Slit was identified in Drosophila embryo as a gene involved in the patterning of larval cuticle.¹ It was later shown that Slit is synthesized in the fly central nervous system by midline glia cells.^2-5 Slit homologues have since been found in C. elegans⁶ and many vertebrate species, from amphibians,⁷ fishes,⁸ birds^9-11 to mammals.^7,12-14 A single slit was isolated in invertebrates, whereas there are three slit genes (slit1-slit3) in mammals, that have around 60% homology.¹² All encodes large ECM glycoproteins of about 200 kDa^15,16 (Fig. 1A), comprising, from their N terminus to their C terminus, a long stretch of four leucine rich repeats (LRR) connected by disulphide bonds, seven to nine EGF repeats, a domain, named ALPS (Agrin, Perlecan, Laminin, Slit) or laminin G-like module (see ref. 17), and a cystein knot (Fig. 1A). Alternative spliced transcripts have been reported for Drosophila Slit2, human Slit2 and Slit3,¹⁴ and Slit1.^18,19 Moreover, two Slit1 isoforms exist in zebrafish as a consequence of gene duplication.²⁰ Last, in mammals, two Slit2 isoforms can be purified from brain extracts, a long 200 kDa one^15,16 and a shorter 150 kDa form (Slit2-N) that was shown to result from the proteolytic processing of full-length Slit2.²¹ Human Slit1 and Slit3 and Drosophila Slit are also cleaved by an unknown protease in a large N-terminal fragment and a shorter C-terminal fragment, suggesting conserved mechanisms for Slit cleavage across species.^12,21-23 Moreover, Slit fragments have different cell association characteristics in cell culture suggesting that they may also have different extents of diffusion, different binding properties, and, hence, different functional activities in vivo. This conclusion is supported by in vitro data showing that full-length Slit2 functions as an antagonist of Slit2-N in the DRG branching assay, and that Slit2-N, not full-length Slit2, causes collapse of OB growth cones.²⁴ In addition, Slit1-N and full-length Slit1 can induce branching of cortical neurons (see below), but only full-length Slit1 repels cortical axons.²³

Figure 1

A) Structure of the slit proteins. B) Diagrammatic comparison of the structure of Robo receptors in invertebrate and vertebrate species. v: vertebrate; d: Drosophila; z: zebrafish. ALPS: domain found in Agrin, Laminin, Perlecan and Slit.

Structure-function analysis in vertebrates and Drosophila demonstrated that the LRRs of Slits are required and sufficient to mediate their repulsive activities in neurons.^24-26 More recent detailed structure function analysis of the LRR domains of Drosophila Slit,²⁷ revealed that the active site of Slit (at least regarding its pro-angiogenic activity) is located on the second of the fourth LRR (LRR2), which is highly conserved between Slits. Slit can also dimerize through the LRR4 domain and the cystein knot.¹⁸ However, a Slit1 spliced-variant that lacks the cystein knot and does not dimerize is still able to repel OB axons.¹⁸

Introduction

The first roundabout gene, robo, was identified in Drosophila during a comprehensive screen for genes regulating midline crossing in the CNS.²⁸ If SAX-3 is the unique robo ortholog in C.elegans,⁶ three robo genes have been found in Drosophila,^5,29,30 zebrafish,^31,32 chick⁹ (Chedotal unpublished data) and mammals.^7,12,33 Robo proteins belong to the immunoglobulin (Ig) superfamily and have five Ig-like domains followed by three fibronectin type III (FNIII) repeats, a transmembrane portion and a long intracellular tail containing up to four conserved cytoplasmic motifs, CC0-CC3, with no obvious catalytic domains (Fig. 1B). The first two Ig domains are the most highly conserved portion and are also found in another protein called Robo4 or magic roundabout that is only expressed by endothelial cells and plays a role in angiogenesis.^34,35 However, Robo4 lacks the last three Ig domains, some FNIII domains and the CC1 and CC3 motifs found in other Robo proteins. Moreover, its capacity to bind Slits is still debated.^36,37

CC0 has no known function but is a site of tyrosine phosphorylation.³⁸ CC1 also contains tyrosine residues that can be phosphorylated and was shown to bind to the P3 domain of the netrin-1 receptor, deleted in colorectal cancer (DCC; see below).

