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Figure 5 a, Axon guidance cues. The four major families of axon guidance cues identified to date are: the netrins, Slits, semaphorins, and ephrins. These molecules interact with specific receptors on the surface of the growth cone to control axon pathfinding. Guidance responses can be modulated by other effectors within the growth cone. b, These guidance cues and their receptors have been highly conserved through evolution, and homologues of each are found in organisms as diverse as C. elegans, Drosophila, and vertebrates. EGF, epidermal growth factor repeat; Ig, immunoglobulin domain; FN, fibronectin type III domain; TK, tyrosine kinase domain; GPI, glycophosphatidylinositol linkage; Sema, Semaphorin domain.

Figure 5 a, Axon guidance cues. The four major families of axon guidance cues identified to date are: the netrins, Slits, semaphorins, and ephrins. These molecules interact with specific receptors on the surface of the growth cone to control axon pathfinding. Guidance responses can be modulated by other effectors within the growth cone. b, These guidance cues and their receptors have been highly conserved through evolution, and homologues of each are found in organisms as diverse as C. elegans, Drosophila, and vertebrates. EGF, epidermal growth factor repeat; Ig, immunoglobulin domain; FN, fibronectin type III domain; TK, tyrosine kinase domain; GPI, glycophosphatidylinositol linkage; Sema, Semaphorin domain.

Goodman, 1996). In vertebrates, two members were initially identified in chick (netrin-1 and netrin-2) and orthologues of netrin-1 have been reported in the mouse, human, frog, and zebra fish (reviewed in Meyerhardt et al., 1999).

The netrin-1 receptors were also initially characterized in C. elegans by studying worm mutants with axon guidance defects. These proteins, UNC-40 and UNC-5, are transmembrane proteins that mediate different responses to netrin-1 in vivo. UNC-40, whose vertebrate homologue is DCC (deleted in colorectal cancer), mediates attraction. In some populations of axons, netrin-1s can also act as a repellent. The UNC-5 receptor, either on its own or in conjunction with UNC-40, mediates these effects (Chan et al., 1996). This is important, as it shows that netrin-1s, though primarily attractants, can also be repulsive. Such bifunctionality is one way in which neural complexity is created during nervous system development.

1.12.3.2.2 Semaphorins The first axonal chemor-epellents to be thoroughly characterized were members of the Semaphorin (Sema) family. The identification of these proteins was the culmination of a study investigating a potent growth cone collapse-inducing molecule from the adult chick brain and another study in which the fascicle-specific expression of proteins was examined in the adult grasshopper CNS (Kolodkin et al., 1992; Luo et al., 1993). Many more Sema proteins have since been isolated, and members of this family are present in vertebrates, invertebrates, and even viruses. Although the Sema proteins form a large and heterogeneous group, they all share a common —420 amino acid region at their NH2-termini, called the Sema domain. Insects and nematodes share a small number of semaphorins that are divided in two subfamilies, transmembrane Semas (class 1) and secreted Semas (class 2). In contrast, vertebrates contain a large number of semaphorins that are divided into five subfamilies based on their common structure. An interesting point is that vertebrate semaphorins are not strict orthologues of invertebrate semaphorins, probably due to the existence of a duplication and a divergence of the semaphorin genes across evolution (reviewed in Chisholm and Tessier-Lavigne, 1999).

How does the growth cone respond to Sema proteins in the extracellular environment? Sema proteins bind to multimeric receptor complexes on the surface of the axonal growth cone. Although the exact makeup of these receptor complexes is in many cases unclear, they will often contain a transmembrane plexin protein. Another well-characterized component of the Sema receptor complex are the neuropilins, which act as receptors for class 3 Semas. Thus, Sema proteins in Drosophila and vertebrates generally act as repellents, with their spatiotemporal expression being used to channel axons through repulsive corridors or to prevent them from entering inappropriate areas, so enabling axons to navigate correctly within the developing CNS (see Section 1.12.4).

