BIOLOGICAL OVERVIEW

The semaphorin gene family includes both secreted and transmembrane proteins that are selectively expressed in unique subsets of neurons in developing invertebrate and vertebrate nervous systems. Semaphorins are defined by the presence of a large phylogenetically conserved 500 amino acid semaphorin (sema) domain. Secreted semaphorins have an immunoglobulin (Ig) domain C-terminal to the sema domain, while transmembrane semaphorins can have either an Ig domain or a thrombospondin type I repeat, or no identified structural motif C-terminal to their sema domain. Transmembrane semaphorins have relatively short cytoplasmic domains that contain no obvious catalytic domains. The existence of both secreted and transmembrane semaphorins suggests that semaphorins function over long and short distances. g Semaphorin I (G-Sema-I, formerly Fasciclin IV), was the first semaphorin to be identified. G-Sema-I is a transmembrane semaphorin containing no obvious structural domains C-terminal to the semaphorin domain and is one of several invertebrate and vertebrate semaphorins with this overall structure. In vivo antibody perturbation experiments show that G-Sema-I plays an important role in establishing the axonal trajectory of the well-characterized Ti1 pioneer neurons (Kolodkin, 1993). In addition to peripheral epithelial localization, G-Sema-I is also found on grasshopper embryonic CNS axons in a highly selective pattern, suggesting that it is also likely to function in axon guidance events during CNS development (Yu, 1998 and references).

Sema-1a is, thus far, the semaphorin in Drosophila most closely related to G-Sema-I. The two proteins are similar in overall structure and Sema-Ia shares 60% amino acid identity with G-Sema-1a over its sema domain. Analysis of Sema-Ia mRNA distribution during Drosophila neurodevelopment has shown that like G-Sema-Ia, Sema-1a mRNA is expressed in the nervous system (Kolodkin, 1993). Sema-1a appears to be expressed in a much larger subset of neurons in Drosophila than is G-Sema-I in the related grasshopper nervous system. Sema-1a is required for the generation of the precise pattern of embryonic neuromuscular connectivity and appears to function at motor neuron pathway choice points in the periphery. Further, Sema I is also required for CNS pathfinding events. Rescue and ectopic expression experiments strongly suggest that Sema-1a can function as a repulsive ligand for motor axons that normally require it to navigate peripheral choice points, supporting the idea that both transmembrane and secreted semaphorins can function in vivo as repulsive guidance cues (Yu, 1998).

The development of the stereotypic pattern of neuromuscular connectivity in embryonic Drosophila abdominal segments has provided an excellent system in which to study axon guidance events that include motor axon fasciculation and defasciculation, target region identification, target recognition and initial synapse formation, and the later events of synaptogenesis. Each hemisegment contains 30 identified muscles that are innervated by 40 motor neurons, several of which have been identified. The pathfinding events that bring these motor axons to their targets involve several discrete steps. These motor neurons initially exit the CNS as part of the ISN (intersegmental nerve) or SN (segmental nerve). They then divide among five major motor neuron branches that target different muscle groups, eventually elaborating synapses upon individual target muscles. This stereotypic pattern of motor axon branches can be easily observed in late stage 16/early stage 17 embryos [Images] by using the monoclonal antibody (mAb) 1D4, which is directed against the axonal glycoprotein Fasciclin II (Fas II) and serves as a robust marker for all motor axons in the periphery (Yu, 1998).

