BIOLOGICAL OVERVIEW

How unique can one muscle and its innervation be? Muscle 33, found in the third thoracic segment (Thoracic 3, or T3) of Drosophila is a quintessential example of such specificity. A brief description of the anatomy of Drosophila musculature is here in order: each side of larval abdominal segments A2 to A7 contains 30 muscles. The ventral muscles in the first abdominal segment (A1) are almost identical to those in A2 to A7; an exception is the addition of Muscle 31. The A1 segment muscles are also missing two muscles otherwise found in A2 to A7. Moving forward one segment to Thoracic 3, one finds larval Muscle 33 stretching across the other thoracic segments to attach to the mouth parts. It is Muscle 33 that expresses a protein, Semaphorin II, not expressed in any other muscle cell in the developing fly (Matthes, 1995).

The position of muscle 33 in T3 is very similar to the position of muscle 31 in A1. Muscles 31 and 33 are located just internal to muscles 7, 6 and 12 (in each of their respective segments): all of them serve as prominent internal ventral longitudinal muscles. All these internal ventral muscles are innervated by branches of segmental nerve b (SNb), including muscle 33 in segment T3. SNb carries multiple motoneurons that innervate specific muscles (Matthes, 1995).

While mutations in Sema-2a are lethal, it is noteworthy that no defects are found in muscle 33 development, nor in the innervation of muscle 33. What then could be the function of Sema-2a, expressed solely in muscle 33? To answer this question Sema-2a was expressed ectopically in a different subset of ventral embryonic muscles during motoneuron pathfinding. A heterologous enhancer (from the Toll gene) was used to express Sema-2a in all segments by some of the ventral muscles that normally do not express it, but that lie adjacent to the normally Semaphorin-2a-expressing muscle 33 in segment T3 (Matthes, 1995).

No gross defects are apparent in muscles of Toll-Sema-2a embryos, suggesting that ectopic Sema II does not alter the differentiation of ventral muscles. However, there were abnormalities in the development of certain branches of the SNb motor nerve that normally innervate muscles 7 and 6 in all abdominal segments as well as muscle 31 in A1. The effects of ectopic Sema II expression are specific to motoneuron SNb, while other motor nerves show normal morphology and branching. SNb carries motorneurons in the RP cluster (RP1, RP3, RP4 and RP5). The innervation of muscles 7 and 6 by RP3 motoneuron is dramatically altered in Toll-Sema-2a embryos. Instead of extending towards muscles 7 and 6, the neuron stalls just external to muscles 7 and 6. Abnormalities are also found in another motoneuron, DC1, which innervates muscle 13 and normally follows the same trajectory as the RP1 motoneuron innervating muscle 13. In most Toll-Sema-2a mutant embryos, DC1 fails to innervate muscle 31. In contrast, motoneuron RP1 is apparently unaffected by Toll-Sema-2a overexpression (Matthes, 1995).

What is a plausable explanation for this result? Apparently certain neurons are allowed to grow into regions where Sema II is expressed, but the protein inhibits the neuronal growth cone from forming a synaptic arborization (branching) to muscles 7 and 6. These results suggest that the RP3 and DC1 growth cones express a Semaphorin II receptor on their surface that confers an inhibitory response to this signal. This putative Semaphorin II receptor on the RP3 and DC1 growth cones can prevent growth cone branching and exploration and inhibit them from forming synaptic arborizations, but it does not appear to deter growth cone pathfinding (Matthes, 1995).

Here, a short digression to discuss Connectin, the axon guidance protein: Connectin is a homophilic cell adhesion molecule expressed in a subset of muscles and the motoneurons that innervate them. In Connectin experiments (Nose, 1994), RP1 changes its trajectory and takes a detour to reach muscle, thus avoiding ectopic connectin-expressing muscles. These results suggest that the RP1 growth cone expresses a functional receptor for Connectin (but not for Semaphorin II) that confers a repulsive role during pathfinding (Nose, 1994). Later studies show that the repulsive function imputed to Connectin can be explained by inappropriate innervation of a neighboring non-target muscle that ectopically expresses CON, thus reconfirming that Connectin function is attractive and homophilic and not repulsive (Nose, 1997). Fas III is also implicated in motoneuronal guidance and target recognition (Chiba, 1995)

The experiments with Connectin and Sema II suggest that neuromuscular specificity is controlled by a combination of attractive signals versus inhibition, repulsion, or both. These signals can either be secreted or cell surface. Different motoneuron growth cones express different combinations of receptors, and these receptor systems can function in either pathfinding or targeting or both. In such a model, different types of inhibitory and repulsive molecules play different roles in establishing the final pattern of axon projections and synaptic connections. The null effect of Sema-2a mutation on pathfinding, suggests that there are multiple redundant signals for the growth of each neuron, and that sufficient cues exist in the absence of one system to assure continued success, even when one set of cues is incomplete. Thus each motoneuron has its own particular response profile in terms of both its pathfinding and targeting preferences, and the signals are provided in a redundant manner assuring pathfinding success even in the absence of individual cues (Matthes, 1995).

