Semaphorin-2a and Semaphorin-2b


EVOLUTIONARY HOMOLOGS (part 3/3)

Neuropilins and other receptors for Semaphorins

Extending axons in the developing nervous system are guided to their targets through the coordinate actions of attractive and repulsive guidance cues. The semaphorin family of guidance cues comprises several members that can function as diffusible axonal chemorepellents. To begin to elucidate the mechanisms that mediate the repulsive actions of Collapsin-1/Semaphorin III/D (Sema III), a search was carried out for Sema III-binding proteins of embryonic rat sensory neurons by expression cloning. Sema III binds with high affinity to the transmembrane protein neuropilin; antibodies to neuropilin block the ability of Sema III to repel sensory axons and to induce collapse of their growth cones. Both the C domain and the semaphorin domain of Sema II can independently bind neuropilin. Neuropilin is a axonal protein present in the developing Xenopus nervous system. Neuropilin comprises in its extracellular domain two domains with similarity to the C1 and C2 domains of coagulation factors V and VIII, a MAM domain, and two CUB motifs (a CUB domain in the metalloproteinase Tolloid, a relative of BMP-1, is suggested to mediate an interaction with the BMP family member Decapentaplegic). These results provide evidence that neuropilin is a receptor or a component of a receptor complex that mediates the effects of Sema III on these axons (He, 1997).

Semaphorin III (Sema III) is a secreted protein that causes in vitro neuronal growth cone collapse and chemorepulsion of neurites, and in vivo is required for correct sensory afferent innervation and other aspects of development. The mechanism of Sema III function, however, is unknown. Neuropilin, a type I transmembrane protein implicated in aspects of neurodevelopment, is a Sema III receptor. Neuropilin-2, a related neuropilin family member is described in this study. Both neuropilin and neuropilin-2 are expressed in overlapping, yet distinct, populations of neurons in the rat embryonic nervous system (Kolodkin, 1997).

Neuropilin is a neuronal cell surface protein that has been shown to function as a receptor for a secreted protein, semaphorin III/D, which can induce neuronal growth cone collapse and repulsion of neurites in vitro. Neuropilin is a type I membrane protein that is highly conserved among vertebrates, can mediate cell adhesion by a heterophilic molecular interaction, and can promote neurite outgrowth in vitro. The roles of neuropilin in vivo, however, are unknown. Neuropilin-deficient mutant mice produced by targeted disruption of the neuropilin gene show severe abnormalities in the trajectory of efferent fibers of the PNS. The trajectory of each cranial nerve is severely disorganized in the neuropilin mutant embryos. The ophthalmic nerve is defasciculated, and overshoots far beyond the growing front of the normal nerve. The distal parts of the maxillary and mandibular nerves are also difasciculated in mutants and spread into almost all parts o the maxillae and mandibula, respectively. The distal parts of the facial nerve, glossopharyngeal and vagus nerves also expand beyond their normal extentions. Spinal nerve fibers at the trunk level show abnormal trajectory and projection in Neuropilin mutants, and limb innervation by the fourth to eighth cervical spinal nerves is abnormal. Neuropilin-deprived dorsal root ganglion neurons are protected from growth cone collapse elicited by semaphorin III/D. These results indicate that neuropilin-semaphorin III/D-mediated chemorepulsive signals play a major role in guidance of PNS efferents (Kitsukawa, 1997).

Neuropilin 1 (NP-1) has been identified as a necessary component of a semaphorin D (SemD) receptor that repulses dorsal root ganglion (DRG) axons during development. SemA and SemE are related to SemD and bind to NP-1, but do not repulse DRG axons. By expressing NP-1 in retinal neurons and NP-2 in DRG neurons, it is demonstrated that neuropilins are sufficient to determine the functional specificity of semaphorin reponsiveness. SemA and SemE block SemD binding to NP-1 and abolish SemD repulsion in axons expressing NP-1. SemA and SemE seem to have a newly discovered protein antagonist capacity to NP-1 receptors, whereas they act as agonists at receptors containing NP-2 (Takahashi, 1998).

The collapsin and semaphorin family of extracellular proteins contributes to axonal path finding by repulsing axons and collapsing growth cones. To explore the mechanism of collapsin-1 action, a truncated collapsin-1-alkaline phosphatase fusion protein (CAP-4) was expressed. This protein retains biological activity as a DRG growth cone collapsing agent and saturably binds to DRG neurons with low nanomolar affinity. Specific CAP-4 binding sites are present on DRG neurons, sympathetic neurons, and motoneurons, but not on retinal, cortical, or brainstem neurons. Outside the nervous system, high levels of CAP-4 binding sites are present in the mesenchyme surrounding major blood vessels and developing bone and in lung. These sites provide a substrate for the collapsin-1-dependent patterning of non-neuronal tissues perturbed in sema III (-/-) mice. The staining patterns for mouse semaphorin D/III and chick collapsin-1 fusion proteins are indistinguishable from one another but quite separate from those for semaphorin B and M-semaphorin F fusion proteins. These data imply that there exists a family of high-affinity semaphorin binding sites similar in complexity to the semaphorin ligand family (Takahashi, 1997).

Neuropilin (neuropilin-1) was recently identified as a receptor for Collapsin-1/Semaphorin III/D (Sema III). A related protein has been identified, neuropilin-2, whose mRNA is expressed by developing neurons in a pattern largely, though not completely, nonoverlapping with that of neuropilin-1. Unlike neuropilin-1, which binds with high affinity to the three structurally related semaphorins Sema III, Sema E, and Sema IV, neuropilin-2 shows high affinity binding only to Sema E and Sema IV, not Sema III. These results identify neuropilins as a family of receptors (or components of receptors) for at least one semaphorin subfamily. They also suggest that the specificity of action of different members of this subfamily may be determined by the complement of neuropilins expressed by responsive cells (Chen, 1997).

Neuropilin-1 and neuropilin-2 show specificity in binding to different class III semaphorins, including Sema III, Sema E, and Sema IV, suggesting that the specificity of action of these semaphorins is dictated by the complement of neuropilins expressed by responsive neurons. In support of this, sympathetic axons have been shown to coexpress neuropilin-1 and -2; their responses to Sema III, Sema E, and Sema IV are affected in predicted ways by antibodies to neuropilin-1, and neuropilin-1 and -2 can form homo- and hetero-oligomers through an interaction involving (at least partly) the neuropilin MAM (meprin, A5, mu) domain. These results support the idea that in sympathetic axons, the Sema III signal is mediated predominantly by neuropilin-1 oligomers; the Sema IV signal by neuropilin-2 oligomers, and the Sema E signal by neuropilin-1 and -2, either as homo- or hetero-oligomers (Chen, 1998).