The establishment of the stereotypic pattern of motor pathways in muscle innervation requires a series of axon pathfinding events that guide growth cones to sites of synapse formation. Central among these are defasciculation events that occur at discrete locations along these motor pathways. Sema-Ia mutants have defects at several of these locations, consistent with motor axons requiring Sema-1a for choice-point defasciculation. Since Sema-1a is found on most, if not all, motor axons, Sema I mutant embryos were examined for defects in the establishment of neuromuscular connectivity. Homozygous Sema-1a ^P1 mutant embryos show dramatic and highly penetrant pathfinding defects in both the ISNb and SNa pathways. Normally, the ISN, ISNb, and ISNd branches exit the CNS as a single pathway. Just lateral to the CNS, in the vicinity of the ventral oblique muscles, the motor neurons of the ISNb and ISNd branches defasciculate from the ISN. The ISNd subsequently innervates muscles 15, 16, and 17, while the ISNb continues to extend dorsally. After encountering muscle 28, the ISNb extends along the external surface of ventral lateral muscles (VLMs) 6 and 7 and the internal surface of VLMs 14 and 30. The ISNb then projects along the internal surface of VLMs 13 and 12. Synaptic arborizations are formed by the defasciculation of several motor axons from the ISNb (the RP3 motor neuron between muscles 6 and 7; the RP1 and RP4 motor neurons on muscle 13, and the RP5 motor neuron on muscle 12). The ISN continues to extend dorsally and contacts its dorsal target muscles, resulting in the formation of three characteristic arborizations in the dorsal muscle field. In the absence of Sema-Ia, ISNb motor neurons often fail to extend from the external to the internal ventral lateral muscle surface, stalling at positions where motor axons normally defasciculate from the ISNb and form synapses on target muscles. The observed defects in ISNb pathway formation are likely not to reflect loss of RP motor neurons, which contribute to ISNb, or any inability on the part of these neurons to extend axons contralaterally across the midline and out of the CNS. Immunohistochemistry using mAb 7G10, which is directed against the axonal glycoprotein Fasciclin III, reveals no abnormalities in the RP fascicles in Sema-1a ^P1 mutant embryos, either within or exiting the CNS, or in RP motor neurons themselves. Thus the defects are due to the stall or defasciculation phenotype and not to a guidance defect (Yu, 1998).

These results show that Sema-1a is required for the generation of the precise pattern of neuromuscular connectivity in the Drosophila embryo. This requirement appears to be for the navigation of specific choice points in motor axon pathways and not a general requirement for process outgrowth. ISN defects are not observed in Sema-Ia^P1 mutants, and the defects observed in the SNa and the ISNb occur at discrete locations along these pathways where motor axons normally defasciculate from their main branch and either extend toward, or elaborate synaptic arborizations on, their target muscles. Analysis of the peripheral sensory neurons with mAb 22C10 (see Futsch) reveals no defects in their mature axonal trajectories, consistent with the fact that no Sema-1a protein is detected on these PNS sensory afferents (Yu, 1998).

Sema-1a is a transmembrane semaphorin with a small cytoplasmic domain. To address how Sema-1a functions in neurons to mediate defasciculation events, rescue experiments were performed using a modified form of Sema-1a protein that lacks the transmembrane and cytoplasmic domains (called Sema-IaEC). The level of ectopic Sema-IaEC, driven by neuronal promoters, assessed immunohistologically, is equal to or higher than that of Sema-1a observed in wild-type embryos. Expression of Sema-IaEC in all neurons results in a partial, but significant, rescue of embryonic neuronal Sema-1a ^P1 phenotypes and adult lethality. For example, there is a 46% reduction (from 92% to 50%) in the fraction of abnormal SNa pathways observed in Sema-1a ^P1 mutant embryos when Sema-IaEC expression is driven ectopically. This reduction is primarily a rescue of the defasciculation defect at position S1. Similar results have been observed using a different ectopic expression protocol in Sema-1a ^P1 mutant embryos. The partial rescue of Sema-1a ^P1 motor axon pathway phenotypes may reflect a requirement for Sema-1a localization or presentation for ISNb and SNa development that is not completely satisfied by the Sema-IaEC protein in the context of the ectopic expression system used here. In addition to partial rescue of SNa and ISNb defasciculation defects, neuronal expression of Sema-IaEC partially but significantly rescues the Sema-1a ^P1 embryonic CNS phenotype, resulting in a 46% reduction in the fraction of hemisegments with a discontinuous Fas II-positive third longitudinal connective, correcting a defect caused by Sema-Ia mutation. Finally, ectopic Sema-IaEC significantly rescues Sema-Ia^P1 adult lethality. However, increasing the dosage of Sema-IaEC results in a reduction in the rescue of Sema-1a ^P1 adult lethality, presumably reflecting deleterious effects of panneuronal Sema-IaEC expression. Taken together, these rescue experiments show that Sema-1a is required in neurons to mediate motor neuron defasciculation events during neurodevelopment. Further, these results strongly suggest that Sema-1a is a ligand for an as yet unidentified receptor on motor axons (Yu, 1998).

