Cellular And Molecular Mechanism Of R7 Target Selection

Eunice Kennedy Shriver National Institute Of Child Health & Human Development

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Abstract

In the fly visual system, the UV-responsive R7 photoreceptor neurons connect to the M6 layer in the medulla neuropil, where they synapse with amacrine neurons Dm8 and projection neurons Tm5. In addition, these connections form a retinotopic map: each R7 axon innervates a single medulla column while preserving their neighbor relationships in the eye. Layer-specific connectivity and retinotopic map are the characteristic features of all complex visual systems. Using a forward genetic approach in Drosophila, we are studying how layer-specific connections and retinotopic map are established during development. We have identified adhesive molecules (such as N-cadherin) and signaling receptors (such as receptor tyrosine phosphatases) that function in R7 photoreceptors to regulate layer-specific targeting. Our investigation of the functions of these molecules strongly supports the notion that R7 growth cones are steered to the correct target layer by regulating actin cytoskeleton and adhesive interactions. In contrast, much less is known about how R7 growth cones, once having reached the correct target layer, form synapses with specific target neurons within their retinotopically correct columns. We and others have previously shown that the retinotopic map in flies is formed in two separate stages. In the larval stage, a coarse topographic map is established, at least in part, by the secreted protein DWnt4 expressed in the target region and its receptor Dfrizzled2 on the photoreceptor axons. In the past years, we studied how the R7 retinotopic map is maintained and further refined at the pupal stages. In a genetic screen based on visual-driven behaviors, we identified pex (premature extension), which affects the refinement of R7 retinotopic map. Pex is a temperature-sensitive allele of baboon, which encodes a type I TGF-beta/activin receptor. Baboon mutant R7 axons target to the appropriate retinotopic medulla column in the correct layer, but they extend collaterals to innervate neighboring medulla columns, indicating that the map formation, but not the layer-specific targeting, is disrupted. In addition, the presynaptic structure of R7s, as visualized by tagged-synaptotagmin, is disrupted in baboon mutants, suggesting that activin signaling plays a role in synaptogenesis. We further identified that the other known components of the canonical activin signaling pathway are required in R7s for retinotopic map formation. Mutations disrupting dSmad2, which encodes the transcription factor downstream of baboon, result in baboon-like R7 phenotypes. In addition, we have identified Important-alpha3 as a new component of the activin signaling pathway. Importi-alpha3 mutants exhibit R7 retinotopic map defects essentially identical to those in baboon/dSmad2 mutants. Importin-alpha3 and dSmad2 are present in the R7 growth cones and they form physical complexes. Removing Importin-alpha3 in R7s disrupts normal nuclear accumulation of dSmad2, suggesting that Importin-alpha3 is required for nuclear import of dSmad2. While activin is expressed in both R7s and subsets of medulla neurons, disrupting activin function only in R7s using RNAi or dominant negative construct results in baboon-like R7 phenotypes. Together, these data suggest that autocrine activin refines R7 retinotopic map through baboon and dSmad2/importin-α3 complexes. We recently uncovered that activin acts not only on R7s (as an autocrine effector) but also on their synaptic target neuron, Dm8s (as a paracrine effector). Mutant Dm8 neurons devoid of Activin signaling were found to have overgrowing dendritic arbors which failed to form synaptic contact with R7s. Based on these observations, we hypothesize that photoreceptor-derived activin is delivered to and activated at the R7-recipient layer to coordinate the development of pre- and post-synaptic partners and to promote mutual synaptogensis.