PROJECT SUMMARY The functions of mammalian organs are maintained by the behaviors and dynamics of individual cells. The ability to systematically map each cell type's temporal dynamics is central to the understanding of many aspects of biological changes that mammals undergo in development. However, conventional methods are restricted by inadequate throughput and the limited range of cellular contents that can be measured. While single-cell genomic techniques have been developed to characterize cell state heterogeneity with high resolution, nearly all such methods capture only a static snapshot at a single time point, with both temporal and spatial information lost during cell isolation. Herein, the proposed projects aim to develop novel methodologies that enable a comprehensive view of single-cell spatiotemporal dynamics across the lifespan of an entire mammalian organism. Specifically, I will expand on the high-throughput single-cell RNA-seq platform (sci-RNA- seq), to develop a novel method for concurrently profiling transcriptome, epigenome, and cellular temporal dynamics (e.g., proliferation, apoptosis) in each of millions of cells. The technique will be employed to investigate how aging regulates the status of a whole mammalian body by systematically monitoring single cell state dynamics across a broad range of tissues in young and aged mice. This approach will be powerful because we can not only visualize in-vivo proliferation and apoptosis behaviors of each cell type but also dissect its connection with internal transcriptome/epigenome states. In addition to the internal molecular programs, cell state dynamics are controlled by aspects of tissue architecture such as cell-cell interactions and extracellular matrix abundance. To profile single cell microenvironment with high throughput and accuracy, we will develop a novel technique called "microtissue-seq", for co-profiling single-cell molecular contents, cellular spatial interactions, and extracellular matrix (ECM) proteins across tens of thousands of spatial locations in a single experiment. We will employ this technique to interrogate how cellular microenvironment regulates organismal-scale cell state dynamics in different age groups of mice. Overall, the proposed projects will establish a technical framework for comprehensive profiling single-cell spatiotemporal dynamics at an unprecedented scale of a whole mammalian organism. By profiling cell-state specific dynamic behaviors across the lifespan of mice, these technologies and experiments would uniquely enable accurate modeling of the exquisite program underlying mammalian system maintenance and breakdown with age at single cell resolution. These multi-pronged approaches also open a new paradigm for understanding the global molecular programs regulating cell states and dynamics during aging, thereby informing potential pathways to delay the aging process as well as the rational design of effective therapies to restore tissue homeostasis for patients with aging-related diseases.

PROJECT NARRATIVE Despite active cell proliferation, differentiation and apoptosis, mammalian organs tightly maintain their overall size and function, indicative of a delicately balanced program that regulates cell population dynamics. This proposal aims to develop powerful single-cell genomic approaches that enable a whole-organismal view of single-cell spatiotemporal dynamics, and apply these methods to investigate how tissue homeostasis is maintained and disrupted during aging at single-cell resolution, thereby contributing to the knowledge of basic biology, as well as opening new therapeutic horizons for aging-related diseases.