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During patterning of the peripheral nervous system, motor axons grow sequentially out of the neural tube in a segmented fashion to ensure functional integration of the motor roots between the surrounding cartilage and bones of the developing vertebrae. This segmented outgrowth is regulated by the intrinsic properties of each segment (somite) adjacent to the neural tube, and in particular by chemical repulsive guidance cues expressed in the posterior half. Yet, knockout models for such repulsive cues still display initial segmentation of outgrowing motor axons, suggesting the existence of additional, yet unknown regulatory mechanisms of axon growth segmentation. As neuronal growth is not only regulated by chemical but also by mechanical signals, we here characterized the mechanical environment of outgrowing motor axons. Using atomic force microscopy-based indentation measurements on chick embryo somite strips, we identified stiffness gradients in each segment, which precedes motor axon growth. Axon growth was restricted to the anterior, softer tissue, which showed lower cell body densities than the repulsive stiffer posterior parts at later stages. As tissue stiffness is known to regulate axon growth during development, our results suggest that motor axons also respond to periodic stiffness gradients imposed by the intrinsic mechanical properties of somites., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Schaeffer, Weber, Thompson, Keynes and Franze.)

Contact repulsion of growing axons is an essential mechanism for spinal nerve patterning. In birds and mammals the embryonic somites generate a linear series of impenetrable barriers, forcing axon growth cones to traverse one half of each somite as they extend towards their body targets. This study shows that protein disulphide isomerase provides a key component of these barriers, mediating contact repulsion at the cell surface in chick half-somites. Repulsion is reduced both in vivo and in vitro by a range of methods that inhibit enzyme activity. The activity is critical in initiating a nitric oxide/S-nitrosylation-dependent signal transduction pathway that regulates the growth cone cytoskeleton. Rat forebrain grey matter extracts contain a similar activity, and the enzyme is expressed at the surface of cultured human astrocytic cells and rat cortical astrocytes. We suggest this system is co-opted in the brain to counteract and regulate aberrant nerve terminal growth., Competing Interests: GC, CS, JS, KW, PJ, AK, EW, CC, SP, PC, GR, MR, NS, AH, AA, SW, RK No competing interests declared, (© 2020, Cook et al.)

The segmented vertebral column comprises a repeat series of vertebrae, each consisting of two key components: the vertebral body (or centrum) and the vertebral arches. Despite being a defining feature of the vertebrates, much remains to be understood about vertebral development and evolution. Particular controversy surrounds whether vertebral component structures are homologous across vertebrates, how somite and vertebral patterning are connected, and the developmental origin of vertebral bone-mineralizing cells. Here, we assemble evidence from ichthyologists, palaeontologists and developmental biologists to consider these issues. Vertebral arch elements were present in early stem vertebrates, whereas centra arose later. We argue that centra are homologous among jawed vertebrates, and review evidence in teleosts that the notochord plays an instructive role in segmental patterning, alongside the somites, and contributes to mineralization. By clarifying the evolutionary relationship between centra and arches, and their varying modes of skeletal mineralization, we can better appreciate the detailed mechanisms that regulate and diversify vertebral patterning., (© 2015. Published by The Company of Biologists Ltd.)

As part of a project to express chondroitinase ABC (ChABC) in neurons of the central nervous system, we have inserted a modified ChABC gene into an adeno-associated viral (AAV) vector and injected it into the vibrissal motor cortex in adult rats to determine the extent and distribution of expression of the enzyme. A similar vector for expression of green fluorescent protein (GFP) was injected into the same location. For each vector, two versions with minor differences were used, giving similar results. After 4 weeks, the brains were stained to show GFP and products of chondroitinase digestion. Chondroitinase was widely expressed, and the AAV-ChABC and AAV-GFP vectors gave similar expression patterns in many respects, consistent with the known projections from the directly transduced neurons in vibrissal motor cortex and adjacent cingulate cortex. In addition, diffusion of vector to deeper neuronal populations led to labelling of remote projection fields which was much more extensive with AAV-ChABC than with AAV-GFP. The most notable of these populations are inferred to be neurons of cortical layer 6, projecting widely in the thalamus, and neurons of the anterior pole of the hippocampus, projecting through most of the hippocampus. We conclude that, whereas GFP does not label the thinnest axonal branches of some neuronal types, chondroitinase is efficiently secreted from these arborisations and enables their extent to be sensitively visualised. After 12 weeks, chondroitinase expression was undiminished., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The growth cone collapse assay has proved invaluable in detecting and purifying axonal repellents. Glycoproteins/proteins present in detergent extracts of biological tissues are incorporated into liposomes, added to growth cones in culture and changes in morphology are then assessed. Alternatively purified or recombinant molecules in aqueous solution may be added directly to the cultures. In both cases after a defined period of time (up to 1 h), the cultures are fixed and then assessed by inverted phase contrast microscopy for the percentage of growth cones showing a collapsed profile with loss of flattened morphology, filopodia, and lamellipodia.