Studies in which Sema-1a was overexpressed suggest that Sema-1a can act as a repulsive axon guidance cue. Sema-1a was overexpressed in all muscles in both wild-type and Sema-1a ^P1 mutant embryos. Dramatic motor axon defects are observed when Sema-1a is expressed in all muscles in wild-type embryos: these defects are sensitive to the endogenous dosage of Sema-Ia. The ISNb phenotypes observed in the presence of ectopic expression of Sema-1a protein on all muscles in wild-type embryos are qualitatively similar to those observed in Sema-1a ^P1 mutant embryos alone. (1) There is a significant increase (from 0% in wild type to 24% when Sema-1a is expressed in all muscles in wild-type embryos) in the fraction of hemisegments with a bypass of ISNb with the ISN. These bypass events include both parallel bypass (PB) events, where the ISNb fails to enter the ventral muscle field and extends dorsally in close proximity to the ISN as a separate pathway, and fusion bypass (FB) events, where the ISNb fails to enter the ventral muscle field and extends dorsally along the ISN. (2) There are a significant number of ISNb stall events that are similar to those observed in Sema-1a^P1 mutant embryos: they occur at discrete locations between either muscles 7 and 6 or between muscles 6 and 13. (3) ISNb pathways with no evidence of synaptic arborizations between muscles 6 and 7, or aberrant synaptic arborizations between muscles 7 and 6 or on muscle 12, are observed in a large percentage of hemisegments. The occurrence of these motor axon pathfinding defects, which appear to reflect defects in motor axon defasciculation, is critically dependent on the dosage of ectopic Sema-1a expressed in muscles. A single copy of Sema-1a transgene does not result in the significant ISNb defects observed with two copies of each transgene. However, these events are sensitive to the endogenous dosage of Sema-1a expressed on axons, since a single copy of each transgene in a Sema-1a ^P1 mutant background results in a dramatic enhancement of the ISNb phenotypes observed in Sema-1a ^P1 mutants alone. For example, there is an increase from 7% to 58% in the fraction of hemisegments showing a bypass of ISNb with the ISN. Unlike the approximately equal numbers of PBs and FBs observed following ectopic Sema-1a expression on muscles in a wild-type background, these bypass events are almost always FBs (Yu, 1998).

Effects qualitatively similar to those described above for ISNb were also observed for SNa following ectopic expression of Sema-1a on all muscles both in wild-type and Sema-1a ^P1 mutant embryos. A significant number of S1 stall events is observed when Sema-1a is ectopically expressed on muscles in wild-type embryos. SNa defects also include a dramatic enhancement of Sema-1a ^P1 SNa phenotypes when Sema-1a is expressed in all muscles in this genetic background using only a single copy of each transgene. These enhanced defects include SNa fusion bypass events in which SNa fails to enter the ventral muscle field and instead extends dorsally along the ISN. SNa FBs are never observed in wild-type or Sema-1a ^P1 mutant embryos in the absence of ectopic Sema-1a muscle expression. In this genetic background, there is a significant increase in the fraction of hemisegments that exhibit a complete loss of the entire major dorsal SNa branch, and in those pathways that do still have this branch a larger fraction stall at the initial bifurcation of this branch in the lateral muscle group (Yu, 1998).

In addition to the enhancement of ISNb and SNa phenotypes observed in Sema-1a ^P1 mutants, ectopic expression of Sema-1a on muscles in this genetic background results in ISN defects. In the vicinity of the dorsal muscles, the ISN normally forms a stereotypic pattern of three arborizations. The formation of these arborizations is not disrupted in Sema-1a ^P1 mutants or by ectopic expression of Sema-1a in wild-type embryos using a single copy of each transgene. In Sema-1a ^P1 mutant embryos that express Sema-1a on all muscles, however, the first and second arborizations of ISN were not observed in 29% and 32% of hemisegments, respectively. No significant stalling of the ISN is observed in this genetic background. These ISN phenotypes are consistent with the observed ISN expression of Sema-1a (Yu, 1998).

The motor axon guidance defects produced by ectopically expressing Sema-1a in all muscles are specific to motor axons; they do not affect peripheral sensory axon pathfinding, and do not appear to be the result of changes in neuronal or muscle cell fate or muscle morphology (assessed as described above for Sema-1a ^P1 mutants). The motor axon phenotypes observed by expressing Sema-1a on all muscles demonstrates an ability of Sema-1a to prevent motor axons from entering regions of Sema-1a expression. This results in a failure of motor axon defasciculation at specific choice points and strongly suggests that Sema-1a can act as a repulsive axon guidance cue (Yu, 1998).

