From the initial stages of axon outgrowth to the formation of a functioning synapse, neuronal growth cones continuously integrate and respond to multiple guidance cues. To investigate the role of semaphorins in the establishment of appropriate axon trajectories, a novel secreted semaphorin in grasshopper, gSema 2a, has been characterized. Sema 2a is a secreted protein that is most similar to Drosophila semaphorin 2a (dSema 2a), with which it shares 67% amino acid identity within the sema domain and 63% within the C terminus. Grasshopper Sema 2a contains a semaphorin domain and an Ig domain; however, similar to dSema 2a, gSema 2a does not have the basic C-terminal domain observed in the vertebrate homologs. Grasshopper Sema 2a shares approximately 34% and 23% amino acid identity with human Sema III/mouse Sem D/chick Coll-1 within the sema and C-terminal domain, respectively. A comparison of the secreted semaphorin gSema 2a and the transmembrane grasshopper semaphorin Sema I reveals the two grasshopper semaphorins share 37% amino acid identity within the sema domain (Isbister, 1999).

In vivo antibody perturbation experiments reveal that g-Sema I, a transmembrane semaphorin expressed in the developing grasshopper limb bud, is important for establishing the peripheral Ti pioneer projection into the CNS; however, it remains unclear whether it performs this role as a repellent or an attractant (Kolodkin, 1992). In addition, evidence suggests that Sema I acts as an attractive guidance cue for later arising neurons in the limb bud (Wong, 1997). Sema 2a is expressed in a gradient in the developing limb bud epithelium during Ti pioneer axon outgrowth. Sema 2a acts as a chemorepulsive guidance molecule critical for axon fasciculation and for determining both the initial direction and subsequent pathfinding events of the Ti axon projection. Interestingly, simultaneous perturbation of both secreted Sema 2a and transmembrane Sema I results in a broader range and increased incidence of abnormal Ti pioneer axon phenotypes, indicating that different semaphorin family members can provide functionally distinct guidance information to the same growth cone in vivo. Given the high levels of gSema 2a protein in muscle during motor neuron outgrowth, it will be interesting to investigate whether gSema 2a is involved in determining neuromuscular synaptic specificity in grasshopper, a function served by Drosophila Sema 2 (Isbister, 1999).

The establishment of the stereotypic Ti pioneer projection requires a series of growth cone pathfinding events beginning with the decision to extend proximally, toward the CNS, along the limb axis. This may be mediated, in part, by the plane of division of the epithelial cell that gives rise to the Ti neurons and to the expression of fasciclin II in the surrounding epithelium. During the axonogenesis of Ti pioneer neurons, there is a graded distribution of Sema 2a, with the highest expression in the distal and dorsal compartments of the limb. Antibody blocking of Sema 2a at this stage results in defects consistent with the Ti pioneer growth cones requiring Sema 2a to direct and maintain their proximal extension. For example, Sema 2a antibody perturbation during the early stages of Ti axonogenesis and outgrowth results in a significant number of axons aberrantly projecting into the distal tip and dorsal regions of the limb, areas expressing high levels of Sema 2a. Therefore, Ti growth cones prefer to migrate down this gradient of Sema 2a protein; this is believed to be the first demonstration of an observable in vivo chemorepulsive gradient (Isbister, 1999). Though repelled by areas of high expression, the Ti growth cones are still able to grow on a substratum of Sema 2a. This is similar to the finding that Drosophila Sema II does not prevent growth cones from exploring their environment, but establishes a threshold that specific attractive signals must overcome in order to permit synapse formation (Winberg, 1998).

In addition to initiating and maintaining directed axon outgrowth, Sema 2a promotes Ti axon fasciculation. Following Sema 2a perturbation, the sibling axons are not fasciculated and they often extended independent aberrant projections. It is likely that Sema 2a provides a surround repulsion that encourages the Ti axons to fasciculate with each other, presumably a less inhibitory interaction. This preference for interaction with neuronal substrata is also illustrated by the close apposition of Ti growth cones with the Tr and Cx1 cells, two preaxonogenesis neurons normally encountered along the Ti projection to the CNS. Furthermore, following Sema 2a perturbation, aberrant dorsal projecting growth cones are often observed to form close interactions with the neuronal Cx2 cells and femoral chordontal organ in the dorsal limb compartment, or even loop around to fasciculate with their own cell bodies. These results suggest that aberrantly projecting Ti growth cones continue to prefer interactions with neuronal substrata rather than limb epithelium, indicating even severely misguided growth cones continue to assess the relative attractiveness of their environment (Isbister, 1999).

