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Role of Slit proteins in the vertebrate brain
Journal of Physiology - Paris 96 (2002) 91–98 www.elsevier.com/locate/jphysparis
Role of Slit proteins in the vertebrate brain Kim T. Nguyen-Ba-Charvet, Alain Che´dotal* INSERM U106, Baˆtiment de Pe´diatrie, Hoˆpital de la Salpeˆtrie`re, 47 Boulevard de l’Hoˆpital, 75013 Paris, France
Abstract Diffusible chemorepellents play a major role in guiding developing axons towards 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) receptors and their secreted ligand Slits. Three distinct slit genes (slit1, slit2 and slit3) and three distinct robo genes (robo1, robo2 and rig1) have been cloned in mammals. In collagen gel co-cultures, Slit1 and Slit2 can repel and collapse olfactory axons. However, there is also some positive effect associated with Slits, as Slit2 stimulates the formation of axon collateral branches by NGF-responsive neurons of the dorsal root ganglia (DRG). Slit2 is a large ECM glycoproteins of about 200 kD, which is proteolytically processed into 140 kD N-terminal and 55–60 kD C-terminal fragments. Slit2 cleavage fragments appear to have different cell association characteristics, with the smaller C-terminal fragment being more diffusible and the larger N-terminal and uncleaved fragments being more tightly cell associated. This suggested that the different fragments might have different functional activities in vivo. We have begun to explore these questions by engineering mutant and truncated versions of hSlit2 representing the two cleavage fragments, N- and C-, and the uncleavable molecule and examining the activities of these mutants in binding and functional assays. We found that an axon’s response to Slit2 is not absolute, but rather is reflective of the context in which the protein is encountered. # 2002 Elsevier Science Ltd. All rights reserved. Keywords: Slit proteins; Slit2; Slit2-N; Slit2-U
1. Introduction Mounting evidence indicates that in the developing central nervous system, growth cones can be guided at a distance by diffusible molecules secreted by non-target cells [42]. Many of these factors function as chemorepellents: they induce growth cone collapse and oriented axonal outgrowth away from the source of the factor. Chemorepulsive molecules are produced in a variety of central nervous system (CNS) regions, such as the ventral spinal cord, the floor plate or the thalamus [5]. Most chemorepulsive factors are members of the semaphorin, netrin and slit families. We have been studying the function of these molecules in the developing telencephalon, and particularly in the developing olfactory system.
2. Chemotropism in the developing olfactory system The organization of axonal projections in the rodent olfactory system has been extensively characterized. * Corresponding author. E-mail address: [email protected] (A. Che´dotal).
Axons from olfactory receptor neurons in the olfactory epithelium project ipsilaterally to glomeruli in the main olfactory bulb, where they synapse on the dendrites of the mitral and tufted cells. These neurons project ipsilaterally to the anterior olfactory nucleus and to higher olfactory centers including the piriform and entorhinal cortex, and some amygdaloid nuclei, collectively referred to as the primary olfactory cortex [36]. The axons of the mitral and tufted cells are located immediately under the pial surface [34] and form the lateral olfactory tract (LOT). The development of bulbofugal projections is still poorly understood. In the rat embryo, isolated fibers start to leave the olfactory bulb by E14, and by E15 a LOT has clearly formed [21,26]. At this stage of development, the great majority of post-mitotic neurons in the olfactory bulb are mitral cells. Therefore, the early LOT is almost solely composed of mitral cell axons. Organotypic co-culture of olfactory bulb and telencephalic vesicles or membranes has shown that the telencephalon contains precisely localized, short-range positional cues that guide LOT axons [7,33,41]. However, the results of other in vitro assays suggest that developing rat LOT axons can also be guided from a distance by some unidentified diffusible chemorepulsive
0928-4257/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved. PII: S0928-4257(01)00084-5
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factors produced by the septum or the neocortex [13,26]. The identity of this septal-derived repulsive activity was unknown. We first investigated whether secreted Semaphorins could play a role in the development of bulbofugal projections [5]. It was already known that the semaphorin receptors neuropilin-1 and neuropilin-2 are highly expressed in the developing olfactory system. First, we showed using collagen gel assay, that the olfactory epithelium releases a diffusible factor that repels olfactory bulb. This repellent effect is mimicked by the secreted semaphorin Sema3F and not by any other secreted Semaphorins or by netrin-1. Moreover Sema3F mRNA is expressed in the olfactory epithelium at the time LOT axons grow [5], suggesting that this molecule could be the epithelial-derived repellent factor (Fig. 1B). In contrast, olfactory bulb axons grow preferentially toward aggregates of COS cells producing another secreted semaphorin, Sema3B, suggesting that chemoattraction could also be involved in the formation of the LOT. In the embryonic rat head, Sema3B mRNA is almost exclusively found in the frontal bone primordium that is apposed to the forebrain parenchyma and therefore immediately adjacent to the LOT. Although direct evidence is still lacking, our results suggest that Sema3B released by the mesenchymal cells could attract in the CNS, axons of the LOT, maintaining it superficially (Fig.1B). This study also indicated that none of the known secreted Semaphorins are expressed in the embryonic septum, suggesting that the septal-derived repulsive factor does not belong to the semaphorin family. We then studied the possible involvement of molecules of the Slit family.
