Robos are required for the correct targeting of retinal ganglion cell axons in the visual pathway of the brain

Axonal projections from the retina to the brain are regulated by molecules including the Slit family of ligands [Thompson, H., Barker, D., Camand, O., Erskine, L., 2006a. Slits contribute to the guidance of retinal ganglion cell axons in the mammalian optic tract. Dev. Biol. 296, 476–484, Thompson, H., Camand, O., Barker, D., Erskine, L., 2006b. Slit proteins regulate distinct aspects of retinal ganglion cell axon guidance within dorsal and ventral retina. J. Neurosci. 26, 8082–8091]. However, the roles of Slit receptors in mammals, (termed Robos), have not been investigated in visual system development. Here we examined Robo1 and 2 mutant mice and found that Robos regulate the correct targeting of retinal ganglion cell (RGC) axons along the entire visual projection. We noted aberrant projections of RGC axons into the cerebral cortex, an area not normally targeted by RGC axons. The optic chiasm was expanded along the rostro-caudal axis (similar to Slit mutant mice, Plump, A.S., Erskine, L., Sabatier, C., Brose, K., Epstein, C.J., Goodman, C.S., Mason, C.A., Tessier-Lavigne, M., 2002. Slit1 and Slit2 cooperate to prevent premature midline crossing of retinal axons in the mouse visual system. Neuron 33, 219–232), with ectopic crossing points, and some axons projecting caudally toward the corticospinal tract. Further, we found that axons exuberantly projected into the diencephalon. These defects were more pronounced in Robo2 than Robo1 knockout animals, implicating Robo2 as the predominant Robo receptor in visual system development.     

Expression of Vema in the developing mouse spinal cord and optic chiasm

A critical phase of nervous system development is the formation of connections between axons and their synaptic targets. Intermediate targets play important roles in axon pathfinding by supplying growing axons with long- and short- range guidance cues at decision points along their trajectory. We recently identified Vema as a novel membrane-associated protein that is expressed at the ventral midline of the developing vertebrate central nervous system (CNS). We report that Vema is expressed in the floor plate, an intermediate target for pathfinding commissural axons located at the ventral midline of the developing mouse spinal cord. Interestingly, Vema expression overlaps with the position of an unique population of neurons situated at the midline of the ventral diencephalon and that function as intermediate targets for pathfinding retinal ganglion cell axons. The distribution of Vema in the developing spinal cord and optic chiasm resembles the expression patterns of a variety of molecules known to play important roles in axon guidance, including Robo2, Neuropilin2, and SSEA. The expression of Vema at two key choice points for pathfinding axons suggests an important role for this protein in regulating axon guidance at the midline of the developing mouse central nervous system.     

Slit-Robo signals regulate pioneer axon pathfinding of the tract of the postoptic commissure in the mammalian forebrain

During early vertebrate forebrain development, pioneer axons establish a symmetrical scaffold descending longitudinally through the rostral forebrain, thus forming the tract of the postoptic commissure (TPOC). In mouse embryos, this tract begins to appear at embryonic day 9.5 (E9.5) as a bundle of axons tightly constrained at a specific dorsoventral level. We have characterized the participation of the Slit chemorepellants and their Robo receptors in the control of TPOC axon projection. In E9.5-E11.5 mouse embryos, Robo1 and Robo2 are expressed in the nucleus origin of the TPOC (nTPOC), and Slit expression domains flank the TPOC trajectory. These findings suggested that these proteins are important factors in the dorsoventral positioning of the TPOC axons. Consistently with this role, Slit2 inhibited TPOC axon growth in collagen gel cultures, and interfering with Robo function in cultured embryos induced projection errors in TPOC axons. Moreover, absence of both Slit1 and Slit2 or Robo1 and Robo2 in mutant mouse embryos revealed aberrant TPOC trajectories, resulting in abnormal spreading of the tract and misprojections into both ventral and dorsal tissues. These results reveal that Slit-Robo signaling regulates the dorsoventral position of this pioneer tract in the developing forebrain.     

