Integrity of developing spinal motor columns is regulated by neural crest derivatives at motor exit points

Spinal motor neurons must extend their axons into the periphery through motor exit points (MEPs), but their cell bodies remain within spinal motor columns. It is not known how this partitioning is established in development. We show here that motor neuron somata are confined to the CNS by interactions with a neural crest subpopulation, boundary cap (BC) cells that prefigure the sites of spinal MEPs. Elimination of BC cells by surgical or targeted genetic ablation does not perturb motor axon outgrowth but results in motor neuron somata migrating out of the spinal cord by translocating along their axons. Heterologous neural crest grafts in crest-ablated embryos stop motor neuron emigration. Thus, before the formation of a mature transitional zone at the MEP, BC cells maintain a cell-tight boundary that allows motor axons to cross but blocks neuron migration.     

Microtubule-associated protein 2 within axons of spinal motor neurons: associations with microtubules and neurofilaments in normal and beta,beta'-iminodipropionitrile-treated axons

We have examined the distribution of microtubule-associated protein 2 (MAP2) in the lumbar segment of spinal cord, ventral and dorsal roots, and dorsal root ganglia of control and beta,beta'-iminodipropionitrile- treated rats. The peroxidase-antiperoxidase technique was used for light and electron microscopic immunohistochemical studies with two monoclonal antibodies directed against different epitopes of Chinese hamster brain MAP2, designated AP9 and AP13. MAP2 immunoreactivity was present in axons of spinal motor neurons, but was not detected in axons of white matter tracts of spinal cord and in the majority of axons of the dorsal root. A gradient of staining intensity among dendrites, cell bodies, and axons of spinal motor neurons was present, with dendrites staining most intensely and axons the least. While dendrites and cell bodies of all neurons in the spinal cord were intensely positive, neurons of the dorsal root ganglia were variably stained. The axons of labeled dorsal root ganglion cells were intensely labeled up to their bifurcation; beyond this point, while only occasional central processes in dorsal roots were weakly stained, the majority of peripheral processes in spinal nerves were positive. beta,beta'- Iminodipropionitrile produced segregation of microtubules and membranous organelles from neurofilaments in the peripheral nervous system portion and accumulation of neurofilaments in the central nervous system portion of spinal motor axons. While both anti-MAP2 hybridoma antibodies co-localized with microtubules in the central nervous system portion, only one co-localized with microtubules in the peripheral nervous system portion of spinal motor axons, while the other antibody co-localized with neurofilaments and did not stain the central region of the axon which contained microtubules. These findings suggest that (a) MAP2 is present in axons of spinal motor neurons, albeit in a lower concentration or in a different form than is present in dendrites, and (b) the MAP2 in axons interacts with both microtubules and neurofilaments.     

Peripheral glia direct axon guidance across the CNS/PNS transition zone.

CNS glia have integral roles in directing axon migration of both vertebrates and insects. In contrast, very little is known about the roles of PNS glia in axonal pathfinding. In vertebrates and Drosophila, anatomical evidence shows that peripheral glia prefigure the transition zones through which axons migrate into and out of the CNS. Therefore, peripheral glia could guide axons at the transition zone. We used the Drosophila model system to test this hypothesis by ablating peripheral glia early in embryonic neurodevelopment via targeted overexpression of cell death genes grim and ced-3. The effects of peripheral glial loss on sensory and motor neuron development were analyzed. Motor axons initially exit the CNS in abnormal patterns in the absence of peripheral glia. However, they must use other cues within the periphery to find their correct target muscles since early pathfinding errors are largely overcome. When peripheral glia are lost, sensory axons show disrupted migration as they travel centrally. This is not a result of motor neuron defects, as determined by motor/sensory double-labeling experiments. We conclude that peripheral glia prefigure the CNS/PNS transition zone and guide axons as they traverse this region.     