When embryos are cultured in the presence of antibodies that perturb the function of Sema 2a, aberrant projections into the ventral compartment of the limb are rarely observed. One explanation for this is the possibility of an attractive cue in the dorsal and distal region of the limb, or a ventral repulsive cue, that Sema 2a is required to counter. In the absence of Sema 2a repulsion, the Ti growth cones project dorsally and/or distally, possibly in response to these cues. While the presence of these additional attractive or repulsive signals may be important for the pathfinding of other axons such as the distally projecting motor neurons, the Ti neurons must usually ignore these cues. Thus, under normal developmental conditions, the balance between these signals is thought to maintain proximal Ti axonal extension along the limb axis towards the Tr cell. Taken together, these axon initiation and pathfinding results and the genetic analysis of guidance molecule interactions during motor neuron target selection illustrate that all stages of axonal development are dependent on the reception and comparison of multiple guidance cues (Isbister, 1999).

In an effort to determine the structural elements necessary for Sema 2a function, function blocking experiments were conducted using antibodies generated against five different regions of Sema 2a. Blocking the semaphorin domain has severe effects on the pathfinding of Ti growth cones, increasing axon pathfinding errors from approximately 10% in controls to 60% following perturbation of the central region of the sema domain. These defects are characterized by aberrant projections into areas of high expression of Sema 2a, indicating that the semaphorin domain is necessary for the chemorepulsive function of Sema 2a. Similarly, in vitro studies have demonstrated that the sema domain does confer functional specificity to secreted semaphorins. For example, the region of specificity within the sema domain of Coll-1 has been narrowed to a 70 amino acid region which, when transplanted into the backbone of any other secreted semaphorin family member, issufficient to determine the biological activity of the new chimeric molecule. A significantly higher number of axonal malformations is found following perturbation of the central region of the sema domain compared to following perturbation of other regions of Sema 2a, suggesting this region is also of particular functional importance for invertebrate secreted semaphorins (Isbister, 1999 and references).

If the semaphorin domain is responsible for the chemorepulsive activity of Sema 2a, what is the function of the C-terminal domain? In vitro studies have shown that members of the vertebrate secreted semaphorins need to be dimerized to be functional and that dimerization is dependent on cysteine residues in the C terminus. In addition, it appears that secreted collapsin family members bind and activate their receptors as preformed dimers. Therefore, the absence of axon abnormalities following perturbation with antibodies directed against the last 172 amino acids of the C terminus of Sema 2a may be a consequence of Sema 2a having been secreted as a covalent dimer. Alternatively, since neither gSema 2a and dSema 2a contain the carboxy-terminal basic domain characteristic of the vertebrate secreted semaphorins, a region proposed to be necessary for regulating the repulsive activity, the C-terminal portion of Sema 2a may not be required for chemorepulsion. In addition to promoting dimerization, the Ig and basic domains of vertebrate secreted semaphorins bind strongly to Neuropilin family members, components of the secreted semaphorin receptor complex. The partial attenuation of Sema 2a repulsion that is observed following perturbation with antibodies directed against the entire C-terminal region, including the Ig domain, may result from a disruption of receptor complex interactions. However, insect neuropilins have not been identified, therefore suggesting the secreted semaphorins may signal through a different mechanism in invertebrates. Nevertheless, it is possible that the conserved sema domain mediates the biological activity of secreted semaphorins, while the Ig and basic domains are involved in dimerizing and binding the ligand to the receptor complex and thereby potentiating its activity. Taken together, the in vivo function blocking data and the vertebrate studies indicate biological activity is domain specific and highly conserved among secreted semaphorins (Isbister, 1999 and references).

