semaphorin-1a

REGULATION

Protein Interactions

The Semaphorins comprise a large family of secreted and transmembrane proteins, some of which function as repellents during axon guidance. Semaphorins fall into seven subclasses. Neuropilins are neuronal receptors for class III Semaphorins. In the immune system, VESPR, a member of the Plexin family, is a receptor for a viral-encoded Semaphorin. Two Drosophila Plexins, both of which are expressed in the developing nervous system, have been identified. Evidence is presented that Plexin A is a neuronal receptor for class I Semaphorins (Sema 1a and Sema 1b), and it is shown that Plexin A controls motor and CNS axon guidance. Plexins, which themselves contain complete Semaphorin domains, may be both the ancestors of classical Semaphorins and binding partners for Semaphorins (Winberg, 1998).

Sema 1a is expressed by neurons and is required for appropriate defasciculation. Loss-of-function analysis for this gene does not indicate whether Sema 1a functions as a ligand or as a receptor. However, misexpressing Sema 1a on muscles repels motor axons, demonstrating that Sema 1a is able to act as a target-derived repulsion cue (Yu, 1998). If PlexA is the receptor for Sema 1a, then reducing PlexA expression levels in the presence of ectopic Sema 1a ligand should suppress the severity of the gain-of-function repulsion phenotypes. Two different GAL4 enhancer trap lines were used to misexpress Sema 1a on muscle subsets. The first, H94-GAL4, is highly expressed by muscle fibers 6 and 13, and moderately by muscle 12 (genetic rescue data suggest that it is also expressed by some motor neurons at a very low level, although this has never been directly visualized. Using H94-GAL4 to drive UAS-Sema1a in these muscles disrupts their innervation by ISNb axons, with the strongest effect seen at muscle 13. A second line, F63-GAL4, was used to drive UAS-Sema1a specifically in muscles 6 and 7, thereby inhibiting innervation at the muscle 6/7 cleft (F63 is not expressed by motor neurons). For both GAL4 lines, the inhibitory effect of muscle-derived Sema 1a is suppressed in PlexA Df heterozygotes. This dominant suppression of the Sema1a gain-of-function suggests that neuronally expressed PlexA acts downstream of Sema 1a in mediating repulsion (Winberg, 1998).

Ectopic expression also provided a means to test for interactions between PlexA and Sema 1b, another Drosophila protein similar to Sema 1a (Yu, 1997). H94-GAL4 and F63-GAL4 were used to test whether Sema 1b can also act as a muscle-derived repellent. Sema 1b was found to be as capable as equally capable Sema 1a in repelling motor axons. Likewise, reducing the gene dose of PlexA suppresses to a similar extent the guidance defects caused by misexpression of Sema 1b. Based on the similarity of structure and sequence, as well as parallel gain-of-function phenotypes and suppression, it is proposed that Sema 1b may serve as an additional ligand for PlexA (Winberg, 1998).

Given the model that PlexA mediates repulsive guidance and thus drives defasciculation, it was asked whether removal of the major motor axon cell adhesion molecule, Fasciclin II, would genetically suppress PlexA mutant phenotypes. In agreement with the model, all of the ISNb motor axon PlexA phenotypes (but not the SNa mutant phenotypes) and the CNS PlexA phenotypes are partially suppressed when one copy of the FasII gene is removed (Winberg, 1998).

Genetic analysis provides strong evidence that PlexA is a necessary component of the Sema 1a signaling pathway and is likely to function as a Sema 1a receptor. Moreover, it suggests that PlexA may have additional ligands, including Sema 1b. These possibilities were tested in a heterologous expression system using alkaline phosphatase (AP) fusion proteins in binding assays on membranes from COS cells transiently transfected with a full-length PlexA construct. Both AP-Sema 1a and AP-Sema 1b bind to PlexA-expressing membranes at significantly higher levels than to mock-transfected membranes or to membranes containing another Drosophila repulsive axon guidance receptor, Robo 1. No specific binding occurs between PlexA and other Semaphorins, including Sema III-AP, AP-Sema E, and AP-Sema B, nor between PlexA and AP-Beaten path. It was also asked whether Sema-Plexin binding, like Plexin homophilic interaction in Xenopus, requires the presence of divalent cations. Binding is eliminated by the inclusion of chelating agent in the ligand supernatant, or by the omission of divalent cations from the wash buffer. Binding is preserved in the presence of either Mg2+ or Ca2+. In contrast, binding of Semaphorins to Neuropilins does not appear to require calcium or magnesium (Winberg, 1998).

DEVELOPMENTAL BIOLOGY

Embryonic

In the central nervous system, Sema-1a protein is first detected in stage 11 [Stages] embryos, just prior to axonogenesis, in a continuous stripe along the midline. At late stage 16, following the establishment of commissural and longitudinal pathways and also the basic pattern of motor projections in the periphery, robust levels of Sema-1a protein are observed in most CNS commissural and longitudinal pathways, as well as in the intersegmental nerve (ISN) and segmental nerve (SN) roots exiting the CNS. Sema-1a protein is observed at low levels in the midline in the ventral portion of CNS, posterior to the posterior commissure and consistent with expression on the ventral unpaired median (VUM) neurons. In the periphery, Sema-1a is found in all five motor axon branches: ISN, ISNb (formally SNb), ISNd (formally SNd), SNa, and SNc. Though the resolution of Sema-1a protein localization in the periphery is somewhat diffuse, Sema-1a protein can still be detected in several of the pathways that make up these motor axon branches. For example, Sema-Ia protein is detected on both dorsal and lateral branches of SNa and on ISNb in branches that include the identified motor axons RP3 and RP5. Sema-1a protein cannot be detected in peripheral sensory neurons of the dorsal or ventral clusters; however, very low levels of Sema-1a are observed in the lateral cluster of chordotonal sensory organs. In addition to neuronal localization, Sema-1a is also observed, starting at stage 14, at segment boundaries in the position of the muscle attachment sites of the ventral lateral muscles 12, 13, 6, and 7. Sema-1a protein or a maternal contribution of Sema-1a mRNA could not be detected in very early wild-type embryos (Yu, 1998).

Larval and Pupal

Semaphorins have been intensively studied for their role in dendritic and axonal pathfinding, but less is known about their potential role in synapse formation. In the adult giant fiber (GF) system of fruit flies, it has been shown that transmembrane Semaphorin 1a is involved in synapse formation in addition to its role in guidance during pathfinding. Cell-autonomous rescue experiments show that Sema-1a is involved in assembly of a central synapse and that it is required both pre- and post-synaptically. Pre- but not post-synaptic gain-of-function Sema-1a is able to disrupt the GF-motor neuron synapse and the phenotype depends on a proline-rich intracellular domain that contains a putative Enabled binding site. Thus Sema-1a may signal via Enabled. It is suggested that transmembrane Sema-1a is part of a bi-directional signaling system that leads to the formation of the GF synapse and possibly acts as both a ligand and a receptor (Godenschwege, 2002).

Thus, in addition to its better-known role in pathfinding, Sema-1a also functions during synapse formation. There were two main phenotypes in the sema-1a loss-of-function mutants. In half of the sema^P1 specimens, the GF axons had pathfinding defects and did not exit the brain. Ectopic expression of three sema-1a constructs in the brain could rescue these pathfinding defects with similar probability, suggesting that Sema-1a acts as a ligand (its well-known role) during giant fiber pathfinding (Godenschwege, 2002).

This study focused on the subset of specimens in which the GFs did exit the brain and reached the target area in the thorax. About half of these GFs either stalled at the site of synapse formation or passed by the target area altogether. These GFs were close to (or in contact with) their postsynaptic target, but the synaptic connectivity between these two neurons was disrupted, suggesting a novel role for Sema-1a in synapse formation. Whereas the GF did not grow along the medial TTMn dendrite [Note: TTM (tergotrochanteral motor neuron is the jump motorneuron)} and thus lacked its characteristic lateral bend, the anatomy of the postsynaptic dendrite was normal (Godenschwege, 2002).

The sema^P1 phenotype in the GF system is superficially similar to the phenotypes described for the ISNb motor neuron in the neuromuscular system of embryos. However, rescue experiments as well as the gain-of-function studies suggest that the functions of Sema-1a at the adult central synapse are distinct from those seen at the embryonic neuromuscular junction (NMJ). (1) As shown by rescue experiments in the embryonic NMJ, Sema-1a is required only presynaptically. In the GF system, it was required both pre- and post-synaptically. (2) There is no gain-of-function phenotype when Sema-1a is overexpressed presynaptically in embryonic motorneurons, but overexpression of Sema-1a in the GF has pronounced effects on the synapse. (3) Ectopic postsynaptic expression in the muscle has a disruptive effect on the anatomy of the motor neuron in embryos. In contrast, postsynaptic overexpression of Sema-1a in the giant fiber system does not disrupt the GF-TTMn synapse. These contradictory results suggest that Sema-1a acts differently in the two systems (Godenschwege, 2002).

