The chromatic photoreceptor neurons, R7 and R8, project their axons to the M3 and M6 layers of the medulla neuropil, respectively, and each pair of R7 and R8 axons innervates a single medulla column. Together these axonal termini tile the entire medulla neuropil. These organizing features, called layer-specific targeting and retinotopic mapping, are the hallmark of all complex visual systems. We have shown previously that during development, R7 and R8 axons project to specific layers and columns in two distinct stages. The sequential targeting of R7 and R8 axons to columns and layers reduces the number of potential synaptic partners (from over sixty medulla neurons to several), effectively reducing wiring complexity. By combining forward genetic screens and behavioral assays, we identified a number of mutants in which layer-specific targeting or retinotopic mapping of R7s is disrupted. We are currently focusing on two loci: overshoot (osh) and premature extension (pex). The hypomorphic osh mutants exhibit a novel phenotype: osh mutant R7s overshoot the target layer M6 and form additional synapses in the deeper medulla layer. Null osh mutant R7 has a much stronger phenotype. The mutant R7s exhibit abnormally enlarged synaptic boutons with excessive presynaptic structural protein, Bruchpilot. The overshoot and enlarged synaptic bouton phenotype appear during the synpatogenesis stage around 80% after pupal formation. Both developmental staging and phenotype suggest that osh play a regulatory role in synaptogenesis. By positional cloning, we have located the osh locus to a genomic region of approximately 50 Kb on the right arm of the second chromosome, and identified a candidate gene by complementation. We are in the process of confirming the identify of the osh gene by genomic rescue approach. Premature extension or pex mutations affects the refinement of R7 retinotopic map. During the larval stage, a gross retinotopic map of R7/R8 termini first forms via a Wnt4-dependent mechanism. During the late pupal stage when the R7 and R8 growth cones regain motility and proceed to their final destined layer, the R7 and R8 growth cones are confined to their retinotopically appropriate columns to form synaptic connections, a process called retinotopic map refinement. Pex is a temperature-sensitive allele of Baboo which encodes for a type I TGF-beta/Activin receptor. Baboon mutant R7 growth cones invades their neighboring columns, forming inappropriate synapses, indicating that Activin signaling is required for confining R7 growth cones to their retinotopically appropriate columns. We further determined that other known components of the canonical Activin signaling pathway, such as the downstream transcription factor Smad2, are required cell-autonomously in R7s for retinotopic map refinement. Immunohistochemistry analysis revealed that Smad2 is physically located at the R7 growth cones for receiving Activin signaling. This raises a question of how the phosphorylated and hence activated Smad2 is transported from growth cones back to nuclear to activate transcription. We found that the transport of Smad2 requires Dynein/Dynactin-dependent retrograde axonal transport system. Disrupting retrograde transport by expressing a dominant negative mutant of p150glued phenocopies baboon. In addition, we found that Improtin-alpha3, a known component of the nuclear import machinery, likely serves an unexpected role as a carrier to transport activated Smad2 from growth cones to nucleus. Importin-alpha3 mutant R7s exhibit retinotopic map defects as seen in baboon mutants. Biochemical analysis further revealed that Importin-alpha3 co-localizes with Smad2 in the R7 growth cones and they form a physical complex. Finally, the nuclear accumulation of Smad2 is disrupted in the absence of Importin-alpha or baboon, indicating that the carrier Importin-alpha3 and Actin-signaling, hence the phosphorylation of Smad2 are both required for transporting Smad2 from growth cones to nucleus. We are now examining the expression profile of baboon and Importin-alpha3 mutants for identifying the transcription targets of Activin signaling in R7s.
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