How does Sema-1a function to direct the navigation of axons past choice points in motor axon pathfinding? The effect of Sema-1a on a wild-type background has a profound, dosage-sensitive effect on motor axon pathfinding. These results show that Sema-1a on muscles can prevent the entry of ISNb axons into the ventral muscle field. Since both ISNb fusion and parallel bypass events are observed, one might predict that endogenous axonal Sema-1a often still functions in this situation to allow ISNb defasciculation from the ISN. This prediction is supported by the observation that lack of endogenous Sema-1a renders the ISNb sensitive to a low dose of ectopic Sema-1a in muscles, which in a wild-type background has no effect. The bypass events observed in this sensitized background are mostly fusion bypasses, not parallel bypasses, and this provides further support for Sema-Ia's role in mediating defasiculation events during normal motor axon pathfinding. Taken together, all of these data support a model in which neuronal Sema-1a acts as a ligand for an as yet unidentified receptor on motor axons to mediate repulsive axon-axon interactions at motor pathway defasciculation choice points (Yu, 1998).

Though the overall penetrance of defective motor axon pathways in Sema-1a ^P1 mutants is very high, the penetrance of specific defects along these pathways is variable. The results presented here strongly suggest that the Sema-1a ^P1 mutant is a null or very severe loss-of-function Sema-Ia allele. Therefore, Sema-1a function is not absolutely required for motor axon defasciculation events to occur, and it is important to consider Sema-1a in the context of the complex panoply of motor axon guidance cues that have already been shown to function in these guidance events. Several genes have been identified that affect defasciculation events in motor pathways. These genes, which are expressed in many motor axon branches, include FasII, which encodes an Ig superfamily member that is a homophilic cell adhesion molecule (CAM), PTP69D and PTP99A, which encode receptor protein tyrosine phosphatases (RPTPs), and beaten path (beat), which encodes Beat, a novel secreted protein expressed by motor neurons. Mutations in the gene sidestep (side), which has yet to be cloned, also affect defasciculation of the ISNb from the ISN. The importance of modulating axon-axon adhesive interactions during motor axon pathfinding is underscored by the genetic interactions between FasII and beat. ISNb axons in beat mutants often fail to defasciculate from the ISN; however, this phenotype can be suppressed by hypomorphic FasII mutations, suggesting that Beat somehow modulates CAM function in axons to promote defasciculation at choice points. Sema-1a is not the only semaphorin in Drosophila capable of acting as a repulsive cue for motor axons. Sema-IaI, a secreted semaphorin, can act as a selective repellent for the RP3 motor axon when ectopically expressed in ventral abdominal muscles. In Sema-IaI mutants, embryonic motor axon guidance appears normal. Sema-IaI expression is very high in a single thoracic ventral muscle. Recent observations show that Sema-IaI is also found at low levels in all muscles, and this general expression is important for motor axon guidance (M. Winberg and C.S. Goodman, personal communication to Yu, 1998). Sema-1a can act as a repulsive guidance cue for motor axons that require it for pathfinding, which suggests that Sema-Ia-mediated repulsive growth cone guidance normally plays an important role in motor axon pathfinding. Future studies will address whether or not Sema-IaI exerts its repulsive effect on RP3 through a different, or the same, signaling pathway as does Sema-1a (Yu, 1998 and references).

PROTEIN STRUCTURE

Amino Acids - 771

Structural Domains

Two new complete insect sequences (Tribolium-Sema-I and Drosophila-Sema-IIa) encode proteins with signal sequences, as does Grasshopper-Sema-I. T-Sema-I and D-Sema-1a have transmembrane domains, as does G-Sema-I. However, D-Sema-IIa has no transmembrane domain, and its C-terminus shows no indication of a potential phospholipid linkage, and thus it is likely to be secreted. In addition, at its C-terminus, D-Sema-II has a single C2-type immunoglobulin (Ig) domain. All four insect Semaphorins share a highly conserved extracellular domain of 500 amino acids that is characterized by 16 conserved cysteines, one conserved potential N-linked glycosylation site, and numerous blocks of conserved amino acids throughout the 500 amino acid domain. In their Semaphorin domains (comprising two-thirds of each protein), the three putative homologs (G-Sema-I, T-Sema-I, and D-Sema-Ia) are most similar to one another and all are more divergent from D-Sema-IaI in terms of both percent identity and the absence of several blocks of amino acids that are found in D-Sema-IaI. The existence of two different sema genes in the same species (Drosophila) suggests that they define a novel gene family encoding both transmembrane and secreted proteins (Kolodkin, 1993).

