Many neurons in both vertebrates and invertebrates innervate multiple targets by sprouting secondary axon collaterals (or branches) from a primary axon shaft. To begin to identify molecular regulators of axon branch initiation or extension, the growth of single sensory axons was studied in an in vitro collagen assay system and an activity was identified in extracts of embryonic spinal cord and of postnatal and adult brain that promotes the elongation and formation of extensive branches by these axons. Biochemical purification of the activity from calf brain extracts led to the identification of an amino-terminal fragment of Slit2 as the main active component and to the discovery of a distinct activity that potentiates its effects. These results indicate that Slit proteins may function as positive regulators of axon collateral formation during the establishment or remodeling of neural circuits (Wang, 1999).

To begin to address the in vivo function of Slit2, its sites of expression were examined by in situ hybridization at the time of sensory axon ingrowth in rats. At E13, a stage before sensory collaterals innervate the spinal cord, Slit2 mRNA is present mainly in floor plate and motor neurons. Then, starting from E14, its expression expands dorsally in a diffuse pattern. At E17, a stage when NGF-dependent sensory axon collaterals are growing into the dorsal spinal cord, Slit2 expression is highest in restricted regions of the ventral spinal cord, intermediate in level in the middle two-thirds, and present but lower in the dorsal-most regions. Expression of Slit2 is also detectable in a punctate pattern in the dorsal route ganglia (DRG) at E17, in contrast to its absence at earlier stages. The distributions of Slit1 and Slit3 mRNAs were also examined, since the other two Slit proteins might also affect sensory axons. Slit1 is expressed in dorsal spinal cord and the DRG at both E14 and, at higher levels, E17. Slit3 is expressed most highly in restricted regions of the ventral spinal cord at both E14 and E17, with lower level diffuse expression in DRG and little expression in dorsal spinal cord. The presence of Slit1 and Slit2 mRNAs in dorsal spinal cord raises the possibility that one or both of these proteins functions as a dorsal spinal cord-derived promoter of elongation or branching of sensory axons in that region. The presence of mRNAs for all three Slit genes in DRG also raises the possibility that one or more Slit proteins derived from DRG may function in autocrine/paracrine fashion. Robo proteins bind Slit proteins, and in Drosophila, Robo1 is implicated in mediating the repellent actions of Drosophila Slit. Robo2 is expressed at high levels in DRG at both E14 and E17, whereas Robo1 is not expressed at significant levels in DRG at these stages. Thus, if a Robo protein mediates the effects of Slit2-N, Robo2 would be the better candidate than Robo1 for the Slit2 receptor (Wang, 1999).

The purified amino-terminal fragment Slit2-N, but not the full-length molecule Slit2, possesses elongation- and branch-promoting activity, indicating that cleavage is important for bioactivity. It is possible that in the absence of cleavage the conformation of the N-terminal moiety is different and fails to lead to receptor binding or activation. Alternatively, the C-terminal fragment may have a dominant inhibitory activity on the N-terminal fragment when presented in cis within the molecule. The full-length molecule may actually function as an antagonist of Slit2-N. More quantitative studies involving mixing purified proteins will be required to address this issue. In addition, a full understanding of Slit2-N function will require identification of the receptor(s) on sensory axons that mediate its elongation- and branch-promoting activity. It is possible that Robo2, whose mRNA is expressed by sensory neurons, is involved in transducing the Slit2-N effect, either alone or in combination with a coreceptor. If so, it will be of interest to determine how Robo receptors elicit positive and negative responses in different neurons. It is also possible, however, that the positive effect of Slit2-N on sensory neurons is transduced by a receptor mechanism not involving Robo proteins (Wang, 1999).

The results raise the possibility that Slit2-N or the amino-terminal fragment of another Slit protein is involved in directing the formation, stabilization, or ingrowth of sensory axon collaterals into the dorsal spinal cord. Sensory axons do not project directly into the spinal cord, but rather first grow to the dorsal root entry zone and bifurcate, sending axons rostrally and caudally parallel to the spinal cord for several days. The ingrowth of sensory axon collaterals is preceded by the development of swellings ('nodes') along the entire intraspinal extent of sensory axons in the dorsal root entry zone, and collaterals develop interstitially from these nodes and project directly to their target laminae inside the gray matter of the spinal cord, never branching into inappropriate laminae en route. The amino-terminal fragment of one or more Slit proteins may play a causal role in directing this process either by stimulating the development of nodes or the subsequent stabilization or ingrowth of collaterals. Slit1 and Slit2 are both expressed in the dorsal spinal cord, increasing in expression level between E14 and E17, suggesting a possible involvement of either or both of these proteins. In addition, mRNAs for all three Slit proteins, but most prominently Slit1 and Slit2, are expressed in the dorsal route ganglia themselves at E17, suggesting that one or more sensory neuron-derived Slit protein could act in autocrine/paracrine fashion to contribute to these processes. In this case, however, the Slit proteins would then presumably not confer directionality on the branches. Perturbation of Slit protein function in vivo will be required to assess these possibilities (Wang, 1999).

Extending axons in the developing nervous system are guided in part by repulsive cues. Genetic analysis in Drosophila (Kidd, 1999) identifies the Slit protein as a candidate ligand for the repulsive guidance receptor Roundabout (Robo). The characterization of three mammalian Slit homologs is described and it is shown 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 (Brose, 1999).

