Dorsally derived netrin 1 provides an inhibitory cue and elaborates the 'waiting period' for primary sensory axons in the developing spinal cord

Dorsal root ganglion (DRG) neurons extend axons to specific targets in the gray matter of the spinal cord. During development, DRG axons grow into the dorsolateral margin of the spinal cord and projection into the dorsal mantle layer occurs after a ;waiting period' of a few days. Netrin 1 is a long-range diffusible factor expressed in the ventral midline of the developing neural tube, and has chemoattractive and chemorepulsive effects on growing axons. Netrin 1 is also expressed in the dorsal spinal cord. However, the roles of dorsally derived netrin 1 remain totally unknown. Here, we show that dorsal netrin 1 controls the correct guidance of primary sensory axons. During the waiting period, netrin 1 is transiently expressed or upregulated in the dorsal spinal cord, and the absence of netrin 1 results in the aberrant projection of sensory axons, including both cutaneous and proprioceptive afferents, into the dorsal mantle layer. Netrin 1 derived from the dorsal spinal cord, but not the floor plate, is involved in the correct projection of DRG axons. Furthermore, netrin 1 suppresses axon outgrowth from DRG in vitro. Unc5c(rcm) mutant shows abnormal invasion of DRG axons as observed in netrin 1 mutants. These results are the first direct evidence that netrin 1 in the dorsal spinal cord acts as an inhibitory cue for primary sensory axons and is a crucial signal for the formation of sensory afferent neural networks.     

Netrin-1 signaling for sensory axons

During development, dorsal root ganglion (DRG) neurons extend their axons toward the dorsolateral part of the spinal cord and enter the spinal cord through the dorsal root entry zone (DREZ). After entering the spinal cord, these axons project into the dorsal mantle layer after a ‘waiting period’ of a few days. We revealed that the diffusible axonal guidance molecule netrin-1 is a chemorepellent for developing DRG axons. When DRG axons orient themselves toward the DREZ, netrin-1 proteins derived from the ventral spinal cord prevent DRG axons from projecting aberrantly toward the ventral spinal cord and help them to project correctly toward the DREZ. In addition to the ventrally derived netrin-1, the dorsal spinal cord cells adjacent to the DREZ transiently express netrin-1 proteins during the waiting period. This dorsally derived netrin-1 contributes to the correct guidance of DRG axons to prevent them from invading the dorsal spinal cord. In general, there is a complete lack of sensory axonal regeneration after a spinal cord injury, because the dorsal column lesion exerts inhibitory activities toward regenerating axons. Netrin-1 is a novel candidate for a major inhibitor of sensory axonal regeneration in the spinal cord; because its expression level stays unchanged in the lesion site following injury, and adult DRG neurons respond to netrin-1-induced axon repulsion. Although further studies are required to show the involvement of netrin-1 in preventing the regeneration of sensory axons in CNS injury, the manipulation of netrin-1-induced repulsion in the CNS lesion site may be a potent approach for the treatment of human spinal injuries.     

Localization of unmyelinated axons in rat skin and mucocutaneous tissue utilizing the isolectin GS-I-B

The a-D-galactose specific isolectin I-B4 from Griffonia simplicifolia (GS-I-B4) labels CNS microglia and certain peripheral neurons, including a subpopulation of small dark, type B dorsal root ganglion cells, some post-ganglionic sympathetic axons, and nearly all peripheral gustatory axons. The innervation patterns of GS-I-B4 reactive sensory ganglion cells are unknown for many peripheral target tissues, including their probable primary target, the skin. The present study describes the distribution of GS-I-B4 reactive axons in hairy and glabrous hindpaw skin and in the glans penis of rats, using both single and double-labelling histochemical techniques. Neuronal processes were identified using (1) histochemistry with horseradish peroxidase conjugated GS-I-B4 or (2) immunohistochemistry against PGP 9.5 to identify all axons, and biotinylated lectin histochemistry with avidin-FITC to identify the subpopulation of GS-I-B4 reactive axons. GS-I-B4 strongly labelled unmyelinated cutaneous sensory afferents, as well as some sympathetic efferents and visceral afferents. Lectin reactive axons were seen to innervate the upper hair shaft epidermis in hairy skin, and were abundant in the shallow dermis in hairy and glabrous skin and glans penis. Lectin reactive axons were also abundant in the lamina propria and distal urethral epithelium of the penis. These results provide new evidence for the cutaneous sensory role of GS-I-B4 reactive primary afferents, as well as evidence to support the contention that the lectin is a specific marker for a subpopulation of unmyelinated axons and not simply a marker for the myelination state of an axon.     