Semaphorin 2a secreted by oenocytes signals through plexin B and plexin A to guide sensory axons in the Drosophila embryo

The semaphorin gene family has been shown to play important roles in axonal guidance in both vertebrates and invertebrates. Both transmembrane (Sema1a, Sema1b, Sema5c) and secreted (Sema2a, Sema2b) forms of semaphorins exist in Drosophila. Two Sema receptors, plexins (PlexinA and PlexinB), have also been identified. Many questions remain concerning the axon guidance functions of the secreted semaphorins, including the identity of their receptors. The well-characterized sensory system of the Drosophila embryo was used to address these problems. Novel sensory axon defects were found in sema2a loss-of-function mutants in which particular axons misproject and follow inappropriate pathways to the CNS. plexB loss-of-function mutants show similar phenotypes to sema2a mutants and sema2a interacts genetically with plexB, supporting the hypothesis that Sema2a signals through PlexB receptors. Sema2a protein is expressed by larval oenocytes, a cluster of secretory cells in the lateral region of the embryo and the sema2a mutant phenotype can be rescued by driving Sema2a in these cells. Ablation of oenocytes results in sensory axon defects similar to the sema2a mutant phenotype. These data support a model in which Sema2a, while being secreted from oenocytes, acts in a highly localized fashion: It represses axon extension from the sensory neuron cell body, but only in regions in direct contact with oenocytes (Bates, 2007).

The detailed knowledge of the cellular basis for axon pathfinding in the sensory system of the Drosophila embryo has been exploited to shed further light on the mechanisms by which semaphorins mediate axon guidance. Analysis of sensory axon trajectories in semaphorin mutants reveals that secreted semaphorins play roles in early pathfinding decisions by these axons. In sema2a loss-of-function mutants, the v'ch1 axon often projects to a nearby, inappropriate pathway, the ISN, instead of the SN. Two other defects are seen at low frequencies in sema2a mutants: one or more of the lateral cluster axons projects aberrantly to the SN and axons of dorsal sensory neurons project anteriorly or posteriorly, instead of growing ventrally towards the lateral sensory cluster (Bates, 2007).

The modest penetrance levels of sema/plex LOF phenotypes suggest that in the sensory system, as in many other situations, axon pathfinding events are likely to involve the simultaneous action of multiple guidance factors that act cooperatively and/or redundantly. Indeed, it has been shown that PlexA and PlexB have partially redundant roles in motor axon pathfinding (Ayoob, 2006) while motor axon branching over muscles is governed by the relative balance of Sema2a, Netrins and FasII, rather than the level of any one of these molecules. Sema2a alone apparently makes only a moderate contribution to sensory axon guidance. Several other molecules have been identified that play a role in the guidance of the same sensory axons that are affected by sema and plex mutations. A future goal of this research is to elucidate how these various guidance factors interact to mediate sensory axon guidance (Bates, 2007).

Overexpression of Sema2a in oenocytes, epidermis or trachea, or ectopic expression in neurons did not result in defective sensory axon morphologies. This finding contrasts with the reported disruption of muscle innervation following overexpression of Sema2a on muscles. Using anti-Sema2a staining, it was cofirmed that the GAL4 driver lines used in these overexpression experiments led to expression of high levels of Sema2a protein in the relevant tissues. Moreover, the partial rescue of the sema2a LOF mutant phenotype observed when the sal-GAL4 line was used to drive UAS-sema2a shows that at least this line can effectively drive expression of functional Sema2a protein. Thus, the absence of sensory axon defects following Sema2a overexpression suggests that sensory axons are less sensitive to semaphorin levels than are motor axons (Bates, 2007).

In contrast to the secreted semaphorins, no evidence was found from analysis of the sema1a loss-of-function mutant and overexpression of sema1a that this transmembrane semaphorin is involved in sensory axon guidance in the periphery (Bates, 2007).

The v'ch1 pathway is a potentially attractive route for lateral cluster axons, as shown by the misprojection of lateral axons along this route in a variety of mutants, including robo, slit and trachealess. However, in wild-type embryos, lateral cluster axons very rarely follow the v'ch1 axon and vice versa. At the onset of sensory axon growth, a cluster of 4 to 7 oenocytes lies between the v'ch1 and lateral cluster neurons. Filopodia extend from the v'ch1 cell body in a variety of directions: those projecting dorsally and anteriorly, towards the oenocytes, are generally short and do not develop into axon branches. These observations suggest that oenocytes form a repulsive zone, preventing axon growth between the v'ch1 and lateral cluster cell bodies. The finding that ablating some of the oenocytes in a hemisegment can result in misprojection of the v'ch1 axon to the lateral cluster neurons and vice versa supports this hypothesis (Bates, 2007).

Oenocytes strongly express Sema2a, and driving expression of Sema2a in these cells rescues the v'ch1 axon misprojection defect seen in sema2a mutants. These findings suggest that Sema2a secreted by oenocytes normally represses axon growth by v'ch1 towards the lateral cluster neurons, and vice versa. Sema2a may act by inhibiting filopodial extension and/or by repressing filopodial dilation and subsequent axon formation. Loss of Sema2a function in sema2a mutants removes that repression, allowing the v'ch1 and lateral cluster axons to misproject in a reciprocal fashion to each other (Bates, 2007).

