InteractiveFly: GeneBrief

off-track : Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References

BIOLOGICAL OVERVIEW

The nervous system in many species consists of multiple neuronal cell layers, each forming specific connections with neurons in other layers or other regions of the brain. How layer-specific connectivity is established during development remains largely unknown. In the Drosophila adult visual system, photoreceptor (R cell) axons innervate one of two optic ganglia layers; R1-R6 axons connect to the lamina layer, while R7 and R8 axons project through the lamina into the deeper medulla layer. The receptor tyrosine kinase Off-track (Otk) is specifically required for lamina-specific targeting of R1-R6 axons. Otk is highly expressed on R1-R6 growth cones. In the absence of otk, many R1-R6 axons connect abnormally to medulla instead of innervating lamina. It is proposed that Otk is a receptor or a component of a receptor complex that recognizes a target-derived signal for R1-R6 axons to innervate the lamina layer (Cafferty, 2004).

The transmembrane protein Off-track associates with Plexin A, the receptor for Sema 1a, and 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 formation of photoreceptor-to-optic-lobe connections in the Drosophila adult visual system is an excellent and simple model to study the molecular mechanisms that control the establishment of layer-specific neuronal connectivity during development. The Drosophila adult visual system is comprised of the compound eye and the optic lobe. The compound eye consists of ~800 ommatidia or single eye units, each containing eight different photoreceptor cells (R cells). R cells project axons into one of two optic ganglion layers in the brain. R1-R6 cells connect to the superficial layer of the optic lobe, the lamina, and are responsible for the absorption of light in the green range. R7 and R8 cells connect to the deeper medulla layer, and are responsible for the absorption of light in the ultraviolet and blue range. The formation of layer-specific R-cell connection pattern begins at the third-instar larval stage. Precursor cells in third-instar larval eye-imaginal discs begin to differentiate into R cells. Within each ommatidium, the R8 precursor cell differentiates first and projects its axon through the optic stalk and the developing lamina into the medulla. Axons from the later differentiated R1-R7 cells within the same ommatidium form a single bundle with the pioneer R8 axon until they encounter a layer of glial cells (i.e. marginal glia) within the lamina layer. There they have to make a binary choice: either stop or keep going into the medulla. The R1-R6 growth cones terminate within the lamina in response to an unknown stop signal from lamina glial cells, their intermediate target at larval stage. By contrast, R7 growth cones extend further to join R8 growth cones in the medulla. During pupation, R1-R6 growth cones undergo further stereotyped rearrangements and subsequently form synaptic connections with lamina neurons (Cafferty, 2004 and references therein).

Recent studies have identified several cell surface proteins that are required for R-cell connectivity. Specifically, N-Cadherin, the receptor tyrosine phosphatase Lar and the Cadherin-related protein Flamingo have each been shown to be required for the establishment of local synaptic connections between R1-R6 axons and lamina cartridge neurons. An additional role for N-Cadherin, Lar and the receptor tyrosine phosphatase PTP69D in R7 axons and Flamingo in R8 axons for forming local connections with target cells within the medulla has also been revealed. However, loss of N-Cadherin or Flamingo does not affect the initial choice between lamina versus medulla target selection. In their absence R1-R6 still connect to the lamina, while R7 and R8 still choose the medulla for establishing synaptic connections. While loss of Ptp69D or Lar does affect the initial projections of R1-R6 axons, the completed pattern of lamina-versus-medulla target selection in adult Ptp69D or Lar mutants remains largely unchanged. These data argue against a direct role for either PTP69D or Lar in specifying lamina-specific targeting of R1-R6 axons. In addition to the above cell surface receptors, two Drosophila receptor tyrosine kinases, the Insulin receptor and Eph receptor, are also required for regulating different aspects of R-cell axon guidance. However, neither has been shown to play a role in regulating layer-specific R-cell connectivity. Thus, it remains unclear how R-cell axons detect layer-specific targeting signals to make the binary decision for choosing either lamina or medulla to establish synaptic connections (Cafferty, 2004 and references therein).