In the embryonic neuromuscular system, the anatomical results are interpreted as evidence for a role of Sema-1a in de-fasciculation of the ISNb motorneurons before they enter the target region and synapse with the muscle. In the GF, it seems unlikely that Sema-1a has a function in de-fasciculation, since overexpression of Sema-1a in the GF does not promote de-fasciculation but rather results in a bendless-like phenotype. The gain-of-function bendless-like phenotype probably results from the destabilizing effect of Sema-1a on the synapse. This was supported by the finding that overexpression of Sema-1a in the GF after synapse formation causes retraction of the GF presynaptic terminal. Therefore, overexpression of Sema-1a throughout giant fiber development might prevent the giant synapse formation by its destabilizing effect on the synapse. In spite of the disruptive presynaptic gain-of-function phenotypes, the rescue experiments suggest a presynaptic requirement in the assembly of the GF-TTMn synapse. Therefore, it is proposed that presynaptic Sema-1a has a function during target recognition but needs to be removed from the GF for synaptogenesis to proceed normally. In sharp contrast to the embryonic NMJ, where Sema-1a is only required presynaptically for normal synapse formation, postsynaptic expression of Sema-1a efficiently rescues GF-TTMn defects in sema^P1 flies. Since this requires physical contact between the GF and the TTMn, these results suggest that Sema-1a in the TTMn has a function in the assembly of the giant synapse after pathfinding (Godenschwege, 2002).

These results suggest that bi-directional signaling is required for assembly of the giant synapse. The clearest evidence for this bi-directionality comes from the cell-autonomous rescue experiments. Full-length and truncated Sema-1a can rescue the GF-TTMn defects by expression either pre- or postsynaptically, indicating that Sema-1a functions as a ligand on both sides of the synapse. This is supported by the finding that the rescue probability upon simultaneous pre- and post-synaptic expression of full-length Sema-1a is increased. The additive nature of pre- and post-synaptic rescue probability argues that the bi-directional interaction is not necessarily homophilic, but rather that Sema-1a serves as a ligand for unknown receptors on both sides of the synapse (Godenschwege, 2002).

The idea that Sema-1a may also function as a receptor is supported by findings in both the rescue and the gain-of-function experiments. (1) Sema^Deltacyt (with a deleted cytoplasmic domain) is less potent than Sema^WT in rescuing the synaptic defects in the target area. This difference may be attributed to the functional requirement of a cytoplasmic domain, suggesting that Sem-1a may work as a receptor during GF-TTMn synapse assembly. (2) The overexpression data in a wild-type background further supports a role for the Sema-1a cytoplasmic domain. The disruptive, bendless-like, gain-of-function phenotype is cell-autonomous and is found only with full-length Sema-1a. (3) The cytoplasmic domain of Sema-1a contains a putative Enabled binding site (LPQP). Enabled family proteins influence growth cone behavior by regulating actin dynamics. A small deletion removing the putative Enabled binding site (LPQP) abolishes Sema-1a's ability to induce the gain-of-function phenotype, suggesting that the presynaptic repulsive effect of Sema-1a may be mediated by signaling via Enabled. A role for Enabled is further supported by the finding that the reduction of ena gene dosage suppresses the Sema-1a gain-of-function phenotype, suggesting that Sema-1a may function as a receptor and signal via Enabled in the giant fiber (Godenschwege, 2002).

Whereas the rescue experiments suggested a requirement of Sema-1a function pre- and postsynaptically, the gain-of-function experiments showed a repulsive effect of Sema-1a presynaptically but not postsynaptically. This shows an asymmetry in Sema-1a function on the two sides of the synapse, which could be due to the fact that Enabled signaling has different effects in the GF and the TTMn. An alternative possibility is that Sema-1a involves different signaling pathways pre- and postsynaptically. In vertebrates, Sema-4c and Sema-4f bind to PSD-95, and Sema-4c interacts with the neurite outgrowth-related protein Norbin as well. Both proteins are involved in the assembly of the post-synapse and synaptic plasticity. Notably, the C-terminal of Sema-1a contains a motif (VYL) that can be recognized by class II PDZ domains and would allow Sema-1a to bind to a variety of cytoplasmic molecules. Therefore, Sema-1a's postsynaptic function may be mediated through a PDZ binding motif, which would be distinct from signaling presynaptically via Enabled (Godenschwege, 2002).

In summary, it is proposed that during target recognition, Sema-1a is present presynaptically as well as postsynaptically. The bi-directional signaling of Sema-1a and unknown receptors on both sides of the synapse may trigger the switch from pathfinding to synaptogenesis. It is speculated that Sema-1a functions presynaptically as a repulsive receptor when it reaches its target, thereby stopping or slowing the GF growth cone. Subsequently, for synaptogenesis to proceed, Sema-1a on the GF needs to be removed or down-regulated from the surface, so as not to disrupt synapse formation with its repulsive effects. In contrast, there is no requirement for removal of Sema-1a from the postsynaptic side, and Sema-1a has a function in the GF-TTMn synapse assembly. Bi-directional signaling of Sema-1a may cause the GF to grow along the TTMn dendrite and thereby promote synapse formation. Thus it ia thought that Sema-1a has a bi-directional and bi-functional role in the assembly of the giant synapse (Godenschwege, 2002).

Targeted expression of shibire^ts and semaphorin 1a reveals critical periods for synapse formation in the giant fiber of Drosophila

In order to determine the timing of events during the assembly of a neural circuit in Drosophila, expression of the temperature-sensitive shibire gene was targeted to the giant fiber system and then endocytosis was disrupted at various times during development. The giant fiber retracts its axon or incipient synapses when endocytosis is blocked at critical times, and four phases were perceived to giant fiber development: an early pathfinding phase, an intermediate phase of synaptogenesis, a late stabilization process and, finally, a mature synapse. By co-expressing shibire^ts and semaphorin 1a, evidence was provided that Semaphorin 1a is one of the proteins being regulated by endocytosis and its removal is a necessary part of the program for synaptogenesis. Temporal control of targeted expression of the semaphorin 1a gene shows that acute excess Semaphorin 1a has a permanent disruptive effect on synapse formation (Murphey, 2003).

The giant fiber (GF) system of the fly consists of a large interneuron that controls the visually evoked escape behavior through its synaptic contacts with thoracic motoneurons. Anatomical studies have provided the outlines of the development of this circuit. The dendrites of the jump motoneuron (TTMn) grow into the target area at the beginning of pupal development, before the axon of the GF reaches the thorax. The GF initiates growth in the late larval stage and the axon leaves the brain and reaches the target area in the second thoracic neuromere by ~25% of pupal development. The growth cone of the GF appears to contact the TTMn dendritic growth cone at this time, and during a synaptogenic phase (~25%-50% of pupal time) the GF elaborates a lateral 'bend' along the TTMn that becomes the presynaptic terminal and by 40% of pupal development the two neurons are dye-coupled (Murphey, 2003).

Blocking endocytosis seems on first examination to be a relatively blunt instrument for the analysis of nervous system development because of the wide variety of functions it might disrupt. However, two distinct effects have helped dissect pupal development of the GF system. First, blocking endocytosis causes retraction and subsequent regeneration of the GF, which alters the timing of axon growth and disrupts the resulting structure and function. Second, a role for membrane trafficking was highlighted by an interaction between sema1a and shibire^ts; this interaction created large vesicles in the axon terminal. Blocking endocytosis at the time of synapse formation appears to enhance the disruptive effect of Sema1a on synapse formation. This supports the idea that Sema1a, in its role as a repulsive receptor, must be removed from the growth cone during synaptogenesis. The interaction with shibire^ts suggests that this is likely to be regulated by dynamin-dependent endocytosis (Murphey, 2003).

Targeted blockade of endocytosis has direct effects on axon growth and retraction, presumably by disrupting the recycling of membrane in a rapidly growing axon terminal. It has been shown that neurons from mutant shi^ts1 animals grown in culture and then shifted to the restrictive temperature undergo collapse of growth cones, cessation of axon outgrowth and axon retraction. Shifting back to the permissive temperature leads to a resumption of growth and a rebound of growth rate. The temperatures used and the temperature shift paradigms employed in vitro are identical to those used to assay the timing of developmental events of the giant fiber in vivo (Murphey, 2003).