This model of Sema2a function differs from prevailing views of the action of secreted semaphorins as diffusible chemo-repellents. Isbister (2003) has shown that sensory axons in the grasshopper limb bud are guided by two orthogonal gradients of Sema2a protein expression which span the entire width and length of the limb bud. The directional growth of the axons is apparently dictated by the fractional change in Sema2a concentration across the limb bud epithelium. Similarly, the secreted vertebrate semaphorin Sema III is proposed to have a long-range guidance function in the spinal cord (Messersmith, 1995). This study concluded that Sema III secreted by ventral spinal cord cells diffuses dorsally, repelling the axons of small diameter sensory afferents and thereby forcing them to terminate in the dorsal horn (Bates, 2007).

In contrast, the current results suggest that the secreted Drosophila semaphorin Sema2a acts in a very local fashion. Sema2a produced by oenocytes in contact with a portion of the surface of the v'ch1 cell body suppresses axon extension specifically from that region of the cell. In this way, Sema2a determines, at least in part, the initial polarity of axon extension from the v'ch1 neuron (Bates, 2007).

Sema2a is also expressed in stripes of epidermal cells at the segment border. This source of Sema2a could conceivably contribute to guidance of the v'ch1 and lateral cluster axons by inhibiting their growth in a posterior direction. However, the absence of aberrant, posteriorly projecting sensory axons on either v'ch1 or lateral cluster neurons in sema2a mutants speaks against this function (Bates, 2007).

Genetic and biochemical evidence points to PlexA being the receptor for the transmembrane class I semaphorins during motor axon guidance in the Drosophila embryo. However, until recently, the receptor(s) for the secreted class II semaphorins and the ligand(s) for PlexB had not been identified. Ayoob (2006) has now provided evidence for a physical and genetic interaction between PlexB and Sema2a during motor axon guidance (Bates, 2007).

The current study provides a number of lines of evidence, suggesting that PlexB acts as a receptor for Sema2a in a different context, regulating the growth of the v'ch1 sensory axon. (1) plexB is expressed in lateral cluster sensory neurons, including v'ch1 at the time of axon outgrowth. (2) plexB loss-of-function mutants show the same v'ch1 axon misprojection phenotypes as sema2a loss-of-function mutants. (3) The v'ch1 axon defects in plexB mutants can be rescued by driving plexB expression in sensory neurons. One caveat with this conclusion is that the P0163-GAL4 line used in these experiments drives GAL4 in the oenocytes as well as the sensory neurons. (4) Halving the gene dose of both sema2a and plexB results in the same v'ch1 axon defects as seen in single sema2a or plexB homozygous mutant embryos (Bates, 2007).

A parallel set of data leads to the tentative suggestion that PlexA may act as a receptor for Sema2a during lateral cluster axon guidance: plexA is expressed in lateral cluster neurons; plexA and sema2a LOF mutants show defects in lateral cluster axon growth, albeit at low penetrance levels; these defects can be rescued by driving PlexA in the sensory neurons; and sema2a^−/+; plexA^−/+ embryos show the same lateral cluster axon defects (Bates, 2007).

While the above results provide support for the idea that sensory axon guidance is mediated by Sema2a signaling through both PlexA and PlexB receptors, the mechanism is likely to be more complex than binding of Sema2a to PlexA and PlexB and consequent independent activation of these two receptors. The increased penetrance of the v'ch1 defect in double homozygous sema2a; plexA and sema2a; plexB mutants, compared to single sema2a, plexA or plexB mutants, suggests that additional ligands are involved. Direct interactions between the PlexA and PlexB receptors, as demonstrated by the co-immuno-precipitation experiments of (Ayoob, 2006), may also contribute to the increased frequency of v'ch1 axon defects observed in double sema2a; plexA and sema2a; plexB mutants compared to single mutants. It is believed that the sensory system provides a valuable platform in which to further investigate this and other issues related to semaphorin function in axon guidance (Bates, 2007).

Drosophila Plexin B is a Sema-2a receptor required for axon guidance

Plexin receptors play a crucial role in the transduction of axonal guidance events elicited by semaphorin proteins. In Drosophila, Plexin A (PlexA) is a receptor for the transmembrane semaphorin semaphorin-1a (Sema-1a) and is required for motor and central nervous system (CNS) axon guidance in the developing embryonic nervous system. However, it remains unknown how Plexin B functions during neural development and which ligands serve to activate this receptor. This study shows that plexB, like plexA, is robustly expressed in the developing CNS and is required for motor and CNS axon pathfinding. PlexB and PlexA serve both distinct and shared neuronal guidance functions. A physical association is observed between these two plexin receptors in vivo, and they can utilize common downstream signaling mechanisms. PlexB does not directly bind to the cytosolic semaphorin signaling component MICAL (molecule that interacts with CasL), but requires MICAL for certain axonal guidance functions. Ligand binding and genetic analyses demonstrate that PlexB is a receptor for the secreted semaphorin Sema-2a, suggesting that secreted and transmembrane semaphorins in Drosophila use PlexB and PlexA, respectively, for axon pathfinding during neural development. These results establish roles for PlexB in central and peripheral axon pathfinding, define a functional ligand for PlexB, and implicate common signaling events in plexin-mediated axonal guidance (Ayoob, 2006).

