VEGF mediates commissural axon chemoattraction through its receptor Flk1

Growing axons are guided to their targets by attractive and repulsive cues. In the developing spinal cord, Netrin-1 and Shh guide commissural axons toward the midline. However, the combined inhibition of their activity in commissural axon turning assays does not completely abrogate turning toward floor plate tissue, suggesting that additional guidance cues are present. Here we show that the prototypic angiogenic factor VEGF is secreted by the floor plate and is a chemoattractant for commissural axons in vitro and in vivo. Inactivation of Vegf in the floor plate or of its receptor Flk1 in commissural neurons causes axon guidance defects, whereas Flk1 blockade inhibits turning of axons to VEGF in vitro. Similar to Shh and Netrin-1, VEGF-mediated commissural axon guidance requires the activity of Src family kinases. Our results identify VEGF and Flk1 as a novel ligand/receptor pair controlling commissural axon guidance.     

Complementary expression of transmembrane ephrins and their receptors in the mouse spinal cord: a possible role in constraining the orientation of longitudinally projecting axons

In the developing spinal cord, axons project in both the transverse plane, perpendicular to the floor plate, and in the longitudinal plane, parallel to the floor plate. For many axons, the floor plate is a source of long- and short-range guidance cues that govern growth along both dimensions. We show here that B-class transmembrane ephrins and their receptors are reciprocally expressed on floor plate cells and longitudinally projecting axons in the mouse spinal cord. During the period of commissural axon pathfinding, B-class ephrin protein is expressed at the lateral floor plate boundaries, at the interface between the floor plate and the ventral funiculus. In contrast, B-class Eph receptors are expressed on decussated commissural axon segments projecting within the ventral funiculus, and on ipsilaterally projecting axons constituting the lateral funiculus. Soluble forms of all three B-class ephrins bind to, and induce the collapse of, commissural growth cones in vitro. The collapse-inducing activity associated with B-class ephrins is likely to be mediated by EphB1. Taken together, these data support a possible role for repulsive B-class Eph receptor/ligand interactions in constraining the orientation of longitudinal axon projections at the ventral midline.     

The morphogen sonic hedgehog is an axonal chemoattractant that collaborates with netrin-1 in midline axon guidance

Developing axons are guided to their targets by attractive and repulsive guidance cues. In the embryonic spinal cord, the floor plate chemoattractant Netrin-1 is required to guide commissural neuron axons to the midline. However, genetic evidence suggests that other chemoattractant(s) are also involved. We show that the morphogen Sonic hedgehog (Shh) can mimic the additional chemoattractant activity of the floor plate in vitro and can act directly as a chemoattractant on isolated axons. Cyclopamine-mediated inhibition of the Shh signaling mediator Smoothened (Smo) or conditional inactivation of Smo in commissural neurons indicate that Smo activity is important for the additional chemoattractant activity of the floor plate in vitro and for the normal projection of commissural axons to the floor plate in vivo. These results provide evidence that Shh, acting via Smo, is a midline-derived chemoattractant for commissural axons and show that a morphogen can also act as an axonal chemoattractant.     

A role for BMP heterodimers in roof plate-mediated repulsion of commissural axons

During spinal cord development, commissural neurons extend their axons ventrally, away from the roof plate. The roof plate is the source of a diffusible repellent that orients commissural axons in vitro and, thus, may regulate the trajectory of commissural axons in vivo. Of three Bmps expressed in the roof plate, BMP7, but not BMP6 or GDF7, mimics the roof plate activity in vitro. We show here that expression of both Bmp7 and Gdf7 by roof plate cells is required for the fidelity of commissural axon growth in vivo. We also demonstrate that BMP7 and GDF7 heterodimerize in vitro and that, under these conditions, GDF7 enhances the axon-orienting activity of BMP7. Our findings suggest that a GDF7:BMP7 heterodimer functions as a roof plate-derived repellent that establishes the initial ventral trajectory of commissural axons.     

Perturbation of neuronal differentiation and axon guidance in the spinal cord of mouse embryos lacking a floor plate: analysis of Danforth's short-tail mutation.