Diversity of contralateral commissural projections in the embryonic rodent spinal cord

In vertebrate embryos, the axons of spinal commissural neurons grow toward and across the floor plate, a specialized structure located at the ventral midline. Although the initial segment of this trajectory has been intensively studied, relatively little is known about commissural axon pathfinding on the contralateral side of the floor plate in higher vertebrates. We recently demonstrated that many embryonic mouse and chick spinal commissural axons follow a complex trajectory once they cross the ventral midline. Here we use focal applications of 1,1'-dioctadecyl-3,3,3',3' tetramethylindocarbocyanine perchlorate (DiI) to identify four different contralateral commissural trajectories, two of which have not previously been described in the embryonic rodent spinal cord. Intermediate longitudinal commissural (ILC) axons travel away from the floor plate along an arcuate trajectory into intermediate regions of the spinal cord. In contrast, medial longitudinal commissural (MLC) axons grow alongside the floor plate, projecting primarily in the rostral direction. Bifurcating longitudinal commissural (BLC) axons branch into rostrally and caudally directed projections. Forked transverse commissural (FTC) axons either execute two orthogonal turns before crossing the floor plate or extend directly across the floor plate. We also show a variation in the relative frequencies of individual contralateral commissural projections along the dorsoventral and anteroposterior axes of the spinal cord. In addition, using a novel culture system, we demonstrate that commissural axons elaborate ILC-, MLC-, BLC-, and FTC-like trajectories in vitro. These results provide a basis for examining the mechanisms that regulate commissural axon pathfinding on the contralateral side of the floor plate in the embryonic rodent spinal cord.     

Spatiotemporal expression patterns of slit and robo genes in the rat brain.

Diffusible chemorepellents play a major role in guiding developing axons toward their correct targets by preventing them from entering or steering them away from certain regions. Genetic studies in Drosophila revealed a repulsive guidance system that prevents inappropriate axons from crossing the central nervous system midline; this repulsive system is mediated by the secreted extracellular matrix protein Slit and its receptors Roundabout (Robo). Three distinct slit genes (slit1, slit2, and slit3) and three distinct robo genes (robo1, robo2, rig-1) have been cloned in mammals. However, to date, only Robo1 and Robo2 have been shown to be receptors for Slits. In rodents, Slits have been shown to function as chemorepellents for several classes of axons and migrating neurons. In addition, Slit can also stimulate the formation of axonal branches by some sensory axons. To identify Slit-responsive neurons and to help analyze Slit function, we have studied, by in situ hybridization, the expression pattern of slits and their receptors robo1 and robo2, in the rat central nervous system from embryonic stages to adult age. We found that their expression patterns are very dynamic: in most regions, slit and robo are expressed in a complementary pattern, and their expression is up-regulated postnatally. Our study confirms the potential role of these molecules in axonal pathfinding and neuronal migration. However, the persistence of robo and slit expression suggests that the couple slit/robo may also have an important function in the adult brain.     

Robo3.1A suppresses Slit‐mediated repulsion by triggering degradation of Robo2

Slits and Robos control the midline crossing of commissural axons, which are not sensitive to the midline repellent Slit before crossing but gain Slit responsiveness to exit the midline and avoid recrossing. Robo3.1A promotes midline crossing of commissural axons by suppressing the axonal responsiveness to the midline repellent Slit, but the underlying mechanism remains unclear. By using a cell surface binding assay and immunoprecipitation, we observed that Robo3.1A did not bind Slit on its own but prevented the specific binding of Slit to the cell surface when it was coexpressed with its close homologue Robo1 or Robo2 (Robo1/2), which are known to mediate the Slit repulsion. Cotransfection with Robo3.1A significantly reduced the protein level of Robo2 in HEK293 cells, and overexpression of Robo3.1A also significantly decreased Robo2 protein level in cerebellar granule cells. Downregulation of endogenous Robo3 by specific small interference RNA (siRNA) significantly increased Robo1 protein level, Slit binding to the cell surface was significantly elevated, and Slit‐triggered growth cone collapse appeared after downregulation of Robo3 in cultured cortical neurons. Immunocytochemical staining showed that Robo2 and Robo3 colocalized in intracellular vesicles positive for the marker of late endosomes and lysosomes, but not trans‐Golgi apparatus and early endosomes. Thus Robo3.1A may prevent the Slit responsiveness by recruiting Robo1/2 into a late endosome‐ and lysosome‐dependent degradation pathway. © 2014 Wiley Periodicals, Inc.     