Several human and mouse expressed sequence tags have been identified that exhibited high homology to Drosophila Slit and these were used to probe both a human fetal brain library and an embryonic day 13 (E13) rat spinal cord library. The clones recovered corresponded to three distinct genes, all with high amino acid identity to Drosophila Slit. The human and rat cDNAs were designated hSlit1, -2, and -3, and rSlit1, -2, and -3, respectively. Recently, three other groups have also reported the cloning of human (hSlit1/MEGF4, hSlit2, and hSlit3/MEGF5) and rat (MEGF4 and MEGF5) slit family members have been identified. The nomenclature of Itoh (1998) was adopted for clarity. All mammalian Slit proteins share a common domain structure and high sequence homology with Drosophila Slit (43.5%, 44.3%, and 41.1% between dSlit and Slit1, -2, and -3, respectively), as well as with one another (60%-66% overall). All of the predicted Slit proteins contain a putative signal peptide, four tandem arrays of leucine rich repeats (LRRs) (which are flanked by conserved amino and carboxy-terminal sequences), a long stretch of EGF repeats, an Agrin-Laminin-Perlecan-Slit (ALPS) conserved spacer motif, and a cysteine knot (a dimerization motif found in several secreted growth factors). Like their Drosophila counterparts, the mammalian Slit proteins lack any hydrophobic sequences that might indicate a transmembrane domain and are thus predicted to encode secreted extracellular proteins. These conserved motifs are also found in a number of other proteins and have been implicated in mediating protein-protein and ECM-protein interactions. Slit1, -2, and -3 differ from Drosophila Slit in that they contain an additional LRR in the third tandem LRR array and two additional EGF repeats. A previously published Drosophila Slit sequence (Rothberg, 1990) lacks an LRR in the first tandem LRR array that is present in vertebrate Slits and in a novel Drosophila Slit sequence isolated by Kidd (1999). Interestingly, the C. elegans Slit homolog, like Drosophila Slit, also contains this additional LRR in LRR-1, has only seven EGF repeats, and is also lacking the extra LRR found in LRR-3 in vertebrates. This suggests that the addition of the EGF repeats occurred after the divergence of the chordate lineage from arthropods and nematodes, during metazoan evolution but prior to the triplication of the ancestral slit gene in the vertebrate lineage (Brose, 1999).

Expression of Slit1, Slit2, and Slit3 mRNAs in comparison to Robo1 and Robo2 mRNAs was determined by in situ hybridization in the developing rat spinal cord at embryonic stages E11-E13, i.e., when commissural axons are migrating to the midline. Rat Robo1 expression overlaps with that of Dcc and TAG-1, known markers for commissural (dorsal) and motor (ventral) neurons at these stages. Beginning at E11, rat Robo2 is also expressed ventrally in the region of the developing motoneuron cell bodies, but in a pattern distinct from that of rat Robo1. In contrast to rRobo1, rRobo2 is expressed in the dorsal root ganglia (DRG) by E13 but is not detected in the region of the commissural neurons overlapping with Dcc and TAG-1. Expression of rRobo2 is, however, detected dorsally in a lateral region of the spinal cord that may comprise the cell bodies of distinct subpopulations of commissural or association neurons (Brose, 1999).

hSlit2 protein can be detected on the surface of living cells with an antibody directed against either the N- or C-terminal tags, indicating that hSlit2 is secreted but remains associated with cell surfaces. Western blots of conditioned media and high salt (1 M NaCl) extracts of membranes from transfected cells reveal a band migrating at 190 kDa, which is slightly higher than the predicted size for hSlit2 and presumably reflects a glycosylated form of the protein. In addition to this 190 kDa isoform, two additional bands are detected: a 140 kDa protein that comprises the amino terminus of hSlit2 and a 55-60 kDa protein that comprises the carboxyl terminus. Since the molecular masses of these two proteins roughly add up to that of full-length hSlit2, it is presumed that they arise from proteolytic cleavage of the full-length protein. The amino- and carboxy-terminal fragments will be referred to as Slit2-N and Slit2-C, respectively. Full-length Slit2, Slit2-N, and Slit2-C exhibit different cell association characteristics. The majority (>90%) of the 190 kDa full-length hSlit2 is found associated with cell surfaces but can be readily extracted from membranes with either high salt (1 M NaCl) or heparin, suggesting its association with cell surfaces via heparan sulfate proteoglycans or other negatively charged moieties. hSlit2-C is more diffusible, partitioning roughly equally between the conditioned medium and cell surfaces. In contrast, hSlit2-N is largely absent from the conditioned medium and is found to be tightly cell associated and more resistant to heparin extraction, requiring several consecutive high salt washes to be fully released. Taken together, these results suggest that mammalian tissue culture cells contain one or more Slit-cleaving proteases, which appear to be absent from or inactive in Drosophila S2 cells. As observed for the mammalian protein, this processing of dSlit also seems to occur in vivo, as the antibody against dSlit detects two similarly sized bands of 190 kDa and 55-60 kDa on Western blots of Drosophila embryo extracts. To begin to identify the cleavage site, recombinant Slit2-C was purified and its amino terminus microsequenced by Edman degradation. The resulting sequence, TSPCDNFD, is found at the beginning of the 6th EGF repeat of hSlit2 and is consistent with the peptide sequences derived from microsequencing of the amino terminal fragment of bovine Slit2 purified from calf brain, all of which map to regions amino-terminal to this site. This sequence is at least partially conserved among Drosophila and mammalian Slit family members, suggesting that the cleavage site may also be conserved between insects and vertebrates. This sequence is not, however, well conserved in the C. elegans Slit homolog (Brose, 1999).

To test whether a mammalian Slit protein can act as a repellent, aggregates of hSlit2-expressing cells were cocultured either in contact with or at a distance from explants of ventral spinal cord from E11 rat embryos. Tissues were cultured in a matrix consisting of a mixture of collagen and matrigel (a partially purified extracellular matrix containing collagen and laminin). In this environment, unlike in a collagen matrix, motor axons grow out of the explants profusely, presumably stimulated by a component in matrigel. When ventral explants are cultured either at a distance (75-200 µm) from or in contact with aggregates of hSlit2-expressing cells, a clear repulsion of motor axons is observed. In many cultures, axons that are originally directed toward the COS cell aggregate appear to turn away. These results indicate that, at least under these culture conditions, hSlit2 can function as a diffusible repellent for developing motor axons (Brose, 1999).

