org Interactive Fly, Drosophila



Control of axon-axon attraction by Semaphorin reverse signaling

Semaphorin family proteins are well-known axon guidance ligands. Recent studies indicate that certain transmembrane Semaphorins can also function as guidance receptors to mediate axon-axon attraction or repulsion. The mechanisms by which Semaphorin reverse signaling modulates axon-surface affinity, however, remain unknown. This study reveals a novel mechanism underlying upregulation of axon-axon attraction by Semaphorin-1a (Sema1a) reverse signaling in the developing Drosophila visual system. Sema1a promotes the phosphorylation and activation of Moesin (Moe), a member of the ezrin/radixin/moesin family of proteins, and downregulates the level of active Rho1 in photoreceptor axons. It is proposed that Sema1a reverse signaling activates Moe, which in turn upregulates Fas2-mediated axon-axon attraction by inhibiting Rho1 (Hsieh, 2014).

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).

Function of the Drosophila receptor guanylyl cyclase Gyc76C in PlexA-mediated motor axon guidance

The second messengers cAMP and cGMP modulate attraction and repulsion mediated by neuronal guidance cues. The Drosophila receptor guanylyl cyclase Gyc76C genetically interacts with Semaphorin 1a (Sema-1a) and physically associates with the Sema-1a receptor plexin A (PlexA). PlexA regulates Gyc76C catalytic activity in vitro, and each distinct Gyc76C protein domain is crucial for regulating Gyc76C activity in vitro and motor axon guidance in vivo. The cytosolic protein dGIPC interacts with Gyc76C and facilitates Sema-1a-PlexA/Gyc76C-mediated motor axon guidance. These findings provide an in vivo link between semaphorin-mediated repulsive axon guidance and alteration of intracellular neuronal cGMP levels (Chak, 2013).

Both membrane-associated and secreted neuronal guidance cues can attract or repel axons and dendrites during neural development, and several families of guidance cues and receptors perform these functions. Modulation of guidance cue activities through intracellular signaling components determines how extrinsic factors are interpreted by extending neuronal processes during development. For example, growth cone turning experiments in vitro demonstrate that attraction mediated by the guidance cue netrin-1 can be converted to repulsion by lowering intracellular cAMP (Ming, 1997), whereas repulsion mediated by the guidance cue Semaphorin 3A (Sema-3A) can be converted to attraction by increasing intracellular cGMP (Song, 1998). Elevated cAMP in cultured DRG neurons neutralizes Sema-3A growth cone collapse, whereas elevated cGMP potentiates it (Dontchev, 2002). The ratio of cAMP to cGMP can determine the sign of a growth cone steering response (Nishiyama, 2003), and Sema-3A induces cGMP production in neuronal growth cones, activating of cGMP-gated calcium channels (CNGCs), Ca2+ influx and repulsion (Togashi, 2008). cAMP and cGMP regulate kinases and phosphodiesterases to direct formation of axons or dendrites in cultured hippocampal neurons (Shelly, 2010). Therefore, coordination of cAMP and cGMP signaling regulates cellular responses to different stimuli in the neurons. Guanylyl cyclases (GCs) include soluble and transmembrane proteins that catalyze the conversion of GTP to cGMP, and they regulate a wide range of diverse cellular and physiological processes (Davies, 2006), including axonal and dendritic guidance (Polleux, 2000; Seidel, 2000; Gibbs, 2001; Nishiyama, 2003). The mammalian receptor guanylyl cyclase GC-B and cGMP-dependent kinase I (cGKI) are essential for proper sensory axon afferent guidance into the CNS, and C-type natriuretic peptide is the GC-B ligand that is crucial for murine sensory axon branching, axon outgrowth and axon attraction (Schmidt, 2009; Zhao, 2009). Yet, how GCs are linked to axon guidance signaling to alter intracellular cGMP levels and modulate growth cone responses in vivo is unclear (Chak, 2013).

The Drosophila transmembrane semaphorin Sema-1a binds to the plexin A (PlexA) receptor to mediate axon-axon repulsion and to control axonal fasciculation in embryonic central and peripheral nervous systems (CNS and PNS). The Drosophila receptor GC Gyc76C is required in motoneurons for Sema-1a-PlexA-mediated axon guidance and is dependent on the integrity of the Gyc76C catalytic cyclase domain (Ayoob, 2004). This study investigated connections between Gyc76C and Sema-1a-PlexA-mediated axon guidance. The findings support the idea that Gyc76C-generated cGMP within neuronal growth cones facilitates axonal repulsion mediated by Sema-1a and PlexA, allowing for the establishment of Drosophila embryonic neuromuscular connectivity (Chak, 2013).

Gyc76C-Sema-1a gain-of-function genetic interactions observed in this study are consistent with previous observations showing that Gyc76C loss and gain of function modifies aberrant CNS midline crossing by FasII+ longitudinal axons in a PlexA gain-of-function genetic background (Ayoob, 2004). Furthermore, robust physical interactions were observed between Gyc76C and PlexA both in vitro and in vivo, raising the possibility that PlexA regulates Gyc76C-mediated signaling. Co-expressing PlexA at high levels in vitro augments cGMP levels produced by Gyc76C. Future work will establish whether extracellular, intracellular, or both, types of protein-protein associations between Gyc76C and PlexA are essential for regulating Gyc76C enzymatic activity (Chak, 2013).

Gyc76C structure-function analyses are consistent with the idea that PlexA binds to the extracellular and intracellular regions of Gyc76C to relieve inhibitory effects on GC activity from of Gyc76C intramolecular interactions, increasing cGMP levels within extending motor axon growth cones and affecting growth cone guidance. This is reminiscent of Sema-3A bath application increasing intracellular cGMP levels in Xenopus spinal neurons in vitro, and the results suggest that intracellular cGMP produced by Gyc76C is required for Sema-1a-mediated repulsion. However, it is possible that signaling by intracellular cGMP is coupled with intracellular cAMP in Sema-1a-mediated repulsive guidance events (Nishiyama, 2003), and future work will determine whether varying the cAMP-to-cGMP ratio modulates Sema-1a-mediated repulsion, or converts it to attraction. Bath application of Sema-1a did not affect Gyc76C-PlexA physical associations or Gyc76C-PlexA-mediated cGMP, suggesting that Sema-1a-dependent regulation of intracellular cGMP levels could involve ligand-gated, dynamic, spatiotemporal regulation of GC activity; visualizing this signaling event will require real-time imaging of cGMP during repulsive growth cone steering (Chak, 2013).

