TGF-??superfamily members have recently been shown to regulate axon pathfinding in a variety of organisms. However, the mechanisms by which TGF-?s influence axons are unclear. The TGF-??superfamily member Activin and its transcription factor effector dSmad2 are required to downregulate the motility of Drosophila R7 photoreceptor axons and thereby restrict them to non-overlapping targets. This proposal will take advantage of the powerful molecular and genetic tools available in Drosophila to determine how the Activin pathway is regulated during axon targeting and how its activation controls axon motility and tiling. A better understanding of this process is of significant clinical importance, as the success of therapies aimed at replacing or repairing damaged neurons will depend on whether axons can be induced to seek and recognize their targets in an adult context. In addition, many of the molecules that control axon motility also regulate cell motility; their misregulation is associated with the invasiveness of metastatic tumor cells, the primary cause of cancer death. The transcriptional repressor Tramtrack69 (Ttk69) normally blocks neuronal differentiation and is therefore downregulated in R7 precursors by Phyllopod (Phyl) to allow them to become neurons. Howvever, Ttk69 is later expressed in R7s and, like the Activin pathway, is required to prevent R7 axons from invading adjacent targets. Ttk69 expression in R7s does not depend on the Activin pathway. Aim 1 will test the hypothesis that Ttk69 acts upstream of the Activin pathway to control R7 growth cone motility and that its late expression in R7s is caused by downregulation of Phyl. Aim 2 will molecularly characterize two new genes that prevent R7 axons from invading adjacent targets and will determine whether they are regulators or targets of Ttk69 or the Activin pathway. Aim 3 will take a novel, efficient approach to identifying additional genes that increase the invasiveness of R7 axons, block their invasiveness, or decrease repulsion among R7 axons, a second mechanism that contributes to preventing overlap. PUBLIC HEALTH RELEVANCE: A better understanding of how axons find their targets is of significant clinical importance, since the success of therapies aimed at replacing or repairing neurons damaged by disease or acute injury will depend on whether axons can be guided to the correct targets. In addition, many of the molecules that control axon motility also control cell motility; their misregulation is associated with the invasiveness of metastatic tumor cells, the primary cause of cancer death. Finally, the Activin signaling pathway that we propose to study is required for a wide variety of processes important for human health, including wound repair and survival of neurons after acute brain injury.