Reciprocal interactions between olfactory receptor axons and olfactory nerve glia cultured from the developing moth Manduca sexta

In olfactory systems, neuron-glia interactions have been implicated in the growth and guidance of olfactory receptor axons. In the moth Manduca sexta, developing olfactory receptor axons encounter several types of glia as they grow into the brain. Antennal nerve glia are born in the periphery and enwrap bundles of olfactory receptor axons in the antennal nerve. Although their peripheral origin and relationship with axon bundles suggest that they share features with mammalian olfactory ensheathing cells, the developmental roles of antennal nerve glia remain elusive. When cocultured with antennal nerve glial cells, olfactory receptor growth cones readily advance along glial processes without displaying prolonged changes in morphology. In turn, olfactory receptor axons induce antennal nerve glial cells to form multicellular arrays through proliferation and process extension. In contrast to antennal nerve glia, centrally derived glial cells from the axon sorting zone and antennal lobe never form arrays in vitro, and growth-cone glial-cell encounters with these cells halt axon elongation and cause permanent elaborations in growth cone morphology. We propose that antennal nerve glia play roles similar to olfactory ensheathing cells in supporting axon elongation, yet differ in their capacity to influence axon guidance, sorting, and targeting, roles that could be played by central olfactory glia in Manduca.     

Human dorsal root ganglion neurons from embryonic donors extend axons into the host rat spinal cord along laminin-rich peripheral surroundings of the dorsal root transitional zone

Following dorsal root crush, the lesioned axons regenerate in the peripheral compartment of the dorsal root, but stop at the boundary between the peripheral and the central nervous system, the dorsal root transitional zone. We have previously shown that fibres from human fetal dorsal root ganglia grafted to adult rat hosts are able to grow into the spinal cord, but were not able to specify the route taken by the ingrowing fibres. In this study we have challenged the dorsal root transitional zone astrocyte boundary with human dorsal root ganglion transplants from 5–8-week-old embryos. By tracing immunolabelled human fibres in serial sections, we found that fibres consistently grow around the dorsal root transitional zone astrocytes in laminin-rich peripheral surroundings, and extend into the host rat spinal cord along blood vessels, either into deep or superficial laminae of the dorsal horn, or into the dorsal funiculus. Human fibres that did not have access to blood vessels grew on the spinal cord surface. These findings indicate, that in spite of a substantial growth capacity by axons from human embryonic dorsal root ganglion cells as well as their tolerance to non-permissive factors in the mature mammalian CNS, these axons are still sensitive to the repellent effects of astrocytes of the mature dorsal root transitional zone. Furthermore, this axonal ingrowth is consistently associated with laminin-expressing structures until the axons reach the host spinal cord.     

Identity, origin, and migration of peripheral glial cells in the Drosophila embryo

Glial cells are crucial for the proper development and function of the nervous system. In the Drosophila embryo, the glial cells of the peripheral nervous system are generated both by central neuroblasts and sensory organ precursors. Most peripheral glial cells need to migrate along axonal projections of motor and sensory neurons to reach their final positions in the periphery. Here we studied the spatial and temporal pattern, the identity, the migration, and the origin of all peripheral glial cells in the truncal segments of wildtype embryos. The establishment of individual identities among these cells is reflected by the expression of a combinatorial code of molecular markers. This allows the identification of individual cells in various genetic backgrounds. Furthermore, mutant analysis of two of these marker genes, spalt major and castor, reveal their implication in peripheral glial development. Using confocal 4D microscopy to monitor and follow peripheral glia migration in living embryos, we show that the positioning of most of these cells is predetermined with minor variations, and that the order in which cells migrate into the periphery is almost fixed. By studying their lineages, we uncovered the origin of each of the peripheral glial cells and linked them to identified central and peripheral neural stem cells.     

Peripherally-derived BDNF promotes regeneration of ascending sensory neurons after spinal cord injury

Background

The blood brain barrier (BBB) and truncated trkB receptor on astrocytes prevent the penetration of brain derived neurotrophic factor (BDNF) applied into the peripheral (PNS) and central nervous system (CNS) thus restrict its application in the treatment of nervous diseases. As BDNF is anterogradely transported by axons, we propose that peripherally derived and/or applied BDNF may act on the regeneration of central axons of ascending sensory neurons.