Semaphorin-3A (Sema3A) is a major guidance cue in the developing nervous system. Previous studies have revealed a dependence of responses to Sema3A on local protein synthesis (PS) in axonal growth cones, but a recent study has called this dependence into question. To understand the basis of this discrepancy we used the growth cone collapse assay on chick dorsal root ganglion neurons. We show that the dependence of growth cone collapse on protein synthesis varies according to Sema3A concentration, from near-total at low concentration (<100 ng/ml) to minimal at high concentration (>625 ng/ml). Further, we show that neuropilin-1 (NP-1) mediates both PS-dependent and PS-independent collapse. Our findings are consistent with the operation of at least two distinct Sema3A signaling pathways: one that is PS-dependent, involving mammalian target of rapamycin, and one that is PS-independent, involving GSK-3β activation and operative at all concentrations of Sema3A examined. The results provide a plausible explanation for the discrepancy in PS-dependence reported in the literature, and indicate that different signaling pathways activated within growth cones can be modulated by changing the concentration of the same guidance cue.

Several diseases and injuries of the central nervous system could potentially be treated by delivery of an enzyme, which might most effectively be achieved by gene therapy. In particular, the bacterial enzyme chondroitinase ABC is beneficial in animal models of spinal cord injury. We have adapted the chondroitinase gene so that it can direct secretion of active chondroitinase from mammalian cells, and inserted it into lentiviral vectors. When injected into adult rat brain, these vectors lead to extensive secretion of chondroitinase, both locally and from long-distance axon projections, with activity persisting for more than 4 weeks. In animals which received a simultaneous lesion of the corticospinal tract, the vector reduced axonal die-back and promoted sprouting and short-range regeneration of corticospinal axons. The same beneficial effects on damaged corticospinal axons were observed in animals which received the chondroitinase lentiviral vector directly into the vicinity of a spinal cord lesion., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Although many eukaryotic proteins have been secreted by transfected bacterial cells, little is known about how a bacterial protein is treated as it passes through the secretory pathway when expressed in a eukaryotic cell. The eukaryotic N-glycosylation system could interfere with folding and secretion of prokaryotic proteins whose sequence has not been adapted for glycosylation in structurally appropriate locations. Here we show that such interference does indeed occur for chondroitinase ABC from the bacterium Proteus vulgaris, and can be overcome by eliminating potential N-glycosylation sites. Chondroitinase ABC was heavily glycosylated when expressed in mammalian cells or in a mammalian translation system, and this process prevented secretion of functional enzyme. Directed mutagenesis of selected N-glycosylation sites allowed efficient secretion of active chondroitinase. As these proteoglycans are known to inhibit regeneration of axons in the mammalian central nervous system, the modified chondroitinase gene is a potential tool for gene therapy to promote neural regeneration, ultimately in human spinal cord injury.

Background: The polarization of somite-derived sclerotomes into anterior and posterior halves underlies vertebral morphogenesis and spinal nerve segmentation. To characterize the full extent of molecular differences that underlie this polarity, we have undertaken a systematic comparison of gene expression between the two sclerotome halves in the mouse embryo., Results: Several hundred genes are differentially-expressed between the two sclerotome halves, showing that a marked degree of molecular heterogeneity underpins the development of somite polarity., Conclusion: We have identified a set of genes that warrant further investigation as regulators of somite polarity and vertebral morphogenesis, as well as repellents of spinal axon growth. Moreover the results indicate that, unlike the posterior half-sclerotome, the central region of the anterior-half-sclerotome does not contribute bone and cartilage to the vertebral column, being associated instead with the development of the segmented spinal nerves.

Aberrant neurite growth is one of the neuropathological signatures of the Alzheimer's disease brain, both around amyloid plaques and in the cortical neuropil. Disruption of neuroinhibitory or repulsive growth and guidance signals, as well as of neurotrophic or permissive signals, may contribute to this dystrophic growth. Hence, therapeutic efforts directed exclusively at restoring neurotrophic activity are unlikely to meet with success. The molecular species responsible for neuroinhibitory effects in the Alzheimer's disease brain are beginning to be elucidated.

The analysis of the outgrowth pattern of spinal axons in the chick embryo has shown that somites are polarized into anterior and posterior halves. This polarity dictates the segmental development of the peripheral nervous system: migrating neural crest cells and outgrowing spinal axons traverse exclusively the anterior halves of the somite-derived sclerotomes, ensuring a proper register between spinal axons, their ganglia and the segmented vertebral column. Much progress has been made recently in understanding the molecular basis for somite polarization, and its linkage with Notch/Delta, Wnt and Fgf signalling. Contact-repulsive molecules expressed by posterior half-sclerotome cells provide critical guidance cues for axons and neural crest cells along the anterior-posterior axis. Diffusible repellents from surrounding tissues, particularly the dermomyotome and notochord, orient outgrowing spinal axons in the dorso-ventral axis ('surround repulsion'). Repulsive forces therefore guide axons in three dimensions. Although several molecular systems have been identified that may guide neural crest cells and axons in the sclerotome, it remains unclear whether these operate together with considerable overall redundancy, or whether any one system predominates in vivo.

The backbone or vertebral column is the defining feature of vertebrates and is clearly metameric. Given that vertebrae arise from segmented paraxial mesoderm in the embryo, this metamerism is not surprising. Fate mapping studies in a variety of species have shown that ventromedial sclerotome cells of the differentiated somite contribute to the developing vertebrae and ribs. Nevertheless, extensive studies in amniote embryos have produced conflicting data on exactly how embryonic segments relate to those of the adult. To date, much attention has focused on the derivatives of the somites, while relatively little is known about the contribution of other tissues to the formation of the vertebral column. In particular, while it is clear that signals from the notochord induce and maintain proliferation of the sclerotome, and later promote chondrogenesis, the role of the notochord in vertebral segmentation has been largely overlooked. Here, we review the established role of the notochord in vertebral development, and suggest an additional role for the notochord in the segmental patterning of the vertebral column.