When endocytosis is blocked during pathfinding the axons retract during the temperature shift and when returned to the permissive temperature regenerate and overgrow the target area (phase I). By contrast, temperature shifts which correspond to the time that the GF is being transformed from growth cone to synapse (phase II) produce a different effect. GFs retract but when examined in adults the axons did not overgrow the target area but rather stopped in the target area and lacked the lateral bends. The initial effects induced by blocking endocytosis during both phase I and II are likely to be caused by the retraction of the axon and the subsequent heterochronic growth of the GF. However, the difference of the responses (overgrowth versus bendless-like) between phase I and II cannot be attributed directly to the block of endocytosis, but are more likely to be attributed to the different developmental states of the GF when heterochronic regeneration occurs. One relevant difference may be that in phase I most GFs have not contacted the target area and in phase II most GFs have contacted the targets. This means that the heterochronic growth induced in phase I results in naïve GFs that approach the target area with a delay, while heterochronic growth in phase II results in the re-generation of 'experienced' GFs. Possibly the GF loses its ability to regenerate the GF-TTMn synapse after it has contacted the target resulting in the bendless-like phenotype (Murphey, 2003).

Finally, blockade of endocytosis in phase III reveals a distinct defect. The function of the synapse is disrupted by temperature shifts although the structure remained normal. This distinguishes a stabilized synapse from a mature synapse. Possibly the block of endocytosis during phase III disrupts trafficking of receptors/ligands that are involved in maturation of the giant synapse. For example, Fasiclin 2 and Wingless/Frazzled have been shown to be required for maturation of the neuromuscular junction and correct dynamin-dependent trafficking is required for normal synaptogenesis (Murphey, 2003).

It has been suggested that Sema1a must be removed from the presynaptic terminal in order for synaptogenesis to proceed correctly, but the exact timing and mechanism for these events have not been examined. The acute presence of Sema1a during synapse formation is shown to have a lasting effect that prevents the regeneration of a functional synapse. The temporal aspects of Sema1a function, independent of endocytosis, were examined by taking advantage of the temperature sensitivity of the UAS constructs. Overexpression of Sema1a during synapse formation (phase II) causes the majority of axons to terminate in bendless-like structure and exhibit weak synaptic connections, while the acute presence of Sema1a in phase I or phase III has only minor effects. This suggests that removal of Sema1a is crucial for synaptogenesis. The sensitivity to Sema1a overexpression overlaps the time that the GF first contacts its targets and becomes dye-coupled to them, suggesting that Sema1a plays a role in the transition from growth cone to synapse. Interestingly, the bendless mutant causes phenotypes similar to those seen when Sema1a is overexpressed in the present experiments. When bendless was cloned and shown to be a ubiquitin conjugase it was speculated that the bendless mutant may be affecting the lifetime of Sema1a on the GF growth cone. The finding that Sema1a trafficking is involved in the assembly of the GF-TTMn synapse and the recent realization that ubiquitin can function to regulate trafficking of membrane proteins suggest that Sema1a trafficking may be regulated by Bendless (Murphey, 2003).

Endocytosis plays an important role in ligand-dependent receptor responses that serve as a mechanism for the regulation of signal strength in a variety of signaling pathways. It is proposed that during the transition from growth cone to synapse, Sema1a, which functions as a receptor on the GF growth cone, encounters its ligand and this slows the progress of the growth cone as a first step in the transition. However, the repulsive signaling of Sema1a must be downregulated because it is disruptive for subsequent events in the formation of the synapse and it is therefore normally removed through a dynamin-dependent receptor-mediated endocytosis. When UAS-sema1a is combined with UAS-shibire^ts in a genetic interaction experiment, simultaneous overexpression of Sema1a and the block of endocytosis exaggerates the disruptive effects of Sema1a. One effect is greater retraction of the axon presumably by enhancing the total amount of the repulsive receptor (Sema1a) present on the surface of the presynaptic cell. A second effect is the accumulation of large vesicles in the axon terminal. An interpretation is that the unusually high levels of Sema1a at the surface activates excessive receptor-mediated endocytosis. This may cause a vesicular 'traffic jam' in the growth cone, thereby disrupting the ability to carry out normal functions. These vesicular traffic jams are consistent with other experiments on the GF system that show similar phenotypes. For example, blocking retrograde transport by expression of a truncated version of the P150^Glued component of the dynein/dynactin motor also causes the formation of large vesicles in the GF terminal. Although the vesicles seen in these various genotypes cannot be directly linked to each other, the common phenotype makes it seem likely that a common membrane trafficking pathway involved in synapse formation is being interrupted. Markers for various aspects of the endosomal system in Drosophila will eventually allow the identification of the origin of these vesicles and link the various genotypes together in a model of receptor trafficking and synapse formation (Murphey, 2003).

In vertebrate neurons, semaphorin signaling has been linked to endocytosis during growth cone guidance and growth cone collapse. Sema3a serves as a ligand for the plexin/neuropilin receptor complex and has been shown to stimulate endocytosis during growth cone collapse. Moreover this is a Rac1-mediated process because Sema3a and Rac1 are associated with vesicles after Sema3a treatment and Rac1 is required for endocytosis of growth cone membrane during growth cone collapse. Although Sema3a is working as a ligand in vertebrate neurons and Sema1a is working as a receptor in the GF, there are a number of striking parallels between the vertebrate work and the Drosophilawork. In both cases, semaphorin and endocytosis are linked and in both cases Rac1 is involved in growth cone structure and behavior. Overexpression of the small GTPase Rac1 disrupts the termination of the GF and caused the accumulation of large vesicles in the terminal. Although Rac1 has not been directly linked to the semaphorin effects, the similarity between the GF phenotypes in these various experiments is consistent with the vertebrate work. The involvement of semaphorins, Rac1 and endocytosis in growth cone repulsion in vertebrate neurons and in the transition to synapse formation in the DrosophilaGF system highlights the similarities between the systems. Since synapse formation requires that growth cones slow or stop as they invade a target region, it seems likely that the growth cone guidance machinery has been commandeered to regulate the initial stages of synaptogenesis (Murphey, 2003).

Surprisingly, the appearance of large vesicles in the GF in the interaction experiment between Sema1a and endocytosis is delayed with respect to the temperature shift since no vesicles are detected immediately after the temperature shift but rather the vesicles emerge as pupal development proceeds. There are numerous suggestions that defects in membrane trafficking are linked to neurodegeneration and that these vesicles in the GF may be a prelude to synaptic degeneration; this possibility is being explored (Murphey, 2003).

Effects of Mutation or Deletion

Sema-Ia^PI and Sema-Ia^P2 are semi-lethal, exhibiting 1% and10% escaping adults, respectively. These escapers do not show observable morphological defects (Yu, 1998).

Since Sema-1a is found in most, if not all, motor axons, Sema-Ia 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. These trajectories were examined in filleted stage 16/17 embryos using mAb 1D4. In Sema-1a ^P1 mutant embryos, 87% of the ISNb pathways are abnormal. 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 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 49% of hemisegments in Sema-Ia^P1 mutant embryos the ISNb stalls, failing to extend from the external surface of VLMs 6 and 7 to the internal surface of VLMs 12 and 13. Most of these stalled ISNb branches terminate between muscles 6 and 13 (39% of hemisegments); however, some ISNb pathways stall at a more ventral position, between muscles 6 and 7 (10% of hemisegments). ISNb stalls are almost always observed to terminate in these discrete locations, and no wandering of aberrant ISNb axons is observed within the ventral muscle field. In a small but significant fraction of hemisegments in SemaI^P1 mutant embryos (7%), ISNb is observed to undergo a fusion bypass with the ISN. In these fusion bypass events, the ISNb bypasses the ventral muscle field and extended along the ISN at least to the dorsal level of the lateral muscles. Synaptic arborizations between muscles 6 and 7 are also abnormal in 35% of hemisegments where they are present. These abnormal arborizations are substantially smaller and thinner than those normally observed in wild-type embryos. Finally, the ISNd branch is defective, either missing or severely truncated and thinner than normal, in 36% of hemisegments in Sema-Ia^P1 mutants as compared to 9% in wild type. Therefore, Sema-Ia is required for correct ISNb and ISNd pathfinding and for the formation of specific synaptic arborizations (Yu, 1998).

In Sema-1a ^P1 mutant embryos, highly penetrant SNa pathway defects are also observed. The SNa and SNc branches constitute the SN branch. After exiting the CNS, the SNc normally defasciculates from the SNa and subsequently innervates ventral external muscles. The SNa then extends dorsally to innervate several lateral muscles (LMs). In the vicinity of the LMs, the SNa contains two major branches. The major dorsal SNa branch extends between muscles 22 and 23 and subsequently bifurcates at a characteristic choice point, extending one process to the posterior, across muscle 23, that then extends dorsally along muscle 24. A second process extends dorsally between muscles 22 and 23. The major lateral branch of SNa extends to the posterior to contact muscles 5 and 8 (Yu, 1998).

