(Verbatim from the Applicant): The vertebrate skeleton is formed through a precise series of developmental events, dependent upon an orchestrated cascade of gene expression regulated by both morphogens and the extracellular matrix (ECM). Within the skeleton, the ECM is composed largely of collagen, in addition to proteoglycans and the linear glycosaminoglycan, hyaluronan (HA). These components define structure and convey strength and flexibility upon the skeleton. What are the true biological roles of HA during skeletal development? We propose that there are many roles for this molecule during skeletal development. First, HA may function in a space-filling capacity, regulating the initial stages of condensation and joint cavitation. Second, HA may play an organizing, structural role, assembling and localizing the cartilage proteoglycan aggregate. Third, HA and its breakdown products may play a role in the regulation of cell behavior through the activation of specific signaling cascades. This may influence chondrocyte proliferation and maturation and may be required for angiogenesis prior to endochondral bone formation. In addition, HA-dependent signals may play a role in the regulation of growth factor expression. HA is synthesized by any one of three plasma membrane HA synthases (HAS), encoded by three separate but related genes. One of these genes, HA synthase 2 (Has2), encodes the dominant embryonic isoform required for embryogenesis. Loss of Has2 function results in embryonic lethality at ElO.5. After ElO.5, Has2 expression becomes progressively localized within the developing embryonic skeleton, being first expressed in the limb bud and craniofacial mesenchyme then becoming restricted to cavitating joints and growth plates. Thus, an understanding of the role and regulation of Has2 will provide critical information regarding the biology of HA during skeletal development. In this proposal, we will specifically investigate Has2 function and the regulation of its expression during skeletal development using mouse molecular genetic approaches. Our major hypotheses are that Has2-dependent HA synthesis is critical to normal cartilage and joint development and that Has2 expression is regulated in part by members of the bone-morphogenetic protein (BMP) family. We will address these hypotheses through the following Specific Aims and major experiments: (1) to investigate the role of Has2-dependent HA biosynthesis in cartilage and joint development. We will use recombinase-dependent conditional inactivation of Has2 in chondrocytes (Col2al-Cre) and in developing joints (Gdf5-Cre) in vivo to investigate the respective effects of loss of Has2 function; and (2) to investigate the regulation of Has2 expression in cartilage and joint. We will use reporter gene constructs (GFP-based), both in cell culture models and in transgenic mice, to determine the minimal sequences necessary for regulated expression of Has2. In particular, we will investigate the potential role of bone morphogenetic proteins (BMPs) in the regulation of Has2 expression. Our long-term goal is to understand the role of HA in skeletal development.
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