The floor plate of the vertebrate nervous system has been implicated in the guidance of commissural axons at the ventral midline. Experiments in chick have also suggested that at earlier stages of development the floor plate induces the differentiation of motor neurons and other neurons of the ventral spinal cord. Here we have examined the development of the spinal cord in a mouse mutant, Danforth's short-tail, in which the floor plate is absent from caudal regions of the neuraxis. In affected regions of the spinal cord, commissural axons exhibited aberrant projection patterns as they reached and crossed the ventral midline. In addition, motor neurons were absent or markedly reduced in number in regions of the spinal cord lacking a floor plate. Our results suggest that the floor plate is indeed an intermediate target in the projection of commissural axons and support the idea that several different mechanisms operate in concert in the guidance of axons to their cellular targets in the developing nervous system. In addition, these experiments suggest that the mechanisms that govern the differentiation of the floor plate and other ventral cell types in the neural tube are common to mammals and lower vertebrates.     

Bilaterally Symmetric Populations of Chicken dI1 (Commissural) Axons Cross the Floor Plate Independently of Each Other

Axons use temporal and directional guidance cues at intermediate targets to set the rate and direction of growth towards their synaptic targets. Our recent studies have shown that disrupting the temporal guidance process, by unilaterally accelerating the rate at which spinal dI1 (commissural) axons grow, resulted in turning errors both in the ventral spinal cord and after crossing the floor plate. Here we investigate a mechanistic explanation for these defects: the accelerated dI1 axons arrive in the ventral spinal cord before necessary fasciculation cues from incoming dI1 axons from the opposite side of the spinal cord. The identification of such an interaction would support a model of selective fasciculation whereby the pioneering dI1 axons serve as guides for the processes of the bilaterally symmetrical population of dI1 neurons. To test this model, we first developed the ability to “double” in ovo electroporate the embryonic chicken spinal cord to independently manipulate the rate of growth of the two bilateral populations of dI1 axons. Second, we examined the requirement for a putative bilateral interaction by unilaterally ablating the dI1 population in cultured explants of chicken embryonic spinal cord. Surprisingly, we find no evidence for a bilateral dI1 axon interaction, rather dI1 axons appear to project independently of each other.     

Guidance of commissural growth cones at the floor plate in embryonic rat spinal cord

The floor plate of the embryonic rat spinal cord has been proposed to act as an intermediate target that plays a role in the pattern of extension of commissural axons. To begin to examine the role of the floor plate in axon guidance at the midline, we have studied the precision of the commissural axon projection to and across the floor plate during development. To delineate the pathway, the fluorescent carbocyanine dye, Di-I, has been used as a probe. We show that commissural axons traverse the floor plate and turn rostrally at its contralateral border with remarkable precision. Axons were not observed to turn ipsilaterally and turned only upon reaching the contralateral edge of the floor plate. Virtually all commissural axons follow this route. The morphology of commissural growth cones was also examined. As they encountered the floor plate, commissural growth cones became larger and increased in complexity. The reorientation of axons in register with the floor plate boundary and the change in the morphological properties of commissural growth cones as they traverse the midline suggest that the floor plate may act as a guidepost with functions similar to cells that have been implicated in axon guidance in invertebrates.     

Ventral midline cells are required for the local control of commissural axon guidance in the mouse spinal cord.

Specialized cells at the midline of the central nervous system have been implicated in controlling axon projections in both invertebrates and vertebrates. To address the requirement for ventral midline cells in providing cues to commissural axons in mice, we have analyzed Gli2 mouse mutants, which lack specifically the floor plate and immediately adjacent interneurons. We show that a Dbx1 enhancer drives tau-lacZ expression in a subpopulation of commissural axons and, using a reporter line generated from this construct, as well as DiI tracing, we find that commissural axons projected to the ventral midline in Gli2(-/-) embryos. Netrin1 mRNA expression was detected in Gli2(-/-) embryos and, although much weaker than in wild-type embryos, was found in a dorsally decreasing gradient. This result demonstrates that while the floor plate can serve as a source of long-range cues for C-axons in vitro, it is not required in vivo for the guidance of commissural axons to the ventral midline in the mouse spinal cord. After reaching the ventral midline, most commissural axons remained clustered in Gli2(-/-) embryos, although some were able to extend longitudinally. Interestingly, some of the longitudinally projecting axons in Gli2(-/-) embryos extended caudally and others rostrally at the ventral midline, in contrast to normal embryos in which virtually all commissural axons turn rostrally after crossing the midline. This finding indicates a critical role for ventral midline cells in regulating the rostral polarity choice made by commissural axons after they cross the midline. In addition, we provide evidence that interactions between commissural axons and floor plate cells are required to modulate the localization of Nr-CAM and TAG-1 proteins on axons at the midline. Finally, we show that the floor plate is not required for the early trajectory of motoneurons or axons of the posterior commissure, whose projections are directed away from the ventral midline in both WT and Gli2(-/-) embryos, although they are less well organized in Gli2(-/-)mutants.     

