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 role of floor plate contact in the elaboration of contralateral commissural projections within the embryonic mouse spinal cord

In vertebrate embryos, commissural axons extend toward and across the floor plate (FP), an intermediate target at the ventral midline (VM) of the spinal cord. After decussating, many commissural axons turn into the longitudinal plane and elaborate diverse projections. FP contact is thought to alter the responsiveness of these axons so that they can exit the FP and adopt new trajectories. However, a requirement for the FP in shaping contralateral commissural projections has not been established in higher vertebrates. Here we further analyze to what extent FP contact is necessary for the elaboration of decussated commissural projections both in cultured, FP-excised spinal cord preparations and in gli2-deficient mice, which lack a FP. In FP-lacking spinal cords, we observe a large number of appropriately projecting contralateral commissural projections in vivo and in vitro. Surprisingly, even though gli2 mutants lack a FP, slit1-3 mRNA and their receptors (Robo1/2) are expressed in a wild-type-like manner. In addition, blocking Robo-Slit interactions in FP-lacking spinal cord explants prevents commissural axons from leaving the VM and turning longitudinally. Thus, compared to FP contact, Slit-Robo interactions are more critical for driving commissural axons out of the VM and facilitating the elaboration of a subset of contralateral commissural projections.     

Adjacent pioneer commissural interneuron growth cones switch from contact avoidance to axon fasciculation after midline crossing

Commissural interneurons (CI) of the vertebrate spinal cord are guided ventrally toward the floor plate, but subsequently cross the midline and select a longitudinal fascicle at specific dorsal-ventral (D-V) positions. We examined at high resolution the detailed behaviors of individual pathfinding CI growth cones on the ipsilateral and contralateral sides of the spinal cord of living Xenopus embryos. We find that pre-crossing CI growth cones exhibit distinct pathfinding behaviors compared to post-crossing axons and that the behavioral switch occurs immediately upon crossing to the contralateral side. Groups of pioneer commissural axons typically extend simultaneously toward the ventral midline following discrete paths with separation between adjacent commissurals apparently maintained through contact inhibition. In contrast, shortly after crossing the midline, commissural axons turn longitudinally and begin to fasciculate with other crossed CIs. However, growth cones of crossed commissurals often select their final D-V longitudinal track through a series of rapid step-like dorsal adjustments that may be due to differential fasciculation with longitudinal axons. Together, our results suggest that guidance of commissural axons is controlled in part through interactions among CIs that switch rapidly from avoidance to fasciculation after midline crossing.     

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.     

Conserved roles for Slit and Robo proteins in midline commissural axon guidance

In Drosophila, Slit at the midline activates Robo receptors on commissural axons, thereby repelling them out of the midline into distinct longitudinal tracts on the contralateral side of the central nervous system. In the vertebrate spinal cord, Robo1 and Robo2 are expressed by commissural neurons, whereas all three Slit homologs are expressed at the ventral midline. Previous analysis of Slit1;Slit2 double mutant spinal cords failed to reveal a defect in commissural axon guidance. We report here that when all six Slit alleles are removed, many commissural axons fail to leave the midline, while others recross it. In addition, Robo1 and Robo2 single mutants show guidance defects that reveal a role for these two receptors in guiding commissural axons to different positions within the ventral and lateral funiculi. These results demonstrate a key role for Slit/Robo signaling in midline commissural axon guidance in vertebrates.     

Neuroanatomical and functional analysis of neural tube formation in notochordless Xenopus embryos; laterality of the ventral spinal cord is lost.

Notochordless Xenopus embryos were produced by u.v. irradiation of the uncleaved fertilized egg. The spinal cords were examined using intermediate filament staining for glial cells, retrograde HRP staining for neuronal morphology and an anti-glycinergic antibody to reveal commissural cells and axons. The floorplate cells of the normal cord appear to be absent and their position along the ventral midline of the cord is occupied by motor neurones, Kolmer-Agduhr cells, radial glial cells and a ventrally placed marginal zone containing the longitudinal axons. Motor neurone number is reduced to 15% of control values, and the sensory extramedullary cell number is increased twentyfold. Commissural axons are still able to cross the ventral cord but do so at abnormal angles and some commissural axons continue to grow circumferentially up the contralateral side of the cord rather than turning to grow longitudinally. Extracellular electrophysiological recordings from motor axons reveal that the normal alternation of locomotor activity on the left and right side of the embryo is lost in notochordless animals. These results suggest that the notochord and/or the normal floor plate structure are important for the development of the laterality of spinal cord connections and may influence motor neurone proliferation or differentiation.     

