Analysis of Cellular Plasticity in White Adipose Tissue.

ABSTRACT The long-term goal of this project is to understand the cellular and molecular mechanisms of adipose tissue establishment, maintenance, and pathophysiological remodeling. Our central hypothesis is that adipose tissue plasticity involves the creation and resolution of dynamic adipogenic niches involving close interactions among specific stromal and immune cell subtypes. While single cell RNA sequencing (scRNA-seq) has identified the stromal and immune phenotypes that are involved in adipose tissue neogenesis, how these cells are recruited to specific locations and interact to create and resolve adipogenic niches has not been addressed. In this continuation we will use cutting edge high-dimension, high-resolution spatial transcriptomics and proteomics to map, order, and test the cellular events underlying adipose tissue neogenesis. Despite recent advances in single cell profiling, the origins of adipocytes and lineage relationships of stromal cells in adipose tissues remains controversial. To address this critical gap in knowledge we will apply newly- developed CRISPR/Cas9-based technologies for unbiased, high-dimension resolution of cell lineages during the life cycle of adipose tissues. This technology will be integrated with complementary techniques of spatial transcriptomics, multiplexed 3-dimensional single molecule fluorescence in situ hybridization and immunofluorescence. This integrated approach will provide comprehensive, unbiased spatial analysis of lineage relationships in postnatal adipose tissue establishment, expansion, aging, and remodeling in vivo. Successful completion of this proposal will provide the first unbiased high-dimension, single-cell resolution map of mouse adipose tissues during establishment, expansion, and aging. We will determine the cellular and molecular mechanisms of adipogenic niche formation and resolution, and resolve long-standing questions of cell lineages in a comprehensive and unbiased fashion.