Fig 5. otd is required for R8 synaptic-layer targeting.

Wild-type (A,A’) and otd ^uvi mutant (B,B’) adult optic lobes expressing the R8 specific marker Rh6-lacZ stained with anti-β-galactosidase (green). In wild-type (A,A’) Rh6-lacZ-positive R8 axons terminate in the M3 layer. otd ^uvi mutants (B-B”) display a strong R8 axon misprojection phenotype. R8 axons specifically overshoot to the M6 layer (boxed in B’ and quantified in Fig 5H) while some R8 axons invade neighbouring columns cross-laterally and terminate in abnormal positions (boxed in B”). Several R8 axons terminals can also be seen to stall at more superficial layers and fail to innervate the medulla (double arrows in b”‘). All panels (C-F) show photoreceptor axon projections stained with the 24B10 antibody (red). R8 axons in wild-type (c,c’) and otd ^uvi mutant (D,D’) optic lobes (40% after puparium formation) are visualized using the ato-τ-myc transgene stained with anti-Myc antibody (green). At this early stage of development, in otd ^uvi mutant retina, R8 growth cones fail to stop in the M1 layer and extend specifically to the R7 temporary layer (n = 438 misprojecting axons of 798 R8 axons quantified in the two temporary layers). The staining for ato-τ-Myc is magnified in (C’,D’) and arrows in (D’) indicate misprojecting R8 axons. boss ¹ mutant (E) and otd ^uvi /boss ¹ double-mutant (F) retina. Adult optic lobes stained with 24B10 antibody. In (E), residual 24B10 staining in the R7 M6 layer is derived from medulla neurons [44]. (E) boss ¹ mutant R8 terminate in the R8 recipient layer M3, with only a few R8 targeting to the M6 layer. (F) Most of the otd ^uvi /boss ¹ double-mutants display a strong R8 mis-projection phenotype in the R7 layer, M6. (G) otd ^uvi mutant flies where Otd expression has been restored specifically in the photoreceptors using the GMRGal4 driver. In this context, staining of the R8 specific marker Rh6-lacZ (green) demonstrates that normal R8 photoreceptor targeting is almost completely restored. (H) Quantification of the misprojections of Rh6-lacZ-positive R8 axons in wild-type, otd ^uvi mutant and otd ^uvi mutant flies where Otd expression has been restored using the GMR-Gal4 driver.