3. Structure and function of Slit proteins The Slits is the most recently discovered family of chemotropic factors [2]. Slit (d-Slit) was first identified in Drosophila embryo as a gene involved in the patterning of larval cuticle. Subsequently, it was shown that dSlit is synthesized in the central nervous system by midline glia cells and that in the absence of slit, longitudinal and commissural axons all converge and coalesce at the midline [2,17,27,37]. More recent works have demonstrated that d-Slit is a chemorepulsive factor and a key regulator of midline crossing and axonal fasciculation [28,38]. Slit homologues have since been found in virtually all vertebrate species, from amphibians [19], fishes [47], birds (A.C, unpublished data) to mammals such as rodents [1,10,19] but also humans [14]. In mammals, three slit genes (slit1-slit3) have been cloned. All encodes large ECM glycoproteins of about 200 kD (Fig. 1A), comprising, from their N terminus to their C terminus, a long stretch of four leucine rich repeats, seven to nine EGF repeats, and a domain, named ALPS [2,31], LNS
[32], or LG module [9]. Slit2 is proteolytically processed into 140 kD N-terminal and 55-60 kD C-terminal fragments in cell culture and in vivo [2]. Likewise, Drosophila Slit also appears to be similarly processed in vitro and in vivo, suggesting conserved mechanisms for Slit proteolytic processing across species [1, 45]. 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 (see later). Like d-Slit, vertebrate Slits have also been shown to be repulsive factors for developing spinal cord axons [1, 51], dentate gyrus [22] and retina [6, 25, 29]. Interestingly, in rodents Slit2 can stimulate axonal elongation and branch formation of sensory axons from the dorsal root ganglia [45]. Moreover, Slit proteins repel migrating muscle precursors in fly embryos [17], mesodermal and neuronal cells in zebrafish embryos [47] and several categories of tangentially migrating rodent telencephalic interneurons in organotypic cultures (Fig. 1C) [12,46,50].
4. Robos are receptors for Slits One major breakthrough toward the understanding of Slit function has been the recent discovery that the Roundabout (robo) proteins are Slit receptors [1,17,19]. The first robo gene, robo1, was identified in Drosophila during a comprehensive screen for genes controlling CNS midline crossing [35]. In robo1 mutants, ipsilateral axons that normally avoid the midline cross it, and commissural axons cross and recross it repeatedly [35]. Robo is an evolutionary conserved family of transmembrane receptors [3,28,37,49]. Robo proteins define a small subgroup within the immunoglobulin superfamily characterized by the presence of five Ig-like domains followed by three fibronectin type III (FNIII) repeats, a transmembrane portion and a long cytoplasmic tail containing robo-specific motifs [18]. So far, three robo genes have been found in flies [18,28,37] and mammals [1,19]. In mammals, CDO is another protein with 5Ig3FNIII [15], but its sequence overall is rather divergent from those of the three Robo receptors, suggesting that CDO probably does not belong to this family. In Drosophila, genetic and biochemical evidence indicates that Slit is a ligand of the robo1-robo3 receptors [18,28,37]. Similarly, in vitro studies have shown that in mammals, Slit1 [19], Slit2 [1] and Slit3 (AC, unpublished data) bind with similar affinity to Robo1 and Robo2. However, it is not known whether Rig-1 [48], the third Robo-like protein, which lacks some cytoplasmic domains found in Robo1 and Robo2, is a receptor for Slit. Robo has recently been shown to interact directly with the netrin-1 receptor DCC, a mechanism that could participate to the control of midline crossing by commissural axons [40].
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Fig. 1. (A) Structure of the Slit and Robo proteins. (B) Schematic representation in horizontal plane of the chemorepulsive and chemoattractive factors that influence the formation of the lateral olfactory tract. The olfactory epithelium (OE) would release a repulsive factor (pink), probably Sema3F, forcing LOT axons to leave the olfactory bulb. Slit1 and Slit2, produced by the septum (S, yellow) would prevent LOT axons to cross the midline. In addition, LOT axons would grow very superficially being attracted by Sema3B secreted by the mesenchyme precursor of the frontal bone (purple). (C) In collagen gel assay, OB axons (right panel) are repelled by Slit expressing cells. Similarly, subventricular zone (SVZ) neurons (in orange) migrate radially when confronted with control cells (middle panel) but away from Slit expressing cells (right panel).
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The potential role of Robo and Slit family members in mediating olfactory bulb axon pathfinding was examined by studying the expression pattern of Robo-1, Robo2, Slit-1, and Slit-2 mRNAs in the rat embryo. Interestingly, from E14, Slit-1 and Slit-2 are expressed by the septum (Fig. 1B) and at that stage mitral and tufted cells express high levels of Robo-2 mRNA (Fig. 1B). In addition, in collagen gel assay, axons from OB explants cultured next to COS cells secreting Slit-2 and Slit-1, are strongly repelled [19,22] (Nguyen-Ba-Chanet and Che´dotal, unpublished data). These data strongly suggest that Slits could represent the septal-derived chemorepulsive activity (Fig. 1C). However, some results have questioned the role of the septum in organizing LOT projections as in organotypic cultures mitral cell axons elongate along their normal pathway in the absence of septum [41] or when Slit signaling is blocked [8].