The Slit gene encodes a secreted molecule essential for neural development in Drosophila embryos. Three Slit homologs have been identified in the mouse. The mouse SLIT1 protein can bind ROBO1, a transmembrane receptor implicated in axon guidance. Both whole-mount and section in situ hybridization studies reveal unique and complementary patterns of expression of the three mouse Slit genes and of Robo1, both within the central nervous system and in other developing tissues. The complementary expression patterns of Slit and Robo1 and their in vitro interaction suggest a ligand-receptor relationship. The expression of all three Slit genes in the floor plate suggests that they are likely to share the same functional properties with their Drosophila homolog in midline neural development and axon guidance. The complementary expression of Slit and Robo1 in different subdivisions of the somites suggests their possible function in axon pathfinding and neural crest cell migration. The unique expression pattern in limb and other organs indicates the potential for additional functions for the Slit gene family (Yuan, 1999).

Commissural axons cross the nervous system midline and then turn to grow alongside it, neither recrossing nor projecting back into ventral regions. In Drosophila, the midline repellent Slit prevents recrossing: axons cross once because they are initially unresponsive to Slit, becoming responsive only upon crossing. Commissural axons in mammals similarly acquire responsiveness to a midline repellent activity upon crossing. Remarkably, they also become responsive to a repellent activity from ventral spinal cord, helping explain why they never reenter that region. Several Slit and Semaphorin proteins, expressed in midline and/or ventral tissues, mimic these repellent activities, and midline guidance defects are observed in mice lacking neuropilin-2, a Semaphorin receptor. Thus, Slit and Semaphorin repellents from midline and nonmidline tissues may help prevent crossing axons from reentering gray matter, squeezing them into surrounding fiber tracts (Zou, 2000).

During development, retinal ganglion cell (RGC) axons either cross or avoid the midline at the optic chiasm. In Drosophila, the Slit protein regulates midline axon crossing through repulsion. To determine the role of Slit proteins in RGC axon guidance, Slit1 and Slit2, two of three known mouse Slit genes, were disrupted. Mice defective in either gene alone exhibit few RGC axon guidance defects, but in double mutant mice a large additional chiasm develops anterior to the true chiasm, many retinal axons project into the contralateral optic nerve, and some extend ectopically -- dorsal and lateral to the chiasm. These results indicate that Slit proteins repel retinal axons in vivo and cooperate to establish a corridor through which the axons are channeled, thereby helping define the site in the ventral diencephalon where the optic chiasm forms (Plump, 2002).

Inferior olivary neurons (ION) migrate circumferentially around the caudal rhombencephalon starting from the alar plate to locate ventrally close to the floor-plate, ipsilaterally to their site of proliferation. The floor-plate constitutes a source of diffusible factors. Among them, netrin-1 is implied in the survival and attraction of migrating ION in vivo and in vitro. A possible involvement of slit-1/2 during ION migration has been explored. slit-1 and slit-2 are coexpressed in the floor-plate of the rhombencephalon throughout ION development. robo-2, a slit receptor, is expressed in migrating ION, in particular when they reach the vicinity of the floor-plate, Using in vitro assays in collagen matrix, netrin-1 exerts an attractive effect on ION leading processes and nuclei, Slit has a weak repulsive effect on ION axon outgrowth and no effect on migration by itself, but when combined with netrin-1, it antagonizes part of or all of the effects of netrin-1 in a dose-dependent manner, inhibiting the attraction of axons and the migration of cell nuclei. The results indicate that slit silences the attractive effects of netrin-1 and participates in the correct ventral positioning of ION, stopping the migration when cell bodies reach the floor-plate (Causeret, 2002).

Axons that carry information from the sensory periphery first elongate unbranched and form precisely ordered tracts within the CNS. Later, they begin collateralizing into their proper targets and form terminal arbors. Target-derived factors that govern sensory axon elongation and branching-arborization are not well understood. Slit2 is a major player in branching-arborization of central trigeminal axons in the brainstem. Embryonic trigeminal axons initially develop unbranched as they form the trigeminal tract within the lateral brainstem; later, they emit collateral branches into the brainstem trigeminal nuclei and form terminal arbors therein. In whole-mount explant cultures of this pathway, embryonic day 15 (E15) rat central trigeminal axons retain their unbranched growth within the tract, whereas E17 trigeminal axons show branching and arborization in the brainstem trigeminal nuclei, much like that seen in vivo. Similar observations were made in E13 and E15 mouse embryos. Slit2-expressing tissues or cells with the whole-mount explant cultures of the central trigeminal pathway derived from embryonic rats or mice were cocultured. When central trigeminal axons are exposed to ectopic Slit2 during their elongation phase, they show robust and premature branching and arborization. Blocking available Slit2 reverses this effect on axon growth. Spatiotemporal expression of Slit2 and Robo receptor mRNAs within the brainstem trigeminal nuclei and the trigeminal ganglion during elongation and branching-arborization further corroborates the experimental results (Ozdinler, 2002).

Although multiple axon guidance cues have been discovered in recent years, little is known about the mechanism by which the spatiotemporal expression patterns of the axon guidance cues are regulated in vertebrates. A homeobox gene, Irx4, is expressed in a pattern similar to that of Slit1 in the chicken retina. Overexpression of Irx4 leads to specific downregulation of Slit1 expression, whereas inhibition of Irx4 activity by a dominant negative mutant leads to induction of Slit1 expression, indicating that Irx4 is a crucial regulator of Slit1 expression in the retina. In addition, by examining axonal behavior in the retinas with overexpression of Irx4 and using several in vivo assays to test the effect of Slit1, it was found that Slit1 acts positively to guide the retinal axons inside the optic fiber layer (OFL). The regulation of Slit1 expression by Irx4 is important for providing intermediate targets for retinal axons during their growth within the retina (Jin, 2003).