The actin-binding protein Canoe/AF-6 forms a complex with Robo and is required for Slit-Robo signaling during axon pathfinding at the CNS midline

Axon guidance is a key process during nervous system development and regeneration. One of the best established paradigms to study the mechanisms underlying this process is the axon decision of whether or not to cross the midline in the Drosophila CNS. An essential regulator of that decision is the well conserved Slit-Robo signaling pathway. Slit guidance cues act through Robo receptors to repel axons from the midline. Despite good progress in our knowledge about these proteins, the intracellular mechanisms associated with Robo function remain poorly defined. In this work, we found that the scaffolding protein Canoe (Cno), the Drosophila orthologue of AF-6/Afadin, is essential for Slit-Robo signaling. Cno is expressed along longitudinal axonal pioneer tracts, and longitudinal Robo/Fasciclin2-positive axons aberrantly cross the midline in cno mutant embryos. cno mutant primary neurons show a significant reduction of Robo localized in growth cone filopodia and Cno forms a complex with Robo in vivo. Moreover, the commissureless (comm) phenotype (i.e., lack of commissures due to constitutive surface presentation of Robo in all neurons) is suppressed in comm, cno double-mutant embryos. Specific genetic interactions between cno, slit, robo, and genes encoding other components of the Robo pathway, such as Neurexin-IV, Syndecan, and Rac GTPases, further confirm that Cno functionally interacts with the Slit-Robo pathway. Our data argue that Cno is a novel regulator of the Slit-Robo signaling pathway, crucial for regulating the subcellular localization of Robo and for transducing its signaling to the actin cytoskeleton during axon guidance at the midline.     

Robo2-Slit and Dcc-Netrin1 Coordinate Neuron Axonal Pathfinding within the Embryonic Axon Tracts

In the embryonic vertebrate brain, early born neurons establish highly stereotyped embryonic axonal tracts along which the neuronal interconnections form. To understand the mechanism underlying neuron axonal pathfinding within the embryonic scaffold of axon tracts, we studied zebrafish anterior dorsal telencephalic (ADt) neuron development. While previous studies suggest the ADt neuronal axons extend along a commissural tract [anterior commissure (AC)] and a descending ipsilateral tract [supraoptic tract (SOT)], it is unclear whether individual ADt neuronal axons choose specific projection paths at the intersection between the AC and the SOT. We labeled individual ADt neurons using a forebrain-specific promoter to drive expression of fluorescent proteins. We found the ADt axonal projection patterns were heterogeneous and correlated with their soma positions. Our results suggest that cell intrinsic differences along the dorsal ventral axis of the telencephalon regulate the axonal projection choices. Next, we determined that the guidance receptors roundabout2 (Robo2) and deleted in colorectal cancer (Dcc) were differentially expressed in the ADt neurons. We showed that knocking down Robo2 function by injecting antisense morpholino oligonucleotides abolished the ipsilateral SOT originating from the ADt neurons. Knocking down Dcc function did not prevent formation of the AC and the SOT. In contrast, the AC was specifically reduced when Netrin1 function was knocked down. Further mechanistic studies suggested that Robo2 responded to the repellent Slit signals and suppressed the attractive Netrin signals. These findings demonstrate how Robo2-Slit and Dcc-Netrin coordinate the axonal projection choices of the developing neurons in the vertebrate forebrain.     