Methodology/Principal Findings

The present study aimed to test the hypothesis by using conditioning lesion of the sciatic nerve as a model to increase the expression of endogenous BDNF in sensory neurons and by injecting exogenous BDNF into the peripheral nerve or tissues. Here we showed that most of regenerating sensory neurons expressed BDNF and p-CREB but not p75NTR. Conditioning-lesion induced regeneration of ascending sensory neuron and the increase in the number of p-Erk positive and GAP-43 positive neurons was blocked by the injection of the BDNF antiserum in the periphery. Enhanced neurite outgrowth of dorsal root ganglia (DRG) neurons in vitro by conditioning lesion was also inhibited by the neutralization with the BDNF antiserum. The delivery of exogenous BDNF into the sciatic nerve or the footpad significantly increased the number of regenerating DRG neurons and regenerating sensory axons in the injured spinal cord. In a contusion injury model, an injection of BDNF into the footpad promoted recovery of motor functions.

Conclusions/Significance

Our data suggest that endogenous BDNF in DRG and spinal cord is required for the enhanced regeneration of ascending sensory neurons after conditioning lesion of sciatic nerve and peripherally applied BDNF may have therapeutic effects on the spinal cord injury.     

Analyzing Murine Schwann Cell Development Along Growing Axons

The development of peripheral nerves is an intriguing process. Neurons send out axons to innervate specific targets, which in humans are often more than 100 cm away from the soma of the neuron. Neuronal survival during development depends on target-derived growth factors but also on the support of Schwann cells (SCs). To this end SC ensheath axons from the region of the neuronal soma (or the transition from central to peripheral nervous system) to the synapse or neuromuscular junction. Schwann cells are derivatives of the neural crest and migrate as precursors along emerging axons until the entire axon is covered with SCs. This shows the importance of SC migration for the development of the peripheral nervous system and underlines the necessity to investigate this process. In order to analyze SC development, a setup is needed which next to the SCs also includes their physiological substrate for migration, the axon. Due to intrauterine development in vivo time-lapse imaging, however, is not feasible in placental vertebrates like mouse (mus musculus). To circumvent this, we adapted the superior cervical ganglion (SCG) explant technique. Upon treatment with nerve growth factor (NGF) SCG explants extend axons, followed by SC precursors migrating along the axons from the ganglion to the periphery. The beauty of this system is that the SC are derived from a pool of endogenous SC and that they migrate along their own physiological axons which are growing at the same time. This system is especially intriguing, because the SC development along axons can be analyzed by time-lapse imaging, opening further possibilities to gain insights into SC migration.     

Formation of the peripheral nervous system during tail regeneration in urodele amphibians: ultrastructural and immunohistochemical studies of the origin of the cells.

In the regenerating newt tail, epimorphic regeneration--which recapitulates morphologically normal embryonic development--proceeds along a rostrocaudal differentiation gradient. Innervation of the new myomeres results from the spinal roots of segments rostral to the amputation plane and from ventral roots emerging from the lateroventral region of the regenerating spinal cord, in which motor neurons are differentiating. Electron microscopy and an indirect immunofluorescence study with anti-glial fibrillary acid protein (GFAP) confirm that the ventrolateral part of the regenerated ependymal tube gives rise to cells of the ventral root sheath and the spinal ganglia. Anti-GFAP and anti-neurofilament antibodies showed that ependymoglial cells and Schwann cells may play a role in neuronal pathfinding by helping guide and stabilize pioneering axons as they extend toward the myomeres. The carbohydrate epitope NC-1 is expressed in the spinal cord, in sheath cells of the spinal ganglia and in the non-myelin-forming Schwann cells of the peripheral nervous system. L1, a Ca++ independent neural cell adhesion molecule, was detected in the axonal compartments of the regenerating spinal cord, on immature and/or non-myelin-forming Schwann cells within the peripheral nervous system (PNS), and on nerve fibers within the regenerate. These immunohistochemical observations collectively support the hypothesis that Schwann cells already present in the blastema could be involved in organizing neural pathways.     

The zebrafish diwanka gene controls an early step of motor growth cone migration.