Here we present ideas connecting the behaviour of the cranial neural crest during development with the venerable, perhaps incorrect, view that gill-supporting cartilages of an ancient agnathan evolved into the skeleton of an early gnathostome's jaw. We discuss the pattern of migration of the cranial neural crest ectomesenchyme in zebrafish, along with the subsequent arrangement of postmigratory crest and head mesoderm in the nascent pharyngeal segments (branchiomeres), in diverse gnathostomes and in lampreys. These characteristics provide for a plausible von Baerian explanation for the problematic inside-outside change in topology of the gills and their supports between these 2 major groups of vertebrates. We consider it likely that the jaw supports did indeed arise from branchiomeric cartilages.

In higher vertebrates, the segmental organization of peripheral spinal nerves is established by a repulsive mechanism whereby sensory and motor axons are excluded from the posterior half-somite. A number of candidate axon repellents have been suggested to mediate this barrier to axon growth, including Sema3A, Ephrin-B, and peanut agglutinin (PNA)-binding proteins. We have tested the candidacy of these factors in vitro by examining their contribution to the growth cone collapse-inducing activity of somite-derived protein extracts on sensory, motor, and retinal axons. We find that Sema3A is unlikely to play a role in the segmentation of sensory or motor axons and that Ephrin-B may contribute to motor but not sensory axon segmentation. We also provide evidence that the only candidate molecule(s) that induces the growth cone collapse of both sensory and motor axons binds to PNA and is not Sema3A or Ephrin-B. By grafting primary sensory, motor, and quail retinal neurons into the chick trunk in vivo, we provide further evidence that the posterior half-somite represents a universal barrier to growing axons. Taken together, these results suggest that the mechanisms of peripheral nerve segmentation should be considered in terms of repellent molecules in addition to the identified molecules., (Copyright 2000 Academic Press.)

The secreted glycoprotein Sonic hedgehog (SHH), a vertebrate homologue of the Drosophila segment polarity gene Hedgehog, is essential for the development of diverse tissues during embryogenesis. Studies of SHH function during neural tube and somite development have focused on its role in specifying the dorsoventral polarity of these structures, but a recent report by Ahlgren and Bronner-Fraser(1) supports the possibility that SHH has additional functions in cell survival and cell proliferation. Perturbation of SHH signaling after the early dorsoventral specification of the cranial neural tube leads to increased cell death in both the neural tube and the neural crest. This implies that SHH is continually required as a trophic and/or mitogenic factor during brain development, and expands the variety of cellular responses to SHH signaling. BioEssays 22:499-502, 2000., (Copyright 2000 John Wiley & Sons, Inc.)

The study of spinal nerve trajectories in higher vertebrate embryos has revealed an inherent polarity within somites along the antero-posterior axis, and provides a simple system in which to study the factors that influence axon pathfinding. We argue that the orientation of spinal axons is determined by the simultaneous operation of two distinct guidance mechanisms, contact repulsion and chemorepulsion. Motor and sensory axons traverse the anterior half of each somite because they are excluded by contact repulsion from the posterior half-somite, and the molecular nature of several candidate contact repellents is reviewed. In contrast, we find that the dorsoventral trajectory of primary sensory axons is oriented by diffusible repellents originating from the notochord medially and dermamyotome laterally. In this system, therefore, repulsion by surrounding tissues ('surround-repulsion') is the main force directing axon growth in three dimensions.

During development of the vertebrate visual system, retinal ganglion cell (RGC) axons follow a precise path toward their midbrain targets. Although much is known about the cues that direct RGC axons once they have left the optic disc, less is known about the guidance of axons at earlier stages, when RGCs first send out their axons to navigate within the developing retina. Using collagen gel coculture experiments, we find that the embryonic lens produces a powerful diffusible repulsive activity for RGC axons. We also find that this activity is localized to the lens epithelium and not the lens fiber layer, while the pigmented epithelium and vitreous humour are devoid of activity. The further observation that the lens also chemorepels primary sensory axons, but does not repel olfactory bulb axons, shows that this activity is specific for subsets of axons. Our experiments have excluded two candidate repellents for RGC axons (collapsin-1/sema III and chondroitin sulfate proteoglycans). These results implicate the lens in the earliest stages of RGC axon guidance. One function of the lens repellent may be to prevent aberrant targeting toward the lens, and it may also be involved in the directional guidance of RGC axons toward the optic disc., (Copyright 1999 Academic Press.)

The segmental arrangement of spinal nerves in higher vertebrate embryos provides a simple system in which to study the factors that influence axon pathfinding. Developing motor and sensory axons are intimately associated with surrounding tissues that direct axon guidance. We argue that two distinct guidance mechanisms, viz. contact repulsion and chemorepulsion, act simultaneously to prescribe spinal axon trajectories by 'surround-repulsion'. Motor and sensory axons grow freely within the anterior half of each mesodermal somite, because they are excluded from posterior half-somites by contact repulsion. By contrast, the dorsoventral trajectory that bipolar sensory axons of the dorsal root ganglia follow is governed by diffusible repellents originating from the notochord medially and dermamyotome laterally. Even though spinal nerve development appears to be a simple system for elucidating axon guidance mechanisms, many distinct candidate guidance molecules have been implicated and their relative contributions remain to be evaluated.