5. Slit2 proteolytic fragments have distinct axon guidance properties As mentioned above, Slit2 is cleaved in vivo and in vitro in two fragments. These 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 possibility was supported by several studies. First, the purification of Slit as a DRG elongation- and branch-promoting activity revealed that only the N-terminal fragment of Slit2 is capable of stimulating elongation and branching [45]. Second, the purification of a repellent activity for migrating SVZ olfactory precursor neurons and of a collapsing activity for retinal ganglion cell axons revealed that they correspond to the full-length fragment of hSlit2 [12,25]. Therefore, it was not clear, however, which fragment(s) were responsible for the other Slit associated activities, nor was it known whether proteolytic processing was required for Slit’s repulsive activity. These questions have been explored by engineering mutant and truncated versions of Slit2 and examining the activities of these mutants in binding and functional assays [4,23,24]. We generated cDNA constructs (Fig. 2A) encoding truncated proteins corresponding to just the N-terminal and the C-terminal cleavage fragments of human Slit2 (with one ending and the other starting at Thr 1118, the putative cleavage site, located at the start of EGF repeat 6; Fig. 2). We also engineered a cDNA construct encoding a presumptive uncleavable form of Slit2. For this, the DNA sequence encoding the nine amino acids preceding Thr 1118 (SPPMVLPRT) was removed from the human Slit2 cDNA. The binding properties of various Slit2 protein fragments with Robo receptors were tested in cell overlay assays. It was found that Slit2-N and Slit2-U both bind
to robo receptors with similar affinity, while Slit2-C does not bind [23]. Therefore, binding to Robo receptors is mediated by amino-terminal Slit2 sequences. This was confirmed by immunoprecipitation studies indicating that the leucine-rich repeats of Slit and the immunoglobulin domain of Robo are sufficient to mediate Slit–Robo interaction [4].
6. Functions and pharmacology of Slit2-N and Slit2-U in repulsion and branching We focused on OB and DRG neurons because they both express Robo2 but not Robo1 mRNAs [22,45] and because they showed dramatically different responses to Slit2, with olfactory axons being repelled [22] and DRG axons stimulated to elongate and branch [45]. Slit2-N and Slit2-U were found to have similar activities in repelling OB axons in the collagen gel repulsion assay (Fig. 2B) [23]. In contrast, the C-terminal portion of Slit2 (Slit2-C) had no repulsive activity. Other studies have shown that the leucine-rich repeats alone can induce repulsion of OB axons and migrating neurons [4]. None of the Slit2 fragment repels DRG axons [23,25], demonstrating that different Robo-expressing neurons exhibit distinct responses to Slit2 fragments. Slit2-U and Slit2-N were also found to have divergent actions in the sensory neuron branching assay. As observed for the native proteins [45], recombinant Slit2N but not Slit2-U stimulates elongation and branching of DRG axons in this assay. Furthermore, Slit2-U functions as an antagonist of Slit2-N, which is coherent with the observation, that native Slit2-N was active only when purified away from full-length Slit2. In addition to repelling olfactory bulb, hippocampal and retinal axons in the collagen gel assay, Slit-2 can also cause the collapse of their growth cones [22,25]. We showed that only Slit2-N, not Slit2-U, causes collapse of OB growth cones. DRG axons are never collapsed by any Slit2 fragment [22,25]. This is surprising, since it is generally expected that factors that can repel also cause collapse. This finding provides a clear demonstration of a dissociation between the two types of activity. This dissociation may be cell type specific, since native fulllength Slit2 has both activities for retinal axons in culture [25]. In conclusion, Slit2-N and Slit2-U, that both bind robo receptors can have different activity in the branching and collapse assays.
7. Substrate-bound Slit2 can guide sensory axons Because of the strong binding of Slit2 fragments to cell membranes, growth cones are very likely to be confronted with immobilized Slit2. We examined whether substrate-bound Slit2 is able to guide developing sensory
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axons in the so-called ‘‘stripe’’ assay [43,44] in which the axons grow parallel to alternating stripes of two different proteins or protein combinations, making it possible to test the axons’ preference for one over the other [24]. We first examined the responses of E15 rat DRG axons to Slit2 protein fragments in stripe assays using as a substrate, membranes from transfected COS cells. These experiments were done using NGF to support DRG cell survival and elicit axon outgrowth. When the axons
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were confronted with alternating lanes of membranes from control (mock-transfected) COS cells and COS cells expressing various Slit2 constructs such as the wildtype Slit2, Slit2-U or Slit2-N, the axons always avoided the Slit2 membranes [24]. In contrast, NT3-responsive axons are not sensitive to Slit2, at least in this assay, suggesting that the results seen with NGF-responsive axons is receptor-mediated and do not reflect simply a masking of positive factors in the COS cells membranes.
