Silencing the siren: guidance cue hierarchies at the CNS midline.

This demonstration by Stein and Tessier-Lavigne (2001)xStein, E and Tessier-Lavigne, M. Science. 2001; 291: 1928–1938Crossref | PubMed | Scopus (410)See all ReferencesStein and Tessier-Lavigne (2001) of a molecular mechanism capable of imparting local and absolute changes in Netrin responsiveness at the midline is particularly satisfying. It explains how a long-range guidance cue such as Netrin can attract axons to a midline target but not distract these same axons as they continue on their way close to the floor plate, much like Odysseus' shipmates silencing the Sirens' alluring song, avoiding a lingering death on the isle of Anthemoessa, and continuing their odyssey. While continued integration of simultaneously presented attractants and repellents may play a role in certain types of axon guidance events where both classes of cues are presented relatively equally all along a developing axonal trajectory, it is appealing to invoke silencing as a general strategy for effecting the rapid and discrete steering events that define the establishment of many neuronal pathways. This study presents compelling biochemical evidence for an absolute hierarchy between these midline guidance cues since Slit-2, not Netrin, mediates the formation of the heteromultimeric Robo/DCC silencing complex, and this fits very well with the observed in vivo behavior of commissural axons at the CNS midline.Clearly, it is imperative to know whether Robo mediates Netrin silencing in vivo. Presumably, this will be addressed by a combination of loss-of-function studies in which silencing is assessed in the absence of these receptors on commissural axons, and in gain-of-function studies where altered DCC and Robo receptors able to mediate steering events in response to Netrin and Slit, but incapable of forming a silencing complex, are expressed on these same axons. It is also essential to address exactly how crossing axons modulate their responses to Slit, since crossing the midline requires that Slit initially not be perceived as a repellent. This strict regulation of Robo's function as a repulsive receptor is underscored by the observation that commissural axons are repelled by Slit-2 only after they have crossed the floor plate (Figures 1D and 1E(Figures 1D and 1E). Regulation of Robo protein levels in the developing vertebrate spinal cord, as has been observed in Drosophila, is an obvious possibility which will be addressed following the generation of appropriate antibody reagents. However, the observation that stage 22–derived Xenopus neurons express functional Robo but are not repelled by Slit, along with the failure at present to identify vertebrate commissureless proteins, suggests alternative possibilities for regulating Slit responsiveness that include developmentally regulated expression of additional signaling components required for Robo repulsion, Robo modifications including phosphorylation and dephosphorylation events, or even silencing of Robo itself.It is likely silencing is applicable to other classes of guidance receptors. For example, vertebrate commissural axons also acquire responsiveness to the class 3 semaphorin chemorepellents Sema3B and Sema3F only after they cross the floor plate (Zou et al., 2000xZou, Y, Stoeckli, E, Chen, H, and Tessier-Lavigne, M. Cell. 2000; 102: 363–375Abstract | Full Text | Full Text PDF | PubMedSee all References(Zou et al., 2000). However, they express neuropilin-2, the obligate coreceptor for these semaphorins, along their entire trajectory and therefore encounter these repellents but do not respond to them prior to crossing the floor plate. It will be interesting to see if class 3 semaphorin receptor complexes, which include neuropilin and plexin proteins, are silenced by other guidance receptors on the ipsilateral portion of commissural axons, and whether this regulation involves active ligand-gated silencing similar to that observed for Robo and DCC, or different interactions that serve to negatively regulate semaphorin receptors in precise regions of axon trajectories. Recent work on secreted semaphorin signaling through neuropilin/plexin complexes supports the notion that plexin extracellular binding interactions have a profound effect on regulating repulsive signaling by the plexin intracellular domain (Takahashi and Strittmatter, 2001xTakahashi, T and Strittmatter, S.M. Neuron. 2001; 29: 429–439Abstract | Full Text | Full Text PDF | PubMed | Scopus (139)See all References(Takahashi and Strittmatter, 2001).An additional principle that emerges from this work and from a companion study from Tessier-Lavigne and colleagues (Stein et al., 2001xStein, E, Zou, Y, Poo, M, and Tessier-Lavigne, M. Science. 2001; 291: 1976–1982Crossref | PubMed | Scopus (166)See all References(Stein et al., 2001), is that ligand-gated homo- and heteromultimeric guidance receptor complex formation involves promotion of associations between cytoplasmic domains of these receptors through discrete cytoplasmic domains. Netrin-mediated attraction requires receptor multimerization via the P3 DCC cytoplasmic domain, Slit silencing of Netrin requires the Robo CC1 and the DCC P3 domains for the formation of a silencing complex, and Netrin-mediated repulsion requires the DB cytoplasmic domain of UNC5 and the DCC P1 domain to form the UNC5/DCC repulsive Netrin receptor complex (Figure 1, Figure 2). Analysis of Drosophila Robo signaling shows cytoplasmic domain–specific interactions with the Abl tyrosine kinase and Ena (Bashaw et al., 2000xBashaw, G.J, Kidd, T, Murray, D, Pawson, T, and Goodman, C.S. Cell. 2000; 101: 703–715Abstract | Full Text | Full Text PDF | PubMedSee all References(Bashaw et al., 2000), proteins intimately involved in neuronal growth cone cytoskeletal reorganization. And finally, recent studies from the Goodman and Dickson laboratories establish that multiple Robo proteins in Drosophila serve unique roles in Slit-mediated midline crossing and in repulsive responses of axons following midline crossing to a Slit gradient (reviewed in Rusch and Van Vactor, 2000xRusch, J and Van Vactor, D. Neuron. 2000; 28: 637–640Abstract | Full Text | Full Text PDF | PubMed | Scopus (17)See all ReferencesRusch and Van Vactor, 2000). Taken together with the observations in Stein and Tessier-Lavigne (2001)xStein, E and Tessier-Lavigne, M. Science. 2001; 291: 1928–1938Crossref | PubMed | Scopus (410)See all ReferencesStein and Tessier-Lavigne (2001) that Slit silences only Netrin-mediated attraction and not outgrowth promotion, and that Slit-mediated repulsion is separable from silencing, it is clear that now the potential exists to dissect the signaling pathways that underlie growth cone behaviors that heretofore have been lumped together for lack of experimental paridigms capable of teasing them apart. Though rapid, modular, and hierarchical interactions among different guidance receptors greatly increase the complexity of understanding at the molecular level how growth cones navigate to their targets, we now have a more complete picture at the CNS midline of how a limited number of cues and receptors interact to insure that echoes of previously encountered guidance cues do not adversely affect future directions.