Mis-expression of L1 on pre-crossing spinal commissural axons disrupts pathfinding at the ventral midline

In vertebrates, spinal commissural axons project along a transverse path toward and across the floor plate (FP). Post-crossing commissural axons alter their responsiveness to FP-associated guidance cues and turn to project longitudinally in a fasciculated manner prior to extending away from the midline. The upregulation of the neural cell adhesion molecule L1 on crossed commissural axon segments has been proposed to facilitate pathfinding on the contralateral side of the FP. To explore this possibility in vivo, we used Math1 regulatory sequences to target L1 to commissural axons before they cross the ventral midline. L1 mis-expression did not alter the distribution of commissural axon-associated markers or the ventral extension of commissural axons toward the midline. However, commissural axons often stalled or inappropriately projected into the longitudinal plane at the ipsilateral FP margin. These observations suggest that L1-mediated pathfinding decisions are normally delayed until axons have crossed the ventral midline (VM).     

The adaptor protein Nck2 mediates Slit1-induced changes in cortical neuron morphology

Slits are multifunctional guidance cues, capable of triggering neurite repulsion, extension, or branching, depending on cell type and developmental context. While the Robo family of Slit receptors is a well-established mediator of axon repulsion, a role for Robos in Slit-mediated neurite growth and branching is not well defined, and the signaling molecules that link Robo to the cytoskeletal changes that drive neurite outgrowth are not well characterized in vertebrates. We show that Slit stimulates cortical dendrite branching, and we report that Slit also triggers a robust increase in the length of cortical axons in vitro. Moreover, neurons derived from Robo1; Robo2 deficient mice do not display an increase in neurite length, indicating that endogenous Robos mediate Slit's growth-promoting effects on both axons and dendrites. We also demonstrate that the SH2/SH3 adaptor proteins Nck1 and Nck2 bind to Robo via an atypical SH3-mediated mechanism. Furthermore, we show that only Nck2 is required for the Slit-induced changes in cortical neuron morphology in vitro. These findings indicate a specific role for Nck2 in linking Robo activation to the cytoskeleton rearrangements that shape cortical neuron morphology.     

Classic axon guidance molecules control correct nerve bridge tissue formation and precise axon regeneration

The peripheral nervous system has an astonishing ability to regenerate following a compression or crush injury;however,the potential for full repair following a transection injury is much less.Currently,the major clinical challenge for peripheral nerve repair come from long gaps between the proximal and distal nerve stumps,which prevent regenerating axons reaching the distal nerve.Precise axon targeting during nervous system development is controlled by families of axon guidance molecules including Netrins,Slits,Ephrins and Semaphorins.Several recent studies have indicated key roles of Netrin1,Slit3 and EphrinB2 signalling in controlling the formation of new nerve bridge tissue and precise axon regeneration after peripheral nerve transection injury.Inside the nerve bridge,nerve fibroblasts express EphrinB2 while migrating Schwann cells express the receptor EphB2.EphrinB2/EphB2 signalling between nerve fibroblasts and migrating Schwann cells is required for Sox2 upregulation in Schwann cells and the formation of Schwann cell cords within the nerve bridge to allow directional axon growth to the distal nerve stump.Macrophages in the outermost layer of the nerve bridge express Slit3 while migrating Schwann cells and regenerating axons express the receptor Robo1;within Schwann cells,Robo1 expression is also Sox2-dependent.Slit3/Robo1 signalling is required to keep migrating Schwann cells and regenerating axons inside the nerve bridge.In addition to the Slit3/Robo1 signalling system,migrating Schwann cells also express Netrin1 and regenerating axons express the DCC receptor.It appears that migrating Schwann cells could also use Netrin1 as a guidance cue to direct regenerating axons across the peripheral nerve gap.Engineered neural tissues have been suggested as promising alternatives for the repair of large peripheral nerve gaps.Therefore,understanding the function of classic axon guidance molecules in nerve bridge formation and their roles in axon regeneration could be highly beneficial in developing engineered neural tissue for more effective peripheral nerve repair.     