As is observed in Sema-1a ^P1 mutant ISNb pathways, these mutant SNa pathways also exhibited discrete defects that reflect an inability of motor axons in this pathway to extend past specific choice points. These defects primarily affect the dorsal, not the lateral, SNa branch. The dorsal branch of the SNa often stalls at a characteristic location between muscles 22 and 23. This is the choice point where SNa motor axons that innervate muscle 24 normally defasciculate from this dorsal branch, extend to the posterior across muscle 23, and then extend dorsally along muscle 24. In some dorsal SNa branches in Sema-1a ^P1 mutant embryos, this choice point is navigated correctly; however, the motor axon that innervates muscle 24 fails to extend dorsally after reaching muscle 24. As was observed in Sema-1a ^P1 mutant ISNb branches, mutant SNa branches do not wander in the vicinity of the lateral muscle field. In addition to these SNa defects, the SNc is observed to be defective in 11% of mutant hemisegments, compared to 1% in wild type. Finally, transverse nerve defects are observed in 20% of hemisegments in Sema-1a ^P1 mutant embryos, in some cases resulting in the establishment of ectopic synapses on VLMs (Yu, 1998).

In addition to motor pathway defects, Sema-1a ^P1 mutants exhibit highly penetrant and specific CNS defects. mAb 1D4 reveals that Fas II is expressed on a subset of CNS longitudinal axon connectives. There are three Fas II-expressing longitudinal connectives: the most medial pCC/MP2, the more lateral MP1, and the most lateral third longitudinal connective. In a wild-type embryo these connectives are continuous between segments and evenly separated from one another over the entire length of the CNS. In Sema-1a ^P1 mutant embryos, the pCC/MP2 and MP1 connectives appear normal; however, the third longitudinal connective is abnormal in 31% of hemisegments, as compared to 1% in wild type. These defective pathways are discontinuous, thin and wavy between segments; often, individual axons are seen to contact the more medial MP1 pathway. Overall CNS organization, as revealed by the mAb BP102 that illuminates all CNS axons, is not altered in Sema-1a ^P1 mutant embryos. Analysis of Sema-1a ^P1 mutant embryos using mAbs 1D4 and 22C10 at stage 16/17, as well as earlier developmental stages, reveals the normal development of several discrete CNS pathways, including the MP1, pCC, RP, and VUM pathways in Sema-1a ^P1 mutants. Connectin-expressing longitudinal pathways also appear normal in Sema-1a ^P1 mutants (Yu, 1998).

One way to check the hypothesis that Plexin A functions as a Sema 1a receptor is to test for dominant genetic interactions between the two genes. In most cases, reducing gene dosage by one copy (thus reducing protein by 50%) has little phenotypic effect. However, simultaneously reducing the dose of two genes whose protein products function together may sufficiently impair their combined function such that phenotypes appear. Such a ''transheterozygous'' phenotype has been demonstrated for various ligand-receptor pairs in Drosophila. Embryos heterozygous for either or both Sema1a and PlexA were examined and significant enhancement was found in embryos in which both were heterozygous. Each of the phenotypes described above for the ISNb, SNa, and CNS is recapitulated in the double heterozygotes. For example, removing one copy of either Sema1a or PlexA permits nearly wild-type levels of ventral muscle innervation by ISNb neurons. Removing both copies of either gene leads to abnormal innervation in most segments. Removing one copy each of Sema1a and PlexA causes the same repertoire of defects in a similar proportion of segments as the single homozygous mutants. Likewise, the rate of defasciculation failures in the dorsal branch of the SNa is almost the same in the transheterozygous combination as it is in the Sema1a or PlexA homozygous mutants alone, roughly 70%. The fraction of affected segments within the CNS is smaller in the transheterozygotes (20% compared to 50% in Sema1a or PlexA) but still much more than would be expected from simple addition (<10%). These results strongly suggest that Sema1a and PlexA are in the same pathway and further suggest a direct physical interaction between the two proteins (Winberg, 1998).

Semaphorin-1a acts in concert with the cell adhesion molecules Fasciclin II and Connectin to regulate axon fasciculation in Drosophila

Semaphorins comprise a large family of phylogenetically conserved secreted and transmembrane glycoproteins, many of which have been implicated in repulsive axon guidance events. The transmembrane semaphorin Sema-1a in Drosophila is expressed on motor axons and is required for the generation of neuromuscular connectivity. Sema-1a can function as an axonal repellent and mediates motor axon defasciculation. By manipulating the levels of Sema-1a and the cell adhesion molecules fasciclin II (Fas II) and connectin (Conn) on motor axons, further evidence is provided that Sema-1a mediates axonal defasciculation events by acting as an axonally localized repellent and that correct motor axon guidance results from a balance between attractive and repulsive guidance cues expressed on motor neurons (Yu, 2000).

The failure of axonal defasciculation observed in Sema1a mutant embryos is likely due to the lack of Sema-1a-mediated repulsion among motor axons along efferent trajectories. Reducing adhesion by removal of attractive cues, the CAMs Fas II and Conn, rescues characteristic hyperfasciculation defects (both ISNb and SNa phenotypes) of Sema1a mutants. In contrast, increasing adhesion by overexpression of the CAM Fas II enhances the hyperfasciculation defects in the ISNb and SNa pathways in Sema1a mutant embryos. In addition, reduction in the level of Fas II will also suppress CNS fasciculation defects in Sema1a mutant embryos. These experiments complement those previous studies showing that FasII loss of function can suppress defasciculation defects observed in PlexA mutants. Further, they show that mutations in genes encoding different classes of CAMs, including both Ig superfamily members and LRR-containing proteins, genetically interact with Sema1a mutants, suggesting that CAM-specific signaling events are not involved in this interaction. Taken together, these results demonstrate that Sema-1a regulates axonal fasciculation at specific choice points by countering the attractive functions of at least two CAMs, Fas II and Conn (Yu, 2000).

How might Sema-1a modulate specific defasciculation events at choice points when it appears to be expressed along the entire motor axon? Sema-1a may serve to negatively regulate motor axon adhesion over the entire trajectory and thereby allow extending motor axon growth cones to respond to target recognition cues. A precedent for a reciprocal role for the CAM Fas II comes from a detailed ultrastructural analysis of FasII mutants, where it was observed that although a loss of Fas II does not compromise the extension of axons during early CNS development, it does result in a lack of fasciculation among individual neurons that normally comprise discrete axon bundles. On the basis of analyses of Sema-1a and analysis of Plex A, it seems likely that Sema-1a on CNS axons acts as a generally expressed repellent. The absence of the CAM Fas II, therefore, changes the balance Fas II and Sema-1a and results in defasciculation of CNS bundles. Therefore, a combination of attractants and repellents may serve to allow individual axons within a developing bundle to respond to cues that may reside either at choice points or in adjacent intermediate or final target regions. However, local regulation of the function of these axonally localized cues may also serve to modulate their function, a possibility supported by the observation that Beaten path, which negatively regulates Fas II in motor axons, appears to be localized at certain ventral motor axon choice points (Yu, 2000 and references therein).

Both Conn and Fas II, in addition to being expressed on motor axons, are also expressed on embryonic muscles. However, the interpretation that the observed genetic interactions reveal a balance of repulsive and attractive axonal cues required to regulate motor axon fasciculation is likely not to be compromised by this muscle CAM expression for the following reasons: (1) if loss of CAM expression on motor neurons and target regions were capable of altering axonal fasciculation through alteration of axon/target interactions, one would expect the effect to be a reduction in CAM-mediated motor axon extension on muscle and an enhancement of motor axon fasciculation -- the opposite of what was observe; (2) the levels of Fas II, and to a lesser extent Conn, on embryonic muscles have been shown to be much lower than high axonal levels of these CAMS observed during the embryonic stages analyzed in this study for motor axon pathfinding (Yu, 2000).

Previous studies support the idea that guidance cues act cooperatively to generate the precise pattern of neuromuscular connectivity in Drosophila. Netrins and a secreted semaphorin expressed on muscles and axonal Fas II function in a complementary fashion to regulate motor axon target selection. The dynamic balance of attractive cues (Net A and B, and Fas II) and repulsive cues (Sema-2a and Net B) has been shown to govern the ability of motor axons to undergo normal target recognition and formation of synaptic arborizations. Indeed, alteration of the degree of axonal attraction mediated by Fas II directly affects the ability of motor axons to respond to target-derived attractants and repellents. The results presented here, in combination with published results on PlexA/FasII interactions, extend these observations to the axon bundle itself and illustrate the importance of the interplay between axonal attractants and repellents for complex axon guidance events. It should be noted that although these results support a repulsive role for Sema-1a in motor axon guidance, the related transmembrane semaphorin Sema-1a in the grasshopper has been shown to act as an attractive cue for peripheral afferents in the developing limb (Wong, 1999). In this situation, however, Sema-1a is target derived and not axonal, and it remains to be seen if transmembrane semaphorin-mediated attraction is also plexin dependent. Finally, the simultaneous expression of transmembrane semaphorins and CAMs on axon pathways undergoing complex pathfinding and defasciculation events is not restricted to invertebrates. For example, in the developing vertebrate olfactory system the Ig superfamily members NCAM and olfactory cell adhesion molecules show selective distribution on main and accessory olfactory neurons, suggesting that these CAMs play an instructive role in matching odorant receptor expression zones in the olfactory epithelium and in the olfactory bulb. In addition, both the transmembrane semaphorin Sema6A and the plexin Plexin A1 are expressed in subsets of primary and accessory olfactory neurons. The defasciculation events involved in directing these neurons to their unique glomerular targets in the main and accessory olfactory bulbs are reminiscent of the motor axon guidance events discussed here, suggesting that the maintenance of a balance on axons between repulsive transmembrane semaphorins and attractive CAMs is phylogenetically conserved (Yu, 2000 and references therein).