The vomeronasal projection conveys information [provided by pheromones and detected by neurons in the vomeronasal organ (VNO)] to the accessory olfactory bulb (AOB) and thence to other regions of the brain such as the amygdala. The VNO-AOB projection is topographically organized such that axons from apical and basal parts of the VNO terminate in the anterior and posterior AOB respectively. Evidence is provided that the Slit family of axon guidance molecules and their Robo receptors contribute to the topographic targeting of basal vomeronasal axons. Robo receptor expression is confined largely to basal VNO axons, while Slits are differentially expressed in the AOB with a higher concentration in the anterior part, which basal axons do not invade. Immunohistochemistry using a Robo-specific antibody reveals a zone-specific targeting of VNO axons in the AOB well before cell bodies of these neurons in the VNO acquire their final zonal position. In vitro assays show that Slit1-Slit3 chemorepel VNO axons, suggesting that basal axons are guided to the posterior AOB due to chemorepulsive activity of Slits in the anterior AOB. These data in combination with recently obtained other data suggest a model for the topographic targeting in the vomeronasal projection where ephrin-As and neuropilins guide apical VNO axons, while Robo/Slit interactions are important components in the targeting of basal VNO axons (Knöll, 2003).

Axon guidance and neuronal migration are critical features of neural development, and it is believed that extracellular gradients of secreted guidance cues play important roles in pathfinding. It has been well documented that the growth cones of extending axons respond to such extracellular gradients by growing toward or away from the source of the secreted cue via asymmetrical extension of a single growth cone. However, it is unclear whether migrating neurons change direction in response to guidance molecules using the same mode of turning as extending axons. This study demonstrates that migrating neurons turn away from the chemo-repellent Slit through repeated rounds of process extension and retraction and do not turn through the reorientation of a single growth cone. Slit increases the rate of somal process formation, and these processes form preferentially on the side of the cell body furthest away from the Slit source. In addition, Slit causes cell turning through asymmetric process selection. Finally, it was shown that multiple types of migrating neurons employ this mode of cell turning in response to a variety of guidance cues. These results show that migrating neurons employ a unique type of turning when faced with secreted guidance cues that is distinct from the type employed by axons (Ward, 2005).

Slit is a secreted protein known to repulse the growth cones of commissural neurons. By contrast, Slit also promotes elongation and branching of axons of sensory neurons. The reason why different neurons respond to Slit in different ways is largely unknown. Islet2 is a LIM/homeodomain-type transcription factor that specifically regulates elongation and branching of the peripheral axons of the primary sensory neurons in zebrafish embryos. PlexinA4, a transmembrane protein known to be a co-receptor for class III semaphorins, was shown to act downstream of Islet2 to promote branching of the peripheral axons of the primary sensory neurons. Intriguingly, repression of PlexinA4 function by injection of the antisense morpholino oligonucleotide specific to PlexinA4 or by overexpression of the dominant-negative variant of PlexinA4 counteracts the effects of overexpression of Slit2 to induce branching of the peripheral axons of the primary sensory neurons in zebrafish embryos, suggesting involvement of PlexinA4 in the Slit signaling cascades for promotion of axonal branching of the sensory neurons. Colocalized expression of Robo, a receptor for Slit2, and PlexinA4 is observed not only in the primary sensory neurons of zebrafish embryos but also in the dendrites of the pyramidal neurons of the cortex of the mammals, and may be important for promoting the branching of either axons or dendrites in response to Slit, as opposed to the growth cone collapse (Miyashita, 2004).

To investigate Slit signalling in forebrain development, Robo1 knockout mice were generated 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 (Andrews, 2006).

Upon arriving at their targets, developing axons cease pathfinding and begin instead to arborize and form synapses. To test whether CNS arborization and synaptogenesis are controlled by Slit-Robo signaling, single retinal ganglion cell (RGC) arbors were followed over time. ast (robo2) mutant and slit1a morphant arbors had more branch tips and greater arbor area and complexity compared to wild-type and concomitantly more presumptive presynaptic sites labeled with YFP-Rab3. Increased arborization in ast was phenocopied by dominant-negative Robo2 expressed in single RGCs and rescued by full-length Robo2, indicating that Robo2 acts cell-autonomously. Time-lapse imaging revealed that ast and slit1a morphant arbors stabilized earlier than wild-type, suggesting a role for Slit-Robo signaling in preventing arbor maturation. Genetic analysis showed that Slit1a acts both through Robo2 and Robo2-independent mechanisms. Unlike previous PNS studies showing that Slits promote branching, these results show that Slits inhibit arborization and synaptogenesis in the CNS (Campbell, 2007).

Neuronal migration and growth-cone extension are both guided by extracellular factors in the developing brain, but whether these two forms of guidance are mechanistically linked is unclear. Application of a Slit-2 gradient in front of the leading process of cultured cerebellar granule cells led to the collapse of the growth cone and the reversal of neuronal migration, an event preceded by a propagating Ca2+ wave from the growth cone to the soma. The Ca2+ wave was required for the Slit-2 effect and was sufficient by itself to induce the reversal of migration. The Slit-2-induced reversal of migration required active RhoA, which was accumulated at the front of the migrating neuron, and this polarized RhoA distribution was reversed during the migration reversal induced by either the Slit-2 gradient or the Ca2+ wave. Thus, long-range Ca2+ signaling coordinates the Slit-2-induced changes in motility at two distant parts of migrating neurons by regulating RhoA distribution (Guan, 2007).

These results support the following scheme of cellular signaling during the reversal of neuronal migration induced by Slit-2. Slit-2 activation of its receptors at the leading growth cone triggers a Ca2+ wave that propagates to the soma, resulting in a front-to-rear gradient of Ca2+ elevation. This somatic Ca2+ gradient in turn causes a gradient of RhoA inhibition that leads to the redistribution of RhoA toward the rear of soma, a process required for establishing the reversed direction of migration. The mechanism by which Ca2+ wave induces the redistribution of RhoA remains to be elucidated. There is evidence suggesting that downregulation of RhoA activity may alter the balance of cortical myosin activity. For the reversal of neuronal migration, a front-to-rear Ca2+ gradient may thus cause a myosin-dependent rearward flow of the cortical F-actin that 'pulls' RhoA to the rear by a process analogous to the cortical flow of proteins during the fertilization-induced zygote polarization. Initial reversal in the distribution of active RhoA may further enhance the backward cortical flow by a positive feedback mechanism. This process of RhoA redistribution may require cumulated effects of the Ca2+ gradient across the cell, leading to the observed delay in the onset of soma reversal following the frontal Slit-2 application (Guan, 2007).

