Craniofacial cartilage differentiation is the result of a complex series of cellular interactions. These interactions initiate specific signaling pathways directing cranial neural crest cell migration, mesenchymal cell proliferation, condensation, and subsequent differentiation into chondrocytes. The secretion of structural and extracellular matrix proteins, including collagens and proteoglycans, results in the formation of cartilaginous structures of reproducible strength, flexibility, size and shape. Central to the process of cartilage differentiation are the signals mediated by transforming growth factor-beta (TGF-b) family receptors, and their corresponding ligands, including bone morphogenetic protein (BMP) family members. The applicant proposes to investigate he expression of BMPs, TGF-b member receptors, and factors with which they interact in normal and craniofacial cartilage differentiation mutant zebrafish. It is hypothesized that TGF-b family member receptors, in mediating signals initiated by BMPs, play a central role in directing craniofacial cartilage differentiation, and that their expression may be altered in zebrafish cartilage differentiation mutants. The vertebrate developmental model system of the zebrafish, Danio rerio, provides experimental, and particularly genetic, manipulability exceeding that available in other systems. This is demonstrated in a recently created collection of zebrafish craniofacial cartilage differentiation mutants, each of which is the result of a single point mutation in one of 13 different loci. In addition to TGF-b receptors and BMPs, a variety of other factors known to influence and participate in cartilage differentiation have been identified: secreted signalling molecules such as sonic hedgehog(SHH); transcription factors such as Msx-1 and Msx-2; fibroblast growth factor (FGF) family members and their receptors. BMPs are regulated by, or in turn regulate, factors. The expression of these additional factors, as well as TGF-b receptors and BMPs, will therefore be analyzed in each craniofacial mutant fish line. The proposed studies will explore the role of TGF-b receptor member signaling during craniofacial cartilage differentiation in the following specific aims: 1) To characterize the cellular nature of each zebrafish cartilage differentiation mutant using detailed histological methods; 2) To map each mutation to one of 25 zebrafish linkage groups using simple sequence length polymorphisms (SSLPs); 3) To map candidate genes, in particular BMPs and TGF-b family member receptors, to zebrafish linkage groups using SSLPs; 4) To characterize the in situ expression of cartilage differentiation markers in normal and mutant zebrafish. The goal of these studies is to elucidate the role of BMPs and TGF-b receptor signaling in craniofacial cartilage development.