The transmembrane protein Off-track associates with plexins and functions downstream of semaphorin signaling during axon guidance

The Plexin family of transmembrane proteins appears to function as repulsive receptors for most if not all Semaphorins. Genetic and biochemical analysis in Drosophila has been used to show that the transmembrane protein Off-track (OTK) associates with Plexin A, the receptor for Sema 1a, and that OTK is a component of the repulsive signaling response to Semaphorin ligands. In vitro, OTK associates with Plexins. In vivo, mutations in the otk gene lead to phenotypes resembling those of loss-of-function mutations of either Sema1a or PlexA. The otk gene displays strong genetic interactions with Sema1a and PlexA, suggesting that OTK and Plexin A function downstream of Sema 1a (Winberg, 2001).

The major projection of the segmental nerve, the SNa, normally extends along the body wall to a lateral position, where it divides into a dorsal and a lateral branch. The dorsal branch then extends further, dividing again and sending fine projections to innervate a group of transverse muscle fibers. In wild-type late stage 16 embryos, the dorsal SNa thus acquires a characteristic 'pitchfork' appearance. In otk loss-of-function or antisense mutants of the same age, these most dorsal growth cones remain fasciculated together in over 60% of segments and extend as a single thicker branch. This is highly similar to the aberrant SNa morphology displayed in Sema1a and PlexA loss-of-function mutant embryos. In contrast, overexpressing Plexin A causes SNa axons to defasciculate prematurely (Winberg, 2001).

The ISNb normally diverges from the main branch of the ISN in a ventral position, termed 'choice point #1'. Within the ventral muscle domain axons of the ISNb then defasciculate from one another: at choice point #2, a single axon splits off to innervate muscle fibers 6 and 7, and at choice point #3, axons either stop and innervate muscle 13 or extend further to muscle 12. By late embryonic stage 16, these growth cones have typically reached their targets and formed rudimentary synaptic contacts along the edges of these muscle fibers. In otk loss-of-function or antisense mutants, growth cones may fail to defasciculate at any of the three choice points. ISNb axons occasionally fail to exit the ISN at choice point #1, instead bypassing their muscle targets completely or else extending small aberrant projections directly from the main branch of the ISN. More often, choice point #1 is navigated correctly but then axons are unable to defasciculate at choice points #2 or #3, resulting in a thickened, stalled nerve and a failure to innervate one or more of the muscles in this domain (Winberg, 2001).

Within the CNS, additional abnormalities are observed. A subset of longitudinal axons is highlighted by monoclonal antibody labeling; in the wild-type, they form neat parallel tracks. In otk mutant embryos, these tracks are variably wavy and defasciculated and occasionally discontinuous. The incidence of 'broken' axon tracks is greater in the antisense embryos than in the loss-of-function embryos (35% versus 15%) (Winberg, 2001).

The abnormalities seen in the SNa and ISNb of embryos lacking otk are qualitatively and quantitatively highly reminiscent of those described for both Sema1a and PlexA mutants. All of these mutants also show qualitatively similar defects in the major axon tracts within the CNS, but, in the case of otk, these defects are less pronounced. Still, the strong resemblance among the phenotypes of all these mutations suggests that these three genes may all be acting in the same genetic pathway, consistent with the hypothesis that OTK positively influences Plexin A function (Winberg, 2001).

Another way to investigate whether these proteins may work together is to test for dominant genetic interactions. For most proteins, reducing gene dose to a single copy (thus reducing the protein level by 50%) produces mild or undetectable defects. However, reducing the gene dose of two different proteins may generate a phenotype if the two proteins normally function together. This 'transheterozygous' genetic test has been applied to several pairs of proteins that have also been shown to interact biochemically: Notch and Delta, Boss and Sevenless, Sema 1a and Plexin A, and Slit and Robo (Winberg, 2001).

Embryos singly and doubly heterozygous for otk and PlexA were examined and strong phenotypic effects due to the combination were observed. Embryos lacking one copy each of both otk and PlexA exhibit the same variety of SNa and ISNb defects as seen in the single homozygous mutants, to nearly the same degree of severity. This provides strong genetic support for the hypothesis that Otk and Plexin A proteins function positively together through direct contact (Winberg, 2001).

Likewise, embryos doubly heterozygous for otk and Sema1a also show phenotypic enhancement beyond additive effects of the single heterozygotes, supporting the idea of a ternary complex of Sema 1a-Plexin A-OTK proteins. However, the severity of phenotypes in the otk, Sema1a combination is somewhat less than in the others. The discrepancy may reflect a true difference between the association of OTK with Sema 1a compared to Plexin A. Alternatively, it may arise from differences in the normal expression levels of the various proteins: if Plexin A were the least abundant component under normal circumstances, then reducing the levels of the other two would be less consequential in this test (Winberg, 2001).

It has been supposed that OTK somehow affects the ability of Plexin A to mediate Sema 1a signaling. However, because all three proteins are expressed by many of the same neurons, the genetic tests above are also consistent with the possibility that OTK may interact directly with Sema 1a in cis. To verify that OTK can act genetically downstream of the signal, use was made of the GAL4 system to misexpress Sema 1a in muscles, thus offering an excess of repulsive target-derived ligand. Ectopic presentation of Sema 1a on specific muscles using UAS-Sema1a and H94-GAL4 turns these muscles into nonpermissive substrates and prevents motoneurons from innervating them correctly. The abnormal innervation of muscle 13 increases from 22% (with H94-GAL4 driver alone) to 49% (with addition of UAS-Sema1a) in this Sema1a gain-of-function experiment. This phenotype is suppressed by removing one copy of otk, reducing neuronal expression levels. Abnormal innervation of muscle 13 is reduced to 26%. It has been shown that the addition of Sema1a increases the percent abnormal from 19% to 53% and removing a single copy of PlexA reduces this frequency of abnormal innervation to 21%. Thus, removal of one copy of otk is nearly as effective in reducing the Sema1a gain-of-function as is removal of one copy of PlexA. Since neuronal OTK is sensitive to muscle-derived Sema 1a, this experiment confirms that OTK is able to act downstream of Sema 1a (Winberg, 2001).

This study has shown that Otk, a transmembrane protein of about 160 kDa, with homology to receptor tyrosine kinases, both associates with Plexins in vitro and appears to function in a Semaphorin-Plexin signaling pathway in vivo to control certain aspects of axon guidance. Biochemical data show that OTK specifically associates with Plexins in vitro. Genetic disruption of otk leads to specific defects resembling those due to lesions in either Sema 1a, a transmembrane Semaphorin that mediates axon defasciculation. These data suggest that all three proteins -- Sema 1a, Plexin A, and OTK -- may function in the same pathway. Genetic interactions suggest that OTK and Plexin A act downstream of Sema 1a. Thus, it appears that OTK and Plexin A can associate as components of a receptor complex that mediates the repulsive signaling in response to Semaphorin ligands (Winberg, 2001).

It is not known whether OTK and Plexins normally associate in vivo in growth cones or whether they might only be brought together by ligand binding. In the absence of ligand in vitro, a tight association is found between the two transmembrane proteins. If transmembrane Semaphorins, like their secreted relatives, function as dimers, then binding of Sema 1a to Plexin A might provide a mechanism for clustering receptor complexes, which by analogy might activate one or more associated kinases and lead to the phosphorylation of Plexin and OTK. Testing such speculations will have to await an appropriate system for testing ligand activation (Winberg, 2001).

Semaphorins have come to be considered as being ligands and Plexins as their receptors. But their roles in axon guidance may not be this simple. On the one hand, some Semaphorins are transmembrane proteins with cytoplasmic domains that appear as if they might be capable of transducing signals. Thus, some Semaphorins might themselves be receptors as well as ligands. On the other hand, Plexins, which are related to Semaphorins and have extracellular Semaphorin domains, can bind to themselves. Thus, some Plexins might be both ligands and receptors. Finally, Plexins associate with OTK, which also can bind homophilically (Winberg, 2001).