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.     

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.     

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.     

Identification of early developing axon projections from spinal interneurons in the chick embryo with a neuron specific beta-tubulin antibody: evidence for a new 'pioneer' pathway in the spinal cord

The early development of interneurons in the chick embryo spinal cord was studied using a monoclonal antibody against a neuron-specific beta-tubulin isoform. Early developing interneurons were divided into two cell groups on the basis of their location and the pattern of growth of their axons. One group is composed of cells that establish a primitive longitudinal pathway (PL-cells), whereas the other group contains cells constituting a circumferential pathway (C-cells). The onset of axonal development in both cell groups occurs at stage (st.) 15 (embryonic day, (E), 2) in the branchial segments, which is prior to axonogenesis of motoneurons. PL-cells develop in the region between the floor plate and the motoneuron nucleus. Their axons are the first neuronal processes ('pioneer axons') to arrive in the ventrolateral marginal zone and they project both rostrally and caudally to establish a primitive longitudinal association pathway at the ventrolateral surface of the neural tube. This pathway is formed before axons of C-cells arrive in the ventrolateral region. The first C-cells are initially located in the most dorsal portion of the neural tube, whereas later appearing C-cells are also located in both intermediate and ventral regions of the neural tube. The axons of C-cells project ventrally, without fasciculating, along the lateral border of the neural tube. Some of their axons enter the ipsilateral ventrolateral longitudinal pathway at st. 17. We often observed apparent contacts and interactions between preexisting axons of PL-cells and newly arriving axons of C-cells. The axons of commissural C-cells first enter the floor plate at st. 17 and cross the midline at st. 18. Axons of C cells begin to join the contralateral ventrolateral longitudinal pathway at st. 18+ to st. 19. In the floor plate region, contacts between growth cones and axons were often observed. However, axons in the floor plate at these stages were not fasciculated. These observations establish the timing and pattern of growth of axons from two specific populations of early developing interneurons in the chick spinal cord. Additionally, we have identified an early and apparently previously undescribed 'pioneer' pathway that constitutes the first longitudinal pathway in the chick spinal cord.     

F-Spondin is required for accurate pathfinding of commissural axons at the floor plate.

The commissural axons project toward and across the floor plate. They then turn into the longitudinal axis, extending along the contralateral side of the floor plate. F-spondin, a protein produced and secreted by the floor plate, promotes adhesion and neurite extension of commissural neurons in vitro. Injection of purified F-spondin protein into the lumen of the spinal cord of chicken embryos in ovo resulted in longitudinal turning of commissural axons before reaching the floor plate, whereas neutralizing antibody (Ab) injections caused lateral turning at the contralateral floor plate boundary. These combined in vitro and in vivo results suggest that F-spondin is required to prevent the lateral drifting of the commissural axons after having crossed the floor plate.     

Zic1 promotes the expansion of dorsal neural progenitors in spinal cord by inhibiting neuronal differentiation

The role of Zic1 was investigated by altering its expression status in developing spinal cords. Zic genes encode zinc finger proteins homologous to Drosophila Odd-paired. In vertebrate neural development, they are generally expressed in the dorsal neural tube. Chick Zic1 was initially expressed evenly along the dorsoventral axis and its expression became increasingly restricted dorsally during the course of neurulation. The dorsal expression of Zic1 was regulated by Sonic hedgehog, BMP4, and BMP7, as revealed by their overexpressions in the spinal cord. When Zic1 was misexpressed on the ventral side of the chick spinal cord, neuronal differentiation was inhibited irrespective of the dorsoventral position. In addition, dorsoventral properties were not grossly affected as revealed by molecular markers. Concordantly, when Zic1 was overexpressed in the dorsal spinal cord in transgenic mice, we observed hypercellularity in the dorsal spinal cord. The transgene-expressing cells were increased in comparison to those of truncated mutant Zic1-bearing mice. Conversely, we observed a significant cell number reduction without loss of dorsal properties in the dorsal spinal cords of Zic1-deficient mice. Taken together, these findings suggest that Zic1 controls the expansion of neuronal precursors by inhibiting the progression of neuronal differentiation. Notch-mediated inhibition of neuronal differentiation is likely to act downstream of Zic genes since Notch1 is upregulated in Zic1-overexpressing spinal cords in both the mouse and the chick.     