Semaphorins, along with other families of guidance cues, play key roles in neural development. Through both repulsion and attraction, semaphorins guide neuronal growth cones and thereby promote the establishment of neuronal connectivity and circuit formation. By relaying guidance information to the growth cone cytoskeleton of responding neurons, plexin proteins serve as central signaling components of many semaphorin receptor complexes. Understanding how neurons integrate a complex palette of guidance cue information through the action of related guidance cue receptors is necessary to reveal the molecular mechanisms underlying the steering of neuronal processes during development and also following nerve injury (Ayoob, 2006).

In vertebrates, nine different plexin proteins are known and they are organized into four distinct classes based upon their degree of evolutionary conservation (Plexin A-D); seven of these plexins belong to classes A and B. In the fruit fly this complexity is not as great as the Drosophila melanogaster genome includes only two plexins, one belonging to class A and one to class B (PlexA and PlexB). PlexA functions as a receptor for the transmembrane semaphorins Sema-1a and Sema-1b (Winberg, 1998). In vivo analyses demonstrate that, through the action of PlexA, Sema-1a regulates the defasciculation of motor axon bundles during embryogenesis. Although gain-of-function (GOF) studies strongly suggest that Drosophila PlexB mediates repulsive guidance events in vivo, and in vitro studies demonstrate that vertebrate plexin-B proteins mediate growth cone and COS cell collapse, the consequences of removing PlexB function in Drosophila, or in vertebrates, have not been determined. It is unclear, therefore, how Plexin B proteins function during neural development (Ayoob, 2006).

It is also unclear whether the different classes of plexins play distinct or redundant roles in the establishment of neuronal connectivity. In Drosophila, plexA and plexB are both expressed throughout the nervous system during development, indicating that they are likely to function within the same neuronal classes (Winberg, 1998). When overexpressed in all neurons, both plexA and plexB can produce similar phenotypes, suggesting that these receptors participate in related signaling events. Interestingly, vertebrate plexin A1 and plexin B1 both modulate R-Ras activation through their intrinsic GTPase activating protein (GAP) domains, and this is essential for semaphorin-mediated repulsion in vitro. These data point towards common, or perhaps redundant, signaling mechanisms that may underlie the in vivo functions of A and B class plexin receptors (Ayoob, 2006).

By contrast, although A and B class plexins are highly conserved, many differences exist among proteins belonging to these two plexin classes. Plexin A proteins are functional receptors for transmembrane class 1 semaphorins in Drosophila and class 6 transmembrane semaphorins in vertebrates. Secreted class 3 semaphorins also signal through class A plexins; however, this requires the assembly of a distinct holo-receptor complex that includes either neuropilin 1 or neuropilin 2, obligate co-receptors that serve to facilitate class 3 semaphorin binding and plexin A activation. Plexin B proteins in vertebrates bind to different transmembrane semaphorin ligands, including those from classes 4 and 5; however, no ligand has been identified for Drosophila PlexB. Differences also exist between the downstream signaling events mediated by A and B class plexins. The cytoplasmic domains of Drosophila PlexA and PlexB share a high degree of amino acid sequence identity, yet they appear to differ with respect to the signaling molecules with which they directly associate. For example, although PlexB directly interacts with the small GTPase Rac, PlexA does not. Likewise MICAL, a large cytosolic oxidoreductase that is crucial for semaphorin-mediated repulsion, associates with PlexA but not PlexB. Therefore, PlexA and PlexB may also serve non-overlapping roles during neural development (Ayoob, 2006 and references therein).

This study examined the consequences of disrupting PlexB function for Drosophila neural development, allowing for a direct comparison between PlexB and PlexA axon guidance functions. Using genetic and biochemical analyses, it has been shown that PlexB and PlexA serve distinct and overlapping roles in motor and CNS axon guidance. The similarities observed in PlexA and PlexB functions may be explained by the findings that these receptors can assemble into a heteromultimeric complex, and also that they employ common downstream signaling components to guide axons during development. Finally, plexin interactions observed with different semaphorin ligands are likely to contribute the distinct roles PlexA and PlexB serve in establishing neuronal connectivity (Ayoob, 2006).

Plexin receptors expressed at the leading edge of navigating axonal growth cones receive and transduce instructive signals encoded by semaphorins. Deciphering how plexins translate external stimuli into intracellular responses is paramount for understanding how the nervous system is wired. Analysis of neural development in Drosophila allows for direct functional comparisons between plexins from distinct classes, as Drosophila contains only two plexin proteins. This study has analyzed plexB mutants and compared the phenotypes observed to those found in plexA mutants, detecting both similarities and differences in the LOF phenotypes of these two genes. A direct physical interaction was detected between PlexA and PlexB, and a convergence of these signaling pathways upon the effector molecule MICAL was demonstrated. These two receptors bind to semaphorin ligands from different classes. These results demonstrate that plexins from different classes can respond in vivo to distinct semaphorin ligands, but have the capacity to work cooperatively using common downstream signaling molecules (Ayoob, 2006).

