Influence of ganglion age,nonneuronal cells and substratum on neurite outgrowth in culture

This study characterizes the outgrowth patterns of superior cervical ganglia (SCG) obtained from embryonic (E15), perinatal (E20–21), and adult (P35) rats when placed in culture on various substrata. Outgrowth morphology, degree of fasciculation, and outgrowth length were examined on collagen (COL), polyornithine (PO), polylysine (PL), fibronectin (FN), and nonneuronal cells (NNCs) from the ganglion. COL and FN supported extensive neuritic outgrowth; PO and PL provided poor support. Outgrowth pattern, degree of fasciculation, neurite growth rate, and the number of NNCs in the outgrowth varied considerably depending upon the COL configuration. When undiluted COL (～5 mg/ml) was air dried, a three-dimensional loose fibrillar network was formed. Upon COL dilution or gelling undiluted COL by ammoniation, an essentially two-dimensional layer was formed. On two-dimensional COL, NNCs were able to proliferate and migrate extensively from ganglia of all ages; their presence influenced the form and extent of neurite growth. E15, E20, and P35 neurites responded differently to their endogenous NNCs. E15 neurites extended in relation to NNC surfaces and were predominantly nonfasciculated. E20 neurites became more fasciculated in the presence of NNCs that exhibited morphological and behavioral differences from those migrating from E15 ganglia. E20 neurite bundles became defasciculated when they extended into E15 outgrowth. Far fewer neurites grew from P35 explants in the presence of their NNCs. Three-dimensional COL greatly slowed NNC migration and thus allowed investigation of neurite outgrowth from ganglia of differing age in the absence of NNCs. We conclude that neuritic outgrowth patterns on varying substrata reflect not only neurite differences depending upon ganglion age but also variation in the behavior of accompanying NNCs.     

Promotion of retinal neurite outgrowth by substratum-bound fibronectin

We have examined conditions under which aggregates of embryonic chick neural retina will extend neurities in vitro. Trypsin-dispersed cells from 7-day embryonic chick neural retina were aggregated in rotation culture for 8 hr and maintained in serum-free medium on a variety of standard culture substrate. Aggregates extend few neurites on untreated plastic, glass, or collagen substrata. However, pretreatment of these substrata with human plasma fibronectin enhances their capacity to support retinal neurite outgrowth. Aggregates cultured on fibronectin-treated substrata extend long, radially oriented neurites within 36 hr in vitro. The morphology of these neurites is distinct from that seen when aggregates are cultured on polylysine-treated substrata. In the latter case, neurites are highly branched and grow concentrically around the aggregate perimeter. Addition of fibronectin to polylysine-treated substrata stimulates radial neurite outgrowth. Promotion of neurite outgrowth is dependent on the amount of fibronectin bound to the culture substratum and on the pH at which binding occurs. The requirements for fibronectin-mediated neurite outgrowth are more stringent than those previously reported for fibroblast attachment and spreading.     

Effect of peripheral nerve on the neurite growth from retinal explants in culture

The effect of peripheral nerve (PN) on neurite outgrowth from retinal explants of adult hamsters was examined.Cultures of retinal explants,and co-cultures of retinal explants and PN were performed using chick retinal basement memebrane (BM) as substrate.The presence of PN increases the number and length of neurite outgrowth.In addition,a high proportion of neurites situated close to PN tend to grow towards it.Since there was no contact between retinal explants and PN,we suggest that PN might secete diffusible substances to attract the neurites to grow towards it.     

Embryonic neural retinal cell response to extracellular matrix proteins: developmental changes and effects of the cell substratum attachment antibody (CSAT)

Cell attachment and neurite outgrowth by embryonic neural retinal cells were measured in separate quantitative assays to define differences in substrate preference and to demonstrate developmentally regulated changes in cellular response to different extracellular matrix glycoproteins. Cells attached to laminin, fibronectin, and collagen IV in a concentration-dependent fashion, though fibronectin was less effective for attachment than the other two substrates. Neurite outgrowth was much more extensive on laminin than on fibronectin or collagen IV. These results suggest that different substrates have distinct effects on neuronal differentiation. Neural retinal cell attachment and neurite outgrowth were inhibited on all three substrates by two antibodies, cell substratum attachment antibody (CSAT) and JG22, which recognize a cell surface glycoprotein complex required for cell interactions with several extracellular matrix constituents. In addition, retinal cells grew neurites on substrates coated with the CSAT antibodies. These results suggest that cell surface molecules recognized by this antibody are directly involved in cell attachment and neurite extension. Neural retinal cells from embryos of different ages varied in their capacity to interact with extracellular matrix substrates. Cells of all ages, embryonic day 6 (E6) to E12, attached to collagen IV and CSAT antibody substrates. In contrast, cell attachment to laminin and fibronectin diminished with increasing embryonic age. Age-dependent differences were found in the profile of proteins precipitated by the CSAT antibody, raising the possibility that modifications of these proteins are responsible for the dramatic changes in substrate preference of retinal cells between E6 and E12.     