Collapsin-1, a member of the semaphorin family, activates receptors on specific growth cones, thereby inhibiting their motility. Neuropilin, a previously cloned transmembrane protein, has recently been identified as a candidate receptor for collapsin-1. The cloning of chick collapsin-3 and -5 has been completed, and collapsin-1, -2, -3, and -5 are known to bind to overlapping but distinct axon tracts. In situ, there are inferred to be distinct receptors with different affinities for collapsin-1, -2, -3, and -5. In contrast, these four collapsins all bind recombinant neuropilin with similar affinities. Strong binding to neuropilin is mediated by the carboxy third of the collapsins, while the semaphorin domain confers their unique binding patterns in situ. It is proposed that neuropilin is a common component of a semaphorin receptor complex, and that additional differentially expressed receptor components interact with the semaphorin domains to confer binding specificity (Feiner, 1997).

To explore a role for chemorepulsive axon guidance mechanisms in the regeneration of primary olfactory axons, the expression of the chemorepellent semaphorin III (sema III), its receptor neuropilin-1, and collapsin response mediator protein-2 (CRMP-2) were examined during regeneration of the olfactory system. In the intact olfactory system, neuropilin-1 and CRMP-2 mRNA expression define a distinct population of olfactory receptor neurons, corresponding to immature (B-50/GAP-43-positive) neurons, and a subset of mature (olfactory marker protein-positive) neurons, located in the lower half of the olfactory epithelium. Sema III mRNA is expressed in pial sheet cells and in second-order olfactory neurons that are the target cells of neuropilin-1-positive primary olfactory axons. These data suggest that in the intact olfactory bulb sema III creates a molecular barrier, which helps restrict ingrowing olfactory axons to the nerve and glomerular layers of the bulb. Both axotomy of the primary olfactory nerve and bulbectomy induce the formation of new olfactory receptor neurons expressing neuropilin-1 and CRMP-2 mRNA. After axotomy, sema III mRNA is transiently induced in cells at the site of the lesion. These cells align regenerating bundles of olfactory axons. In contrast to the transient appearance of sema III-positive cells at the lesion site after axotomy, sema III-positive cells increase progressively after bulbectomy, apparently preventing regenerating neuropilin-1-positive nerve bundles from growing deeper into the lesion area. The presence of sema III in scar tissue and the concomitant expression of its receptor neuropilin-1 on regenerating olfactory axons suggests that semaphorin-mediated chemorepulsive signal transduction may contribute to the regenerative failure of these axons after bulbectomy (Pasterkamp, 1998).

The semaphorins are the largest family of repulsive axon guidance molecules. Secreted semaphorins bind neuropilin receptors and repel sensory, sympathetic and motor axons. CA1, CA3 and dentate gyrus axons from E15-E17 mouse embryo explants are selectively repelled by entorhinal cortex and neocortex. The secreted semaphorins Sema III and Sema IV and their receptors Neuropilin-1 and -2 are expressed in the hippocampal formation during appropriate stages. Sema III and Sema IV strongly repel CA1, CA3 and dentate gyrus axons; entorhinal axons are only repelled by Sema III. An antibody against Neuropilin-1 blocks the repulsive action of Sema III and the entorhinal cortex, but has no effect on Sema IV-induced repulsion. Thus, chemorepulsion plays a role in axon guidance in the hippocampus, secreted semaphorins are likely to be responsible for this action, and the same axons can be repelled by two distinct semaphorins via two different receptors (Chedotal, 1998).

Somatosensory axon outgrowth is repulsed when soluble semaphorin D (semD) binds to growth cone neuropilin-1 (Npn-1). SemD ligand binding studies of Npn-1 mutants demonstrate that the sema domain binds to the amino-terminal quarter, or complement-binding (CUB) domain, of Npn-1. By herpes simplex virus- (HSV-) mediated expression of Npn-1 mutants in chick retinal ganglion cells, it has been shown that semD-induced growth cone collapse requires two segments of the ectodomain of Npn-1: the CUB domain and the juxtamembrane portion, or MAM (meprin, A5, mu) domain. In contrast, the transmembrane segment and cytoplasmic tail of Npn-1 are not required for biologic activity. These data imply that the CUB and MAM ectodomains of Npn-1 interact with another transmembrane growth cone protein, which in turn transduces a semD signal into axon repulsion (Nakamura, 1998).

Neuropilins bind secreted members of the semaphorin family of proteins. Neuropilin-1 is a receptor for Sema III. Neuropilin-2 is a receptor for the secreted semaphorin Sema IV and acts selectively to mediate repulsive guidance events in discrete populations of neurons. neuropilin-2 and semaIV are expressed in strikingly complementary patterns during neurodevelopment. The extracellular complement-binding (CUB) and coagulation factor domains of neuropilin-2 confer specificity to the Sema IV repulsive response, and these domains of neuropilin-1 are necessary and sufficient for binding of the Sema III semaphorin (sema) domain. The coagulation factor domains alone are necessary and sufficient for binding of the Sema III immunoglobulin- (Ig-) basic domain and the unrelated ligand, vascular endothelial growth factor (VEGF). Lastly, neuropilin-1 can homomultimerize and form heteromultimers with neuropilin-2. These results provide insight into how interactions between neuropilins and secreted semaphorins function to coordinate repulsive axon guidance during neurodevelopment (Giger, 1998b).

The olfactory system provides an example of the complex expression pattern of Sema IV and Neuropilin-2. In the developing olfactory system, SemaIV is expressed in regions apical to the VNR neurons of the vomeronasal organ (VNO). Equally strong expression of neuropilin-2 is observed in the V2R neurons of the basal VNO. The V2R neurons initially project basally into the accessory olfactory nerve, away from the region of SemaIV expression in the VNO, commensurate with the idea that Sema IV serves to direct the initial projections of these neurons. The accessory olfactory bulb (OB), unlike the main OB, does not express high levels of SemaIV, and this lack of semaIV expression may serve to help segregate VNO projections to the accessory OB from olfactory epithelium (OE) projections to the main olfactory bulb. Neuropilin-2 is strongly expressed by both VNO neurons and their target region in the accessory OB. It is possible that homophilic Neuropilin-2 interactions may serve to help establish appropriate connectivity between the VNO and the accessory olfactory bulb and may even function to regulate fasciculation of accessory olfactory neurons. SemaIV is expressed in the main OE, with stronger expression observed in the basal OE, and since subsets of main olfactory receptor neurons (ORNs) also express Neuropilin-2, Sema IV may play a role as well in directing the initial outgrowth of ORNs to the olfactory nerve. In addition, SemaIV is expressed in the main OB in the periglomerular, mitral, and tufted cells and could prevent the main ORNs from overshooting their glomerular targets in the main OB (Giger, 1998b).