In a search for genes that are required for R-cell projections in the developing visual system, the receptor tyrosine kinase Otk was identified as a key determinant in specifying the binary lamina versus medulla target selection. While Otk was originally isolated based on its homology with the trk family of neurotrophin receptors in vertebrates (Pulido, 1992), more recent studies suggest strongly that Otk is not a homolog of the vertebrate Trk A receptor (Kroiher, 2001). It has been shown that in vitro Otk mediates cell-cell adhesion in a Ca2+-independent homophilic manner (Pulido, 1992), while in vivo it functions downstream of Semaphorin-1a (Sema-1a) to regulate motor axon guidance at the embryonic stage (Winberg, 2001). Otk is predominantly localized to R1-R6 growth cones in the fly visual system and is specifically required for lamina-specific targeting of R1-R6 axons. It is proposed that Otk recognizes a lamina-derived signal for R1-R6 targeting (Cafferty, 2004).

R1-R6 targeting errors in otk mutants are first observed at third-instar larval stage when R cells begin to project axons into the developing optic lobe. Many R1-R6 growth cones pass through the lamina and extend into the medulla instead. This initial R1-R6 targeting error is not corrected at later developmental stages, since many R1-R6 axons remain within the medulla in adult otk mutants. While otk is necessary for lamina-specific R1-R6 targeting, it is not required in R7 axons for establishing connections with local target cells within the medulla. Both the presence of Otk on R1-R6 growth cones and the specific otk loss-of-function phenotype support a key role for Otk in R1-R6 growth cones to specify their lamina-specific targeting decision (Cafferty, 2004).

The role of Otk in R1-R6 growth cones appears to be different from that of PTP69D, the only other cell surface receptor that has also been shown to be required for the initial termination of R1-R6 axons within the lamina. In Ptp69D mutants, although ~25% of ommatidia projected one or more R1-R6 axons into the medulla at larval stage, only a few axon bundles (32 mistargeted R1-R6 axons or axon bundles in a total of 34 hemispheres examined) remained within the medulla at adult stage. In addition, mutations in Ptp69D also disrupt R7 targeting. Many R7 axons do not project into their normal M6 layer, but instead stay with the pioneer R8 axon at the superficial M3 layer within the medulla. These observations have led to the suggestion that PTP69D plays a permissive role in R1-R6 targeting: that is, PTP69D may mediate defasciculation between R1-R6 and the pioneer R8 axon in the lamina and between R7 and R8 axons in the medulla, thus allowing them to respond to a targeting signal. While this possibility for the action of Otk cannot be entirely excluded, it appears unlikely that R1-R6 targeting error in otk mutants is simply caused by defects in R-cell defasciculation. Unlike that in Ptp69D mutants, severe R1-R6 targeting errors (one or more mistargeted R1-R6 axons in ~42% of total ommatidial axon bundles) were also observed in otk adult mutants, whereas R7 target selection remains normal. Moreover, although mutations in the trio or pak gene caused a severe hyper-fasciculation phenotype, they do not affect the completed pattern of R1-R6 connectivity. Thus, a model is favored in which Otk is actively involved in detecting a targeting signal for R1-R6 axons to select the lamina layer (Cafferty, 2004).

While in otk mutants a large number of R1-R6 axons connect abnormally to medulla, many R1-R6 axons still select the lamina for establishing synaptic connections. One probable explanation is that the absence of Otk may be partially compensated by another receptor that also plays a role in specifying R1-R6 targeting. Partial redundancy is not uncommon for genes that regulate axon guidance. For instance, it has been shown that four neural-specific receptor tyrosine phosphastes (i.e. PTP10D, LAR, PTP69D and PTP99A) are partially redundant with each other in regulating axon guidance in the fly embryo. In mammals, recent studies demonstrate that the floor-plate-derived morphogen sonic hedgehog cooperates with netrin to guide commissural axons toward the ventral midline in the developing spinal cord (Cafferty, 2004).