Like Drosophila slit, which is expressed by cells at the midline of the Drosophila nervous system, the mRNAs for all three rat Slit proteins are expressed by floor plate cells at the ventral midline of the spinal cord. From E11-E13, Slit1 is expressed at high levels in the floor plate and at lower levels in other regions of the spinal cord, including the region of commissural and association neurons and the motor column. Likewise, Slit2 is also expressed by the floor plate, but in a different pattern: whereas Slit1 is expressed broadly by both floor plate cells and cells in the ventral portion of the ventricular zone, Slit2 is restricted to the most basal and medial region of the floor plate. Slit2 is expressed at high levels in the developing motor column and the roof plate, as well as very weakly in the region of the commissural neurons in the dorsal spinal cord. Like Slit1 and -2, Slit3 is also expressed by the floor plate but at significantly lower levels than Slit1 and Slit2. In addition, whereas the spatial distributions of Slit1 and Slit2 in the spinal cord remain largely constant between E11 and E13, Slit3 expression is dynamic: at E11 and E12 Slit3 is expressed in the motor column, but by E13, the stage at which most commissural axons have crossed the floor plate and have turned rostrally, Slit3 expression in the motor column is significantly diminished and expression is largely restricted to the floor plate. It is striking that, in addition to having specific additional sites of expression, the three mammalian Slit genes, like their counterpart in Drosophila, are all expressed by cells at the ventral midline of the nervous system. This expression is consistent with a potential role for Slit proteins in repulsive axon guidance at the midline mediated by Robo proteins (Brose, 1999).

The development of an assay in which spinal commissural axons are first made to cross the floor plate before being confronted with tissues or guidance cues has enabled dissection of the changes in responsiveness of these axons during midline crossing. Paralleling previous studies in Drosophila, it has been shown that commissural axons acquire responsiveness to a midline repellent activity upon crossing the midline and that Slit proteins may contribute to this activity. These observations are extended, however, by showing that ventral spinal cord tissue also secretes a repellent activity -- perhaps involving Slit-2 -- to which the axons become responsive upon midline crossing, providing an explanation for why the axons do not reenter the ventral spinal cord. While Slit proteins may contribute to the repellent activities in both floor plate and ventral spinal cord, this study also implicates the class 3 Semaphorins Sema3B and Sema3F, which are high-affinity ligands for neuropilin-2 receptors on commissural axons, in mediating the repellent actions of floor plate and ventral spinal cord. The finding of projection defects in a neuropilin-2 knockout mouse supports this hypothesis. Taken together, these results suggest that midline recrossing in vertebrates is prevented not just by the loss of responsiveness to positive factors at the midline, but also the acquisition of responsiveness to negative factors. They also support a model in which commissural axons are forced, or squeezed, out of the gray matter of the nervous system into surrounding fiber tracts by repellents secreted by both the floor plate and the ventral spinal cord (Zou, 2000).

A pleasing result from this study is that, as in Drosophila, spinal commissural axons acquire responsiveness to at least one Slit protein (and perhaps all three) upon midline crossing. Whether this involves upregulation of vertebrate Robo receptor expression on the commissural axons after midline crossing remains to be determined. A surprising aspect of these results, however, was the finding that the class 3 Semaphorin Sema3B likely contributes to the repulsive floor plate activity as well, since its mRNA is expressed by floor plate cells and it repels post-crossing axons in this assay. This repulsive action is likely to be mediated by the high-affinity Sema3B receptor neuropilin-2, which is expressed by these axons. (Neuropilin-2 is likely only the ligand binding portion of the Sema3B receptor, with signaling presumably mediated by a plexin family member such as plexin-A3, which is expressed by these neurons). Thus, in contrast to Drosophila, where a single Slit protein is thought to account for all the midline repulsive activity, in vertebrates the task of repulsion of post-crossing axons by midline cells appears to be shared by at least three Slit proteins and one Semaphorin (Zou, 2000).

These studies also revealed for the first time in any organism that crossing axons also acquire responsiveness to a repellent activity from the ventral portion of the nervous system. This is the terrain that the axons have traversed immediately before reaching the midline and which is therefore permissive for growth prior to crossing; after crossing, however, it becomes repulsive to the axons. This repulsive activity again appears to involve both Slit and Semaphorin proteins, since Slit-2 is expressed in the motor column and since Sema3F (another high-affinity neuropilin-2 ligand) is expressed throughout the mantle layer of the entire spinal cord (including the ventral spinal cord but excluding the floor plate). The existence of this repulsive activity should help prevent the axons from reentering the ventral portions of the nervous system. In fact, the repellent actions of the floor plate and the ventral spinal cord together should help squeeze the commissural axons out of the gray matter of the spinal cord entirely after they have crossed the midline. If Slit-2 and/or Sema3F proteins are also displayed on motor axons, then they might also help organize post-crossing commissural axons within the regions of the fiber tracts that motor axons traverse, a possibility suggested for Slit-2 (Zou, 2000).

The analysis of a neuropilin-2 knockout mouse supports the involvement of the class 3 Semaphorins in regulating midline crossing of commissural axons. A frequent defect observed in the mutants is the apparent stalling out of the axons in the floor plate, which is consistent with the existence of insufficient inhibitory activity within the floor plate to help push the axons out of the midline region once they have started crossing it. Interestingly, in these cases of stalling, many or all the axons stall out at the contralateral floor plate edge; this is reminiscent of the situation in robo mutants in Drosophila, where the axons can recross the midline but do not stall out in the middle, apparently because of the operation of a weaker repulsive mechanism (also involving Slit but mediated by some other receptor, perhaps Robo-2). The presence of residual inhibition at the midline in the neuropilin-2 knockout mice might similarly explain why axons grow to the contralateral edge of the floor plate (Zou, 2000).

