Unlike in controls, 27% of Rh6-lacZ-positive R8 axons stalled at the medulla neuropil border, while 8% terminated incorrectly in the M1/M2 layers (265 axons, n = 13) ( Figures 8B–8F). Second, NetBcd8 was directed to a subset of ectopic layers using MH502-Gal4, a driver active in lamina Selleckchem MK0683 neurons L1/L2, ascending T1 medulla neurons and C2/C3 neurons throughout development. Transient expression in R cells during larval and pupal development was suppressed using ey3.5-Gal80 and lGMR-Gal80 transgenes ( Figures 8G and S7). Expression analysis at 55 hr confirmed that high levels of NetB are present in layers M1/M2 ( Figure 8H). Despite the presence of endogenous Netrins

in the M3 layer, 32% of Rh6-lacZ-positive R8 axons stalled at the medulla neuropil border, while 19% stopped in the M1/M2 layers (227 axons, n = 12) ( Figures 8I–8J). Using NP1086-Gal4 ( Rister

et al., 2007), we also expressed NetBcd8 in T1 neurons, which extend dendrites into layer M2 and axons into the lamina. Membrane-tethered ligand was not detected in the medulla, but in the lamina, and consistently, R8 axon targeting to layer M3 was unaffected ( Figures S7L–S7M′). This confirms that axons are the primary site of NetB release, and ectopic ligand expression using MH502-Gal4 can be mainly attributed to lamina neurons L1 and L2. The increased percentage of redirected axons to defined NetB-expressing layers with MH502-Gal4 compared to the effects of wide ectopic expression using ap-Gal4 supports the model FK228 in vivo that layer-specific localization of Netrins is sufficient for R8 axon targeting. Recent studies identified at least four molecular mechanisms that control layer-specific targeting in the nervous system by cell-cell interactions independently of neural activity. First, combinatorial expression of homophilic cell surface molecules promotes the recognition and stabilization of contacts between matching branches of pre- and postsynaptic neuron subsets. For instance, four members of the immunoglobulin MRIP superfamily of

cell adhesion molecules, Sidekick 1 and 2 and Dscam and DscamL, are expressed and required in subsets of bipolar, amacrine, and retinal ganglion cells for targeting to different inner plexiform sublayers (IPLs) in the chick retina (Yamagata and Sanes, 2008). In Drosophila, the leucine-rich repeat protein Caps may play an analogous role, as it is specifically expressed in R8 cells and target layers M1–M4 and, thus, could promote homophilic interactions to stabilize connections within correct columns and layers ( Shinza-Kameda et al., 2006). Second, concise temporal transcriptional control is used to regulate the levels of ubiquitous cell surface molecules and, thus, adhesiveness of afferent and target neurons to balance branch growth and targeting.