The small GTPase Rac and its downstream effector p21 activated kinase (PAK) can regulate receptor GCs to raise cellular cGMP levels in fibroblasts in vitro, and the kinase domain of PAK interacts with the cyclase domain of receptor GC-E (Guo, 2007; Guo, 2010); PAK, therefore, may associate with Gyc76C and regulate this receptor GC during axon pathfinding (Chak, 2013).

The deletion of the Gyc76C PDZ-binding motif (PBM) strongly suppresses cGMP production by FL Gyc76C, and Gyc76C variants lacking the PBM motif exhibit low cell-surface expression levels, suggesting dGIPC regulates Gyc76C cell-surface localization. PDZ-containing proteins could form a protein scaffold required for plasma membrane localization of the Sema-1a-PlexA/Gyc76C signaling complex, and the PDZ domain-containing dGIPC protein was found to interact with Gyc76C. In vertebrates, GIPC regulates protein trafficking, subcellular localization and various signaling events. Gyc76C cell-surface localization is enhanced in vitro in the presence of dGIPC, and this may serve to regulate Gyc76C-mediated signaling. dGIPC genetic analyses show that dGIPC plays a neuronal role in motor axon guidance, and this likely occurs through interactions that modulate Gyc76C-mediated cGMP signaling. Mammalian GIPC forms dimers and multimeric complexes. dGIPC may be a part of a molecular scaffold that couples Gyc76C to cell membrane trafficking machinery or anchors Gyc76C to the plasma membrane. Alternatively, dGIPC may be essential for activating or transducing Gyc76C-mediated cGMP signaling in axon guidance events. Future genetic and biochemical analyses will reveal the downstream signaling components that respond to changes in cGMP levels in vivo and direct discrete neuronal growth cone steering responses (Chak, 2013).



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 semaP1 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 semaP1 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 semaP1 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) SemaDeltacyt (with a deleted cytoplasmic domain) is less potent than SemaWT 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 shibirets 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 shibirets 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 shibirets; 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 shibirets 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 shits1 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-shibirets 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 P150Glued 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-IaPI and Sema-IaP2 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-IaP1 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 SemaIP1 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-IaP1 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).

Matrix metalloproteinases promote motor axon fasciculation in the Drosophila embryo; defasciculation observed when MMP activity is compromised is suppressed by otherwise elevating interaxonal adhesion - either by overexpressing Fas2 or by reducing Sema-1a dosage

Matrix metalloproteinases (MMPs) are a large conserved family of extracellular proteases, a number of which are expressed during neuronal development and upregulated in nervous system diseases. Primarily on the basis of studies using pharmaceutical inhibitors, MMPs have been proposed to degrade the extracellular matrix to allow growth cone advance during development and hence play largely permissive roles in axon extension. This study shows that MMPs are not required for axon extension in the Drosophila embryo, but rather are specifically required for the execution of several stereotyped motor axon pathfinding decisions. The Drosophila genome contains only two MMP homologs, Mmp1 and Mmp2. Mmp1 was isolated in a misexpression screen to identify molecules required for motoneuron development. Misexpression of either MMP inhibits the regulated separation/defasciculation of motor axons at defined choice points. Conversely, motor nerves in Mmp1 and Mmp2 single mutants and Mmp1 Mmp2 double mutant embryos are loosely bundled/fasciculated, with ectopic axonal projections. Quantification of these phenotypes reveals that the genetic requirement for Mmp1 and Mmp2 is distinct in different nerve branches, although generally Mmp2 plays the predominant role in pathfinding. Using both an endogenous MMP inhibitor and MMP dominant-negative constructs, it was demonstrated that MMP catalytic activity is required for motor axon fasciculation. In support of the model that MMPs promote fasciculation, it was found that the defasciculation observed when MMP activity is compromised is suppressed by otherwise elevating interaxonal adhesion - either by overexpressing Fas2 or by reducing Sema-1a dosage. These data demonstrate that MMP activity is essential for embryonic motor axon fasciculation (Miller, 2008).

Motor axons navigate an extracellular environment rich with potentially competing attractive and repulsive cues. Remarkably, motor axon growth cones are able to both interpret and integrate the signals present in this complex environment en route to their individual muscle targets. The particular axonal trajectory taken by any given motoneuron depends on the nature of the extracellular cues encountered by the extending axon as well as the complement of receptor or adhesion molecules expressed on its growth cone. In addition, several molecules required for either the activation or distribution of extracellular guidance molecules have recently been implicated in axon guidance (Miller, 2008).

The number and diversity of molecules implicated in motor axon pathfinding suggest that work in genetic model systems will continue to be essential to identify and tease apart the relative contributions of proteins involved in this process. In particular, the Drosophila embryo provides an important model for the study of motor axon pathfinding as a result of the small number of motoneurons, their defined trajectories and invariant muscle targets. Work by a number of groups has led to the identification and characterization of molecules critical for pathfinding and target recognition by Drosophila motor axons. An underlying principle to emerge from these studies is that in order for axons to reach their muscle targets, the activity of adhesion molecules that promote the fasciculation and/or bundling of motor axons must be precisely balanced with repulsive signals that trigger the defasciculation and/or separation of the extending axons (Miller, 2008).

