We therefore tested whether Netrins could act in a similar manner within the visual system by examining

R cell projections of flies, which have been modified by homologous recombination to solely express membrane-tethered NetB (NetBTM) at near-endogenous levels ( Brankatschk and Dickson, 2006). We observed that membrane-bound NetB was strongly enriched in the emerging M3 layer. While a small CB-839 mw percentage of R8 axons abnormally projected past the distal medulla neuropil border at 24 and 42 hr APF (n = 6 for each stage), projections were unaffected at 55 hr (n = 5) and in adults (n = 9) ( Figures 6A–6I). In flies in which fra has been knocked down in the target area, NetBTM levels remained high, further supporting the

notion that target-associated Fra prevents soluble NetB from diffusion ( Figures S5F and S5F′). Together, these findings suggest that target layer recognition of R8 axons depends on locally acting Netrins in layer M3. Furthermore, we examined the morphology of single R8 growth cones Selleckchem CP673451 using the Flybow FB1.1 approach ( Hadjieconomou et al., 2011a) in conjunction with the R cell-specific pGMR-Gal4 driver during pupal development ( Figures 6J–6M). At 42–44 hr APF (n = 11), R8 growth cones spread along the distal medulla neuropil border as they pause in their temporary layer. At 48–50 hr (n = 19), they extended a single thin filopodium along the R7 axon shaft toward the NetB-positive emerging M3 layer. At 52–55 hr (n = 7), the growth cone core at the medulla neuropil border was decreased in size, while the filopodium increased in thickness to eventually develop into a mature terminal. Thus, filopodial extensions of R8 axons could bridge the distance to the NetB-positive layer M3 to mediate short-range interactions. To test whether lamina neurons L3 indeed provide the local Netrin signal, we next conducted rescue experiments using MH56-Gal4, a driver with strong activity secondly in lamina neurons L3 throughout pupal development ( Figures 7A and S6). Overexpression of NetB with

MH56-Gal4 did not interfere with R8 axon targeting ( Figures 7B–7C′). Strikingly, expression of NetB in a NetABΔ background significantly rescued R8 axon-targeting defects ( Figures 7D–7F). Only 8% of Rh6-lacZ-expressing neurons stalled at the medulla neuropil border or terminated in the distal M1/M2 layers (248 axons, n = 12) compared to 61% in mutant siblings lacking UAS-NetB (91 axons, n = 5). While we cannot exclude a contribution of other neuron subtypes, these findings indicate that NetB in lamina neurons L3 is sufficient to control layer-specific targeting of R8 growth cones. Finally, we tested whether layer-specific localized Netrins could play an instructive role in controlling R8 axon targeting. For this purpose we assessed the effects of ectopically expressing membrane-tethered NetB using a UAS-NetBcd8 transgene ( Figure 8A) under two conditions.