In Drosophila, PlexA is required for the proper defasciculation of motor and CNS axon bundles (Winberg, 1998). This axon-axon repulsion enables individual axons to overcome the adhesive forces holding them together, to separate from each other, and to innervate their appropriate targets. This study examined the role played by PlexB in motor and CNS axon pathfinding during Drosophila embryogenesis and found that plexB mutants display defects in axon fasciculation that dramatically affect pathfinding. Similar to what has been observed in plexA mutants, plexB mutants display a failure of ISNb motor axons to initially separate from the main ISN bundle or, at later stages of ISNb pathway formation, to separate from other ISNb axons. Defasciculation errors similar to those observed in plexA mutants are also observed for the dorsal branch of the SNa in plexB mutants. However, other plexB mutant SNa axon bundles display navigation phenotypes not seen in plexA mutants. These dorsal SNa axons follow an aberrant trajectory to their target, muscle 24, and as a consequence are often unable to reach this post-synaptic partner. In the CNS, however, PlexB and PlexA play distinct roles. Loss of plexA disrupts the contiguity of the outermost bundle of axons, whereas losing plexB causes excessive defasciculation of the medial tract. This differential requirement for plexins in medial and lateral FasII-postive CNS axon bundles is strikingly reminiscent of the specific requirements for differential expression of roundabout (Robo) proteins to regulate the formation of the inner, medial and lateral FasII-positive axon tracts. Determining whether the positioning and consolidation of CNS longitudinal tracts by Robos and plexins are separate or integrated processes will lend insight into how axons respond simultaneously to distinct guidance influences that serve to regulate neuropil organization (Ayoob, 2006).

Although unique axonal fasciculation and pathfinding defects are observed in plexA and plexB mutants, ISNb motor axon phenotypes in these mutants are remarkably similar. This suggests that plexins from different classes may function collaboratively to pattern certain neuronal trajectories. Drosophila provides a robust experimental model with which to examine this issue. As there are only two Drosophila plexins, cross-rescue experiments were performed with plexA and plexB. Expression of plexA in a plexB mutant background significantly reduces the severity of plexA ISNb defects, although it does not fully rescue these defects. plexA expression is, however, unable to rescue the SNa and CNS phenotypes that were observed in plexB, but not plexA, mutants. In the reciprocal experiment, PlexB cannot replace any PlexA function, either in motor axon pathways or in the CNS. Immunoprecipitation and genetic interaction experiments provide an explanation for why PlexB cannot substitute for PlexA. When epitope-tagged versions of PlexA and PlexB were expressed in vivo, immunoprecipitating PlexA brings down PlexB, indicating that these two receptors can associate in a complex in vivo. Furthermore, for ISNb pathway phenotypes, genetic interactions were observed between plexB and MICAL heterozygotes, strongly supporting a requirement for MICAL in PlexB signaling, although these two proteins do not interact directly. It is proposed that PlexB gains access to MICAL through its association with PlexA. Because MICAL is a crucial downstream signaling component for plexin-mediated axonal repulsion, PlexA may be able to substitute in a limited fashion for PlexB through its ability to recruit MICAL and mediate repulsion of ISNb axons. However, the inability of PlexB to substitute at all for PlexA may stem from its inability to directly recruit MICAL (Ayoob, 2006).

Two other transmembrane proteins play important roles in PlexA-mediated axon guidance events and may facilitate the formation of complexes that contain PlexB and PlexA. The catalytically inactive receptor tyrosine kinase Off-track (Otk), which binds to and functions with PlexA in Sema-1a signaling, is also able to associate with two vertebrate plexins from classes A and B. It is unknown whether Otk binds to Drosophila PlexB. However, in the Drosophila CNS, Otk may function separately with PlexA and PlexB. Otk mutants display a disrupted outer Fas-II-positive fascicle, a phenotype specific to plexA, and also a defasciculated middle Fas-II-positive axon bundle, a phenotype specific to plexB. Overexpression of another PlexA signaling component produces phenotypes also seen in plexB mutants. Increasing in all neurons the levels of Gyc76C, a receptor guanylyl cyclase involved in PlexA signaling, produces an SNa pathfinding defect very similar to the 'double turn' SNa phenotype seen in plexB mutants. Future work will reveal whether either of these transmembrane proteins involved in PlexA signaling serve as co-receptors for PlexB ligands and participate in the PlexB signaling cascade (Ayoob, 2006).

There are five semaphorins in Drosophila. Sema-1a and Sema-1b, two class 1 transmembrane semaphorins, bind to PlexA. This study found that AP-tagged versions of the extracellular domains of these transmembrane semaphorins do not bind to PlexB in vitro. However, robust binding of AP-tagged Sema-2a, a secreted semaphorin, was observed to insect cells expressing PlexB. Genetic analysis shows that this interaction is indeed functional, since plexB LOF suppresses a Sema-2a GOF phenotype. These data also suggest that there are additional PlexB ligands. plexB mutants show more severe and complex phenotypes than do the low-penetrance phenotypes reported for Sema-2a mutants. Sema-2b, the other Drosophila secreted semaphorin, is a likely candidate PlexB ligand. Sema-2b resides at cytolocation 53C4 on chromosome 2 and is only separated from Sema-2a by a few genes. Sema-2a and Sema-2b share 70% amino acid identity (84% similarity), and it seems likely this semaphorin duo is a product of a genetic duplication and that these two secreted semaphorins share certain neuronal signaling functions. Sema-2b is expressed in a small subset of neurons within the CNS suggesting that, alone, or in combination with Sema-2a, it is responsible for maintaining the medial bundle of longitudinally projecting CNS axons as a tight fascicle. Consistent with findings for class A and B plexins in vertebrates, it was found that PlexA and PlexB in Drosophila serve as receptors for different classes of semaphorins. This specificity provides a basis for postulating distinct functions for the two Drosophila plexins in motor and CNS axon guidance. Because secreted semaphorins are not tethered to their substrate, as are transmembrane semaphorins, the range over which these cues might act is greater, enabling PlexB to mediate not only axonal defasciculation, but also growth cone steering and surround repulsion (Ayoob, 2006).

