Longitudinal axons are guided by Slit/Robo signals from the floor plate

Longitudinal axons grow long distances along precise pathways to connect major CNS regions. However, during embryonic development, it remains largely undefined how the first longitudinal axons choose specific positions and grow along them. Here, we review recent evidence identifying a critical role for Slit/Robo signals to guide pioneer longitudinal axons in the embryonic brain stem. These studies indicate that Slit/Robo signals from the floor plate have dual functions: to repel longitudinal axons away from the ventral midline, and also to maintain straight longitudinal growth. These dual functions likely cooperate with other guidance cues to establish the major longitudinal tracts in the brain.     

Robo-Slit interactions regulate longitudinal axon pathfinding in the embryonic vertebrate brain

The early network of axons in the embryonic brain provides connectivity between functionally distinct regions of the nervous system. While many of the molecular interactions driving commissural pathway formation have been deciphered, the mechanisms underlying the development of longitudinal tracts remain unclear. We have identified here a role for the Roundabout (Robo) family of axon guidance receptors in the positioning of longitudinally projecting axons along the dorsoventral axis in the embryonic zebrafish forebrain. Using a loss-of-function approach, we established that Robo family members exhibit complementary functions in the tract of the postoptic commissure (TPOC), the major longitudinal tract in the forebrain. Robo2 acted initially to split the TPOC into discrete fascicles upon entering a broad domain of Slit1a expression in the ventrocaudal diencephalon. In contrast, Robo1 and Robo3 restricted the extent of defasciculation of the TPOC. In this way, the complementary roles of Robo family members balance levels of fasciculation and defasciculation along this trajectory. These results demonstrate a key role for Robo-Slit signaling in vertebrate longitudinal axon guidance and highlight the importance of context-specific guidance cues during navigation within complex pathways.     

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.     

Robo1 and Robo2 have distinct roles in pioneer longitudinal axon guidance

Pioneer longitudinal axons grow long distances parallel to the floor plate and precisely maintain their positions using guidance molecules released from the floor plate. Two receptors, Robo1 and Robo2, are critical for longitudinal axon guidance by the Slit family of chemorepellents. Previous studies showed that Robo1^−/−;2^−/− double mutant mouse embryos have disruptions in both ventral and dorsal longitudinal tracts. However, the role of each Robo isoform remained unclear, because Robo1 or 2 single mutants have mild or no errors. Here we utilized a more sensitive genetic strategy to reduce Robo levels for determining any separate functions of the Robo1 and 2 isoforms. We found that Robo1 is the predominant receptor for guiding axons in ventral tracts and prevents midline crossing. In contrast, Robo2 is the main receptor for directing axons within dorsal tracts. Robo2 also has a distinct function in repelling neuron cell bodies from the floor plate. Therefore, while Robo1 and 2 have some genetic overlap to cooperate in guiding longitudinal axons, each isoform has distinct functions in specific longitudinal axon populations.     

Lateral neuron--glia interactions steer the response of axons to the Robo code

Glia are required for axon pathfinding along longitudinal trajectories, but it is unknown how this relates to the molecular paradigm of axon guidance across the midline. Most interneuron axons in bilateral organisms cross the midline only once. Preventing them from recrossing the midline requires the expression of Robo receptors on the axons. These sense the repulsive signal Slit, which is produced by the midline. The lateral positioning of longitudinal axons depends on the response to Slit by the combination of Robo receptors expressed by the axons, on selective fasciculation, and on longitudinal (lateral) glia. Here, we analyse how longitudinal glia influence reading of the 'Robo code' by axons. We show that whereas loss of robo1 alone only affects the most medial axons, loss of both glial cells missing (gcm) and robo1 causes a severe midline collapse of longitudinal axons, similar to that caused by the loss of multiple Robo receptors. Furthermore, whereas ectopic expression of robo2 is sufficient to displace the medial MP2 axons along a more lateral trajectory, this does not occur in gcm-robo1 double-mutant embryos, where axons either do not extend at all or they misroute exiting the CNS. Hence, lateral neuron-glia interactions steer the response of axons to the Robo code.     