Fig. 2. (A) Schematic representation of the Slit2 constructs. Slit2-full represents the native full length Slit2. The nine EGF repeats and the ALPS domain are represented, and the nine amino acids sequence deleted to generate Slit2-U is indicated (arrowhead).B, In collagen gel assay, OB axons grow symmetrically when confronted with control cells or Slit2-C expressing cells. In contrast they are repelled by cells expressing Slit2-N or Slit2U.C-E, DRG axons show different preferences between the different Slit2 fragments and fibronectin or laminin in stripe assays. DRG explants were cultured on substrates patterned with alternating stripes of Slit2-U (U) or Slit2-N (N) and fibronectin (F) or laminin (L). Explants were immunolabeled for classIII-b-tubulin and the Slit2 stripes were labeled with fluorescein conjugated-BSA. (C) When DRG axons are presented a choice between Slit2-N and laminin, they constantly grow on laminin. When grown on stripe of Slit2-U and fibronectin (D), DRG axons show a clear preference for fibronectin. In contrast, when confronted with alternating stripe of Slit2-N and fibronectin (E), DRG axons grow on Slit2-N. Bar, 60 mm.
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When confronted with alternating lanes of membranes from COS cells expressing Slit2-N and those from cells expressing Slit2-U, the DRG axons invariably selected the membranes containing Slit2-N [24]. This result confirms that in some conditions, Slit2-U and Slit2-N do not have the same axon guidance properties.
8. ECM molecules influence sensory axon response to substrate-bound Slit2 fragments Given these results, we were curious to examine the response of DRG axons to purified Slit2 proteins in the stripe assay (Fig. 2C,D,E) [24]. Because ECM molecules, specially laminin-1 have been shown to influence the response of retinal axons to netrin-1, another chemotropic molecules [11], we compared the activity of Slit2 fragments in the presence of two different ECM molecules laminin or fibronectin which are both excellent substrates for DRG axons in culture. As expected, when Slit2-U or Slit2-N were mixed with laminin, DRG axons always preferred to grow on laminin alone than on any of the two mixtures, a result very similar to what was found using membrane extracts. When DRG axons were confronted with alternating stripes of fibronectin alone and fibronectin mixed with Slit2-U they also avoided Slit2-U. Surprisingly, they consistently preferred a mixture of fibronectin and Slit2-N to fibronectin alone. We also tested the behavior of rat E15 OB axons when confronted with substrate-bound Slit2-N in the stripe assay. In contrast to DRG axons, Slit2-N was always avoided by OB axons in the stripe assay, when given a choice with fibronectin or laminin. These observations are coherent with the results of the repulsion assay and confirm that different axons exhibit different responses to Slit2 fragments. What can explain the uniformity of response in the presence of laminin? Laminin-1 may trigger a second messenger pathway in the axons that alters their responses to the various proteins, such that all the responses become negative (repulsive). A precedent for this idea is provided by the finding that exposure of Xenopus retinal axons to laminin converts the responses of these axons to netrin-1 from attraction to repulsion, apparently by lowering cAMP levels in these growth cones [11]. The level of cyclic nucleotides in the growth cone in part determines the action of many guidance cues. When the level is above the critical range, the guidance cue causes attraction and conversely, when the level is below the critical range, the cue induces repulsion [39]. We tested this hypothesis on DRG axons. It was found that Slit2-N could be converted to a negative effect by lowering cGMP levels. Interestingly, as it is the case for netrin-1, a laminin-1 peptide was able to convert Slit2-N growth-promoting action into an inhibition [24]. This can probably explain the homogeneity of
axons response to Slit2 proteolytic fragments avoidance) in the presence of laminin. For netrin-1, laminin-1 effect is at least in part mediated by b-integrins, we still don’t know if this is the case for Slit2. These results underscore the importance of the molecular context in which the axons are confronted with Slit2 protein fragments on axonal responses to those fragments. This also supports the notion that ECM molecules, such as laminin and fibronectin can modulate growth cone response to a variety of chemotropic molecules.
9. Conclusion Although extensive progress has been made toward the understanding of Slit function in the developing CNS there are still many unanswered questions. For instance, could Slit2-C have a role in axon guidance? Although the sole purpose of the cleavage could be to generate bioactive Slit2-N, there are nonetheless reasons, based on its structure, for thinking that Slit2-C is also bioactive. However, it had been shown that in the brain, Slit-2 is a ligand for the glycosylphosphatidylinositol-anchored heparan sulfate proteoglycan glypican-1 [20]. More recently, it was determined that Slit2 binding to glypican-1 is mediated by the Cterminal portion of Slit2, and most likely by the ALPS domain [30]. In the rat embryo, glypican-1 is highly expressed in several types of neurons, including motor neurons [16] suggesting that Slit2-C could influence growing axons via this proteoglycan. It is still unknown whether Slit1 and Slit3 are cleaved and in the case the identity of the protease which cleaves Slit2 will have to be determined. It also remains to be proven whether in mammals, Slit functions, including its repulsive action on tangentially migrating interneurons, are mediated by Robos. Our results suggest that some difference exists in the receptor mechanisms mediating axonal repulsion and branching. Many answers should come from the analysis of Slit and Robo knock-outs.
Acknowledgements This work is supported by the Institut de la Sante´ et de le Recherche Me´dicale, the Ministe`re de la Recherche et de la Technologie (ACI) and the Association pour la Recherche sur le Cancer (No.5249). K N-B-C is supported by the Fondation de France.
