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).
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).
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).
Reference names in red indicate recommended papers.
Cafferty, P., Yu, L. and Rao, Y. (2004)
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
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
date revised: 15 January 2005
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