Integration of Shallow Gradients of Shh and Netrin-1 Guides Commissural Axons

During nervous system development, gradients of Sonic Hedgehog (Shh) and Netrin-1 attract growth cones of commissural axons toward the floor plate of the embryonic spinal cord. Mice defective for either Shh or Netrin-1 signaling have commissural axon guidance defects, suggesting that both Shh and Netrin-1 are required for correct axon guidance. However, how Shh and Netrin-1 collaborate to guide axons is not known. We first quantified the steepness of the Shh gradient in the spinal cord and found that it is mostly very shallow. We then developed an in vitro microfluidic guidance assay to simulate these shallow gradients. We found that axons of dissociated commissural neurons respond to steep but not shallow gradients of Shh or Netrin-1. However, when we presented axons with combined Shh and Netrin-1 gradients, they had heightened sensitivity to the guidance cues, turning in response to shallower gradients that were unable to guide axons when only one cue was present. Furthermore, these shallow gradients polarized growth cone Src-family kinase (SFK) activity only when Shh and Netrin-1 were combined, indicating that SFKs can integrate the two guidance cues. Together, our results indicate that Shh and Netrin-1 synergize to enable growth cones to sense shallow gradients in regions of the spinal cord where the steepness of a single guidance cue is insufficient to guide axons, and we identify a novel type of synergy that occurs when the steepness (and not the concentration) of a guidance cue is limiting.     

BMPs as mediators of roof plate repulsion of commissural neurons

During spinal cord development, commissural (C) neurons, located near the dorsal midline, send axons ventrally and across the floor plate (FP). The trajectory of these axons toward the FP is guided in part by netrins. The mechanisms that guide the early phase of C axon extension, however, have not been resolved. We show that the roof plate (RP) expresses a diffusible activity that repels C axons and orients their growth within the dorsal spinal cord. Bone morphogenetic proteins (BMPs) appear to act as RP-derived chemorepellents that guide the early trajectory of the axons of C neurons in the developing spinal cord: BMP7 mimics the RP repellent activity for C axons in vitro, can act directly to collapse C growth cones, and appears to serve an essential function in RP repulsion of C axons.     

Leaving the midline

In the developing nervous system, pathfinding axons navigate through a series of intermediate targets in order to form synaptic connections. Vertebrate spinal commissural axons extend toward and across the floor plate (FP), a key intermediate target located at the ventral midline (VM). Subsequently, post-crossing commissural axons grow either alongside or significant distances away from the floor plate (FP), but never re-cross the VM. Consistent with this behavior, post-crossing commissural axons lose responsiveness to the FP-associated chemoattractants, Netrin-1 and SHH, and gain responsiveness to Slits, which are potent midline repellents, in vitro. In addition, the results of several in vivo studies suggest that the upregulation of Slit-binding repulsive Robo receptors, Robo1/2, alters the responsiveness of decussated commissural axons to midline guidance cues. Nevertheless, in vertebrates, it is unclear whether Robo1/2 are the sole or major repellent receptors responsible for driving these commissural axons away from the VM and preventing their re-entry into the FP. We recently re-visited these issues in the chick spinal cord by assessing the consequences of manipulating Robo expression on commissural axons in ovo. Our findings suggest that, at least in chick embryos, the upregulation of repulsive Robos on post-crossing axons alters the responsiveness of these axons to midline repellents and facilitates their expulsion from, but is not likely to have a significant role in preventing their re-entry into the VM.     