Although the mechanisms responsible for limiting defasciculation to defined choice points in the periphery are not clear, a number of molecules necessary for proper defasciculation have been identified. In particular, repulsive signaling mediated by the Semaphorin-Plexin (Sema-Plex) pathway is essential for motor axon defasciculation. In wild-type embryos, axons of the intersegmental nerve branch b (ISNb) defasciculate from the primary ISN pathway and innervate the ventrolateral muscle (VLM) field. In embryos with reduced Sema-Plex pathway activity, however, ISNb axons fail to reach their targets and often remain bundled with the primary ISN branch - a phenotype consistent with diminished interaxonal repulsion. Furthermore, embryos with loss-of-function (LOF) mutations in nervy and protein kinase A RII, two genes that have been proposed to antagonize Sema-Plex signaling, exhibit premature and excessive motor axon defasciculation. By contrast, LOF mutations in the genes for cell adhesion molecules Fasciclin II (FasII) or Connectin (Con) suppress LOF mutations in Sema-1a and plexA, arguing that Sema-1a and PlexA stimulate defasciculation by overcoming axon-axon adhesion maintained by FasII and Con. These genetic interaction studies demonstrate the importance of balancing attractive and repulsive forces to enable correct fasciculation and pathfinding (Miller, 2008).

To understand how the precise balance of attraction and repulsion is achieved, the roles of additional molecules capable of modulating fasciculation of extending motor axons must be characterized. A number of studies have investigated the roles of metalloproteinases in axon extension and guidance. The metzincin metalloproteinases are zinc-dependent extracellular proteases that are subdivided into four subfamilies based on structure: astacins, serralysins, matrix metalloproteinases (MMPs) and adamlysins - a subfamily that includes the ADAMs (a disintegrin and a metalloproteinase). Classic models of metalloproteinase function in neuronal development proposed that they acted to degrade extracellular matrix (ECM) in order to clear a path for advancing axons. Recently, the roles of metalloproteinases in axonogenesis have been revisited in a number of experimental systems. These studies indicate that relevant neuronal metalloproteinase substrates include molecules directly involved in mediating axon pathfinding, including guidance receptors and their ligands. Among the metalloproteinases, the ADAM family is most strongly implicated in the regulation of axon guidance. For instance, ADAM10 terminates the interaction between ephrin A2 and EphA by cleaving ephrin A2, thereby facilitating axon retraction in vitro. Analyses of Drosophila embryos mutant for the ADAM family homolog kuzbanian (kuz) further support the idea that ADAMs regulate particular guidance events; kuz mutations display genetic interactions with mutations in the repulsive midline factor slit. Interestingly, independent work from several groups has recently provided evidence that tolloid-related 1 (tlr1; also known as tolkin - FlyBase), a Drosophila astacin-family metalloproteinase, acts through its TGFβ ligand Dawdle to regulate motor axon guidance in the embryo (Miller, 2008).

As a family, MMPs are able to cleave nearly every component of the ECM, as well as numerous signaling molecules and cell surface receptors. In the CNS, investigations of MMP function have largely centered on the roles of these proteases in nervous system disease, as MMPs are known to be dramatically upregulated in a host of CNS diseases, as well as following nervous system injury. However, in large part due to issues of redundancy and compensation among the twenty-four vertebrate MMP family members, the normal physiological roles of MMPs in the nervous system have remained largely elusive. Notably, a number of vertebrate MMPs display neuronal expression patterns in the embryo, suggesting that they may be involved in normal nervous system development. In support of this model, studies of Xenopus retinal ganglion cell axon guidance using MMP pharmaceutical inhibitors suggest that MMPs are required for specific pathfinding decisions. Drosophila affords an attractive genetic model system in which to study MMP function since there are only two MMP family members in the fly, Mmp1 and Mmp2. Whereas Mmp1 is a secreted protein, Mmp2 contains a GPI-anchor sequence and has been shown to be membrane-bound in tissue culture cells (Miller, 2008).

This work presents an analysis of MMP function during Drosophila embryonic neuronal development. Both LOF and gain-of-function (GOF) analyses support the model that MMP activity promotes motor axon fasciculation in the embryo. Misexpression of either Mmp1 or Mmp2 drives excessive motor axon fasciculation. By contrast, aberrant defasciculation was found in MMP LOF mutants. Although Mmp1 mutants display relatively mild pathfinding defects, many motor axons separate prematurely and aberrantly in Mmp2 single mutants and Mmp1 Mmp2 double mutants, indicating that Mmp2 plays a primary role in motor axon fasciculation. The embryonic expression of both MMPs was analyzed, and it was found that whereas Mmp1 exhibits a limited embryonic expression profile, Mmp2 is expressed in neurons and glia - supporting a primary role for Mmp2 in embryonic neuronal development. Importantly, aberrant motor axon defasciculation was found in embryos misexpressing the endogenous MMP inhibitor Timp and in embryos misexpressing MMP dominant-negative constructs, indicating that MMP catalytic activity is essential for pathfinding. Finally, it was shown that the defasciculation phenotype exhibited by MMP LOF mutants are dominantly suppressed by LOF mutations in Sema-1a, arguing that MMP activity normally acts to promote fasciculation by antagonizing Sema-1a function. Together, these results indicate that MMPs are not required for motor axon extension per se, but instead may modulate the responses of the axons of defined neuronal populations to specific guidance cues (Miller, 2008). To further investigate the possibility that MMP activity plays a role in neuronal development, the embryonic expression patterns of Mmp1 and Mmp2 were characterized. Previous studies have established that both genes are embryonically expressed. Using anti-Mmp1 antibodies, Mmp1 protein was found to be expressed in essentially the same spatiotemporal expression profile as has been described for Mmp1 RNA. The most prominent embryonic expression of Mmp1 is in the proventriculus and hindgut. Consistent with previous studies, Mmp1 CNS expression was found to be restricted to small clusters of segmentally repeating cells at the CNS midline. Mmp1 expression was also detected in the chordotonal organs of the peripheral nervous system and in two cells situated in the ventral mesodermal region. This expression is undetectable in Mmp1-null mutant embryos, (Mmp12/Mmp1Q112*), confirming antibody specificity (Miller, 2008).

