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. 2015 Jun 26;11(6):e1005303. doi: 10.1371/journal.pgen.1005303

Fig 6. otd and sens are required for R8 synaptic layer targeting.

Fig 6

All panels (A-F) show photoreceptor cell projections stained with 24B10 (red). (A) Expression of UAS-sens RNAi (GFP-negative ommatidia, encircled by a dotted line) in wild-type tissue (GFP positive) 48 h after clone induction using the tub>GFP>Gal4 system. UAS-sens RNAi expressing cells show a clear reduction in Sens protein levels (blue). (B) Expression of UAS-sens RNAi transgene (GFP-negative ommatidia, encircled by a dotted line) in wild-type tissue (GFP positive) 48 h after clone induction using the tub>GFP>Gal4 system. Otd expression (red) is unaffected in UAS-sens RNAi expressing R8 cells (indicated by asterisks). (C) Rh6-lacZ-positive R8 axons in sens RNAi (R8-specific driver-Gal4 109–68;UAS-sens RNAi) and in otd uvi mutant combined with sens RNAi retina (D), stained with anti-β-galactosidase (green). sens knockdown in the retina leads to a few defects in R8 axon projection, however the combination of the sens knockdown and otd uvi mutant leads to a complete failure of R8 layer-specific targeting, indicating that sens and otd act in parallel. (E,F) Layer-specific targeting of the R7 photoreceptors is assessed using the R7 specific transgene Rh4-lacZ. As in wild-type (E), Rh4-lacZ positive R7 terminals (green) correctly target to the M6 layer in otd uvi mutants flies (F). (G) Quantification of the misprojections of Rh6-lacZ-positive R8 axons in wild-type, otd uvi mutant and otd uvi mutant flies where either caps, fmi or gogo expression has been restored. Since Caps is present only in R8, UAS-caps is expressed under the control of an R8-specific Gal4 driver, while UAS-gogo and UAS-fmi are expressed using the pan-photoreceptor GMR-Gal4 driver. Restoring caps expression in otd-mutant R8 photoreceptors partially rescues the otd uvi mutant R8 mis-targeting phenotype (from 59% R8 misprojection in otd uvi to 25% in otd uvi + UAS-caps). Similarly, expression of fmi and gogo in otd uvi mutant photoreceptors partially suppress the R8 misprojection phenotype (36% R8 axons misprojecting in otd uvi + UAS-fmi, n = 374 of 1039; 40% R8 axons misprojecting in otd uvi + UAS-gogo, n = 436 of 1090). Percentages indicate quantitative assessment of R8 axons as detected in M3 and M6 layers only. In wild-type, all R8 neurons target to the M3 layer, thus the mistargeting percentage is zero.