Semaphorin Signaling
Authors: Hung-Hsiang Yu, Alex L Kolodkin
Journal: Neuron (1999)

Recent advances in our understanding of axon guidance during neural development are highlighted by the characterization of several gene families containing members capable of functioning as repellents. These include ephrins, netrins, and semaphorins. Though future work will undoubtedly result in the identification of still more repellents, the wealth of information now in hand concerning the cellular and molecular basis of repulsive guidance demands that we turn our attention toward how these repulsive cues actually direct discrete steering events. Pharmacological experiments are beginning to provide important insights into the basis of repulsive guidance signaling mechanisms in cell culture (13Song H.-j Ming G.-l He Z Lehmann M McKerracher L Tessier-Lavigne M Poo M.-m Science. 1998; 281: 1515-1518Crossref PubMed Scopus (712) Google Scholar). However, the characterization of endogenous receptors through which these cues transduce extracellular signals is paramount to addressing this issue in vivo. The knowledge that Eph receptor tyrosine kinases (RTKs) function as ephrin receptors and that UNC40 and UNC5 gene family members function as netrin receptors provides important clues for characterizing the intracellular signaling pathways activated by these guidance cues. The identification of semaphorin receptors, however, is only now beginning to point the way toward elucidating the signaling pathways used by semaphorins during neural development to direct extending growth cones away from inappropriate targets. The semaphorins are a large family of phylogenetically conserved secreted and transmembrane proteins (see Figure 1 for description of the semaphorin family), many members of which can mediate repulsive guidance events during neural development (9Mark M.D Lohrum M Püschel A.W Cell Tissue Res. 1997; 290: 299-306Crossref PubMed Scopus (86) Google Scholar). All semaphorins have a conserved ∼500 amino acid extracellular semaphorin (Sema) domain. To date within the semaphorin family, secreted and transmembrane semaphorins belonging to classes 1, 2, and 3 have been best characterized functionally at the cellular level. For example, extensive work from many groups demonstrates that semaphorin III/collapsin-1 (Sema III) in vertebrates can function as a potent chemorepellent both in vitro and in vivo and strongly suggests that other class 3 semaphorins are likely to function in a similar fashion. In Drosophila, the class 1 transmembrane semaphorin Sema 1a has also been shown to function as a repellent during neural development, demonstrating that both secreted and transmembrane semaphorins can act as repulsive guidance cues (19Yu H.H Araj H.H Ralls S.A Kolodkin A.L Neuron. 1998; 20: 207-220Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). In addition, both the class 1 semaphorin G/Sema I in grasshopper and the vertebrate class 3 semaphorins Sema III and Sema E can function as permissive or attractive axon guidance cues, showing that semaphorins are bifunctional (17Wong J.T Yu W.T O'Connor T.P Development. 1997; 124: 3597-3607PubMed Google Scholar, 1Bagnard D Lohrum M Püschel A Bolz J Development. 1998; 125: 5043-5053PubMed Google Scholar, 13Song H.-j Ming G.-l He Z Lehmann M McKerracher L Tessier-Lavigne M Poo M.-m Science. 1998; 281: 1515-1518Crossref PubMed Scopus (712) Google Scholar). Several class 3 semaphorins bind to and require neuropilins, a small family of transmembrane proteins (Figure 1), for transducing repulsive guidance signals (2Chen H.C He Z Tessier-Lavigne M Nat. Neurosci. 1998; 1: 436-438Crossref PubMed Scopus (48) Google Scholar). Neuropilins, however, have very short cytoplasmic domains that contain no obvious signaling motifs and that are dispensable for repulsive semaphorin guidance (10Nakamura F Tanaka M Takahashi T Kalb R.G Strittmatter S.M Neuron. 1998; 21: 1093-1100Abstract Full Text Full Text PDF PubMed Scopus (235) Google Scholar). This strongly suggests that neuropilins serve an essential role in assembling a receptor complex that includes an as yet unknown transmembrane signaling component. Indeed, neuropilin-1 also serves as a receptor component for an isoform of vascular endothelial growth factor (VEGF165; unrelated to semaphorins), augmenting a functional response to VEGF165 by the RTK KDR/Flk-1 (12Soker S Takashima S Miao H.