DRG Axon Elongation and Growth Cone Collapse Rate Induced by Sema3A are Differently Dependent on NGF Concentration

Regeneration of embryonic and adult dorsal root ganglion (DRG) sensory axons is highly impeded when they encounter neuronal growth cone-collapsing factor semaphorin3A (Sema3A). On the other hand, increasing evidence shows that DRG axon’s regeneration can be stimulated by nerve growth factor (NGF). In this study, we aimed to evaluate whether increased NGF concentrations can counterweight Sema3A-induced inhibitory responses in 15-day-old mouse embryo (E15) DRG axons. The DRG explants were grown in Neurobasal-based medium with different NGF concentrations ranging from 0 to 100 ng/mL and then treated with Sema3A at constant 10 ng/mL concentration. To evaluate interplay between NGF and Sema3A number of DRG axons, axon outgrowth distance and collapse rate were measured. We found that the increased NGF concentrations abolish Sema3A-induced inhibitory effect on axon outgrowth, while they have no effect on Sema3A-induced collapse rate.     

Independent development of sensory and motor innervation patterns in embryonic chick hindlimbs

Previous studies suggest that sensory axon outgrowth is guided by motoneurons, which are specified to innervate particular target muscles. Here we present evidence that questions this conclusion. We have used a new approach to assess the pathfinding abilities of bona fide sensory neurons, first by eliminating motoneurons after neural crest cells have coalesced into dorsal root ganglia (DRG) and second by challenging sensory neurons to innervate muscles in a novel environment created by shifting a limb bud rostrally. The resulting sensory innervation patterns mapped with the lipophilic dyes DiI and DiA showed that sensory axons projected robustly to muscles in the absence of motoneurons, if motoneurons were eliminated after DRG formation. Moreover, sensory neurons projected appropriately to their usual target muscles under these conditions. In contrast, following limb shifts, muscle sensory innervation was often derived from inappropriate segments. In this novel environment, sensory neurons tended to make more "mistakes" than motoneurons. Whereas motoneurons tended to innervate their embryologically correct muscles, sensory innervation was more widespread and was generally from more rostral segments than normal. Similar results were obtained when motoneurons were eliminated in embryos with limb shifts. These findings show that sensory neurons are capable of navigating through their usual terrain without guidance from motor axons. However, unlike motor axons, sensory axons do not appear to actively seek out appropriate target muscles when confronted with a novel terrain. These findings suggest that sensory neuron identity with regard to pathway and target choice may be unspecified or quite plastic at the time of initial axon outgrowth.     

Roundabout gene family functions during sensory axon guidance in the drosophila embryo are mediated by both Slit-dependent and Slit-independent mechanisms

roundabout (robo) family genes play key roles in axon guidance in a wide variety of animals. We have investigated the roles of the robo family members, robo, robo2, and robo3, in the guidance of sensory axons in the Drosophila embryo. In robo(-/-), slit(-/-), and robo(-/+) slit(-/+) mutants, lateral cluster sensory neurons misproject to cells and axons in the nearby ventral' (v') cluster. These phenotypes, together with the normal expression pattern of Slit and Robo, suggest that Slit ligand secreted from the epidermis interacts with Robo receptors on lateral cluster sensory growth cones to limit their exploration of nearby attractive substrates. The most common sensory axon phenotype seen in robo2(-/-) mutants was misprojection of dorsal cluster sensory axons away from their normal growth substrate, the transverse connective of the trachea. slit appears to play no role in this aspect of sensory axon growth. Robo2 is expressed, not on the dorsal sensory axons, but on the transverse connective. These results suggest a novel, non-cell-autonomous mechanism for axon guidance by robo family genes: Robo2 expressed on the trachea acts as an attractant for the dorsal sensory growth cones.     

