Axons in the Chick Embryo Follow Soft Pathways Through Developing Somite Segments.

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.

Regulation of nerve growth and patterning by cell surface protein disulphide isomerase.

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.

Building the backbone: the development and evolution of vertebral patterning.

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.

Growth cone collapse assay.

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.

AAV vector-mediated secretion of chondroitinase provides a sensitive tracer for axonal arborisations.

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.

Differing semaphorin 3A concentrations trigger distinct signaling mechanisms in growth cone collapse.

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-3ß 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.

Lentiviral vectors express chondroitinase ABC in cortical projections and promote sprouting of injured corticospinal axons.

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.

Modification of N-glycosylation sites allows secretion of bacterial chondroitinase ABC from mammalian cells.

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.

Extensive molecular differences between anterior- and posterior-half-sclerotomes underlie somite polarity and spinal nerve segmentation.

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.

Somite polarity and segmental patterning of the peripheral nervous system.

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.