Role of Slit proteins in the vertebrate brain

doi:10.1016/S0928-4257(01)00084-5

Journal of Physiology-Paris

Volume 96, Issues 1–2, January–March 2002, Pages 91-98

https://doi.org/10.1016/S0928-4257(01)00084-5 Get rights and content

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 rig-1) 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.

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.

Section snippets

Chemotropism in the developing olfactory system

The organization of axonal projections in the rodent olfactory system has been extensively characterized. 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

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 d-Slit 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

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

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

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

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 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

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

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

Acknowledgements

This work is supported by the Institut de la Santé et de le Recherche Médicale, the Ministère 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

Abstract

Introduction

Section snippets

Chemotropism in the developing olfactory system

Structure and function of Slit proteins

Robos are receptors for Slits

Slit2 proteolytic fragments have distinct axon guidance properties

Functions and pharmacology of Slit2-N and Slit2-U in repulsion and branching

Substrate-bound Slit2 can guide sensory axons

ECM molecules influence sensory axon response to substrate-bound Slit2 fragments

Conclusion

Acknowledgements

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