During vertebrate embryogenesis different classes of motor axons exit the spinal cord and migrate on common axonal paths into the periphery. Surprisingly little is known about how this initial migration of spinal motor axons is controlled by external cues. Here, we show that the diwanka gene is required for growth cone migration of three identified subtypes of zebrafish primary motoneurons. In diwanka mutant embryos, motor growth cone migration within the spinal cord is unaffected but it is strongly impaired as motor axons enter their common path to the somites. Chimera analysis shows that diwanka gene activity is required in a small set of myotomal cells, called adaxial cells. We identified a subset of the adaxial cells to be sufficient to rescue the diwanka motor axon defect. Moreover, we show that this subset of adaxial cells delineates the common axonal path prior to axonogenesis, and we show that interactions between these adaxial cells and motor growth cones are likely to be transient. The studies demonstrate that a distinct population of myotomal cells plays a pivotal role in the early migration of zebrafish motor axons and identify the diwanka gene as a somite-derived cue required to establish an axonal path from the spinal cord to the somites.     

Ddx20, an Olig2 binding factor,governs the survival of neural and oligodendrocyte progenitor cells via proper Mdm2 splicing and p53 suppression

Olig2 is indispensable for motoneuron and oligodendrocyte fate-specification in the pMN domain of embryonic spinal cords, and also involved in the proliferation and differentiation of several cell types in the nervous system, including neural progenitor cells (NPCs) and oligodendrocytes. However, how Olig2 controls these diverse biological processes remains unclear. Here, we demonstrated that a novel Olig2-binding protein, DEAD-box helicase 20 (Ddx20), is indispensable for the survival of NPCs and oligodendrocyte progenitor cells (OPCs). A central nervous system (CNS)-specific Ddx20 conditional knockout (cKO) demonstrated apoptosis and cell cycle arrest in NPCs and OPCs, through the potentiation of the p53 pathway in DNA damage-dependent and independent manners, including SMN complex disruption and the abnormal splicing of Mdm2 mRNA. Analyzes of Olig2 null NPCs showed that Olig2 contributed to NPC proliferation through Ddx20 protein stabilization. Our findings provide novel mechanisms underlying the Olig2-mediated proliferation of NPCs, via the Ddx20-p53 axis, in the embryonic CNS.Subject terms: Oligodendrocyte, Experimental models of disease     

A Global In Vivo Drosophila RNAi Screen Identifies a Key Role of Ceramide Phosphoethanolamine for Glial Ensheathment of Axons

Glia are of vital importance for all complex nervous system. One of the many functions of glia is to insulate and provide trophic and metabolic support to axons. Here, using glial-specific RNAi knockdown in Drosophila, we silenced 6930 conserved genes in adult flies to identify essential genes and pathways. Among our screening hits, metabolic processes were highly represented, and genes involved in carbohydrate and lipid metabolic pathways appeared to be essential in glia. One critical pathway identified was de novo ceramide synthesis. Glial knockdown of lace, a subunit of the serine palmitoyltransferase associated with hereditary sensory and autonomic neuropathies in humans, resulted in ensheathment defects of peripheral nerves in Drosophila. A genetic dissection study combined with shotgun high-resolution mass spectrometry of lipids showed that levels of ceramide phosphoethanolamine are crucial for axonal ensheathment by glia. A detailed morphological and functional analysis demonstrated that the depletion of ceramide phosphoethanolamine resulted in axonal defasciculation, slowed spike propagation, and failure of wrapping glia to enwrap peripheral axons. Supplementing sphingosine into the diet rescued the neuropathy in flies. Thus, our RNAi study in Drosophila identifies a key role of ceramide phosphoethanolamine in wrapping of axons by glia.     

NGF controls axonal receptivity to myelination by Schwann cells or oligodendrocytes

Axons dictate whether or not they will become myelinated in both the central and peripheral nervous systems by providing signals that direct the development of myelinating glia. Here we identify the neurotrophin nerve growth factor (NGF) as a potent regulator of the axonal signals that control myelination of TrkA-expressing dorsal root ganglion neurons (DRGs). Unexpectedly, these NGF-regulated axonal signals have opposite effects on peripheral and central myelination, promoting myelination by Schwann cells but reducing myelination by oligodendrocytes. These findings indicate a novel role for growth factors in regulating the receptivity of axons to myelination and reveal that different axonal signals control central and peripheral myelination.     