In addition, the defects are only partially penetrant and also seem to be corrected as the embryo matures, indicating the operation of redundant guidance mechanisms. These mechanisms presumably include the Slit proteins but also possibly other nondiffusible guidance cues, such as ephrinB2, which a recent descriptive analysis has suggested might be involved in regulating midline guidance as well. EphrinB2 might, in fact, be a good candidate for the short-range repellent activity of floor plate cells documented in chick, to which commissural axons appear to be sensitive even prior to crossing (Zou, 2000 and references therein).

The exiting of spinal commissural axons into the ventral funiculus from the gray matter after midline crossing is representative of the behavior of large numbers of other axons up and down the neuraxis, which grow to their targets by coursing through the gray matter to some exit point where they join and grow in fiber tracts, only later leaving the tracts to reenter the gray matter and to connect with their target cells. It is suggested that the mechanism described in this study may be representative of those operating throughout the nervous system to propel axons out of the gray matter into fiber tracts. It may be true quite generally that as axons leave the gray matter, they acquire responsiveness to both midline and gray matter repellent activities. It is intriguing in this regard that Sema3F and Sema3B, between them, are expressed throughout much of the gray matter and midline. In fact, the finding that Sema3F is expressed throughout the mantle layer, essentially everywhere where axons grow within the spinal cord (and in other brain regions as well), is hard to square with a role in guidance within the mantle layer. Rather, it seems more likely that it functions to prevent axons from entering or reentering the mantle layer and thus helps keep them in fiber tracts. The Slit proteins may also play such a role quite generally, since their mRNAs, after initially being most highly expressed in midline tissues, later become more widely expressed throughout the gray matter (Zou, 2000 and references therein).

After axons have grown in fiber tracts, what permits them to reenter the gray matter? This is an issue studied recently in the context of sensory axon collateral ingrowth into the spinal cord. Remarkably, that study implicated Slit proteins as positive regulators of sensory axon branching and ingrowth into the spinal cord gray matter. Slit proteins might function generally to permit axon ingrowth into gray matter from adjacent fiber tracts. Putting together these two suggestions, a global hypothesis suggests itself: axons that leave the gray matter are kept out because they acquire responsiveness to a repellent activity made by gray matter that involves Slit proteins (and Semaphorin proteins), and when they later branch back into the gray matter, they may do so because they acquire responsiveness to an attractive or permissive activity made by gray matter that may also involve Slit proteins (and perhaps also Semaphorin proteins?). Thus, in the most extreme version of this hypothesis, the axons may initially be able to grow through the gray matter because they are impervious to Slit and Semaphorin proteins and then acquire repulsive responses to these factors as they leave the gray matter, only reentering the gray matter when their responses to Slit and Semaphorin proteins switch from being repulsive to attractive. The ability of growth cones to rapidly switch their responsiveness between repulsion and attraction has been demonstrated for several types of cues, including Semaphorins, in tissue culture experiments using Xenopus neurons. Future experiments will test whether the initial exit and subsequent reentry of the gray matter is controlled by such a neatly choreographed series of changes in growth cone responsiveness - from no response, to repulsion, to attraction - to guidance cues of the Slit and Semaphorin families and help elucidate what other mechanisms are at play in regulating gray matter entry and exit (Zou, 2000 and references therein).

The ventral midline of the nervous system is an important choice point at which growing axons decide whether to cross and project contralaterally or remain on the same side of the brain. In Drosophila, the decision to cross or avoid the CNS midline is controlled, at least in part, by the Roundabout receptor on the axons and its ligand, Slit, an inhibitory extracellular matrix molecule secreted by the midline glia. Vertebrate homologs of these molecules have been cloned and have also been implicated in regulating axon guidance. Using in situ hybridization, the expression patterns have been determined of robo1,2 and slit1,2,3 in the mouse retina and in the region of the developing optic chiasm, a ventral midline structure in which retinal ganglion cell (RGC) axons diverge to either side of the brain. The receptors and ligands are expressed at the appropriate time and place, in both the retina and the ventral diencephalon, to be able to influence RGC axon guidance. In vitro, slit2 is inhibitory to RGC axons, with outgrowth of both ipsilaterally and contralaterally projecting axons being strongly affected. When presented alone in vitro, Slit2 does not have a differential effect on ipsilaterally and contralaterally projecting RGC axons; both are strongly inhibited. One possibility is that Slit2 alone is not enough to direct divergence but that additional factors are required. Other axon guidance molecules, such as Nr-CAM and Eph/ephrin receptors and ligands are expressed on the glial cells present at the ventral midline of the mouse diencephalon. In the future, it will be important to determine whether these molecules can synergize with Slit2 and thereby control RGC axon divergence at the midline. Overall, these results indicate that Robos and Slits alone do not directly control RGC axon divergence at the optic chiasm and may additionally function as a general inhibitory guidance system involved in determining the relative position of the optic chiasm at the ventral midline of the developing hypothalamus (Erskine, 2000).

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 was shown to divert 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 (Farmer, 2008).

In addition to being critical for apoptosis, components of the apoptotic pathway, such as caspases, are involved in other physiological processes in many types of cells, including neurons. However, very little is known about their role in dynamic, nonphysically destructive processes, such as axonal arborization and synaptogenesis. This study shows that caspases are locally active in vivo at the branch points of young, dynamic retinal ganglion cell axonal arbors but not in the cell body or in stable mature arbors. Caspase activation, dependent on Caspase-3, Caspase-9, and p38 mitogen-activated protein kinase (MAPK), rapidly increased at branch points corresponding with branch tip addition. Time-lapse imaging revealed that knockdown of Caspase-3 and Caspase-9 led to more stable arbors and presynaptic sites. Genetic analysis showed that Caspase-3, Caspase-9, and p38 MAPK interacted with Slit1a-Robo2 signaling, suggesting that localized activation of caspases lie downstream of a ligand receptor system, acting as key promoters of axonal branch tip and synaptic dynamics to restrict arbor growth in vivo in the central nervous system (Campbell, 2013).