Previous studies show that mutations in the brakeless (bks) (aka scribbler) gene causes a more severe R1-R6 targeting phenotype. Most, if not all, R1-R6 axons in bks mutants projected aberrantly into the medulla. The bks gene encodes a nuclear protein expressed in all R cells. Additional studies have indicated that Bks functions in R-cell growth-cone targeting by repressing the expression of another nuclear protein, Runt, in R2 and R5 cells. These studies thus raise the interesting possibility that Bks and Runt are components of a gene expression regulatory pathway, which controls the expression of specific cell surface receptors on R1-R6 growth cones for detecting a stop signal from the target region. To examine if the expression of Otk in R1-R6 cells is dependent on Bks, the level of the Otk protein was examined in bks mutants. However, no alteration in the expression level of Otk was detected, arguing against Otk as a downstream target of the Bks pathway (Cafferty, 2004).

Although otk is necessary for lamina-specific targeting of R1-R6 axons, its expression in R7 axons is not sufficient to target R7 axons to the lamina. There are several possible explanations for this result. Otk may need to collaborate with another cell surface protein that is present on R1-6 but not R7 growth cones to mediate the lamina-specific targeting decision, and thus act as a component of a receptor complex. This situation may be similar to that of the Nogo (Rtn4 -- Mouse Genome Informatics) receptor complex, which is involved in inhibiting neurite outgrowth in mammals (Wang, 2002). Upon ligand binding, the Nogo receptor initiates an inhibitory response only in the presence of p75 (Ngfr -- Mouse Genome Informatics), another cell surface receptor. Alternatively, the signaling components that function downstream of Otk in R1-6 growth cones may not be present in R7 growth cones. Alternatively, the presence of some inhibitory mechanisms within R7 growth cones may prevent them from responding to an Otk-mediated lamina-targeting signal. The possibility that Otk plays a permissive but not instructive role in R1-R6 growth-cone targeting cannot be excluded either (Cafferty, 2004).

Previous studies have demonstrated that Otk forms a receptor complex with Plexin A, which functions downstream of Sema-1a during motor axon guidance in the fly embryo (Winberg, 2001). In the fly adult visual system, however, the sema-1a phenotype appears quite different from that of otk, since the R1-R6 targeting pattern remain largely normal in sema-1a mutants. The simplest interpretation of this data is that otk functions in a different pathway in R1-R6 growth cones for specifying lamina-specific targeting decision. An alternative explanation is that Sema-1a may function redundantly with other proteins (for instance, other members of the Semaphorin protein family), to regulate the function of Otk during R1-R6 targeting. The present data do not allow distinguishing among these possibilities (Cafferty, 2004).

Otk belongs to the evolutionarily conserved CCK-4 family of `dead' receptor tyrosine kinases (Kroiher, 2001). Members of this family carry alterations in several evolutionarily conserved residues within the kinase domain that have been shown to be essential for the activity of most (if not all) active tyrosine kinases. Indeed, several of them have been shown to be inactive kinases by biochemical analysis (Miller, 2000). How does a defective receptor tyrosine kinase such as Otk transduce targeting signals for specifying layer-specific R-cell connectivity? One possibility is that Otk associates with an unknown active tyrosine kinase, which induces tyrosine phosphorylation on Otk upon ligand binding. One precedent for this is the dead kinase ErbB3, a member of the vertebrate EGFR family. Although the kinase activity of ErbB3 is greatly impaired, it can transduce mitogenic signals by forming a heterodimer receptor complex with another EGFR family member (e.g., ErbB2) carrying an active kinase domain. ErbB2 then induces tyrosine phosphorylation in the cytoplasmic domain of ErbB3, which serves as a docking site for downstream signaling proteins. Interestingly, it has been shown that Otk is phosphorylated on tyrosine residues in both fly and mammalian cultured cells (Pulido, 1992; Winberg, 2001). It is highly possible that in response to a targeting signal these phosphorylation sites recruit downstream signaling proteins, which then transduce the signal into the termination of R1-R6 growth cones within the lamina. In this context, it is notable that the intracellular signaling protein Dreadlocks (Dock), a SH2/SH3 adapter protein, also plays a role in lamina-specific targeting of R1-R6 axons. Dock contains a single SH2 domain that can bind to specific phosphorylated tyrosine residues on activated proteins. Previous studies suggest that a Dock-mediated signal activates the Ste20-like kinase Msn, which in turn phosphorylates the cytoskeletal regulator Bif, leading to the termination of R1-R6 growth cones in the lamina (Ruan, 2002; Ruan, 1999). Experiments were performed to investigate the potential interaction between Otk and Dock during R1-R6 targeting. However, no genetic interaction was observed between them. Moreover, quantification of the R1-R6 targeting phenotype in adults shows that the phenotype in dock mutants is less severe than that in otk mutants. While these data appear inconsistent with the notion that Otk and Dock function in the same pathway, it does not exclude the possibility that Dock cooperates with another SH2-containing protein to transduce the signal from the activation of Otk to downstream effectors for lamina-specific targeting of R1-R6 axons. Further studies will be necessary to critically address this matter (Cafferty, 2004).

