Secondary motoneuron axons localize DM-GRASP on their fasciculated segments

Cell surface adhesion molecules are thought to play a necessary role in axon guidance and fasciculation in the developing nervous system. We have studied a potential adhesion molecule using the zn-5 monoclonal antibody, which recognizes the surfaces of zebrafish spinal motoneurons. We show that zn-5 recognizes zebrafish DM-GRASP. DM-GRASP is a cell adhesion molecule of the immunoglobulin superfamily that mediates homophilic adhesion and neurite outgrowth in vitro. It is necessary for correct axon routing and fasciculation in the Drosophila visual system. In zebrafish, primary motoneurons pioneer the peripheral motor nerve pathways, and the axons of secondary motoneurons follow the routes established by the primary motoneuron axons. We show that, of the two classes of zebrafish spinal motoneurons, only the later growing secondary motoneurons express DM-GRASP. The secondary motoneurons restrict DM-GRASP protein to their cell bodies and fasciculated segments of their axons. Expression of DM-GRASP is transient: The protein is present during the period of axonal growth and disappears after axons have reached their muscle targets. Thus, homophilic adhesion mediated by DM-GRASP may play a role in fasciculation of secondary motoneuron axons but not in pathfinding by the pioneer axons of the primary motoneurons or in guidance of secondary motoneuron axons to their targets.     

The L1-type cell adhesion molecule Neuroglian is necessary for maintenance of sensory axon advance in the Drosophila embryo

Background

Cell adhesion molecules have long been implicated in the regulation of axon growth, but the precise cellular roles played by individual cell adhesion molecules and the molecular basis for their action are still not well understood. We have used the sensory system of the Drosophila embryo to shed light on the mechanism by which the L1-type cell adhesion molecule Neuroglian regulates axon growth.

Results

We have found a highly penetrant sensory axon stalling phenotype in neuroglian mutant embryos. Axons stalled at a variety of positions along their normal trajectory, but most commonly in the periphery some distance along the peripheral nerve. All lateral and dorsal cluster sensory neurons examined, except for the dorsal cluster neuron dbd, showed stalling. Sensory axons were never seen to project along inappropriate pathways in neuroglian mutants and stalled axons showed normal patterns of fasciculation within nerves. The growth cones of stalled axons possessed a simple morphology, similar to their appearance in wild-type embryos when advancing along nerves. Driving expression of the wild-type form of Neuroglian in sensory neurons alone rescued the neuroglian mutant phenotype of both pioneering and follower neurons. A partial rescue was achieved by expressing the Neuroglian extracellular domain. Over/mis-expression of Neuroglian in all neurons, oenocytes or trachea had no apparent effect on sensory axon growth.

Conclusion

We conclude that Neuroglian is necessary to maintain axon advance along axonal substrates, but is not required for initiation of axon outgrowth, axon fasciculation or recognition of correct growth substrates. Expression of Neuroglian in sensory neurons alone is sufficient to promote axon advance and the intracellular region of the molecule is largely dispensable for this function. It is unlikely, therefore, that Nrg acts as a molecular 'clutch' to couple adhesion of F-actin within the growth cone to the extracellular substrate. Rather, we suggest that Neuroglian mediates sensory axon advance by promoting adhesion of the surface of the growth cone to its substrate. Our finding that stalling of a pioneer sensory neuron is rescued by driving Neuroglian in sensory neurons alone may suggest that Neuroglian can act in a heterophilic fashion.     

An Analysis of an Axonal Gradient of Phosphorylated MAP 1B in Cultured Rat Sensory Neurons