Projections of the locus coeruleus and adjacent pontine tegmentum in the cat.

The projections of the locus coeruleus and adjacent pontine tegmentum have been studied using anatomical and physiological methods in the cat. Axonal trajectories were traced using either the Fink-Heimer I method following electrolytic lesions, or the autoradiographic method after injection of tritiated proline into the nucleus. Results with both methods were similar. Axons of locus noeruleus neurons ascended ipsilaterally through the mesencephalon lateral to the medial longitudinal fasiculus, ventrolateral to the central gray. In the caudal diencephalon, the ascending fibers entered the centrum medianum-parafascicular complex where they diverged into two fascicles: a dorsal fascicle which terminated in the intralaminar nuclei of the thalamus, and a ventral fascicle which gave off fibers to the ventrobasal complex and reticular nucleus of the thalamus while continuing centrolaterally into the lateral hypothalamus medial to the internal capsule. Fibers of the ventral fascicle ascended in the lateral hypothalamus and zona incerta and were traced through the preoptic region into the septum. Fibers could not be consistently traced to the cerebral cortex, and were not seen at all in the cerebellum. Throughout the ascending course of the path from the locus coeruleus, axons were given off to the pretectal area, the medial and lateral geniculate nuclei and the amygdala; fibers passed contralaterally through the posterior commissure, the midline thalamus, and the supraoptic commissure. Fibers descending from the locus coeruleus surrounded the intramedullary portion of the facial nerve and further caudally were observed ventrolateral to the hypoglossal and dorsal vagal nuclei. The axonal trajectories visualized with degeneration and autoradiographic methods followed closely those previously shown for reticular formation neurons, but were also similar to locus coeruleus projections revealed by histofluorescence methods. After injections of horseradish peroxidase into the centrum medianum-parafascicular complex, lateral hypothalamus or preoptic region, labeled neurons were located in the locus coeruleus, nucleus subcoeruleus, and lateral parabrachial nucleus. Reticular formation neurons were not labeled. Neurons in locus coeruleus and adjacent pontine tegmentum could be antidromically activated by stimulation in the rostral midbrain or caudal diencephalon. Our data indicate that both adrenergic and non-adrenergic neurons of the dorsolateral pontine tegmentum have similar projections.     

Dynamic expression of Slit1–3 and Robo1–2 in the mouse peripheral nervous system after injury

The Slit family of axon guidance cues act as repulsive molecules for precise axon pathfinding and neuronal migration during nervous system development through interactions with specific Robo receptors.Although we previously reported that Slit1–3 and their receptors Robo1 and Robo2 are highly expressed in the adult mouse peripheral nervous system,how this expression changes after injury has not been well studied.Herein,we constructed a peripheral nerve injury mouse model by transecting the right sciatic nerve.At 14 days after injury,quantitative real-time polymerase chain reaction was used to detect mRNA expression of Slit1–3 and Robo1–2 in L4–5 spinal cord and dorsal root ganglia,as well as the sciatic nerve.Immunohistochemical analysis was performed to examine Slit1–3,Robo1–2,neurofilament heavy chain,F4/80,and vimentin in L4–5 spinal cord,L4 dorsal root ganglia,and the sciatic nerve.Co-expression of Slit1–3 and Robo1–2 in L4 dorsal root ganglia was detected by in situ hybridization.In addition,Slit1–3 and Robo1–2 protein expression in L4–5 spinal cord,L4 dorsal root ganglia,and sciatic nerve were detected by western blot assay.The results showed no significant changes of Slit1–3 or Robo1–2 mRNA expression in the spinal cord within 14 days after injury.In the dorsal root ganglion,Slit1–3 and Robo1–2 mRNA expression were initially downregulated within 4 days after injury;however,Robo1–2 mRNA expression returned to the control level,while Slit1–3 mRNA expression remained upregulated during regeneration from 4–14 days after injury.In the sciatic nerve,Slit1–3 and their receptors Robo1–2 were all expressed in the proximal nerve stump;however,Slit1,Slit2,and Robo2 were barely detectable in the nerve bridge and distal nerve stump within 14 days after injury.Slit3 was highly ex-pressed in macrophages surrounding the nerve bridge and slightly downregulated in the distal nerve stump within 14 days after injury.Robo1 was upregulated in vimentin-positive cells and migrating Schwann cells inside the nerve bridge.Robo1 was also upregulated in Schwann cells of the distal nerve stump within 14 days after injury.Our findings indicate that Slit3 is the major ligand expressed in the nerve bridge and distal nerve stump during peripheral nerve regeneration,and Slit3/Robo signaling could play a key role in peripheral nerve repair after injury.This study was approved by Plymouth University Animal Welfare Ethical Review Board (approval No.30/3203) on April 12,2014.     