In summary, the present study demonstrates an essential role for Otk in specifying R-cell connectivity. It is proposed that Otk is involved in recognizing a layer-specific signal for R1-R6 axons to select the lamina for synaptic connections. Further biochemical, molecular and genetic dissection of the Otk pathway will help to understand the action of Otk in R-cell growth cones and shed light on the general mechanisms controlling the establishment of layer-specific neuronal connectivity in the nervous system (Cafferty, 2004).

REGULATION

Protein Interactions

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).

Immunoprecipitated human Plexins A3 and B1 copurify a number of proteins from BOSC-23 cell extracts, some of which became tyrosine phosphorylated in an in vitro kinase assay. Western blotting has indicated that this activity is not due to the presence of Met, Ron, Abl, or Src tyrosine kinases. The most prominent labeled band other than Plexins is approximately of 160 kDa (Winberg, 2001).

To identify the putative Plexin-associated protein, candidates in Drosophila were considered. Several proteins with homology to receptor-tyrosine kinases have been identified that are expressed in the CNS and could potentially interact with Plexins. However, in the cases where the loss-of-function phenotypes have been assayed, there is not a notable similarity with those described for Semaphorins or Plexins, suggesting an unrelated function (e.g., EGFR, FGFR, Derailed). In other cases, in vivo functional data are yet lacking, but some of these candidates may be considered less probable on the basis of molecular weight (e.g., Dror, Nrk). A leading remaining contender is the Drosophila protein Off-track (OTK; previously called Dtrk) (Winberg, 2001).

Based on its molecular weight, the observation that it can be tyrosine phosphorylated, and its expression on axons at the appropriate time in development to play a role in axon guidance, OTK seemed like a good candidate for possible interaction with Plexin. BLAST searches of protein databases, using either the cytoplasmic kinase or extracellular domain, indicate that the closest relatives of OTK are the chick protein KLG and its human homolog CCK4/PTK7 (Winberg, 2001).

As a first test of OTK protein function, molecular association was examined in COS cells. Epitope-tagged versions of both OTK and a variety of Drosophila and mammalian Plexins (DPlexA, PlexA3, and PlexB1) were generated and tested for expression. The cytoplasmic domains of Plexins are highly conserved, and, thus, binding relationships are likely to be conserved across phylogeny. Cells were cotransfected to express both proteins, and the formation of complexes was analyzed by immunopurification and Western blotting (Winberg, 2001).

Drosophila PlexA (HA tagged) copurifies with immunoprecipitated OTK (myc tagged). Moreover, mammalian PlexA3 and PlexB1 (VSV tagged) also copurify with immunoprecipitated OTK (myc tagged). OTK can copurify all three Plexins but not an unrelated protein, the netrin receptor DCC. In addition, OTK (myc tagged) copurifies with immunoprecipitated mammalian Plexin A3 and B1 (VSV tagged). OTK is copurified in a similar fashion with immunoprecipitated Drosophila Plexin A (HA tagged). These experiments identify OTK as a transmembrane protein that can constitutively associate with both Drosophila and mammalian Plexins in transfected cells, raising the possibility that OTK might play a role in either up- or down-regulating Plexin activity or mediating Semaphorin-Plexin signaling. To determine whether this association reflects a true functional interaction, genetic analysis was performed of OTK in Drosophila (Winberg, 2001).