The present study investigated the cellular distribution of a developmentally regulated phosphorylated form of MAP 1B recognized by monoclonal antibody (mAb) 150 in cultures of dorsal root ganglia. The cell soma and the whole axon, when it first appears, are labelled, but longer axons label with a proximodistal gradient, such that the cell soma and proximal axon become unlabelled, whilst the distal axon and growth cone label strongly. Double-labelling experiments with mAb 150 and a polyclonal antibody (N1–15) that recognizes all forms of MAP 1B demonstrated that MAP 1B is distributed along the entire length of axons with gradients, so the gradient of phosphorylated MAP 1B is not due to a loss or absence of MAP 1B from the proximal axon. The proportion of axons from 20 h cultures that were labelled with a mAb 150 gradient was at least 80% and this proportion was independent of the nerve growth factor concentration of the culture medium. Analysis of axons ranging in length from 100 to 700 μm and labelled with a gradient showed that the unlabelled proximal portions of axons increased in length more slowly than the labelled distal axon. Axons labelled along their entire length accounted for no more than 19% of the axonal population and analysis of these showed them to be frequently <400 μm long. After simultaneously fixing and detergent-extracting cultures this proportion rose significantly to 93%, suggesting that in the proximal axon the mAb 150 epitope is masked by some factor(s) that is removed by detergent extraction. The possibility that mAb 150 could not access the epitope in the proximal axon was discounted because another IgM, mAb 125, which recognizes a different phosphorylation epitope on MAP 1B, labelled the proximal axon of conventionally fixed cultures. In growth cones of fixed and extracted neurons examined by immunofluorescence, the mAb 150 labelling strongly colocalized to bundled microtubules in the distal axon shaft and the C-domain. In the P-domain, mAb 150 staining was weaker and more widely distributed than the microtubules. Immunogold electron microscopy confirmed that antibody N1–15 and mAb 150 strongly labelled the bundled microtubules in the C-domain and also showed that individual microtubules in the P-domain, some of which lie alongside actin filament bundles of filopodia, were labelled lightly and discontinuously with both antibodies. This suggests that the phosphorylated isoform of MAP 1B recognized by mAb 150 may be involved in bundling microtubules in the proximal region of the growth cone and in the interaction between microtubules and actin filaments in the P-domain.     

Differential adhesion and the initial assembly of the mammalian olfactory nerve

During the initial assembly of the olfactory pathway, the behavior of olfactory axons changes as they grow from the olfactory epithelium toward the telencephalic vesicle. The axons exit the epithelium singly or in small fascicles, and their growth cones are simple and bullet-shaped. Outside the epithelium, they make a sharp dorsal turn and fasciculate into a single nerve; the growth cones remain simple. Upon entering the ventromedial telencephalon, the axons defasciculate, branch extensively, and end in complex, lamellate growth cones which extend toward the ventrolateral aspect of the telencephalic vesicle. The distribution of laminin, collagen-IV, and fibronectin varies in register with these changes in olfactory axon and growth cone behavior. Each of these extracellular matrix molecules influences olfactory neurite outgrowth and growth cone morphology in vitro consistent with its distribution in vivo. The distribution of E-cadherin, L1, neural cell adhesion molecule (NCAM) and the polysialated form of NCAM also varies in register with changes in olfactory axon behavior. In vitro, L1 modulates embryonic olfactory neurite outgrowth and growth cone morphology consistent with its distribution in vivo. Thus, olfactory axon trajectory, fasciculation, and growth cone morphology change within distinct adhesive environments in the nascent olfactory pathway, and some of the molecules that characterize these environments have differential effects upon olfactory neurite growth and growth cone morphology. Consequently, the patterned expression and activity of extracellular matrix and cell surface adhesion molecules may contribute to the initial assembly of the olfactory pathway. © 1996 Wiley-Liss, Inc.     

Expression and putative role of lactoseries carbohydrates present on NCAM in the rat primary olfactory pathway

Primary olfactory neurons project axons from the olfactory neuroepithelium lining the nasal cavity to the olfactory bulb in the brain. These axons grow within large mixed bundles in the olfactory nerve and then sort out into homotypic fascicles in the nerve fiber layer of the olfactory bulb before terminating in topographically fixed glomeruli. Carbohydrates expressed on the cell surface have been implicated in axon sorting within the nerve fiber layer. We have identified two novel subpopulations of primary olfactory neurons that express distinct alpha-extended lactoseries carbohydrates recognised by monoclonal antibodies LA4 and KH10. Both carbohydrate epitopes are present on novel glycoforms of the neural cell adhesion molecule, which we have named NOC-7 and NOC-8. Primary axon fasciculation is disrupted in vitro when interactions between these cell surface lactoseries carbohydrates and their endogenous binding molecules are inhibited by the LA4 and KH10 antibodies or lactosamine sugars. We report the expression of multiple members of the lactoseries binding galectin family in the primary olfactory system. In particular, galectin-3 is expressed by ensheathing cells surrounding nerve fascicles in the submucosa and nerve fiber layer, where it may mediate cross-linking of axons. Galectin-4, -7, and -8 are expressed by the primary olfactory axons as they grow from the nasal cavity to the olfactory bulb. A putative role for NOC-7 and NOC-8 in axon fasciculation and the expression of multiple galectins in the developing olfactory nerve suggest that these molecules may be involved in the formation of this pathway, particularly in the sorting of axons as they converge towards their target.     