The data presented it this study demonstrate a role for OTK downstream from a Semaphorin on the receiving side of a signaling event. The best evidence for this conclusion is the genetic suppression data. Removing one copy of otk suppresses a Sema 1a gain-of-function phenotype. The most parsimonious interpretation of this result is that OTK functions downstream of Sema 1a. It is not known to what degree OTK binding and function is ligand gated. Moreover, it is not known whether OTK responds directly to Semaphorins, to some other ligand, or alternatively whether it simply binds to Plexins as part of a Semaphorin signaling complex. It will be interesting in the future to determine how these different Semaphorin, Plexin, and OTK proteins associate, modulate Semaphorin-mediated signal transduction, and thus control axon guidance (Winberg, 2001).

The Drosophila receptor Guanylyl cyclase Gyc76C is required for Semaphorin-1a-Plexin A-mediated axonal repulsion

Cyclic nucleotide levels within extending growth cones influence how navigating axons respond to guidance cues. Pharmacological alteration of cAMP or cGMP signaling in vitro dramatically modulates how growth cones respond to attractants and repellents, although how these second messengers function in the context of guidance cue signaling cascades in vivo is poorly understood. Using a novel Sema-1a-dependent forward genetic screening approach, it was found that Drosophila receptor-type guanylyl cyclase :Gyc76C, a protein possessing a single transmembrane domain, is required for semaphorin-1a (Sema-1a)-plexin A repulsive axon guidance of motor axons in vivo. Genetic analyses define a neuronal requirement for Gyc76C in axonal repulsion. Additionally, it was found that the integrity of the Gyc76C catalytic cyclase domain is critical for Gyc76C function in Sema-1a axon repulsion. These results support a model in which cGMP production by Gyc76C facilitates Sema-1a-plexin A-mediated defasciculation of motor axons, allowing for the generation of neuromuscular connectivity in the developing Drosophila embryo (Ayoob, 2005 ).

These experiments provide an important molecular link between semaphorin-mediated repulsion and cGMP signaling in vivo. Gyc76C is critical for Sema-1a-Plexin A-mediated selective defasciculation of axon bundles in the developing Drosophila neuromuscular system. A conserved amino acid residue within the Gyc76C cyclase domain, a residue required for receptor guanylyl cyclase (rGC) catalytic activity, is also required in Gyc76C for correct motor axon pathfinding. The identification of Gyc76C as an essential component of the Sema-1a-PlexA repulsive axon guidance signaling pathway provides insight into how cyclic nucleotide production is linked to the cascade of events downstream of semaphorin-mediated repulsion. These observations also provide a potential target for modulating repulsive semaphorin signaling by alterations of cGMP levels directly through rGCs (Ayoob, 2005).

These analyses demonstrate a role for the rGC Gyc76C in Sema-1a-mediated axon-axon repulsion. LOF mutations were generated in the Gyc76C gene and highly penetrant phenotypes were observed similar to the motor axon guidance defects observed in sema1a, plexA, and mical mutants. Micals are a family of conserved flavoprotein oxidoreductases that function in Plexin-mediated axonal repulsion (Terman, 2002). Neuronal expression of a Gyc76C cDNA restores the wild-type innervation pattern in gyc76C mutant embryos and also restores viability to the lethal gyc76C mutant line, demonstrating a requirement for Gyc76C in neurons for correct axonal pathfinding. Neuronal overexpression of wild-type Gyc76C also results in phenotypes resembling PlexA GOF phenotypes. The genetic interaction analyses confirm a role for Gyc76C in Sema-1a-PlexA repulsive signaling. Embryos heterozygous for both Gyc76C and other members of this signaling cascade, including Sema-1a, PlexA, and MICAL, display motor axon pathway disruptions. These phenotypes are qualitatively similar to LOF mutant phenotypes observed in sema1a, plexA, and mical LOF mutants and are seen at comparable frequencies. In addition to suppressing the Sema-1a- dependent midline phenotype, loss of Gyc76C function also suppresses a PlexA- dependent phenotype. However, increasing the levels of Gyc76C enhances this PlexA GOF phenotype. Finally, a Gyc76C transgene lacking a key conserved aspartate residue required for cyclase catalytic activity does not rescue either the gyc76C embryonic motor axon guidance defects or the lethality associated with gyc76C mutants and appears to function in a dominant-negative manner. Taken together, these results link Gyc76C to the proper generation of neuromuscular connectivity in Drosophila through its role in mediating semaphorin-plexin signaling events associated with axonal repulsion. In addition, these results strongly suggest that cGMP production is critical for Gyc76C participation in Sema1a neuronal signaling events (Ayoob, 2005).

Initial in vitro observations demonstrating the importance of cGMP levels in semaphorin-mediated repulsion shows that increasing cGMP signaling reverses the repulsive signal from the secreted vertebrate semaphorin Sema3A, resulting in Sema3A acting as an attractant in the single growth cone steering assay. Recent studies show that Sema3A growth cone collapse requires increased cGMP signaling and also that cAMP signaling acts in opposition to cGMP signaling in the modulation of Sema3A-mediated growth cone collapse. Support for cAMP signaling cascades modulating semaphorin-mediated repulsion in vivo is provided by a demonstration that the A-kinase anchoring protein Nervy serves to antagonize Sema-1a-mediated axonal repulsion in Drosophila motor axons. Presumably, Nervy acts by localizing cAMP activation of PKA to the Plexin receptor and decreases Sema-1a repulsive signaling (Terman, 2004). The identification of Gyc76C as a positive effector in vivo of Sema-1a-PlexA-mediated repulsion is consistent with these Sema3A growth cone collapse studies. A model recently proposed for cyclic nucleotide modulation of netrin-1-mediated attraction and repulsion provides insight into how cGMP might effect semaphorin-mediated steering, collapse, and in vivo axonal repulsion. Using the in vitro growth cone steering assay, Nishiyama (2004) has shown that the [cAMP]/[cGMP] ratio determines whether netrin-1 acts in an attractive or a repulsive manner: high ratios promote attraction, whereas lower ratios promote repulsion. Importantly, a basal level of cGMP signaling is required for both netrin-mediated attractive and repulsive responses in this system. Although it remains to be determined, it is tempting to speculate that, like the observations for netrin-1-mediated guidance, the [cAMP]/[cGMP] ratio also serves to modulate semaphorin signaling events. In Drosophila motor axons, Gyc76C and Nervy could function antagonistically to regulate Sema-1a signaling in this manner. Gyc76C production of cGMP would lower a [cAMP]/[cGMP] ratio and thus promote repulsion, whereas increases in cAMP levels would decrease repulsion through PKA tethered to PlexA by Nervy. A loss of Gyc76C altogether would result in abolition of Sema-1a repulsion because of a cGMP requirement for any guidance response, and this is what was observed in the gyc76C mutants. Future experiments will determine how raising or lowering Gyc76C activity affects the guidance response to Sema-1a in vivo (Ayoob, 2005).

This study describes a role for a receptor-type guanylyl cyclase in axon guidance as an effector of transmembrane Sema1a axonal repulsion. Soluble guanylyl cyclases in both vertebrates and invertebrates have been implicated in axonal and dendritic guidance. However, in a GOF genetic screen for Sema-1a signaling components, genomic regions containing genes encoding all of the identified Drosophila soluble guanylyl cyclase subunits were assayed, including one known to be expressed in the nervous system, yet heterozygosity at these loci did not suppress or enhance the Sema-1a GOF phenotype. This may reflect a requirement for cGMP production at or near the PlexA receptor to provide a local increase in cGMP levels essential for semaphorin-mediated axonal repulsion and suggests that basal cGMP signaling provided by soluble gyanylyl cyclases is not essential for semaphorin-mediated repulsion. The initial genetic screen covered an additional two of the seven Drosophila rGCs, however, neither of the deficiencies that remove these rGCs genetically interacted with the Sema-1a GOF phenotype. Taken together, these results from the genetic screen suggest that Gyc76C is an integral component of the semaphorin signaling cascade and that cGMP production by other sources may not contribute to this repulsion. These results also motivate future experiments to investigate specific interactions between Gyc76C and PlexA (Ayoob, 2005).

Vertebrate receptor guanylyl cyclases that have a single transmembrane domain like Gyc76C are best known for their roles as receptors for natriuretic peptides that regulate blood pressure and volume and also for their role in the visual phototransduction cascade. The other vertebrate rGCs, however, have no known ligands or functions. In addition, very little is known about what roles, if any, these vertebrate rGCs play during neural development. It will be of great interest to investigate whether any vertebrate rGCs participate in semaphorin repulsive signaling (Ayoob, 2005).

Because Gyc76C is a multidomain protein, it is likely that regions other than the cyclase domain are important for its function. Interestingly, like the transmembrane protein Off-track, which is also required for Sema1a-mediated motor axon repulsion in Drosophila, Gyc76C contains a catalytically inactive kinase homology domain (KHD). In the vertebrate receptor guanylyl cyclase GC-A, this region has been shown to play a regulatory role by inhibiting the catalytic cyclase domain. The KHD of Gyc76C, or possibly Off-track, may function as an important modulator of cyclase activity (Ayoob, 2005).

