plexin A

DEVELOPMENTAL BIOLOGY

Embryonic

Embryonic expression of PlexA and PlexB was examined by Northern analysis and by mRNA in situ hybridization. PlexA probes detect a single transcript of about 7.5 kb; PlexB probes detect two transcripts, approximately 8 and 12 kb. PlexA and PlexB mRNAs show largely similar in situ localization: both are maternally deposited and are broadly distributed during early embryogenesis. Beginning with germband retraction, both transcripts show a reduction in general expression, while remaining highly expressed in the CNS. After embryonic stage 15, during the period in which many motor axons are reaching their targets, PlexA and PlexB transcripts are largely confined to the CNS, where they are expressed by many neurons. Although transcripts are found in myoblasts, expression does not persist in muscle fibers (Winberg, 1998).

Larval

Drosophila metamorphosis is characterized by diverse developmental phenomena, including cellular proliferation, tissue remodeling, cell migration, and programmed cell death. Cells undergo one or more of these processes in response to the hormone 20-hydroxyecdysone (ecdysone), which initiates metamorphosis at the end of the third larval instar and before puparium formation (PF) via a transcriptional hierarchy. Additional pulses of ecdysone further coordinate these processes during the prepupal and pupal phases of metamorphosis. Larval tissues such as the gut, salivary glands, and larval-specific muscles undergo programmed cell death and subsequent histolysis. The imaginal discs undergo physical restructuring and differentiation to form rudimentary adult appendages such as wings, legs, eyes, and antennae. Ecdysone also triggers neuronal remodeling in the central nervous system (White, 1999).

Wild-type patterns of gene expression in D. melanogaster during early metamorphosis were examined by assaying whole animals at stages that span two pulses of ecdysone. Microarrays were constructed containing 6240 elements that included more than 4500 unique cDNA expressed sequence tag (EST) clones along with a number of ecdysone-regulated control genes having predictable expression patterns. These ESTs represent approximately 30% to 40% of the total estimated number of genes in the Drosophila genome. In order to gauge expression levels, microarrays were hybridized with fluorescent probes derived from polyA⁺ RNA isolated from developmentally staged animals. The time points examined are relative to PF, which last approximately 15 to 30 min, during which time the larvae cease to move and evert their anterior spiracles. Nineteen arrays were examined representing six time points relative to PF: one time point before the late larval ecdysone pulse; one time point just after the initiation of this pulse (4 hours BPF), and time points at 3, 6, 9, and 12 hours after PF (APF). The prepupal pulse of ecdysone occurs 9 to 12 hours APF (White, 1999).

In order to manage, analyze, and disseminate the large amount of data, a searchable database was constructed that includes the average expression differential at each time point. The analysis set consists of all elements that reproducibly fluctuate in expression threefold or more at any time point relative to PF, leaving 534 elements containing sequences represented by 465 ESTs and control genes. More than 10% of the genes represented by the ESTs display threefold or more differential expression during early metamorphosis. This may be a conservative estimate of the percentage of Drosophila genes that change in expression level during early metamorphosis, because of the stringent criteria used for their selection (White, 1999).

To interpret these data, genes were grouped according to similarity of expression patterns by two methods. The first relied on pairwise correlation statistics, and the second relied on the use of self-organizing maps (SOMs). Differentially expressed genes fall into two main categories. The first category contains genes that are expressed at >18 hours BFP (before the late larval ecdysone pulse) but then fall to low or undetectable levels during this pulse. These genes are potentially repressed by ecdysone and make up 44% of the 465 ESTs identified in this set. The second category consists of genes expressed at low or undetectable levels before the late larval ecdysone pulse but then are induced during this pulse. These genes are potentially induced by ecdysone and make up 31% of the 465 ESTs. Consequently, 75% of genes that changed in expression by threefold or more do so during the late larval ecdysone pulse that marks the initial transition from larva to prepupa. This result is consistent with the extreme morphological changes that are about to occur in these animals. There are clearly discrete subdivisions of gene expression within these categories (White, 1999).

In contrast to the histolyzing larval muscles, the CNS undergoes dramatic differentiation and restructuring during early metamorphosis. The majority of the CNS is composed of adult-specific neurons that reorganize at this time by extending processes and establishing new connections. Several genes known to be involved in neuronal-specific processes are differentially regulated during the late larval ecdysone pulse (see Developmental control genes induced during metamorphosis.) For example, the Drosophila neurotactin and plexin A genes are induced. These genes are involved in axonal pathfinding and in establishing synaptic connections. The neurotactin (nrt) gene product is involved in growth cone guidance and is localized to the cell surface at points of interneuronal cell contact in the presumptive imaginal neurons within the larval CNS. Nerve cord condensation does not occur normally in the late third instar CNS of nrt mutant animals. In prepupae, nrt is expressed in a tissue- and cell type-specific manner: it is restricted to a small set of ocellar pioneer neurons in the brain, photoreceptors of the eye, and some sensory neurons in the developing wing. It is suggested that nrt, like the control genes induced from >18 hours BPF to PF, is regulated by the late larval ecdysone pulse. The plexin A gene belongs to a family of genes that encode Ca²⁺-dependent homophilic cell adhesion molecules first identified in the vertebrate CNS and PNS. Drosophila Plexin A also acts as a receptor for class I semaphorins, and both loss of function and overexpression experiments demonstrate that Plexin A is involved in axon guidance and repulsion of adjacent neurons (defasciculation). Many neurons defasciculate in response to ecdysone during nervous system remodeling, and it is suggested that an increase in plexin A expression may be partly responsible for this response. Several more differentially expressed neuronal-specific molecules are shown at The Drosophila Microarray Project. These genes provide several new candidates for factors that are involved in the neuronal outgrowth and morphological remodeling responses to ecdysone (White, 1999).

