The central theme and primary objective of this research is to understand the systemic and local regulation of osteoclast ontogeny in fetal skeletal development. We will test the following hypothesis of osteoclast ontogeny: Stem Cell -> multipotent Progenitor -> Committed Progenitor -> Early Precursor -> Late Precursor -> Osteoclast. Explants of fetal mouse metatarsals, at either 14-days (E14) or 15-days (E15) of embryonic development, cultured in serumless medium, will be employed as a model system since it affords the opportunity to test the hypothesis by starting with in situ progenitors and proceeding through osteoclast ontogeny in the normal physiologic environment. Sensitive morphologic and molecular techniques including electron microscopy, 125I-CT binding, and localization of carbonic anhydrase II mRNA using in situ hybridization will be utilized to further characterize the phenotype and help to define the formation of the osteoclast, its precursors and progenitors. The relationship between skeletal development and osteoclast formation will be examined by attempting to optimize the conditions for skeletal growth and cartilage differentiation in serumless medium using insulin, somatotropin, thyroxine, and phosphate and determining concomitant effects on osteoclast formation. Protocols will be defined to study the stages of osteoclast ontogeny and then IL-6, GM-CSF, TGF-beta, calcitonin, 1,25-dihydroxyvitamin D39 and interferon-gamma, representative factors which have been demonstrated to affect bone resorption and influence osteoclast-like cell formation in other model systems, will be evaluated as to their effect on specific stages of osteoclast ontogeny in the model system proposed. It is anticipated that knowledge obtained from these experiments will be applied to the diagnosis and treatment of skeletal pathology and disorders of calcium homeostasis. These include: skeletal dysplasias, fracture repair, hypercalcemia associated with neoplasia, and bone loss associated with chronic arthritis and periodontal disease. In addition, the model system and experimental protocol developed in this program of study will provide a suitable paradigm for testing the effects of various potential therapeutic agents on osteoclast differentiation and function, both as a means of predicting their usefulness in treating skeletal disease, as well as assessing their potential hazard as skeletal teratogens.