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.     

Slit proteins bind Robo receptors and have an evolutionarily conserved role in repulsive axon guidance

Extending axons in the developing nervous system are guided in part by repulsive cues. Genetic analysis in Drosophila, reported in a companion to this paper, identifies the Slit protein as a candidate ligand for the repulsive guidance receptor Roundabout (Robo). Here we describe the characterization of three mammalian Slit homologs and show that the Drosophila Slit protein and at least one of the mammalian Slit proteins, Slit2, are proteolytically processed and show specific, high-affinity binding to Robo proteins. Furthermore, recombinant Slit2 can repel embryonic spinal motor axons in cell culture. These results support the hypothesis that Slit proteins have an evolutionarily conserved role in axon guidance as repulsive ligands for Robo receptors.     

Slit-mediated repulsion is a key regulator of motor axon pathfinding in the hindbrain

The floor plate is known to be a source of repellent signals for cranial motor axons, preventing them from crossing the midline of the hindbrain. However, it is unknown which molecules mediate this effect in vivo. We show that Slit and Robo proteins are candidate motor axon guidance molecules, as Robo proteins are expressed by cranial motoneurons, and Slit proteins are expressed by the tissues that delimit motor axon trajectories, i.e. the floor plate and the rhombic lip. We present in vitro evidence showing that Slit1 and Slit2 proteins are selective inhibitors and repellents for dorsally projecting, but not for ventrally projecting, cranial motor axons. Analysis of mice deficient in Slit and Robo function shows that cranial motor axons aberrantly enter the midline, while ectopic expression of Slit1 in chick embryos leads to specific motor axon projection errors. Expression of dominant-negative Robo receptors within cranial motoneurons in chick embryos strikingly perturbs their projections, causing some motor axons to enter the midline, and preventing dorsally projecting motor axons from exiting the hindbrain. These data suggest that Slit proteins play a key role in guiding dorsally projecting cranial motoneurons and in facilitating their neural tube exit.     

Robo1 regulates the development of major axon tracts and interneuron migration in the forebrain

The Slit genes encode secreted ligands that regulate axon branching, commissural axon pathfinding and neuronal migration. The principal identified receptor for Slit is Robo (Roundabout in Drosophila). To investigate Slit signalling in forebrain development, we generated Robo1 knockout mice by targeted deletion of exon 5 of the Robo1 gene. Homozygote knockout mice died at birth, but prenatally displayed major defects in axon pathfinding and cortical interneuron migration. Axon pathfinding defects included dysgenesis of the corpus callosum and hippocampal commissure, and abnormalities in corticothalamic and thalamocortical targeting. Slit2 and Slit1/2 double mutants display malformations in callosal development, and in corticothalamic and thalamocortical targeting, as well as optic tract defects. In these animals, corticothalamic axons form large fasciculated bundles that aberrantly cross the midline at the level of the hippocampal and anterior commissures, and more caudally at the medial preoptic area. Such phenotypes of corticothalamic targeting were not observed in Robo1 knockout mice but, instead, both corticothalamic and thalamocortical axons aberrantly arrived at their respective targets at least 1 day earlier than controls. By contrast, in Slit mutants, fewer thalamic axons actually arrive in the cortex during development. Finally, significantly more interneurons (up to twice as many at E12.5 and E15.5) migrated into the cortex of Robo1 knockout mice, particularly in both rostral and parietal regions, but not caudal cortex. These results indicate that Robo1 mutants have distinct phenotypes, some of which are different from those described in Slit mutants, suggesting that additional ligands, receptors or receptor partners are likely to be involved in Slit/Robo signalling.     

