This project covers not only cartilage but also tooth and craniofacial development. Our objectives are to define the mechanisms of chondrocyte differentiation 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 animal models and cell culture, as well as in human disorders. We identify novel genes relevant to cartilage, 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 identified several new genes that were preferentially hybridized to tooth germ mRNA by differential hybridization using tooth germ cDNA micorroarrays. One of the clones, which we named epiprofin, encodes a member of the KLF/Sp zinc-finger family and homologue to Sp6. In situ hybridization revealed that epiprofin mRNA is expressed by proliferating dental epithelium and also hair follicle matrix epithelium. Epiprofin is also transiently expressed in the apical ectodermal ridge in developing limb and genital organ. Transfection of an epiprofin expression vector showed that epiprofin promotes differentiation of dental epithelium into an ameloblast phenotype and enhances promoter activity of the enamel matrix-specific ameloblastin gene. To determine the role of epiprofin in ectodermal organ development, we have created gene knockout mice for epiprofin. Our preliminary data indicate that knockout mice survive with growth retardation and develop with enamel hypoplasia, an excess number of teeth (hyperdontia), severe defects in hair formation, thickening of epidermis, and digit fusions. These results suggest diverse functions of epiprofin that are essential for the development of these ectodermal organs.? ? At the early stage of tooth development, the basement membrane separates two key tissues that ultimately form enamel and dentin, but the precise role of the basement membrane and the underlying mechanism of its involvement in tooth morphogenesis are not clear. Laminin alpha-5, a component of laminin-10/11, is the major laminin alpha chain in tooth basement membrane. We examined the role of laminin alpha-5 in early tooth using gene knockout mice for laminin alpha-5. Mutant mice develop a small tooth germ with no cusps, in which the inner dental epithelium is not polarized and enamel knot formation is defective. In normal mice, laminin alpha-5 interacts with a cell surface receptor integrin alpha-6/beta-4 on the epithelium that is in contact with the basement membrane, whereas in mutant mice, cell polarity of the dental epithelium is lost in the absence of laminin alpha-5, preventing the interaction with integrin receptors and leading to deformities in tooth development. We found that laminin alpha-5 is required for proper cell proliferation, spreading, and filopodia-like microspike formation, which are involved in cell polarity, and we elucidated the cell signaling molecules involved in these processes. We also found that laminin 10/11 promotes spreading and filopodia-like microspike formation of dental epithelium and that the interaction of laminin alpha-5 and integrin alpha-6/beta-4 mediates these cellular changes through PI 3 kinase-Cdc42/Rac signaling. These results demonstrate that laminin alpha-5 is critical for the proliferation and differentiation of the dental epithelium and suggest that laminin alpha-5 in the basement membrane interacts with integrin alpha-6/beta-4 on the epithelial-cell surface to regulate the size and shape of the tooth germ. ? ? Cartilage Genes? Cartilage, a highly specialized connective tissue, contains an extensive extracellular matrix, and provides mechanical strength to resist compression in joints. In development, cartilage serves as the template for the growth and development of most bones. During chondrocyte differentiation, a unique set of ECM molecules including type II, XI and X collagens, link protein, and perlecan, are expressed in a temporal and location-specific manner. 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. ? ? Perlecan, a heparan sulfate proteoglycan, is present in all basement membranes and also in cartilage where there is no basement membrane. We previously showed that perlecan deficiency in mice and humans causes perinatal lethal chondrodysplasia, indicating that perlecan is essential for cartilage development. However, the functions of perlecan in cartilage development are unknown. We propose at least two potential functions of perlecan in normal cartilage development: 1. modulation of growth factor activity, such as FGF/FGFR3c, and 2. formation of the extracellular matrix in the hypertrophic zone. FGFR3c, the FGF receptor specific to chondrocytes, regulates cartilage development by inhibiting chondrocyte proliferation and expression of Indian hedgehog (Ihh). Activating mutations of FGFR3c, which cause Thanatophoric dysplasia, the most common human lethal chondrodysplasia, result in a reduced proliferative zone and a shortened growth plate, similar to perlecan-null mice, whereas Fgfr3-null mice develop an opposite phenotype, i.e., expansion of the proliferative and hypertrophic zones and survival. To test the modulation of Fgfr3c activity by perlecan, we used limb organ cultures from KO mice and Fgfr inhibitors. We also created double-KO mice for perlecan and Fgfr3 to examine if the abnormal phenotypes of perlecan KO cartilage can be restored in the absence of Fgfr3. Our data indicated that the inhibition of Fgfr in the growth plate cartilage of KO mice increased chondrocyte proliferation and restored the Ihh-expressing prehypertrophic chondrocyte zone, but they failed to differentiate into hypertrophic chondrocytes. These results suggest that in normal cartilage development, perlecan inhibits Fgfr3c activity, probably by trapping Fgf in the matrix. The modulation of Fgfr3c activity by perlecan allows the appropriate size and expansion of the growth plate. Further, perlecan plays a critical role in matrix formation in the hypertrophic zone. Without the matrix, cells fail to form an organized columnar structure and cannot differentiate well. Since perlecan is predominantly located in the pericellular space of hypertrophic chondrocytes, it is possible that perlecan interacts with both ECM and a cell surface receptor and stabilizes the interaction of ECM and cells to form organized columnar cell structure in the hypertrophic zone.
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