Distinct subpopulations of sensory afferents require F11 or axonin-1 for growth to their target layers within the spinal cord of the chick.

Dorsal root ganglion neurons project axons to specific target layers in the gray matter of the spinal cord, according to their sensory modality. Using an in vivo approach, we demonstrate an involvement of the two immunoglobulin superfamily cell adhesion molecules axonin-1/TAG-1 and F11/F3/contactin in subpopulation-specific sensory axon guidance. Proprioceptive neurons, which establish connections with motoneurons in the ventral horn, depend on F11 interactions. Nociceptive fibers, which target to layers in the dorsal horn, require axonin-1 for pathfinding. In vitro NgCAM and NrCAM were shown to bind to both axonin-1 and F11. However, despite this fact and despite their ubiquitous expression in the spinal cord, NgCAM and NrCAM are selective binding partners for axonin-1 and F11 in sensory axon guidance. Whereas nociceptive pathfinding depends on NgCAM and axonin-1, proprioceptive fibers require NrCAM and F11.     

Anterograde Transport of Herpes Simplex Virus Proteins in Axons of Peripheral Human Fetal Neurons: an Immunoelectron Microscopy Study

Herpes simplex virus (HSV) reactivates from latency in the neurons of dorsal root ganglia (DRG) and is subsequently transported anterogradely along the axon to be shed at the skin or mucosa. Although we have previously shown that only unenveloped nucleocapsids are present in axons during anterograde transport, the mode of transport of tegument proteins and glycoproteins is not known. We used a two-chamber culture model with human fetal DRG cultivated in an inner chamber, allowing axons to grow out and penetrate an agarose barrier and interact with autologous epidermal cells in the outer chamber. After HSV infection of the DRG, anterograde transport of viral components could be examined in the axons in the outer chamber at different time points by electron and immunoelectron microscopy (IEM). In the axons, unenveloped nucleocapsids or focal collections of gold immunolabel for nucleocapsid (VP5) and/or tegument (VP16) were detected. VP5 and VP16 usually colocalized in both scanning and transmission IEM. In contrast, immunolabel for glycoproteins gB, gC, and gD was diffusely distributed in axons and was rarely associated with VP5 or VP16. In longitudinal sections of axons, immunolabel for glycoprotein was arrayed along the membranes of axonal vesicles. These findings provide evidence that in DRG axons, virus nucleocapsids coated with tegument proteins are transported separately from glycoproteins and suggest that final assembly of enveloped virus occurs at the axon terminus.     

Invariant Sema5A inhibition serves an ensheathing function during optic nerve development

Retinal axon pathfinding from the retina into the optic nerve involves the growth promoting axon guidance molecules L1, laminin and netrin 1, each of which governs axon behavior at specific regions along the retinal pathway. In identifying additional molecules regulating this process during embryonic mouse development, we found that transmembrane Semaphorin5A mRNA and protein was specifically expressed in neuroepithelial cells surrounding retinal axons at the optic disc and along the optic nerve. Given that growth cone responses to a specific guidance molecule can be altered by co-exposure to a second guidance cue, we examined whether retinal axon responses to Sema5A were modulated by other guidance signals axons encountered along the retinal pathway. In growth cone collapse, substratum choice and neurite outgrowth assays, Sema5A triggered an invariant inhibitory response in the context of L1, laminin, or netrin 1 signaling, suggesting that Sema5A inhibited retinal axons throughout their course at the optic disc and nerve. Antibody-perturbation studies in living embryo preparations showed that blocking of Sema5A function led to retinal axons straying out of the optic nerve bundle, indicating that Sema5A normally helped ensheath the retinal pathway. Thus, development of some CNS nerves requires inhibitory sheaths to maintain integrity. Furthermore, this function is accomplished using molecules such as Sema5A that exhibit conserved inhibitory responses in the presence of co-impinging signals from multiple families of guidance molecules.     