Robo2 is required for Slit-mediated intraretinal axon guidance

The developing optic pathway has proven one of the most informative model systems for studying mechanisms of axon guidance. The first step in this process is the directed extension of retinal ganglion cell (RGC) axons within the optic fibre layer (OFL) of the retina towards their exit point from the eye, the optic disc. Previously, we have shown that the inhibitory guidance molecules, Slit1 and Slit2, regulate two distinct aspects of intraretinal axon guidance in a region-specific manner. Using knockout mice, we have found that both of these guidance activities are mediated via Robo2. Of the four vertebrate Robos, only Robo1 and Robo2 are expressed by RGCs. In mice lacking robo1 intraretinal axon guidance occurs normally. However, in mice lacking robo2 RGC axons make qualitatively and quantitatively identical intraretinal pathfinding errors to those reported previously in Slit mutants. This demonstrates clearly that, as in other regions of the optic pathway, Robo2 is the major receptor required for intraretinal axon guidance. Furthermore, the results suggest strongly that redundancy with other guidance signals rather than different receptor utilisation is the most likely explanation for the regional specificity of Slit function during intraretinal axon pathfinding.     

Distinct roles for Robo2 in the regulation of axon and dendrite growth by retinal ganglion cells

Guidance factors act on the tip of a growing axon to direct it to its target. What role these molecules play, however, in the control of the dendrites that extend from that axon’s cell body is poorly understood. Slits, through their Robo receptors, guide many types of axons, including those of retinal ganglion cells (RGCs). Here we assess and contrast the role of Slit/Robo signalling in the growth and guidance of the axon and dendrites extended by RGCs in Xenopus laevis. As Xenopus RGCs extend dendrites, they express robo2 and robo3, while slit1 and slit2 are expressed in RGCs and in the adjacent inner nuclear layer. Interestingly, our functional data with antisense knockdown and dominant negative forms of Robo2 (dnRobo2) and Robo3 (dnRobo3) indicate that Slit/Robo signalling has no role in RGC dendrite guidance, and instead is necessary to stimulate dendrite branching, primarily via Robo2. Our in vitro culture data argue that Slits are the ligands involved. In contrast, both dnRobo2 and dnRobo3 inhibited the extension of axons and caused the misrouting of some axons. Based on these data, we propose that Robo signalling can have distinct functions in the axon and dendrites of the same cell, and that the specific combinations of Robo receptors could underlie these differences. Slit acts via Robo2 in dendrites as a branching/growth factor but not in guidance, while Robo2 and Robo3 function in concert in axons to mediate axonal interactions and respond to Slits as guidance factors. These data underscore the likelihood that a limited number of extrinsic factors regulate the distinct morphologies of axons and dendrites.     

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.     

Possible roles of robo1+ ensheathing cells in guiding dorsal‐zone olfactory sensory neurons in mouse

In the mouse olfactory system, the anatomical locations of olfactory sensory neurons (OSNs) correlate with their axonal projection sites along the dorsoventral axis of the olfactory bulb (OB). We have previously reported that Neuropilin‐2 expressed by ventral‐zone OSNs contributes to the segregation of dorsal and ventral OSN axons, and that Slit is acting as a negative land mark to restrict the projection of Robo2⁺, early‐arriving OSN axons to the embryonic OB. Here, we report that another guidance receptor, Robo1, also plays an important role in guiding OSN axons. Knockout mice for Robo1 demonstrated defects in targeting of OSN axons to the OB. Although Robo1 is colocalized with dorsal‐zone OSN axons, it is not produced by OSNs, but instead by olfactory ensheathing cells. These findings indicate a novel strategy of axon guidance in the mouse olfactory system during development. © 2013 Wiley Periodicals, Inc. Develop Neurobiol 73:828–840, 2013     

Robo2 is required for establishment of a precise glomerular map in the zebrafish olfactory system