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Role of Slit proteins in the vertebrate brain Kim T. Nguyen-Ba-Charvet, Alain Che´dotal* INSERM U106, Baˆtiment de Pe´diatrie, Hoˆpital de la Salpeˆtrie`re, 47 Boulevard de l’Hoˆpital, 75013 Paris, France
Abstract Diffusible chemorepellents play a major role in guiding developing axons towards 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) receptors and their secreted ligand Slits. Three distinct slit genes (slit1, slit2 and slit3) and three distinct robo genes (robo1, robo2 and rig1) have been cloned in mammals. In collagen gel co-cultures, Slit1 and Slit2 can repel and collapse olfactory axons. However, there is also some positive effect associated with Slits, as Slit2 stimulates the formation of axon collateral branches by NGF-responsive neurons of the dorsal root ganglia (DRG). Slit2 is a large ECM glycoproteins of about 200 kD, which is proteolytically processed into 140 kD N-terminal and 55–60 kD C-terminal fragments. Slit2 cleavage fragments appear to have different cell association characteristics, with the smaller C-terminal fragment being more diffusible and the larger N-terminal and uncleaved fragments being more tightly cell associated. This suggested that the different fragments might have different functional activities in vivo. We have begun to explore these questions by engineering mutant and truncated versions of hSlit2 representing the two cleavage fragments, N- and C-, and the uncleavable molecule and examining the activities of these mutants in binding and functional assays. We found that an axon’s response to Slit2 is not absolute, but rather is reflective of the context in which the protein is encountered. # 2002 Elsevier Science Ltd. All rights reserved. Keywords: Slit proteins; Slit2; Slit2-N; Slit2-U
1. Introduction Mounting evidence indicates that in the developing central nervous system, growth cones can be guided at a distance by diffusible molecules secreted by non-target cells [42]. Many of these factors function as chemorepellents: they induce growth cone collapse and oriented axonal outgrowth away from the source of the factor. Chemorepulsive molecules are produced in a variety of central nervous system (CNS) regions, such as the ventral spinal cord, the floor plate or the thalamus [5]. Most chemorepulsive factors are members of the semaphorin, netrin and slit families. We have been studying the function of these molecules in the developing telencephalon, and particularly in the developing olfactory system.
2. Chemotropism in the developing olfactory system The organization of axonal projections in the rodent olfactory system has been extensively characterized. * Corresponding author. E-mail address: [email protected] (A. Che´dotal).
Axons from olfactory receptor neurons in the olfactory epithelium project ipsilaterally to glomeruli in the main olfactory bulb, where they synapse on the dendrites of the mitral and tufted cells. These neurons project ipsilaterally to the anterior olfactory nucleus and to higher olfactory centers including the piriform and entorhinal cortex, and some amygdaloid nuclei, collectively referred to as the primary olfactory cortex [36]. The axons of the mitral and tufted cells are located immediately under the pial surface [34] and form the lateral olfactory tract (LOT). The development of bulbofugal projections is still poorly understood. In the rat embryo, isolated fibers start to leave the olfactory bulb by E14, and by E15 a LOT has clearly formed [21,26]. At this stage of development, the great majority of post-mitotic neurons in the olfactory bulb are mitral cells. Therefore, the early LOT is almost solely composed of mitral cell axons. Organotypic co-culture of olfactory bulb and telencephalic vesicles or membranes has shown that the telencephalon contains precisely localized, short-range positional cues that guide LOT axons [7,33,41]. However, the results of other in vitro assays suggest that developing rat LOT axons can also be guided from a distance by some unidentified diffusible chemorepulsive
0928-4257/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved. PII: S0928-4257(01)00084-5
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factors produced by the septum or the neocortex [13,26]. The identity of this septal-derived repulsive activity was unknown. We first investigated whether secreted Semaphorins could play a role in the development of bulbofugal projections [5]. It was already known that the semaphorin receptors neuropilin-1 and neuropilin-2 are highly expressed in the developing olfactory system. First, we showed using collagen gel assay, that the olfactory epithelium releases a diffusible factor that repels olfactory bulb. This repellent effect is mimicked by the secreted semaphorin Sema3F and not by any other secreted Semaphorins or by netrin-1. Moreover Sema3F mRNA is expressed in the olfactory epithelium at the time LOT axons grow [5], suggesting that this molecule could be the epithelial-derived repellent factor (Fig. 1B). In contrast, olfactory bulb axons grow preferentially toward aggregates of COS cells producing another secreted semaphorin, Sema3B, suggesting that chemoattraction could also be involved in the formation of the LOT. In the embryonic rat head, Sema3B mRNA is almost exclusively found in the frontal bone primordium that is apposed to the forebrain parenchyma and therefore immediately adjacent to the LOT. Although direct evidence is still lacking, our results suggest that Sema3B released by the mesenchymal cells could attract in the CNS, axons of the LOT, maintaining it superficially (Fig.1B). This study also indicated that none of the known secreted Semaphorins are expressed in the embryonic septum, suggesting that the septal-derived repulsive factor does not belong to the semaphorin family. We then studied the possible involvement of molecules of the Slit family.