Analysis of Drosophila photoreceptor axon guidance in eye-specific mosaics

During development of the adult Drosophila visual system, axons of the eight photoreceptors in each ommatidium fasciculate together and project as a single bundle towards the optic lobes of the brain. Within the brain, individual photoreceptor axons from each bundle then seek specific targets in distinct layers of the optic lobes. The axons of photoreceptors R1-R6 terminate in the lamina, while R7 and R8 axons pass through the lamina to terminate in separate layers of the medulla. To identify genes required for photoreceptor axon guidance, including those with essential functions during early development, we have devised a strategy for the simple and efficient generation of genetic mosaics in which mutant photoreceptor axons innervate a predominantly wild-type brain. In a large-scale saturation mutagenesis performed using this system, we recovered new alleles of the gene encoding the receptor tyrosine phosphatase PTP69D. PTP69D has previously been shown to function in the correct targeting of motor axons in the embryo and R1-R6 axons in the visual system. Here, we show that PTP69D is also required for correct targeting of R7 axons. Whereas mutant R1-R6 axons occasionally extend beyond their normal targets in the lamina, mutant R7 axons often fail to reach their targets in the medulla, stopping instead at the same level as the R8 axon. These targeting errors are difficult to reconcile with models in which PTP69D plays an instructive role in photoreceptor axon targeting, as previously proposed. Rather, we suggest that PTP69D plays a permissive role, perhaps reducing the adhesion of R1-R6 and R7 growth cones to the pioneer R8 axon so that they can respond independently to their specific targeting cues.     

Semaphorin 2a secreted by oenocytes signals through plexin B and plexin A to guide sensory axons in the Drosophila embryo

The semaphorin gene family has been shown to play important roles in axonal guidance in both vertebrates and invertebrates. Both transmembrane (Sema1a, Sema1b, Sema5c) and secreted (Sema2a, Sema2b) forms of semaphorins exist in Drosophila. Two Sema receptors, plexins (Plex) A and B, have also been identified. Many questions remain concerning the axon guidance functions of the secreted semaphorins, including the identity of their receptors. We have used the well-characterized sensory system of the Drosophila embryo to address these problems. We find novel sensory axon defects in sema2a loss-of-function mutants in which particular axons misproject and follow inappropriate pathways to the CNS. plexB loss-of-function mutants show similar phenotypes to sema2a mutants and sema2a interacts genetically with plexB, supporting the hypothesis that Sema2a signals through PlexB receptors. Sema2a protein is expressed by larval oenocytes, a cluster of secretory cells in the lateral region of the embryo and the sema2a mutant phenotype can be rescued by driving Sema2a in these cells. Ablation of oenocytes results in sensory axon defects similar to the sema2a mutant phenotype. These data support a model in which Sema2a, while being secreted from oenocytes, acts in a highly localized fashion: It represses axon extension from the sensory neuron cell body, but only in regions in direct contact with oenocytes.     

Npn-1 contributes to axon-axon interactions that differentially control sensory and motor innervation of the limb

The initiation, execution, and completion of complex locomotor behaviors are depending on precisely integrated neural circuitries consisting of motor pathways that activate muscles in the extremities and sensory afferents that deliver feedback to motoneurons. These projections form in tight temporal and spatial vicinities during development, yet the molecular mechanisms and cues coordinating these processes are not well understood. Using cell-type specific ablation of the axon guidance receptor Neuropilin-1 (Npn-1) in spinal motoneurons or in sensory neurons in the dorsal root ganglia (DRG), we have explored the contribution of this signaling pathway to correct innervation of the limb. We show that Npn-1 controls the fasciculation of both projections and mediates inter-axonal communication. Removal of Npn-1 from sensory neurons results in defasciculation of sensory axons and, surprisingly, also of motor axons. In addition, the tight coupling between these two heterotypic axonal populations is lifted with sensory fibers now leading the spinal nerve projection. These findings are corroborated by partial genetic elimination of sensory neurons, which causes defasciculation of motor projections to the limb. Deletion of Npn-1 from motoneurons leads to severe defasciculation of motor axons in the distal limb and dorsal-ventral pathfinding errors, while outgrowth and fasciculation of sensory trajectories into the limb remain unaffected. Genetic elimination of motoneurons, however, revealed that sensory axons need only minimal scaffolding by motor axons to establish their projections in the distal limb. Thus, motor and sensory axons are mutually dependent on each other for the generation of their trajectories and interact in part through Npn-1-mediated fasciculation before and within the plexus region of the limbs.     

