Project Summary/Abstract Osteoclasts are the cells in the body responsible for bone resorption. Osteoclasts in the craniofacial region participate in several skeletal site-specific processes that involve bone resorption such as tooth eruption and orthodontic tooth movement. Much of what is known about the activity of craniofacial osteoclasts in current literature is based on our understanding of osteoclasts derived from the appendicular skeleton and long bones such as the femur. This proposal aims to further elucidate the unique characteristics that exist between the osteoclasts derived from the mandibular and the femur bone marrow. Evidence shows that these two populations of osteoclasts are different, but the conclusions drawn about these differences are unclear. Using single-cell RNA sequencing to identify unique cell clusters present within mandible derived marrow cells as compared to long bone-derived bone marrow I have determined that mandible derived monocytes have decreased expression of genes involved in proliferation but an increase in inflammatory gene expression. Bulk RNA-Seq and ATAC-Seq of monocytes from the mandible and femur derived cells will be used to further determine changes in chromatin accessibility (Aim1). Additionally, siRNA knockdown experiments will further explore the impact of key genes identified by single-cell sequencing on osteoclast development. In addition to a lack of mechanism for the existent differences between mandibular and long bone derived osteoclasts, it is also unknown which osteoclast precursors are recruited to the site of injury during bone healing in the mandible. Proposed experiments will work to determine if CX3CR1+ cells contribute to mandible bone repair. It will be investigated if embryonic circulating and/or adult myeloid precursors, defined by expression of the CX3CR1 or Vav1-reporters respectively, provide osteoclasts within the mandible following a single cortex bone injury (Aim 2). Concurrently, this work will further the understanding of osteoclasts role in craniofacial site-specific processes as well as inform the future development of improved healing and regenerative treatments in the craniofacial region. These research goals will be enriched by the training goals of this application. The training goals of this application include learning techniques such as single cell RNA sequencing analysis, bulk RNA sequencing, ATAC-sequencing, as well as mouse husbandry and micro-computed tomography at the University of Minnesota School of Dentistry.