Chemical toolbox for multiscale, integrative imaging: Connecting cellular gene expression to organ-scale phenotype

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PROJECT SUMMARY The biology of multicellular organisms is organized on multiple levels. Molecular abundance and interactions regulate cell function; Communication of cells with nearby cells and environments further shapes cellular states and functions; Cells form highly interconnected functional networks organ-wide. Thus, a function or a dysfunction of an organ is manifested through the orchestrated action of individual cells comprising the organ. To mechanistically comprehend how a disease develops, we need to understand how the abnormal alteration in a cell is translated into system-level dysfunctions. With remarkable progress in sequencing, imaging, and genetic manipulation, researchers are now a step closer to decipher how cellular genotype gives rise to system-level phenotype in vivo. Single-cell sequencing probes the genetic profile of individual cells comprising a tissue. Organ-scale phenotyping, such as CLARITY, probes the detailed morphology of cells, cellular wiring, and the spatial organization of cells throughout an organ. CRISPR-based genetic perturbation establishes causal links between genes and phenotype in vitro at unprecedented throughput. However, these technologies mostly probe a single facet of a complex biological system. This limitation makes it challenging to integrate information obtained from different molecular types and scales, and to extract the mechanistic underpinning of system-level phenotype, especially in vivo. We aim to address this critical gap by developing a transformational, multiscale, multimodal imaging platform that screens a large tissue volume to identify cells with abnormal phenotype and characterize the complete and quantitative molecular contents or the abnormal cells as well as nearby cells. This platform will identify how the abnormal genetic change in a cell alters its phenotype, affects nearby cells, and contributes to disease development. Despite its immense potential, streamlining organ-scale proteomic phenotyping and in situ single cell transcriptomics is impossible due to the incompatibility of chemistry and imaging requirements. We propose to develop a series of chemical tools to enable multiscale, integrative profiling of proteins and RNAs: reversible protection of RNAs in an intact tissue; tissue transformation chemistry for multi- omic profiling; and quantum dot-based NIR imaging platform for thick-tissue imaging. Integrating these tools, we will develop and implement the multiscale, integrative imaging platform to characterize phenotypic abnormalities in autistic brains, such as ectopic neuronal connections, and profile cellular transcriptome at the region of phenotypic defects. Such study will provide a holistic view of diseased tissues to decipher pathogenic mechanisms behind a phenotypic abnormality at a molecular level, through the identification of altered gene expression patterns near an abnormal phenotype, intercellular communication network that leads to the system- level phenotype, and the spatial organization of differential cell types in healthy versus diseased tissues. In addition to enabling new biological studies, the newly developed chemical tools will drive innovations in a wide range of biomedical science, including RNA biology, genetics, imaging, and tissue engineering.