Neuropilin-1 (Npn-1), a receptor for semaphorin III, mediates the guidance of growth cones on extending neurites. The molecular mechanism of Npn-1 signaling remains unclear. A yeast two-hybrid system has been used to isolate a protein that interacts with the cytoplasmic domain of Npn-1. This Npn-1-interacting protein (NIP) contains a central PSD-95/Dlg/ZO-1 (PDZ) domain and a C-terminal acyl carrier protein domain. The physiological interaction of Npn-1 and NIP is supported by co-immunoprecipitation of these two proteins in extracts from a heterologous expression system and from a native tissue. The C-terminal three amino acids of Npn-1 (S-E-A-COOH), which is conserved from Xenopus to human, is responsible for interaction with the PDZ domain-containing C-terminal two-thirds of NIP. NIP as well as Npn-1 are broadly expressed in mice as assayed by Northern and Western analysis. Immunohistochemistry and in situ hybridization experiments reveal that NIP expression overlaps that of Npn-1. NIP has been independently cloned as RGS-GAIP-interacting protein (GIPC): it was identified by virtue of its interaction with the C terminus of RGS-GAIP and has been suggested to participate in clathrin-coated vesicular trafficking. It is suggested that NIP and GIPC may participate in regulation of Npn-1-mediated signaling as a molecular adapter that couples Npn-1 to membrane trafficking machinery in the dynamic axon growth cone (Cai, 1999).

Neuropilin-1 is a membrane protein that is expressed in developing neurons and functions as a receptor or a component of the receptor complex for the class 3 semaphorins, which are inhibitory axon guidance signals. Targeted inactivation of the neuropilin-1 gene in mice induces disorganization of the pathway and projection of nerve fibers, suggesting that neuropilin-1 mediates semaphorin-elicited signals and regulates nerve fiber guidance in embryogenesis. Neuropilin-1 is also expressed in endothelial cells and binds vascular endothelial growth factor (VEGF), a potent regulator for vasculogenesis and angiogenesis. However, the roles of neuropilin-1 in vascular formation have been unclear. neuropilin-1 mutant mouse embryos exhibit various types of vascular defects, including impairment in neural vascularization, agenesis and transposition of great vessels, insufficient aorticoplumonary truncus (persistent truncus arteriosus), and disorganized and insufficient development of vascular networks in the yolk sac. The vascular defects induced by neuropilin-1 deficiency in mouse embryos suggest that neuropilin-1 plays roles in embryonic vessel formation, as well as nerve fiber guidance (Kawasaki, 1999).

Neuropilin 1 is the specific receptor for Sema3A and plays a role in nerve fiber guidance. Neuropilin 1 and Sema3A mutant mouse embryos, generated by targeted gene disruption, show displacement of sympathetic neurons and their precursors and abnormal morphogenesis in the sympathetic trunk. Sema3A suppresses the cell migration activity of sympathetic neurons from wild-type but not neuropilin 1 mutant embryos in vitro and promotes sympathetic neuron accumulation into compact cell masses and fasciculation of their neurites. The cell aggregation activity of Sema3A may play a role in the aggregation of differentiated sympathetic neurons into ganglia and probably the aggregation of sympathetic neuron precursors into compact cell masses at the dorsal aorta. In the Sema3A and neuropilin 1 mutant embryos, sympathetic neuron precursors may fail to be aggregated but disperse widely and give rise to sympathetic neurons at ectopic positions. The differentiated sympathetic neurons may migrate further or be pulled by growing spinal nerve fibers into places far from the original target sites, such as the upper arm and the abdominal wall. These findings suggest that the neuropilin 1-mediated Sema3A signals regulate arrest and aggregation of sympathetic neuron precursors and sympathetic neurons themselves at defined target sites and axon fasciculation to produce the stereotyped sympathetic nerve pattern (Kawasaki, 2002).

Neuropilins are receptors for class 3 secreted semaphorins, most of which can function as potent repulsive axon guidance cues. Mice have been generated with a targeted deletion in the neuropilin-2 (Npn-2) locus. Many Npn-2 mutant mice are viable into adulthood, allowing for an assessment of the role of Npn-2 in axon guidance events throughout neural development. Npn-2 is required for the organization and fasciculation of several cranial nerves and spinal nerves. In addition, several major fiber tracts in the brains of adult mutant mice are either severely disorganized or missing. These results show that Npn-2 is a selective receptor for class 3 semaphorins in vivo and that Npn-1 and Npn-2 are required for development of an overlapping but distinct set of CNS and PNS projections (Giger, 2000).

A striking finding in the present study is that many Npn-2 mutant mice survive into adulthood despite the existence of numerous neurological deficits. Also noteworthy is the observation that some of the neural defects observed in Npn-2 mutant mice are complementary to those observed previously for Npn-1 mutant mice. This point is well illustrated by comparing cranial nerve defects in the two neuropilin mutants. In mice lacking Npn-1, many cranial nerves are disorganized and/or defasciculated, including all branches of the trigeminal nerve, the facial nerve, the glossopharyngeal nerve, and the vagus nerve. Importantly, in Npn-1 mutant animals the oculomotor and trochlear nerves appear normal. In dramatic contrast, Npn-2 mutants show severe defasciculation of the oculomotor nerve and absence of the peripheral projection of the trochlear nerve. Interestingly, in vitro coculture experiments have shown that oculomotor motor axons are not repelled by Sema3A, though in this same study trochlear motor axons were observed to be repelled by Sema3A. Sema3A or Sema3C repulsion of trochlear neurons was not observed, however, a clear repulsive response by these neurons to Sema3F was observed. Though the oculomotor nucleus and trochlear nucleus do not initially express Npn-1 at high levels, in the present study in the rat, Npn-2 expression was observed in these cranial nuclei at E13. The dramatic trochlear nerve phenotype in Npn-2 mutant embryos, coupled with embryonic Sema3F expression in the CNS and in the periphery, suggest an important role for Sema3F and Npn-2 in establishing this trajectory. Sema3F in the CNS is likely to provide a strong repulsive cue for trochlear neurons in their initial dorsal trajectory. Together with previous work showing that netrin-1 can repel trochlear neurons in vitro, this suggests that semaphorins and netrins work in concert to guide trochlear motor neurons within the CNS. Following their dorsal decussation and exit from the CNS at the hindbrain-midbrain junction, peripheral Sema3F is likely to channel trochlear motor neurons, promoting their fasciculation and defining their peripheral trajectory. Interestingly, a dramatic misalignment of the pupils in eyes of some Npn-2 mutant adult mice has been observed, suggesting that defects in eye muscle innervation resulting from aberrant Sema3F-Npn-2 signaling may underlie certain types of strabismus (Giger, 2000).

Neuropilin-1 and neuropilin-2 bind differentially to different class 3 semaphorins and are thought to provide the ligand-binding moieties in receptor complexes mediating repulsive responses to these semaphorins. The function of neuropilin-2 has been studied through analysis of a neuropilin-2 mutant mouse, which is viable and fertile. Repulsive responses of sympathetic and hippocampal neurons to Sema3F but not to Sema3A are abolished in the mutant. Marked defects are observed in the development of several cranial nerves, in the initial central projections of spinal sensory axons, and in the anterior commissure, habenulo-interpeduncular tract, and the projections of hippocampal mossy fiber axons in the infrapyramidal bundle. These results show that neuropilin-2 is an essential component of the Sema3F receptor and identify key roles for neuropilin-2 in axon guidance in the PNS and CNS (Chen, 1999).

