In both vertebrates and invertebrates, the interneuronal connections are often organized into columns and layers, which facilitate information processing and propagating. We use the Drosophila visual system as a model to study circuit assembly and focus on the mechanisms guiding R7 axons into specific layers and columns during development. In a large genetic screen based on a R7-dependent behavior, we identified two loci, baboon and importin-alpha3, which are required for the establishment of a precise R7 retinotopic map. Baboon encodes for a type I Activin receptor and importin-alpha3 for a component of nuclear import machinery. Removing Baboon or Importin-alpha3 in single R7s resulted in their axons invading neighboring columns, indicating that Baboon and Importin-alpha3 are required cell-autonomously in R7s to restrict their growth cones to retinotopically appropriate columns. In addition, the synaptic boutons of baboon or importin-alph3 mutant R7s appeared to be smaller and more irregular than those of the wild-type, suggesting that these two gene products are involved in synaptogenesis. Examining other known components of the Activin pathways, including the ligand Activin, the downstream transcription factor Smad2, revealed that the canonical Activin signaling pathway is required for restricting R7 growth cones to their retinotopically appropriate columns. Interestingly, Activin is functionally required in R7s, suggesting that Activin serves as an autocrine ligand it is secreted from and act on R7 growth cones.[unreadable] Several lines of evidence indicate that Importin-alph3 is a new component of the Activin signaling pathway. First, Smad2 and Importin-alph3 form a physical complex in the growth cones and axons. Second, nuclear accumulation of Smad2 depends on Importin-alpha3. Most importantly, these observations raise the intriguing possibility that Importin-alph3 plays a role in the retrograde axonal transport of Smad2. A similar role for Importins in transporting transcription factors from axons/dendrites to nuclei has been recently proposed in vertebrate neurons, suggesting this function of Importins is conserved in both flies and vertebrates. In summary, our results support a novel model for Activin signaling in R7s: Autocrine Activin activates Baboon on R7 growth cones and results in the phosphorylation of the downstream transcription factor Smad2, which together with Importin-alpha3 is transported from the growth cones back to the nuclei to regulate transcription. This model is further supported by our observation that blocking retrograde axonal transport in R7s phenocopied baboon/importin-alpha3 phenotypes. [unreadable] Removing Importin-alpha3 or Baboon resulted in incomplete penetrance of R7 phenotypes: only 12-30% of mutant R7 axons invaded their neighboring columns. This suggests the existence of an additional mechanism that functions redundantly to the Activin signaling pathway in restricting R7 growth cones to their retinotopically appropriate columns. To test whether repulsive interactions among neighboring R7s play a role to restrict R7 termini in appropriate columns, we genetically ablated most of the R7s and examined the targeting of the remaining R7s. We found that wild-type R7 axons form normal synaptic boutons in retinotopically correct columns even in a largely empty R7 terminal field. By contrast, removing neighboring R7s greatly increased the tendency of importin-alpha3 or baboon mutant R7s to invade adjacent columns. These results suggest that importin-alpha3 and baboon mutant R7 are still responsive to repulsion by neighboring R7s and these repulsive interactions account for their incomplete penetrance of phenotype. To determine the molecular nature of the R7-R7 interactions, we examined a number of candidate genes, whose products are known to mediate repulsive interactions. Among these, we identified the protocadherin Flamingo. Removing Flamingo alone in single R7s did not cause any obvious phenotype but the invasiveness of baboon mutant R7s was greatly enhanced by the removal of Flamingo in the neighboring R7s. In summary, at least two redundant mechanisms restrict R7 termini to the correct columns: (i) an intrinsic mechanism mediated by autocrine Activin signaling; and (ii) an extrinsic mechanism by Flamingo-mediated repulsion among R7s.