The world’s smallest vertebrate brain: A new model for nervous system function and regeneration research

Project Summary / Abstract Regeneration is widespread and variable in the animal kingdom. Regenerative capacity of the human central nervous system (CNS) following injury or disease is poor, yet several mechanisms exist in several vertebrate organisms that lead to functional regeneration of the CNS. What is different between what happens at injury sites in these organisms and in humans? Are lost neuron types and neuronal wiring patterns restored upon regeneration of neural circuitry in animals that can achieve this feat? I propose to develop a novel experimental vertebrate regeneration system that will enable identification of mechanisms that naturally promote nervous system regeneration. Specifically, I plan to focus on Danionella cerebrum, a miniature and transparent fish species that has the smallest vertebrate brain on record, with ~650,000 neurons in total. Because of its minute size, optical transparency, complex behavioral repertoire, and genetic tractability Danionella cerebrum is potentially unparalleled as a vertebrate for tissue-wide single-cell RNA sequencing (scRNA-seq), and for whole- body and -brain imaging applications targeted to evaluate cellular dynamics in the adult state. I have two main aims: In aim 1, I plan to generate a complete Danionella cerebrum scRNA-seq atlas that includes homeostatic and regenerating states. Generation of a single-cell transcriptome atlas of the whole body of a vertebrate will be a powerful resource for a myriad of central problems in vertebrate biology and neuroscience. The proposed resource combined with the small size of the Danionella nervous system will enable utilization of scRNA-seq experiments in the future to probe a host of problems that involve body-wide manipulation of neural development and circuit activity, and recovery following injury. This dataset will also allow characterization and manipulation of circuit components within distinct areas of the central and peripheral nervous system, and will identify genes with appropriate cell-specific expression to enable genetic cell ablations, cell-specific labeling, and imaging of regenerating neural circuits. Because of its optical transparency, I have complete access to the Danionella nervous system for validation and future functional imaging studies. In aims 2 and 3, I propose to develop strategies to study and manipulate neural dynamics during regeneration, and in the absence of it (i.e. following inhibition of regeneration). This aim will include generation of cell type-specific nitroreductase expression-based genetic cell ablation lines, surgical injury strategies, learning and memory paradigms, and a calcium imaging platform to enable characterization of functional regeneration in the nervous system. Success with this aim will enable future work elucidating the “rules” for functional integration of new neurons following acute and chronic ablation in the nervous system. The molecular and cellular insights gained from the proposed aims will accelerate discovery and understanding of fundamental aspects of neural regeneration with potential for developing approaches that could be taken to generate therapeutic intervention in the cases of nervous system injury and degeneration.