Strategies to repair lost sensory connections to the spinal cord

Failure of injured axons to regenerate in the central nervous system (CNS) is the main obstacle for repair of stroke and traumatic injuries to the spinal cord and sensory roots. This regeneration failure is high-lighted at the dorsal root transitional zone (DRTZ), the boundary between the peripheral (PNS) and central nervous system where sensory axons enter the spinal cord. Injured sensory axons regenerate in the PNS compartment of the dorsal root but are halted as soon as they reach the DRTZ. The failure of regenerating dorsal root axons to re-enter the mature spinal cord is a reflection of the generally nonpermissive nature of the CNS environment, in contrast to the regeneration supportive properties of the PNS. The dorsal root injury paradigm is therefore an attractive model for studying mechanisms underlying CNS regeneration failure in general and how to overcome the hostile CNS environment. Here we review the main lines that have been pursued to achieve growth of injured dorsal root axons into the spinal cord: (i) modifying the inhibitory nature of the DRTZ by breaking down or blocking the effect of growth repelling molecules, (ii) stimulate elongation of injured dorsal root axons by a prior conditioning lesion or administration of specific growth factors, (iii) implantation of olfactory ensheathing cells to provide a growth supportive cellular terrain at the DRTZ, and (iv) replacing the regeneration deficient adult dorsal root ganglion neurons with embryonic neurons or neural stem cells.     

Ectopic myelinating oligodendrocytes in the dorsal spinal cord as a consequence of altered semaphorin 6D signaling inhibit synapse formation

Different types of sensory neurons in the dorsal root ganglia project axons to the spinal cord to convey peripheral information to the central nervous system. Whereas most proprioceptive axons enter the spinal cord medially, cutaneous axons typically do so laterally. Because heavily myelinated proprioceptive axons project to the ventral spinal cord, proprioceptive axons and their associated oligodendrocytes avoid the superficial dorsal horn. However, it remains unclear whether their exclusion from the superficial dorsal horn is an important aspect of neural circuitry. Here we show that a mouse null mutation of Sema6d results in ectopic placement of the shafts of proprioceptive axons and their associated oligodendrocytes in the superficial dorsal horn, disrupting its synaptic organization. Anatomical and electrophysiological analyses show that proper axon positioning does not seem to be required for sensory afferent connectivity with motor neurons. Furthermore, ablation of oligodendrocytes from Sema6d mutants reveals that ectopic oligodendrocytes, but not proprioceptive axons, inhibit synapse formation in Sema6d mutants. Our findings provide new insights into the relationship between oligodendrocytes and synapse formation in vivo, which might be an important element in controlling the development of neural wiring in the central nervous system.     

Tissue-specific neuro-glia interactions determine neurite differentiation in ganglion cells

Guided formation and extension of axons versus dendrites is considered crucial for structuring the nervous system. In the chick visual system, retinal ganglion cells (RGCs) extend their axons into the tectum opticum, but not into glial somata containing retina layers. We addressed the question whether the different glia of retina and tectum opticum differentially affect axon growth. Glial cells were purified from retina and tectum opticum by complement-mediated cytolysis of non-glial cells. RGCs were purified by enzymatic delayering from flat mounted retina. RGCs were seeded onto retinal versus tectal glia monolayers. Subsequent neuritic differentiation was analysed by immunofluorescence microscopy and scanning electron microscopy. Qualitative and quantitative evaluation revealed that retinal glia somata inhibited axons. Time-lapse video recording indicated that axonal inhibition was based on the collapse of lamellipodia- and filopodia-rich growth cones of axons. In contrast to retinal glia, tectal glia supported axonal extension. Notably, retinal glia were not inhibitory for neurons in general, because in control experiments axon extension of dorsal root ganglia was not hampered. Therefore, the axon inhibition by retinal glia was neuron type-specific. In summary, the data demonstrate that homotopic (retinal) glia somata inhibit axonal outgrowth of RGCs, whereas heterotopic (tectal) glia of the synaptic target area support RGC axon extension. The data underscore the pivotal role of glia in structuring the developing nervous system.     