The Semaphorins are a family of secreted and transmembrane proteins known to elicit growth cone repulsion and collapse. A putative null mutant of the C. elegans gene semaphorin-2a (Ce-sema-2a)was made and characterized. This mutant fails to complement mutants of mab-20. In addition to low-frequency axon guidance errors, mab-20 mutants have unexpected defects in epidermal morphogenesis. A detailed study of the phenotype of mab-20 mutants has revealed that C. elegans Semaphorin-2a regulates the formation or stabilization of contacts between epithelial cells during epithelial morphogenesis. To put the morphogenetic defects of mab-20 mutants into context, a brief review of the major morphogenetic events of C. elegans development is presented. C. elegans hypodermal morphogenesis can be divided into several steps. (1) Several short-range migrations and rearrangements of ectoblasts produce six longitudinal rows of hypodermal cells that sit on the embryo in a dorsal-posterior locale, leaving the ventral neuroblasts uncovered. (2) While the two dorsal rows of hypodermal cells intercalate, the leader cells of the ventral row of hypodermal cells initiate a migration toward the ventral midline to cover the rest of the body of the embryo in hypodermis in a process called ventral enclosure. It is not known how the head becomes covered with hypodermis. (3) Microfilament bundles within the hypodermis align in parallel along the circumferential contour after ventral enclosure. The fourfold elongation of the embryo is mediated by the threefold contraction of these circumferential microfilaments. (4) Six P cells become vulva precursor cells, some of which undergo several divisions, movements and fusions, and then evert to form a vulva by adulthood. (5) The three posterior seam cells on both sides of males undergo extra rounds of division in the third and fourth larval stages to generate the ray precursor cells. (6) Finally, an anterior-directed retraction of the entire male tail at the end of the fourth larval stage results in the formation of male tail sensory rays embedded within a cuticular fan. Except for dorsal hypodermal intercalation, head and vulva morphogenesis, and the anterior retraction of the male tail, Ce-Sema-2a is involved in each of these processes (Roy, 2000).

The earliest phenotypic trait of mab-20 mutants is the clustering of lateral hypodermal seam cells with each other. During the longitudinal alignment of the seam cell rows, numerous migrations and rearrangements of seam cells and their precursors occur. A comparison of the seam cell positions in mab-20 mutant embryos to the wild type suggests that migrating seam cells fail to reach their normal anteroposterior (AP) positions, resulting in the observed mutant seam cell clusters. The next defect observed in mab-20 mutants is the formation of ectopic contacts between non-neighboring ipsilateral ventral hypodermal cells as they move ventrally. These early ectopic contacts likely persist and are later observed as ectopic contacts between P cells after the completion of ventral enclosure. The third defect observed in mab-20 embryos is the extrusion of internal contents from the ventral surface during embryonic elongation. Contraction of the circumferential hypodermal microfilaments presumably forces internal contents through the incompletely enclosed ventral surface of mutant embryos It has been suggested that Ce-Sema-2a prevents ectopic contacts between ventral hypodermal cells during enclosure. Thus, mab-20 mutants most likely burst from the ventral side during elongation because abnormal pocket cell contacts either sterically hinder pocket closure or weaken the contacts formed between the contralateral pocket cells at the ventral midline. Those mab-20 mutant embryos that escape lethality frequently develop severe bulges and constrictions during embryonic elongation. Body wall bulges are always coincident with circumferentially misaligned microfilaments contained within the clustered seam cells of mab-20 mutants. The last observed morphogenetic defect in mab-20 mutants is the fusion of normally distinct sensory rays of the male tail. These phenotypic traits are explained by the formation of inappropriate contacts between cells and suggest that Ce-Sema-2a may normally prevent formation or stabilization of ectopic adhesive contacts between these cells (Roy, 2000).

The plexin family transmembrane proteins are putative receptors for semaphorins, which are implicated in the morphogenesis of animal embryos, including axonal guidance. Putative null mutants of the C. elegans plexinA gene, plx-1, have been generated and characterized. plx-1 mutants exhibit morphological defects: displacement of ray 1 and discontinuous alae. The epidermal precursors for the affected organs are aberrantly arranged in the mutants, and a plx-1::gfp transgene is expressed in these epidermal precursor cells as they undergo dynamic morphological changes. Suppression of C. elegans transmembrane semaphorins, Ce-Sema-1a and Ce-Sema-1b, by RNA interference causes a displacement of ray 1 similar to that of plx-1 mutants, whereas mutants for the Ce-Sema-2a/mab-20 gene, which encodes a secreted-type semaphorin, exhibits phenotypes distinct from those of plx-1 mutants. A heterologous expression system has shown that Ce-Sema-1a, but not Ce-Sema-2a, physically binds to PLX-1. These results indicate that PLX-1 functions as a receptor for transmembrane-type semaphorins, and, though Ce-Sema-2a and PLX-1 both play roles in the regulation of cellular morphology during epidermal morphogenesis, they function rather independently (Fujii, 2002).