Effects of Mutation or Deletion

If Plexins are indeed Semaphorin receptors, then lesions in the PlexA gene might be expected to show similar axon guidance defects as are displayed by embryos mutant for either Sema1a (Yu, 1998 ) or Sema2a (Winberg, 1998). In embryos lacking PlexA, axon guidance defects were found both in the CNS and in the projections of motor nerves to their muscle targets in the periphery. These embryos do not show any morphological abnormalities, muscle defects, or cell fate changes. Embryos from the Df(4)G strain (that does not remove PlexA) display normal axon guidance, indicating that the observed phenotypes are closely linked with the PlexA gene. To test whether axon guidance phenotypes associated with the deficiency are actually due to the lack of PlexA, expression was restored using a transgenic construct, UAS-PlexA, under the transcriptional control of the neuron-specific driver, elav-GAL4. This combination rescues motor and CNS axon guidance defects in homozygous deficiency embryos. Some segments are not completely restored to wild type, but rather than displaying loss-of-function phenotypes, these segments display gain-of-function phenotypes for PlexA. Thus, it is concluded that the aberrations seen in the deficiency strain do indeed result from the lack of PlexA. Moreover, neuron-specific replacement of PlexA is sufficient to rescue the observed guidance phenotypes (Winberg, 1998).

PlexA-deficient embryos show axon guidance phenotypes that markedly resemble defects seen in Sema1a loss-of-function mutants (Yu, 1998). Such defects are seen in the 'b' branch of the intersegmental nerve (the ISNb), which innervates ventral muscles. ISNb axons normally exit the CNS as part of the ISN; they defasciculate from and exit the ISN at ISNb choice point 1, entering the ventral muscle region as a fasciculated bundle. At ISNb choice point 2, a single axon leaves the ISNb to innervate muscle fibers 6 and 7. At ISNb choice point 3, certain growth cones extend further dorsally to innervate muscle 12, while others stop and innervate muscle 13. In the absence of PlexA, ISNb growth cones often fail to defasciculate from one another at any or all three of the ISNb choice points. Occasionally they fail to exit the ISN and thus bypass the ventral muscles; in some cases they innervate their ventral muscle targets via small projections made directly from the main branch of the ISN. More often, they exit the ISN but then fail to defasciculate from each other at choice points 2 and/or 3, leading to a thickened, stalled nerve branch and failure to innervate muscles 6 and 7 and/or 12 (Winberg, 1998).

In addition to the ISNb, the segmental nerve (SN) is also frequently abnormal, with defects resembling those of Sema1a mutants (Yu, 1998 ). In wild-type embryos, the SN exits the CNS, and its main branch, the SNa, extends past the ventral muscle domain to the lateral muscle region. The SNa then divides into a lateral and a dorsal branch at SNa choice point 1, and then further dorsally, bifurcates again at SNa choice point 2. In PlexA-deficient embryos examined at late stage 16, axons at the second choice point failed to defasciculate from one another in roughly 70% of segments and instead extended dorsally as a single branch (Winberg, 1998).

Projections within the CNS are also abnormal in both Sema1a and PlexA mutants. Three major longitudinal axon fascicles on each side of the CNS can be detected with the 1D4 monoclonal antibody against Fas II. In wild-type embryos these tracts are evenly spaced and show fairly uniform thickness. In embryos lacking PlexA, the outermost longitudinal Fas II-positive fascicle is often disrupted, being thinner in some segments, discontinuous in others, and sometimes fused with the middle Fas II-positive fascicle (Winberg, 1998).

This analysis of phenotypes in PlexA Df mutant embryos (and their rescue by a PlexA transgene) clearly indicates an important role for PlexA in axon guidance. The phenotypes for PlexA in the projections of the ISNb, the SNa, and within the CNS are strikingly similar, both qualitatively and quantitatively, to those reported for null mutations of Sema1a (Yu, 1998 ). The high degree of phenotypic correspondence strongly suggests that these two genes encode components of the same pathway. Because Sema 1a has been described as a repulsive ligand for growth cone guidance, it is proposed that PlexA functions as a Sema 1a receptor for these guidance events (Winberg, 1998).

A further loss-of-function phenotype for PlexA is seen in the projection of the transverse nerve (TN). This nerve is composed of two parts, a peripheral neuron that extends an axon toward the CNS, and a pair of central neurons that project outward. These two projections normally extend toward one another along a shared mesodermal substrate, meeting and fasciculating near muscle 7. In PlexA-deficient embryos, growth cones from the TN extend ectopic projections onto ventral muscles in 36.5% of segments, compared with 2.8% in the PlexA rescue background. This phenotype is not seen in Sema1a mutants, raising the possibility that PlexA may also interact with one or more additional ligands (Winberg, 1998).

One way to check the hypothesis that PlexA 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).

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, 2004).

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, 2004).

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, 2004).

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, 2004).

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, 2004).

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, 2004).

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, 2004).

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, 2004).

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, 2004).

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, 2004).

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).

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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date revised: 10 April 2008

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