Nerve growth cone migration onto Schwann cells involves the calcium-dependent adhesion molecule, N-cadherin

The role of calcium-dependent adhesion molecules in the migration of nerve growth cones onto the top of Schwann cells was probed by examination of sensory growth cone-Schwann cell interactions in medium containing either 1.0 mM Ca2+ or 0.1 mM Ca2+. In the presence of 1.0 mM Ca2+ growth cones rapidly migrated onto Schwann cells, spread, and remained for extended periods. However, in 0.1 mM Ca2+ growth cones still made frequent contacts with Schwann cells, but migration onto the upper cell surface was much reduced. This contrast in growth cone-Schwann cell interactions could be switched rapidly by changing the Ca2+ concentration of the culture medium. Growth cones of retinal neurons showed similar calcium-dependence in their migration onto Schwann cells. Antibodies to the calcium-dependent adhesion molecule, N-cadherin, also blocked growth cone migration onto Schwann cells, but antibodies to another neuronal adhesion molecule, L1, had no effect on growth cone-Schwann cell interactions. Immunocytochemical staining for N-cadherin and L1 indicated that growth cones and Schwann cells have N-cadherin on their surfaces, while L1 is present only on axons and growth cones. These results provide two kinds of evidence that N-cadherin is important in the initial interactions of growth cones and Schwann cells.     

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.     

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 2mM 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.     

Detection of brain-derived neurotrophic factor-like activity in fibroblasts and Schwann cells: inhibition by antibodies to NGF

mRNA coding for brain-derived neurotrophic factor (BDNF) has been detected in cultured L929 fibroblasts, rat dermal fibroblasts, and sciatic nerve Schwann cells, as well as in rat skin. Medium conditioned by cultured fibroblasts and Schwann cells also stimulates neurite growth from retinal explants and promotes the survival in culture of BDNF-responsive sensory neurons; biological activity is abolished by antibodies raised against NGF. These results suggest that molecules with BDNF-like activity may be produced by cells in the peripheral nervous system and that the BDNF-like activity in fibroblasts and Schwann cells is derived from molecules immunologically related to NGF. In support of this concept, antibodies against NGF have been found to reduce the biological activity of recombinant BDNF in culture and to cross-react with BDNF on Western blots.     

Spontaneous immortalisation of Schwann cells in culture: short-term cultured Schwann cells secrete growth inhibitory activity.

In the developing peripheral nerve, Schwann cells proliferate rapidly and then become quiescent, an essential step in control of Schwann cell differentiation. Cell proliferation is controlled by growth factors that can exert positive or inhibitory influences on DNA synthesis. It has been well established that neonatal Schwann cells divide very slowly in culture when separated from neurons but here we show that when culture was continued for several months some cells began to proliferate rapidly and non-clonal lines of immortalised Schwann cells were established which could be passaged for over two years. These cells had a similar molecular phenotype to short-term cultured Schwann cells, except that they expressed intracellular and cell surface fibronectin. The difference in proliferation rates between short- and long-term cultured Schwann cells appeared to be due in part to the secretion by short-term cultured Schwann cells of growth inhibitory activity since DNA synthesis of long-term, immortalised Schwann cells was inhibited by conditioned medium from short-term cultures. This conditioned medium also inhibited DNA synthesis in short-term Schwann cells stimulated to divide by glial growth factor or elevation of intracellular cAMP. The growth inhibitory activity was not detected in the medium of long-term immortalised Schwann cells, epineurial fibroblasts, a Schwannoma (33B), astrocytes or a fibroblast-like cell-line (3T3) and it did not inhibit serum-induced DNA synthesis in epineurial fibroblasts, 33B cells or 3T3 cells. The activity was apparently distinct from transforming growth factor-beta, activin, IL6, epidermal growth factor, atrial natriuretic peptide and gamma-interferon and was heat and acid stable, resistant to collagenase and destroyed by trypsin treatment. We raise the possibility that loss of an inhibitory autocrine loop may contribute to the rapid proliferation of long-term cultured Schwann cells and that an autocrine growth inhibitor may have a role in the cessation of Schwann cell division that precedes differentiation in peripheral nerve development.     