-Q Neufeld G Klagsbrun M Cell. 1998; 92: 735-745Abstract Full Text Full Text PDF PubMed Scopus (1996) Google Scholar). At present, there are no clear predictions about the nature of the transmembrane signaling component that interacts with a class 3 semaphorin/neuropilin complex to initiate growth cone–steering responses; CRMP and Rac1, however, have been implicated as downstream cytosolic Sema III signaling components (5Goshima Y Nakamura F Strittmatter P Strittmatter S.M Nature. 1995; 376: 509-514Crossref PubMed Scopus (606) Google Scholar, 7Jin Z Strittmatter S.M J. Neurosci. 1997; 17: 6256-6263Crossref PubMed Google Scholar). Recently, a major advance in our understanding of semaphorin-mediated guidance events has been made with the observation that a family of transmembrane proteins distinct from neuropilins, the plexins, includes members capable of serving as semaphorin receptors both in the immune system and in the nervous system (3Comeau M.R Johnson R DuBose R.F Peterson M Gearing P VandenBos T Park L Farrah T Buller R.M Cohen J.I et al.Immunity. 1998; 8: 473-482Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar, 15Winberg M.L Noordermeer J.N Tamagnone L Comoglio P.M Spriggs M.K Tessier-Lavigne M Goodman C.S Cell. 1998; 95: 903-916Abstract Full Text Full Text PDF PubMed Scopus (367) Google Scholar). This work provides an exciting new line of inquiry directed toward linking extracellular semaphorin guidance cues with cytoskeletal alterations in the growth cone. Several secreted viral semaphorins have been identified that are composed almost entirely of a single Sema domain. To identify host immune system regulators, 3Comeau M.R Johnson R DuBose R.F Peterson M Gearing P VandenBos T Park L Farrah T Buller R.M Cohen J.I et al.Immunity. 1998; 8: 473-482Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar undertook a search for a receptor for the vaccinia virus A39R semaphorin (Figure 1), a secreted protein that is found in supernatants from virally infected, but not uninfected, cells. When added to human monocytes, A39R induces a robust response that includes cell aggregation, the induction of the proinflammatory cytokines IL-6 and IL-8, and the increased expression of the monocyte cell surface marker CD54 (intracellular adhesion molecule 1 [ICAM-1]). Using a chimeric A39RFc protein, the receptor for A39R was detected on the surface of a human B cell line and subsequently affinity purified. This receptor, called viral-encoded semaphorin protein receptor (VESPR), is expressed on a variety of hematopoietic cells. A39R binds to recombinant VESPR with high affinity (∼6 nM), accounting for the high-affinity binding of A39R to B cells. These results strongly suggest that inflammatory responses induced by poxvirus-infected hosts result from the action of viral semaphorins through VESPR. Further, since viral immune modulators often are closely related to mammalian genes involved in host immune system regulation, there are likely to be A39R-related mammalian proteins that also use VESPR as a receptor. Sema K1, a class 7 semaphorin that can bind to specific immune cell lines, is more closely related to semaphorins in the V class than to any other semaphorins, raising the possibility that it may be a ligand for VESPR (18Xu X Ng S Wu Z.-L Nguyen D Homburger S Seidel-Dugan C Ebens A Luo Y J. Biol. Chem. 1998; 273: 22428-22434Crossref PubMed Scopus (104) Google Scholar). Though the amino acid sequence of VESPR does not immediately suggest how it transduces the cellular effects of viral semaphorins, it does provide a clear link between the function of semaphorins in the immune system and in neural development. VESPR is a divergent member of the plexin family, whose members to date have been best characterized with respect to their expression and function in the developing vertebrate nervous system (4Fujisawa H Ohta K Kameyama T Murakami Y Dev. Neurosci. 1997; 19: 101-105Crossref PubMed Scopus (18) Google Scholar). Xenopus Plexin-1 (Plex-1) was the first member of the plexin family to be characterized. It was found in a screen for antigens whose expression pattern in the optic tectum suggested they might be involved in matching optic nerve fibers with their tectal targets. Subsequently, three mouse plexins (plexins 1, 2, and 3) were identified by homology with plexin-1 (4Fujisawa H Ohta K Kameyama T Murakami Y Dev. Neurosci. 