Growth cones and axon trajectories of a sensory pathway in the amphibian spinal cord

Central axons of sensory ganglion (SG) neurons of the Xenopus tail enter the spinal cord via the ventral roots and travel dorsally and rostrally following a diagonal course within the lateral marginal zone (LMZ) to reach the dorsolateral fasciculus (DLF) (Nordlander et al.: Brain Res., 440:391-395, 1988). Axons are dispersed as they cross the cord. At the DLF they turn and travel together rostrally, sharing the fascicle with axons of primary sensory neurons (Rohon-Beard cells) already present in the tract. In this paper we analyze the growth patterns of the central projections of SG axons in the tail by using HRP applied to proximal branches of tail spinal nerves. Growth cones of the diagonal route are variable in configuration, often bearing processes that spread within the LMZ. Once the DLF, growth cones change shape, becoming distinctly linear. While growth cones navigating the diagonal part of the route never contact or fasciculate with other diagonal SG axons, SG growth cones and axons of the DLF are more closely associated with their fellows. Measurements of the slopes of SG axons in the diagonal route indicated a limited range with a mean of 23 degrees with respect to the cord axis. On the basis of these observations, we conclude that 1) navigational patterns for growth cones of this pathway differ for the diagonal versus the DLF part of its course, and 2) fasciculation is not a mechanism used by SG axons to reach the DLF, but that instead, each axon is able to find its way independently.     

Chondroitin sulfate proteoglycan expression pattern in hippocampal development: potential regulation of axon tract formation

A variety of molecular influences in the extracellular matrix (ECM) interact with developing axons to guide the formation of hippocampal axon pathways. One of these influences may be chondroitin sulfate proteoglycans (CSPGs), which are known to inhibit axonal extension during development and following central nervous system injury. In this study, we examined the role of CSPGs and cell adhesion molecules in the regulation of axon tract formation during hippocampal development. We used indirect immunofluorescence to examine the developmental pattern of CSPG expression relative to axon tracts that express the cell adhesion molecule L1. Additionally, we used dissociated and explant cell cultures to examine the effects of CSPGs on hippocampal axon development in vitro. In vivo, we found that the CSPG neurocan is expressed throughout the alveus, neuropil layers, and parts of the dentate gyrus from E16 to P2. The CSPG phosphacan is expressed primarily in the neuropil layers at postnatal stages. After E18, intense labeling of neurocan was observed in regions of the alveus surrounding L1-expressing axon fascicles. In vitro, axons from brain regions that project through the alveus during development would not grow across CSPG substrata, in a concentration-dependent manner. In addition, hippocampal axons from dissociated neuron cultures only traveled across CSPG substrata as fasciculated axon bundles. These findings implicate CSPG in the regulation of axon trajectory and fasciculation during hippocampal axon tract formation.     

Axonal growth cones in the developing amphibian spinal cord

Axonal growth cones in the spinal cord of embryonic and larval Xenopus (stages 24-48) were filled with the anatomical tracer horseradish peroxidase (HRP). Growth cones of lateral and ventral marginal zones, including those of descending spinal and supraspinal pathways, were labeled by application of tracer to the caudal medulla or to one of several levels of the spinal cord. Central axons of sensory neurons were filled via their peripheral processes. Growth cone configuration varied widely but fell into five general categories: complex with both filopodia and veils, filopodia only, lamellipodia only, clavate, and fusiform. Several general patterns emerged from the distribution of these various configurations. Growth cones of younger animals generally were more complex than those of older ones. Growth cones closer to the leading edge of descending fiber bundles were more elaborate than those that followed. Growth cones of the dorsolateral fascicle, which carries ascending central processes of Rohon-Beard and sensory ganglion neurons, were very simple and followed a straight course rostrally, whereas those of descending axons of the lateral fiber areas were more complex and sometimes spread over almost the entire lateral marginal zone. Growth cones of Rohon-Beard central ascending axons were fusiform or clavate, while those of sensory ganglion axons showed several fine filopodia at their tips. Growth cones of both types of sensory axons change configuration as they approached the hindbrain, becoming more complex. This study demonstrates that the configurations of growth cones belonging to the same axonal pathway vary with age and with position along their routes, and that growth cones of different neuron classes exhibit characteristic ranges of morphological variation.     

