The development of precise connectivity patterns during the establishment of the nervous system depends on the regulated action of diverse, conserved families of guidance cues and their neuronal receptors. Determining how these signaling pathways function to regulate axon growth and guidance is fundamentally important to understanding wiring specificity in the nervous system and will undoubtedly shed light on many neural developmental disorders. Considerable progress has been made in defining the mechanisms that regulate the correct spatial and temporal distribution of guidance receptors and how these receptors in turn signal to the growth cone cytoskeleton to control steering decisions. This review focuses on recent advances in our understanding of the mechanisms mediating growth cone guidance with a particular emphasis on the control of guidance receptor regulation and signaling.

Determining how axon guidance receptors transmit signals to allow precise pathfinding decisions is fundamental to our understanding of nervous system development and may suggest new strategies to promote axon regeneration after injury or disease. Signaling mechanisms that act downstream of four prominent families of axon guidance cues-netrins, semaphorins, ephrins, and slits-have been extensively studied in both invertebrate and vertebrate model systems. Although details of these signaling mechanisms are still fragmentary and there appears to be considerable diversity in how different guidance receptors regulate the motility of the axonal growth cone, a number of common themes have emerged. Here, we review recent insights into how specific receptors for each of these guidance cues engage downstream regulators of the growth cone cytoskeleton to control axon guidance.G enerating precise patterns of connectivity depends on the regulated action of conserved families of guidance cues and their neuronal receptors. Activation of specific signaling pathways can promote attraction, repulsion, result in growth cone collapse, or affect the rate of axon extension through signaling events that act locally to modulate cytoskeletal dynamics in the growth cone. Here, we review recent insights into how specific guidance receptors from each of the four "classic" guidance pathways engage downstream regulators of the growth cone cytoskeleton with a particular emphasis on Rho family small GTPases. We begin with a consideration of how events at the growth cone plasma membrane, including endocytosis and proteolytic processing, influence guidance receptor activation and signaling and then discuss how bidirectional links between receptors and cytoplasmic signaling molecules control axon guidance responses. ENDOCYTOSISEndocytosis may be a necessary aspect of guidance receptor activation and signaling. In the case of membrane-associated ephrins, endocytosis of the ephrin-Eph complex is required for efficient cell detachment ( parallel to proteolytic cleavage, see the following). Vav family guanine nucleotide exchange factors (GEFs) have been implicated as regulators of Eph receptor

Protein trafficking requires coat complexes that couple recognition of sorting motifs in transmembrane cargos with biogenesis of transport carriers. The mechanisms of cargo transport through the endosomal network are poorly understood. Here, we identify a sorting motif for endosomal recycling of cargos including the cation-independent mannose-6-phosphate receptor and semaphorin 4C by the membrane tubulating BAR domain-containing sorting nexins SNX5 and SNX6. Crystal structures establish that this motif folds into a β-hairpin that binds a site in the SNX5/SNX6 phox homology domains. Over sixty cargos share this motif and require SNX5/ SNX6 for their recycling. These include cargos involved in neuronal migration and a Drosophila snx6 mutant displays defects in axonal guidance. These studies identify a sorting motif and provide molecular insight into an evolutionary conserved coat complex, the 'Endosomal SNX-Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:

Drosophila Roundabout (Robo) is the founding member of a conserved family of repulsive axon guidance receptors that respond to secreted Slit proteins. Here we present evidence that the SH3-SH2 adaptor protein Dreadlocks (Dock), the p21-activated serine-threonine kinase (Pak), and the Rac1/Rac2/Mtl small GTPases can function during Robo repulsion. Loss-of-function and genetic interaction experiments suggest that limiting the function of Dock, Pak, or Rac partially disrupts Robo repulsion. In addition, Dock can directly bind to Robo's cytoplasmic domain, and the association of Dock and Robo is enhanced by stimulation with Slit. Furthermore, Slit stimulation can recruit a complex of Dock and Pak to the Robo receptor and trigger an increase in Rac1 activity. These results provide a direct physical link between the Robo receptor and an important cytoskeletal regulatory protein complex and suggest that Rac can function in both attractive and repulsive axon guidance.

have undertaken a genetic and biochemical analysis of the Roundabout (Robo) repulsive axon guidance recep- † Samuel Lunenfeld Research Institute Mt. Sinai Hospital tor in Drosophila (Kidd et al., 1998a, 1998b). Drosophila Robo is the founding member of a con-Toronto, Ontario M5G 1X5 Canada served family of repulsive axon guidance receptors (Kidd et al., 1998a; Zallen et al., 1998) that respond to secreted Slit proteins (Brose et al., 1999; Kidd et al., 1999; Li et al., 1999). Robo is a member of the immuno-Summary globulin (Ig) superfamily and has an ectodomain with five Ig domains and three fibronectin (FN) type III repeats, a Drosophila Roundabout (Robo) is the founding memsingle transmembrane domain, and a long 457 amino ber of a conserved family of repulsive axon guidance acid cytoplasmic tail. The cytoplasmic domain does not receptors that respond to secreted Slit proteins. Little contain any obvious catalytic signaling motif, but it does is known about the signaling mechanisms which funchave proline-rich regions, potential phosphorylation tion downstream of Robo to mediate repulsion. Here, sites, and other short stretches of evolutionarily conwe present genetic and biochemical evidence that the served sequences (Kidd et al., 1998a). robo was identi-Abelson (Abl) tyrosine kinase and its substrate Enabled fied in a mutant screen for genes that control the deci-(Ena) play direct and opposing roles in Robo signal sion by axons to cross or not to cross the CNS midline transduction. Genetic interactions support a model (Seeger et al., 1993). In robo mutant embryos, too many in which Abl functions to antagonize Robo signaling, axons cross and recross the midline. while Ena is required in part for Robo's repulsive out-How does Robo transmit its repulsive signal in reput. Both Abl and Ena can directly bind to Robo's sponse to Slit? A comparison of the Drosophila and cytoplasmic domain. A mutant form of Robo that inter-Human Robo sequences revealed four short blocks of feres with Ena binding is partially impaired in Robo conserved cytoplasmic sequence, which were called function, while a mutation in a conserved cytoplasmic CC0, CC1, CC2, and CC3 (CC for conserved cytotyrosine that can be phosphorylated by Abl generates plasmic). CC2 (LPPPP) is a consensus binding site for a hyperactive Robo receptor. the Ena-VASP-homology (EVH1) domain of Drosophila Enabled (Ena). Ena was originally identified in a screen for suppressors of mutations in the abelson(abl) tyrosine