In vitro studies on the control of nerve fiber growth by the extracellular matrix of the nervous system

1. Cultured neurons from embryonic chick sympathetic ganglia or dorsal root ganglia grow nerve fibers extensively on simple substrata containing fibronectin, collagens (types I, III, IV), and especially laminin. 2. The same neurons cultured on substrata containing glycosaminoglycans grow poorly. Glycosaminoglycans (heparin) inhibit nerve fiber growth on fibronectin substrata. 3. Proteolytic fragments of fibronectin support nerve fiber growth only when the cell attachment region is intact. For example, a 105 kD fragment, encompassing the cell attachment region, supports growth when immobilized in a substratum, but a 93 kD subfragment, lacking the cell attachment region, is unable to support fiber growth. When it is added to the culture medium, the 105 kD fragment inhibits fiber growth on substrata containing native fibronectin. 4. In culture medium lacking NGF, DRG neurons extend nerve fibers only on laminin and not on fibronectin, collagen or polylysine. Studies with radioiodinated laminin indicate that laminin binds with a relatively high affinity (kd approximately equal to 10(-9) M) to DRG neurons, and to a variety of other neural cells (NG108 cells, PC12 cells, rat astrocytes, chick optic lobe cells). We have isolated a membrane protein (67 kD) by affinity chromatography on laminin columns and are characterizing this putative laminin receptor. 5. Dissociated DRG neurons or ganglionic explants cultured on complex substrata consisting of tissue sections of CNS or PNS tissues extend nerve fibers onto the PNS (adult rat sciatic nerve) but not CNS (adult rat optic nerve) substrata. Other tissue substrata which support fiber growth in vivo (embryonic rat spinal cord, goldfish optic nerve) support growth in culture. While substrata from adult CNS, which support meager regeneration in vivo (adult rat spinal cord) support little fiber growth in culture. 6. Ganglionic explants cultured in a narrow space between a section of rat sciatic nerve and optic nerve grow preferentially onto the sciatic nerve suggesting that diffusible growth factors are not responsible for the differential growth on the two types of tissues. 7. Dissociated neurons adhere better to sections of sciatic nerve than optic nerve. Laminin, rather than fibronectin or heparan sulfate proteoglycan, is most consistently identifiable by immunocytochemistry in tissues (sciatic nerve, embryonic spinal cord, goldfish optic nerve) which support nerve fiber growth. Taken together, these data suggest that ECM adhesive proteins are important determinants of nerve regeneration.     

Effects of Bone Marrow Stromal Cell-conditioned Medium on Primary Cultures of Peripheral Nerve Tissues and Cells

Implantation of bone marrow stromal cells (MSCs) produces an improved functional outcome of peripheral nerve repair. In this study, rat dorsal root ganglion (DRG) explants, rat DRG neurons, and rat Schwann cells (SCs) were treated with monkey MSC-conditioned medium, respectively, and then subjected to MTT assay, Bromodeoxyuridine/Hoechst 33342 double staining, flow cytometry, immunohistochemistry, real-time quantitative PCR, and Western blot analysis, respectively. The results showed that MSC-conditioned medium enhanced axon growth and neurogenesis in cultured DRG explants, augmented cell survival of and expression of NF and GAP-43 by cultured DRG neurons, promoted cell survival and proliferation of cultured SCs, and increased the expression of NGF, BDNF, and bFGF in cultured SCs. We also found that mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (Erk) 1/2 pathway was involved in the enhanced cell proliferation of SCs evoked by MSC-conditioned medium. The data of this study might help the understanding of MSCs-based treatment for peripheral nerve repair.     

Chick sensory neuronal growth cones distinguish fibronectin from laminin by making substratum contacts that resemble focal contacts

The adhesive interactions of nerve growth cones stabilize elongating nerve fibers and mediate transmembrane signaling to regulate growth cone behaviors. We used interference reflection microscopy and immunocytochemistry to examine the dynamics and composition of substratum contacts that growth cones of chick sensory neurons make with extracellular adhesive glycoproteins, fibronectin and laminin. Interference reflection microscopy indicated that sensory neuronal growth cones on fibronectin-treated substrata, but not on laminin, make contacts that have the appearance and immobility of fibroblastic focal contacts. Interference reflection microscopy and subsequent immunocytochemical staining showed that β1 integrin and phosphotyrosine residues were concentrated at growth cone sites that resemble focal contacts. Two other components of focal contacts, paxillin and zyxin, were also co-localized with concentrated phosphotyrosine residues at sites that resemble focal contacts. Such staining patterns were not observed on laminin-treated substrata. Growth cone migration on fibronectin-treated substrata was inhibited by herbimycin A, a tyrosine kinase inhibitor. We conclude that sensory neuronal growth cones distinguish fibronectin from laminin by making contacts with distinct organization and regulation of cytoskeletal components at the adhesive sites. This finding suggests that growth cone interactions with different adhesive molecules lead to distinctive transmembrane organization and signaling to regulate nerve fiber elongation. © 1996 John Wiley & Sons, Inc.     

