PROJECT SUMMARY / ABSTRACT The skeleton is an essential part of vertebrate anatomy, serving to protect vital soft tissues and organs, as well as a functional system allowing for stability and movement. The development and maintenance of skeletal elements requires coordinated interactions between tissue types of diverse origin and function. Understanding the mechanisms underlying these biological processes is essential to both our understanding of how the body develops and for clinical interventions in response to malformations or injuries. Cartilage is crucial to the development of skeletal features, laying down models as the foundation of future bone. Differentiated cartilage cells, or chondrocytes, undergo cellular hypertrophy to rapidly increase cellular size as a means to facilitate elongation of the limbs. Hypertrophic chondrocytes also serve in unique and important capacities in developing vasculature and recruiting hematopoietic stem cells to produce blood cells. Despite their importance in multiple developmental processes, the cellular mechanisms underlying chondrocyte hypertrophy are not well understood. The objective of this proposal is to understand how hypertrophic chondrocytes undergo cellular changes over the course of skeletal development. In order to answer this fundamental biological question, the following aims are proposed: 1) Characterize the fate of hypertrophic chondrocytes using a live-imaging approach; 2) Investigate modes of hypertrophy in chondrocytes exhibiting different cellular fates. This study will employ a novel imaging system (Stimulated Raman Scattering microscopy) to quantify dry-mass density within hypertrophic chondrocytes in the intact growth plate, thereby characterizing modes of cell size increase during endochondral ossification. Further, I will use ex vivo imaging of live explants to perform a longitudinal study of hypertrophic chondrocyte-to-osteoblast transdifferentiation. The ability of differentiated cells to transition to other lineages is a largely unexplored biological phenomenon and will be of interest across broader disciplines. Taken together, the insight gained from this work will lead to a clearer understanding of cell size regulation in hypertrophic chondrocytes and their influence on the development of bone. Importantly, this work will inform on cellular mechanisms relevant to human health by clarifying the role of hypertrophic chondrocytes in patterning, growth and maintenance of the skeletal system, which will be applicable both for developmental abnormalities as well as injury repair.