Retinal neurons are evenly spaced across the retina, a pattern known as a mosaic. Even spacing arises during development through contact-mediated repulsion that occurs specifically between neurons of the same type. The molecular mechanisms that allow homotypic neurons to recognize each other, and consequently to avoid each other, are not known. The objective here is to learn how homotypic recognition signals are initiated, received, and translated into signals that adjust cell position. The central hypothesis s that the transmembrane proteins MEGF10 and MEGF11 constitute a receptor-ligand system that: 1) confers homotypic recognition through binding upon cell-cell contact; and 2) triggers intracellular signaling pathways that produce mutual cell-cell repulsion, thereby creating mosaic spacing. The rationale for this work is that it will provide the first mechanistic explanation of mosaic formation, by revealing how the first identified set of recognition molecules (i.e. MEGF10/11) positions neurons. The mechanisms thus revealed are expected to provide general insight into how retinal neurons recognize and avoid each other, opening the way to understanding both mosaics as well as other neuronal patterning events that influence visual function. To this end, the following Specific Aims are proposed: 1) Determine the intercellular molecular interactions that initiate recognition signals. Preliminary data suggest that MEGF10 and 11 mediate these interactions by binding to themselves and acting as both receptors and ligands. To test this hypothesis the binding specificity of each molecule will be determined biochemically, and their receptor/ligand function will be confirmed in vivo using Megf10 and Megf11 mutant mice. 2) Determine how recognition signals are reported in the cell. Preliminary data show that MEGF10 is required to transduce recognition signals. Using biochemical and in vivo genetic experiments, this Aim will test the hypothesis that ITAM phosphotyrosine motifs in the MEGF10 intracellular domain mediate these recognition signals. 3) Determine how recognition signals alter cellular behavior to produce mosaic spacing.
This aim will test the hypothesis that recognition alters the behavior of dendrites. Specifically, it is proposed that recognition causes homotypic dendritic repulsion, through which neurons stake out unique territories that allow them to avoid their neighbors. Recognition will be abrogated genetically in Megf10; Megf11 double mutant mice and dendritic repulsion will be assessed by live imaging of retinal explants. Together, the experiments proposed in these three Aims are expected to reveal for the first time 1) the cell-surface molecules that bind to each other when cells of the same type touch; and 2) how these molecules trigger repulsion in order to specify neuronal position. The approach is innovative because it deploys novel tools and methods to enable the first molecular studies of homotypic recognition in mosaic patterning. The contribution will be significant because molecular events that determine the precise locations of neurons are important for circuit function, both in the retina and throughout the nervous system.
The proposed research is relevant to public health because retinal mosaics are an important example of how developing neurons use cell-cell recognition to integrate into the appropriate neural circuit. Knowledge of the molecules that neurons use to do this could be used to design rational strategies for integrating regenerated neurons into existing circuits, thereby facilitating regenerative strategies for treating retinal disease or other neurodegenerative disorders.
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