The adult mammalian central nervous system (CNS) lacks the capacity to support axonal regeneration. There is increasing evidence to suggest that astrocytes, the major glial population in the CNS, may possess both axon-growth promoting and axon-growth inhibitory properties and the latter may contribute to the poor regenerative capacity of the CNS. In order to examine the molecular differences between axon-growth permissive and axon-growth inhibitory astrocytes, a panel of astrocyte cell lines exhibiting a range of axon-growth promoting properties was generated and analysed. No clear correlation was found between the axon-growth promoting properties of these astrocyte cell lines with: (i) the expression of known neurite-outgrowth promoting molecules such as laminin, fibronectin and N-cadherin; (ii) the expression of known inhibitory molecules such tenascin and chondroitin sulphate proteoglycan; (iii) plasminogen activator and plasminogen activator inhibitor activity; and (iv) growth cone collapsing activity. EM studies on aggregates formed from astrocyte cell lines, however, revealed the presence of an abundance of extracellular matrix material associated with the more inhibitory astrocyte cell lines. When matrix deposited by astrocyte cell lines was assessed for axon-growth promoting activity, matrix from permissive lines was found to be a good substrate, whereas matrix from the inhibitory astrocyte lines was a poor substrate for neuritic growth. Our findings, taken together, suggest that the functional differences between the permissive and the inhibitory astrocyte cell lines reside largely with the ECM.

Repulsive or inhibitory interactions between growth cones and their environment are now widely implicated in neural development and regeneration. Over the past year, descriptive studies of the various neuronal systems in which repulsion may participate have clarified its biology. Molecular and genetic studies have also provided the necessary entry point for further experimental manipulations, and are beginning to yield important clues regarding the function of repulsion in vivo. Although candidate second messengers underlying the growth cone response have been identified, they have yet to be incorporated into a comprehensive mechanism.

There is increasing evidence that axons are guided by repulsion in several regions of the developing nervous system, although this has yet to be confirmed directly in vivo. As more candidate repulsion molecules are identified, it is becoming clear that collapse of the growth cone in vitro may be mediated by more than one intracellular mechanism. The present emphasis on molecular cloning of the ligands and their receptors should enable a proper definition of their function during development.

Sulfated polysaccharides have been shown to inhibit human immunodeficiency virus (HIV) infection in vitro. Dextrin sulfate, fucoidan, and dextran sulfate fail to neutralize virions directly, but interact with target cells to inhibit virus entry. Ionic interactions of sulfated polyanions with oppositely charged cell surface components, including CD4, have been assumed to be the inhibitory mechanism. It is shown that the sulfated polysaccharides inhibit infection of both CD4+ and CD4- cell lines by HIV and also that they inhibit HTLV-1 and, to a lesser extent, the simian retrovirus, MPMV, which use receptors other than CD4. One binding site for radiolabeled fucoidan on the surface of human T cells is an 18 kD protein, but its significance is not yet clear.

Evacuated muscle is a possible substitute for nerve autografts in the repair of damaged peripheral nerves. Previous experiments have shown that killed or evacuated muscle grafts are as effective as nerve autografts for bridging gaps of up to 4 cm between proximal and distal nerve stumps. Evacuated muscle grafts are made of extracellular matrix components, which are good substrates for axon growth in vitro. However, experiments in vivo have generally demonstrated that live Schwann cells are essential for successful axon regeneration. In the present experiments we have used immunohistochemical techniques with anti-S100 and anti-neurofilament antibodies to visualize axon growth and Schwann cell migration into muscle grafts over the first 10 days following grafting. We only saw axons growing into grafts accompanied by Schwann cells, and most though not all Schwann cells were associated with axons. Schwann cell migration from the proximal stump in association with axons was much faster and more extensive than from the distal stump. We examined muscle grafts over the first 20 days after grafting by electron microscopy. Regenerating axons were always associated with Schwann cells, which were mostly in the basal lamina-lined tubes left by the evacuated myofibrils. A comparison between evacuated muscle grafts and grafts in which the muscle had been killed but not evacuated revealed that 7 days after grafting there were more than twice as many regenerated axons in and distal to the evacuated grafts, but that by 20 days the numbers of axons were similar in the two groups.

We have investigated whether the neuromeres of the developing chick spinal cord (myelomeres) are manifestations of intrinsic segmentation of the CNS by studying the patterns of cell proliferation and neuronal differentiation. Treatment of 2-day embryos with colchicine does produce exaggerated myelomeres, in confirmation of Källén (Z. Anat. Entwickl.-Gesch. 123, 309-319, 1962). However, this does not imply that myelomeres are segmental proliferation centres: the undulations caused by colchicine are irregular alongside the unsegmented mesoderm, and another mitotic inhibitor, bromodeoxyuridine, has no such effects. In contrast to lower vertebrate embryos, there is no evidence for segmental groups of primary motor neurons in the chick: the earliest motor neurons express cholinesterase, and project their axons into the adjacent sclerotome, at random positions in relation to the somite boundaries. The population of motor neurons projecting HRP-labelled axons into a single somite lies out of phase with both myelomere and somite, and is placed symmetrically about the anterior half-sclerotome. The earliest intrinsic spinal cord neurons, as stained with zinc iodide-osmium tetroxide or anti-68 x Mr neurofilament antibody, show no segmental patterns of differentiation. We conclude that, in contrast to the rhombomeres of the developing hindbrain, myelomeres are not matched by segmental groupings of differentiating nerve cells, and result from mechanical moulding of the neuroepithelium by the neighbouring somites.