CC2 is a proline-rich sequence that matches the consensus binding site for Drosophila Enabled (Ena; see below), CC3 is also a polyproline stretch.²⁹ Drosophila Robo2 and Robo3 lack the CC2 and CC3 domains^5,30 and the second half of CC2 is also not conserved in mouse and zebrafish Robo3. Furthermore, mouse Robo3/Rig-1 (Rig-1 for retinoblastoma inhibiting gene 1³⁹) lacks the CC1 motif^33,39 but zebrafish Robo3 has it.³² In addition, some spliced variants of mouse Robo3/Rig-1, including a secreted form, may exist.³⁹ Last, Robo 1 can be cleaved in transfected cells.²⁶

In Drosophila, genetic and biochemical evidence demonstrated that Slit is a ligand of the Robo1-Robo3 receptors.^4,5,29,30,40 Likewise, mammalian Slits can bind to all Robo receptors with comparable affinity.^7,12,41 Slit cleavage fragments appear to have different cell association characteristics, with the smaller C-terminal fragment being more diffusible and the larger N-terminal and full length fragments being more tightly cell-associated.²¹ In addition, the C-fragment does not bind to Robo.²⁴ More recent studies have shown that in Drosophila all three Robo receptors compete for a single active binding site in the second LRR of Slit²⁷ and that neither the FNIII domains nor Robo dimerization are required for Slit binding. The major Robo1-3 binding site of Slit is in the second of the four LRRs, is evolutionary conserved and has a similar affinity for all Robos. However, Slit affinity is higher when all LRRs are present, probably due to its dimerization. On the receptor side, several results suggest that the first two Ig domains of Robos are required for Slit binding. First, the genetic deletion of Ig1 and Ig2 results in abnormal lung development.⁴² Second, antibodies against Robo Ig1 inhibit tumor growth in mice⁴³ and neurite outgrowth in vitro.⁴⁴ Third, Robo1 Ig1-2 are important for Slit binding and function in vitro.⁴⁵

Several studies suggest that Slit can bind to other proteins than Robo, in particular heparan sulfate glycosaminoglycans that are negatively charged carbohydrates found on the cell surface. Slit1 and Slit2 were shown to bind to heparin column^21,46 and to Glypican-1,⁴⁶ a glycosyl phosphatidyl inositol (GPI)-anchored heparan sulfate proteoglycan known to interact with positively charged molecules. Biochemical data suggest that Slit binds to glypican-1 through its C-terminus.⁴⁷ Moreover, heparinase III treatment reduces Slit2 activity and binding to Robo1.⁴⁸ In Drosophila, expression of the transmembrane heparan sulfate proteoglycan syndecan in target cells appears to be required for Slit signaling.⁴⁹ There is also genetic evidence in mouse supporting interaction between Slit and heparan sulfates in vivo.⁵⁰ Heparan sulfates could help stabilizing the Robo/Slit complex or function as coreceptors presenting Slits to Robos or to alternative receptors.

Robo Partners

The analysis of Frazzled-Robo chimeric proteins in Drosophila, first revealed that the cytoplasmic domain of Robo is required to control the lateral positioning of post-crossing axons.⁵¹ Genetic and biochemical studies have then led to the identification of a number of transmembrane and cytoplasmic proteins that may participate or modulate Slit signaling through Robo receptors (Fig. 2). However, only a few of these proteins have been shown to directly participate to Robo signaling upon binding Robo CC domains.

Figure 2

A central role for Abelson tyrosine kinase in Robo repulsion. Abl can inactivate Robo signaling through phosphorylation of CC0 and CC1. Other data suggest that Abl is recruited following Robo activation. Abl interacts with multiple effectors such as Enabled (more...)

Rho Family of Small GTPases

Rho family of small GTP-binding proteins (Rho GTPases) are major modulators of the actin cytoskeleton and play a central role in axonal growth and cell migration.⁵⁷ Rho GTPases are activated upon GTP binding and inactive when bound to GDP. The switch from their active to their inactive state is controlled by two families of proteins: the guanine nucleotide exchange factors (GEFs) and the GTPase activating proteins (GAPs). GEFs activate Rho GTPAses while GAPs inactivate them by inducing GTP hydrolysis.⁵⁷

Many studies have shown that Rho GTPases play an important role in the modulation of Slit function (Fig. 3). First, activation of RhoA in Drosophila causes axons to cross the midline.⁵⁸ Second, In this system, Rac1 inactivation or Cdc42 activation can overcome the effect of constitutively active Robo suggesting that Cdc42 and Rac1 function downstream of Robo.⁵⁹ Likewise, in vertebrate neurons, a constitutively active Cdc42 blocks the repulsive effect of Slit.⁶⁰ Biochemical studies have shown that the SH3-SH2 adaptator protein Dock, can bind directly to Robo and that this interaction requires the SH3 domain of Dock and the CC2 and CC3 motifs of Robo.^61,62 Dock is known to interact with key regulators of the actin cytoskeleton such as p21-activated protein kinase (Pak), a serine-threonine kinase which in turn can interact with RhoGTPases such as Rac1 and Cdc42. Slit binding to Robo increases the level of Dock-Robo association, recruits Pak and stimulates Rac (in particular Rac1). Thus Slit regulates the assembly of a multiproteic complex composed of Dock, Pak and Rac that couples Robo receptor activation to the regulation of the actin cytoskeleton.

Figure 3

Rho GTPases in Slit/Robo signaling. Rho family of small GTP-binding proteins (Rho GTPases) are activated upon GTP binding and inactive when bound to GDP. Rho GTPases play an important role in the modulation of Slit function. The SH3-SH2 adaptator protein (more...)

