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.     

Plasmin-mediated release of the guidance molecule F-spondin from the extracellular matrix

Serine proteases are implicated in a variety of processes during neurogenesis, including cell migration, axon outgrowth, and synapse elimination. Tissue-type plasminogen activator and urokinase-type activator are expressed in the floor plate during embryonic development. F-spondin, a gene also expressed in the floor plate, encodes a secreted, extracellular matrix-attached protein that promotes outgrowth of commissural axons and inhibits outgrowth of motor axons. F-spondin is processed in vivo to yield an amino half protein that contains regions of homology to reelin and mindin, and a carboxyl half protein that contains either six or four thrombospondin type I repeats (TSRs). We have tested F-spondin to see whether it is subjected to processing by plasmin and to determine whether the processing modulates its biological activity. Plasmin cleaves F-spondin at its carboxyl terminus. By using nested deletion proteins and mutating potential plasmin cleavage sites, we have identified two cleavage sites, the first between the fifth and sixth TSRs, and the second at the fifth TSR. Analysis of the extracellular matrix (ECM) attachment properties of the TSRs revealed that the fifth and sixth TSRs bind to the ECM, but repeats 1-4 do not. Structural functional experiments revealed that two basic motives are required to elicit binding of TSR module to the ECM. We demonstrate further that plasmin releases the ECM-bound F-spondin protein.     

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.     

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.     

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.     

Proteolysis and membrane capture of F-spondin generates combinatorial guidance cues from a single molecule

The formation of neuronal networks is governed by a limited number of guidance molecules, yet it is immensely complex. The complexity of guidance cues is augmented by posttranslational modification of guidance molecules and their receptors. We report here that cleavage of the floor plate guidance molecule F-spondin generates two functionally opposing fragments: a short-range repellent protein deposited in the membrane of floor plate cells and an adhesive protein that accumulates at the basement membrane. Their coordinated activity, acting respectively as a short-range repellant and a permissive short-range attractant, constricts commissural axons to the basement membrane beneath the floor plate cells. We further demonstrate that the repulsive activity of the inhibitory fragment of F-spondin requires its presentation by the lipoprotein receptor-related protein (LRP) receptors apolipoprotein E receptor 2, LRP2/megalin, and LRP4, which are expressed in the floor plate. Thus, proteolysis and membrane interaction coordinate combinatorial guidance signaling originating from a single guidance cue.     

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.     

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.     

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.     

The neuronal class 2 TSR proteins F-spondin and Mindin: a small family with divergent biological activities

F-spondin and Mindin are members of a subgroup of the thrombospondin type 1 (TSR) class molecules, defined by two domains of homology, the FS1/FS2 and TSR domains. The TSRs of F-spondin proteins are typical of class 2 TSRs. F-spondin and Mindin are evolutionarily conserved proteins. The embryonic expression of the vertebrate genes is enriched in the nervous system, mainly at the floor plate and the hippocampus. Similar to thrombospondin, F-spondin and Mindin are extracellular matrix attached molecules that promote neurite outgrowth and inhibit angiogenesis. Analysis of gain and loss of function experiments reveal that F-spondin is required for accurate pathfinding of embryonic axons. F-spondin plays a dual role in patterning axonal trajectories: it promotes the outgrowth of commissural and inhibits the outgrowth of motor axons. Macrophages of Mindin-deficient mice exhibit defective responses to a broad spectrum of microbial stimuli. This may implicate Mindin and F-spondin in inflammatory processes in the nervous system.     

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.     

Molecular and cellular characterization of the glial roof plate of the spinal cord and optic tectum: a possible role for a proteoglycan in the development of an axon barrier.

Certain types of glial structures, located at strategic positions along axon pathways, may provide the mechanical and/or chemical elements for the construction of barriers which can grossly direct the elongation of axons during development. The roof plate, a putative axon barrier, is located along the dorsal midline of the developing spinal cord and may be important for the guidance of the commissural and dorsal column axons. We examined the roof plate to determine the developmental morphology of the region and to determine which molecules were correlated with the barrier function when axons were growing nearby. Light and electron microscopic observations of the roof plate revealed that this glial domain undergoes a dramatic change in shape from a "wedge" with large extracellular spaces between the cell apices at E12.5 to a thin, dense septum with reduced extracellular space at E15.5. Immunocytochemical techniques demonstrated that highly sialylated neural cell adhesion molecule (N-CAM), the carbohydrate recognized by L2 monoclonal antibody, cholinesterase, stage-specific embryonic antigen 1, and a ligand that binds tetragonolobus purpureas agglutinin are expressed by the roof plate. These molecules, however, were also found in other regions of the spinal cord which are permissive or attractive to axon growth. A molecule which is unique to the roof plate when axons grow close to, but do not cross, the dorsal midline is a glycosaminoglycan (GAG), keratan sulfate. Keratan sulfate is also present in the tectal midline and in other noninnervated regions such as the outer epidermis and developing cartilage. Our data suggest that keratan sulfate, alone or in combination with other molecules expressed by the roof plate, may be responsible, in part, for the inhibition of axon elongation through the roof plate in the embryonic spinal cord.     