Expression of the cell adhesion molecule axonin-1 in neuromeres of the chicken diencephalon

Axonin-1/TAG-1, a member of the immunoglobulin (Ig) superfamily of adhesion molecules, has been shown to be selectively expressed by a subset of neurons and fiber tracts in the developing nervous system of vertebrates. Axonin-1/TAG-1 is thought to play a role in the outgrowth, guidance, and fasciculation of neurites. In the present study, we map the expression of axonin-1 in the diencephalon of the chicken brain at early and intermediate stages of development [2–8 days of incubation; embryonic day (E)2–E8] by immunohistochemical methods. Results show that axonin-1 is first expressed at about E2.5 by postmitotic neurons scattered throughout most of the diencephalon. During the neuromeric stage of brain development (about E3–E5), axonin-1⁺ nerve cell bodies are predominantly found in two neuromeric subdivisions: 1) in the alar plate of the precommissural pretectum and dorsal thalamus and 2) in the posterior preoptic region of the hypothalamus. The axonin-1⁺ fiber bundles emerging from these areas grow across segmental boundaries. For example, axonin-1⁺ neurites originating in the dorsal thalamus cross the zona limitans intrathalamica at a right angle to project to the striatum. Later, the axonin-1⁺ neuromere areas give rise to particular axonin-1⁺ gray and white matter structures. Most of these structures correspond to the structures described to express TAG-1 in rodents. In conclusion, axonin-1 can be used as a marker to study aspects of the transition from the early neuromeric structure to the mature anatomy of the chicken brain.J. Comp. Neurol. 381:230-252, 1997. © 1997 Wiley-Liss, Inc.     

L1 and GAD65 are expressed on dorsal commissural axons in embryonic rat spinal cord

Using immunocytochemical methods, the cell adhesion molecule L1 was detected on axons crossing in the dorsal commissure of developing rat spinal cord. Immunoreactive axons were found in locations similar to fiber bundles illustrated by Ramón y Cajal and designated the anterior, middle and posterior bundles of the dorsal commissure. L1-immunoreactive dorsal commissural axons were first observed on embryonic day 17 (E17), appeared more numerous by E19, and remained detectable in early postnatal ages. The massive middle axon bundles extended bilaterally from the dorsolateral funiculi towards the midline and crossed in the central part of the commissure. In horizontal sections, bundles of L1-labeled middle axons were observed to traverse the dorsal commissure in a periodic pattern along the entire rostrocaudal extent of the spinal cord. Bundles of glutamic acid decarboxylase (GAD65)-positive axons were detected crossing in the middle and posterior regions of the dorsal commissure between E17 and E20. Results from double-labeling experiments demonstrated that GAD65-positive fibers were embedded in larger bundles of L1-labeled axons and that some dorsal commissural axons were double-labeled. To determine if there were axons crossing in the dorsal commissure that did not express L1, double-labeling experiments were conducted using neurofilament and L1 antibodies. Results indicated that bundles of axons identified with anti-neurofilament antibodies were also L1-positive, whereas individually coursing axons within the commissure were L1-negative. The predominance of L1 on fiber bundles traversing the dorsal commissure adds to the growing evidence that this molecule may play a role in axon outgrowth and fasciculation.     

Morphology of developing olfactory axons in the olfactory bulb of the rabbit (Oryctolagus cuniculus): a Golgi study

Transient expression of axon collaterals plays an important role in enabling neurons to find appropriate targets during development. In the olfactory bulb, the numbers of both sensory neurons and their targets, the glomeruli, increase markedly during the postnatal period. In the present study, the morphology of developing olfactory axons in the olfactory bulb of 1-21-day-old rabbits was analyzed using stereological methods and the rapid Golgi technique. The findings demonstrated a change in axon morphology from the olfactory nerve layer to the glomeruli suggestive of a sequence in axon development. In the olfactory nerve layer, axons typically had knob-like growth cones and a few collateral branches. Close to glomeruli, axons increased in thickness, formed rather complex and irregular growth cones, and typically gave off many collaterals. Within glomeruli, the axons formed terminal branches and boutons. Extraglomerular branches were apparently removed once axons had entered a glomerulus, insofar as these branches often displayed morphological signs of degeneration. In contrast, collateral branches ending in the same glomerulus remained, indicating that formation of collaterals may assist olfactory axons in locating glomerular targets.     