Growth cone interactions with a glial cell line from embryonic Xenopus retina

We have isolated a nonneuronal cell line from Xenopus retinal neuroepithelium (XR1 cell line). On the basis of immunocytochemical characterization using monoclonal antibodies generated in our laboratory as well as several other glial-specific antibodies, we have established that the XR1 cells are derived from embryonic astroglia. A monolayer of XR1 cells serves as an excellent substrate upon which embryonic retinal explants attach and elaborate neurites. This neurite outgrowth promoting activity appears not to be secreted into the medium, as medium conditioned by XR1 cells is ineffective in promoting outgrowth. Cell-free substrates were prepared to examine whether outgrowth promoting activity is also associated with the XR1 extracellular matrix (ECM). Substrates derived from XR1 cells grown on collagen are still capable of promoting outgrowth following osmotic shock and chemical extraction. This activity does not appear to be associated with laminin or fibronectin. Scanning electron microscopy was used to examine growth cones of retinal axons on XR1 cells and other substrates that supported neurite outgrowth. Growth cones and neurites growing on a monolayer of XR1 cells, or on collagen conditioned by XR1 cells, closely resemble the growth cones of retinal ganglion cells in vivo. A polyclonal antiserum (NOB1) generated against XR1 cells effectively and specifically inhibits neurite outgrowth on XR1-conditioned collagen. We therefore propose that neurite outgrowth promoting factors produced by these cells are associated with the extracellular matrix and may be glial specific.     

A glial cell line promotes the outgrowth of neurites from embryonic Xenopus retina

A glial cell line (XR1 cell line) derived from Xenopus retinal neuroepithelium was examined for neurite outgrowth promoting activity. A monolayer of the XR1 cells serves as an excellent substrate upon which embryonic retinal explants attach and freely elaborate neurites. The XR1 neurite outgrowth promoting activity is not secreted into the medium, but is laid down directly on the substrate where it remains active after lysing the cells by hypoosmotic shock. A polyclonal antiserum raised against membranes of the XR1 cells was effective in blocking neurite outgrowth on XR1 conditioned collagen. It is proposed that the neurite outgrowth promoting factors produced by the XR1 cells are associated with the extracellular matrix and possibly glial specific.     

Laminin and fibronectin modulate inner ear spiral ganglion neurite outgrowth in an in vitro alternate choice assay

Extracellular matrix (ECM) molecules have been shown to function as cues for neurite guidance in various populations of neurons. Here we show that laminin (LN) and fibronectin (FN) presented in stripe micro-patterns can provide guidance cues to neonatal (P5) inner ear spiral ganglion (SG) neurites. The response to both ECM molecules was dose-dependent. In a LN versus poly-L-lysine (PLL) assay, neurites were more often observed on PLL at low coating concentrations (5 and 10 microg/mL), while they were more often on LN at a high concentration (80 microg/mL). In a FN versus PLL assay, neurites were more often on PLL than on FN stripes at high coating concentrations (40 and 80 microg/mL). In a direct competition between LN and FN, neurites were observed on LN significantly more often than on FN at both 10 and 40 microg/mL. The data suggest a preference by SG neurites for LN at high concentrations, as well as avoidance of both LN at low and FN at high concentrations. The results also support a potential model for neurite guidance in the developing inner ear in vivo. LN, in the SG and osseus spiral lamina may promote SG dendrite growth toward the organ of Corti. Within the organ of Corti, lower concentrations of LN may slow neurite growth, with FN beneath each row of hair cells providing a stop or avoidance signal. This could allow growth cone filopodia increased time to sample their cellular targets, or direct the fibers upward toward the hair cells.     