3. Structure and function of Slit proteins The Slits is the most recently discovered family of chemotropic factors [2]. Slit (d-Slit) was first identified in Drosophila embryo as a gene involved in the patterning of larval cuticle. Subsequently, it was shown that dSlit is synthesized in the central nervous system by midline glia cells and that in the absence of slit, longitudinal and commissural axons all converge and coalesce at the midline [2,17,27,37]. More recent works have demonstrated that d-Slit is a chemorepulsive factor and a key regulator of midline crossing and axonal fasciculation [28,38]. Slit homologues have since been found in virtually all vertebrate species, from amphibians [19], fishes [47], birds (A.C, unpublished data) to mammals such as rodents [1,10,19] but also humans [14]. In mammals, three slit genes (slit1-slit3) have been cloned. All encodes large ECM glycoproteins of about 200 kD (Fig. 1A), comprising, from their N terminus to their C terminus, a long stretch of four leucine rich repeats, seven to nine EGF repeats, and a domain, named ALPS [2,31], LNS
[32], or LG module [9]. Slit2 is proteolytically processed into 140 kD N-terminal and 55-60 kD C-terminal fragments in cell culture and in vivo [2]. Likewise, Drosophila Slit also appears to be similarly processed in vitro and in vivo, suggesting conserved mechanisms for Slit proteolytic processing across species [1, 45]. 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 (see later). Like d-Slit, vertebrate Slits have also been shown to be repulsive factors for developing spinal cord axons [1, 51], dentate gyrus [22] and retina [6, 25, 29]. Interestingly, in rodents Slit2 can stimulate axonal elongation and branch formation of sensory axons from the dorsal root ganglia [45]. Moreover, Slit proteins repel migrating muscle precursors in fly embryos [17], mesodermal and neuronal cells in zebrafish embryos [47] and several categories of tangentially migrating rodent telencephalic interneurons in organotypic cultures (Fig. 1C) [12,46,50].
4. Robos are receptors for Slits One major breakthrough toward the understanding of Slit function has been the recent discovery that the Roundabout (robo) proteins are Slit receptors [1,17,19]. The first robo gene, robo1, was identified in Drosophila during a comprehensive screen for genes controlling CNS midline crossing [35]. In robo1 mutants, ipsilateral axons that normally avoid the midline cross it, and commissural axons cross and recross it repeatedly [35]. Robo is an evolutionary conserved family of transmembrane receptors [3,28,37,49]. Robo proteins define a small subgroup within the immunoglobulin superfamily characterized by the presence of five Ig-like domains followed by three fibronectin type III (FNIII) repeats, a transmembrane portion and a long cytoplasmic tail containing robo-specific motifs [18]. So far, three robo genes have been found in flies [18,28,37] and mammals [1,19]. In mammals, CDO is another protein with 5Ig3FNIII [15], but its sequence overall is rather divergent from those of the three Robo receptors, suggesting that CDO probably does not belong to this family. In Drosophila, genetic and biochemical evidence indicates that Slit is a ligand of the robo1-robo3 receptors [18,28,37]. Similarly, in vitro studies have shown that in mammals, Slit1 [19], Slit2 [1] and Slit3 (AC, unpublished data) bind with similar affinity to Robo1 and Robo2. However, it is not known whether Rig-1 [48], the third Robo-like protein, which lacks some cytoplasmic domains found in Robo1 and Robo2, is a receptor for Slit. Robo has recently been shown to interact directly with the netrin-1 receptor DCC, a mechanism that could participate to the control of midline crossing by commissural axons [40].
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Fig. 1. (A) Structure of the Slit and Robo proteins. (B) Schematic representation in horizontal plane of the chemorepulsive and chemoattractive factors that influence the formation of the lateral olfactory tract. The olfactory epithelium (OE) would release a repulsive factor (pink), probably Sema3F, forcing LOT axons to leave the olfactory bulb. Slit1 and Slit2, produced by the septum (S, yellow) would prevent LOT axons to cross the midline. In addition, LOT axons would grow very superficially being attracted by Sema3B secreted by the mesenchyme precursor of the frontal bone (purple). (C) In collagen gel assay, OB axons (right panel) are repelled by Slit expressing cells. Similarly, subventricular zone (SVZ) neurons (in orange) migrate radially when confronted with control cells (middle panel) but away from Slit expressing cells (right panel).
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The potential role of Robo and Slit family members in mediating olfactory bulb axon pathfinding was examined by studying the expression pattern of Robo-1, Robo2, Slit-1, and Slit-2 mRNAs in the rat embryo. Interestingly, from E14, Slit-1 and Slit-2 are expressed by the septum (Fig. 1B) and at that stage mitral and tufted cells express high levels of Robo-2 mRNA (Fig. 1B). In addition, in collagen gel assay, axons from OB explants cultured next to COS cells secreting Slit-2 and Slit-1, are strongly repelled [19,22] (Nguyen-Ba-Chanet and Che´dotal, unpublished data). These data strongly suggest that Slits could represent the septal-derived chemorepulsive activity (Fig. 1C). However, some results have questioned the role of the septum in organizing LOT projections as in organotypic cultures mitral cell axons elongate along their normal pathway in the absence of septum [41] or when Slit signaling is blocked [8].