Primary sensory neurons are located in the olfactory epithelium that is derived from the olfactory placode. The olfactory placode invaginates from the surface of the chick embryo to form the nasal pit beginning at stage 18. The first olfactory axons begin to grow out of the olfactory epithelium and into the adjacent mesenchyme by late stage 19. These axons have reached the surface of the telencephalon by E5. The vast majority of olfactory sensory axons do not enter the CNS at this time but, instead, halt at the outside surface of the telencephalon where the olfactory bulb will form. A small number of axons do penetrate the telencephalon transiently, accompanied by cells that originate in the olfactory epithelium and migrate along the olfactory nerve. Olfactory axons continue to project from the olfactory epithelium and accumulate on the surface of the telencephalon. The bulb forms beneath them over the next several days in chicks and in other species. Olfactory axons cover the surface of the nascent olfactory bulb to form the olfactory nerve fiber layer (ONL) by E9. They then leave the ONL to make connections in deeper layers of the bulb. Thus, during a waiting period of several days, olfactory axons arrive and accumulate outside the CNS while the bulb differentiates beneath them. Semaphorin-3A is expressed in the telencephalon during this period and has been proposed to prevent their entry into the CNS. The misexpression of a dominant-negative neuropilin-1 that blocks SEMA-3A-mediated signaling in olfactory sensory axons induces many of them to enter the telencephalon prematurely and to overshoot the olfactory bulb. These results suggest that chemorepellents can prevent the premature innervation of immature targets (Renzi, 2000).

Neuropilin 2 is a receptor for class III semaphorins and for certain members of the vascular endothelial growth factor family. Targeted inactivation of the neuropilin 2 gene (Nrp2) has shown its role in neural development. Neuropilin 2 expression in the vascular system is restricted to veins and lymphatic vessels. Homozygous Nrp2 mutants show absence or severe reduction of small lymphatic vessels and capillaries during development. This correlates with a reduction of DNA synthesis in the lymphatic endothelial cells of the mutants. Arteries, veins and larger, collecting lymphatic vessels developed normally, suggesting that neuropilin 2 is selectively required for the formation of small lymphatic vessels and capillaries (Yuan, 2002).

Mutations in the L1 gene induce a spectrum of human neurological disorders due to abnormal development of several brain structures and fiber tracts. Among its binding partners, L1 immunoglobulin superfamily adhesion molecule (Ig CAM) associates with neuropilin-1 (NP-1) to form a semaphorin3A (Sema3A) receptor and soluble L1 converts Sema3A-induced axonal repulsion into attraction. Using L1 constructs containing missense pathological mutations, it has been shown that this reversion is initiated by a specific trans binding of L1 to NP-1, but not to L1 or other Ig CAMs. This binding leads to activation of the NO/cGMP pathway. The L1-NP-1-binding site has been identified in a restricted sequence of L1 Ig domain 1, since a peptide derived from this region could reverse Sema3A repulsive effects. A pathological L1 missense mutation located in this sequence specifically disrupts both L1-NP-1 complex formation and Sema3A reversion, suggesting that the cross-talk between L1 and Sema3A might participate in human brain development (Castellani, 2002).

Trochlear motor axons project dorsally along the midbrain-hindbrain boundary (MHB) to decussate at the dorsal midline. Neuropilin 2 and its ligands control this trajectory. In chick embryos, neuropilin 2 is expressed in the neuroepithelium of the dorsal isthmus in addition to the trochlear neurons, and Sema3F transcripts are localized along the caudal margin of the midbrain. Misexpression of Sema3F demonstrates that Sema3F displays repulsive activity in vivo that guides the trochlear motor axons along the MHB. An unexpected result was that misexpression of neuropilin 2 cancels the midbrain-evoked repulsion, allowing trochlear motor axons to cross the MHB and invade the tectum. A binding assay with neuropilin 2 ectodomain revealed the existence of neuropilin 2 ligands in the midbrain, which were masked by ectopic neuropilin 2. It is therefore proposed that neuropilin 2 neutralizes the repulsive activity in order to steer trochlear motor axons towards the dorsal decussation point. Taken together, these results suggest that the interaction of neuropilin 2 with its ligands has crucial roles for establishing trochlear trajectory along the MHB (Watanabe, 2004).

The medial habenular nuclei of the zebrafish diencephalon, which lie bilateral to the pineal complex, exhibit left-right differences in their neuroanatomy, gene expression profiles and axonal projections to the unpaired midbrain target - the interpeduncular nucleus (IPN). Efferents from the left habenula terminate along the entire dorsoventral extent of the IPN, whereas axons from the right habenula project only to the ventral IPN. How this left-right difference in connectivity is established and the factors involved in differential target recognition are unknown. Prior to IPN innervation, only the left habenula expresses the zebrafish homologue of Neuropilin1a (Nrp1a), a receptor for class III Semaphorins (Sema3s). Directional asymmetry of nrp1a expression relies on Nodal signaling and the presence of the left-sided parapineal organ. Loss of Nrp1a, through parapineal ablation or depletion by antisense morpholinos, prevents left habenular neurons from projecting to the dorsal IPN. Selective depletion of Sema3D, but not of other Sema family members, similarly disrupts innervation of the dorsal IPN. Conversely, Sema3D overexpression results in left habenular projections that extend to the dorsal IPN, as well as beyond the target. The results indicate that Sema3D acts in concert with Nrp1a to guide neurons on the left side of the brain to innervate the target nucleus differently than those on the right side (Kuan, 2007).

The olfactory system of the mouse includes several subsystems that project axons from the olfactory epithelium to the olfactory bulb. Among these is a subset of neurons that do not express the canonical pathway of olfactory signal transduction, but express guanylate cyclase-D (GC-D). These GC-D-positive (GC-D+) neurons are not known to express odorant receptors. Axons of GC-D+ neurons project to the necklace glomeruli, which reside between the main and accessory olfactory bulbs. To label the subset of necklace glomeruli that receive axonal input from GC-D+ neurons, two strains of mice were generated with targeted mutations in the GC-D gene (Gucy2d). These mice co-express GC-D with an axonal marker, tau-ß-galactosidase or tauGFP, by virtue of a bicistronic strategy that leaves the coding region of the Gucy2d gene intact. With these strains, the patterns of axonal projections of GC-D+ neurons to necklace glomeruli can be visualized in whole mounts. Deficiency of one of the neuropilin 2 ligands of the class III semaphorin family, Sema3f, but not Sema3b, phenocopies the loss of neuropilin 2 (Nrp2) for axonal wiring of GC-D+ neurons. Some glomeruli homogeneously innervated by axons of GC-D+ neurons form ectopically within the glomerular layer, across wide areas of the main olfactory bulb. Similarly, axonal wiring of some vomeronasal sensory neurons is perturbed by a deficiency of Nrp2 or Sema3f, but not Sema3b or Sema3c. These findings provide genetic evidence for a Nrp2-Sema3f interaction as a determinant of the wiring of axons of GC-D+ neurons into the unusual configuration of necklace glomeruli (Walz, 2007).