Denervation and age modify neuromuscular positional selectivity

The rostrocaudal position of neurons within the spinal motor pool maps systematically onto the surface of several muscles in mammals. In an effort to understand the mechanisms that generate such maps, we have been studying choices made by embryonic spinal cord neurons on muscle membrane substrates in the in vitro stripe assay. In this report we explore the effects of postnatal age of the muscle on neurite choice, and how prior denervation modifies this choice. Our results further differentiate rostral from caudal motor neurons in preferring one substrate to another. First, caudal neurites prefer to grow on P6 neonatal caudal over rostral membranes, but lose this ability to distinguish axial position of origin in older muscles. Rostral neurites prefer growth on rostral membranes, but this preference also diminishes with age. Second, when adult muscles have been denervated, both rostral and caudal neurites regain their positional growth selectivity. Third, caudal neurites are particularly sensitive to substrate choice. When growing on a preferred substrate (gluteus) caudal neurites prefer neonatal over adult membranes. These results support the concept of fundamental differences in the growth preferences of rostral and caudal spinal neurites. These differences will assist in the identification of molecular guidance cues that determine the formation of neuromuscular positional maps.     

The role of cell adhesion molecules in neurite outgrowth on Müller cells

The roles of neural cell adhesion molecule (NCAM), L1, N-cadherin, and integrin in neurite outgrowth on various substrates were studied. Antibodies against these cell surface molecules were added to explants of chick retina and the neurites from retinal ganglion cells were examined for effects of the antibodies on neurite length and fasciculation. On laminin, an anti-integrin antibody completely inhibited neurite outgrowth. The same antibody did not inhibit neurite outgrowth on polylysine or Müller cells. Antibodies to NCAM, L1, and N-cadherin did not significantly inhibit neurite outgrowth on laminin but produced significant inhibition on Müller cells. The inhibition of neurite outgrowth on glia by anti-L1 antibodies supports the hypothesis that L1 is capable of acting in a heterophilic binding mechanism. On laminin, both anti-N-cadherin and anti-L1 caused defasciculation of neurites from retinal ganglion cells, while anti-NCAM did not. None of these antibodies produced defasciculation on Müller cells. The results indicate that these three cell adhesion molecules may be very important in interactions with glia as axons grow from the retina to the tectum and may be less important in axon-axon interactions along this pathway. No evidence was found supporting the role of integrins in axon growth on Müller cells.     

Steps in the formation of a bipolar outgrowth pattern by cultured neurons, and their substrate dependence.

We studied the steps in the formation of the bipolar outgrowth pattern of cultured adult Anterior Pagoda (AP) neurons of the leech growing on a central nervous system (CNS) homogenate as substrate. This pattern, which consists of two primary neurites directed in opposite directions plus some bifurcations, resembles their embryonic pattern but is different from the patterns they develop in culture on leech laminin or Concanavalin A as substrates. In eight neurons that were studied, one primary neurite formed and branched several hours before the second one. Time-lapse video analysis showed that between 12 and 36 h of growth, the more proximal branch of the early neurite migrated retrogradely, rotated, and formed the second primary branch. Both neurites elongated until the total neurite length reached 130-160 microm, when the elongation of primary neurites became synchronous with the retraction of secondary processes, suggesting competition. The substrate dependence of these events was tested by plating AP neurons on leech laminin. On this substrate AP neurons produced multiple independent primary neurites with branches. Retraction of some large branches was followed by their regrowth, and did not correlate with the changes in other neurites. We propose that the dynamics in the formation of the bipolar outgrowth pattern of AP neurons arise from inhibitory extracellular matrix molecules, which reduce the synthesis of precursors for neurite formation.     

Tenascin is accumulated along developing peripheral nerves and allows neurite outgrowth in vitro.

The extracellular matrix protein, tenascin, appears in a restricted pattern during organ morphogenesis. Here we studied the expression of tenascin along developing peripheral nerves in chick embryos and tested its activity as a substrate for cultured neurons. Motor axons grow out through the tenascin-rich, anterior part of the sclerotome. Shortly after, tenascin surrounds axon fascicles of ventral roots. At the limb levels, outgrowing axons accumulate in the tenascin-containing girdle region forming a plexus. In the limb, tenascin first appears in bracket-like structures that surround the precartilage cell condensations of the femur and humerus, respectively. These regions coincide with the channels along which axons first grow in from the girdle plexus to form the limb nerves. Later, the major tenascin staining is associated with the cartilage and tendon primordia, and not with the limb nerves. We used tenascin as a substrate for cultured neural explants and single cells in order to test for its function in neurite outgrowth. Dissociated embryonic neurons of various types attached to mixed polylysine/tenascin substrates and sprouted rapidly after a lag of several hours. Outgrowth was inhibited and neurites were detached by anti-tenascin antibodies. On substrates coated with tenascin alone, neurite outgrowth was achieved from 3 day spinal cord explants. Whereas growth cones were well spread and rapidly moving, the neurites were poorly attached, straight and rarely branched. We speculate that in vivo tenascin allows axonal outgrowth, but inhibits branching and supports fasciculation of newly formed axons.     