Semaphorins share a common ~500-amino-acid semaphorin domain and are grouped into six classes. Class I consists of transmembrane semaphorins, including Drosophila Sema-Ia. Class II has a single member, Drosophila Sema-II. Class III consists of secreted vertebrate semaphorins, including Sema D (Coll-I/Sema-IaII), Sema A (Sema V), Sema E (Cooll-3), Sema-IV, Coll-2 and Sema H (Coll-5), that all possess an immunoglobulin doman (Ig) and a basic C-terminal domain, lacking in Drosophila Sema-IaI. Class IV semaphorins are also transmembrane proteins that possess an extracellular Ig domain. Class V semaphorins, including Sema F are transmembrane proteins with a set of tandem thrombosponding type I (TSP domains). Class VI is defined by the recently described Sema KI, a glycosylphosphatidylinositol (GPI)-anchored protein (Chen, 1998; Takahashi, 1998).

EVOLUTIONARY HOMOLOGS

For information on other Semaphorin family members see Semaphorin II.

Drosophila Semaphorins

Transmembrane and secreted glycoproteins of the semaphorin family are typically classified as inhibitory neuronal guidance molecules. However, although chemorepulsive activity has been demonstrated for several semaphorin family members, little is known about the function of the numerous transmembrane semaphorins identified to date. The extracellular semaphorin domain of a transmembrane semaphorin, semaphorin-1a, can actively perturb axon pathfinding in vivo when presented homogenously as a recombinant freely soluble factor. When ectopic overexpression is limited to defined epithelial regions, semaphorin-1a can directly steer axons by acting as an attractive guidance molecule (Wong, 1999).

The sequence and expression analysis of two new Drosophila members of the Semaphorin family is reported. Both proteins show the presence of Semaphorin domains and transmembrane domains. Both genes are expressed maternally and in embryos, and reveal distinct expression patterns much earlier than the onset of neurogenesis. Elements of two novel Semaphorin-like sequences were observed in the STS and EST databases of Drosophila. Cloning and sequence analyses for these two cDNAs reveals the presence of the Sema domain with its conserved cysteine residues. This led to their classification as members of the Semaphorin family. A conceptual open reading frame (ORF) for the first gene contains 759 amino acid residues. The initiating methionine is followed by a stretch containing structural regions characteristic of a secretory signal sequence. A second long hydrophobic stretch at the C terminus suggests a transmembrane domainfollowed by a short cytoplasmic tail. The protein shows the closest resemblance to Drosophila Sema 1a with 46% identity within the Sema domain, and has therefore been named Sema 1b. The second Semaphorin is a protein of 1081 amino acids. As in Sema 1b, the N-terminus of this molecule shows the presence of a signal sequence and the C-terminus has transmembrane spanning segment and a short cytoplasmic tail (Khare, 2000).

Sequence comparison reveals no Drosophila counterpart, but it is most homologous to murine Sema 5B with 41% identity within the Sema domain. Therefore the Drosophila protein has been named Sema 5c. It contains seven thrombospondin type I (TspI) and thrombospondin-like repeats that are characteristic of class V Semaphorins. Phylogenetic analysis of all known Semaphorins using the Sema domain for comparison shows that the family can be grouped into 3 classes. Class I and II Semas exist in duplicates, while only one member of class V Sema has been detected in the Drosophila genome so far. A comparison between Drosophila and C. elegans Semas shows that most likely class I Semaphorins constitute the ancestral form because they can also be found in C. elegans. C. elegans does not contain any class II or class V Semaphorins. Class V Semaphorins appear more closely related to class I than class II. Class II Semas appear as a distinct group, with two closely related molecules, which may have arisen by a recent gene duplication. Both Semas reside within the same chromosomal sub-band, and are likely to be less than 250 kb apart. Furthermore, the duplication must have occurred after the separation of class II and class V Semas (Khare, 2000).

In Northern analyses, Sema 1b transcripts are expressed more widely during embryogenesis than 5c. Both genes are expressed maternally, are virtually absent during third larval stage and reappear in late pupa during the last stage of metamorphosis. Sema 1b is first expressed in early oocytes, and staining is observed in ovarioles and nurse cells. In stage 2 embryos staining is uniform due to the presence of a smaller, putatively maternal RNA species. At stage 5, two lateral stripes, one to two cells wide, appear. These meet at stage 8 due to ventral furrow invagination as part of gastrulation. This movement of cells during this period is similar to the migration of the meso-ectodermal cells that line the ventral neurogenic region. A similar pattern has also been seen in the expression of the single-minded gene (sim). Interestingly, Sema 1b expression decays rapidly at the time point when sim expression becomes apparent, suggesting that Sema 1b may induce sim expression. No altered Sema 1b pattern was observed in sim mutants, suggesting that Sema 1b is acting upstream or in parallel to sim. At later embryonic stages, the expression is seen primarily in the ectoderm and becomes diffused as development proceeds. No staining is observed in either the PNS or the CNS in any of the later stages suggesting that this molecule is probably not involved directly in neural functioning (Khare, 2000).