Identification of the major proteins that promote neuronal process outgrowth on Schwann cells in vitro

Schwann cells have a unique role in regulating the growth of axons during regeneration and presumably during development. Here we show that Schwann cells are the best substrate yet identified for promoting process growth in vitro by peripheral motor neurons. To determine the molecular interactions responsible for Schwann cell regulation of axon growth, we have examined the effects of specific antibodies on process growth in vitro, and have identified three glycoproteins that play major roles. These are the Ca2+-independent cell adhesion molecule (CAM), L1/Ng-CAM; the Ca2+-dependent CAM, N-cadherin; and members of the integrin extracellular matrix receptor superfamily. Two other CAMs present on neurons and/or Schwann cells-N-CAM and myelin-associated glycoprotein-do not appear to be important in regulating process growth. Our results imply that neuronal growth cones use integrin-class extracellular matrix receptors and at least two CAMs--N-cadherin and L1/Ng-CAM-for growth on Schwann cells in vitro and establish each of these glycoproteins as a strong candidate for regulating axon growth and guidance in vivo.     

Effects of neurotrophic factors and cell substrates on the differentiation of a sympathoadrenal progenitor cell line

The detailed spatial organization of cytoskeletal proteins in an immortalized sympathoadrenal precursor cell line, termed MAH, was studied when the cells were grown on cellular substrates and when treated with combinations of recombinant nerve growth factor, ciliary neurotrophic factor and basic fibroblast growth factor. In response to growth factors, MAH cells expressed appropriate distributions of phosphorylated and non-phosphorylated neurofilaments, and dendrite and axon specific microtubule associated proteins. Sequential stages of maturation and axon formation were identified as the MAH cells established neuronal polarity and developed into sympathetic-like neurons. Combinations of the growth factors initiated growth associated protein-43 expression in processes and promoted the MAH cells to acquire sympathetic-like neuron characteristics with long, thin processes that branched and often terminated in elaborate growth cones. When treated with the three trophic factors, 15% of the MAH cells differentiated into sympathetic-like neurons, in contrast to less than 10% when cultured with ciliary neurotrophic factor plus nerve growth factor. An enhanced cholinergic phenotype was evident in the MAH cells when grown with ciliary neurotrophic factor. MAH cells also expressed neuron-specific markers when co-cultured on enriched substrates of smooth muscle, fibroblasts or Schwann cells. The results indicate that this sympathoadrenal cell lineage, carrying the v-myc oncogene, can express appropriate cytoskeletal markers in the process of neuronal differentiation when induced by neurotrophic factors or by specific cellular conditions in vitro.     

Schwann cell extracellular matrix molecules and their receptors

The major cellular constituents of the mammalian peripheral nervous system are neurons (axons) and Schwann cells. During peripheral nerve development Schwann cells actively deposit extracellular matrix (ECM), comprised of basal lamina sheets that surround individual axon-Schwann cell units and collagen fibrils. These ECM structures are formed from a diverse set of macromolecules, consisting of glyco-proteins, collagens and proteoglycans. To interact with ECM, Schwann cells express a number of integrin and non-integrin cell surface receptors. The expression of many Schwann cell ECM proteins and their receptors is developmentally regulated and, in some cases, dependent on axonal contact. Schwann cell ECM acts as an organizer of peripheral nerve tissue and strongly influences Schwann cell adhesion, growth and differentiation and regulates axonal growth during development and regeneration.     

