The overall goal of this R29 application is to understand whether differential expression of alternatively spliced type II procollagen isoforms is necessary for the normal development of craniofacial cartilage and bone. Previous work by the Principal Investigator in the chick, and by others in different species, has shown that the type II collagen gene encodes two isoforms of mRNA that are the result of alternative splicing and that are expressed differentially during skeletogenesis. One form (IIA) includes exon 2 that encodes a cysteine-rich globular domain of the amino (N)-propeptide and is expressed in prechondrogenic mesenchyme and embryonic calvaria at low levels. The type IIB gene excludes exon 2, and is expressed at high levels by well-differentiated chondrocytes. The change in splicing pattern during chondrogenesis is accompanied by markedly increased type II collagen mRNA levels resulting in deposition of type II collagen-containing cartilage matrix. The proposed studies will address the hypotheses that (1) the cysteine-rich globular domain of type IIA procollagen functions in maintaining low levels of type II collagen synthesis in embryonic calvaria as well as prechondrogenic mesenchyme by a negative feedback mechanism, and thus splicing out of this domain is a necessary step for upregulation of type II collagen synthesis during cartilage formation, and (2) type IIA procollagen regulates fibrillogenesis in embryonic calvaria by forming heterotypic fibrils with type I collagen.
The specific aims are: (1) To investigate the role of the alternatively spliced cysteine-rich globular domain in regulation of type II collagen production in chondrocytes and calvarial cells; (2) To define the role of type IIA procollagen in fibrillogenesis in developing calvaria; and (3) To analyze the effects of targeted overexpression of type IIA procollagen N-propeptide and abrogation of type IIA procollagen in the developing craniofacial skeleton of chick embryos using replication-competent retroviral (RCAS) constructs. These studies should strengthen our understanding of normal and abnormal development of the craniofacial skeleton.
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