InteractiveFly: GeneBrief

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


Gene name - off-track

Synonyms - Dtrk

Cytological map position - 48D6--7

Function - receptor

Keywords - axon guidance, Plexin receptor complex

Symbol - otk

FlyBase ID: FBgn0004839

Genetic map position - 2R

Classification - CCK-4 family of 'dead' receptor tyrosine kinases, Ig domains

Cellular location - surface transmembrane



NCBI links: Entrez Gene
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 RacGTP 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).


EFFECTS OF MUTATION

To identify genes that are required for layer-specific targeting of R-cell axons, R-cell projection pattern were examined in available mutants, including novel P-element insertions from the Berkeley Drosophila Genome Project as well as mutations that disrupt known genes that are expressed specifically in the nervous system. Among them, mutations in the otk gene were found to cause a specific R-cell projection phenotype. Since the null mutation otk3 in which the putative translational start codon and part of the signal peptide is deleted causes embryonic lethality (Winberg, 2001), genetic mosaic analysis was performed to examine axonal projections from otk3 mutant R-cell clones. otk homozygous mutant tissues were generated in an otherwise heterozygous or wild-type eye-imaginal disc by eye-specific mitotic recombination using the eyFLP/FRT system. By examining mutant clones in adult mosaic eyes, It was establised that ~80-90% of ommatidia in each mosaic eye examined were otk mutant clones, which was consistent with the absence of most anti-Otk immunoreactivity in the lamina in all otk3 mosaic third-instar eye-brain complexes examined (see below) (Cafferty, 2004).

R-cell projection pattern in otk mosaic larvae was examined using monoclonal antibody 24B10, which visualizes all R-cell axons in the developing optic lobe, R1-R6 growth cones terminated within the lamina and then expanded significantly in size; this was seen as a continuous layer of 24B10 immunoreactivity within the lamina, whereas expanded R7 and R8 growth cones form a highly organized pattern within the medulla. In otk3 mosaic individuals, small gaps were frequently observed in R1-R6 terminal field. The terminal field within the medulla was also disorganized: thicker bundles were frequently observed within the medulla. Unlike some known mutations (e.g., dock and pak) that affect R-cell guidance, loss of otk did not cause an obvious defect in the overall organization of R-cell axons within the developing optic lobe. The formation of topographic map also appeared normal (Cafferty, 2004).

To determine if the above defect is caused by mistargeting of R1-R6 axons, the larval R2-R5 marker ro-tau-lacZ was used to assess the initial targeting of a subset of R1-R6 axons at third-instar larval stage. In wild type, the vast majority of R2-R5 axons stop within the lamina, and only a few labeled axons (average three mistargeted axons or axon bundles per hemisphere) projected into the medulla. In otk3 mosaic individuals, however, more than 32% (average 33 axons or axon bundles per hemisphere) of ommatidia projected one or more R2-R5 axons or axon bundles aberrantly into the medulla. A similar mistargeting phenotype (average 18 mistargeted R2-R5 axons or axon bundles per hemisphere, n=19 hemispheres) was also observed in otk3/otkEP(2)2017 transheterozygous larvae. However, the phenotype was less severe than that in otk3 mosaic larvae; this was probably due to the hypomorphic nature of the otkEP(2)2017 allele. To further determine if the above phenotype was indeed due to the lesion in the otk gene locus, transgene rescue experiments were performed. It was found that eye-specific expression of an otk transgene completely rescued the R1-R6 mistargeting phenotype in otk mutants. The average number of mistargeted R2-R5 axons or axon bundles was reduced to three in otk3/otkEP(2)2017 transheterozygous larvae expressing the otk transgene, which is similar to that in wild type. This result, taken together with that from eye-specific genetic mosaic analysis, indicates that Otk is required in the eye for lamina-specific targeting of R1-R6 growth cones (Cafferty, 2004).

