The proper development of mature, functional osteoblasts from mesenchymal stem cells (MSCs) is critical to the bone health of millions of Americans suffering from osteoporosis. Osteoblasts play a key role in bone homeostasis as they are responsible for the formation and mineralization of new bone tissue. A potential treatment for osteoporosis is to promote osteoblast activity in order to combat the loss of bone material that occurs in many patients. However, the developmental progression and differentiation of osteoblasts is inadequately characterized at a molecular level to identify potential new drug targets. In this study, we propose to measure whole genome transcriptional dynamics using RNA-seq technology throughout the differentiation process from MSC to mature osteoblast in purified cell cultures derived from 5 diverse strains of laboratory mouse. In parallel, we will measure in vitro mineralization dynamics of our cultures, as well as in vivo histomorphometric measures of bone formation in mice of the same strains. We will analyze these RNA-seq data to indentify the key transcriptional events of osteoblastogenesis, including significant expression changes common to all examined strains and unique to different genetic backgrounds. By integrating these expression data with existing in vivo phenotypic data as well as the in vitro and in vivo phenotypes that will be measured in this project, we will correlate transcriptional changes with clinically important phenotypes. The genes and proteins identified through this process will include potential drug targets that can be further evaluated and studied in future projects. PUBLIC HEALTH RELEVANCE: It is predicted that currently 43.7 million Americans over the age of 50 already have or are at serious risk of developing osteoporosis. Based on a recent surgeon general's report, in the United States there are over 1.5 million osteoporotic fractures each year, including 280,000 hip fractures and 500,000 vertebral fractures. Two out of every 10 women and 3 out of every 10 men who suffer a fracture of the hip will die within 1 year of that fracture, half will never return to their previous living situation, and 70-80% will never walk again unaided. The annual direct health care costs for treatment of osteoporosis in the United States is over $18.3 billion. Increasing evidence shows that proper differentiation of mesenchymal stem cells (MSCs) into functional osteoblasts plays a key role in bone health and homeostasis. Studies in both mouse and human have shown that with age, the number of osteoblasts in marrow decreases and the degree of marrow adiposity increases. One of the most promising therapeutic avenues for the treatment of osteoporosis is the development of anabolic agents which promote osteoblast function. Currently, only a single osteoblast promoting drug is approved by the FDA (teriparatide), but this compound carries significant risks and is thus indicated for use for no more than two years. In order to better understand the genetic bases of osteoporosis and to develop new treatments, we must understand the process of osteoblast differentiation from both a molecular and genetic perspective, which is the goal of this proposal.