Structural characterization of a ribose-5-phosphate isomerase B from the pathogenic fungus Coccidioides immitis

Background

F-spondin is a multi-domain extracellular matrix (ECM) protein and a contact-repellent molecule that directs axon outgrowth and cell migration during development. The reelin_N domain and the F-spondin domain (FS domain) comprise a proteolytic fragment that interacts with the cell membrane and guides the projection of commissural axons to floor plate. The FS domain is found in F-spondins, mindins, M-spondin and amphiF-spondin.

Results

We present the crystal structure of human F-spondin FS domain at 1.95Å resolution. The structure reveals a Ca²⁺-binding C2 domain variant with an 8-stranded antiparallel β-sandwich fold. Though the primary sequences of the FS domains of F-spondin and mindin are less than 36% identical, their overall structures are very similar. The unique feature of F-spondin FS domain is the presence of three disulfide bonds associated with the N- and C-termini of the domain and a highly conserved N-linked glycosylation site. The integrin-binding motif found in mindin is not conserved in the F-spondin FS domain.

Conclusion

The structure of the F-spondin FS domain completes the structural studies of the multiple-domain ECM molecule. The homology of its core structure to a common Ca²⁺- and lipid-binding C2 domain suggests that the F-spondin FS domain may be responsible for part of the membrane targeting of F-spondin in its regulation of axon development. The structural properties of the FS domain revealed in this study pave the way for further exploration into the functions of F-spondin.     

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.     

The expression of tissue and urokinase-type plasminogen activators in neural development suggests different modes of proteolytic involvement in neuronal growth.

Tissue and urokinase-type plasminogen activators are serine proteases with highly restricted specificity, their best characterised role being to release the broad specificity protease plasmin from inactive plasminogen. It has frequently been suggested that these, and similar proteases, are involved in axonal growth and tissue remodelling associated with neural development. To help define what this role might be, we have studied the expression of the plasminogen activators in developing rat nervous tissue. Urokinase-type plasminogen activator mRNA is strongly expressed by many classes of neurons in peripheral and central nervous system. We have analysed its appearance in spinal cord and sensory ganglia, and found the mRNA is detectable by in situ hybridisation very early in neuronal development (by embryonic day 12.5), at a stage compatible with it playing a role in axonal or dendritic growth. Tissue plasminogen activator mRNA, on the other hand, is expressed only by cells of the floor plate in the developing nervous system, from embryonic day 10.5 and thereafter. Immunohistochemical and enzymatic analysis showed that active tissue plasminogen activator is produced by, and retained within, the floor plate. A mechanism is suggested by which high levels of tissue plasminogen activator produced by the stationary cells of the floor plate could influence the direction of growth of commissural axons as they pass through this midline structure.     

Slit proteins are not dominant chemorepellents for olfactory tract and spinal motor axons.

Members of the Slit family are large extracellular glycoproteins that may function as chemorepellents in axon guidance and neuronal cell migration. Their actions are mediated through members of the Robo family that act as their receptors. In vertebrates, Slit causes chemorepulsion of embryonic olfactory tract, spinal motor, hippocampal and retinal ganglion cell axons. Since Slits are expressed in the septum and floor plate during the period when these tissues cause chemorepulsion of olfactory tract and spinal motor axons respectively, it has been proposed that Slits function as guidance cues. We have tested this hypothesis in collagen gel co-cultures using soluble Robo/Fc chimeras, as competitive inhibitors, to disrupt Slit interactions. We find that the addition of soluble Robo/Fc has no effect on chemorepulsion of olfactory tract and spinal motor axons when co-cultured with septum or floor plate respectively. Thus, we conclude that although Slits are expressed in the septum and floor plate, their proteins do not contribute to the major chemorepulsive activities emanating from these tissues which cause repulsion of olfactory tract and spinal motor axons.     

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.     

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.     

Immunohistochemical properties of the "floor plate glycogen body" of the human embryonic spinal cord and brain stem

Prominent glycogen accumulations have been found in the floor plate radial glial cells of the human spinal cord and brain stem during the 6th to 13th week of intrauterine life. These glycogen-rich cells are totally negative to indirect immunoperoxidase staining with an antibody to glial fibrillary acidic protein, a protein that is strongly expressed in the remaining radial glial cells that border the central canal of the spinal cord. The glycogen-rich floor plate radial glial cells are, on the other hand, heavily stained by a monoclonal antibody against a vimentin-related protein. The neighbouring lateral radial glial cells do not express this protein. These and other distinctive features of the floor plate radial glial cells indicate an organoid specialisation of the floor plate during limited periods of intrauterine life. The function(s) of this specialised tissue remains obscure, but it may be related to cortico-spinal fibres crossing the midline through the floor plate, or to the transport of substances in both directions between blood vessels and the central canal.