The R1-R6 mistargeting phenotype may reflect a direct role for Otk in regulating R-cell growth-cone targeting. Alternatively, the defect might be caused by abnormal R-cell differentiation or cell fate determination; for instance, the transformation of a R1-R6 cell into a R7 or R8 fate. To distinguish among those possibilities, R-cell development was examined by using R-cell-specific developmental markers. Differentiating R7 and R8 cells in the developing eye disc were identified with anti-Prospero and anti-Boss antibodies, respectively. As in wild type, only one R7 and one R8 were observed in each ommatidium in all otk3 mosaic eye discs examined. Consistently, examination of otk adult mosaic eyes did not reveal any defect in either the number or the organization of R cells in all otk3 mutant ommatidia examined. Thus, otk is not required for R-cell differentiation and cell fate determination (Cafferty, 2004).

Previous studies demonstrate a dynamic interaction between R-cell axons and lamina glial cells, the intermediate target of R1-R6 axons at larval stage. Lamina glial cells produce an unknown stop signal to induce the initial termination of R1-R6 growth cones within the lamina. In addition, R-cell axons produce an unknown signal to induce the migration of lamina glial cells into the R1-R6 target region. To determine if the expression of Otk in R-cell axons is necessary for lamina glial cell differentiation and/or migration, the development of lamina glial cells was examined in otk mutants. Glial cells were visualized using a monoclonal antibody that recognizes the glia-specific nuclear protein Repo. In wild type, R1-R6 axons stop in the lamina and expand their growth cones in between two layers of lamina glial cells (i.e. epithelial and marginal glia). Although lamina glial cells in otk mutants appeared less organized than those in wild type, the number of lamina glial cells surrounding the lamina plexus in otk mutants was similar to that in wild type, indicating that the migration of lamina glial cells occurs normally in otk mutants (Cafferty, 2004).

Studies have shown that Otk interacts with Plexin A in mediating a Sema-1a-induced repulsive response during motor axon guidance at embryonic stage (Winberg, 2001), raising the possibility that the role of Otk in R1-R6 growth cones is also dependent on Sema-1a signaling. If so, one would predict that loss of Plexin A or Sema-1a should cause a similar R1-R6 targeting phenotype. Unfortunately, it was not possible to assess the role of plexin A during R1-R6 growth-cone targeting, sinced the available plexin A mutation causes early lethality; the plexin A gene is located on the fourth chromosome and thus not amenable to FLP/FRT-mediated mosaic analysis. However, R-cell projections could be examined in both sema-la homozygous null mutants (i.e., semaP1) and sema-la eye-specific mosaic animals in which large clones of semaP1 mutant eye tissues were generated similarly using the eyFLP/FRT system. Labeling of R-cell axons with MAb 24B10 staining revealed an R-cell projection phenotype in both semaP1 homozygous mutant and mosaic larvae. The R1-R6 terminal field in the lamina was severely disrupted; clumps and loop-like structures were frequently observed in sema mutants. In comparison, otk mutations caused only relatively mild defects in the organization of R-cell axons within the lamina (Cafferty, 2004).

To specifically assess the potential effect of sema-1a mutations on R1-R6 targeting, the ro-tau-lacZ marker was used to label R2-R5 axons in semaP1 homozygous mutant larvae. Surprisingly, although the organization of R-cell axons within the lamina was severely disrupted in semaP1 mutants, lamina-specific targeting of R2-R5 axons occurred in a largely normal fashion. In semaP1 homozygous mutants, the average number of mistargeted axons or axon bundles in each hemisphere is seven, a few more than that in wild type (i.e., three), but much fewer than that in otk3 mosaic animals (i.e., 33). Those observations argue against the possibility that Otk is regulated by Sema-1a for targeting R1-R6 axons to the lamina (Cafferty, 2004).

To determine the effect of the otk mutation on the completed pattern of R-cell-to-brain connectivity in adults, R-cell axonal projections were examined in otk mosaic heads. Again, large clones of otk3 mutant tissues were generated in the compound eye by eye-specific mitotic recombination. The completed R-cell projection pattern in adults was examined by staining frozen sections of otk mosaic heads with MAb 24B10. Although R-cell axons appear to project into correct topographic locations, an increase in the number of axon terminals within the medulla was observed in all sections examined, suggesting that many mistargeted R1-R6 axons remain within the medulla (Cafferty, 2004).