Ephrins and semaphorins regulate a wide variety of developmental processes, including axon guidance and cell migration. The roles of the ephrin EFN-4 and the semaphorin MAB-20 have been studied in patterning cell-cell contacts among the cells that give rise to the ray sensory organs of Caenorhabditis elegans. In wild-type, contacts at adherens junctions form only between cells belonging to the same ray. In efn-4 and mab-20 mutants, ectopic contacts form between cells belonging to different rays. Ectopic contacts also occur in mutants in regulatory genes that specify ray morphological identity. efn-4 and mab-20 reporters were used to investigate whether these ray identity genes function through activating expression of efn-4 or mab-20 in ray cells. mab-20 reporter expression in ray cells is unaffected by mutants in the Pax6 homolog mab-18 and the Hox genes egl-5 and mab-5, suggesting that these genes do not regulate mab-20 expression. mab-18 is found to be necessary for activating efn-4 reporter expression, but this activity alone is not sufficient to account for mab-18 function in controlling cell-cell contact formation. In egl-5 mutants, efn-4 reporter expression in certain ray cells is increased, inconsistent with a simple repulsion model for efn-4 action. The evidence indicates that ray identity genes primarily regulate ray morphogenesis by pathways other than through regulation of expression of semaphorin and ephrin (Hahn, 2003).

Semaphorins and ephrins are axon guidance cues. In C. elegans, semaphorin-2a/mab-20 and ephrin-4/efn-4/mab-26 also regulate cell sorting to form distinct rays in the male tail. Several erf (enhancer of ray fusion) mutations were identified in a mab-20 enhancer screen. Mutants of plexin-2 (plx-2) and unc-129, which encodes a divergent axon guiding TGF-β, were also found to be erfs. Genetic analyses show that plx-2 and mab-20 function in the same pathway, as expected if PLX-2 is a receptor for MAB-20. Surprisingly, MAB-20 also signals in a parallel pathway that requires efn-4. This signal utilizes a non-plexin receptor. The expression of plx-2, efn-4, and unc-129 in subsets of 3-cell sensory ray clusters likely mediates the ray-specific cell sorting functions of the ubiquitously expressed mab-20. A model is presented for the integrated control of TGF-β, semaphorin, and ephrin signaling in the sorting of cell clusters into distinct rays in the developing male tail (Ikegami, 2004).

The male tail in C. elegans is characterized by nine distinct, linearly arranged sensory rays visible as finger-like protrusions on each side of the animal. Each ray comprises three cells that derive from a common ray precursor cell (Rn cell where n = 1-9). Each Rn cell divides to form Rn.p, a hypodermal cell, and Rn.a. The Rn.a descendants form a pre-ray cluster of three lineally related cells that eventually form two neurons (RnA and RnB) and a structural support cell (Rnst), which encases the sensory endings of these neurons to form an adult ray. Initially, the Rn.a descendants (3-cell cluster) of one ray contact the Rn.a descendants destined to form neighboring rays. They then undergo dynamic shape and position changes that separate each cluster of three lineally related cells from neighboring 3-cell clusters to ultimately form the distinct ray sensillae of the adult male. These changes are orchestrated by active cellular mechanisms involving specific cell-cell interactions within and between the 3-cell clusters destined to form rays. The final positions of the adult rays are determined by the site of attachment of the structural support cell Rnst (one of the Rn.a descendants in each cluster) to the basal surface of the cuticle in L4 male larvae (Ikegami, 2004 and references therein).

In mutants of mab-20, neighboring Rn.a descendants for each ray frequently fail to separate from each other, resulting in fused rays. Mutants of ephrin-4 (efn-4/mab-26) are known to share a similar male tail phenotype with mutants of mab-20. efn-4 encodes an ephrin-related protein that fails to bind to VAB-1, the only known ephrin receptor in C. elegans. As shown by the nonadditive ray fusion defects of mab-20 and efn-4 null mutants, it was proposed that these two genes function in a common pathway to sort the 3-cell clusters destined to form individual rays. The functional relatedness of efn-4 to mab-20 suggests the existence of crosstalk and a possible ancestral link between ephrin and semaphorin signaling mechanisms (Ikegami, 2004 and references therein).

