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First published online 29 September 2004
doi: 10.1242/dev.01406

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The receptor tyrosine kinase Off-track is required for layer-specific neuronal connectivity in Drosophila

McGill Centre for Research in Neuroscience, and Department of Neurology and Neurosurgery, McGill University Health Centre, 1650 Cedar Avenue, Montreal, Quebec H3G 1A4, Canada

View larger version (88K): [in a new window]   Fig. 1. The effect of otk mutations on R-cell projection pattern at larval stage. All R-cell axons in third-instar larvae (A,B) were stained with MAb 24B10. R2-R5 axons in third-instar larvae (C-F) were labeled with the larval R2-R5 marker ro--lacZ. In wild type (A), after exiting the optic stalk (os), R7 and R8 growth cones passed through the lamina into the medulla, whereas R1-R6 growth cones stop within the lamina, which could be identified as a continuous line of MAb 24B10 immunoreactivity. In B, an otk3 mosaic individual in which 80-90% eye tissues were homozygous otk3 mutant ommatidia, displayed defects in R-cell projections. The lamina plexus was uneven with the presence of small gaps. Abnormal thicker bundles were observed within the medulla. In wild type (C), ro--lacZ labeled R2-R5 axons terminated within the lamina. In an otk3 mosaic individual (D), many labeled R2-R5 axons projected aberrantly into the medulla. Similar mistargeting phenotype was also observed in otk3/otkEP(2)2017 transheterozygous larvae (E). In an otk3/otkEP(2)2017 transheterozygous larvae expressing an UAS-otk transgene in R cells under control of the GMR-GAL4 driver (Mismer and Rubin, 1987) (F), most labeled R2-R5 axons now terminated within the lamina. Scale bar: 20 µm.

View larger version (117K): [in a new window]   Fig. 2. Otk is highly enriched in R1-R6 growth cones. Third-instar eye–brain complexes were double-stained with MAb 24B10 (green) and anti-Otk antibody (red). In a wild type optic lobe (A-C), anti-Otk immunoreactivity was detected in both lamina and medulla. The strongest staining was detected in the lamina plexus, consisting mainly of R1-R6 growth cones at this developmental stage. In an otk3 eye-specific mosaic individual in which most R-cell axons were otk3 mutant axons (D-F), anti-Otk immunoreactivity was largely absent in the lamina plexus. Scale bar: 20 µm.

View larger version (135K): [in a new window]   Fig. 3. otk is not required for R-cell differentiation and cell fate determination. Third-instar eye-imaginal discs were stained with anti-Prospero (A,B) or anti-Boss antibody (C,D). In wild type, each ommatidium contains only a single R7 (R7 nucleus indicated by arrow in A) and a single R8 cell (cell apical surface indicated by arrow in C). In an otk3 mosaic eye-imaginal disc, only one R7 (B) and one R8 (D) were present in each ommatidium. Tangential sections of wild-type (E) and otk3 adult mosaic eyes (F) did not reveal any defect in either the number or the organization of R cells within each ommatidium. The arrow in F indicates a wild-type ommatidium surrounded by dense pigment granules. The arrowhead in F indicates an otk3 mutant ommatidium that can be recognized by the absence of pigment granules. Scale bar: 10 µm.

View larger version (83K): [in a new window]   Fig. 4. Lamina glial cell migration occurs normally in otk3 mutants. Third-instar eye–brain complexes were double-stained with anti-Repo (red) and anti-ß-galactosidase antibody (green). Anti-ß-galactosidase antibody was used to visualize all R-cell axons in both wild type and otk3 mosaic individuals that carry a glass-lacZ transgene, in which the expression of lacZ is under control of the eye-specific glass promoter (Mismer and Rubin, 1987). Anti-Repo recognizes the nuclear protein Repo expressed in all types of glial cells. In wild type (A), all R1-R6 growth cones (green) stop in between two layers of glial cells (red), epithelial (eg) and marginal glia (mg), forming the lamina plexus (lp). In an otk3 mosaic individual (B), lamina glial cells were still present at the R1-R6 termination site. Scale bar: 20 µm.

View larger version (91K): [in a new window]   Fig. 5. R1-R6 targeting appears largely normal in sema mutants. Third-instar eye-brain complexes of wild type (A,D), otk3 eye-specific mosaic (B,E), sema-1aP1 eye-specific mosaic (C) and homozygous mutants (F) were stained with MAb 24B10 (A-C) or with anti-ß-galactosidase antibody (D-F). Individuals in D-F carried the ro--lacZ marker, which labels R2-R5 axons at larval stage. Although sema-1aP1 caused a defect in the organization of R-cell axons within the lamina (C) that was more severe than that caused by the otk3 mutation (B), it did not significantly affect R1-R6 targeting (compare F with E). Scale bar: 20 µm.

View larger version (107K): [in a new window]   Fig. 6. The completed pattern of R1-R6 connectivity was severely disrupted in adult otk mutants. Cryostat sections of wild-type (A,C) and otk3 eye-specific mosaic heads (B,D) were stained with MAb 24B10 to visualize all R-cell axons. In wild type (A), R7 and R8 axons projected into different layers within the medulla. Individual R7 and R8 axon terminals could be readily identified. In an otk3 mosaic head (B), the medulla appeared to be innervated by an increased number of R-cell axons. C and D are higher magnification views of the boxed region in A and B, respectively. Whole-mount wild-type (E) and otk3 mosaic brains (F) carrying an adult R1-R6 specific marker, Rh1-LacZ, were stained with anti-ß-galactosidase antibody. In wild type (E), LacZ staining was exclusively observed in the lamina, as all R1-R6 axons terminate within this layer. In an otk3 mosaic brain (F), a large number of R1-R6 axons (arrow) connected abnormally to the medulla. Scale bar: 20 µm in A,B,E,F; 5 µm in C,D.

View larger version (51K): [in a new window]   Fig. 7. otk is not required for R7 targeting. Cryostat sections of wild-type (A) and otk3 eye-specific mosaic heads (B) were double-stained with MAb 24B10 (red) and anti-GFP antibody (green). Both wild-type and otk3 mosaic individuals carried the adult R7 marker PANR7-GAL4::UAS-Synaptobrevin-GFP, in which the expression of UAS-Synaptobrevin-GFP was controlled by the R7-specific driver PANR7-GAL4 (Lee et al., 2001). In wild type (A), all labeled R7 axons innervated a region that is deeper than the R8 targeting layer within the medulla. Although R7 axons in an otk3 eye-specific mosaic head (B) appeared less organized than that in wild type (A), they still projected into the correct target region. Scale bar: 20 µm.

View larger version (75K): [in a new window]   Fig. 8. The Expression of Otk in R7 did not retarget R7 growth cones to the lamina. Otk was expressed in R7 (B) under control of the PM181-GAL4 driver. Third-instar eye–brain complexes were stained with anti-ß-galactosidase. Individuals in A and B carried the PM181-GAL4 driver and an UAS-lacZ transgene. Individuals in B also carried an UAS-otk transgene. In an individual expressing Otk in R7 axons (B), like that in wild type (A), all labeled R7 axons projected correctly through the lamina (la) into the medulla (me). Scale bar: 20 µm.

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