In vitro growth properties of Xenopus retinal neurons undergo developmental modulation

To determine whether Xenopus retinal neurons undergo intrinsic developmental changes in growth properties, retinal explants from embryos and tadpoles of different stages were grown on laminin, fibronectin, and collagen I in serum-free media. Growth was assayed in terms of a neurite growth index (NGI) and the appearance of clockwise bundles, or a clockwise growth index (CGI). The first neurites from stage 25 optic vesicles are pioneers and display a unique growth phenotype; they emerge rapidly, survive for a short time, show little substrate preferences for growth (they grow almost as well on BSA as they do on laminin and fibronectin), and form no clockwise bundles under any conditions. Neurites from progressively older retinas (stages 32-37) share with stage 25 neurites the rapid outgrowth pattern, but begin to show substrate preferences and clockwise growth. From stage 40 to 50, the mature growth pattern is expressed; a lag in initial outgrowth, long-term survival, distinct substrate preferences (they grow 10 times better on laminin and fibronectin than on BSA) and display robust clockwise growth patterns on laminin and fibronectin. The acquisition of clockwise growth is independent of optic fiber contact with the tectum or exposure to diffusible factors from mature brain tissues. The results suggest that retinal neurons undergo developmental modulation of surface adhesive properties and/or cytoskeletal organization.     

Steps in the formation of a bipolar outgrowth pattern by cultured neurons,and their substrate dependence

We studied the steps in the formation of the bipolar outgrowth pattern of cultured adult Anterior Pagoda (AP) neurons of the leech growing on a central nervous system (CNS) homogenate as substrate. This pattern, which consists of two primary neurites directed in opposite directions plus some bifurcations, resembles their embryonic pattern but is different from the patterns they develop in culture on leech laminin or Concanavalin A as substrates. In eight neurons that were studied, one primary neurite formed and branched several hours before the second one. Time‐lapse video analysis showed that between 12 and 36 h of growth, the more proximal branch of the early neurite migrated retrogradely, rotated, and formed the second primary branch. Both neurites elongated until the total neurite length reached 130–160 μm, when the elongation of primary neurites became synchronous with the retraction of secondary processes, suggesting competition. The substrate dependence of these events was tested by plating AP neurons on leech laminin. On this substrate AP neurons produced multiple independent primary neurites with branches. Retraction of some large branches was followed by their regrowth, and did not correlate with the changes in other neurites. We propose that the dynamics in the formation of the bipolar outgrowth pattern of AP neurons arise from inhibitory extracellular matrix molecules, which reduce the synthesis of precursors for neurite formation. © 2002 Wiley Periodicals, Inc. J Neurobiol 50: 106–117, 2002; DOI 10.1002/neu.10017     

Purification and characterization of an 82-kD membrane protein as a neurite outgrowth factor binding protein: possible involvement of NOF binding protein in axonal outgrowth in developing retina

Neurite outgrowth factor (NOF) is a glycoprotein isolated from an extract of gizzard that induces neurite outgrowth from cultured retinal or ciliary ganglionic (CG) neurons. We have reported that a glycoprotein of approximately 82 kD solubilized from gizzard muscles binds to NOF (ligand blotting) and inhibits the neurite promoting activity of NOF (inhibition assay). The 82-kD protein (NOF binding protein) was purified from gizzard muscle membranes as a doublet band on SDS-PAGE and a polyclonal antibody was raised against it. An NOF binding protein in developing retina exhibited the same physicochemical properties as that of the gizzard muscle. Quantitative decrease in NOF binding protein in embryonic retinas was observed after day 11 by the inhibition assay, ligand blotting, and immunoblotting, its decrease being parallel with reduction of NOF-induced neurite outgrowth of embryonic retinas. In an immunohistochemical study, the antibody stained only the optic fiber layers of the retinas of 8-d embryos, and this staining was no longer detectable in retinas of 18-d embryos. These results suggest that the 82-kD protein is a novel membrane protein that behaves as an NOF receptor and that the loss of neuritic response of the retinal neurons to NOF reflects a decrease in NOF receptor molecules.     

