Axon Growth and Guidance

Alain Chédotal, Chédotal, Alain

Slit was identified in Drosophila embryo as a gene involved in the patterning of larval cuticle. It was later shown that Slit is synthesized in the fly central nervous system by midline glia cells. Slit homologues have since been found in C. elegans and many vertebrate species, from amphibians, fishes, birds to mammals. A single slit was isolated in invertebrates, whereas there are three slit genes (slit1-slit3) in mammals, that have around 60% homology. All encodes large ECM glycoproteins of about 200 kDa (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, and Slit1. Moreover, two Slit1 isoforms exist in zebrafish as a consequence of gene duplication. Last, in mammals, two Slit2 isoforms can be purified from brain extracts, a long 200 kDa one and a shorter 150 kDa form (Slit2-N) that was shown to result from the proteolytic processing of full-length Slit2. Human Slit 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. 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. In addition, Slit1-N and full-length Slit1 can induce branching of cortical neurons (see below), but only full-length Slit1 repels cortical axons. Structure-function analysis in vertebrates and Drosophila demonstrated that the LRRs of Slits are required and sufficient to mediate their repulsive activities in neurons. More recent detailed structure function analysis of the LRR domains of Drosophila Slit, 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.However, a Slit1 spliced-variant that lacks the cysteine knot and does not dimerize is still able to repel OB axons.