Gonadotropin-releasing hormone (GnRH) neurons are neuroendocrine cells that are born in the nasal placode during embryonic development and migrate through the nose and forebrain to the hypothalamus, where they regulate reproduction. Many molecular pathways that guide their migration have been identified, but little is known about the factors that control the survival of the migrating GnRH neurons as they negotiate different environments. It has been reported that the class 3 semaphorin SEMA3A signals through its neuropilin receptors, NRP1 and NRP2, to organise the axons that guide migrating GnRH neurons from their birthplace into the brain. By combining analysis of genetically altered mice with in vitro models, this study shows that the alternative neuropilin ligand VEGF164 promotes the survival of migrating GnRH neurons by co-activating the ERK and AKT signalling pathways through NRP1. Survival signalling relies on neuronal, but not endothelial, NRP1 expression, and it occurs independently of KDR, the main VEGF receptor in blood vessels. Therefore, VEGF164 provides survival signals directly to developing GnRH neurons, independently of its role in blood vessels. Finally, it was shown that VEGF164-mediated neuronal survival and SEMA3A-mediated axon guidance cooperate to ensure that migrating GnRH neurons reach the brain. Thus, the loss of both neuropilin ligands leads to an almost complete failure to establish the GnRH neuron system (Cariboni, 2011).

Plexins are receptors for Semaphorins

In Drosophila, plexin A is a functional receptor for semaphorin-1a. Plexins encode large transmembrane proteins whose cysteine-rich extracellular domains share regions of homology with the scatter factor receptors (encoded by the Met gene family). The extracellular domains of plexins also contain ~500 amino acid semaphorin domains. However, the highly conserved cytoplasmic moieties of plexins (~600 amino acids), have no homology with the Met tyrosine kinase domain nor with any other known protein. Met-like receptors and their ligands, the scatter factors, mediate a complex biological program including dissociation of cell-cell contacts, motility, and invasion. During embryogenesis, scatter factor-1 and Met promote the dissociation of cell layers in the somites and drive the migration of myogenic cells to their appropriate location. Met and scatter factor-1 are also involved in controlling neurite outgrowth and axonal guidance. The human plexin gene family comprises at least nine members in four subfamilies. Plexin-B1 is a receptor for the transmembrane semaphorin Sema4D (CD100), and plexin-C1 is a receptor for the GPI-anchored semaphorin Sema7A (Sema-K1). Secreted (class 3) semaphorins do not bind directly to plexins, but rather plexins associate with neuropilins, coreceptors for these semaphorins. Plexins are widely expressed: in neurons, the expression of a truncated plexin-A1 protein blocks axon repulsion by Sema3A. The cytoplasmic domain of plexins associates with a tyrosine kinase activity. Plexins may also act as ligands mediating repulsion in epithelial cells in vitro. It is concluded that plexins are receptors for multiple (and perhaps all) classes of semaphorins, either alone or in combination with neuropilins, and trigger a novel signal transduction pathway controlling cell repulsion (Tamagnone, 1999).

Class 1 and 3 semaphorins repulse axons but bind to different cell surface proteins. The two known semaphorin-binding proteins, plexin 1 (Plex 1) and neuropilin-1 (NP-1), form a stable complex. Plex 1 alone does not bind semaphorin-3A (Sema3A), but the NP-1/Plex 1 complex has a higher affinity for Sema3A than does NP-1 alone. While Sema3A binding to NP-1 does not alter nonneuronal cell morphology, Sema3A interaction with NP-1/Plex 1 complexes induces adherent cells to round up. Expression of a dominant-negative Plex 1 in sensory neurons blocks Sema3A-induced growth cone collapse. Sema3A treatment leads to the redistribution of growth cone NP-1 and plexin into clusters. Thus, physiologic Sema3A receptors consist of NP-1/plexin complexes (Takahashi, 1999).

Commissural axons cross the nervous system midline and then turn to grow alongside it, neither recrossing nor projecting back into ventral regions. In Drosophila, the midline repellent Slit prevents recrossing: axons cross once because they are initially unresponsive to Slit, becoming responsive only upon crossing. Commissural axons in mammals similarly acquire responsiveness to a midline repellent activity upon crossing. Remarkably, they also become responsive to a repellent activity from ventral spinal cord, helping explain why they never reenter that region. Several Slit and Semaphorin proteins, expressed in midline and/or ventral tissues, mimic these repellent activities, and midline guidance defects are observed in mice lacking neuropilin-2, a Semaphorin receptor. Thus, Slit and Semaphorin repellents from midline and nonmidline tissues may help prevent crossing axons from reentering gray matter, squeezing them into surrounding fiber tracts (Zou, 2000).

The development of an assay in which spinal commissural axons are first made to cross the floor plate before being confronted with tissues or guidance cues has enabled dissection of the changes in responsiveness of these axons during midline crossing. Paralleling previous studies in Drosophila, it has been shown that commissural axons acquire responsiveness to a midline repellent activity upon crossing the midline and that Slit proteins may contribute to this activity. These observations are extended, however, by showing that ventral spinal cord tissue also secretes a repellent activity -- perhaps involving Slit-2 -- to which the axons become responsive upon midline crossing, providing an explanation for why the axons do not reenter the ventral spinal cord. While Slit proteins may contribute to the repellent activities in both floor plate and ventral spinal cord, this study also implicates the class 3 Semaphorins Sema3B and Sema3F, which are high-affinity ligands for neuropilin-2 receptors on commissural axons, in mediating the repellent actions of floor plate and ventral spinal cord. The finding of projection defects in a neuropilin-2 knockout mouse supports this hypothesis. Taken together, these results suggest that midline recrossing in vertebrates is prevented not just by the loss of responsiveness to positive factors at the midline, but also the acquisition of responsiveness to negative factors. They also support a model in which commissural axons are forced, or squeezed, out of the gray matter of the nervous system into surrounding fiber tracts by repellents secreted by both the floor plate and the ventral spinal cord (Zou, 2000).