In addition to being repulsive axon guidance receptors, plexins also interact homophilically. Therefore, it is possible that in some instances PlexB might function in a semaphorin ligand-independent manner, perhaps even as an adhesive molecule. Proteolytic processing may also regulate PlexB function. The extracellular domains of B-class plexins contain a protease site, located close to the plasma membrane, that is cleaved by a subtilisin-like proprotein convertase. In western blots of Myc-PlexB extracts, in addition to full-length PlexB at 250 kDa, a smaller protein is detected at 150 kDa. The size of this protein is equal to that of the PlexB ectodomain and correlates well with a predicted PlexB protease cleavage product. These bands were also observed in the lysates of S2R+ cells transfected with Myc-PlexB. Conditioned media from these transfected cells contains only the smaller (150 kDa) form of PlexB, presumably the ectodomain released from the membrane and into the media. This proteolytic processing of the PlexB receptor may play a role in the modulation of its activity (Ayoob, 2006).

In conclusion, evidence is presented that plexin B receptors, like plexin A receptors, are crucial for the generation of neuronal connectivity in vivo. The results show that A and B class plexins can regulate similar axon guidance events collaboratively, whereas interactions with distinct classes of semaphorin ligands are likely to mediate receptor-specific functions. Further analysis of how these guidance receptors function in Drosophila will allow for a better understanding of the complex roles played by plexins during neural development, and will define plexin-mediated convergent and divergent signaling events (Ayoob, 2006).

Orr, B. O., Fetter, R. D. and Davis, G. W. (2017). Retrograde semaphorin-plexin signalling drives homeostatic synaptic plasticity. Nature 550(7674): 109-113. PubMed ID: 28953869

Retrograde semaphorin-plexin signalling drives homeostatic synaptic plasticity

Homeostatic signalling systems ensure stable but flexible neural activity and animal behaviour. Presynaptic homeostatic plasticity is a conserved form of neuronal homeostatic signalling that is observed in organisms ranging from Drosophila to human. Defining the underlying molecular mechanisms of neuronal homeostatic signalling will be essential in order to establish clear connections to the causes and progression of neurological disease. During neural development, semaphorin-plexin signalling instructs axon guidance and neuronal morphogenesis. However, semaphorins and plexins are also expressed in the adult brain. This study shows that semaphorin 2b (Sema2b) is a target-derived signal that acts upon presynaptic plexin B (PlexB) receptors to mediate the retrograde, homeostatic control of presynaptic neurotransmitter release at the neuromuscular junction in Drosophila. Further, Sema2b-PlexB signalling regulates presynaptic homeostatic plasticity through the cytoplasmic protein Mical and the oxoreductase-dependent control of presynaptic actin. It is proposed that semaphorin-plexin signalling is an essential platform for the stabilization of synaptic transmission throughout the developing and mature nervous system. These findings may be relevant to the aetiology and treatment of diverse neurological and psychiatric diseases that are characterized by altered or inappropriate neural function and behaviour (Orr, 2017).

Semaphorins are a large family of secreted or membrane-associated signalling proteins and plexins serve as signal-transducing semaphorin receptors. Semaphorin-plexin signalling was initially described as mediating growth cone collapse. But, semaphorin-plexin signalling is far more diverse. Notably, semaphorins and plexins continue to be expressed in the mature brain, where their function remains mostly unknown. Semaphorins have been shown to be synaptic signalling proteins, but the activity of semaphorins has been limited to the control of neuroanatomical synapse formation and elimination. This study demonstrates that semaphorin-plexin signalling achieves retrograde, trans-synaptic control of presynaptic neurotransmitter release and homeostatic plasticity (Orr, 2017).

A well-documented assay was used to induce presynaptic homeostatic plasticity (PHP), applying a sub-blocking concentration of the glutamate-receptor antagonist philanthotoxin-433 (PhTx; 15 μM) to significantly decrease the amplitude of average miniature excitatory postsynaptic potentials (mEPSPs; 0.3 μM [Ca2+]e) or miniature excitatory postsynaptic currents (mEPSCs; 1.5 μM [Ca2+]e). This postsynaptic perturbation induces a significant increase in presynaptic neurotransmitter release (the quantal content) that offsets the postsynaptic perturbation and restores normal muscle excitation. This offsetting increase in presynaptic neurotransmitter release is characteristic of PHP1. When this assay was used in larvae containing a null mutation in either the sema2b gene (sema2bC4) or the PlexB gene (PlexB^KG0088), PHP was blocked. Consistent with this being a loss-of-function phenotype, heterozygous mutations (either sema2b/+ or PlexB/+) have normal PHP. Remarkably, a double-heterozygous mutant combination of sema2b/+ and PlexB/+ blocks PHP, consistent with both genes acting in concert to drive the expression of PHP (Orr, 2017).