A direct in vivo test of OTK function was aided by the discovery of a P element insertional mutation near the otk gene, designated EP2017. This mutant strain was obtained from the collection of the Berkeley Drosophila Genome Project and was examined for axon guidance defects in homozygous embryos. Indeed, some defects were found, but they were subtle in nature and poorly penetrant. However, the element is located upstream of the coding sequence and may not completely disrupt gene function. Attempts were made to generate complete loss-of-function otk alleles through imprecise excision of the P element (Winberg, 2001).

The EP2017 element is inserted 30 bp upstream of the 5' end of the published otk cDNA. Since the OTK transcript is ~900 bp longer than the cDNA (Pulido, 1992), it is likely that the insert is in the 5' UTR. Ten excision lines were genetically characterized; eight were homozygous lethal and two homozygous viable (the starting strain is semilethal), suggesting that otk is an essential gene. Molecular analysis indicates that the viable strains otk² and otk⁸ are precise excisions. In contrast, the lethal strain otk³ carries a 3 kb deletion that extends downstream of the EP2017 element, apparently disrupting otk but not upstream genes. The otk³ lesion removes the putative translational start codon and part of the signal peptide and thus likely represents a complete loss-of-function allele. Subsequent examination of axon guidance defects has shown that otk³ and three other lethal alleles are similar to one another in the variety and severity of their phenotypes, which are more pronounced than those displayed by the original EP2017 strain. In comparison, otk² is in the range of wild-type (Winberg, 2001).

These reagents allowed for a test of another property of the EP2017 insert. The EP series of P elements contains a UAS gene-regulatory sequence that, in combination with a GAL4 driver, permits transcription of sequences flanking the insertion site of the P element. In the present case, EP2017 is oriented such that GAL4-regulated expression yields short antisense OTK transcripts. In conjunction with elav-GAL4, one copy of EP2017 produces axon guidance abnormalities comparable with homozygous mutant otk¹ or otk³ strains, suggesting that this antisense transcription from EP2017 confers a neuron-specific dominant loss-of-function phenotype (Winberg, 2001).

If OTK is important for Plexin A function, then loss-of-function mutations in otk might show guidance phenotypes similar to other mutations in the pathway. Specifically, if OTK is a positive activator or effector of Plexin A, then loss-of-function phenotypes of one should resemble loss-of-function phenotypes of the other. However, if OTK is a negative regulator of Plexin A, then the loss of OTK might lead to similar phenotypes as the overproduction of Plexin A protein. Indeed, embryos mutant for otk display axon guidance defects in the CNS and in the projections of the motor nerves, with abnormalities that are similar to those previously reported for PlexA and Sema1a loss-of-function mutants. The projections of motor neurons to their muscle targets are more obviously affected, disrupted in a way that suggests individual growth cones are not always able to defasciculate from pioneer neurons when they should. The most telling examples are provided by the dorsal projections of the segmental nerve (SN) and the ventrolateral or 'b' branch of the intersegmental nerve (ISNb) (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).