Schwann cell mediated trophic effects by differentiated mesenchymal stem cells

Mesenchymal stem cells were isolated from the bone marrow of rats and differentiated to provide a functional substitute for slow growing Schwann cells for peripheral nerve regeneration. To assess the properties of the differentiated mesenchymal stem cell, the cells were co-cultured with dorsal root ganglia and the secretion of the neurotrophic factors and the neurite outgrowth was evaluated. The neurite outgrowth of the dorsal root ganglia neurons was enhanced in co-culture with the differentiated stem cells compared to the undifferentiated stem cells. Differentiated stem cells like Schwann cells were responsible for the stimulation of longer and branched neurites. Using enzyme-linked immunosorbant assays and blocking antibodies, we have shown that this effect is due to the release of brain derived neurotrophic factor and nerve growth factor, which were up-regulated in differentiated mesenchymal stem cells following co-culture. The relevance of the tyrosine kinase receptors was confirmed by the selective tyrosine kinase inhibitor, K252a which abolished the neurite outgrowth of the dorsal root ganglia neurons when co-cultured with the differentiated mesenchymal stem cells similar to Schwann cells. The results of the study further support the notion that mesenchymal stem cells can be differentiated and display trophic influences as those of Schwann cells.     

Neuronal nets and nerve cell interactions in vitro in insect systems

Summary The development of a synthetic medium that supports growth and differentiation of insect embryonic tissues afforded the possibility of studying the interactions between nerve and other cell types in long term cultures. The mechanical dissociation of embryonic nerve tissues results in survival of nerve cells but not of glial cells. The dissociated glial-free neurons produce a dense fibrillar network in the presence, but not in the absence, of foregut explants or other tissues from same donors. Nerve fiber bundles outgrowing from dissociated neurons enter foregut segments and establish synaptic connections with muscle cells. Foregut explants undergo differentiation and become contractile in long term cultures when innervated by dissociated nerve cells. The progressive deterioration of similar foregut tissues cultured alone contrasts with the excellent condition of innervated explants and suggests that this is due to trophic factors released by nerve fibers. The same in vitro systems provided the opportunity of studying the interaction between nerve fibers produced by the autonomic ingluvial ganglion, which adheres to the surface of the alimentary tract, and muscle cells. Multiple esophagus explants from cockroach embryos become interconnected by fibers emerging from ingluvial ganglia, when the explants are combined in vitro at short distance from each other. Muscle cells migrating from the esophagi line up on axons branching out in the medium, or form contractile ribbons which, in turn, establish connections with nerve fibers. The thigmotropism of muscle cells and strong affinity for nerve fibers reveal a new aspect of muscle cells-to-fibers interaction, amenable to further analysis in vitro. This work was supported in part by United States Public Health Service grant NS-03777 and grant GB-16330 X from the National Science Foundation     

Uptake of Endoneurial Lipoprotein into Schwann Cells and Sensory Neurons Is Mediated by Low Density Lipoprotein Receptors and Stimulated After Axonal Injury

During Wallerian degeneration of rat sciatic nerve, the expression of apolipoprotein E increases and apolipoprotein E-containing endoneurial lipoproteins accumulate in the distal nerve segment. In established primary cultures dissociated from dorsal root ganglia, Schwann cells and sensory neurons internalized rhodamine-labeled lipoproteins isolated from crushed rat sciatic nerve as well as low density lipoprotein (LDL) from human serum. The uptake of endoneurial lipoproteins could be inhibited by an excess of LDL or at low temperature (4 degrees C). After transection of nerve fibers in dorsal root ganglia explant cultures, the uptake of lipoproteins was markedly stimulated in Schwann cells that were in close proximity to degenerating neurites. A specific monoclonal antibody directed to the bovine LDL receptor (clone C7) was shown to cross-react with LDL receptor preparations of rat endoneurial cells. LDL receptor immunoreactivity was expressed by cell bodies and processes of cultured Schwann cells, sensory neurons, and fibroblasts from dorsal root ganglia. Incubation of Schwann cells and neurons with the LDL receptor antibody strongly inhibited the uptake of endoneurial lipoproteins. Our results provide direct evidence for the important role of the LDL receptor-mediated pathway to internalize endoneurial lipoproteins into Schwann cells and peripheral neurons required for reuse of cholesterol and other lipids in myelin and plasma membrane biogenesis during nerve repair.     