DM-GRASP, a novel immunoglobulin superfamily axonal surface protein that supports neurite extension

We have identified a 95 kd cell surface protein, DM-GRASP, that is expressed on a restricted population of axons. Its expression begins early in chick embryogenesis, and within the spinal cord it is localized to axons in the dorsal funiculus, midline floorplate cells, and motoneurons. Antibodies to DM-GRASP impair neurite extension on axons, and purified DM-GRASP supports neurite extension from chick sensory neurons. We have cloned and sequenced the cDNA corresponding to this protein and find that it is a new member of the immunoglobulin superfamily of adhesion molecules. Consequently we have named this protein DM-GRASP, since it is an immunoglobulin-like restricted axonal surface protein that is expressed in the dorsal funiculus and ventral midline of the chick spinal cord.     

Orientation of commissural axons in vitro in response to a floor plate-derived chemoattractant

Developing axons are guided to their targets by molecular cues in their local environment. Some cues are short-range, deriving from cells along axonal pathways. There is also increasing evidence for longer-range guidance cues, in the form of gradients of diffusible chemoattractant molecules, which originate from restricted populations of target cells. The guidance of developing commissural axons within the spinal cord depends on one of their intermediate cellular targets, the floor plate. We have shown previously that floor plate cells secrete a diffusible factor(s) that can alter the direction of commissural axon growth in vitro. Here we show that the factor is an effective chemoattractant for commissural axons. It can diffuse considerable distances through a collagen gel matrix and through dorsal and ventral neural epithelium in vitro to reorient the growth of virtually all commissural axons. The orientation of axons occurs in the absence of detectable effects on the survival of commissural neurons or on the rate of commissural axon extension. The regionally restricted expression of the factor suggests that it is present in the embryonic spinal cord in a gradient with its high point at the floor plate. These observations support the idea that the guidance of commissural axons to the ventral midline of the spinal cord results in part from the secretion of a chemoattractant by the floor plate.     

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.     

Pathfinding during spinal tract formation in the chick-quail chimera analysed by species-specific monoclonal antibodies.

In order to analyse the spinal tract formation at early stages of development in avian embryos, chick-quail spinal cord chimeras were prepared and species-specific monoclonal antibodies (MAb) were developed. MAbs CN, QN and CQN uniquely stained chick, quail, and both chick and quail nervous tissues, respectively. All three antibodies appeared to bind to the same membrane molecule, but to different epitopes. Cord reversal revealed the features of axonal growth of both cord interneurons and dorsal root ganglion cells. Quail cord interneurons grew along an originally ventral marginal layer in the quail cord transplanted in a reversed position, then turned toward the ventral side at the boundary between the graft and the host, and grew along the host chick ventral marginal layer. Central axons of dorsal root ganglia were restricted to the ventrolateral region of the cord which originally formed the dorsal funiculus. These results suggest that cord interneurons and dorsal root ganglion cells actively select to grow along specific regions of the cord and that spinal tract formation appears to be determined by cord cells, and not by sclerotome cells.     

Ephrin A/EphA controls the rostral turning polarity of a lateral commissural tract in chick hindbrain

Most post-crossing commissural axons turn into longitudinal paths to make synaptic connections with their targets. Mechanisms that control their rostrocaudal turning polarity are still poorly understood. We used the hindbrain as a model system to investigate the rostral turning of a laterally located commissural tract, identified as the caudal group of contralateral cerebellar-projecting second-order vestibular neurons (cC-VC). We found that the caudal hindbrain possessed a graded non-permissive/repulsive activity for growing cC-VC axons. This non-permissiveness/repulsion was in part mediated by glycosyl-phosphatidylinositol (GPI)-anchored ephrin A. We further demonstrated that ephrin A2 was distributed in a caudal-high/rostral-low gradient in the caudolateral hindbrain and cC-VC axons expressed EphA receptors. Finally, perturbing ephrin A/EphA signalling both in vitro and in vivo led to rostrocaudal pathfinding errors of post-crossing cC-VC axons. These results suggest that ephrin A/EphA interactions play a key role in regulating the polarity of post-crossing cC-VC axons as they turn into the longitudinal axis.