A pleasing result from this study is that, as in Drosophila, spinal commissural axons acquire responsiveness to at least one Slit protein (and perhaps all three) upon midline crossing. Whether this involves upregulation of vertebrate Robo receptor expression on the commissural axons after midline crossing remains to be determined. A surprising aspect of these results, however, was the finding that the class 3 Semaphorin Sema3B likely contributes to the repulsive floor plate activity as well, since its mRNA is expressed by floor plate cells and it repels post-crossing axons in this assay. This repulsive action is likely to be mediated by the high-affinity Sema3B receptor neuropilin-2, which is expressed by these axons. (Neuropilin-2 is likely only the ligand binding portion of the Sema3B receptor, with signaling presumably mediated by a plexin family member such as plexin-A3, which is expressed by these neurons). Thus, in contrast to Drosophila, where a single Slit protein is thought to account for all the midline repulsive activity, in vertebrates the task of repulsion of post-crossing axons by midline cells appears to be shared by at least three Slit proteins and one Semaphorin (Zou, 2000).

These studies also revealed for the first time in any organism that crossing axons also acquire responsiveness to a repellent activity from the ventral portion of the nervous system. This is the terrain that the axons have traversed immediately before reaching the midline and which is therefore permissive for growth prior to crossing; after crossing, however, it becomes repulsive to the axons. This repulsive activity again appears to involve both Slit and Semaphorin proteins, since Slit-2 is expressed in the motor column and since Sema3F (another high-affinity neuropilin-2 ligand) is expressed throughout the mantle layer of the entire spinal cord (including the ventral spinal cord but excluding the floor plate). The existence of this repulsive activity should help prevent the axons from reentering the ventral portions of the nervous system. In fact, the repellent actions of the floor plate and the ventral spinal cord together should help squeeze the commissural axons out of the gray matter of the spinal cord entirely after they have crossed the midline. If Slit-2 and/or Sema3F proteins are also displayed on motor axons, then they might also help organize post-crossing commissural axons within the regions of the fiber tracts that motor axons traverse, a possibility suggested for Slit-2 (Zou, 2000).

The analysis of a neuropilin-2 knockout mouse supports the involvement of the class 3 Semaphorins in regulating midline crossing of commissural axons. A frequent defect observed in the mutants is the apparent stalling out of the axons in the floor plate, which is consistent with the existence of insufficient inhibitory activity within the floor plate to help push the axons out of the midline region once they have started crossing it. Interestingly, in these cases of stalling, many or all the axons stall out at the contralateral floor plate edge; this is reminiscent of the situation in robo mutants in Drosophila, where the axons can recross the midline but do not stall out in the middle, apparently because of the operation of a weaker repulsive mechanism (also involving Slit but mediated by some other receptor, perhaps Robo-2). The presence of residual inhibition at the midline in the neuropilin-2 knockout mice might similarly explain why axons grow to the contralateral edge of the floor plate (Zou, 2000).

In addition, the defects are only partially penetrant and also seem to be corrected as the embryo matures, indicating the operation of redundant guidance mechanisms. These mechanisms presumably include the Slit proteins but also possibly other nondiffusible guidance cues, such as ephrinB2, which a recent descriptive analysis has suggested might be involved in regulating midline guidance as well. EphrinB2 might, in fact, be a good candidate for the short-range repellent activity of floor plate cells documented in chick, to which commissural axons appear to be sensitive even prior to crossing (Zou, 2000 and references therein).

The exiting of spinal commissural axons into the ventral funiculus from the gray matter after midline crossing is representative of the behavior of large numbers of other axons up and down the neuraxis, which grow to their targets by coursing through the gray matter to some exit point where they join and grow in fiber tracts, only later leaving the tracts to reenter the gray matter and to connect with their target cells. It is suggested that the mechanism described in this study may be representative of those operating throughout the nervous system to propel axons out of the gray matter into fiber tracts. It may be true quite generally that as axons leave the gray matter, they acquire responsiveness to both midline and gray matter repellent activities. It is intriguing in this regard that Sema3F and Sema3B, between them, are expressed throughout much of the gray matter and midline. In fact, the finding that Sema3F is expressed throughout the mantle layer, essentially everywhere where axons grow within the spinal cord (and in other brain regions as well), is hard to square with a role in guidance within the mantle layer. Rather, it seems more likely that it functions to prevent axons from entering or reentering the mantle layer and thus helps keep them in fiber tracts. The Slit proteins may also play such a role quite generally, since their mRNAs, after initially being most highly expressed in midline tissues, later become more widely expressed throughout the gray matter (Zou, 2000 and references therein).

After axons have grown in fiber tracts, what permits them to reenter the gray matter? This is an issue studied recently in the context of sensory axon collateral ingrowth into the spinal cord. Remarkably, that study implicated Slit proteins as positive regulators of sensory axon branching and ingrowth into the spinal cord gray matter. Slit proteins might function generally to permit axon ingrowth into gray matter from adjacent fiber tracts. Putting together these two suggestions, a global hypothesis suggests itself: axons that leave the gray matter are kept out because they acquire responsiveness to a repellent activity made by gray matter that involves Slit proteins (and Semaphorin proteins), and when they later branch back into the gray matter, they may do so because they acquire responsiveness to an attractive or permissive activity made by gray matter that may also involve Slit proteins (and perhaps also Semaphorin proteins?). Thus, in the most extreme version of this hypothesis, the axons may initially be able to grow through the gray matter because they are impervious to Slit and Semaphorin proteins and then acquire repulsive responses to these factors as they leave the gray matter, only reentering the gray matter when their responses to Slit and Semaphorin proteins switch from being repulsive to attractive. The ability of growth cones to rapidly switch their responsiveness between repulsion and attraction has been demonstrated for several types of cues, including Semaphorins, in tissue culture experiments using Xenopus neurons. Future experiments will test whether the initial exit and subsequent reentry of the gray matter is controlled by such a neatly choreographed series of changes in growth cone responsiveness — from no response, to repulsion, to attraction — to guidance cues of the Slit and Semaphorin families and help elucidate what other mechanisms are at play in regulating gray matter entry and exit (Zou, 2000 and references therein).

Hippocampal mossy fibers project preferentially to the stratum lucidum, the proximal-most lamina of the suprapyramidal region of CA3. The molecular mechanisms that govern this lamina-restricted projection are still unknown. This study examined the projection pattern of mossy fibers in mutant mice for semaphorin receptors plexin-A2 and plexin-A4, and their ligand, the transmembrane semaphorin Sema6A. plexin-A2 deficiency causes a shift of mossy fibers from the suprapyramidal region to the infra- and intrapyramidal regions, while plexin-A4 deficiency induces inappropriate spreading of mossy fibers within CA3. The plexin-A2 loss-of-function phenotype is genetically suppressed by Sema6A loss of function. Based on these results, a model is proposed for the lamina-restricted projection of mossy fibers: the expression of plexin-A4 on mossy fibers prevents them from entering the Sema6A-expressing suprapyramidal region of CA3 and restricts them to the proximal-most part, where Sema6A repulsive activity is attenuated by plexin-A2 (Suto, 2007).