Selective inhibition of ventral temporal but not dorsal nasal neurites from mouse retinal explants during contact with chondroitin sulphate

We have determined whether chondroitin sulphate (CS) glycosaminoglycans are sufficient to direct a selective inhibition of neurite growth from ventral temporal (VT) but not from dorsal nasal (DN) retina in mouse embryos; this may underlie the formation of axon divergence in the optic chiasm. Explants from the retinal region of embryonic day-14 mouse were grown on a laminin–polylysine substrate near to a circular spot coated with CS. In control cultures, in which no CS was added to the spot, both VT and DN retinal neurites grew extensively into the coated territory. When presented with spots coated with 10 mg/ml CS, neurite growth from the VT retina into the CS territory was dramatically reduced but that from the DN retina was not significantly affected. The selective inhibition to VT neurites was completely abolished by treatment with chondroitinase ABC, indicating a specific contribution of CS glycosaminoglycan in this regionally specific behaviour. This differential behaviour was not observed in explants presented with a lower or higher concentration of CS or in explants grown on substrate coated with a different laminin concentration. Thus, a critical ratio of CS to laminin seems to be essential to induce this differential behaviour in retinal neurites towards contact with CS. Furthermore, this behavior was not observed in explants cultured directly on a CS-rich substrate, suggesting that contact with growth-promoting molecules is necessary for the selective responses of retinal neurites during subsequent contact with CS. We concluded that CS glycosaminoglycan is sufficient to drive selective inhibition of VT but not DN neurites and that, together with a critical combination of growth-promoting factors, it may control the axon divergence process at the mouse optic chiasm.This project is partially supported by a grant from the Research Grant Council of the Hong Kong Special Administrative Region (project no. CUHK 4417/03M) and a Direct Grant from the Chinese University of HK (project no. 2004.1.051).     

Neurite outgrowth in response to transfected N-CAM changes during development and is modulated by polysialic acid

We have used monolayers of control 3T3 cells and 3T3 cells transfected with a cDNA encoding human N-CAM as a culture substrate for embryonic chick retinal ganglion cells (RGCs). At embryonic day 6 (E6), but not at E11, RGCs extended longer neurites on monolayers of N-CAM-transfected cells. This loss of RGC responsiveness was not associated with substantial changes in the level of N-CAM expression on RGC growth cones. The neurite outgrowth response from E6 RGCs could be inhibited by removal of N-CAM from the monolayer, by removal of alpha 2-8-linked polysialic acid from neuronal N-CAM, or by antibodies that bind exclusively to chick (neuronal) N-CAM. In contrast, the response was not dependent on neuronal beta 1 integrin function. These data provide substantive evidence for a homophilic binding mechanism directly mediating N-CAM-dependent neurite outgrowth, and suggest that changes in polysialic acid expression on neuronal N-CAM may modulate N-CAM-dependent axonal growth during development.     

Vitronectin and thrombospondin promote retinal neurite outgrowth: developmental regulation and role of integrins.

Extracellular matrix (ECM) glycoproteins regulate neuronal development and axonal growth. In this paper, the ECM glycoprotein vitronectin was identified and localized in the embryonic chick neuroretina. To identify potentially important neurite outgrowth-promoting molecules, responses of embryonic chick retinal neurons to vitronectin and thrombospondin, another retinal ECM constituent, were examined. These neurons were shown to attach and extend neurites on either glycoprotein. Integrins containing the alpha v or beta 1 subunits mediate both responses to vitronectin and neurite outgrowth on thrombospondin. Attachment to thrombospondin was inhibited by heparin, suggesting that neurons also utilize a proteoglycan or sulfated glycolipid as a receptor for this glycoprotein. Thus, retinal neurons use specific receptors to interact with vitronectin and thrombospondin, two glycoproteins present in the embryonic neuroretina, suggesting roles for these ligands and their receptors in retinal development.     