The fact that only mutants of mab-20/sema-2A and efn-4/mab-26 were identified in large-scale screens for male ray fusion defects suggests that the signaling mechanisms that are regulated by MAB-20/Sema-2A and EFN-4/MAB-26 are encoded largely by genes that are either essential for viability or are redundant. Modifier screens have proven to be especially useful for revealing such genes. Therefore, in order to identify novel semaphorin signaling pathways and novel components of known semaphorin signaling pathways, and to better understand how ephrin and semaphorin signaling are integrated, a large-scale screen was undertaken for mutations that enhance a weak allele of mab-20. This screen identified mutants of the C. elegans plexin-2 (plx-2) gene, which encodes a presumed receptor for Sema-2A/MAB-20, plus mutants of at least six erf genes (erf-1 to erf-5 plus plx-2) that enhance both the body morphology and male ray fusion defects of mab-20(bx61^ts). unc-129, which encodes a TGF-β that regulates axon guidance in C. elegans, was also found to enhance ray fusion defects of a mab-20 weak allele. Genetic and phenotypic characterization of these mutants has begun to reveal hierarchical pathways of semaphorin and ephrin function involved in several aspects of C. elegans morphogenesis. For example, even though putative null alleles of plx-2 do not cause a male ray fusion phenotype resembling the mab-20 mutants, genetic interactions between plx-2, efn-4, unc-129, and erf mutations have revealed the existence of redundant mechanisms that regulate the signaling of mab-20 and has suggested a role (in the context of a functional network) for efn-4, plx-2, and unc-129 in integrating these pathways. The integrated regulation of semaphorin, ephrin, and TGF-β signaling in the sorting of Rn.a descendants into distinct rays in C. elegans may have implications for regulating differential cell adhesions involved in a variety of cell movements and cell shape changes that occur during animal development (Ikegami, 2004).

Plexins are functional receptors for Semaphorin axon guidance cues. Previous studies have established that some Plexins directly bind RAC^GTP and RHO. Recent work in C. elegans has shown that semaphorin 1 (smp-1 and smp-2) and plexin 1 (plx-1) are required to prevent anterior displacement of the ray 1 cells in the male tail. plx-1 is shown genetically to be part of the same functional pathway as smp-1 and smp-2 for male ray positioning. RAC GTPase genes mig-2 and ced-10 probably function redundantly, whereas unc-73, which encodes a GEF for both of these GTPases, is required cell autonomously for preventing anterior displacement of ray 1 cells. RNAi analysis indicates that rho-1-encoded RHO GTPase, plus let-502 and K08B12.5-encoded RHO-kinases, are also required to prevent anterior displacement of ray 1 cells, suggesting that different kinds of RHO-family GTPases act similarly in ray 1 positioning. At low doses of wild-type mig-2 and ced-10, the Semaphorin 1 proteins no longer act through PLX-1 to prevent anterior displacements of ray 1, but have the opposite effect, acting through PLX-1 to mediate anterior displacements of ray 1. These results suggest that Plexin 1 senses levels of distinct RHO and RAC GTPases. At normal levels of RHO and RAC, Semaphorin 1 proteins and PLX-1 prevent a forward displacement of ray 1 cells, whereas at low levels of cycling RAC, Semaphorin 1 proteins and PLX-1 actively mediate their anterior displacement. Endogenously and ectopically expressed SMP-1 and SMP-2 suggest that the hook, a major source of Semaphorin 1 proteins in the male tail, normally attracts PLX-1-expressing ray 1 cells (Dalpé, 2004).

Members of the Semaphorin family of glycoproteins play an important role in axonal pathfinding by functioning as inhibitory guidance cues. A transmembrane form of Semaphorin (Semaphorin I), which is expressed by bands of epithelial cells in the developing grasshopper limb bud, functions as an attractive/permissive cue for the growth cones of the subgenual organ. In the developing limb bud, the first projection towards the CNS is established by the Ti1 pioneer neurons between 30-34% of development. Later in development, neurons arising in distal regions of the limb bud fasciculate with and extend along the Ti1 pathway into the CNS. One such group of neurons, the mechanoreceptor neurons of the subgenual organ, arise at approximately 38% of development, distal to the Ti1 cell bodies. Semaphorin I is needed for initial axonal outgrowth from the subgenual organ. These results may explain the previously reported arrest of the proximal extension of the subgenual organ growth cones in the absence of the Ti1 pioneer pathway (Wong, 1997).