The expression pattern of Mmp2 was characterized via whole-mount RNA in situ hybridization. In contrast to Mmp1, Mmp2 is widely expressed in the embryonic CNS. To identify the neuronal cells, wild-type embryos were double labeled with Mmp2 RNA and markers for specific neural and glial populations. It was found that Mmp2 is expressed in midline glia as Mmp2 RNA is co-expressed with Wrapper in these cells. Next whether Mmp2 is expressed in additional glial populations by was ested by co-labeling embryos with Mmp2 RNA and the glial marker anti-Repo. At stage 15, Mmp2 and Repo are co-expressed in approximately three glial cells per hemisegment situated at the base of motor nerve roots. The position and morphology of these cells suggest they correspond to exit glia, a group of peripheral glia originating within the CNS before migrating into the periphery during embryogenesis along extending motor axons. To confirm that these Mmp2-expressing cells are glia, it was asked whether they are absent in embryos mutant for glial cells missing (gcm), in which the number of glial cells is greatly reduced. In support of this conclusion, gcm mutant embryos specifically lack the Mmp2-expressing cells situated at the boundary between the CNS and periphery (Miller, 2008).

The observation that Mmp2-positive cells within the CNS do not co-express Repo suggested that they are probably neurons. To determine whether they correspond to well-characterized subsets of motoneurons or interneurons, embryos were double labeled with Mmp2 RNA and antibodies specific for particular neuronal populations. Co-expression between Mmp2 and Islet, a marker for distinct motoneuron and interneuron populations, was detected in three neurons per hemisegment in the lateral CNS. It was next asked whether these Mmp2-expressing neurons are Hb9-positive motoneurons. No co-expression was detected between Hb9 and Mmp2 RNA, suggesting that the Mmp2-positive neurons in the lateral CNS are Islet-positive interneurons. In sum, whereas Mmp1 exhibits a limited neuronal expression pattern, Mmp2 is expressed in stereotyped populations of neurons and glia, consistent with a role for Mmp2 in neuronal development (Miller, 2008).

This work demonstrates that the level of MMP catalytic activity dictates the degree of motor axon fasciculation in the Drosophila embryo. MMP misexpression is sufficient to inhibit separation of motor axons during outgrowth, but both of the primary embryonic motor nerve branches display striking defasciculation in MMP LOF mutants. The opposing axonal phenotypes observed in MMP LOF and GOF embryos indicates that the level of MMP activity is critical for pathfinding and further suggests that the relevant MMP substrate(s) plays an instructive role in motor axon guidance. In support of the hypothesis that MMPs influence axon outgrowth by modulating the activity of established guidance cues, Mmp2 LOF mutants were shown to be dominantly suppressed by a null mutation in Sema-1a, arguing that MMP function is tightly coupled to guidance decisions. Possible substrates for Mmp2 in motor axon pathfinding are considered and these findings are put in the context of proposed neural functions for metalloproteinases in vertebrates and invertebrates (Miller, 2008).

Both fly MMPs were previously shown to be expressed in the embryonic CNS, suggesting that they regulate aspects of neuronal development. However, the finding that both MMP single mutants and the Mmp1 Mmp2 double mutant survived embryogenesis called into question the extent of any possible roles for the MMPs in embryogenesis. This work presents genetic evidence that MMP catalytic activity is essential for motor axon fasciculation. Whereas Mmp1 mutants display subtle fasciculation errors, it was found that motor axons in Mmp2 mutants are markedly defasciculated, with many embryonic nerves appearing frayed and poorly organized. Consistent with this phenotypic analysis, the CNS expression profile of Mmp2 is considerably broader than that of Mmp1: Mmp2 is expressed in midline glia, in clusters of interneurons and in peripheral/exit glia but CNS expression of Mmp1 is limited to the midline. The prominent expression of Mmp1 and Mmp2 at the CNS midline prompted an examination of whether either MMP might be required for proper guidance there. However, no alteration was found in the behavior of axons at the midline in either MMP LOF or GOF mutant backgrounds, and no genetic interactions were found between Mmp2 and Slit or Mmp1 and Robo. These data indicate that MMPs do not contribute significantly to embryonic midline guidance in the fly (Miller, 2008).

Although the Mmp1 and Mmp2 LOF phenotypes are distinct, several pieces of evidence suggest that they have overlapping substrate specificities and can cleave the same guidance cue(s). First, misexpression of either Mmp1 or Mmp2 yields qualitatively indistinguishable guidance phenotypes with many motor axons remaining inappropriately bundled together. Second, misexpression of an Mmp1 dominant-negative transgene gives phenotypes nearly identical to those observed with a dominant negative Mmp2. Furthermore, the phenotypes observed with these constructs are stronger and more penetrant than the phenotypes of Mmp1 LOF mutants, suggesting that the Mmp1 dominant-negative transgene affects motor axon pathfinding by interfering with Mmp2 function by binding to the relevant Mmp2 substrate(s). Lastly, if Mmp1 and Mmp2 cleave the same substrate(s), they might be expected to be genetically redundant, since removal of one would be compensated for by the presence of the other. In fact, Mmp1 and Mmp2 show partially redundant roles in SNa pathfinding; the double mutant phenotype is significantly stronger than the phenotype observed in either single mutant. These results are in agreement with analyses of enzymatic activity of vertebrate MMPs that suggest that there is overlap between the substrates cleaved by individual MMPs (Miller, 2008).