5. Slit2 proteolytic fragments have distinct axon guidance properties As mentioned above, Slit2 is cleaved in vivo and in vitro in two fragments. These 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 possibility was supported by several studies. First, the purification of Slit as a DRG elongation- and branch-promoting activity revealed that only the N-terminal fragment of Slit2 is capable of stimulating elongation and branching [45]. Second, the purification of a repellent activity for migrating SVZ olfactory precursor neurons and of a collapsing activity for retinal ganglion cell axons revealed that they correspond to the full-length fragment of hSlit2 [12,25]. Therefore, it was not clear, however, which fragment(s) were responsible for the other Slit associated activities, nor was it known whether proteolytic processing was required for Slit’s repulsive activity. These questions have been explored by engineering mutant and truncated versions of Slit2 and examining the activities of these mutants in binding and functional assays [4,23,24]. We generated cDNA constructs (Fig. 2A) encoding truncated proteins corresponding to just the N-terminal and the C-terminal cleavage fragments of human Slit2 (with one ending and the other starting at Thr 1118, the putative cleavage site, located at the start of EGF repeat 6; Fig. 2). We also engineered a cDNA construct encoding a presumptive uncleavable form of Slit2. For this, the DNA sequence encoding the nine amino acids preceding Thr 1118 (SPPMVLPRT) was removed from the human Slit2 cDNA. The binding properties of various Slit2 protein fragments with Robo receptors were tested in cell overlay assays. It was found that Slit2-N and Slit2-U both bind
to robo receptors with similar affinity, while Slit2-C does not bind [23]. Therefore, binding to Robo receptors is mediated by amino-terminal Slit2 sequences. This was confirmed by immunoprecipitation studies indicating that the leucine-rich repeats of Slit and the immunoglobulin domain of Robo are sufficient to mediate Slit–Robo interaction [4].
6. Functions and pharmacology of Slit2-N and Slit2-U in repulsion and branching We focused on OB and DRG neurons because they both express Robo2 but not Robo1 mRNAs [22,45] and because they showed dramatically different responses to Slit2, with olfactory axons being repelled [22] and DRG axons stimulated to elongate and branch [45]. Slit2-N and Slit2-U were found to have similar activities in repelling OB axons in the collagen gel repulsion assay (Fig. 2B) [23]. In contrast, the C-terminal portion of Slit2 (Slit2-C) had no repulsive activity. Other studies have shown that the leucine-rich repeats alone can induce repulsion of OB axons and migrating neurons [4]. None of the Slit2 fragment repels DRG axons [23,25], demonstrating that different Robo-expressing neurons exhibit distinct responses to Slit2 fragments. Slit2-U and Slit2-N were also found to have divergent actions in the sensory neuron branching assay. As observed for the native proteins [45], recombinant Slit2N but not Slit2-U stimulates elongation and branching of DRG axons in this assay. Furthermore, Slit2-U functions as an antagonist of Slit2-N, which is coherent with the observation, that native Slit2-N was active only when purified away from full-length Slit2. In addition to repelling olfactory bulb, hippocampal and retinal axons in the collagen gel assay, Slit-2 can also cause the collapse of their growth cones [22,25]. We showed that only Slit2-N, not Slit2-U, causes collapse of OB growth cones. DRG axons are never collapsed by any Slit2 fragment [22,25]. This is surprising, since it is generally expected that factors that can repel also cause collapse. This finding provides a clear demonstration of a dissociation between the two types of activity. This dissociation may be cell type specific, since native fulllength Slit2 has both activities for retinal axons in culture [25]. In conclusion, Slit2-N and Slit2-U, that both bind robo receptors can have different activity in the branching and collapse assays.
7. Substrate-bound Slit2 can guide sensory axons Because of the strong binding of Slit2 fragments to cell membranes, growth cones are very likely to be confronted with immobilized Slit2. We examined whether substrate-bound Slit2 is able to guide developing sensory
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axons in the so-called ‘‘stripe’’ assay [43,44] in which the axons grow parallel to alternating stripes of two different proteins or protein combinations, making it possible to test the axons’ preference for one over the other [24]. We first examined the responses of E15 rat DRG axons to Slit2 protein fragments in stripe assays using as a substrate, membranes from transfected COS cells. These experiments were done using NGF to support DRG cell survival and elicit axon outgrowth. When the axons
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were confronted with alternating lanes of membranes from control (mock-transfected) COS cells and COS cells expressing various Slit2 constructs such as the wildtype Slit2, Slit2-U or Slit2-N, the axons always avoided the Slit2 membranes [24]. In contrast, NT3-responsive axons are not sensitive to Slit2, at least in this assay, suggesting that the results seen with NGF-responsive axons is receptor-mediated and do not reflect simply a masking of positive factors in the COS cells membranes.