Segregation and pathfinding of callosal axons through EphA3 signaling

The corpus callosum, composed of callosal axons, is the largest structure among commissural connections in eutherian animals. Axon pathfinding of callosal neurons has been shown to be guided by intermediate targets, such as midline glial structures. However, it has not yet been understood completely how axon-axon interactions, another major mechanism for axon pathfinding, are involved in the pathfinding of callosal neurons. Here, we show that callosal axons from the medial and lateral regions of the mouse cerebral cortex pass through the dorsal and ventral parts, respectively, of the corpus callosum. Using an explant culture system, we observed that the axons from the medial and lateral cortices were segregated from each other in vitro, and that this segregation was attenuated by inhibition of EphA3 signaling. We also found that knockdown of EphA3, which is preferentially expressed in the lateral cortex, resulted in disorganized segregation of the callosal axons and disrupted axon pathfinding in vivo. These results together suggest the role of axonal segregation in the corpus callosum, mediated at least in part by EphA3, in correct pathfinding of callosal neurons.     

Dynamic expression patterns of Robo (Robo1 and Robo2) in the developing murine central nervous system

The Robo family of molecules is important for axon guidance across the midline during central nervous system (CNS) development in invertebrates and vertebrates. Here we describe the patterns of Robo protein expression in the developing mouse CNS from embryonic day (E) 9.5 to postnatal day (P) 4, as determined by immunohistochemical labeling with an antibody (S3) raised against a common epitope present in the Robo ectodomain of Robos 1 and 2. In the spinal cord, midline-crossing axons are initially (at E11) S3-positive. At later times, midline Robo expression disappears, but is strongly upregulated in longitudinally running postcrossing axons. It is also strongly expressed in noncrossing longitudinal axons. Differential expression of Robo along axons was also found in axons cultured from E14 spinal cord. These findings resemble those from the Drosophila ventral nerve cord and indicate that in vertebrates a low level of Robo expression occurs in the initial crossing of the midline, while a high level of expression in the postcrossing fibers prevents recrossing. Likewise, Robo-positive ipsilateral axons are prevented from crossing at all. However, in the brain different rules appear to apply. Most commissural axons including those of the corpus callosum are strongly S3-positive along their whole length from their time of formation to postnatal life, but some have more complex age-dependent expression patterns. S3 labeling of the optic pathway is also complex, being initially strong in the retinal ganglion cells, optic tract, and chiasma but thereafter being lost except in a proportion of postchiasmal axons. The corticospinal tract is strongly positive throughout its course at all stages examined, including its decussation, formed at about P2 in the central part of the medulla oblongata.     

