A Single-Cell Transcriptome Atlas for Zebrafish Development

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The Problem: Different diseases affect different cell types, each defined by its unique set of expressed genes: its transcriptome. Single cell transcriptome sequencing (scRNA-seq) is a powerful way to identify cell types in health and disease by identifying clusters of cells that share similar transcriptomes. scRNA- seq methods, however, generally dissociate samples, thus losing information about where each cell cluster resides in the body. To learn where each cluster resides in an intact animal, the best current method is in situ hybridization to whole mounts or tissue sections. The problem is that traditional in situ hybridization protocols seldom achieve sufficient resolution to locate rare cell types in the body. The solution is Single molecule Fluorescent In Situ Hybridization (smFISH), a method to detect and count specific individual RNA molecules inside cells. The Specific Aim of this Administrative Supplement request is to purchase a Keyence BZ-X800E fluorescence microscope to enable smFISH experiments. This equipment will more effectively achieve the aims of the parent grant within the scope of the funded project. Proposed work does not overlap with funded work in the parent award because we cannot perform smFISH effectively without the requested equipment. This supplement request aligns with the parent grant’s main goals: to develop a single-cell transcriptomic atlas for zebrafish and to validate cluster identification by in situ hybridization. Commercially available smFISH kits have recently become available, but our current fluorescence microscope is insufficient for smFISH. In contrast, the Keyence fluorescence microscope can efficiently validate scRNA- seq cell clusters by smFISH. It combines features of a formal microscope, a plate reader, a slide scanner, and a confocal fluorescence microscope that will enable validation experiments on zebrafish not possible with our current equipment. Requested equipment will impact the full range of NIH Institutes and Centers because we perform scRNA-seq and smFISH on intact animals encompassing all organ systems. Affected research fields include genetics, developmental biology, cell biology, and pathology. The timeline for completion includes immediate purchase of the microscope on award notification, delivery two weeks later, followed directly by smFISH experiments. All funds will be spent in the current period. Requested equipment will improve an important resource for animal models by localizing cell types in the body of a premiere animal model of human disease, thus enhancing knowledge of cellular mechanisms in health and disease.