Fig. 2. (A) Schematic representation of the Slit2 constructs. Slit2-full represents the native full length Slit2. The nine EGF repeats and the ALPS domain are represented, and the nine amino acids sequence deleted to generate Slit2-U is indicated (arrowhead).B, In collagen gel assay, OB axons grow symmetrically when confronted with control cells or Slit2-C expressing cells. In contrast they are repelled by cells expressing Slit2-N or Slit2U.C-E, DRG axons show different preferences between the different Slit2 fragments and fibronectin or laminin in stripe assays. DRG explants were cultured on substrates patterned with alternating stripes of Slit2-U (U) or Slit2-N (N) and fibronectin (F) or laminin (L). Explants were immunolabeled for classIII-b-tubulin and the Slit2 stripes were labeled with fluorescein conjugated-BSA. (C) When DRG axons are presented a choice between Slit2-N and laminin, they constantly grow on laminin. When grown on stripe of Slit2-U and fibronectin (D), DRG axons show a clear preference for fibronectin. In contrast, when confronted with alternating stripe of Slit2-N and fibronectin (E), DRG axons grow on Slit2-N. Bar, 60 mm.
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When confronted with alternating lanes of membranes from COS cells expressing Slit2-N and those from cells expressing Slit2-U, the DRG axons invariably selected the membranes containing Slit2-N [24]. This result confirms that in some conditions, Slit2-U and Slit2-N do not have the same axon guidance properties.
8. ECM molecules influence sensory axon response to substrate-bound Slit2 fragments Given these results, we were curious to examine the response of DRG axons to purified Slit2 proteins in the stripe assay (Fig. 2C,D,E) [24]. Because ECM molecules, specially laminin-1 have been shown to influence the response of retinal axons to netrin-1, another chemotropic molecules [11], we compared the activity of Slit2 fragments in the presence of two different ECM molecules laminin or fibronectin which are both excellent substrates for DRG axons in culture. As expected, when Slit2-U or Slit2-N were mixed with laminin, DRG axons always preferred to grow on laminin alone than on any of the two mixtures, a result very similar to what was found using membrane extracts. When DRG axons were confronted with alternating stripes of fibronectin alone and fibronectin mixed with Slit2-U they also avoided Slit2-U. Surprisingly, they consistently preferred a mixture of fibronectin and Slit2-N to fibronectin alone. We also tested the behavior of rat E15 OB axons when confronted with substrate-bound Slit2-N in the stripe assay. In contrast to DRG axons, Slit2-N was always avoided by OB axons in the stripe assay, when given a choice with fibronectin or laminin. These observations are coherent with the results of the repulsion assay and confirm that different axons exhibit different responses to Slit2 fragments. What can explain the uniformity of response in the presence of laminin? Laminin-1 may trigger a second messenger pathway in the axons that alters their responses to the various proteins, such that all the responses become negative (repulsive). A precedent for this idea is provided by the finding that exposure of Xenopus retinal axons to laminin converts the responses of these axons to netrin-1 from attraction to repulsion, apparently by lowering cAMP levels in these growth cones [11]. The level of cyclic nucleotides in the growth cone in part determines the action of many guidance cues. When the level is above the critical range, the guidance cue causes attraction and conversely, when the level is below the critical range, the cue induces repulsion [39]. We tested this hypothesis on DRG axons. It was found that Slit2-N could be converted to a negative effect by lowering cGMP levels. Interestingly, as it is the case for netrin-1, a laminin-1 peptide was able to convert Slit2-N growth-promoting action into an inhibition [24]. This can probably explain the homogeneity of
axons response to Slit2 proteolytic fragments avoidance) in the presence of laminin. For netrin-1, laminin-1 effect is at least in part mediated by b-integrins, we still don’t know if this is the case for Slit2. These results underscore the importance of the molecular context in which the axons are confronted with Slit2 protein fragments on axonal responses to those fragments. This also supports the notion that ECM molecules, such as laminin and fibronectin can modulate growth cone response to a variety of chemotropic molecules.
9. Conclusion Although extensive progress has been made toward the understanding of Slit function in the developing CNS there are still many unanswered questions. For instance, could Slit2-C have a role in axon guidance? Although the sole purpose of the cleavage could be to generate bioactive Slit2-N, there are nonetheless reasons, based on its structure, for thinking that Slit2-C is also bioactive. However, it had been shown that in the brain, Slit-2 is a ligand for the glycosylphosphatidylinositol-anchored heparan sulfate proteoglycan glypican-1 [20]. More recently, it was determined that Slit2 binding to glypican-1 is mediated by the Cterminal portion of Slit2, and most likely by the ALPS domain [30]. In the rat embryo, glypican-1 is highly expressed in several types of neurons, including motor neurons [16] suggesting that Slit2-C could influence growing axons via this proteoglycan. It is still unknown whether Slit1 and Slit3 are cleaved and in the case the identity of the protease which cleaves Slit2 will have to be determined. It also remains to be proven whether in mammals, Slit functions, including its repulsive action on tangentially migrating interneurons, are mediated by Robos. Our results suggest that some difference exists in the receptor mechanisms mediating axonal repulsion and branching. Many answers should come from the analysis of Slit and Robo knock-outs.
Acknowledgements This work is supported by the Institut de la Sante´ et de le Recherche Me´dicale, the Ministe`re de la Recherche et de la Technologie (ACI) and the Association pour la Recherche sur le Cancer (No.5249). K N-B-C is supported by the Fondation de France.
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