This project covers not only cartilage but also tooth and craniofacial development. Our objectives are to define the mechanisms for activating chondrocyte-specific genes and to elucidate the molecular basis of cartilage, tooth, and craniofacial development. We determine the function of protein factors in vivo and in vitro for these tissues using an animal model and cell culture as well as in human disorders. We identify novel genes relevant to tooth and craniofacial development. Oral and Craniofacial Genes Craniofacial birth defects with anomalies of the mouth, neck, and head are of major public concern. Many vertebrate organs begin their development by inductive interactions between an epithelium and a mesenchyme. Tooth development is a classic example of this process and provides a useful experimental system for understanding the molecular mechanisms of organogenesis. The early morphogenetic event of mouse tooth development occurs in the embryo by invagination of the oral ectoderm into the underlying neural crest-derived mesenchyme, which later differentiates into enamel-secreting ameloblasts and dentin secreting odontoblasts. We previously identified a new enamel matrix protein, which we named ameloblastin, by cloning from an incisor cDNA library. To identify in vivo functions of ameloblastin, we created gene knockout mice for ameloblastin. Ameloblastin-deficient mice showed severe enamel hypoplasia. In mutant tooth, the dental epithelium differentiated into enamel-secreting ameloblasts, but the cells were detached from the matrix and subsequently lost cell polarity, resumed proliferation, and formed multicell layers. Expression of differentiation marker factors such as Msx2, p27, and p75 was deregulated in mutant ameloblasts, the phenotypes of which were reversed to undifferentiated epithelium. We found that recombinant ameloblastin adhered specifically to ameloblasts and inhibited dental epithelial cell proliferation. In addition, the mutant mice developed an odontogenic tumor of dental epithelium origin. Thus, ameloblastin is a cell adhesion molecule essential for amelogenesis, and it plays a role in maintaining the differentiation state of secretory stage ameloblasts by binding to ameloblasts and inhibiting proliferation. We also identified epiprofin, a Kruppel-like-factor containing three characteristic C2H2-type zinc-finger motifs by differential hybridization. Epiprofin is highly cell-type specific, primarily expressed by epithelium of developing tooth, hair follicles and limb bud. To examine the in vivo function of epiprofin, we have created epiprofin knockout mice. Cartilage Genes Cartilage provides mechanical strength to resist compression in joints and also serves as the template for the growth and development of most bones. Cartilage development is initiated by mesenchymal cell condensation followed by a series of chondrocyte maturation processes including resting, proliferative, and hypertrophic chondrocytes. Cartilage contains an extensive extracellular matrix that includes type II, IX, and XI collagens. Type II collagen, a major collagen in cartilage, forms collagen fibrils and provides a structural framework for cartilage matrix. Type XI collagen co-assembles stoichiometrically with type II collagen in the fibrils, whereas type IX collagen is associated with the surface of the fibrils. Type XI collagen is composed of three chains, alpha-1(XI), alpha-2(XI) and alpha-3(XI), and plays a critical role in the formation of cartilage collagen fibrils and in skeletal morphogenesis. We previously reported that the promoter segment of the alpha-2(XI) collagen gene (Col11a2) was sufficient for cartilage-specific expression and that a 24-bp sequence from this segment was able to switch promoter activity from neural tissues to cartilage in transgenic mice when this sequence was placed in the heterologous neurofilament light gene (NFL) promoter. We also identified a protein factor, NT2, that binds the 24-bp sequence of the Col11a2 promoter. NT2 is a zinc-finger protein containing a Kruppel-associated box (KRAB). We found that NT2 is expressed in the mesenchyme cells and hypertrophic chondrocytes, and we demonstrate that NT2 functions as a negative regulator of Col11a2. In collaboration with Dr. Rausher, we demonstrated by chromatin immunoprecipitation experiments that the Col11a2 silencing site is the target site for NT2 and other chromatin-associated repressor proteins, suggesting that NT2 suppresses Col11a2 transcription by binding the Col11a2-promoter via its zinc-finger motif and recruiting the suppressor protein via its KRAB domain. To study the role of NT2 in cartilage development, we prepared a Cre-inducible NT2 activation vector to create conditional ectopic NT2 expression in mice and also a Cre-mediated NT2 targeted vector to create conditional knockout mice. Perlecan, a large heparan sulfate proteoglycan, is present in all basement membranes and some other tissues such as cartilage. We previously created perlecan knockout (perl-/-) mice, which developed severe chondrodysplasia, and the majority of them died immediately after birth due to respiratory failure. We have created transgenic mice expressing recombinant perlecan under the control of the Col2a1 promoter and enhancer and crossed them with perl+/- mice. These mice show normal cartilage development and survive. To identify domains and sequences of perlecan important for cartilage development, we have created several Col2a1-mutant perlecan constructs and created transgenic mice expressing truncated perlecan molecules.
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