Although studies of netrin-1s have shown the remarkable molecular and functional conservation of guidance molecules in axon pathfinding, studies on the semaphorin receptors provide an example of a strong evolutionary divergence. Indeed, the neu-ropilin receptors are not conserved across species as they have not been found in C. elegans and Drosophila. It is thought that in invertebrates, instead of the neuropilin receptor, the plexin protein mediates the repulsive action of semaphorins. However, the reason why, during evolution, the plexin proteins present in invertebrates have been replaced by the neuropilin receptors functioning in vertebrates remains a mystery (reviewed in Chisholm and Tessier-Lavigne, 1999).

1.12.3.2.3 Slits and their receptors Another highly conserved family of guidance molecules are the Slits. In a screen for cuticular defects in Drosophila conducted over 20 years ago, one of the many mutations identified was dubbed Slit. In this mutation the longitudinal axon scaffold of the ventral midline collapsed into a single, fused tract, suggesting the absence of an unknown midline repellent in the mutant flies. This repellent was identified as the product of the Slit gene, a large, secreted protein with multiple protein-protein-binding motifs (Kidd et al., 1999). Slit has three vertebrate homologues, which also primarily act as repellents during nervous system development. The Slits signal via interactions with the Roundabout (Robo) family of transmembrane receptors (in Drosophila Robo mutants, axons recross the mid-line multiple times, forming loops, hence Roundabout), which are expressed on the surface of the axon and growth cone. Like netrin-1s, Slits have been implicated in guiding axons from many developing neuronal populations. However, two developmental systems in particular have provided insights into the mechanisms underlying Slit-induced repulsion: the Drosophila ventral midline and the vertebrate optic chiasm (see Section 1.12.4).

1.12.3.2.4 Ephrins and Eph receptors Eph receptors were first found in a screen for tyrosine kinases in a cultured cell line, as their name reflects (ery-thropoietin-producing hematoma cell line). The ligands for these receptors, named ephrins, were identified using the extracellular domain of the Eph receptor for affinity chromatography, screening expression libraries, and in a search for axon guidance cues in the tectum. Based on their amino acid sequence similarities, both Eph receptors and ephrins are grouped into two subclasses: A- and B-type. There are eight EphA receptors (EphA1-EphA8) and six EphB receptors (EphB1-EphB6). The ephrinAs (ephrinA1-ephrinA6) are glycosyl-phosphatidyl (GPI)-anchored and ephrinBs are transmembrane-anchored proteins. Eph receptors and ephrins have been found in several species. The highest diversity of expression is found in chick, mouse, and human, followed by Xenopus and zebra fish, although family members have also been identified in both flies and worms, indicating that the ephrins and their receptors are evolutiona-rily ancient. Indeed, the large diversity of vertebrate ephrins and their receptors and the high sequence homology of these proteins within vertebrate phyla suggest a major expansion of the Ephs and ephrins has occurred. This may have accompanied the evolution of vertebrates, which might have been critical in the construction of increasingly complex brains (reviewed in Flanagan and Vanderhaeghen, 1998; Wilkinson, 2000).

High-affinity binding usually occurs between ligands and receptors of the same subtype (ephrinA activating EphA, ephrinB activating EphB), although there is increasing evidence for a cross activation between subclasses (for example, EphA4 activated by ephrinB2, and recently it was found that ephrinA5 can activate EphB2; Himanen et al., 2004). Ligand binding results in a clustering of the receptors, autophosphorylation, and activation of the intracellular pathways, finally leading to re-arrangements of the cytoskeleton. Both ligands and receptors are enriched in lipid rafts providing a platform for clustering and signaling. One interesting aspect of the signaling is the finding that both A- and B-type ephrins and Ephs can act as receptors and ligands, a fact described as bidirectional signaling. This signaling can occur in the forward (ephrin) or reverse (Eph) direction.

The most prominent role of this family is its role in cell contact repulsion. Eph receptors and their ligands act as stop signals at boundaries to prevent overshooting specific targets, or to channel axon growth. They are also involved in the formation of topographic maps in the retinotectal projection and in other pathfinding events, such as in the vomer-onasal projection (critical for pheromone detection in rodents), in the hippocamposeptal projection (involved in learning and memory), and in the connection of motor neurons with their muscle targets.

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