During development, retinal neurons make exquisitely precise connections with specific synaptic partners. These synaptic choices impact the computational capacity of retinal circuits, and thereby influence visual per- ception. Cell-surface recognition molecules mediate synaptic choices by encoding two kinds of trans-cellular signals: 1) attractive signals that connect neurons with their circuit partners; 2) repulsive signals that shun non- target cells. Both types of cues are needed for precise retinal wiring, but the molecular mechanisms underlying rejection of inappropriate synaptic partners are unknown. The objective here is to identify recognition mecha- nisms that prevent connections between inappropriate synaptic partners. Our central hypothesis is that FLRT and UNC5 families of cell-surface molecules mediate repulsive receptor-ligand interactions that prevent cross- circuit synapse formation. The rationale for this work is that it will reveal a new class of synaptic choice recog- nition molecules that act through repulsive mechanisms. Understanding how the wrong synapses are avoided is a necessary step towards ultimately deciphering the molecular logic underlying synaptic partner choice. To this end, the following Specific Aims are proposed: 1) Identify ligands that prevent retinal neurons from se- lecting inappropriate synaptic partners. Retinal circuits occupy parallel sublayers within the inner plexiform layer (IPL) neuropil. This arrangement facilitates synapse specificity by bringing together arbors of circuit part- ners in a defined location where they are segregated from non-target cells. In preliminary studies using the mouse direction-selective (DS) circuit as a model, we obtained preliminary evidence that the UNC5C cell sur- face protein is a repulsive ligand that confines DS circuit arbors to their appropriate sublayers. This hypothesis will be tested using Unc5c mutant mice and Unc5c misexpression in vivo. 2) Identity receptor-mediated mo- lecular mechanisms that enforce synaptic specificity. Preliminary studies led us to hypothesize that the cell surface protein FLRT2, which is expressed by DS circuit neurons, serves as an UNC5C receptor that con- fines DS circuit arbors to their appropriate sublayers. This hypothesis will be tested using biochemical and in vivo genetic approaches. 3) Determine cellular mechanisms by which retinal neurons shun inappropriate synaptic partners. During dendrite growth, many exploratory branches are eliminated. Our preliminary data suggest that elimination of mistargeted arbors is impaired in Flrt2 and Unc5c mutants. We therefore hypothe- size that UNC5C-FLRT2 repulsion eliminates errant branches to prevent neurons from accessing inappropriate synaptic partners. This idea will be tested by time-lapse imaging of nascent DS circuit dendrites and synapses in Flrt2 and Unc5c mutants. Completion of these Aims is expected to define cellular and molecular mecha- nisms by which neurons avoid incorrect synaptic choices. This contribution will be significant because, once repulsive mechanisms for synapse specificity are known, it will become possible to comprehend how repulsion and attraction work together to produce the overarching molecular logic of synaptic partner choice.
Insight into the mechanisms of retinal synaptic partner choice will be relevant to public health because, once we understand these mechanisms, it may become possible to manipulate them for therapeutic benefit. For ex- ample, knowledge about these cell-cell recognition molecules could be used to design rational strategies for integrating new neurons into existing circuits. Such strategies could ultimately lead to new therapeutic options for treating retinal degenerative disease, or even neurodegenerative disorders in other regions of the central nervous system.