Olfactory sensory neurons (OSNs) expressing a given odorant receptor project their axons to specific glomeruli, creating a topographic odor map in the olfactory bulb (OB). The mechanisms underlying axonal pathfinding of OSNs to their precise targets are not fully understood. Here, we demonstrate that Robo2/Slit signaling functions to guide nascent olfactory axons to the OB primordium in zebrafish. robo2 is transiently expressed in the olfactory placode during the initial phase of olfactory axon pathfinding. In the robo2 mutant, astray (ast), early growing olfactory axons misroute ventromedially or posteriorly, and often penetrate into the diencephalon without reaching the OB primordium. Four zebrafish Slit homologs are expressed in regions adjacent to the olfactory axon trajectory, consistent with their role as repulsive ligands for Robo2. Masking of endogenous Slit gradients by ubiquitous misexpression of Slit2 in transgenic fish causes posterior pathfinding errors that resemble the ast phenotype. We also found that the spatial arrangement of glomeruli in OB is perturbed in ast adults, suggesting an essential role for the initial olfactory axon scaffold in determining a topographic glomerular map. These data provide functional evidence for Robo2/Slit signaling in the establishment of olfactory neural circuitry in zebrafish.     

The divergent Robo family protein rig-1/Robo3 is a negative regulator of slit responsiveness required for midline crossing by commissural axons

Commissural axons in vertebrates and insects are initially attracted to the nervous system midline, but once they reach this intermediate target they undergo a dramatic switch, becoming responsive to repellent Slit proteins at the midline, which expel them onto the next leg of their trajectory. We have unexpectedly implicated a divergent member of the Robo family, Rig-1 (or Robo3), in preventing premature Slit sensitivity in mammals. Expression of Rig-1 protein by commissural axons is inversely correlated with Slit sensitivity. Removal of Rig-1 results in a total failure of commissural axons to cross. Genetic and in vitro analyses indicate that Rig-1 functions to repress Slit responsiveness similarly to Commissureless (Comm) in Drosophila. Unlike Comm, however, Rig-1 does not produce its effect by downregulating Robo receptors on precrossing commissural axon membranes. These results identify a mechanism for regulating Slit repulsion that helps choreograph the precise switch from attraction to repulsion at a key intermediate axonal target.     

Slit/Robo信号在血管新生中的功能研究

分泌型糖蛋白Slit及其受体Roundabout(Robo)最初是作为一类重要的发育中神经元轴突导向分子而被发现的。目前为止对Slit/Robo信号对神经系统发育过程中轴突吸引或排斥的导向功能研究比较多,而对在发育中生长方式与其非常相似的血管发生过程中研究比较少。现有研究提示两者在发育过程中可能存在共同的信号调控机制,是Slit/Robo信号通路在血管新生中充当着重要的角色。该文就Slit/Robo信号对血管内皮细胞迁移的调节、对血管新生的作用及其可能介导的信号通路进行综述,以期进一步推动Slit/Robo信号通路在血管发生中的研究。     

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

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

Short-range and long-range guidance by Slit and its Robo receptors: a combinatorial code of Robo receptors controls lateral position

Slit is secreted by midline glia in Drosophila and functions as a short-range repellent to control midline crossing. Although most Slit stays near the midline, some diffuses laterally, functioning as a long-range chemorepellent. Here we show that a combinatorial code of Robo receptors controls lateral position in the CNS by responding to this presumptive Slit gradient. Medial axons express only Robo, intermediate axons express Robo3 and Robo, while lateral axons express Robo2, Robo3, and Robo. Removal of robo2 or robo3 causes lateral axons to extend medially; ectopic expression of Robo2 or Robo3 on medial axons drives them laterally. Precise topography of longitudinal pathways appears to be controlled by a combination of long-range guidance (the Robo code determining region) and short-range guidance (discrete local cues determining specific location within a region).     

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.     

Slit2-Mediated chemorepulsion and collapse of developing forebrain axons

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 novel repulsive guidance system that prevents inappropriate axons from crossing the CNS midline; this repulsive system is mediated by the Roundabout (Robo) receptor and its secreted ligand Slit. In rodents, Robo and Slit are expressed in the spinal cord and Slit can repel spinal motor axons in vitro. Here, we extend these findings into higher brain centers by showing that Robo1 and Robo2, as well as Slit1 and Slit2, are often expressed in complementary patterns in the developing forebrain. Furthermore, we show that human Slit2 can repel olfactory and hippocampal axons and collapse their growth cones.