Collapsin-1/semaphorin D is a repellent for chick ganglion of Remak axons.

Chick collapsin-1/human semaphorin III/mouse semaphorin D is believed to guide the extension of specific axons by a repellent mechanism. Here we examine its role in the guidance of axons of the ganglion of Remak (Remak) in the developing chick intestine. Early in embryogenesis Remak axons extend parallel to, but do not enter, the intestine when collapsin-1 is expressed in the adjacent rectal wall. Remak axons later penetrate the peripheral portions of the rectal wall when collapsin-1 expression retreats from the outer muscle layer to the more internal submucosal and mucosal layers of the rectum. Extension of Remak neurites is repelled in vitro by rectum explants and also by 293T cells expressing collapsin-1. The rectal chemorepellent activity is blocked by anti-collapsin-1 antibodies. Our results suggest that collapsin-1 may help prevent Remak axons from projecting into the intestinal wall at early developmental times and later restricts Remak axon trajectories to the outer part of the intestinal muscle layer.     

Netrin-1 signaling for sensory axons: Involvement in sensory axonal development and regeneration

During development, dorsal root ganglion (DRG) neurons extend their axons toward the dorsolateral part of the spinal cord and enter the spinal cord through the dorsal root entry zone (DREZ). After entering the spinal cord, these axons project into the dorsal mantle layer after a “waiting period” of a few days. We revealed that the diffusible axonal guidance molecule netrin-1 is a chemorepellent for developing DRG axons. When DRG axons orient themselves toward the DREZ, netrin-1 proteins derived from the ventral spinal cord prevent DRG axons from projecting aberrantly toward the ventral spinal cord and help them to project correctly toward the DREZ. In addition to the ventrally derived netrin-1, the dorsal spinal cord cells adjacent to the DREZ transiently express netrin-1 proteins during the waiting period. This dorsally derived netrin-1 contributes to the correct guidance of DRG axons to prevent them from invading the dorsal spinal cord. In general, there is a complete lack of sensory axonal regeneration after a spinal cord injury, because the dorsal column lesion exerts inhibitory activities toward regenerating axons. Netrin-1 is a novel candidate for a major inhibitor of sensory axonal regeneration in the spinal cord; because its expression level stays unchanged in the lesion site following injury, and adult DRG neurons respond to netrin-1-induced axon repulsion. Although further studies are required to show the involvement of netrin-1 in preventing the regeneration of sensory axons in CNS injury, the manipulation of netrin-1-induced repulsion in the CNS lesion site may be a potent approach for the treatment of human spinal injuries.Key words: netrin-1, dorsal root ganglion, axon guidance, chemorepellent, Unc5, spinal cord, axon regenerationDeveloping axons navigate to their targets by responding to attractive and repulsive guidance cues working in a contact-dependent or diffusible fashion in their environment (reviewed in ref. ¹). During early development of the primary sensory system, centrally projecting sensory axons from dorsal root ganglion (DRG) neurons extend toward the dorsolateral region of the spinal cord (Fig. 1A and C), where they enter the spinal cord exclusively through the dorsal root entry zone (DREZ), and never orient themselves toward the notochord or the ventral spinal cord (Fig. 1A; reviewed in ref. ²). We previously showed that the notochord but not the ventral spinal cord secretes semaphorin 3A (Sema3A), which is known to be a chemorepellent for DRG axons at early developmental stages (Fig. 1A).³ This is the reason why DRG axons never project toward the notochord. Along the same line, it is highly possible that the ventral spinal cord may secrete some chemorepulsive cue other than Sema3A for DRG axons.Open in a separate windowFigure 1Netrin-1 plays a critical role in sensory axonal guidance as an axon chemorepellent. (A) A schematic diagram of a thoracic transverse section of an E10 mouse embryo, summarizing the possible mechanism of netrin-1 action in early DRG axonal guidance. When DRG axons project toward the DREZ in the dorsal spinal cord (dSC), ventrally derived netrin-1 chemorepels DRG axons to prevent them from orienting aberrantly toward the ventral spinal cord (vSC) (upper). NC; notochord. In netrin-1-deficient embryos, some DRG axons misorient themselves toward the ventral spinal cord, because of the absence of netrin-1 proteins in the ventral spinal cord (lower). (B) At E12.5 when DRG axons grow to the marginal zone of the spinal cord longitudinally (arrows) to form the dorsal funiculus (DF), netrin-1 proteins are transiently expressed in a subpopulation of dorsal spinal cord cells adjacent to the dorsal funiculus (upper). In netrin-1-deficient embryos, the dorsal funiculus is disorganized because DRG axons are no longer waiting for invading the dorsal mantle layer (lower). (C) Gain-of-function experiments by electroporation confirm the repulsive activity of netrin-1 toward DRG axons. When netrin-1 is misexpressed in the dorsal spinal cord, the number of DRG axons that enter the DREZ is significantly reduced compared with the control, because some DRG axons fail to project toward the DREZ and turn in the wrong direction.After entering the spinal cord, DRG axons grow to the marginal zone of the spinal cord longitudinally to form the dorsal funiculus without projecting to the dorsal mantle layer for a few days (this delay of the axonal projection to the mantle layer is referred to as the ‘waiting period;’ Fig. 1B). A few days later, proprioceptive afferents of DRGs begin to send collaterals into the dorsal layers, and cutaneous afferents project ventrally through the dorsal layers.