Pioneer longitudinal axons navigate using floor plate and Slit/Robo signals

Longitudinal axons transmit all signals between the brain and spinal cord. Their axon tracts through the brain stem are established by a simple set of pioneer axons with precise trajectories parallel to the floor plate. To identify longitudinal guidance mechanisms in vivo, the overall role of floor plate tissue and the specific roles of Slit/Robo signals were tested. Ectopic induction or genetic deletion of the floor plate diverted longitudinal axons into abnormal trajectories. The expression patterns of the diffusible cues of the Slit family were altered in the floor plate experiments, suggesting their involvement in longitudinal guidance. Genetic tests of Slit1 and Slit2, and the Slit receptors Robo1 and Robo2 were carried out in mutant mice. Slit1;Slit2 double mutants had severe longitudinal errors, particularly for ventral axons, including midline crossing and wandering longitudinal trajectories. Robo1 and Robo2 were largely genetically redundant, and neither appeared to specify specific tract positions. However, combined Robo1 and Robo2 mutations strongly disrupted each pioneer tract. Thus, pioneer axons depend on long-range floor plate cues, with Slit/Robo signaling required for precise longitudinal trajectories.     

Growth cone guidance by floor plate cells in the spinal cord of zebrafish embryos.

The spinal cord of early zebrafish embryos contains a small number of neuronal classes whose growth cones all follow stereotyped, cell-specific pathways to their targets. Two classes of spinal neurons make cell-specific turns at or near the ventral midline of the spinal cord, which is occupied by a single row of midline floor plate cells. We tested whether these cells guide the growth cones by examining embryos missing the midline floor plate cells due either to laser ablation of the cells or to a mutation (cyc-1). In these embryos the growth cones followed both normal and aberrant pathways once near the ventral midline. This suggests that normally the midline floor plate cells do provide guidance cues, but that these cues are not obligatory.     

Signals from the cerebellum guide the pathfinding of inferior olivary axons

During development, inferior olivary axons cross the floor plate and project from the caudal to the rostral hindbrain, whence they grow into the cerebellar plate. We have investigated the axon guidance signals involved in the formation of this projection in vitro. When the cerebellar plate was grafted ectopically along the margin of the hindbrain in organotypic cultures, inferior olivary axons could pathfind to the ectopic cerebellum, establishing a topographically normal projection. Following rostrocaudal reversal of a region of tissue in the axon pathway between the inferior olive and the cerebellum, olivary axons still navigated towards the cerebellum. Moreover, olivary axons could cross a bridging tissue explant (spinal cord) to reach a cerebellar explant. In collagen gel cultures of inferior olive explants, olivary axon outgrowth increased significantly in the presence of cerebellar explants and axons deflected towards the cerebellar tissue. These results show that the cerebellum is a source of diffusible axon guidance signals for olivary axons. We also found that, in organotypic cultures, olivary axons which had crossed the floor plate showed an increased tendency to respond to cerebellar cues. Taken together, these results indicate that the cerebellum is the source of cues that are chemoattractant and growth-promoting for inferior olivary axons; prior exposure to the floor plate increases responsiveness to these cues.     

Commissural axon pathfinding on the contralateral side of the floor plate: a role for B-class ephrins in specifying the dorsoventral position of longitudinally projecting commissural axons.

In both invertebrate and lower vertebrate species, decussated commissural axons travel away from the midline and assume positions within distinct longitudinal tracts. We demonstrate that in the developing chick and mouse spinal cord, most dorsally situated commissural neuron populations extend axons across the ventral midline and through the ventral white matter along an arcuate trajectory on the contralateral side of the floor plate. Within the dorsal (chick) and intermediate (mouse) marginal zone, commissural axons turn at a conserved boundary of transmembrane ephrin expression, adjacent to which they form a discrete ascending fiber tract. In vitro perturbation of endogenous EphB-ephrinB interactions results in the failure of commissural axons to turn at the appropriate dorsoventral position on the contralateral side of the spinal cord; consequently, axons inappropriately invade more dorsal regions of B-class ephrin expression in the dorsal spinal cord. Taken together, these observations suggest that B-class ephrins act locally during a late phase of commissural axon pathfinding to specify the dorsoventral position at which decussated commissural axons turn into the longitudinal axis.     