To confirm this, R1-R6 axons were specifically labeled using an adult R1-R6 marker Rh1-lacZ. To accurately count the total number of axons that project abnormally into the medulla, whole-mount staining of the brain was performed instead of staining frozen sections. In wild type, all Rh1-lacZ-labeled axons connected to the lamina. In all 11 wild-type hemispheres examined, no labeled axons projected into the medulla. In otk mosaic heads, however, a large number of R1-R6 axons were present in the medulla in otk mosaic animals (16 out of 17 hemispheres). Among 16 otk mosaic hemispheres that displayed the mistargeting phenotype, 13 hemispheres were mounted properly such that the total number of mistargeted R1-R6 axons or axon bundles could be accurately counted. The average number of mistargeted R1-R6 axons or axon bundles per hemisphere was 336 (ranging from 119 to 363 in different hemispheres). Mistargeted axons were distributed evenly within the medulla. By dividing the average number of mistargeted R1-R6 axons or axon bundles (i.e., 336) by 800 (the approximately total number of ommatidial fascicles within an adult eye), it is estimated that approximately 42% of ommatidia projected one or more R1-R6 axons aberrantly into the medulla. This is in marked contrast to that in Ptp69D adult mutants, in which only a few mistargeted R1-R6 axon bundles (<5%) were observed within the medulla (Cafferty, 2004).

To determine if loss of otk also affects the targeting of other R cells, the adult R7 marker PanR7-GAL4::UAS-Synaptobrevin-GFP was used to specifically assess the projections of R7 axons in otk adult mosaic heads in which the vast majority of R cells are otk mutant cells. In wild type, R7 axons projected into a region (i.e., M6 layer) in the medulla that is deeper than the R8 terminal field (i.e., M3 layer). In all sections examined, labeled R7 axons still projected into the correct locations within the medulla. Thus, unlike loss of Ptp69D or Lar, mutations in otk do not affect R7 targeting (Cafferty, 2004).

In a previous studies (Ruan, 2002), it was shown that the expression of the Ste20-like ser/thr kinase Misshapen (Msn) or the cytoskeletal regulator Bifocal (Bif) in R7 cells under control of a larval R7-specific driver PM181-GAL4 caused some R7 growth cones to target into the lamina. To determine if the expression of Otk alone is sufficient for specifying lamina-specific targeting of R-cell axons, the effect of expressing Otk in R7 axons was examined using the PM181-GAL4 driver. In wild type, all labeled R7 axons projected through the lamina and terminated within the medulla. In all larvae expressing Otk in R7 cells, R7 axons still extended normally into the medulla (Cafferty, 2004).


EVOLUTIONARY HOMOLOGS

Protein tyrosine kinase-7 (PTK7) is a receptor protein tyrosine kinase (RPTK)-like molecule that contains a catalytically inactive tyrosine kinase domain. The genomic structure of the human PTK7 gene is reported by screening a BAC library and DNA sequencing. The PTK7 gene is organized into 20 exons. All of the splicing junctions followed the conserved GT/AG rule. The exon-intron structure of the PTK7 gene in the region that encodes the catalytic domain is distinct from those of other RPTKs with strong homology. The 5'-flanking sequence of the PTK7 gene contains two GC boxes that concatenate Sp1 binding motifs, but does not contain either the TATA or CAAT consensus sequence. Using a luciferase reporter assay, the 883-bp 5'-flanking sequence was demonstrated to be functional as a promoter of the PTK7 gene. Four new splicing variants were identified in testis that could be derived from alternative splicing of exons 8-10, exon 10, a part of exons 12 and 13, and exon 16. The expression patterns of the splicing variants in the hepatoma and colon cancer cells were different from those of the testis. These findings suggest that PTK7 is evolutionarily distinct from other RPTKs, and that the alternative splicing of PTK7 mRNA may contribute to its diverse function in cell signaling (Jung, 2002).