⁴ This evidence raises the possibility that some repulsive cues transiently prevent the collaterals of DRGs from penetrating the dorsal spinal cord during this waiting period.Netrins are a family of secreted proteins that play a key role in axonal guidance, cell migration, morphogenesis and angiogenesis.⁵ Netrin-1 is a bifunctional axonal guidance cue, attracting some axons including commissural axons via the Deleted in Colorectal Cancer (DCC) receptor and repelling others via Unc5 receptors (reviewed in ref. ⁶). However, it has not been clear whether netrin-1 plays a role in sensory axonal guidance during development.Several observations strongly suggest a role for netrin-1 in DRG axonal guidance as a repulsive guidance cue during development.^7,8 First, in the mouse embryo at embryonic day (E) 10–11.⁵ when many DRG axons orient themselves to reach the DREZ, netrin-1 is strongly expressed in the floor plate of the ventral spinal cord but not in the dorsal spinal cord (Fig. 1A). Second, at E12.5 when DRG neurons extend their axons longitudinally along the dorsolateral margin of the spinal cord, netrin-1 is expressed in the dorsolateral region adjacent to the DREZ (Fig. 1B), but its expression is down-regulated in the dorsal spinal cord at E13.5 when many collaterals have entered the mantle layer. Third, repulsive netrin-1 receptor Unc5c is expressed in the DRG neurons during development.These observations motivated us to explore whether netrin-1/Unc5c signaling contributes to DRG axonal guidance. We used cell and tissue cultures combined with tissues from netrin-1-deficient mice. We clearly showed that netrin-1 exerts a chemorepulsive activity toward developing DRG axons and that the ventral spinal cord-derived repulsive activity depends on netrin-1 in vitro.⁸ Additional evidence for a chemorepulsive role of netrin-1 came from the observation of DRG axonal trajectories in netrin-1-deficient mice.^7,8 In netrin-1-deficient embryos at E10, we showed that some DRG axons became misoriented toward the ventral spinal cord, probably because of the absence of netrin-1 proteins in the ventral spinal cord (Fig. 1A). In addition, at E12.5 when DRG axons grow to the marginal zone of the spinal cord longitudinally to form the dorsal funiculus, the dorsal funiculus is disorganized in netrin-1-deficient embryos, because in the absence of netrin-1 DRG axons are not waiting for invading the dorsal mantle layer adjacent to the dorsal funiculus (Fig. 1B). Gain-of-function experiments further confirmed the repulsive activity of netrin-1 toward DRG axons (Fig. 1C). These lines of evidence lead us to the conclusion that dorsally derived netrin-1 plays an important role in providing the ‘waiting period’ for extension of collaterals from sensory afferents and that ventrally derived netrin-1 prevents sensory axons from misorienting themselves toward the ventral spinal cord.At later developmental stages (E13.5), DRG axons still possess a weak responsiveness to the chemorepulsive activity of netrin-1 in vitro.⁸ In addition, both postnatal and adult DRG neurons respond to netrin-1-induced axon inhibition.⁹ Consistent with these results, DRG neurons at not only later developmental stages (E13.5) but also postnatal stages express the repulsion-mediating netrin-1 receptor Unc5c.^8,9Generally, lesioning of the dorsal column projection of sensory axons results in a complete lack of regeneration. The possible explanation for the complete lack of regeneration is that the environment, the lesion site itself and/or oligodendrocytes adjacent to the lesion, may be non-permissive for regenerating axons.¹⁰ Sema3A and chondroitin sulfate proteoglycans (CSPGs) are candidates as major inhibitors of sensory axonal regeneration in the spinal cord, because they are expressed in the lesion site and can inhibit DRG axonal growth in vitro.^3,11–14 Recently, Kaneko et al. showed that a selective inhibitor of Sema3A also enhances axonal regeneration and functional recovery in a subpopulation of sensory neurons after lesioning of the dorsal column.¹² More recently, McMahon''s group clearly demonstrated that enzymatic degradation of CSPGs on the dorsal column lesion of the spinal cord promotes sensory axonal regeneration and functional recovery.^13,14 Although these treatments greatly improved functional recovery, complete sensory axonal growth and functional recovery have not been yet achieved after the spinal cord injury. To promote further recovery of sensory axonal regeneration in the CNS, we should focus on other candidate inhibitors of CNS injury sites.Following spinal cord injury, the expression of the attraction- mediating netrin-1 receptor DCC decreases, while the expression level of the repulsive receptor Unc5c returns to normal.¹⁵ Levels of netrin-1 expression also stay unchanged in neurons and oligodendrocytes adjacent to the lesion site. Together with the in vitro evidence described above, these data strongly suggest a possible role for netrin-1 as a novel inhibitor of CNS myelin for regenerating DRG axons in the dorsal column-lesioned spinal cord. Further studies will be required to show directly the functional recovery of sensory axons in the spinal cord by perturbation of netrin-1 in and around the lesion site after spinal cord injury.     