A cytomechanical investigation of neurite growth on different culture surfaces

We have examined the relationship between tension, an intrinsic stimulator of axonal elongation, and the culture substrate, an extrinsic regulator of axonal elongation. Chick sensory neurons were cultured on three substrata: (a) plain tissue culture plastic; (b) plastic treated with collagen type IV; and (c) plastic treated with laminin. Calibrated glass needles were used to increase the tension loads on growing neurites. We found that growth cones on all substrata failed to detach when subjected to two to threefold and in some cases 5-10-fold greater tensions than their self-imposed rest tension. We conclude that adhesion to the substrate does not limit the tension exerted by growth cones. These data argue against a "tug-of-war" model for substrate-mediated guidance of growth cones. Neurite elongation was experimentally induced by towing neurites with a force-calibrated glass needle. On all substrata, towed elongation rate was proportional to applied tension above a threshold tension. The proportionality between elongation rate and tension can be regarded as the growth sensitivity of the neurite to tension, i.e., its growth rate per unit tension. On this basis, towed growth on all substrata can be described by the simple linear equation: elongation rate = sensitivity x (applied tension - tension threshold) The numerical values of tension thresholds and neurite sensitivities varied widely among different neurites. On all substrata, thresholds varied from near zero to greater than 200 mudynes, with some tendency for thresholds to cluster between 100 and 150 mudynes. Similarly, the tension sensitivity of neurites varied between 0.5 and 5.0 microns/h/mudyne. The lack of significant differences among sensitivity or threshold values on the various substrata suggest to use that the substratum does not affect the internal "set points" of the neurite for its response to tension. The growth cone of chick sensory neurons is known to pull on its neurite. The simplest cytomechanical model would assume that both growth cone-mediated elongation and towed growth are identical as far as tension input and elongation rate are concerned. We used the equation above and mean values for thresholds and sensitivity from towing experiments to predict the mean growth cone-mediated elongation rate based on mean rest tensions. These predictions are consistent with the observed mean values.     

Neurite outgrowth on cryostat sections of innervated and denervated skeletal muscle

To localize factors that guide axons reinnervating skeletal muscle, we cultured ciliary ganglion neurons on cryostat sections of innervated and denervated adult muscle. Neurons extended neurites on sections of muscle (and several other tissues), generally in close apposition to sectioned cell surfaces. Average neurite length was greater on sections of denervated than on sections of innervated muscle, supporting the existence of functionally important differences between innervated and denervated muscle fiber surfaces. Furthermore, outgrowth was greater on sections of denervated muscle cut from endplate-rich regions than on sections from endplate-free regions, suggesting that a neurite outgrowth-promoting factor is concentrated near synapses. Finally, 80% of the neurites that contacted original synaptic sites (which are known to be preferentially reinnervated by regenerating axons in vivo) terminated precisely at those contacts, thereby demonstrating a specific response to components concentrated at endplates. Together, these results support the hypothesis that denervated muscles use cell surface (membrane and matrix) molecules to inform regenerating axons of their state of innervation and proximity to synaptic sites.     

J1/tenascin in substrate-bound and soluble form displays contrary effects on neurite outgrowth

The influence of J1/tenascin adsorbed to polyornithine-conditioned plastic (substrate-bound J1/tenascin) and J1/tenascin present in the culture medium (soluble J1/tenascin) on neurite outgrowth was studied with cultured single cells from hippocampus and mesencephalon of embryonic rats. Neurons at low density grew well on J1/tenascin substrates and extended neurites that were approximately 40% longer than on the polyornithine control substrate after 24 h in vitro. The neurite outgrowth promoting effect of substrate bound J1/tenascin was largely abolished in the presence of mAb J1/tn2, but not by mAb J1/tn1. In contrast to the neurite growth-promoting effects of substrate bound J1/tenascin, neurite outgrowth on polyornithine, laminin, fibronectin, or J1/tenascin as substrates was inhibited by addition of soluble J1/tenascin to the cultures. Neither of the two mAbs neutralized the neurite outgrowth-inhibitory properties of soluble J1/tenascin. In contrast to their opposite effects on neurite outgrowth, both substrate-bound and soluble J1/tenascin reduced spreading of the neuronal cell bodies, suggesting that the neurite outgrowth-promoting and antispreading effects are mediated by two different sites on the molecule. This was further supported by the inability of the mAb J1/tn2 to neutralize the antispreading effect. The J1/tn2 epitope localizes to a fibronectin type III homology domain that is presumably distinct from the putative Tn68 cell-binding domain of chicken tenascin for fibroblasts, as shown by electronmicroscopic localization of antibody binding sites. We infer from these experiments that J1/tenascin contains a neurite outgrowth promoting domain that is distinguishable from the cell-binding site and presumably not involved in the inhibition of neurite outgrowth or cell spreading. Our observations support the notion that J1/tenascin is a multifunctional extracellular matrix molecule.