Cell-cell signalling of semaphorin ligands through interaction with plexin receptors is important for the homeostasis and morphogenesis of many tissues and is widely studied for its role in neural connectivity, cancer, cell migration and immune responses. SEMA4D and Sema6A exemplify two diverse vertebrate, membrane-spanning semaphorin classes (4 and 6) that are capable of direct signalling through members of the two largest plexin classes, B and A, respectively. In the absence of any structural information on the plexin ectodomain or its interaction with semaphorins the extracellular specificity and mechanism controlling plexin signalling has remained unresolved. This study presents crystal structures of cognate complexes of the semaphorin-binding regions of plexins B1 and A2 with semaphorin ectodomains (human PLXNB1(1-2)-SEMA4D(ecto) and murine PlxnA2(1-4)-Sema6A(ecto)), plus unliganded structures of PlxnA2(1-4) and Sema6A(ecto). These structures, together with biophysical and cellular assays of wild-type and mutant proteins, reveal that semaphorin dimers independently bind two plexin molecules and that signalling is critically dependent on the avidity of the resulting bivalent 2:2 complex (monomeric semaphorin binds plexin but fails to trigger signalling). In combination, the data favour a cell-cell signalling mechanism involving semaphorin-stabilized plexin dimerization, possibly followed by clustering, which is consistent with previous functional data. Furthermore, the shared generic architecture of the complexes, formed through conserved contacts of the amino-terminal seven-bladed β-propeller (sema) domains of both semaphorin and plexin, suggests that a common mode of interaction triggers all semaphorin-plexin based signalling, while distinct insertions within or between blades of the sema domains determine binding specificity (Janssen, 2010).

UNC-33 homologs: Collapsin-response mediator proteins

Mutations in the unc-33 gene of the nematode C. elegans lead to severely uncoordinated movement, abnormalities in the guidance and outgrowth of the axons of many neurons, and a superabundance of microtubules in neuronal processes. Three unc-33 messages, transcribed from a genomic region of at least 10 kb, have been identified and characterized. The three messages have common 3' ends and identical reading frames. The three putative polypeptides encoded by the three messages overlap in C-terminal sequence but differ by the positions at which their N termini begin; none has significant similarity to any other known protein (Li, 1992)

Collapsin, a semaphorin family member, contributes to axonal pathfinding during neural development by inhibiting growth cone extension. The mechanism of Collapsin action is poorly understood. A Xenopus oocyte expression system was used to identify molecules involved in collapsin signaling. A Collapsin response mediator protein required for collapsin-induced inward currents in frog oocytes was isolated. Called CRMP-62, it shares homology with UNC-33, a nematode neuronal protein required for appropriately directed axonal extension. CRMP-62 is localized exclusively in the developing chick nervous system. Introduction of anti-CRMP-62 antibodies into dorsal root ganglion neurons blocks collapsin-induced growth cone collapse. CRMP-62 appears to be an intracellular component of a signaling cascade initiated by an unidentified transmembrane collapsin-binding protein (Goshima, 1995).

Rat gene TOAD-64 (Turned On After Division, 64 kDa), is expressed immediately after neuronal birth and is dramatically downregulated in the adult. TOAD-64 shows homology to the unc-33 gene from C. elegans, mutations that lead to aberrations in axon outgrowth. TOAD-64 mRNA is enriched in the nervous system and is developmentally regulated in parallel with the protein. The expression of the TOAD-64 protein and gene coincident with initial neuronal differentiation and the downregulation when the majority of axon growth is complete suggests a role in axon elaboration. Three additional lines of evidence support this possibility: TOAD-64 is upregulated following neuronal induction of P19 and PC12 cells; the protein is found in lamellipodia and filopodia of growth cones; and axotomy of the sciatic nerve induces reexpression. While the sequence of TOAD-64 lacks a signal sequence and therefore is likely to encode a cytoplasmic protein, biochemical experiments demonstrate that the protein is tightly, but noncovalently, associated with membranes. The data suggest that TOAD-64 could be a central element in the machinery underlying axonal outgrowth and pathfinding, perhaps playing a role in the signal transduction processes that permit growing axons to choose correct routes and targets (Minturn, 1995).

Transmembrane Semiphorins: Class IV and Class V

This paper reports the identification of a mouse semaphorin cDNA, termed Sema VIb. Although Sema VIb contains the extracellular semaphorin domain, it lacks the immunoglobulin domain or thrombospondin repeats that are present in other described vertebrate (but not invertebrate) transmembrane semaphorins. During development, Sema VIb mRNA is expressed in subregions of the nervous system and is particularly prominent in muscle. In adulthood, Sema VIb mRNA is expressed ubiquitously. The cytoplasmic domain of Sema VIb contains several proline-rich potential SH3 domain binding sites. Sema VIb is shown to bind specifically the SH3 domain of the protooncogene c-src. In transfected COS cells Sema VIb coimmunoprecipitates with c-src. These results, along with evidence that Sema VIb can form dimers, suggests that the semaphorin family not only serves as ligands but may include members, especially those that are transmembrane, that serve as receptors, triggering intracellular signaling via an src-related cascade (Eckhardt, 1997).

The semaphorins comprise a large family of membrane-bound and secreted proteins, some of which have been shown to function in axon guidance. A transmembrane semaphorin, Sema W, has been cloned that belongs to the class IV subgroup of the semaphorin family. The mouse and rat forms of Sema W show 97% amino acid sequence identity with each other, and each shows about 91% identity with the human form. The gene for Sema W is divided into 15 exons, up to 4 of which are absent in the human cDNAs sequenced in this study. Unlike many other semaphorins, Sema W is expressed at low levels in the developing embryo but is expressed at high levels in the adult central nervous system and lung. Functional studies with purified membrane fractions from COS7 cells transfected with a Sema W expression plasmid show that Sema W provides growth-cone collapse activity against retinal ganglion-cell axons. This indicates that vertebrate transmembrane semaphorins, like secreted semaphorins, can collapse growth cones. Genetic mapping of human SEMAW with human/hamster radiation hybrids localizes the gene to chromosome 2p13. Genetic mapping of mouse Semaw with mouse/hamster radiation hybrids localizes the gene to chromosome 6, and physical mapping places the gene on bacteria artificial chromosomes carrying microsatellite markers D6Mit70 and D6Mit189. This localization places Semaw within the locus for motor neuron degeneration 2, making it an attractive candidate gene for this disease (Encinas, 1999).