Sensory activity affects sensory axon development in C. elegans

The simple nervous system of the nematode C. elegans consists of 302 neurons with highly reproducible morphologies, suggesting a hard-wired program of axon guidance. Surprisingly, we show here that sensory activity shapes sensory axon morphology in C. elegans. A class of mutants with deformed sensory cilia at their dendrite endings have extra axon branches, suggesting that sensory deprivation disrupts axon outgrowth. Mutations that alter calcium channels or membrane potential cause similar defects. Cell-specific perturbations of sensory activity can cause cell-autonomous changes in axon morphology. Although the sensory axons initially reach their targets in the embryo, the mutations that alter sensory activity cause extra axon growth late in development. Thus, perturbations of activity affect the maintenance of sensory axon morphology after an initial pattern of innervation is established. This system provides a genetically tractable model for identifying molecular mechanisms linking neuronal activity to nervous system structure.     

Ectoderm removal prevents cutaneous nerve formation and perturbs sensory axon growth in the chick hindlimb

Target tissues are thought to provide important cues for growing axons, yet there is little direct evidence that they are essential for axonal pathfinding. Here we examined whether target ectoderm is necessary for the formation of cutaneous nerves, and for the normal growth and guidance of cutaneous axons as they first enter the limb plexus. To do this, we removed a patch of ectoderm from the chick hindlimb at various times during early axon outgrowth. We find there is a critical period when cutaneous nerve formation requires target ectoderm. When the ectoderm is absent during this time, axons progress into the limb more slowly and, although a few sensory axons occasionally diverge a short distance from the plexus, they do not form a discrete nerve that travels to the skin. A few days later, when the nerve pattern is mature, axons normally destined for the 'deprived' cutaneous nerve are not segregated appropriately within the plexus. Some cutaneous axons are instead misdirected along an inappropriate cutaneous nerve, while others have seemingly failed to reach their correct target, or a suitable alternative, and died. These results demonstrate that the target ectoderm is necessary for normal sensory axon growth and guidance in the hindlimb.     