Interestingly, despite its homology with receptor tyrosine kinases and the observation that immunoprecipitates of Drosophila OTK possess tyrosine kinase activity (Pulido, 1992), OTK itself is probably not an active tyrosine kinase. The OTK sequence suggests that it belongs to a family of kinase 'dead' receptors. The catalytic domain of OTK, like other members of this family, is altered in a few key conserved residues that are implicated in autophosphorylation (the conserved DFG motif substituted by YPA). Vertebrate family members bear similar alterations in the DFG motif and apparently do not have kinase activity. Modest tyrosine phosphorylation of OTK has been observed in 293T cells but no significant increase in Plexin phosphorylation has been observed upon coexpression with OTK. Thus, OTK either possesses a weak catalytic activity, which is barely detectable in the tested experimental conditions, or like other members of the CCK-4 subfamily of receptor tyrosine kinases, OTK might be kinase dead. In the latter case, some other active kinase would be expected to be present in or recruited to the OTK/Plexin complex in order to account for the observed tyrosine phosphorylation of these proteins. This situation is reminiscent of the interleukin receptors, which are heterodimers composed of a ligand binding subunit and a signal transducing subunit known as gp130. Neither subunit possesses a catalytic activity; rather, gp130 associates with the Janus kinases. Upon ligand binding, the receptors multimerize, resulting in activation of the Janus kinases and tyrosine phosphorylation of the receptor (Winberg, 2001).

Another receptor tyrosine kinase carrying mutations in conserved DFG catalytic residues, h-Ryk/d-Derailed, appears also to be kinase inactive. Nevertheless, Ryk/Derailed is crucially involved in axon guidance. Thus, at least two highly conserved receptor tyrosine kinases, both of which are members of families which are kinase dead -- OTK and Derailed -- have been shown to function in axon guidance. In the case of OTK, it functions apparently by associating with Plexins and helps to mediate their output (Winberg, 2001).

The signal transduction pathway activated by Semaphorins is beginning to be clarified. The cytoplasmic domains of Plexins do not have any obvious signal transduction motif such as a kinase or phosphatase domain. However, the cytoplasmic domains of Plexin B receptors bind directly to the Rac GTPase in a GTP-dependent manner. It has been confirmed that the cytoplasmic domain of Plexin B (PlexB) indeed binds directly to the active, GTP-bound form of the Rac GTPase and, in addition, that a different region of PlexB binds to RhoA. The genetic and biochemical evidence suggests a model whereby PlexB mediates repulsion in part by coordinately regulating two small GTPases in opposite directions: PlexB binds to Rac^GTP and downregulates its output by blocking its access to PAK and, at the same time, binds to and increases the output of RhoA. While the contribution of OTK to this signaling pathway has not yet been investigated, by analogy with other tyrosine-phosphorylated receptor complexes, one hypothesis to test is that a Rho exchange factor is recruited to the activated Plex/OTK complex, providing local activation of Rho (Winberg, 2001).

Prior to the identification of Plexins as Semaphorin receptors and the implication of OTK as a Plexin-associated kinase, both proteins were shown to be capable of mediating cell aggregation in vitro. These studies led to the suggestion that both Plexins and OTK might function as homophilic cell adhesion molecules. Whether either or both of them normally functions in a homophilic fashion in vivo is unknown (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).

DEVELOPMENTAL BIOLOGY

Embryonic

OTK is a glycoprotein of apparent molecular weight 160 kDa whose extracellular domain, with its six immunoglobulin (Ig) repeats, shows similarity to cell adhesion proteins. In vitro studies have shown that OTK can mediate homophilic adhesion, which results in tyrosine phosphorylation of the intracellular domain (Pulido, 1992). In early Drosophila embryos, OTK transcript is broadly distributed, consistent with both maternal loading and zygotic expression. In later stages, the protein is detected on neuronal cell bodies and axons within the CNS and in the projections of motor neurons as they extend to muscle fibers in the periphery. Because of this axonal localization and in vitro adhesion, OTK has been suggested to play a role in selective fasciculation and axon guidance (Pulido, 1992).