Mmp2 contains a predicted GPI anchor and is membrane associated in Drosophila tissue culture cells. Thus, the expression pattern of Mmp2 in the embryo would be expected to reflect the locations of Mmp2-dependent proteolysis. Mmp2 RNA was found to be expressed in restricted populations of interneurons and peripheral glia, but not in motoneurons. Peripheral glia originate at the lateral edge of the CNS and migrate into the periphery along elongating motor axons. By the end of embryogenesis, they extend cytoplasmic processes and wrap axon bundles in a manner similar to vertebrate non-myelinating Schwann cells. It is proposed that peripheral glial-derived Mmp2 modulates the activity of factors required for pathfinding. This model implies that peripheral glia play a significant role in regulating motor axon fasciculation. This finding contrasts slightly with previous results showing subtle errors in the motor axon projection pattern when peripheral glia were genetically ablated. One possible explanation for the weaker phenotypes in the peripheral glia-ablated embryos relative to Mmp2 LOF mutants is that peripheral glia express several factors that influence axon pathfinding in opposing directions - for example, proteins that both inhibit and stimulate fasciculation. In this way, peripheral glia would somewhat resemble midline glia which express both an axonal attractant (Netrin) and repellent (Slit). Therefore, ablation of the entire cellular population would be expected to yield different phenotypes than mutating individual molecules. Another possibility is that although Mmp2 is likely to act locally, its substrate might be secreted and could regulate motor axon guidance at a distance. In this case, Mmp2 need not be expressed at the site of fasciculation decisions, and either midline or interneuron-derived Mmp2 might provide the relevant proteolytic activity (Miller, 2008).

In principle, since MMP cleavage might either activate or inhibit the function of a molecule required for axon guidance, the motor axon phenotypes observed in MMP mutants could be expected to be identical to or opposite that of the phenotypes displayed by substrate mutations. Based solely on phenotypic considerations, several guidance molecules could be considered candidate MMP substrates. For example, LOF mutations in a number of genes give hyperfasciculation and/or stalled motor axon phenotypes. These include beaten path (beat) and sidestep (side), two immunoglobulin superfamily proteins required for proper defasciculation of both ISNb and SNa. There are also five CNS-expressed receptor protein tyrosine phosphatases (RPTPs) that have combinatorial roles in the regulation of motor axon pathfinding. A number of these RPTPs, in particular LAR, are involved in ISNb defasciculation decisions. Additionally, Plexin proteins and their receptors, the semaphorins, are critical regulators of motor axon fasciculation. Sema-Plex pathway activity promotes inter-axonal repulsion so that LOF mutations in Sema-Plex pathway components result in ISNb stall phenotypes. Importantly, it has also been shown that for axons to remain tightly bundled during normal axon outgrowth, Sema-Plex signaling must be actively antagonized, as LOF mutations in two downstream inhibitors, nervy and Protein kinase A, give aberrant defasciculation phenotypes similar to that observed in MMP mutations. Hence, levels of Sema-plex activity must be tightly controlled to ensure that defasciculation occurs properly at guidance choice points. And similar to what is described in this study for MMPs, reciprocal GOF and LOF mutations in the pathway can result in opposing hyper- and hypo-fasciculation phenotypes (Miller, 2008).

The MMP family as a whole does not cleave a conserved amino acid sequence in their targets, meaning that Drosophila substrates must be determined empirically, not computationally. One identified Mmp1 substrate, Ninjurin A (NijA), represented an appealing candidate in motor axon guidance as it is a signaling protein that regulates cell adhesion whose vertebrate homologs are upregulated in response to nerve injury. However, no aberrations to motor axon pathfinding were found in either NijA LOF or GOF mutants, indicating that NijA is unlikely to be a relevant substrate in this context. Although few other Drosophila MMP substrates have been identified, the Drosophila homologs of several putative vertebrate MMP substrates make appealing candidates for MMP targets in embryonic CNS development. For instance, vertebrate membrane type MMP1 (MT1-MMP), has been shown to interact with the transmembrane heparan sulfate proteoglycan Syndecan 1 and trigger Syndecan 1 ectodomain shedding. Syndecan 1 processing stimulated cell migration on collagen, suggesting that this cleavage has functional consequences in vivo. Interestingly, Drosophila Syndecan (Sdc) has been identified as a ligand for the LAR RPTP. Accordingly, genetic interaction studies indicate that Sdc and LAR act in concert to regulate ISNb pathfinding. As it is currently unknown whether LAR binds membrane-bound or soluble Sdc, MMP activity could potentially regulate the LAR/Sdc interaction. In addition, MT1-MMP has also recently been shown to be required for ectodomain shedding of Semaphorin 4D in a model of tumor-induced angiogenesis - a processing event required for the induction of blood vessel growth in vivo. Semaphorin signaling plays a well-documented role in regulating motor axon behavior. Furthermore, since Sema-1a mutations display strong genetic interactions with Mmp2 mutations in this system, it is conceivable that MMPs directly modulate Sema-Plex signaling activity (Miller, 2008).

MMP expression levels are highly elevated in a number of neuronal pathologies and after nervous system injury. MMP upregulation in CNS disease states raises the issue of whether MMP induction has an overall positive or negative effect on disease outcome. There is substantial evidence that the net effect of high MMP expression in some diseases is detrimental. For example, treatment with broad-spectrum metalloproteinase inhibitors is able to alleviate or prevent experimental autoimmune encephalomyelitis (EAE), a mouse multiple sclerosis model. There is also, however, growing recognition of beneficial functions for MMPs following CNS injury. The diverse functions for MMPs in disease states have become increasingly apparent as investigators have moved beyond the use of general metalloproteinase inhibitors to the study of particular MMPs. For example, increased expression of individual MMPs has been shown to correlate with periods of regeneration and repair following nervous system injury. The functional significance of elevated MMP expression on regenerating axons has not been established, though in some regeneration models treatment with active MMPs promotes axon outgrowth. In regeneration, it is thought that MMPs influence axon growth by degrading chondroitin sulphate proteoglycans (CSPGs), which normally inhibit regrowth beyond the glial scar (Miller, 2008).