Ultrastructural observations of the migration and early development of trigeminal motoneurons in chick embryos

The migration of immature chick trigeminal (V) motoneurons from a miclline medial column to a lateral nucleus was studied between days 3 and 7 of incubation. The somatic translocation of these cells is preceded by the lateral outgrowth of their axons. At days 3–4 the axons grow within extracellular spaces dorsal to pial end feet at the external limiting membrane. At later ages the axons grow deeper within the intermediate zone. Upon reaching the medial border of the V sensory tract the axons turn ventrally, exit from the neural tube, and grow toward the mandibular arch. The medial column somas rotate from their original radial orientation to lie parallel to the pial surface. They migrate laterally in close association with fiber bundles composed of axonal and trailing processes of medial column and other migrating V motoneurons. Several punctate contacts are made between the migrating cells and the axon bundles; these contacts may stabilize the somas while the growing tips of their axons elongate. During migration the cytoplasm of the V motoneuron matures, acquiring rough endoplasmic retic-ulum, lysosomes, and multivesicular bodies. Immature synapses contact premigratory, migratory, and postmigratory cells on day 4. Their maturation on day 5 coincides with the onset of active jaw movements. Some V motoneurons secondarily migrate dorsally from the lateral nucleus while their trailing axons are in the head mesenchyme. Few punctate contacts are observed between these cells and their substrate, the longitudinal fibers of the lateral longitudinal fasciculus. Postmigratory dorsal V motoneurons are organelle poor and have few synaptic contacts by day 7. This undifferentiated state may reflect a later onset of the spontaneous movement of their target, the levator palpebrae muscle.     

Neurite growth on different substrates: permissive versus instructive influences and the role of adhesive strength.

Growing axons use environmental cues to guide them to their targets. One class of cues is thought to be adhesion molecules on cells and in the extracellular matrix that axons interact with as they grow to their targets. In choosing between two possible pathways, the relative adhesiveness of the two substrates could be an important factor in controlling neurite growth. We conducted experiments in vitro to study how naturally occurring adhesion molecules influence neurite growth. Neurite growth rates, the degree of neurite fasciculation, the choices neurites make between two substrates, and the relative adhesiveness of different substrates were examined. We found that the relative adhesiveness of a substrate was a poor predictor of either axon growth rate or the degree of fasciculation. Furthermore, neurites showed little selectivity between three different naturally occurring substrates, L1, N-cadherin, and laminin. These results suggest that some adhesion molecules may serve as permissive substrates in that they can define axonal pathways but they do not provide information about which path to take at a choice point or about which direction to go along the path. Finally, these results suggest that substrates in vivo may not exert their effects on axon guidance principally via relative adhesiveness.     

Guidance of neuronal growth cones in the grasshopper embryo. I. Recognition of a specific axonal pathway by the pCC neuron

The selective affinities that growth cones display for specific axonal surfaces give rise to stereotyped patterns of selective fasciculation. Previous studies on cell recognition by neuronal growth cones in the grasshopper embryo led to the proposal and initial experimental testing of the labeled-pathways hypothesis. Here we report on a further experimental analysis of this hypothesis, using the first 3 longitudinal axon fascicles, which initially contain only the axons of 7 identified neurons. We describe and experimentally test the selective affinity of the pCC growth cone for the MP1 and dMP2 axons in the MP1/dMP2 fascicle. The pCC growth cone appears to demonstrate an absolute, rather than hierarchical, preference for the MP1/dMP2 fascicle, as compared with other longitudinal axon fascicles, which supports the notion that the surfaces of the MP1 and dMP2 axons have some special distinguishing label that guides the pCC growth cone onto and along them.     