The 3.8-kb full-length mouse Ptk7 cDNA encoding a defective receptor protein tyrosine kinase was cloned by reverse transcription-PCR of mouse liver mRNA. The mouse PTK7 polypeptide shows 92.6% identity to human PTK7. The mouse Ptk7 gene consists of 20 exons and has exactly the same exon structure as the human PTK7 gene. Mouse PTK7 is no phosphorylated either by itself or by other protein tyrosine kinases. In addition, its expression does not affect the phospho-tyrosine level of cellular proteins in COS-1 cells. The mouse Ptk7 mRNA is expressed at high levels in lung and un-pregnant uterus among adult tissues, and in the tail, limbs, somites, gut, and craniofacial regions among embryonic tissues. These data suggest that mouse PTK7, an orthologue of human PTK7, plays multiple roles in embryonic development (Jung, 2004).

In addition to the apical-basal polarity pathway operating in epithelial cells, a planar cell polarity (PCP) pathway establishes polarity within the plane of epithelial tissues and is conserved from Drosophila to mammals. In Drosophila, a 'core' group of PCP genes including frizzled (fz), flamingo/starry night, dishevelled (dsh), Van Gogh/strabismus and prickle, function to regulate wing hair, bristle and ommatidial polarity. In vertebrates, the PCP pathway regulates convergent extension movements and neural tube closure, as well as the orientation of stereociliary bundles of sensory hair cells in the inner ear. A mutation in the mouse protein tyrosine kinase 7 (PTK7) gene, which encodes an evolutionarily conserved transmembrane protein with tyrosine kinase homology, disrupts neural tube closure and stereociliary bundle orientation, and shows genetic interactions with a mutation in the mouse Van Gogh homologue vangl2. PTK7 is dynamically localized during hair cell polarization, and the Xenopus homologue of PTK7 is required for neural convergent extension and neural tube closure. These results identify PTK7 as a novel regulator of PCP in vertebrates (Lu, 2004).

PTK7 recruits dsh to regulate neural crest migration

PTK7 regulates planar cell polarity (PCP) signaling during vertebrate neural tube closure and establishment of inner ear hair cell polarity; however, its signaling mechanism is unknown. This study demonstrates a new function for PTK7 in Xenopus neural crest migration and uses this system in combination with in vitro assays to define the intersection of PTK7 with the non-canonical Wnt signaling pathway that regulates PCP. In vitro, using Xenopus ectodermal explants, it was shown that PTK7 recruits dishevelled (dsh) to the plasma membrane, a function that is dependent on the PDZ domain of dsh, as well as on the conserved kinase domain of PTK7. Furthermore, endogenous PTK7 is required for frizzled7-mediated dsh localization. Immunoprecipitation experiments confirm that PTK7 can be found in a complex with dsh and frizzled7, suggesting that it cooperates with frizzled to localize dsh. To evaluate the in vivo relevance of the PTK7-mediated dsh localization, Xenopus neural crest migration was analyzed, since loss-of-function of PTK7 inhibits neural crest migration in whole embryos as well as in transplanted neural crest cells. Supporting the in vivo role of PTK7 in the localization of dsh, a PTK7 deletion construct deficient in dsh binding inhibits neural crest migration. Furthermore, the PTK7-mediated membrane localization of a dsh deletion mutant lacking PCP activity inhibits neural crest migration. Thus, PTK7 regulates neural crest migration by recruiting dsh, providing molecular evidence of how PTK7 intersects with the PCP signaling pathway to regulate vertebrate cell movements (Shnitsar, 2008).