The portion of Gyc76C that is C terminal to the conserved cyclase domain is unique among rGC family members; it is much longer than the same region in other rGCs and shares no amino acid similarity with these regions or with sequences of any known proteins. However, the last four amino acids of Gyc76C fit the consensus for a PDZ (PSD-95, Discs-large, zona occludens-1) domain binding motif. A similar motif is also found in MICAL, another component of the Sema-1a signaling cascade (Terman, 2002), raising the possibility that, as has been observed for other assemblages of signaling components, PDZ domain-containing scaffolding proteins may serve an important role in semaphorin signaling (Ayoob, 2005).

Gyc76C may provide a direct physical link between the leading edge of the growth cone and the motile machinery of the actin cytoskeleton. Vertebrate rGCs in photoreceptors are able to bind actin filaments, and the C-terminal domains of intestinal rGCs have also been implicated in interactions with the actin cytoskeleton. Perhaps the large C-terminal extension of Gyc76C functions in a similar manner to bridge the regions of signal reception and output. Whether or not Gyc76C cyclase activity is ligand gated remains unknown, and like all other Drosophila rGCs and the majority of vertebrate rGCs, Gyc76C is an orphan receptor. Future experiments will address whether Sema-1a triggers Gyc76C catalytic activity and also whether Gyc76C is indeed part of the receptor complex for Sema-1a (Ayoob, 2005).

In conclusion, using a novel genetic screening paradigm for identifying semaphorin signaling cascade components, an in vivo link was found between Sema-1a-mediated repulsive guidance and cGMP signaling pathways. Characterization of other candidates from this screen will likely provide additional insight into the mechanisms of repulsive axon guidance signaling (Ayoob, 2005).

Semaphorin-1a controls receptor neuron-specific axonal convergence in the primary olfactory center of Drosophila

In the olfactory system of Drosophila, 50 functional classes of sensory receptor neurons (ORNs) project in a highly organized fashion into the CNS, where they sort out from one another and converge into distinct synaptic glomeruli. The transmembrane molecule Semaphorin-1a (Sema-1a) has been identified as an essential component to ensure glomerulus-specific axon segregation. Removal of sema-1a in ORNs does not affect the pathfinding toward their target area but disrupts local axonal convergence into a single glomerulus, resulting in two distinct targeting phenotypes: axons either intermingle with adjacent ORN classes or segregate according to their odorant receptor identity into ectopic sites. Differential Sema-1a expression can be detected among neighboring glomeruli, and mosaic analyses show that sema-1a functions nonautonomously in ORN axon sorting. These findings provide insights into the mechanism by which afferent interactions lead to synaptic specificity in the olfactory system (Lattemann, 2007).

Directing primary afferent projections to their distinct central targets is essential for generating precise sensory maps. However, the mechanisms how olfactory receptor neurons establish an accurate map in their primary synaptic target region in the CNS are poorly understood. This study describes the function of the signaling molecule Semaphorin-1a in controlling the development of synaptic specificity in the Drosophila olfactory system. The following main results suggest that Sema-1a mediates a local interaction between ORN axons during glomerular targeting. First, sema-1a-deficient ORNs in an otherwise wild-type olfactory system project normally from the sensory epithelium to the CNS target region but fail to segregate their axons in a class-specific manner. Second, sema-1a functions primarily nonautonomously onto neighboring ORN classes during axon targeting, as revealed in 'forward' and 'reverse MARCM' analyses. Third, based on a strong genetic interaction sema-1a controlled axonal signaling is mediated via plexinA receptor function. Finally, the postsynaptic dendrite organization and synaptic specificity of corresponding antennal lobe (AL) target neurons appear to be unaffected following the removal of Sema-1a from ORN axons, indicating a significant independent patterning ability of the olfactory afferents during sensory map formation. The fact that ectopic axons do not simply 'spill over' into neighboring glomeruli but extend into a distinct direction, normally occupied by another ORN class with a different Sema-1a expression level, suggests a short-range signaling mechanism that mediates the sorting of axons with different OR identity (Lattemann, 2007).

Out of the 44 described ORN classes in the adult olfactory system, 22 were tested in sema-1a mosaics and roughly half of them display local axonal convergence defects. There is no obvious link between the requirement of sema-1a and other features like sensillum type (as both trichoid and basiconic ORNs are affected) or position of the ORNs in the periphery or their target glomerulus in the AL. A class-specific ORN targeting defect in large mosaic clones has been described for other genes involved in olfactory system connectivity, like Dscam, N-Cadherin, or Acj6. In contrast to these genes, which show a ubiquitous expression in the developing AL, Sema-1a is the first signaling molecule described so far that displays a complex spatial expression pattern in newly formed, neighboring glomeruli. However, there is no simple correlation apparent between the Sema-1a expression level and expressivity of the ORN targeting phenotype for all ORN classes analyzed. In same cases, highly expressing glomeruli appear to define regions that are avoided by neighboring Sema-1a-sensitive axons, thereby restricting their convergence area. This model would be in agreement with data suggesting a strong nonautonomous function of sema-1a onto neighboring axons for some of the ORN classes. Furthermore, ORN47b and the adjacent ORN88a axons display the same Sema-1a expression level and axon termini of these ORN classes do not intermingle in sema-1a mutants. But five AL areas were found in which highly expressing ORN classes extend into regions of low Sema-1a expression in sema-1a mutants. In general, local axon targeting defects are observed for ORN classes that project to the same AL regions but express different levels of Sema-1a. In contrast, none of the tested ORN classes projecting into the Sema-1a-negative dorsomedial AL is affected in mutant mosaics. Finally, the differential Sema-1a expression levels of developing glomeruli could also be partially caused by the expression of sema-1a in AL target neurons. Unfortunately, due to technical limitations (lack of early ORN class specific markers), it is not possible to distinguish between an axonal and dendritic contribution. Nevertheless, this differential spatiotemporal expression pattern of Sema-1a is likely to provide local positional information during class-specific axonal convergence (Lattemann, 2007).

The development of neuronal connections in the vertebrate olfactory system seems to occur in distinct phases leading to a progressive refinement that ultimately results in the precise matching of pre- and postsynaptic partner neurons. In Drosophila, the global spatial organization of ORN classes in the antenna is also maintained in their projections onto the AL. Mutations in the guidance receptor Dscam and the signaling adaptor molecules Dock and Pa affect the topographic pattern but the developmental mechanism controlling zonal segregation is unclear. Once restricted to a target field subcompartment, signaling between ORN axons carrying distinct molecular identities allows overlapping axons to sort out via homotypic adhesion and heterotypic repulsion. In vertebrates, misexpression of various odorant receptor variants lead to distinct axonal segregation and convergence phenotypes, indicating that these molecules are an essential part of the ORN class-specific axonal identity code. In contrast, different EphrinA class signaling molecules have been shown to affect the local positioning of glomeruli without changing the ORN class identity. From this analysis of sema-1a function, a two-step mechanism is proposed controlling local ORN axon segregation in the Drosophila olfactory system. First, as shown in earlier studies, the initial ORN axon targeting occurs over a broader domain of the early AL followed by receptor class-specific axon sorting in the process of protoglomerulus formation. At this stage, overlapping axon termini segregate from another via intraclass adhesion (N-Cadherin-dependent) and interclass repulsion (Sema-1a-dependent), which leads to protoglomerular domains with different OR identities. However, the boundaries between adjacent protoglomeruli are still unshaped and intertwined due to innate variations in the initial axon in-growth. In the second step, repulsive signals between presorted protoglomeruli lead to the mature glomerulus arrangement with their characteristic size, shape, and position. The fact that the loss of sema-1a affects axon targeting of some ORN classes at the first step ('type 1 convergence defect') whereas others sort out correctly but fail to perform the subsequent protoglomerular patterning ('type 2 convergence defect') indicates the existence of class-specific targeting mechanisms. Following these afferent-afferent interactions, ORN axons and PN dendrites have to assemble into a functional glomerulus. The finding that PN dendrites in sema-1a mutant project to ectopic glomeruli in a class-specific manner is a strong indication that a final axon-dendrite matching mechanism is able to adapt to local perturbances in the protoglomerular field. Similar adjustments of the axonal targeting have recently been reported following the local disruption of the dendritic field, implying a sophisticated recognition code between the pre- and postsynaptic components (Lattemann, 2007).