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 have 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, these findings are extended 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. Human Slit2 can repel olfactory and hippocampal axons and collapse their growth cones (Nguyen Ba-Charvet, 1999).

To determine the potential role of Robo and Slit family members in mediating axon pathfinding in the forebrain, the expression patterns of Robo1, Robo2, Slit1, and Slit2 mRNAs in the developing telencephalon were examined at the time olfactory and hippocampal projections are forming, near the end of the second week of gestation. In the rat olfactory system (the expression pattern in the mouse is identical), fibers begin to leave the olfactory bulb (OB) by E14, and by E15 the lateral olfactory tract is clearly formed. OB axons project ipsilaterally, never cross the midline, and avoid the septal area. In vitro, the septum has been shown to repel olfactory tract axons. As early as E12, Slit2, but not Slit1, is highly expressed in the midline of the telencephalon, including the region of the presumptive septum. By E14 and continuing until at least E18, both Slit1 and Slit2 are expressed by the septum. At these stages, mitral cells and tufted cells of the OB express high levels of Robo2 mRNA, while Robo1 is almost undetectable. During this time period, Slit1 is also found in a subset of mitral cells, but Slit2 is completely absent from the OB. Thus, their expression patterns in the olfactory system are complementary (Nguyen Ba-Charvet, 1999).

Tracing studies in mouse hippocampus have shown that hippocampal afferents invade their target territories in a highly specific fashion. Such stereotyped, directed growth suggests the involvement of long-range and short-range guidance cues, and previous studies have focused on the role of Semaphorins in shaping this trajectory. To explore potential contributions of Slit and Robo proteins in patterning hippocampal connections, their expression patterns in this region were examined. The earliest entorhinal axons leave the entorhinal cortex by E14 and reach the hippocampus by E15, where they first project onto neurons in the CA1 and CA3 subfields and later in the dentate gyrus (DG). In rodents, most of the development of the DG occurs postnatally; however, a DG primordium can clearly be observed by E16 in the mouse (Nguyen Ba-Charvet, 1999).

In the rat, Robo and Slit mRNAs are highly expressed in the developing hippocampal formation. From E12-E13, Slit2 can be detected in the so-called 'cortical hem', a neuroepithelial structure that forms a boundary between the hippocampus, the most medial part of the cerebral cortex, and the telencephalic choroid plexus. This expression persists until at least E15, when it can also be observed in the entorhinal cortex. At E18, Slit2 mRNA is found in the DG, in a portion of the CA3 subfield, and in the entorhinal cortex but is not present in the neocortex. Slit2 is also detectable in the choroid plexuses and in cells lining the telencephalic ventricles. Neither Slit1, Robo1, nor Robo2 can be observed in the hippocampal formation until E14, and by E18 Slit1 is found at the level of the hippocampal plate, in the presumptive pyramidal cells of the Ammon horn, and in most of the other hippocampal-related structures, with the exception of the DG. In contrast to Slit2, Slit1 expression is strong in the neocortex, where it is largely restricted to the cortical plate. By P3, Slit1 is expressed almost exclusively in CA3 and in the subiculum, while Slit2 can still be detected throughout the hippocampus proper and presubiculum. At E20, both Robo1 and Robo2 are expressed in the hippocampus in relatively similar structures, with Robo1 at a higher level, but only Robo1 is found in the dentate granule cell layer. Interestingly, Robo2 is also expressed by Cajal-Retzius cells, which have been shown to have a role in guiding the entorhinal cortex projections. At this stage, both Robo genes are expressed in the entorhinal cortex and neocortex, but they remain confined to distinct and complementary layers: Robo1 mRNA is found in neurons of the cortical plate and marginal zone, while Robo2 is localized to the intermediate zone. By P3, the expression patterns of Robo1 and Robo2 have changed, such that Robo1 expression has now disappeared from the subiculum and Robo2 expression has increased in the subiculum. In addition, at P3 both genes are also expressed in the Ammon horn pyramidal cell layer and in the hilus. Moreover, Robo1 expression is maintained at a low level in the DG granule cell layer, and Robo2 expression persists in Cajal-Retzius cells. Thus, although there is some overlap, Slit and Robo expression patterns in the developing hippocampus are to a large extent complementary (Nguyen Ba-Charvet, 1999).

When cultured in a three-dimensional collagen gel matrix, E14 rat OB axons can be repelled by a diffusible factor released by the septum. At that stage, mitral cells in the OB express Robo2, while its ligand Slit2 and putative ligand Slit1 are found in the septum and telencephalic midline. Since it is known that COS cells secreting hSlit2 can repel spinal motor axons, a test was performed to see whether Slit2 can also act as a repulsive molecule in the olfactory system by culturing E14-E15 OB explants with aggregates of hSlit2-expressing cells. When cultured either directly adjacent to or at a distance (up to 200 µm) from control COS cells, axons extend from all OB explants in a radial pattern. In contrast, axons from 98% of OB explants cultured at a distance from COS cells secreting hSlit2 are repelled. In addition, in the distal quadrant, both the number of OB neurite bundles and the area covered by OB neurites are significantly larger than in the proximal quadrant, facing hSlit2-expressing cells. As early as 18-24 hr in culture, an asymmetric pattern of outgrowth can be observed, with far fewer axons in the quadrant proximal to the cell aggregate. This repulsive effect lasts until at least 36-48 hr in culture. At these later time points, only a few axons can be observed in the proximal quadrant, but most axons are clearly directed away from the hSlit2-expressing cells. Explants were not cultured for more than 48 hr, because transfected COS cells start to die after this point. These results indicate that hSlit2 is a chemorepellent for OB axons (Nguyen Ba-Charvet, 1999).