Robo also controls the activity of Rho GTPases through a family of Slit/Robo specific GAPs, SrGAP1-3, that were identified using yeast two hybrid and Robo1 CC3 domain as a bait. SrGAPs consist of a RhoGAP domain, a SH3 domain and a Fes/CIP4 (FCH) homology domain. SrGAP1 and srGAP3 bind to the CC3 domain of Robo1 through their SH3 domain. Slit2 increases Robo1 binding to srGAP1 and its activity and this regulation requires CC3. In turn, SrGAP1 inactivates Cdc42 and activates RhoA but not Rac1.⁶⁰ Contrary to srGAP1, srGAP3 is mainly a repressor of Rac1.⁶³ Recently, it was also shown that in Drosophila, Slit/ Robo signaling could also control Rac activity upon binding the GAP Vilse/CrossGAP, that is conserved in vertebrates.^64,65 Genetic evidence showed that CrossGAP may be involved in Robo-dependent axonal repulsion and tracheal cell migration and that it specifically inactivates Rac. Moreover, CrossGAP WW domains can directly bind to the CC2 domain of Robo and thus may not function downstream of other Robo receptors that lack the CC2 domain.⁶⁴ Moreover, the two proteins can be coimmunoprecipitated from brain extracts.⁶⁵ Although it inactivates Rac, Vilse seems to have a positive role in Robo repulsion.⁶⁴

Molecular Control of Slit and Robo Expression

Multiple Functions for Slit/Robo in the Nervous System

Slits play a major role in axon guidance in many systems and animal species. In most cases Slits act as repellents but there is some evidence that they may act positively on some axons.^71,77

A Role for Slit and Robo in Neurological Disorders?

There is relatively little direct evidence so far indicating that Slit and Robo may be involved in pathological processes except in cancers.¹²⁸ However, several patients suffering from a rare congenital syndrome named Horizontal gaze palsy with progressive scoliosis and hindbrain dysplasia (HGPPS) were recently shown to bear mutations in the ROBO3 gene. In these patients, the pyramidal tract and the dorsal column-medial lemniscus are uncrossed.³³ Moreover, there is a reduced pontine nucleus and abducens nuclei. As shown in robo3 knockout mice the pontine nucleus defect is probably caused by an abnormal migration during development.⁹¹ It is still unknown if the other human brain defects are also present in mice lacking Robo3 and as those die at birth, behavioral analysis are not possible.

One of the Slit/Robo GAP, SrGAP3 has a putative role in idiopathic mental retardation⁶³ as it is mutated in patients with X chromosome-linked MR. However, the cellular basis for these defects and the normal function of SrGAP3 in the CNS are unknown.

The incapacity of adult axons to regenerate in the CNS of mammals is known to rely for a large extent on the existence of inhibitors of axonal growth expressed either in the glial scar or in myelin. Several studies have shown that repulsive axon guidance molecules may be responsible for some of the inhibition.¹²⁹ Slit an Robo expression after injury has not been extensively studied so far. However, in a model of cryoinjury, Slit2 was found to be expressed in reactive astrocytes together with glypican-1.¹³⁰ In addition, adult DRG neurons also express mRNAs for Robo2 and Slit1 but their expression does not change after sciatic nerve transection or dorsal column lesion in the spinal cord.¹³¹ In contrast, Glypican-1 and SrGAP2 expression are upregulated after such lesions.^131,132 Thus, it is still unclear if Slit and Robo play any role in preventing axonal regeneration.

Perspectives

There is mounting evidence that Slits regulate a large range of biological functions, from axon guidance, neuronal migration, immune response¹³³ to cell differentiation most likely through Robo signaling. However there are still many open questions.

First, could Robo functions independently of Slit in particular through Robo/Robo interaction and what are the signaling pathways involved. Thus, in the Drosophila PNS, Robo2 expressed on visceral mesoderm binds Slit and present it to Robo expressing chordotonal sensory neurons. This may also involve Robo-Robo2 direct interaction.^81,134 In vitro experiments also showed that the growth of retinal and olfactory Robo expressing axons is stimulated on Robo expressing cells, suggesting that Robo might work as cell-adhesion molecule to regulate outgrowth.⁴⁴ In Drosophila, Robo and Robo2 can dimerize in vitro⁴⁰ and ectopic expression of low level of Robo2 causes a Robo-like phenotype suggesting that Robo2 could interfere with Robo function.⁵

The function of Slits and Robos to in the normal adult brain and in pathological condition also remains to be clarified. Many data support a role for these molecules in tumorigenesis, in particular in gliomas¹²⁸ but this needs to be further demonstrated. As all Slits and Robos are expressed in adult neurons it is likely that they modulate synaptic transmission as shown recently for other secreted axon guidance molecules of the semaphorin family.¹³⁵ Many answers to these questions should come from the analysis of mouse deficient for one or several of these proteins.

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