Temporal target restriction of olfactory receptor neurons by Semaphorin-1a/PlexinA-mediated axon-axon interactions

Axon-axon interactions have been implicated in neural circuit assembly, but the underlying mechanisms are poorly understood. In the Drosophila antennal lobe, early-arriving axons of olfactory receptor neurons (ORNs) from the antenna are required for the proper targeting of late-arriving ORN axons from the maxillary palp (MP). Semaphorin-1a is required for targeting of all MP but only half of the antennal ORN classes examined. Sema-1a acts nonautonomously to control ORN axon-axon interactions, in contrast to its cell-autonomous function in olfactory projection neurons. Phenotypic and genetic interaction analyses implicate PlexinA as the Sema-1a receptor in ORN targeting. Sema-1a on antennal ORN axons is required for correct targeting of MP axons within the antennal lobe, while interactions amongst MP axons facilitate their entry into the antennal lobe. It is proposed that Sema-1a/PlexinA-mediated repulsion provides a mechanism by which early-arriving ORN axons constrain the target choices of late-arriving axons (Sweeney, 2007).

Genetic mosaic analyses of the POU transcription factor Acj6 have suggested hierarchical interactions among different classes of ORNs contribute to their axon targeting. However, it has been unclear what molecules mediate these interactions and under what cellular and developmental context these interactions take place. This study provides mechanisms to address both questions. A 'temporal target restriction' model is presented. Antennal ORN axons reach and start to pattern the developing antennal lobe before the arrival of MP axons. These early-arriving antennal axons express a high level of Sema-1a. Late-arriving MP axons express the repulsive receptor PlexinA and are repelled by Sema-1a expressed on the antennal axons. Thus, antennal ORN axons restrict MP ORN axon targeting to the proper antennal lobe region. The target glomeruli of MP classes are indeed clustered in a small area in the adult antennal lobe, surrounded by target glomeruli of antennal ORNs (Sweeney, 2007).

Multiple lines of evidence support the temporal target-restriction model. First, pioneering axons of the antennal ORNs reach the antennal lobe ~12 hr prior to those of the MP ORNs. Second, loss of antennal ORN axons results in mistargeting of MP axons, but not vice versa. Third, both Sema-1a and its known receptor PlexinA are expressed in ORN axons at appropriate developmental stages. Fourth, extensive genetic mosaic analyses of sema-1a indicate that Sema-1a is required for axon targeting of all MP ORN classes and acts non-cell-autonomously as a ligand. Fifth, knockdown of PlexinA in ORNs results in MP mistargeting phenotypes similar to those of sema-1a mosaics and those resulting from loss of antennal axons. Lastly, MP axon targeting within the antennal lobe predominantly relies on Sema-1a on antennal axons (Sweeney, 2007).

This model makes a few additional predictions that have not been directly tested due to technical limitations: (1) PlexinA should act cell autonomously in MP ORNs; (2) Sema-1a/PlexinA should mediate repulsion between antennal and MP axons; (3) the sequential arrival of antennal and MP axon innervation should be essential for their interactions. The first prediction is supported by previous findings that PlexinA acts as a receptor for Sema-1a in embryonic motor axon guidance, and PlexinA acts in ORNs and genetically interacts with Sema-1a. The second prediction is suggested by MP axon mistargeting to normal targets of antennal axons in sema-1a^−/− and plexinA RNAi conditions and is consistent with the well-documented repulsive functions of Sema-1a in Drosophila embryos and of Semaphorins more generally from insects to mammals. Finally, the temporal evidence remains correlative rather than causal, since it is currently not possible to specifically alter the sequence of axon arrival (Sweeney, 2007).

Although a central focus of this study is the axon-axon interaction between antennal and MP ORNs, it is likely that similar axon-axon interactions take place between different classes of antennal axons to regulate their targeting. The following data support this extrapolation. Antennal ORN axons express both Sema-1a and PlexinA; certain classes of antennal ORN axons require Sema-1a non-cell-autonomously; and PlexinA is required for proper targeting of many antennal ORN classes examined. A rigorous test of this extrapolation will require the identification of ORN class-specific promoters that are expressed early during development. This will allow for the examination of axon arrival timing and genetic manipulations of specific antennal ORN classes (Sweeney, 2007).

Axon-axon interactions among ORN axons likely represent one of multiple mechanisms that enable ~50 classes of ORNs to target their axons to ~50 glomeruli. In the phenotypic analyses described in this study for sema-1a and plexinA, although the severity of phenotypes varies depending on classes and genetic manipulations, the normal glomerular targets are often still innervated. This could be rationalized by the mosaic nature of sema-1a loss-of-function analyses, the partial knockdown of PlexinA by RNAi, or contributions of other ligand-receptor pairs to antenna-MP axon-axon interactions. However, even in the extreme cases of smo clones where both antennae fail to develop and all antennal axons are presumably missing, the MP axon mistargeting phenotype is only partially penetrant. These observations suggest that axon-axon interactions contribute to the fidelity of axon targeting together with other mechanisms. It is envisioned that global cues expressed in the antennal lobe act first to direct pioneering ORN axons to different general areas of the antennal lobe, axon-axon interactions then act to constrain the coarse targeting of later-arriving axons, and pre- and postsynaptic recognition contributes to the final target selection (Sweeney, 2007).

Genetic mosaic analyses indicate that Sema-1a- and PlexinA-mediated axon-axon interactions are also used among MP axons to regulate their entry into the antennal lobe. A disruption of MP-MP interactions results in occasional MP axon termination before entering the antennal lobe. This phenotype is quite analogous to the failure of motor axons to defasciculate from their fascicles upon reaching their muscle field observed in sema-1a or plexinA mutant Drosophila embryos; this embryonic phenotype has been interpreted as a defect in Sema-1a-PlexinA mediated axon-axon repulsion, which normally would facilitate defasciculation of individual axons from the rest of the fascicle. Similarly, MP-MP axon-axon repulsion mediated by Sema-1a and PlexinA may serve to loosen the individual MP axons within the bundle, allowing them to dissociate from each other and facilitate their entry into the antennal lobe (Sweeney, 2007).

A separate study shows that at an earlier stage during development, Sema-1a acts cell autonomously as a receptor (in response to an unknown ligand) in olfactory PNs for their dendritic targeting. It is thus of interest that Sema-1a acts in two different modes to regulate targeting specificity of PNs and ORNs that eventually become synaptic partners. This finding also raises the possibility that in addition to acting as a receptor for PN dendritic targeting, Sema-1a on PN dendrites might also act as a ligand for targeting of ORN axons that express and require PlexinA. However, preliminary studies have not yielded positive evidence to support this hypothesis (Sweeney, 2007).

Semaphorins and their receptors have various functions in wiring the nervous system, including olfactory systems. In mice, Semaphorin3F-Neuropilin2 signaling restricts ORN axon termination to the glomerular layer, preventing axon overshoot into deeper layers of the olfactory bulb. Moreover, Semaphorin3A-Neuropilin1 contributes to the broad organization of ORN axon targeting. Semaphorin3A, expressed in a broad compartment of the olfactory bulb by glial cells, repels Neuropilin1-expressing ORNs from this area. Sema3A-Neuropilin1 signaling has a different function in chick ORN targeting: it prevents ORNs from prematurely entering, and subsequently overshooting, the olfactory bulb. The current findings are conceptually and qualitatively distinct from these previous reports: Sema-1a mediates the interactions between axons with temporally distinct innervation patterns, rather than the interaction between axons and their targets (Sweeney, 2007).

Clear examples that temporal sequence plays an important role in neuronal wiring come from numerous studies on pioneering axons from insects to mammals. Early axons lay down the path for late ones to follow, presumably through axon-axon adhesion and fasciculation. Axon-axon interactions have also been proposed to play a role in final target selection. For example, in Drosophila photoreceptor axon targeting, R1-R6 axons from the same ommatidium, upon reaching the laminar layer, select six distinct cartridges to send their final terminal branches. Hierarchical interactions among photoreceptors contribute to their target selections, although the mechanism is unknown. In the establishment of the retinotopic map of the vertebrate visual system, relative rather than absolute EphA receptor levels on retinal ganglion cells determine the anterior-posterior positions of their axon termination at the target, likely through axon-axon interactions and competition. In mouse ORN axon targeting, axon-axon interactions have been proposed to allow ORNs expressing the same OR to converge and stabilize and to provide comparisons and discriminations of different ORN classes. The mechanisms by which these axon-axon interactions regulate targeting specificity are not well understood, and the role of temporal sequences has not been explored in these systems. A difficulty is to unravel where these neurons interact, whether at cell bodies, axon paths, or target areas. The Drosophila olfactory system provides an excellent model to explore the molecular and cellular basis of these axon-axon interactions. In particular, the physical separation of ORN cell bodies into two sensory organs, the antenna and the maxillary palp, allows assessment of afferent-afferent interactions exclusively at their final target area -- a feature exploited in this study to dissect the cellular and molecular basis of ORN axon-axon interactions (Sweeney, 2007).

Examples of a common target area innervated by multiple input axons, whether arriving simultaneously or sequentially, are ample in developing nervous systems. It is proposed that target restriction through axon-axon interactions as described here could contribute widely to establishing neuronal wiring specificity (Sweeney, 2007).

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date revised: 15 October 2007

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