Evidence for a role of srGAP3 in the positioning of commissural axons within the ventrolateral funiculus of the mouse spinal cord

Slit-Robo signaling guides commissural axons away from the floor-plate of the spinal cord and into the longitudinal axis after crossing the midline. In this study we have evaluated the role of the Slit-Robo GTPase activating protein 3 (srGAP3) in commissural axon guidance using a knockout (KO) mouse model. Co-immunoprecipitation experiments confirmed that srGAP3 interacts with the Slit receptors Robo1 and Robo2 and immunohistochemistry studies showed that srGAP3 co-localises with Robo1 in the ventral and lateral funiculus and with Robo2 in the lateral funiculus. Stalling axons have been reported in the floor-plate of Slit and Robo mutant spinal cords but our axon tracing experiments revealed no dorsal commissural axon stalling in the floor plate of the srGAP3 KO mouse. Interestingly we observed a significant thickening of the ventral funiculus and a thinning of the lateral funiculus in the srGAP3 KO spinal cord, which has also recently been reported in the Robo2 KO. However, axons in the enlarged ventral funiculus of the srGAP3 KO are Robo1 positive but do not express Robo2, indicating that the thickening of the ventral funiculus in the srGAP3 KO is not a Robo2 mediated effect. We suggest a role for srGAP3 in the lateral positioning of post crossing axons within the ventrolateral funiculus.     

Nr-CAM promotes neurite outgrowth from peripheral ganglia by a mechanism involving axonin-1 as a neuronal receptor.

Nr-CAM is a neuronal cell adhesion molecule (CAM) belonging to the immunoglobulin superfamily that has been implicated as a ligand for another CAM, axonin-1, in guidance of commissural axons across the floor plate in the spinal cord. Nr-CAM also serves as a neuronal receptor for several other cell surface molecules, but its role as a ligand in neurite outgrowth is poorly understood. We studied this problem using a chimeric Fc-fusion protein of the extracellular region of Nr-CAM (Nr-Fc) and investigated potential neuronal receptors in the developing peripheral nervous system. A recombinant Nr-CAM-Fc fusion protein, containing all six Ig domains and the first two fibronectin type III repeats of the extracellular region of Nr-CAM, retains cellular and molecular binding activities of the native protein. Injection of Nr-Fc into the central canal of the developing chick spinal cord in ovo resulted in guidance errors for commissural axons in the vicinity of the floor plate. This effect is similar to that resulting from treatment with antibodies against axonin-1, confirming that axonin-1/Nr-CAM interactions are important for guidance of commissural axons through a spatially and temporally restricted Nr-CAM positive domain in the ventral spinal cord. When tested as a substrate, Nr-Fc induced robust neurite outgrowth from dorsal root ganglion and sympathetic ganglion neurons, but it was not effective for tectal and forebrain neurons. The peripheral but not the central neurons expressed high levels of axonin-1 both in vitro and in vivo. Moreover, antibodies against axonin-1 inhibited Nr-Fc-induced neurite outgrowth, indicating that axonin-1 is a neuronal receptor for Nr-CAM on these peripheral ganglion neurons. The results demonstrate a role for Nr-CAM as a ligand in axon growth by a mechanism involving axonin-1 as a neuronal receptor and suggest that dynamic changes in Nr-CAM expression can modulate axonal growth and guidance during development.     

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.     

Contacts between the commissural axons and the floor plate cells are mediated by nectins

During development of the central nervous system (CNS), commissural axons grow toward the ventral midline. After crossing the floor plate, they abruptly change their trajectory from the circumferential to the longitudinal axis. The contacts between the commissural axons and the floor plate cells are involved in this axonal guidance, but their mechanisms or structures have not fully been understood. In this study, we found that nectin-1 and -3, immunoglobulin-like cell-cell adhesion molecules, asymmetrically localized at the contact sites between the commissural axons and the floor plate cells, respectively. In vitro perturbation of the endogenous trans-interaction between nectin-1 and -3 caused abnormal fasciculation of the commissural axons and impairment of the contacts, and resulted in failure in longitudinal turns of the commissural axons at the contralateral sites of the rat hindbrain. These results indicate that the contacts between the commissural axons and the floor plate cells are mediated by the hetero-trans-interaction between nectin-1 and -3 and involved in regulation of the trajectory of the commissural axons.