We have investigated whether the developing spinal cord is intrinsically segmented in its rostrocaudal (anteroposterior) axis by mapping the spread of clones derived from single labelled cells within the neural tube of the chick embryo. A single cell in the ventrolateral neural tube of the trunk was marked in situ with the fluorescent tracer lysinated rhodamine dextran (LRD) and its descendants located after two days of further incubation. We find that clones derived from cells labelled before overt segmentation of the adjacent mesoderm do not respect any boundaries within the neural tube. Those derived from cells marked after mesodermal segmentation, however, never cross an invisible boundary aligned with the middle of each somite, and tend to be elongated along the mediolateral axis of the neural tube. When the somite pattern is surgically disturbed, neighbouring clones derived from neuroectodermal cells labelled after somite formation behave like clones derived from younger cells: they no longer respect any boundaries, and are not elongated mediolaterally. These results indicate that periodic lineage restrictions do exist in the developing spinal cord of the chick embryo, but their maintenance requires the presence of the adjacent somite mesoderm.

The guidance of axons during embryonic development is likely to involve both adhesive and repulsive interactions between growth cones and their environment. We are characterising the role and mechanism of repulsion during the segmental outgrowth of motor and sensory axons in the somite mesoderm of chick embryos. Axons are confined to the anterior half of each somite by the expression in the posterior half of a glycoconjugate system (48 x 10(3) M(r) and 55 x 10(3) M(r)) that causes the collapse of dorsal root ganglion growth cones when applied in vitro. Enzymatic cleavage of this fraction with specific combinations of endo- and exoglycosidases removes collapse activity, suggesting that carbohydrate residues are involved in the execution of collapse. A similar activity is also detectable in normal adult grey matter, suggesting roles for repulsion beyond the development of spinal nerve segmentation.

Although most proto-oncogenes such as c-myc are involved in cell proliferation, being expressed in a wide range of tissues as well as in progenitors of transformed cells, others may normally function in cellular differentiation. We now report on a gene on human chromosome 11, at the junction of the T-cell tumour-associated chromosomal translocation t(11; 14) (p15; q11) and known as the 11p15 gene or Ttg, which is believed to be involved in the pathogenesis of the tumour. It has two transcriptional promoters (both retained by the translocated allele) and is expressed in tumour cells with neuro-endocrine properties, suggesting that normal expression may occur in nerve cells. Using fusion constructs of one 11p15 promoter and lacZ in transgenic mice, we found that the gene is expressed in a segment-specific manner in rhombomeres of the developing mouse hind-brain. During subsequent development, the gene is more widely expressed, again in precisely defined regional patterns, but in post-mitotic neurons confined to the central nervous system. Thus, this presumptive T-cell oncogene is both developmentally regulated and segmentally restricted in a tissue different from that in which the original tumour arose.

The segmented pattern of peripheral spinal nerves in higher vertebrates is generated by interactions between nerve cells and somites. Neural crest cells, motor axons, and sensory axons grow exclusively through anterior-half sclerotome. In chick embryos, posterior cells bind the lectins peanut agglutinin (PNA) and Jacalin. When liposomes containing somite extracts are applied to cultures of chick sensory neurons, growth cones collapse abruptly, recovering within 4 hr of liposome removal. Collapse activity is eliminated by immobilized PNA, and SDS-PAGE demonstrates two major components (48K and 55K), which are absent from anterior-half sclerotome. Rabbit polyclonal antibodies against these components recognize only posterior cells and may also be used to eliminate collapse activity. We suggest that spinal nerve segmentation is produced by inhibitory interactions between these components and growth cones.

We have investigated the interactions between the cells of the rostral and caudal halves of the chick somite by carrying out grafting experiments. The rostral half-sclerotome was identified by its ability to support axon outgrowth and neural crest cell migration, and the caudal half by the binding of peanut agglutinin and the absence of motor axons and neural crest cells. Using the chick-quail chimaera technique we also studied the fate of each half-somite. It was found that when half-somites are placed adjacent to one another, their interactions obey a precise rule: sclerotome cells from like halves mix with each other, while those from unlike halves do not; when cells from unlike halves are adjacent to one another, a border is formed. Grafting quail half-somites into chicks showed that the fates of the rostral and caudal sclerotome halves are similar: both give rise to bone and cartilage of the vertebral column, as well as to intervertebral connective tissue. We suggest that the rostrocaudal subdivision serves to maintain the segmental arrangement when the mesenchymal sclerotome dissociates, so that the nervous system, vasculature and possibly vertebrae are patterned correctly.