Differential expression of the mRNAs of the axonal glycoproteins axonin-1 and NgCAM in the developing chick retina

Cell adhesion molecules expressed on the axonal membrane have been thought to be involved in the guidance of axons to their target area. In the chick, axonin-1 and NgCAM have been shown to promote, through reciprocal interactions, neurite outgrowth in vitro. We have recently shown that chick retinal ganglion cells (RGC) express both proteins as early as the axonal elongation begins. Their expression continues throughout the development of the retinotectal system synchronously with the chronotopic spread of axons. To further investigate the spatiotemporal distribution of axonin-1 and NgCAM in the retina, we have analysed the expression of their mRNAs in the present study. From stage 36 (E10) until hatching photoreceptors express axonin-1 but not NgCAM. In the inner nuclear layer groups of amacrine cells were strongly labelled with both probes but they seemed to belong to different subgroups. These patterns of expression might indicate a differential influence of the two proteins on the development of the local neural circuits of the retina.     

Pioneer growth cone steering along a series of neuronal and non-neuronal cues of different affinities

We have analyzed the morphology of over 5000 Ti1 pioneer growth cones labeled with anti-HRP, which reveals the disposition of axons, growth cone branches, and filopodia. Ti1 axon pathways typically consist of a sequence of 7 characteristically oriented segments, with a single, distinct reorientation point between each segment. Growth cones exhibit the same orientations and reorientations in a given region as do axon segments at later stages. The single, distinct reorientations suggest that growth cones make discrete switches between guidance cues as they grow. Ti1 growth cones are guided by various types of cues. A set of 3 immature identified neurons serves as nonadjacent guidepost cells and lies at the proximal end of 3 of the axon segments. To form another segment, growth cones reorient along a limb segment boundary within the epithelium. Growth cones also respond consistently to, and orient toward, a specific mesodermal cell, which may be a muscle pioneer. Thus, growth cones respond to at least 3 different types of cells in the leg. Ti1 growth cones exhibit a hierarchy of affinity for these cues. Guidepost neurons are the dominant cues in that contact with them reorients growth cones from guidance by the other types of cues. Growth cone branches are exclusively oriented to specific cues. Growth cones reorient by extending a branch directly to the cue of highest affinity and by withdrawing any branches that are extended to a cue of lesser affinity. A single filopodium in direct contact with a guidepost neuron can reorient a growth cone that still has multiple filopodia or even prominent branches specifically oriented to a previous cue of lesser affinity. These observations suggest that growth cone steering may not result simply from passive adhesion and filopodial traction, but may involve more active processes.     

Chick wing innervation. II. Morphology of motor and sensory axons and their growth cones during early development

The development and distribution of neuronal projections to the developing chick wing was studied using anterograde transport of horseradish peroxidase (HRP). Small injections of HRP were made into motor or sensory neuronal populations in order to visualize individual axons and their associated growth cones. Motor growth cones were observed in different regions of the embryo at different stages, in a proximal-to-distal pattern of distribution which paralleled the process of axon outgrowth and nerve formation. Different growth cone morphologies were associated with differing regions of the developing projection. In the spinal nerves, axons destined for the limb were unbranched and terminated in simply shaped growth cones. As axons approached the developing limb and entered the plexus region, their growth cones became more complex and larger primarily because of widening, and they sometimes branched, producing processes which could extend tens of microns from a tricorne branch point on the parent axon. Both motor and sensory fibers showed similar morphological changes in the plexus region. A distinctively shaped growth cone expanded on its leading edge was observed, sequentially apparent in the distal spinal nerves, in the plexus region, in the loosely organized axonal sheets projecting to the uncleaved dorsal or ventral muscle masses, and where muscle nerves diverged from nerve trunks and within muscle nerves. It is likely that some of these are transitional growth cones preparing to branch, because complex and branched growth cones were also observed in these regions. Branched axons oriented along the anteroposterior axis were similarly observed in the plexus region and distal to the plexus when axons first projected to the limb bud. At somewhat older stages when the basic peripheral nerve branching pattern had formed, motor growth cones were observed in common nerve trunks and in individual muscle nerves, but they were no longer found in the plexus region. Branched axons were likewise restricted to these peripheral Imations. Taken together, these observations suggest that one of the ways in which axons navigate is by exploration in the form of growth cone widening, and in some cases terminal bifurcation which may produce axon branches. Selection of the most appropriately directed growth cone process and/or precocious axonal branches may be one of the ways in which axons respond to specific growth cues which guide axons into the limb bud. Alternatively, this precocious branching may be an early neurotrophic response to developing muscle and play no significant role in axon navigation. © 1995 Wiley-Liss, Inc.