All six eukaryotic Semaphorin proteins share the 500 amino acid Sema domain. Two viral sequences (vaccinia virus A39R ORF Sema IV and variola virus compiled ORF Sema IV) encode truncated and more divergent Sema domains. Insect Sema I proteins possess a C-terminal transmembrane domain with a short cytoplasmic tail. Drosophila Sema II, chick collapsin, and Human Sema III have neither transmembrane domains nor any other potential membrane linkage, but rather have a single C-terminal Ig domain followed by another short stretch of amino acids (Kolodkin, 1993).

The secreted mouse semaphorin D (SemD) is synthesized as an inactive precursor (proSemD) and becomes repulsive for sensory and sympathetic neurites upon proteolytic cleavage. ProSemD processing can be blocked completely by an inhibitor selective for furin-like endoproteases or mutagenesis of three conserved dibasic cleavage sites. Its C-terminal pro-peptide contains a processing signal that is essential for SemD to acquire its full repulsive activity. SemD processing is regulated during the embryonic development of the mouse and determines the magnitude of its repulsive activity. Similar to SemD, the secreted semaphorins SemA and SemE display repulsive properties that are regulated by processing. These data suggest that differential proteolytic processing determines the repulsive potency of secreted semaphorins and implicate proteolysis as an important regulatory mechanism in axonal pathfinding (Adams, 1997).

Chick collapsin-1, the first identified vertebrate member of the semaphorin family of axon guidance proteins, repels specific growth cones. Like all family members, collapsin-1 contains within its sequence a semaphorin domain that is necessary for specifying activity. Two additional structural domains of collapsin-1, the immunoglobulin (Ig) domain and the basic tail, each potentiate collapsin-1 activity. In this study another structural feature of collapsin-1 is identified that is necessary for its function. Collapsin-1 covalently dimerizes, and dimerization is necessary for collapse activity. This dimerization is mediated through a cysteine at residue 723, between the Ig domain and basic tail. The semaphorin domain alone is not active since it cannot dimerize. The collapsing activity of the semaphorin domain can be reconstituted when made as a chimeric construct with an immunoglobin Fc domain, which promotes dimerization (Koppel, 1998).

Class III (SemD) and class IV semaphorins (SemB) are shown to form homodimers linked by intermolecular disulfide bridges. In addition to the 95-kDa form of SemD [SemD(95k)], proteolytic processing of SemD creates a 65-kDa isoform [SemD(65k)] that lacks the 33-kDa carboxyl-terminal domain. Although SemD(95k) forms dimers, the removal of the carboxyl-terminal domain results in the dissociation of SemD homodimers to monomeric SemD(65k). Mutation of cysteine 723, one of four conserved cysteine residues in the 33-kDa fragment, reveals its requirement both for the dimerization of SemD and its chemorepulsive activity. It is suggested that dimerization is a general feature of semaphorins that depends on class-specific sequences and is important for their function (Klostermann, 1998).

Chick collapsin-1 is a repellent for specific growth cones. Two other secreted members of the semaphorin family, collapsin-2 and -3, are structurally similar to collapsin-1 but have different biological activities. The semaphorin domain of collapsin-1 is shown to be both necessary and sufficient for biological activity. The semaphorin domain contains a 70 amino acid region that specifies the biological activity of the three family members. The positively charged carboxy terminus potentiates activity without affecting specificity. It is proposed that semaphorins interact with their receptors through two independent binding sites: one that mediates the biological response and one that potentiates it (Koppel, 1997).

Alterations in neuronal connectivity of the mature central nervous system (CNS) appear to depend on a delicate balance between growth-promoting and growth-inhibiting molecules. To begin to address a potential role for the secreted chemorepulsive protein semaphorin(D)III/collapsin-1 (semaIII/coll-1) in structural plasticity during adulthood, high-resolution nonradioactive in situ hybridization was used to identify neural structures that express semaIII/coll-1 mRNA in the mature rat and human brain. SemaIII/coll-1 is expressed in distinct but anatomically and functionally linked structures of the adult nervous system. The olfactory-hippocampal pathway displays semaIII/coll-1 expression in a continuum of neuronal structures, including mitral and tufted cells of the olfactory bulb, olfactory tubercle and of piriform cortex, and distinct nuclei of the amygdaloid complex, the superficial layers of the entorhinal cortex, and the subiculum of the hippocampal formation. In addition, prominent labeling is found in neuronal components of the motor system, particularly in cerebellar Purkinje cells and in subpopulations of cranial and spinal motoneurons. Retrograde tracing combined with in situ hybridization also reveals that the staining of semaIII/coll-1 within the entorhinal cortex is present in the stellate neurons that project via the perforant path to the molecular layer of the dentate gyrus. As in the rat, the human brain displays discrete expression of semaIII/coll-1. Among the structures examined, the most prominent staining is observed in the cellular islands of the superficial layers of the human entorhinal cortex. The constitutive expression of the chemorepellent semaIII/coll-1 in discrete populations of neurons in the mature rat and human CNS raises the possibility that, in addition to its function as repulsive axon guidance cue during development, semaIII/coll-1 might be involved in restricting structural changes that occur in the wiring of the intact CNS (Giger,1998a).

