Beyond ephrins: unbiased discovery of novel signaling pathways regulating topographic map formation and maturation in vivo.

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PROJECT SUMMARY The brain efficiently processes information thanks to the precise organization of its neuronal networks. In most circuits, axonal projections are organized into topographic maps based on the spatial organization of the neu- rons they originate from. This is especially true in the visual system, where retinal projections transmit a pre- cise and continuous representation of the external world to the brain by maintaining the neighboring relation- ship of the retinal ganglion cells (RGCs) they originate from in the retina. Studies over the past decades have demonstrated that topographic maps are initially coarsely established and then refine in an activity-dependent manner to become more precise. Maps are first generated by specific axon-target interactions, whereby axons with a unique profile of receptors interpret guidance cues distributed in a gradient at the target. In the visual system, the guidance cues ephrins and their receptors Ephs have been identified as the “master regulators” of retinotopic mapping. Along the antero-posterior axis, for instance, ephrinAs and EphAs are expressed in coun- ter-gradients across the nasal-temporal axis in the retina and rostro-caudal axis in the brain target, thereby providing a spatial code instructing map development. Yet, an increasing number of studies including ours indi- cates that ephrins do not act alone to generate visual maps. Several receptors and adhesion molecules have indeed been preferentially detected in nasal or temporal RGCs and shown to regulate retinotopic mapping. Yet, technical limitations have so far prevented the full identification and characterization of the signaling path- ways other than ephrins/Ephs that generate retinotopic maps. We notably lack a comprehensive profile of the secreted factors, adhesion molecules and receptors that are differentially expressed in RGCs and control reti- notopy. Here, we propose to address that major gap by identifying the receptors and ligands that are differen- tially expressed in nasal vs temporal RGCs, and testing their function in retinotopic map formation and matura- tion in vivo. We will achieve these objectives by taking advantage of a new zebrafish “retinotopic” transgenic line we have recently generated, in which nasal and temporal RGCs selectively express different fluorescent markers. In Aim 1, we will use single-cell RNA sequencing (scRNA-seq) to generate a transcriptional profile of the secreted factors and plasma membrane molecules preferentially expressed in nasal or temporal RGCs over time. We will also use in situ hybridization to test whether these candidates are indeed differentially ex- pressed in the retina, and quantify their expression across the RGC layer. In Aim 2, we will use a CRISPR/ Cas9-mediated mutation screen to test the function of the secreted factors and trans-membrane proteins iden- tified in aim 1. We will notably analyze the area covered by nasal and temporal retinal projections at the brain target over time, as well as the sharpening of the retinotopic map in mutants. Altogether, our innovative ap- proach will identify new players controlling retinotopy and revisit the classical model of topographic map devel- opment, thereby providing new perspectives on the mechanisms underlying circuit wiring and maturation.