1997; 19: 101-105Crossref PubMed Scopus (18) Google Scholar), and their human homologs (NOV, OCT, and SEX) were independently identified as MET-related proteins (see below; 8Maestrini E Tamagnone L Longati P Cremona O Gulisano M Bione S Tamanini F Neel B.G Toniolo D Comoglio P.M Proc. Natl. Acad. Sci. USA. 1996; 93: 674-678Crossref PubMed Scopus (145) Google Scholar). Given the observations that vertebrate plexins constitute a novel family of neuronal transmembrane proteins and that the divergent plexin VESPR is a receptor for viral semaphorins, 15Winberg M.L Noordermeer J.N Tamagnone L Comoglio P.M Spriggs M.K Tessier-Lavigne M Goodman C.S Cell. 1998; 95: 903-916Abstract Full Text Full Text PDF PubMed Scopus (367) Google Scholar sought to determine if plexins are functional semaphorin receptors in the nervous system. Two plexins were found in Drosophila, Plex A and Plex B. Both fly plexins are quite similar to vertebrate plexins with respect to the type, number, spacing, and identity at the amino acid level over their entire length (Figure 1). The plexin extracellular domains include three cysteine-rich repeats called Met-related sequences (MRS) and three motifs rich in glycine and proline residues (G-P repeats). These repeats are found in all vertebrate and invertebrate plexins. These repeats are also found, though in different numbers and with altered spacing, in the MET RTK and the MET-related RTKs SEA and RON (11Ohta K Mizutani A Kawakami A Murakami Y Kasuya Y Takagi S Tanaka H Fujisawa H Neuron. 1995; 14: 1189-1199Abstract Full Text PDF PubMed Scopus (117) Google Scholar, 8Maestrini E Tamagnone L Longati P Cremona O Gulisano M Bione S Tamanini F Neel B.G Toniolo D Comoglio P.M Proc. Natl. Acad. Sci. USA. 1996; 93: 674-678Crossref PubMed Scopus (145) Google Scholar). The intracellular portions of all plexin family members, including VESPR, are highly conserved and contain two large blocks of high homology that are separated by a variable linker region. Thus, though the degree of homology between the extracellular portion of VESPR and all other plexins is rather low, their cytoplasmic domains are substantially more closely related, pointing toward the likelihood that a conserved mode of intracellular signaling is used by all plexins. In addition to these conserved motifs, however, additional sequence analyses of plexins (3Comeau M.R Johnson R DuBose R.F Peterson M Gearing P VandenBos T Park L Farrah T Buller R.M Cohen J.I et al.Immunity. 1998; 8: 473-482Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar, 15Winberg M.L Noordermeer J.N Tamagnone L Comoglio P.M Spriggs M.K Tessier-Lavigne M Goodman C.S Cell. 1998; 95: 903-916Abstract Full Text Full Text PDF PubMed Scopus (367) Google Scholar) reveal a fascinating structural feature: the N-terminal portion of the plexin extracellular region is in fact a divergent Sema domain, sharing ∼17% amino acid identity with semaphorin Sema domains. Further, members of the MET subfamily of RTKs also contain similarly located and even more divergent Sema domains (∼10% identical to semaphorin Sema domains). Though divergent, these plexin and MET RTK Sema domains have most of the conserved cysteine residues and many short peptide sequences that define semaphorin Sema domains. Most of what is known about the function of vertebrate plexins in the nervous system comes from the analysis of Xenopus Plex-1 (4Fujisawa H Ohta K Kameyama T Murakami Y Dev. Neurosci. 1997; 19: 101-105Crossref PubMed Scopus (18) Google Scholar). In the visual system, Plex-1 is expressed in distinct plexiform layers of the optic tectum and in the inner plexiform layer (IPL) of the neural retina. Plex-1 distribution during neural development eventually becomes restricted to sublaminae of the IPL. IPL formation was severely impaired when embryonic neural retinas were cultured in the presence of anti-plexin-1 antibodies, demonstrating a role for Plex-1 in assembly of the IPL. In the developing olfactory system, Plex-1 is expressed in the neuropil of the accessory olfactory bulb, on axons of a subset of primary olfactory receptor neurons, and on axons of vomeronasal receptor neurons. Axons of Plex-1-expressing primary olfactory receptor neurons join the olfactory nerve and remain fasciculated until they segregate into their specific glomerular targets in the olfactory bulb. The vomeronasal axons are derived from neurons in the vomeronasal organ, extend in the vomeronasal nerve, and contact their targets in the accessory olfactory bulb. Plex-1 distribution in both the visual system and olfactory system suggested a role for this protein in fasciculation and target recognition events involving homophilic Plex-1 interactions. Plex-1 can in fact function as a divalent cation-dependent homophilic cell adhesion molecule in both cell aggregation and adhesion assays (4Fujisawa H Ohta K Kameyama T Murakami Y Dev. Neurosci. 1997; 19: 101-105Crossref PubMed Scopus (18) Google Scholar). It remains to be shown, however, that Plex-1 functions in vivo as an adhesion molecule. 