The part played by basement membrane in the guidance of peripheral nerve growth in vivo has been assessed by examining the capacity of degenerating mouse muscle to support the regeneration of the cut sciatic and saphenous nerves. Ethanol and formaldehyde-fixed gluteus maximus muscles were implanted around the contralateral cut nerves. The subsequent nerve growth into the degenerating muscle was assessed by silver staining after 3, 4 and 10 days. By 4 days, linear axonal growth was seen, parallel to the length of the muscle fibres, and coinciding with the onset of degeneration of the sarcoplasm. Transverse sections of the 10 day preparations showed that over 90% of linearly growing axons were located inside the remaining sheaths of muscle fibre basement membrane. This relationship was confirmed by electron microscopy of ruthenium red-stained preparations. Both motor and sensory axons were able to grow in this manner, for electrophysiological testing revealed the presence of motor axons from the sciatic nerve, while the saphenous nerve contains only sensory axons. Identical growth was seen at 10 days in muscles caused to degenerate by incubation in distilled water. However, linear growth did not occur in live-innervated and glutaraldehyde-fixed muscles, in which muscle fibre architecture was preserved. It is concluded that basement membrane derived from muscle can promote peripheral nerve regeneration. Furthermore, both motor and sensory axons show a strong preference for growth along its inner surface, the basal lamina.

Six asthmatic subjects and six matched controls performed a standardized exercise test in which resting and post-exercise blood samples were assayed radioenzymatically for catecholamines. Plasma adrenaline one minute after exercise rose over threefold in the controls whereas in the asthmatic subjects there was no increase. Plasma noradrenaline increased to five times the basal concentration in the controls, but to less than half this level in the asthmatic subjects. These results agree with a recent study (Barnes et al. 1981) and suggest there is a marked abnormality of the sympathoadrenal response to exercise in asthmatic subjects.

After neural processes emerge from the neural tube in the chick embryo, their growth is restricted to the cranial halves of the neighbouring somites. In this study we have developed an in vitro system to model the interactions between these tissue types. Pioneer neurites display a hierarchy of preferences in terms of the substrates they can grow on. As expected, tissue culture plastic does not support neural outgrowth, but this can be overcome by coating the plastic substrate with either collagen or poly-L-lysine. Neural crest, cranial half somite, and a number of other tissues support growth well, while caudal half somite and tail bud mesenchyme do so to a much smaller extent. The binding pattern of a variety of lectins was assessed in cryostat sections of embryos and in cultured cells of the above tissues. It was found that peanut agglutinin can discriminate between cranial and caudal sclerotome both in vitro and in the embryo, since it binds preferentially to caudal sclerotome in both cases. This difference is expressed as soon as the sclerotome forms. The significance of these findings is twofold: first, they show that the interactions that take place during peripheral neural segmentation can be modelled in vitro; second, they represent the first instance of a molecular difference between the cranial and caudal halves of the sclerotome, detectable both in culture and in the embryo.

1. A study was made of short- and long-term effects of paralysis induced by type A botulinum toxin on the development of innervation of mouse muscles. The toxin was injected locally over the tensor fasciae latae (t.f.l.) and gluteus muscles at various times after birth, and the innervation was later examined by intracellular recording and by a histological technique using a reduced silver stain for axons.2. Paralysis induced at 0-4 days of age delayed but did not prevent the eventual elimination of nearly all focal multiple innervation in gluteus muscle fibres, whereas in t.f.l. up to 50% of the fibres remained focally innervated by more than one axon for at least 120 days. There was an associated reduction in the number of muscle fibres in t.f.l. of between 50 and 70%. The biggest reduction in the number of gluteus fibres was under 40%.3. In the t.f.l., paralysis begun at 6-9 days caused the extent of single-site polyneuronal innervation to increase above the level existing at the time of paralysis. Histologically this increase was seen to be due at least in part to the stimulation of nodal sprout growth from a limited number of nodes.4. Motor nerve terminal sprouts were evoked by paralysis at all ages. In mice injected before 4 days of age recovery from the toxin occurred rapidly and without the formation of ectopic synapses by sprouted motor terminals; however, intrafusal motor nerves also sprouted and established a permanent ectopic innervation on surrounding extrafusal muscle fibres.5. The following conclusions are drawn. (a) In some but not all muscles, neonatally induced paralysis can not only temporarily halt elimination of polyneuronal innervation but actually lead to an increase; it is suggested tentatively that this occurs only during the neonatal period because of the availability at that time of endoneurial pathways associated with normal polyneuronal innervation, rather than because of any special neuronal growth potential then. (b) Permanent establishment of focal polyneuronal innervation is due not to stability of presynaptic elements maintained past a critical developmental stage but is associated with substantial losses of muscle fibres, leading to a high ratio of nerve fibres to muscle fibres.