Prox1 Inhibits Proliferation and Is Required for Differentiation of the Oligodendrocyte Cell Lineage in the Mouse

Central nervous system injury induces a regenerative response in ensheathing glial cells comprising cell proliferation, spontaneous axonal remyelination, and limited functional recovery, but the molecular mechanisms are not fully understood. In Drosophila, this involves the genes prospero and Notch controlling the balance between glial proliferation and differentiation, and manipulating their levels in glia can switch the response to injury from prevention to promotion of repair. In the mouse, Notch1 maintains NG2 oligodendrocyte progenitor cells (OPCs) in a progenitor state, but what factor may enable oligodendrocyte (OL) differentiation and functional remyelination is not understood. Here, we asked whether the mammalian homologue of prospero, Prox1, is involved. Our data show that Prox1 is distributed in NG2+ OPCs and in OLs in primary cultured cells, and in the mouse spinal cord in vivo. siRNA prox1 knockdown in primary OPCs increased cell proliferation, increased NG2+ OPC cell number and decreased CC1+ OL number. Prox1 conditional knockout in the OL cell lineage in mice increased NG2+ OPC cell number, and decreased CC1+ OL number. Lysolecithin-induced demyelination injury caused a reduction in CC1+ OLs in homozygous Prox1-/- conditional knockout mice compared to controls. Remarkably, Prox1-/- conditional knockout mice had smaller lesions than controls. Altogether, these data show that Prox1 is required to inhibit OPC proliferation and for OL differentiation, and could be a relevant component of the regenerative glial response. Therapeutic uses of glia and stem cells to promote regeneration and repair after central nervous system injury would benefit from manipulating Prox1.     

Vitamin D Promotes Remyelination by Suppressing c-Myc and Inducing Oligodendrocyte Precursor Cell Differentiation after Traumatic Spinal Cord Injury

Demyelination due to oligodendrocytes loss occurs after traumatic spinal cord injury (TSCI). Several studies have suggested the therapeutic potential of vitamin D (VitD) in demyelinating diseases. However, experimental evidence in the context of TSCI is limited, particularly in the presence of prior VitD-deficiency. In the present study, a contusion and a transection TSCI rat model were used, representing mild and severe injury, respectively. Motor recovery was assessed in rats with normal VitD level or with VitD-deficiency after 8 weeks'' treatment post-TSCI (Cholecalciferol, 500 IU/kg/day). The impact on myelin integrity was examined by transmission electron microscopy and studied in vitro using primary culture of oligodendrocytes. We found that VitD treatment post-TSCI effectively improved hindlimb movement in rats with normal VitD level irrespective of injury severity. However, cord-transected rats with prior deficiency did not seem to benefit from VitD supplementation. Our data further suggested that having sufficient VitD was essential for persevering myelin integrity after injury. VitD rescued oligodendrocytes from apoptotic cell death in vitro and enhanced their myelinating ability towards dorsal root axons. Enhanced myelination was mediated by increased oligodendrocyte precursor cells (OPCs) differentiation into oligodendrocytes in concert with c-Myc downregulation and suppressed OPCs proliferation. Our study provides novel insights into the functioning of VitD as a regulator of OPCs differentiation as well as strong preclinical evidence supporting future clinical testing of VitD for TSCI.     

Cells of the oligodendroglial lineage, myelination, and remyelination

Myelin is critical in maintaining electrical impulse conduction in the central nervous system. The oligodendrocyte is the cell type responsible for myelin production within this compartment. The mutual supply of trophic support between oligodendrocytes and the underlying axons may indicate why demyelinated axons undergo degeneration more readily; the latter contributes to the neural decline in multiple sclerosis (MS). Myelin repair, termed remyelination, occurs in acute inflammatory lesions in MS and is associated with functional recovery and clinical remittances. Animal models have demonstrated that remyelination is mediated by oligodendrocyte progenitor cells (OPCs) which have responded to chemotactic cues, migrated into the lesion, proliferated, differentiated into mature oligodendrocytes, and ensheathed demyelinated axons. The limited remyelination observed in more chronic MS lesions may reflect intrinsic properties of neural cells or extrinsic deterrents. Therapeutic strategies currently under development include transplantation of exogenous OPCs and promotion of remyelination by endogenous OPCs. All currently approved MS therapies are aimed at dampening the immune response and are not directly targeting neural processes.     

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.