Formation of the transverse nerve in moth embryos : I. A scaffold of nonneuronal cells prefigures the nerve

We have studied the embryonic development of the transverse nerve (TN), an unpaired segmental nerve of the moth Manduca sexta. Two identified motor neurons and 16 identified neuroendocrine neurons project axons within the larval TN; therefore, the TN is both a peripheral nerve and a neurohaemal organ. At 33% of embryogenesis, and prior to the arrival of any neuronal growth cones, the position, shape, and trajectory of the TN are anticipated by two groups of nonneuronal cells that we call the strap and the bridge. At this time the strap and the bridge together consist of approximately 100 cells, all of which express a cell surface epitope recognized by the monoclonal antibody TN-1. As development proceeds, both the number of nonneuronal cells within the strap and the bridge and the fraction that expresses the TN-1 antigen(s) decrease. Moreover, individual cells within the strap become morphologically identifiable before the arrival of the neuronal growth cones. Most of the axons that project to the TN also express the TN-1 antigen(s) during their period of outgrowth. The two motor neuron growth cones are the first to reach the environment of the strap and the bridge, doing so at approximately 37%; having encountered these cellular structures, the growth cones restrict their navigation to this preexisting scaffolding, until they reach their muscle target. The neuroendocrine growth cones arrive later and also grow within the confines of the strap and the bridge (J.N. Carr and P.H. Taghert, 1988, Dev. Biol, 130, 500-512). In this first paper we describe the development of the strap and the bridge, and the interactions of the motor neuron growth cones with these structures. The observations are novel in documenting the extent and precision to which a peripheral nerve pathway is prefigured by a contiguous assemblage of nonneuronal cells.     

Nestin-Expressing Stem Cells Promote Nerve Growth in Long-Term 3-Dimensional Gelfoam?-Supported Histoculture

We have previously reported that hair follicles contain multipotent stem cells which express nestin. The nestin-expressing cells form the hair follicle sensory nerve. In vitro, the nestin-expressing hair follicle cells can differentiate into neurons, Schwann cells, and other cell types. In the present study, the sciatic nerve was excised from transgenic mice in which the nestin promoter drives green fluorescent protein (ND-GFP mice). The ND-GFP cells of the sciatic nerve were also found to be multipotent as the ND-GFP cells in the hair follicle. When the ND-GFP cells in the mouse sciatic nerve cultured on Gelfoam® and were imaged by confocal microscopy, they were observed forming fibers extending the nerve. The fibers consisted of ND-GFP-expressing spindle cells, which co-expressed the neuron marker β-III tubulin, the immature Schwann-cell marker p75^NTR and TrkB which is associated with neurons. The fibers also contain nestin-negative spherical cells expressing GFAP, a Schwann-cell marker. The β-III tubulin-positive fibers had growth cones on their tips expressing F-actin, indicating they are growing axons. When the sciatic nerve from mice ubiquitously expressing red fluorescent protein (RFP) was co-cultured on Gelfoam® with the sciatic nerve from ND-GFP transgenic mice, the interaction of nerves was observed. Proliferating nestin-expressing cells in the injured sciatic nerve were also observed in vivo. Nestin-expressing cells were also observed in posterior nerves but not in the spinal cord itself, when placed in 3-D Gelfoam® culture. The results of the present report suggest a critical function of nestin-expressing cells in peripheral nerve growth and regeneration.     

Repulsive axon guidance molecule Sema3A inhibits branching morphogenesis of fetal mouse lung

Semaphorin III/collapsin-1 (Sema3A) guides a specific subset of neuronal growth cones as a repulsive molecule. In this study, we have investigated a possible role of non-neuronal Sema3A in lung morphogenesis. Expression of mRNAs of Sema3A and neuropilin-1 (NP-1), a Sema3A receptor, was detected in fetal and adult lungs. Sema3A-immunoreactive cells were found in airway and alveolar epithelial cells of the fetal and adult lungs. Immunoreactivity for NP-1 was seen in fetal and adult alveolar epithelial cells as well as endothelial cells. Immunoreactivity of collapsin response mediator protein CRMP (CRMP-2), an intracellular protein mediating Sema3A signaling, was localized in alveolar epithelial cells, nerve tissue and airway neuroendocrine cells. The expression of CRMP-2 increased during the fetal, neonate and adult periods, and this pattern paralleled that of NP-1. In a two-day culture of lung explants from fetal mouse lung (E11.5), with exogenous Sema3A at a dose comparable to that which induces growth cone collapse of dorsal root ganglia neurons, the number of terminal buds was reduced in a dose-dependent manner when compared with control or untreated lung explants. This decrease was not accompanied with any alteration of the bromodeoxyuridine-positive DNA-synthesizing fraction. A soluble NP-1 lacking the transmembrane and intracellular region, neutralized the inhibitory effect of Sema3A. The fetal lung explants from neuropilin-1 homozygous null mice grew normally in vitro regardless of Sema3A treatment. These results provide evidence that Sema3A inhibits branching morphogenesis in lung bud organ cultures via NP-1 as a receptor or a component of a possible multimeric Sema3A receptor complex.     