Chiasmatic neurons in the ventral diencephalon of mouse embryos--changes in arrangement and heterogeneity in surface antigen expression

We have investigated the changes in arrangement of the SSEA-1 immunoreactive chiasmatic neurons in the mouse ventral diencephalon from embryonic day (E) 9 to the end of gestation. A regionally specific staining of SSEA-1 was first detected in the ventricular layer of the caudal diencephalon at E10 and later at E11 on the cells in the subventricular layer. At E12, these cells formed the characteristic V-shaped configuration caudal to the optic axons in the chiasm. At E13-E15, this neuronal array changes gradually to a configuration that facilitates contact with the optic axons only at the midline and the initial segment of the optic tract. Colocalization studies showed that CD44 was localized strongly on the neurons in the central but not lateral domains of the array, suggesting existence of heterogeneity in these neurons in terms of surface antigen presentation. This difference between the central and lateral domains raises the possibility that the chiasmatic neurons may regulate the patterning of axon orders at the midline and the optic tract through presentation of distinct combination of guidance cues at these strategic positions in the optic pathway. Furthermore, exogenous Lewis-x/SSEA-1 inhibited neurite outgrowth from the E14 retinal explants; this inhibition was observed in neurites from both ventral temporal and dorsal nasal retina. These findings suggest an action of this surface carbohydrate on the control of axon growth and guidance in the mouse optic pathway.     

Axonal trajectories and distribution of GABAergic spinal neurons in wildtype and mutant zebrafish lacking floor plate cells.

The role of the midline floor plate cells in the neuronal differentiation of the spinal cord was examined by comparing putative GABAergic neurons in wildtype zebrafish embryos with those in cyc-1 mutant embryos. The mutation produces a pleiotropic recessive lethal phenotype and is severe in rostral brain regions, but its direct effect in the caudal hindbrain and the spinal cord is apparently restricted to the depletion of the midline floor plate cells. In wildtype embryos, an antibody against the neurotransmitter GABA labeled the cell bodies, axons, and growth cones of three classes of previously identified neurons; dorsal longitudinal neurons (DoLA), commissural secondary ascending neurons (CoSA), and ventral longitudinal neurons (VeLD). A novel ventral cell type, Kolmer-Agduhr (KA) neurons, was also labeled. In the cyc-1 mutant, abnormalities were observed in some, but not all, of the GABAreactive CoSA, VeLD, and KA axons, while the axonal trajectories of DoLA neurons were not affected. Furthermore, the number of KA cells was reduced in the mutant while the numbers of the other GABAreactive cells were unperturbed. These observations corroborate our earlier hypothesis that the floor plate cells are one of several guidance cues that direct axonal outgrowth near the ventral midline of the spinal cord. They also suggest that the floor plate cells may play a role in the cellular differentiation of the spinal cord of zebrafish embryos.     

Semaphorins 3A, 3C, and 3F in mesencephalic dopaminergic axon pathfinding

By analyzing the mechanisms that govern dopaminergic axon pathfinding from the midbrain to the striatum in embryonic rat brains, we identified neuroepithelial regions that exert chemotropic effects on mesencephalic dopaminergic axons. Explants from the pretectum and the striatum showed an attractive effect, whereas those from the midhindbrain boundary, the dorsal thalamus, and the ventral thalamus had no effect. Expression of semaphorin (Sema) 3C and Sema3F in the pretectum and of Sema3A in the striatum suggested a role for these axon guidance molecules in dopaminergic axon pathfinding. When expressed in HEK293 cell aggregates, Sema3C had an attractive effect and enhanced axon growth, Sema3A enhanced axon growth, and Sema3F had a repulsive effect on dopaminergic axons. Antineuropilin-1 and antineuropilin-2 antibodies reduced attraction by the pretectum, whereas attraction by the striatum was not affected by the presence of antineuropilin-1 antibodies. Moreover, neuropilin-1- and neuropilin-2-soluble Fc chimeras reduced the attraction by the pretectum. These results suggest that semaphorins may help to establish the dopaminergic projection from the midbrain to the striatum during embryonic development.