The interaction of human fetal neurons and epidermal cellsin vitro

Summary the interaction of autologous human fetal neurons with epidermal cells was studied by culturing fetal dorsal root ganglia (DRG) in the center of a dual chamber system with epidermal explants in the outer chamber. The two chambers were separated by two concentric stainless steel annular rings adherent to the substratum by silicon grease and agarose. Axons from the DRG penetrated the agarose barrier, growing into the exterior chamber by 10 din vitro (DIV) and extended past sparse peripheral fibroblasts to interact specifically with epidermal cells by 12 to 16 DIV. Scanning electron microscopy (SEM) showed single or multiple neuronal fascicles terminating on epidermal cells with spatular, veillike or bulbous axon termini. Transmission electron microscopy (TEM) showed fine axonal termini between epidermal cells, separated by an intercellular gap. The specificity of axonal targeting for epidermal cells rather than fibroblasts was also demonstrated by infecting the DRG with Herpes simplex virus type-1 (HSV-1). Specific anterograde transport of HSV-1 along axons to keratin-expressing epidermal cells was demonstrated by immunofluorescence and immunoperoxidase staining using monoclonal antibodies to viral glycoprotein D. This model allows the study of the mechanism of the specific interactions between neurons and epidermal cells analogous to those in fetal development and after cutaneous nerve regeneration.     

Genetic Study of Axon Regeneration with Cultured Adult Dorsal Root Ganglion Neurons

It is well known that mature neurons in the central nervous system (CNS) cannot regenerate their axons after injuries due to diminished intrinsic ability to support axon growth and a hostile environment in the mature CNS^1,2. In contrast, mature neurons in the peripheral nervous system (PNS) regenerate readily after injuries³. Adult dorsal root ganglion (DRG) neurons are well known to regenerate robustly after peripheral nerve injuries. Each DRG neuron grows one axon from the cell soma, which branches into two axonal branches: a peripheral branch innervating peripheral targets and a central branch extending into the spinal cord. Injury of the DRG peripheral axons results in substantial axon regeneration, whereas central axons in the spinal cord regenerate poorly after the injury. However, if the peripheral axonal injury occurs prior to the spinal cord injury (a process called the conditioning lesion), regeneration of central axons is greatly improved⁴. Moreover, the central axons of DRG neurons share the same hostile environment as descending corticospinal axons in the spinal cord. Together, it is hypothesized that the molecular mechanisms controlling axon regeneration of adult DRG neurons can be harnessed to enhance CNS axon regeneration. As a result, adult DRG neurons are now widely used as a model system to study regenerative axon growth^5-7.Here we describe a method of adult DRG neuron culture that can be used for genetic study of axon regeneration in vitro. In this model adult DRG neurons are genetically manipulated via electroporation-mediated gene transfection^6,8. By transfecting neurons with DNA plasmid or si/shRNA, this approach enables both gain- and loss-of-function experiments to investigate the role of any gene-of-interest in axon growth from adult DRG neurons. When neurons are transfected with si/shRNA, the targeted endogenous protein is usually depleted after 3-4 days in culture, during which time robust axon growth has already occurred, making the loss-of-function studies less effective. To solve this problem, the method described here includes a re-suspension and re-plating step after transfection, which allows axons to re-grow from neurons in the absence of the targeted protein. Finally, we provide an example of using this in vitro model to study the role of an axon regeneration-associated gene, c-Jun, in mediating axon growth from adult DRG neurons⁹.     

Cell-type specific modular regulation of derailed in the Drosophila nervous system.

The derailed (drl) gene encodes a receptor tyrosine kinase (RTK) that governs aspects of axon guidance and muscle-epidermal interactions in the Drosophila embryo. To determine the types of neurons that express drl, we have examined a series of drl promoter fusions to axon-targeted reporters. We have identified enhancers that drive reporter expression in four distinct subtypes of embryonic neurons, all of which project axons in the anterior commissure of the developing nervous system. We also identified enhancers driving expression in the drl-expressing muscles and epidermal attachment cells. These enhancers define the classes of neurons projecting in the anterior commissure and can be used to precisely define axon pathfinding errors in drl and other mutants.