Retinal axon pathfinding from the retina into the optic nerve involves the growth promoting axon guidance molecules L1, laminin and netrin 1, each of which governs axon behavior at specific regions along the retinal pathway. In identifying additional molecules regulating this process during embryonic mouse development, it was found that transmembrane Semaphorin5A mRNA and protein is specifically expressed in neuroepithelial cells surrounding retinal axons at the optic disc and along the optic nerve. Given that growth cone responses to a specific guidance molecule can be altered by co-exposure to a second guidance cue, axons encountered along the retinal pathway were examined in order to determine whether retinal axon responses to Sema5A are modulated by other guidance signals. In growth cone collapse, substratum choice and neurite outgrowth assays, Sema5A triggers an invariant inhibitory response in the context of L1, laminin, or netrin 1 signaling, suggesting that Sema5A inhibits retinal axons throughout their course at the optic disc and nerve. Antibody-perturbation studies in living embryo preparations show that blocking of Sema5A function leads to retinal axons straying out of the optic nerve bundle, and indicate that Sema5A normally helped ensheath the retinal pathway. Thus, development of some CNS nerves requires inhibitory sheaths to maintain integrity. Furthermore, this function is accomplished using molecules such as Sema5A that exhibit conserved inhibitory responses in the presence of co-impinging signals from multiple families of guidance molecules (Oster, 2003).

Semaphorins and their receptors, plexins, carry out important functions during development and disease. In contrast to the well-characterized plexin A family, however, very little is known about the functional relevance of B-type plexins in organogenesis, particularly outside the nervous system. This study demonstrates that plexin B1 and its ligand Sema4d are selectively expressed in epithelial and mesenchymal compartments during key steps in the genesis of some organs. This selective expression suggests a role in epithelial-mesenchymal interactions. Importantly, using the developing metanephros as a model system, it was observed that endogenously expressed and exogenously supplemented Sema4d inhibits branching morphogenesis during early stages of development of the ureteric collecting duct system. The results further suggest that the RhoA-ROCK pathway, which is activated downstream of plexin B1, mediates these inhibitory morphogenetic effects of Sema4d and suppresses branch-promoting signalling effectors of the plexin B1 signalling complex. Finally, mice that lack plexin B1 show early anomalies in kidney development in vivo. These results identify a novel function for plexin B1 as a negative regulator of branching morphogenesis during kidney development, and suggest that the Sema4d-plexin B1 ligand-receptor pair contributes to epithelial-mesenchymal interactions during organogenesis via modulation of RhoA signalling (Korostylev, 2008).

Class 6 Semaphorins: A GPI-anchored protein

Semaphorin K1 (sema K1) is described as the first semaphorin known to be associated with cell surfaces via a glycosylphosphatidylinositol linkage. Sema K1 is highly homologous to a viral semaphorin and can interact with specific immune cells, suggesting that like its viral counterpart, sema K1 could play an important role in regulating immune function. Sema K1 does not bind to neuropilin-1 or neuropilin-2, the two receptors implicated in mediating the repulsive action of several secreted semaphorins, and thus it likely acts through a novel receptor. In contrast to most previously described semaphorins, sema K1 is only weakly expressed during development but is present at high levels in postnatal and adult tissues, particularly brain and spinal cord (Xu, 1998).

On and off retinal circuit assembly by divergent molecular mechanisms

Direction-selective responses to motion can be to the onset (On) or cessation (Off) of illumination. This study shows that the transmembrane protein semaphorin 6A and its receptor plexin A2 are critical for achieving radially symmetric arborization of On starburst amacrine cell (SAC) dendrites and normal SAC stratification in the mouse retina. Plexin A2 is expressed in both On and Off SACs; however, semaphorin 6A is expressed in On SACs. Specific On-Off bistratified direction-selective ganglion cells in semaphorin 6A-/- mutants exhibit decreased tuning of On directional motion responses. These results correlate the elaboration of symmetric SAC dendritic morphology and asymmetric responses to motion, shedding light on the development of visual pathways that use the same cell types for divergent outputs (Sun, 2013).

This study demonstrates that Sema6A, a classical axon guidance cue, is a molecular determinant that distinguishes On from Off visual pathways. Sema6A, together with PlexA2, regulates SAC dendritic stratification, On SAC dendritic morphology, and functional assembly of retinal direction-selective circuitry. In the neonatal murine retina, repulsive Sema6A-PlexA2 signaling disentangles On and Off SAC dendritic processes, providing the anatomical organization critical for the emergence and separation of On and Off direction-selective circuitry. Despite the observation of SAC inner plexiform layer stratification defects in all Sema6A−/− and PlexA2−/− mutant retinas examined, there remain normally stratified SAC processes in these mutants such that most SAC dendrites are still confined to their normal ChAT+ sublaminae, suggesting that additional dendritic stratification mechanisms function in parallel to Sema6A-PlexA2 signaling to ensure proper SAC dendrite stratification. The in vitro observation that exogenous Sema6A protein repels SAC neurites expressing PlexA2 but not Sema6A (corresponding to Off SACs), but does not affect SAC neurites expressing both PlexA2 and Sema6A (corresponding to On SACs), suggests that Sema6A and PlexA2 use in trans repulsion to facilitate correct SAC stratification. The lack of a repulsive response to exogenous Sema6A by SACs that express both Sema6A and PlexA2 likely reflects the silencing of PlexA2 by ligand expressed in cis, as has been observed in murine sensory neurons that express both Sema6A and PlexA4 and do not respond to exogenous Sema6A in vitro. The data suggest that On SAC dendritic processes in vivo are not repelled by exogenous Sema6A, so defects in their laminar stratification in the inner plexiform layer may occur as a secondary consequence of Off SAC stratification defects or as a result of distinct Sema6A-PlexA2 signaling interactions (Sun, 2013).

Sema6a and Plxna2 mediate spatially regulated repulsion within the developing eye to promote eye vesicle cohesion

Organs are generated from collections of cells that coalesce and remain together as they undergo a series of choreographed movements to give the organ its final shape. Little is known about the cellular and molecular mechanisms that regulate tissue cohesion during morphogenesis. Extensive cell movements underlie eye development, starting with the eye field separating to form bilateral vesicles that go on to evaginate from the forebrain. What keeps eye cells together as they undergo morphogenesis and extensive proliferation is unknown. This study shows that plexina2 (Plxna2), a member of a receptor family best known for its roles in axon and cell guidance, is required alongside the repellent semaphorin 6a (Sema6a) to keep cells integrated within the zebrafish eye vesicle epithelium. sema6a is expressed throughout the eye vesicle, whereas plxna2 is restricted to the ventral vesicle. Knockdown of Plxna2 or Sema6a results in a loss of vesicle integrity, with time-lapse microscopy showing that eye progenitors either fail to enter the evaginating vesicles or delaminate from the eye epithelium. Explant experiments, and rescue of eye vesicle integrity with simultaneous knockdown of sema6a and plxna2, point to an eye-autonomous requirement for Sema6a/Plxna2. A novel, tissue-autonomous mechanism of organ cohesion is proposed, with neutralization of repulsion suggested as a means to promote interactions between cells within a tissue domain (Ebert, 2014).

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Semaphorin-2a and Semaphorin-2b: Biological Overview | Developmental Biology | Effects of Mutation | References

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