It was next examined whether Slit proteins are chemorepellents for axons of the hippocampal formation. During development, hippocampal axons never invade the adjacent entorhinal cortex, whereas axons from the entorhinal cortex project massively to the DG. Axons from the DG, CA3, and CA1 subfields of the embryonic hippocampus can be repelled by the entorhinal cortex. Cells secreting either Semaphorin III or IV can mimic this activity. Since Slit2 mRNA and Robo1 and Robo2 mRNAs are expressed at high levels in the entorhinal cortex and in the DG, respectively, attempts were made to determine whether Slit2 could repel DG axons. DG axons grow symmetrically in 82% of the cases when confronted with control COS cells. In contrast, in 93% of the explants cultured with COS cells expressing hSlit2, axons preferentially grow away from the cell aggregates. Repulsion could be observed after 24 hr and is maintained for one more day in vitro. In the distal quadrant, both the number of DG neurite bundles and the area covered by DG neurites are significantly larger than in the proximal quadrant, facing hSlit2-expressing cells. This demonstrates that Slit2 is also a chemorepellent molecule for DG axons. It will be interesting to study Slit1 function using similar assays, but Slit1 expression constructs are not available yet (Nguyen Ba-Charvet, 1999).

The afferent axonal projections of the rodent hippocampus are very precisely organized, with subsets of axons arborizing in specific layers and synapsing on specific portions of the dendritic trees of their target neurons. These afferent axons project directly to their proper target layers, suggesting that their final targeting involves layer-specific positional cues. In rodents, the DG consists of three layers: the molecular layer, the granule cell layer, and the hilus or polymorphic layer. The principal cells of the DG are the granule cells, but other neuronal subtypes, such as pyramidal basket cells, stellate cells, and mossy cells, can be found scattered thought the molecular layer and hilus. The major afferents to the DG arise from the entorhinal cortex and innervate the superficial two-thirds of the molecular layer via the perforant pathway, beginning at E19 in the mouse. The inner third of the molecular layer is innervated postnatally (around P2) by commissural/associational axons originating from the mossy cells from the hilus. Interestingly, both Robo1 and Robo2, as well as their candidate ligands, Slit1 and Slit2, are expressed in somewhat complementary patterns in the embryonic hippocampus when these hippocampal connections are being formed. In addition, the entorhinal cortex secretes a diffusible repellent for axons of the DG, and hSlit2-expressing cells can mimic this activity. Granule cell axons, the mossy fibers, project to the CA3 subfield of the hippocampus proper, where they are restricted to the proximal segment of the pyramidal cell stem dendrites, while entorhinal axons occupy their distal segment. Therefore, Slit2 produced by entorhinal axons could block the invasion of the upper dendritic segment by mossy fibers. Robust expression of Slit2 mRNA was also found in CA3 pyramidal neurons. A heterogeneous distribution of Slit2 on the surface of pyramidal cell dendrites could also participate in the dendritic segregation of mossy fiber afferents. In contrast with other hippocampal projection neurons in CA1 and CA3, mossy fibers never cross the telencephalic midline, which expresses high levels of Slit2 mRNA from very early stages. These results suggests that Slit2 may have a similar function in pushing noncommissural axons away from the midline of the forebrain, as has been proposed for Robo and Slit at the Drosophila ventral midline (Nguyen Ba-Charvet, 1999 and references).

Semaphorins function as chemorepellents when presented chronically to growth cones but induce growth cone collapse when presented acutely. Since hSlit2 can repel axons of a variety of classes of neurons, it was next examined whether hSlit2, like Semaphorins, could induce growth cone collapse. Membranes from hSlit2-expressing cells were used. Explants from OB or DG were cultured on a poly-L-lysine/laminin substrate. After a day in culture, individual axons extending from these explants could be observed. Membrane extracts prepared from hSlit2-expressing COS cells or control COS cells were applied to the OB and DG cultures for 1-2 hr. Cultures were fixed and collapsed growth cones, identified by a lack of lamellipodia and filopodia, were quantified in blinded experiments. Four independent experiments gave similar results. Application of hSlit2 membrane extracts to OB growth cones results in a marked and significant increase in the percentages of axons with collapsed growth cones, as compared with addition of control membranes. hSlit2 membranes can also induce collapse of 58% of DG growth cones, although in this case the difference between hSlit2 and control membranes is less significant than for OB growth cones. Western blots of membrane extracts reveal that the hSlit2-containing membrane extracts consist primarily of the full-length and N-terminal cleavage fragments, with most, if not all, of the C-terminal cleavage fragment presumably being lost during treatment. These results suggest that the collapse-inducing activity is contained in the full-length or N-terminal fragments (Nguyen Ba-Charvet, 1999).

In addition to acting as a chemorepellant for cortical axons, Slit1 regulates dendritic development. Slit1 is expressed in the developing cortex, and exposure to Slit1 leads to increased dendritic growth and branching. Conversely, inhibition of Slit-Robo interactions by Robo-Fc fusion proteins or by a dominant-negative Robo attenuates dendritic branching. Stimulation of neurons transfected with a Met-Robo chimeric receptor with Hepatocyte growth factor leads to a robust induction of dendritic growth and branching, suggesting that Robo-mediated signaling is sufficient to induce dendritic remodeling. These experiments indicate that Slit-Robo interactions may exert a significant influence over the specification of cortical neuron morphology by regulating both axon guidance and dendritic patterning (Whitford, 2002).

It is interesting to note that all three Slits have layer-restricted expression during postnatal development and in adulthood. The layer-specific expression during early postnatal development may be important in regulating layer-specific apical dendrite collateral branching or layer-specific axon terminations. For example, slit3 is expressed at high levels in most layers at P10 except layers IV and VI, which are the recipient layers for thalamic axons. It will be interesting to know whether a repulsive effect of Slit3 prevents innervation of layers II/III and V by these axons. It is also striking that all three slits as well as robos are expressed at high levels in the adult cortex, which suggests that Slit-Robo interactions must influence cellular events other than axon guidance and dendritic patterning. Although not much is known about how Slits are secreted, based on the in situ hybridization studies and the results of Robo-Fc blocking experiments in essentially pure neuronal cultures, it would appear that Slit proteins are secreted by neurons (Whitford, 2002).