1. The properties of denervated and fully innervated but paralysed soleus and extensor digitorum longus (e.d.l.) muscles were compared in mice.2. Muscles were paralysed by single or repeated injections of botulinum toxin into the right lower leg. Contralateral muscles were denervated at the same time by sciatic nerve section. Muscles were excised 3-20 days later and direct and indirect isometric twitch and tetanic tensions measured in vitro. ACh sensitivity was assessed from isometric contractions to various concentrations of bath-applied ACh perchlorate.3. Denervated muscles as expected from the work of others developed sensitivity to ACh earlier and more rapidly than paralysed muscles. A transient peak of sensitivity was reached after 7 days of denervation in soleus and after 8 days in e.d.l. However 2 days later the sensitivity of muscles fully paralysed to nerve twitch and tetanic stimuli was not significantly different from that of denervated muscles.4. In both e.d.l. and soleus the direct tetanus/twitch ratios declined in paralysed and denervated muscles. The decline was initially more rapid in denervated than paralysed soleus, but the ratios for paralysed and denervated muscles were not significantly different after 10 days in either muscle. By this time there were no differences in the absolute strengths of the paralysed and denervated muscles.5. It is concluded that the ACh sensitivity and dynamic properties of fully paralysed mouse muscles become equal to those of denervated muscles once the acute effects associated with nerve degeneration are past. This result is consistent with the idea that normal neuronal control of extrajunctional mammalian skeletal muscle properties is mediated solely by means of activity.

Administration to mice of a 0.1% solution of pyronin G in their drinking water caused an acceleration both of axonal sprouting from nodes of Ranvier in partly denervated gluteus maximus muscles, and of motor nerve regeneration following a crush to the soleus nerve. Sprouting from soleus motor nerve terminals in response to botulinum toxin-induced paralysis was, however, unaffected. Removal of degenerating axons following nerve section was also accelerated by pyronin treatment. Pyronin is therefore likely to act upon the process of Wallerian degeneration, rather than upon intact motor nerves directly.

Treatment of the mouse gluteus maximus with the organophosphorus anticholinesterase ecothiopate, 4-aminopyridine and caffeine caused necrosis of the endplate region of the muscle fibres. When these drugs were given 12 h prior to partial denervation of the muscle there was a significant reduction in the amount of nodal sprouting seen 48 h after nerve section. No such reduction was seen if the drugs were given immediately after nerve section. In the former case both innervated and denervated fibres are necrotic; in the latter, only innervated fibres are necrotic. The amount of terminal sprouting was significantly increased in the former group, showing that the drug treatment and necrosis were not directly inhibitory to nerve growth. The reduction of nodal sprouting is therefore likely to be due to necrosis of denervated fibres; this supports the hypothesis that the denervated endplate is the source of a stimulus to nodal sprouting of motoneurones.

The embryonic origin of peripheral nerve Schwann/sheath cells is still uncertain. Although the neural crest is known to be an important source, it is not clear whether the ventral neural tube also contributes a progenitor population for motor axons. We have used the techniques of immunohistochemistry, electron microscopy and quail-chick grafting to examine this problem. Immunohistochemistry with monoclonal antibody HNK-1 identified a cluster of immunoreactive cells in the sclerotome, at the site of the future ventral root. With the electron microscope, nucleated cells could not be seen breaching the basal lamina of the neural tube, exclusively in the region of the ventral root and preceding axon outgrowth. After grafting a length of crest-ablated quail neural tube in place of host chick neural tube, a population of quail cells was found localized to the ventral root exit zone, associated with the ventral root axons. Taken together, these observations support the possibility of a neural tube origin for ventral root sheath cells, although we found no evidence for a more extensive migration of these cells. The ventral root cells share certain phenotypic traits, such as HNK-1 immunoreactivity, with neural-crest-derived Schwann cells, but are not necessarily identical to them. We argue that while they may help motor axons to exit the neural tube at the correct position, they are unlikely to guide axons beyond the immediate vicinity of the neural tube.

We have studied the pathway of migration of neural crest cells through the somites of the developing chick embryo, using the monoclonal antibodies NC-1 and HNK-1 to stain them. Crest cells, as they migrate ventrally from the dorsal aspect of the neural tube, pass through the lateral part of the sclerotome, but only through that part of the sclerotome which lies in the rostral half of each somite. This migration pathway is almost identical to the path which presumptive motor axons take when they grow out from the neural tube shortly after the onset of neural crest migration. In order to see whether the ventral root axons are guided along this pathway by neural crest cells, we surgically excised the neural crest from a series of embryos, and examined the pattern of axon outgrowth approximately 24 h later. In somites which contained no neural crest cells, ventral root axons were still found only in the rostral half of the somite, although axonal growth was slightly delayed. These axons were surrounded by sheath cells, which had presumably migrated out of the neural tube, to a point about 50 micron proximal to the growth cones. With appropriate antibodies we found that the extracellular matrix components fibronectin and laminin are evenly distributed between the rostral and caudal halves of the somite. Neither of these molecules therefore plays a critical role in determining the specific pathway of neural crest cells or motor axons through the rostral half of the somite.

A reduced silver stain was used to examine the development of complexity of motor nerve terminals in the postnatal period. Terminals in three histochemically different muscles were examined in mice aged 12 days to 3 years. The total number and total length of intraterminal axon branches increases with age, but only until animals are 3 months old. Terminals become longest and most branched in the histochemically glycolytic tensor fascia latae (TFL) muscle, shortest and least branched in the oxidative diaphragm, and intermediate in the histochemically mixed gluteus muscle. In addition, myelinated terminal branches develop in TFL and to a lesser extent in the gluteus between 1 and 3 months of age. These myelinated branches appear to be produced by nodal sprouting from the penultimate node of Ranvier of the terminal axon, and also by myelination of pre-existing terminal branches. The diameter of muscle fibres also increases until animals are 3 months old, and there is a good correlation between mean fibre diameter and either mean terminal length or mean number of terminal branches when all muscles at all ages are compared. This suggests that terminal growth could be determined by muscle fibre growth; however, within any given muscle there is little or no correlation between the diameter of a muscle fibre and either the length or number of branches of its nerve terminal, suggesting that terminal morphology is not controlled solely by muscle fibre growth. The presence of a myelinated branch in a nerve terminal is also unrelated to fibre diameter within a given muscle, but again when means are compared there are good, but significantly different correlations for the three different muscles. Thus some kind of muscle or nerve type-specific property additional to a general effect of muscle fibre size influences the development of myelinated terminal branches. Between 3 and 12 months of age terminal complexity remains constant or may decrease slightly. At 19 months or older, when mice are becoming senile, a large proportion of synapses have terminal sprouts and muscle fibres become innervated by two or more distinct axons. These changes can be attributed to the death of some motor neurons and sprouting of the remaining axons.

