CRCNS US-German Research Proposal - The diversification of retinal ganglion cells: A combined transcriptomic, genome engineering and imaging approach

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The brain contains a multitude of types of neuronal cells that assemble into elaborate circuits that underlie sensation, perception, and behavior. A fundamental question in neuroscience is how the developing brain generates such an impressive array of diverse neuronal types. Specifically, the grand challenge is to determine the networks of genes whose activity restricts immature “precursor” neurons to adopt specific terminal fates. The neural retina, which is an outpost of the brain residing in the back of the eye, is an ideal system for addressing this question due to its experimental accessibility and well-defined census of neuronal diversity in several species. Pioneering studies beginning in the 1980s used the then available experimental tools in frogs, rodents and fish to gain valuable insights into the process by which retinal cells become restricted to broadly defined “classes”. However, some retinal classes contain several (20-50) distinct neuronal types, and how cells commit to specific types within a class is unknown, and cannot be studied using classical tools. This project brings together an experimental neuroscientist and computational researcher to address this question combining several recently developed technologies to study the process of cell-type specification in the retina of the zebrafish in unprecedented detail. The approaches developed in this work will be useful for understanding neuronal development and maturation in other brain regions and species, and pinpoint the genes whose dysregulation may lead to developmental abnormalities. The project will combine high-throughput single-nucleus RNA-sequencing (snRNA-seq), advanced computational methods, genome engineering and live imaging to understand how ~35-50 types of retinal ganglion cells (RGCs), the output neurons of the eye, emerge in the developing and growing zebrafish retina. By leveraging the experimental advantages of zebrafish, which permits direct live imaging of developing cells in vivo, the researchers will study how postmitotic neuronal differentiation unfolds at a level of detail that is not possible in mice, the most commonly used vertebrate model. We will map the transcriptional landscapes of differentiating RGCs using snRNA-seq profiles collected at six developmental stages of zebrafish,and use computational methods to reconstruct lineage relationships among transcriptional clusters across development. To test predictions from genomic analysis, the researchers will label molecularly defined immature RGCs by CRISPR/Cas9-based genome engineering and image them to visualize their initial differentiation and transdifferentiation from embryonic stages into adulthood. Taken together, the efforts will lead to new insights into the patterning of a complex neurobiological system.This project is funded jointly by the Neural Systems Cluster in the Directorate for Biological Sciences and the Engineering Biology and Health Cluster in the Directorate for Engineering. A companion project is being funded by the German Federal Ministry of Education and Research (BMBF).This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.