMP-Fc and OBCAM-Fc. Neither DRG nor sympathetic neurones extended neurites on a protein A/IgLON-Fc substrate and overexpression of CEPU-1-GFP in DRG neurones also failed to stimulate neurite outgrowth on an IgLON-Fc substrate. DRG neurones adhered to and extended neurites equally on transfected and non-transfected cell lines and the recombinant proteins did not modulate the outgrowth of neurones on laminin. In contrast to previous reports we suggest that IgLONs may not have a primary role in axon guidance but may be more important for cell-cell adhesion and recognition.