This study provides evidence that cells destined to segment together into somites have a degree of cell division synchrony. We have measured the duration of the cell division cycle in somite and segmental plate cells of the chick embryo as 9.5 h using [3H]thymidine pulse- and-chase. Treatment of embryos with any of a variety of inhibitors known to affect the cell division cycle causes discrete periodic segmental anomalies: these anomalies appear about 6-7 somites after treatment and, in some cases, a second anomaly is observed 6 to 7 somites after the first. Since somites take 1.5 h to form, the 6- to 7- somite interval corresponds to about 9-10 h, which is the duration of the cell cycle as determined in these experiments. The anomalies are similar to those seen after heat shock of 2-day chick embryos. Heat shock and some of the other treatments induce the expression of heat-shock proteins (hsp); however, since neither the expression nor the distribution of these proteins relate to the presence or distribution of anomalies seen, we conclude that hsps are not responsible for the pattern of segmental anomalies observed. The production of periodic segmental anomalies appears to be linked to the cell cycle. A simple model is proposed, in which we suggest that the cell division cycle is involved directly in gating cells that will segment together.

In all higher vertebrate embryos the sensory ganglia of the trunk develop adjacent to the neural tube, in the cranial halves of the somite-derived sclerotomes. It has been known for many years that ganglia do not develop in the most cranial (occipital) sclerotomes, caudal to the first somite. Here we have investigated whether this is due to craniocaudal variation in the neural tube or crest, or to an unusual property of the sclerotomes at occipital levels. Using the monoclonal antibody HNK-1 as a marker for neural crest cells in the chick embryo, we find that the crest does enter the cranial halves of the occipital sclerotomes. Furthermore, staining with zinc iodide/osmium tetroxide shows that some of these crest-derived cells sprout axons within these sclerotomes. By stage 23, however, no dorsal root ganglia are present within the five occipital sclerotomes, as assessed both by haematoxylin/eosin and zinc iodide/osmium tetroxide staining. Moreover, despite this loss of sensory cells, motor axons grow out in these segments, many of them later fasciculating to form the hypoglossal nerve. The sclerotomes remain visible until stages 27/28, when they dissociate to form the base of the skull and the atlas and axis vertebrae. After grafting occipital neural tube from quail donor embryos in place of trunk neural tube in host chick embryos, quail-derived ganglia do develop in the trunk sclerotomes. This shows that the failure of occipital ganglion development is not the result of some fixed local property of the neural crest or neural tube at occipital levels. We therefore suggest that in the chick embryo the cranial halves of the five occipital sclerotomes lack factors essential for normal sensory ganglion development, and that these factors are correspondingly present in all the more caudal sclerotomes.

The suitability of muscle basal lamina as a graft material for the repair of peripheral nerves was investigated. Grafts were prepared by evacuating the myoplasm from muscles excised from rats and rabbits. This produced a material consisting mainly of basal lamina and connective tissue, with the basal lamina arranged as parallel tubes. Rat- and rabbit-derived graft material in 0.5-cm lengths was sutured into rat sciatic nerves, and 4-cm lengths of rabbit-derived graft material were interposed into rabbit sciatic nerves. For controls, 0.5-cm nerve autografts were grafted into rats and 4-cm autografts into rabbits. After 2 to 3 months, the success of the grafts was assessed functionally, electrophysiologically, and anatomically. By all these criteria the basal lamina grafts were as successful as nerve autografts; essentially the same number of axons of the same size grew through both graft types, animals recovered their limb function equally well, and the nerve conduction velocities and relative refractory periods were the same in both groups of animals. In rats, following both basal lamina and nerve autografts, the number of axons distal to the grafts was approximately the same as that proximal to them, but axon diameter and speed of conduction were significantly less than normal. The authors conclude that muscle basal lamina grafts are as effective as nerve autografts for repairing severed rat or rabbit peripheral nerves, and suggest that grafts prepared in this way may prove to be useful for nerve repair in humans.

Treatment of mice with daily intraperitoneal gangliosides (50 mg/kg) enhanced both the number and mean length of nodal sprouts 4 days after partial denervation of the gluteus maximus muscle. Terminal sprouting in the soleus muscle 14 days after botulinum toxin paralysis was also enhanced by gangliosides, as assessed by zinc iodide-osmium tetroxide staining. Functional recovery from paralysis, assessed electrophysiologically, was simultaneously accelerated; this may have resulted from the enlarged terminal area, rather than reflecting an additional action of gangliosides upon synaptogenesis.