Larval

Previous studies have demonstrated that Otk is specifically expressed in the nervous system at the embryonic stage (Pulido, 1992; Winberg, 2001). To determine if Otk is also expressed in the developing adult visual system at larval stage, third-instar larval eye-brain complexes were stained with an affinity purified anti-Otk antibody (Pulido, 1992). In wild type, anti-Otk staining was detected on R-cell axons in the developing optic lobe. In the lamina, the staining overlapped largely with 24B10 immunoreactivity, which reflects the expression pattern of Chaoptin, a cell surface adhesion molecule expressed exclusively on all R cells and their axons. The strongest staining was observed in the lamina plexus, comprised primarily of R1-R6 growth cones. Although anti-Otk immunoreactivity was also detected in the developing medulla, it was not possible tell if Otk is present on R7 and R8 growth cones due to the uniform staining pattern in the medulla neuropil, which consists of both R-cell and non-R-cell axons. The specificity of anti-Otk staining was supported by the fact that the staining within the lamina was largely absent in otk³ mosaic larvae. It is concluded that Otk is expressed in developing R cells and is localized predominantly to R1-R6 growth cones (Cafferty, 2004).

REFERENCES

Search PubMed for articles about Drosophila off-track

Caddy, J., et al. (2010). Epidermal wound repair is regulated by the planar cell polarity signaling pathway. Dev. Cell 19(1): 138-47. PubMed Citation: 20643356

Cafferty, P., Yu, L. and Rao, Y. (2004). The receptor tyrosine kinase Off-track is required for layer-specific neuronal connectivity in Drosophila. Development 131(21): 5287-95. 15456725

Jung, J. W., Ji, A. R., Lee, J., Kim, U. J. and Lee, S. T. (2002). Organization of the human PTK7 gene encoding a receptor protein tyrosine kinase-like molecule and alternative splicing of its mRNA. Biochim. Biophys. Acta 1579(2-3): 153-63. 12427550

Jung, J. W., Shin, W. S., Song, J. and Lee, S. T. (2004). Cloning and characterization of the full-length mouse Ptk7 cDNA encoding a defective receptor protein tyrosine kinase. Gene 328: 75-84. 15019986

Kroiher, M., Miller, M. A. and Steele, R. E. (2001). Deceiving appearances: signaling by 'dead' and 'fractured' receptor protein-tyrosine kinases. Bioessays 23: 69-76. 1113531

Lu, X., Borchers, A. G., Jolicoeur, C., Rayburn, H., Baker, J. C. and Tessier-Lavigne, M. (2004). PTK7/CCK-4 is a novel regulator of planar cell polarity in vertebrates. Nature 430(6995): 93-8. 15229603

Miller, M. A. and Steele, R. E. (2000). Lemon encodes an unusual receptor protein-tyrosine kinase expressed during gametogenesis in Hydra. Dev. Biol. 224: 286-298. 10926767

Pulido, D., Campuzano, S., Koda, T., Modolell, J. and Barbacid, M. (1992). Dtrk, a Drosophila gene related to the trk family of neurotrophin receptors, encodes a novel class of neural cell adhesion molecule. EMBO J. 11: 391-404. 1371458

Ruan, W., Pang, P. and Rao, Y. (1999). The SH2/SH3 adaptor protein dock interacts with the Ste20-like kinase misshapen in controlling growth cone motility. Neuron 24: 595-605. 10595512

Ruan, W., Long, H., Vuong, D. H. and Rao, Y. (2002). Bifocal is a downstream target of the Ste20-like serine/threonine kinase misshapen in regulating photoreceptor growth cone targeting in Drosophila. Neuron 36: 831-842. 12467587

Savory, J. G., et al. (2011). Cdx mediates neural tube closure through transcriptional regulation of the planar cell polarity gene Ptk7. Development 138(7): 1361-70. PubMed Citation: 21350009

Shnitsar, I. and Borchers, A. (2008). PTK7 recruits dsh to regulate neural crest migration. Development 135(24): 4015-24. PubMed Citation: 19004858

Wang, K. C., Kim, J. A., Sivasankaran, R., Segal, R. and He, Z. (2002). P75 interacts with the Nogo receptor as a co-receptor for Nogo, MAG and OMgp. Nature 420: 74-78. 12422217

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

Yen, W. W., et al. (2009). PTK7 is essential for polarized cell motility and convergent extension during mouse gastrulation. Development 136(12): 2039-48. PubMed Citation: 19439496

date revised: 22 May 2023

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