The long-term maintenance of PHP was investigated and the involvement of other semaphorin or Plexin gene family members. Deletion of a non-essential glutamate-receptor subunit (GluRIIA) induces a long-lasting form of PHP1. Long-term PHP is blocked in a sema2b;GluRIIA double mutant as well as in GluRIIA larvae expressing transgenic RNA interference (RNAi) to knockdown PlexB selectively in motor neurons. Next, the effect of mutations was separately tested in all of the remaining semaphorin and Plexin genes encoded in the Drosophila genomet. The sema2b and PlexB mutants are the only mutants that show disruption of PHP (Orr, 2017).

Tissue-specific RNAi and transgenic rescue experiments were performed. Expression of UAS-Sema2b-RNAi in motor neurons (OK371-Gal4) had no effect on PHP, whereas expression in muscle (BG57-Gal4) blocked PHP. In addition, expression of UAS-sema2b in muscle rescues PHP in the sema2b-mutant background. Consistent with these data, sema2b was found to be expressed in muscle and Sema2b protein, expressed under endogenous promoter sequences, concentrates at postsynaptic membranes. Next, it was shown that motor neuron-specific expression of UAS-PlexB-RNAi blocks PHP, whereas muscle-specific expression does not. Motor neuron-specific expression of a previously characterized UAS-PlexBDN dominant-negative transgene, lacking the intracellular signalling domain, blocks PHP. RNA-sequencing analysis of purified motor neurons demonstrates PlexB expression in motor neurons. Finally, motor neuron-specific expression of a PlexB-myc transgene shows that PlexB traffics to the presynaptic nerve terminal. Taken together, these data indicate that Sema2b is a ligand originating in the muscle that acts via presynaptic PlexB to drive expression of PHP (Orr, 2017).

If Sema2b is a retrograde signal that acts upon the presynaptic PlexB receptor, then it should be possible to reconstitute this retrograde signalling by acute application of Sema2b protein. Purified Sema2b protein was acutely applied to the neuromuscular junction (NMJ) of sema2b mutants following PhTx treatment to induce PHP. Sema2b protein (100 nM) was found to completely restores PHP in the sema2b mutant, but fails to restore PHP in the PlexB mutant. In addition, application of Sema2b protein is sufficient to potentiate baseline release, and this effect is also dependent upon PlexB. Finally, a membrane-tethered UAS-sema2b transgene, expressed in muscle, fails to rescue PHP, even though it is concentrated on the postsynaptic membranes. Together, these results indicate that Sema2b is a secreted, postsynaptic ligand that acts upon presynaptic PlexB to enable the expression of PHP. The possibility is acknowledged that PlexB could require a presynaptic co-receptor of, as yet, unknown identity (Orr, 2017).

Given that acute application of Sema2b protein rescues PHP in the sema2b mutant, the failure of PHP in sema2b-mutant larvae cannot be a secondary consequence of altered NMJ development. Nonetheless, Sema2b-PlexB signalling is required for normal NMJ growth. Axon-targeting errors are rare at muscles 6/7, analysed at the third instar larval stage. This study demonstrated that the NMJs in sema2b and PlexB mutants are composed of fewer, larger synaptic boutons with no change in total NMJ area. The abundance of the active-zone-associated protein Bruchpilot (Brp) is unaltered in the sema2b mutant and the sema2b/+;;PlexB/+ double-heterozygous larvae, both of which block PHP. There is a significant decrease in total Brp staining in the PlexB mutant, an effect of unknown consequence. Qualitatively, the ring-like organization of Brp staining was similar across all genotypes, indicative of normal active-zone organization. Finally, there is no consistent difference in synapse ultrastructure across genotypes. Therefore, the Sema2b-PlexB-dependent control of bouton size may be a separate function of Sema2b-PlexB signalling, analogous to anatomical regulation by semaphorins in mammalian systems (Orr, 2017).

PHP occurs through the potentiation of the readily releasable pool (RRP) of synaptic vesicles. Application of PhTx induces a doubling of the apparent RRP in wild-type larvae, an effect that is disrupted in both sema2b and PlexB mutants. Failure to potentiate the RRP is also shown as a failure to maintain the cumulative EPSC amplitude after PhTx application. A strong genetic interaction was subsequently shown with a mutation in the presynaptic scaffolding gene rab3-interacting molecule (rim), a PHP gene. Heterozygous mutations in rim, or in sema2b or PlexB have no effect on PHP. However, double-heterozygous combinations of rim/+ with either sema2b/+ or PlexB/+ strongly impaired the expression of PHP (sema2b/+,rim/+) or abolished PHP (rim/+;;PlexB/+). These data do not, however, reflect direct signalling between PlexB and Rim (Orr, 2017).