3Comeau M.R Johnson R DuBose R.F Peterson M Gearing P VandenBos T Park L Farrah T Buller R.M Cohen J.I et al.Immunity. 1998; 8: 473-482Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar and 15Winberg M.L Noordermeer J.N Tamagnone L Comoglio P.M Spriggs M.K Tessier-Lavigne M Goodman C.S Cell. 1998; 95: 903-916Abstract Full Text Full Text PDF PubMed Scopus (367) Google Scholar provide compelling evidence for plexins serving as receptors for class V and I semaphorins, proteins whose extracellular domains contain Sema domains and no other obvious structural motifs. This raises the intriguing possibility that many additional layers of complexity may underlie homotypic and heterotypic interactions between various semaphorins. Both PlexA and PlexB are expressed primarily in the embryonic Drosophila CNS during the time when both central and peripheral pathfinding events are taking place. Though a precise assessment of the distribution of Plex A and B proteins awaits the generation of antibodies, the expression patterns of the plexin transcripts suggested that genetic analyses should yield information about the role played by plexins in both central and peripheral axon guidance events. PlexA loss-of-function mutations do indeed provide important clues about how Plex A mediates repulsive guidance signals, and additional genetic and biochemical experiments show that this involves direct interactions with the class 1 semaphorin Sema 1a. To establish mature axonal trajectories, axon fasciculation and defasiculation must be tightly regulated. This regulation results from a balance between attractive and repulsive guidance cues, many of which are located on axons themselves while others are distributed in the surrounding environment (14Tessier-Lavigne M Goodman C.S Science. 1996; 274: 1123-1133Crossref PubMed Scopus (2590) Google Scholar). The generation of neuromuscular connectivity in the Drosophila embryo provides one of the best examples of these forces at work, and it is here that Plex A can most easily be seen to function as a receptor for Sema 1a. During Drosophila neural development, motoneurons that innervate ventral muscles exit from the CNS in a large fascicle called the intersegmental nerve (ISN) (Figure 2A). These motoneurons defasciculate from the ISN and extend into the ventral muscle field in a single pathway called the ISNb. Subsequent defasiculation events result in individual motoneurons leaving the ISNb and elaborating synaptic arborizations on their target muscles. For example, the RP3 motoneuron axon defasciculates from the ISNb at muscles 6 and 7 and establishes contacts with these targets, while axons from other motoneurons extend further dorsally before leaving the ISNb and contacting their muscle targets. PlexA mutants show specific defasciculation defects that include a failure of RP3 to leave the ISNb and contact muscles 6 and 7. In addition, more dorsally extending motor axons fail to defasciculate and contact their targets, which results in stalling of these axons between muscles 6 and 13 (Figure 2B). Remarkably, these defasiculation defects (along with other defects in the PNS and CNS not discussed here) are identical to those seen in Sema1a mutants (19Yu H.H Araj H.H Ralls S.A Kolodkin A.L Neuron. 1998; 20: 207-220Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). This suggests, especially in light of the fact that Sema 1a is found on all motor axons, that Plex A and Sema 1a function in a common pathway required for axon defasiculation. To test this idea, embryos heterozygous for both PlexA and Sema1a were examined for dominant genetic interactions. While neither heterozygote alone showed defasiculation defects, removing one dose of both PlexA and Sema1a resulted in defects similar to either single mutant alone (Figure 2B). Additional support for a Plex A/Sema 1a interaction in repulsive motor axon guidance comes from experiments showing that removal of one copy of the PlexA gene can suppress the failure of RP3 innervation observed when Sema 1a is ectopically expressed on muscles 6 and 7 (Figure 2C and Figure 2D). Motor axon defasciculation defects in PlexA mutants can also be partially restored by the simultaneous removal of an attractive cue located on motor axons, the cell adhesion molecule fasciclin II (Figure 2A). Since fasciclin II has been shown to promote axon fasciculation, this provides further support for the essential role played by Plex A in repulsive guidance events leading to defasiculation of motor axons. Finally, overexpression of Plex A in