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).

GENE STRUCTURE

cDNA clone length - 4640

Bases in 5' UTR - 883

Exons - 6

Bases in 3' UTR - 655

PROTEIN STRUCTURE

Amino Acids - 1033

Structural Domains

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).

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 (Cafferty, 2004).

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).

date revised: 15 January 2005

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