In the context of neuronal development, there is substantial support for the idea that metalloproteinases, and in particular the ADAM subfamily, regulate axon outgrowth and pathfinding. Early work in the field suggested that metalloproteinases play a largely permissive role in axon outgrowth - by degrading the ECM in order to clear a path for extending axons. In support of a role for MMPs in outgrowth, it has been shown that a number of MMPs are expressed on the growth cones of vertebrate neurites extending in vitro. More recent work has demonstrated that in vitro, metalloproteinases are capable of modulating the interactions between guidance cues and their receptors. For example, the interaction between ephrin A2 and Eph receptor is terminated by ephrin A2 cleavage via ADAM10 (also known as Kuzbanian-like - FlyBase) and/or Kuz. Functionally, this cleavage allows growth cone withdrawal of hippocampal neurons in culture, as a cleavage-inhibiting mutation delays axon retraction. Metalloproteinases have also been implicated in DCC (deleted in colorectal carcinoma) receptor activity as broad-spectrum metalloproteinase inhibitors inhibit ectodomain shedding of DCC and potentiate netrin-mediated axon outgrowth. In vivo support for the role of ADAM proteases in axon outgrowth and pathfinding comes from work in Drosophila. kuz mutant embryos display ectopic axon crossing at the midline suggesting that kuz is required for repulsive signaling mediated by Slit-Roundabout (Robo). Supporting this idea, kuz and slit mutations genetically interact, and Kuz appears to be required for the clearance of the Robo receptor from commissural axons (Miller, 2008).

Although a number of vertebrate MMPs display neuronal expression patterns in the embryo, until relatively recently there was little direct evidence supporting a role for this metalloproteinase subclass in axon pathfinding. Studies of retinal ganglion cell (RGC) pathfinding in frogs argue that MMP activity is required for axon guidance at several defined choice points. MMP-specific inhibitor was used to demonstrate that MMPs are required for RGC guidance decisions both at the optic chiasm and tectum. Hehr work suggested that MMPs are normally required for axon guidance during vertebrate development, though the particular MMPs involved in RGC pathfinding remain to be identified. Exploiting the relative simplicity of the Drosophila model system, this study has established that individual MMPs play critical and distinct roles in well-defined axon pathfinding decisions during development. To extend this work to more complex vertebrate systems, it will be critical to analyze axon outgrowth and pathfinding in MMP single and compound mutant mice (Miller, 2008).

The Drosophila L1CAM homolog Neuroglian signals through distinct pathways to control different aspects of mushroom body axon development

The spatiotemporal integration of adhesion and signaling during neuritogenesis is an important prerequisite for the establishment of neuronal networks in the developing brain. This study describes the role of the L1-type CAM Neuroglian protein (NRG) in different steps of Drosophila mushroom body (MB) neuron axonogenesis. Selective axon bundling in the peduncle requires both the extracellular and the intracellular domain of NRG. A novel role was uncovered for the ZO-1 homolog Polychaetoid (PYD) in axon branching and in sister branch outgrowth and guidance downstream of the neuron-specific isoform NRG-180. Furthermore, genetic analyses show that the role of NRG in different aspects of MB axonal development not only involves PYD, but also TRIO, SEMA-1A and RAC1 (Goossens, 2011).

This study demonstrates a requirement for Neuroglian signaling in different steps of mushroom body (MB) axonogenesis, namely (1) axonal projection into the peduncle, and (2) branching, outgrowth and guidance of axonal sister branches. The two steps in mushroom body axonogenesis are genetically separable and seem to involve distinct NRG signaling complexes (Goossens, 2011).

In peduncle formation, NRG signaling does not rely on the NRG-180-specific intracellular domain, but on the extracellular domain and the part of the cytoplasmic domain common to both NRG isoforms. The extracellular domain contributes intercellular adhesive properties, necessary for axon fasciculation into a peduncle. This conclusion is supported by the defective adhesive properties of the NRG849 mutant protein in cell aggregation assays, and by the fact that Nrg849 hemizygotes frequently lack the peduncle. Interaxonal fasciculation in the peduncle probably involves binding to and stabilization by the actin cytoskeleton network via the ankyrin-binding domain shared by the two NRG isoforms. This conclusion is supported by previous aggregation experiments in Drosophila S2 cells in which it was shown that homophilic binding of NRG leads to recruitment of ankyrin to the contact sites and by the observation that RNAi-mediated knockdown of neuron-specific ank2-RNA results in MB phenotypes similar to those seen in Nrg mutants (Goossens, 2011).

MB lobe development, on the other hand, requires the NRG-180-specific intracellular fragment. This study showed that PYD acts downstream of NRG-180 during the formation of α and γ lobes. Consistent with this, axon stalling defects (i.e. lack of peduncle formation) were never observed in pyd mutants, whereas defects were observed in lobe outgrowth, branching and guidance. Furthermore, the neuron-specific NRG-180 isoform can bind directly to the first PDZ-domain of this MAGUK protein. The observation that NRG and PYD interact to mediate proper sister neurite projections defines a novel role for the ZO-1 homolog PYD in axonogenesis. Thus far, the best-known role of MAGUKs in the nervous system has been in synapse development and function, as is the case for one of the prototypic MAGUKs, Drosophila Discs Large 1 (Dlg1), whereas PYD is known as a component of adherens junctions (Goossens, 2011).

Sema-1a, trio and Rac1 were also found to be a part of the genetic network that interacts with Nrg. The observation that heterozygosity for mutations in Sema-1a and trio both suppress NRG-180 overexpression induced MB phenotypes indicates that Sema-1a and trio are genetically downstream of Nrg and possibly in the same pathway. By contrast, the introduction of a Rac1 mutation in a Nrg gain- or loss-of-function background results in both cases in enhancement of MB phenotypes. This argues against a one-to-one signaling model between NRG and RAC1, in which RAC1 acts only downstream of NRG-180. Consistent with the genetic data, no direct physical interaction could be detected between NRG-180 and RAC1, but preliminary co-immunoprecipitation data suggest that NRG-180 can bind to TRIO. Further experiments will be necessary to assess whether this binding also occurs in vivo, and whether it is instrumental for NRG-180-dependent modulation of RAC1 signaling (Goossens, 2011).

Contrary to the observed genetic interaction between Nrg and Sema-1a, this study found no evidence for interaction between Nrg and two genes that code for well-characterized Semaphorin receptors, plexin A and plexin B. This is an unexpected observation in light of the fact that Sema-1a and plexin A and plexin B interact during mushroom body development. Therefore, this suggests that during mushroom body development Sema-1a acts both in a plexin-dependent and a plexin-independent way. A plexin-independent role in axon outgrowth has previously been described for vertebrate Sema7a (Goossens, 2011).