To define how PlexB could modulate the RRP, known downstream signalling elements were tested. Mical is necessary for PHP. In Drosophila a single mical gene encodes a highly conserved multi-domain cytoplasmic protein that mediates actin depolymerization, achieved through redox modification of a specific methionine residue (Met44) in actin. Notably, prior genetic evidence has placed Mical downstream of both PlexA and PlexB signalling during axon guidance (Orr, 2017).

An analysis of multiple mical mutations in larvae as well as transgenic rescue animals demonstrates that mical is necessary presynaptically for PHP. Mical protein is present presynaptically and presynaptic expression of a Mical-resistant UAS-Actin5C transgene, which interferes with Mical-mediated actin depolymerization, blocks PHP. This transgenic protein also concentrates within presynaptic boutons. Additional experiments reveal that the homeostatic expansion of RRP is blocked in mical mutants and when Mical-resistant UAS-Act5 is expressed presynaptically. Strong genetic interactions were found between mical and both the PlexB and rim mutants. Finally, anatomical experiments demonstrate that active zones are normal in the mical mutant, including in both light and electron microscopy experiments. It is proposed that Mical enables PlexB-mediated control of the RRP through the regulation of presynaptic actin (Orr, 2017).

For half a century, evidence has underscored the importance of target-derived, retrograde signalling that controls presynaptic neurotransmitter release1. Gene discovery, based on forward genetics, indicates that PHP is controlled by the coordinated action of at least three parallel signalling systems. These data regarding Sema2b, PlexB and Mical can be generalized, then semaphorin-plexin signalling could represent a platform for retrograde, trans-synaptic, homeostatic control of presynaptic release, thereby stabilizing synaptic transmission and information transfer throughout the nervous systems of organisms ranging from Drosophila to humans (Orr, 2017).

In Vivo Calcium Signaling during Synaptic Refinement at the Drosophila Neuromuscular Junction

Neural activity plays a key role in pruning aberrant synapses in various neural systems, including the mammalian cortex, where low frequency (0.01 Hz) calcium oscillations refine topographic maps. However, the activity-dependent molecular mechanisms remain incompletely understood. Activity-dependent pruning also occurs at embryonic Drosophila neuromuscular junctions (NMJs), where low frequency Ca2+ oscillations are required for synaptic refinement and the response to the muscle-derived chemorepellant Sema2a. This study examined embryonic growth-cone filopodia in vivo to directly observe their exploration and to analyze the episodic Ca2+ oscillations involved in refinement. Motoneuron filopodia repeatedly contacted off-target muscle fibers over several hours during late embryogenesis, with episodic Ca2+ signals present in both motile filopodia as well as in later-stabilized synaptic boutons. The Ca2+ transients matured over several hours into regular low frequency (0.03Hz) oscillations. In vivo imaging of intact embryos of both sexes revealed that the formation of ectopic filopodia is increased in Sema2a heterozygotes. Genetic evidence is provided suggesting a complex presynaptic Ca2+-dependent signaling network underlying refinement that involves the phosphatases Calcineurin and PP1, as well the serine/threonine kinases CaMKII and PKA. Significantly, this network influenced the neuron's response to the muscle's Sema2a chemorepellant, critical for the removal of off-target contacts (Vonhoff, 2017).

As Ca2+ signals were detected at both ectopic and native motoneuron terminals during the critical period for synaptic refinement, the effect of knocking-down Ca2+ channels on synaptic connectivity and development was tested. In separate experiments, RNAi constructs were expressed pan-neurally during embryonic and larval development to target each of the known genes that encode α subunits of voltage-gated Ca2+ channels (VGCCs): the Ca(v)1 channel gene Dmca1D, the Ca(v)2.1 gene cacophony (also known as Dmca1A), and Ca(v)3 gene Dmca1G. Compared with control larvae, animals expressing RNAi-knockdowns of either cacophony or Dmca1G had an elevated frequency of ectopic contacts. By contrast, RNAi-knockdown of Dmca1D did not have a miswiring phenotype, although Dmca1D-RNAi has been shown to reduce Ca2+ currents in Drosophila larval motoneurons. An elevated ectopic frequency was also observed in larvae expressing the RNAi construct for the auxiliary Ca2+ channel β subunit that is required for channel function (Vonhoff, 2017).

PROTEIN STRUCTURE

Amino Acids - 724

Structural Domains and Evolutionary Homologs

Four Semaphorin family members have been identified in insects: G-Sema I from grasshopper, T-Sema I from Tribolium, and Sema I and Sema II from the fly. All four insect Semaphorins share a highly conserved extracellular domain of 500 amino acids characterized by 16 conserved cysteines, one conserved potential N-linked glycosylation site, and numerous blocks of conserved amino acids. Each protein begins with a signal sequence and is likely to encode a transmembrane or secreted protein. Drosophila Sema I protein is characterized by the presence of a transmembrane domain, while Sema II has no transmembrane domain but in its place a single C2 type immunoglobulin domain (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 semaphorin I . Class II has a single member, Drosophila Sema-II. Class III consists of secreted vertebrate semaphorins, including Sema D (Coll-I/Sema-III), 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-II. 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).

date revised: 22 November 2022

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