neurons disrupts motor axon pathfinding, in some cases causing phenotypes opposite to those observed in PlexA mutants. The genetic interactions between PlexA and Sema1a suggest that Plex A functions downstream from Sema 1a as a receptor. This is supported by direct evidence of Plex A and Sema 1a binding interactions. A Sema 1a alkaline phosphatase fusion protein binds to membranes from Plex A expressing COS cells with a Kd of ∼2 nM. Similar to homophilic vertebrate Plex-1 interactions, this interaction requires divalent cations and further distinguishes plexin/class 1 semaphorin associations from those observed between neuropilins and class 3 semaphorins, which are independent of divalent cations. Plex A can also bind to a class 1 Drosophila semaphorin related to Sema 1a called Sema 1b, and Sema 1b is capable of functioning as a repellent for motor axons. In combination with the observation that there are some additional neuronal defects in PlexA mutants that are not observed in Sema1a mutants, this implies that Plex A may serve as a receptor for more than one semaphorin. Taken together, these biochemical and genetic data provide evidence that Plex A is a functional Sema 1a receptor and show that axonally distributed transmembrane semaphorins and their receptors play an essential role in mediating defasiculation events required for the generation of neural networks. The demonstration that plexins can function as receptors for class 1 and V semaphorins is a key finding on the road to understanding how the diverse semaphorin family of proteins affects a variety of neuronal and nonneuronal cell types during development. A rigorous assessment of the binding interactions among all known plexins and semaphorins will begin to address whether or not these recently demonstrated functional plexin/semaphorin relationships can be extended to interactions between plexins and other semaphorin classes. For example, it will be interesting to determine the function of class 6 Sema VIa in the developing olfactory system and whether this depends upon interactions with Plex-1. This seems likely for several reasons: (1) class 6 semaphorins are structurally most similar to invertebrate class 1 semaphorins; (2) Plex-1 in vertebrates has been shown to be selectively expressed in developing primary and accessory olfactory receptor neurons; (3) SemaVIa is also expressed in the developing olfactory system (20Zhou L White F.A Lentz S.I Wright D.E Fisher D.A Snider W.D Mol. Cell. Neurosci. 1997; 9: 26-41Crossref PubMed Scopus (43) Google Scholar); and (4) the fasciculation and defasciculation events involved in directing these neurons to their targets are reminiscent of those observed in the generation of motoneuron connectivity in Drosophila. PlexA mutants exhibit certain neuronal defects that are not found in Sema1a mutants; these defects, however, are characteristic of Sema2a mutant embryos, which lack the secreted class 2 semaphorin Sema 2a (16Winberg M.L Mitchell K.J Goodman C.S Cell. 1998; 93 (a): 581-591Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar). This suggests that Plex A may also serve as a receptor for secreted semaphorins. Since invertebrate class 3 semaphorins and neuropilins have not yet been found, it is possible that this particular secreted semaphorin/coreceptor relationship is restricted to chordates and that invertebrate and vertebrate plexins can associate with multiple semaphorin classes. Finally, it is possible that semaphorins act to antagonize plexin/plexin associations and that transmembrane semaphorins themselves may function as semaphorin receptors. This is suggested by the observation that plexins and semaphorins both contain sema domains, in combination with the fact that plexins are semaphorin receptors and that plexins can also function as homophilic cell adhesion molecules. The class 4 transmembrane semaphorin CD100 appears to have some immune system functions consistent with its functioning as a receptor (6Hall K.T Boumsell L Schultze J.L Boussiotis V.A Dorfman D.M Cardoso A.A Bensussan A Nadler L.M Freeman G.J Proc. Natl. Acad. Sci. USA. 1996; 93: 11780-11785Crossref PubMed Scopus (222) Google Scholar), and it remains to be determined whether this or other transmembrane semaphorins act similarly in neurons. Regardless of complexity in semaphorin/plexin interactions, however, it is clear that much will be learned about the mechanisms of repulsive guidance from analysis of how the conserved plexin cytoplasmic domains interact with cytosolic signal–transducing components following activation by semaphorin binding.