Sema III expression was examined in the developing nerve cord. Centrally, dorsal route ganglion axons enter the spinal cord by embryonic (E) stage 11 and branch into the gray matter by E15 in brachial and thoracic regions. Laminar specific targets are reached by E17. Between E13 and E17, Sema III mRNA is expressed at high levels in the entire ventral half of the spinal cord except the floor plate. This pattern suggests that Sema III may inhibit non-proprioceptive sensory axons from penetrating the ventral spinal cord. Peripherally, sensory axons have entered the anterior sclerotome by E11 at all rostrocaudal levels. At this age, Sema III mRNA is already expressed in the dermamyotome and ventral aspect of the posterior sclerotome, areas that axons pass between but where they do not penetrate en route to their peripheral targets. From E12 to E15, the axons lengthen and branch into smaller fascicles which extend toward peripheral targets. During this time, Sema III mRNA is expressed by many mesodermal structures surrounding the axon fascicles, with highest levels observed in the dermamyotome, perinotochordal mesenchyme, pelvic girdle, and limb. As development proceeds, Sema III mRNA expression is quickly downregulated before disappearing by birth. Taken together these results demonstrate that the gene for Sema III is expressed in central and peripheral regions that are avoided by growing DRG axons. These findings are consistent with the idea that Sema III inhibits growth and branching of axons into inappropriate areas during development (Wright, 1995).

Five new members of the semaphorin family (Sem A-Sem E) have been characterized. The murine semaphorin genes are differentially expressed in mesoderm and neuroectoderm before and during the time when axons select their pathways in the embryo. In explant cultures, recombinant Sem D/collapsin converts a matrix permissive for axonal growth into one that is inhibitory for neurites of peripheral ganglia (Püschel, 1995).

A chick brain glycoprotein, collapsin, a member of the semaphorin family, has been shown to be a good candidate for a repulsive guidance cue. Four new molecules related to collapsin have been characterized in chick brains. All contain a semaphorin domain. One of these, collapsin-2, is structurally very similar to collapsin but is only 50% identical in its amino acid sequence. The collapsin-related genes exhibit distinct but overlapping patterns of mRNA expression in the developing spinal cord and the developing visual system (Luo, 1995).

Two additional members of the human semaphorin family [human Semaphorin A(V) and human Semaphorin IV] have been identified in chromosome region 3p21.3, where several small cell lung cancer (SCLC) cell lines exhibit homozygous deletions indicative of a tumor suppressor gene. Human Semaphorin A(V) has 86% amino acid homology with murine Semaphorin A, whereas Semaphorin IV is most closely related to murine Semaphorin E, with 50% homology. These semaphorin genes are approximately 70 kb apart, flanking two GTP-binding protein genes, GNAI-2 and GNAT-1. In contrast, other human semaphorin gene sequences (human Semaphorin III and homologues of murine Semaphorins B and C) are not located on chromosome 3. Human Semaphorin A(V) is translated in vitro into a 90-kDa protein, which accumulates at the endoplasmic reticulum. The human Semaphorin A(V) (3.4-kb mRNA) and IV (3.9- and 2.9-kb mRNAs) genes are expressed abundantly but differentially in a variety of human neural and nonneural tissues. Human Semaphorin A(V) is expressed in only 1 out of 23 SCLCs and 7 out of 16 non-SCLCs, whereas Semaphorin IV is expressed in 19 out of 23 SCLCs and 13 out of 16 non-SCLCs. Mutational analysis in Semaphorin A(V) reveals mutations (germ line in one case) in 3 of 40 lung cancers. Determination of the function of human Semaphorins A(V) and IV in nonneural tissues and their role in the pathogenesis of lung cancer should be of interest (Sekido, 1996).