The distinct requirement for NRG in peduncle and lobe formation is reminiscent of what has been shown for DSCAM. This protein has an early and essential role for selective fasciculation of young axons in the peduncle and is subsequently required for bifurcation and branch segregation. In light of this, it is interesting to note that the different cell-adhesion molecules that have been implicated in MB development have a different MB expression pattern or temporal requirement for MB development. NRG is expressed in the MBs throughout its entire development, but no essential function was found for Neuroglian in larval MB development. By contrast, DSCAM expression disappears with fiber maturation and mutants have larval MB phenotypes. Likewise, Fas2 mutants display larval lobe defects, whereas no lobe defects were found in adult mutants. Taken together, these observations suggest that different steps in MB axonogenesis depend on combinations not only of isoforms of the same cell-adhesion molecule (e.g. DSCAM) but also of different cell surface molecules (e.g. NRG, FAS2 and SEMA-1A). Jointly, the cell-surface molecule complement of any given axon combined with guidance signals will then control the signaling required for proper neural circuit formation in the MBs (Goossens, 2011).


Ayoob, J. C., Yu, H.-H. Terman, J. R. and Kolodkin, A. L. (2005). The Drosophila receptor Guanylyl cyclase Gyc76C is required for Semaphorin-1a-Plexin A-mediated axonal repulsion. J. Neurosci. 24(30): 6639-6649. 15282266

Bagnard, D., et al. (1998). Semaphorins act as attractive and repulsive guidance signals during the development of cortical projections. Development 125(24): 5043-5053. PubMed Citation: 9811588

Cai, H. and Reed, R. R. (1999). Cloning and characterization of Neuropilin-1-interacting protein: A PSD-95/Dlg/ZO-1 domain-containing protein that interacts with the cytoplasmic domain of Neuropilin-1. J. Neurosci. 19(15): 6519-6527. PubMed Citation: 10414980

Chak, K. and Kolodkin, A. L. (2013). Function of the Drosophila receptor guanylyl cyclase Gyc76C in PlexA-mediated motor axon guidance. Development 141(1): 136-47. PubMed ID: 24284209

Chedotal, A., et al. (1998). Semaphorins III and IV repel hippocampal axons via two distinct receptors. Development 125(21): 4313-4323. PubMed Citation: 9753685

Chen, H., He, Z., and Tessier-Lavigne, M. (1998). Axon guidence mechanisms: semaphorins as simultaneous repellents and anti-repellents. Nature Neurosci. 1: 436-439. PubMed Citation: 10196539

Dalpe, G., Brown, L., Culotti, J. G. (2005). Vulva morphogenesis involves attraction of plexin 1-expressing primordial vulva cells to semaphorin 1a sequentially expressed at the vulva midline. Development 132(6): 1387-400. 15716342

Davies, S. A. (2006). Signalling via cGMP: lessons from Drosophila. Cell Signal 18: 409-421. PubMed ID: 16260119

Dontchev, V. D. and Letourneau, P. C. (2002). Nerve growth factor and semaphorin 3A signaling pathways interact in regulating sensory neuronal growth cone motility. J Neurosci 22: 6659-6669. PubMed ID: 12151545

Eickhoff, R. and Bicker, G. (2012). Developmental expression of cell recognition molecules in the mushroom body and antennal lobe of the locust Locusta migratoria. J Comp Neurol 520(9): 2021-2040. PubMed ID: 22173776

Gibbs, S. M., Becker, A., Hardy, R. W. and Truman, J. W. (2001). Soluble guanylate cyclase is required during development for visual system function in Drosophila. J Neurosci 21: 7705-7714. PubMed ID: 11567060

Ginzburg, V. E., Roy, P. J. and Culotti, J. G. (2002). Semaphorin 1a and semaphorin 1b are required for correct epidermal cell positioning and adhesion during morphogenesis in C. elegans. Development 129: 2065-2078. 11959817

Godenschwege, T. A., Hu, H., Shan-Crofts, X., Goodman, C. S. and Murphey, R. K. (2002). Bi-directional signaling by Semaphorin 1a during central synapse formation in Drosophila. Nat Neurosci. 5(12): 1294-301. 12436113

Goossens, T., et al. (2011). The Drosophila L1CAM homolog Neuroglian signals through distinct pathways to control different aspects of mushroom body axon development. Development 138(8): 1595-605. PubMed Citation: 21389050

Guo, D., Tan, Y. C., Wang, D., Madhusoodanan, K. S., Zheng, Y., Maack, T., Zhang, J. J. and Huang, X. Y. (2007). A Rac-cGMP signaling pathway. Cell 128: 341-355. PubMed ID: 17254971

Guo, D., Zhang, J. J. and Huang, X. Y. (2010). A new Rac/PAK/GC/cGMP signaling pathway. Mol Cell Biochem 334: 99-103. PubMed ID: 19937092

Hsieh, H. H., Chang, W. T., Yu, L. and Rao, Y. (2014). Control of axon-axon attraction by Semaphorin reverse signaling. Proc Natl Acad Sci U S A. 111(31):11383-8. PubMed ID: 25049408

Khare, N., et al. (2000). Expression patterns of two new members of the Semaphorin family in Drosophila suggest early functions during embryogenesis. Mech. Dev. 91: 393-97. PubMed Citation: 10704872

Lattemann. M., et al. (2007). Semaphorin-1a controls receptor neuron-specific axonal convergence in the primary olfactory center of Drosophila. Neuron 53: 169-184. Medline abstract: 17224401

Matsuoka, R. L., Nguyen-Ba-Charvet, K. T., Parray, A., Badea, T. C., Chedotal, A. and Kolodkin, A. L. (2011). Transmembrane semaphorin signalling controls laminar stratification in the mammalian retina. Nature 470: 259-263. PubMed ID: 21270798