Axon initiation by ciliary neurons in culture

A nerve culture system for the study of axon initiation is described. A population of individual chick embryo ciliary neurons, free from contact with other cells and attached to a polyornithinecoated culture dish, is exposed to heart cell-conditioned medium (HCM). Within 30 min after the addition of HCM the majority of neurons have formed growth cones, and by 90 min more than 80% of the neurons bear at least one axon longer than 15 μm. Before the addition of HCM, ciliary neurons generate membrane ruffles and extend filopodia around the entire periphery of the rounded cell body. Axon initiation, following addition of HCM, consists of two distinctive changes in the cell surface: (1) organization of the randomly distributed surface movements into localized highly active growth cones, which then form axons; and (2) the cessation of surface movements elsewhere on the cell periphery. Heart cell-conditioned medium may induce these changes by increasing the adhesion between parts of the nerve cell surface and the substratum.     

Elongation of mouse spinal cord axons in isogenic grafts of the sciatic nerve, skeletal muscle and submaxillary gland

Isografts of sciatic nerve, skeletal muscle, submaxillary gland and, as control experiments, of optice nerve, were transplanted into the non transected spinal cord of young albino mice, through a punctiform pial aperture. Under these conditions, local cellular reactions were reduced and the sensori motor behavior of the operated animals remained apparently undisturbed throughout the experimental period. Within a few days, axonal sprouts issuing mainly from the terminal clubs of intraspinal nerve fibres severed by the grafting procedure were seen elongating and growing into--and presumably throughout--the nervous as well as the muscular and glandular transplants. The Schwann cells of these grafts, either sedentary or migrating towards the cord and intermingling with host reactive glial cells, appeared to guide the growth of the axonal sprouts they ensheathed (from day 3 to day 10) and generally myelinated (as early as day 6). Optic nerve transplants, lacking Schwann cells, were never reinnervated. Furthermore, in control microinjuries without grafting, limited growth of axonal sprouts was observed only when a few host Schwann cells were present. Mouse spinal neurons, therefore, demonstrate a marked capacity for regrowth when minimal damage to the spinal cord is associated with an adequate supply of Schwann cells. In contrast, host as well as transplanted glial cells, were unable, at least when they were not associated with Schwannian elements, to promote regenerative expression of these central neurons.     

Pluripotent Hair Follicle Neural Crest Stem-Cell-Derived Neurons and Schwann Cells Functionally Repair Sciatic Nerves in Rats

In this paper, we constructed a novel acellular nerve xenograft (ANX) seeded with neurons and Schwann cells to bridge long-distance gaps in rat sciatic nerves. The neurons and Schwann cells were induced from Sprague Dawley (SD) rat hair follicle neural crest stem cells with sonic hedgehog/retinoic acid and neuregulin 1, respectively. Fifty male SD rats were randomly divided into two groups (n = 25): ANX + cells group and ANX group. A 4-cm-long sciatic nerve defect was created on the right hind limb and bridged with cell-seeded ANX in ANX + cells group or ANX alone in ANX group. We found that the implanted neurons and Schwann cells could survive by 4 weeks and as far as 52 weeks posttransplantation. In implanted grafts, chemical synaptic structures were also found under transmission electron microscope and confirmed with immunostaining of synapsin 1, a synaptic marker. The number of regenerated axons in ANX + cells group was higher than that in ANX group (P < 0.01). This novel implantation of neurons and Schwann cells via acellular nerve graft may provide an alternative way for repairing peripheral nerve defect.