The Sema 5c RNA is present right from the earliest developmental stages. The weak expression in the stage 10 oocyte and uniform expression in the stage 2 embryo suggests the presence of maternal contribution. At the blastoderm stage, a pattern of six stripes emerges: in this pattern the anterior three stripes are considerably stronger than the posterior three stripes. In addition, dorsal lateral extensions of the stripes are observed. During germband extension, the stripe pattern refines to 12 stripes that also become weaker. At this stage, expression is also observed in the region of amnioserosa. At stage 10, expression is most prominent in the mesoderm. Late stages of embryogenesis show strong expression at muscle attachment sites, the visceral mesoderm of the anteriormost part of the midgut and in the dorsal vessel. Several genes have been reported to be expressed in two bilateral stripes: ventral nervous system defective (vnd), intermediate neuroblast defective (ind), and the Enhancer of split E(spl) genes. Of these, only the E(spl) genes show overlapping expression with sema 1b. Sema1b has been shown to bind biochemically to plexins that are neuronal surface molecules. When expressed ectopically, it can also interact genetically with plexins. These data do not show any substantial expression of Sema 1b in the CNS. It is possible, however, that the levels of Sema 1b transcripts are below the level of detection, and that the genetics of interaction is much more sensitive. However, it is also plausible to assume that one Sema can replace the function of another; consequently, another Sema of the same class expressed at the right location could exert the expected function (Khare, 2000).

C. elegans semaphorins

The semaphorin family comprises secreted and transmembrane proteins involved in axon guidance and cell migration. Deletion mutants of C. elegans semaphorin 1a (Ce-sema-1a or smp-1) and semaphorin 1b (Ce-sema-1b or smp-2) genes have been isolated and characterized. Both mutants exhibit defects in epidermal functions. For example, the R1.a-derived ray precursor cells frequently fail to change anterior/posterior positions completely relative to their sister tail lateral epidermal precursor cell R1.p, causing ray 1 to be formed anterior to its normal position next to ray 2. The ray cells, which normally separate from the lateral tail seam cell (SET) at the end of L4 stage, remain connected to the SET cell even in adult mutant males. The ray 1 defects are partially penetrant in each single Ce-sema-1 mutant at 20°C, but are greatly enhanced in Ce-sema-1 double mutants, suggesting that Ce-Sema-1a and Ce-Sema-1b function in parallel to regulate ray 1 position. Both mutants also have defects in other aspects of epidermal functions, including head and tail epidermal morphogenesis and touch cell axon migration, whereas, smp-1 mutants alone have defects in defecation and brood size. A feature of smp-1 mutants that is shared with mutants of mab-20 (which encodes Sema-2a) is the abnormal perdurance of contacts between epidermal cells (Ginzburg, 2002).

Vulva development in C. elegans involves cell fate specification followed by a morphogenesis phase in which homologous mirror image pairs within a linear array of primordial vulva cells form a crescent shape as they move sequentially towards a midline position within the array. The homologous pairs from opposite half vulvae in fixed sequence fuse with one another at their leading tips to form ring-shaped (toroidal) cells stacked in precise alignment one atop the other. The semaphorin 1a SMP-1, and its plexin receptor PLX-1, are required for the movement of homologous pairs of vulva cells towards this midline position. SMP-1 is upregulated on the lumen membrane of each primordial vulva cell as it enters the forming vulva and apparently attracts the next flanking homologous PLX-1-expressing vulva cells towards the lumen surface of the ring. Consequently, a new ring-shaped cell forms immediately ventral to the previously formed ring. This smp-1- and plx-1-dependent process repeats until seven rings are stacked along the dorsoventral axis, creating a common vulva lumen. Ectopic expression of SMP-1 suggests it has an instructive role in vulva cell migration. At least two parallel acting pathways are required for vulva formation: one requires SMP-1, PLX-1 and CED-10; and another requires the MIG-2 Rac GTPase and its putative activator UNC-73 (Dalpe, 2005).

date revised: 30 October 98

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