Miller, C. M., Page-McCaw, A. and Broihier, H. T. (2008). Matrix metalloproteinases promote motor axon fasciculation in the Drosophila embryo. Development 135(1): 95-109. PubMed Citation: 18045838

Ming, G. L., Song, H. J., Berninger, B., Holt, C. E., Tessier-Lavigne, M. and Poo, M. M. (1997). cAMP-dependent growth cone guidance by netrin-1. Neuron 19: 1225-1235. PubMed ID: 9427246

Nern, A., Zhu, Y. and Zipursky, S. L. (2008). Local N-cadherin interactions mediate distinct steps in the targeting of lamina neurons. Neuron 58: 34-41. PubMed ID: 18400161

Nishiyama, M., et al. (2003). Cyclic AMP/GMP-dependent modulation of Ca2+ channels sets the polarity of nerve growth-cone turning. Nature 424: 990-995. 12827203

Kolodkin, A. L., Matthes, D. J. and Goodman, C. S. (1993). The semaphorin genes encode a family of transmembrane and secreted growth cone guidance molecules. Cell 75(7): 1389-99. 94094332

Murphey, R. K., et al. (2003). Targeted expression of shibirets and semaphorin 1a reveals critical periods for synapse formation in the giant fiber of Drosophila. Development 130: 3671-3682. 12835384

Pecot, M. Y., Tadros, W., Nern, A., Bader, M., Chen, Y. and Zipursky, S. L. (2013). Multiple interactions control synaptic layer specificity in the Drosophila visual system. Neuron 77: 299-310. PubMed ID: 23352166

Polleux, F., Morrow, T. and Ghosh, A. (2000). Semaphorin 3A is a chemoattractant for cortical apical dendrites. Nature 404: 567-573. PubMed ID: 10766232

Schmidt, H., Stonkute, A., Juttner, R., Koesling, D., Friebe, A. and Rathjen, F. G. (2009). C-type natriuretic peptide (CNP) is a bifurcation factor for sensory neurons. Proc Natl Acad Sci U S A 106: 16847-16852. PubMed ID: 19805384

Seidel, C. and Bicker, G. (2000). Nitric oxide and cGMP influence axonogenesis of antennal pioneer neurons. Development 127: 4541-4549. PubMed ID: 11023858

Shelly, M., Lim, B. K., Cancedda, L., Heilshorn, S. C., Gao, H. and Poo, M. M. (2010). Local and long-range reciprocal regulation of cAMP and cGMP in axon/dendrite formation. Science 327: 547-552. PubMed ID: 20110498

Song, H., Ming, G., He, Z., Lehmann, M., McKerracher, L., Tessier-Lavigne, M. and Poo, M. (1998). Conversion of neuronal growth cone responses from repulsion to attraction by cyclic nucleotides. Science 281: 1515-1518. PubMed ID: 9727979

Sun, L. O., Jiang, Z., Rivlin-Etzion, M., Hand, R., Brady, C. M., Matsuoka, R. L., Yau, K. W., Feller, M. B. and Kolodkin, A. L. (2013). On and off retinal circuit assembly by divergent molecular mechanisms. Science 342: 1241974. Abstract

Sweeney, L. B., et al. (2007). Temporal target restriction of olfactory receptor neurons by Semaphorin-1a/PlexinA-mediated axon-axon interactions. Neuron 53: 185-200. Medline abstract: 17224402

Takahashi, T., et al. (1998). Semaphorins A and E act as antagonists of neuropilin-1 and agonsists of neuropilin-2 receptors. Nature Neurosci. 1: 487-493. PubMed Citation: 10196546

Terman, J. R., Mao, T., Pasterkamp, R.J., Yu, H. H. and Kolodkin, A. L. (2002). MICALs, a family of conserved flavoprotein oxidoreductases, function in Plexin-mediated axonal repulsion. Cell 109: 887-900. 12110185

Terman, J. R. and Kolodkin, A. L. (2004). Nervy links protein kinase A to plexin-mediated semaphorin repulsion. Science 303: 1204-1207. 14976319

Timofeev, K., Joly, W., Hadjieconomou, D. and Salecker, I. (2012). Localized netrins act as positional cues to control layer-specific targeting of photoreceptor axons in Drosophila. Neuron 75: 80-93. PubMed ID: 22794263

Togashi, K., von Schimmelmann, M. J., Nishiyama, M., Lim, C. S., Yoshida, N., Yun, B., Molday, R. S., Goshima, Y. and Hong, K. (2008). Cyclic GMP-gated CNG channels function in Sema3A-induced growth cone repulsion. Neuron 58: 694-707. PubMed ID: 18549782

Winberg, M. L., et al. (1998). Plexin A is a neuronal semaphorin receptor that controls axon guidance. Cell 95(7): 903-16. PubMed Citation: 9875845

Winberg, M. L., et al. (2001). The transmembrane protein Off-track associates with plexins and functions downstream of semaphorin signaling during axon guidance. Neuron 32: 53-62. 11604138

Wong, J. T., Wong, S. T. and O'Connor, T. P. (1999). Ectopic semaphorin-1a functions as an attractive guidance cue for developing peripheral neurons. Nat. Neurosci. 2: 798-803. PubMed Citation: 10461218

Yu, H. H., et al. (1998). The transmembrane Semaphorin Sema-I is required in Drosophila embryonic motor and CNS axon guidance. Neuron 20(2): 207-20. 98150947

Yu, H.-H., Huang, A. S. and Kolodkin, A. L. (2000). Semaphorin-1a acts in concert with the cell adhesion molecules Fasciclin II and Connectin to regulate axon fasciculation in Drosophila. Genetics 156: 723-731. PubMed Citation: 11014819

Zhao, Z. and Ma, L. (2009). Regulation of axonal development by natriuretic peptide hormones. Proc Natl Acad Sci U S A 106: 18016-18021. PubMed ID: 19805191

semaphorin-Ia : Biological Overview | Regulation | Developmental Biology | Effects of Mutation

date revised: 30 April 2017

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