PTK7 is essential for polarized cell motility and convergent extension during mouse gastrulation

Despite being implicated as a mechanism driving gastrulation and body axis elongation in mouse embryos, the cellular mechanisms underlying mammalian convergent extension (CE) are unknown. This study shows, with high-resolution time-lapse imaging of living mouse embryos, that mesodermal CE occurs by mediolateral cell intercalation, driven by mediolaterally polarized cell behavior. The initial events in the onset of CE are mediolateral elongation, alignment and orientation of mesoderm cells as they exit the primitive streak. This cell shape change occurs prior to, and is required for, the subsequent onset of mediolaterally polarized protrusive activity. In embryos mutant for PTK7, a novel cell polarity protein, the normal cell elongation and alignment upon leaving the primitive streak, the subsequent polarized protrusive activity, and CE and axial elongation all failed. The mesoderm normally thickens and extends, but on failure of convergence movements in Ptk7 mutants, the mesoderm underwent radial intercalation and excessive thinning, which suggests that a cryptic radial cell intercalation behavior resists excessive convergence-driven mesodermal thickening in normal embryos. When unimpeded by convergence forces in Ptk7 mutants, this unopposed radial intercalation resulted in excessive thinning of the mesoderm. These results show for the first time the polarized cell behaviors underlying CE in the mouse, demonstrate unique aspects of these behaviors compared with those of other vertebrates, and clearly define specific roles for planar polarity and for the novel planar cell polarity gene, Ptk7, as essential regulators of mediolateral cell intercalation during mammalian CE (Yen, 2009).

Epidermal wound repair is regulated by the planar cell polarity signaling pathway

The mammalian PCP pathway regulates diverse developmental processes requiring coordinated cellular movement, including neural tube closure and cochlear stereociliary orientation. This study shows that epidermal wound repair is regulated by PCP signaling. Mice carrying mutant alleles of PCP genes Vangl2, the flamingo homolog Celsr1, off-track homolog PTK7, and Scrb1, and the Grainyhead transcription factor Grhl3, interact genetically, exhibiting failed wound healing, neural tube defects, and disordered cochlear polarity. Using phylogenetic analysis, ChIP, and gene expression in Grhl3-/- mice, RhoGEF19, a homolog of a RhoA activator involved in PCP signaling in Xenopus, was identified as a direct target of GRHL3. Knockdown of Grhl3 or RhoGEF19 in keratinocytes induced defects in actin polymerization, cellular polarity, and wound healing, and re-expression of RhoGEF19 rescued these defects in Grhl3-kd cells. These results define a role for Grhl3 in PCP signaling and broadly implicate this pathway in epidermal repair (Caddy, 2010).

Cdx mediates neural tube closure through transcriptional regulation of the planar cell polarity gene Ptk7

The vertebrate Cdx genes (Cdx1, Cdx2 and Cdx4) encode homeodomain transcription factors with well-established roles in anteroposterior patterning. To circumvent the peri-implantation lethality inherent to Cdx2 loss of function, the Cre-loxP system has been used to ablate Cdx2 at post-implantation stages, and a crucial role for Cdx2 function was confirmed in events related to axial extension. As considerable data suggest that the Cdx family members functionally overlap, this analysis was extended to assess the consequence of concomitant loss of both Cdx1 and Cdx2. This study report that Cdx1-Cdx2 double mutants exhibit a severely truncated anteroposterior axis. In addition, these double mutants exhibit fused somites, a widened mediolateral axis and craniorachischisis, a severe form of neural tube defect in which early neurulation fails and the neural tube remains open. These defects are typically associated with deficits in planar cell polarity (PCP) signaling in vertebrates. Consistent with this, it was found that expression of Ptk7 (Protein tyrosine kinase 7), which encodes a gene involved in PCP (a homolog of Drosophila Off-track), is markedly reduced in Cdx1-Cdx2 double mutants, and is a candidate Cdx target. Genetic interaction between Cdx mutants and a mutant allele of Scrib, a gene involved in PCP signaling, is suggestive of a role for Cdx signaling in the PCP pathway. These findings illustrate a novel and pivotal role for Cdx function upstream of Ptk7 and neural tube closure in vertebrates (Savory, 2011).


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


Biological Overview

date revised: 20 June 2012

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