The aim of this project is to understand the mechanisms underlying cartilage and craniofacial development. We have studied gene defects in diseases with skeletal and craniofacial abnormalities and identified genes involved in normal development of cartilage and cranofacial tissues. Mouse cartilage matrix deficiency (cmd) is an autosomal recessive mutation characterized by cleft palate, short limbs, tail and snout, and homozygous mice dye just after birth. Cartilage from cmd mice has normal levels of collagen II and link protein, but lacks aggrecan. We mapped the aggrecan gene close to mouse chromosome 7 to which cmd had been mapped. The levels of mRNA for aggrecan from cmd mice was low compared to that from normal mice. We identified a 7 bp deletion in exon 5 of the aggrecan gene of the cmd mice resulting a truncated molecule. We found a new enhancer sequence in the first intron of the collagen II gene which increased the promoter activity of the gene in chondrocytes. The minimum size of the enhancer was about 100 bp and contained a sequence homologous to a sequence in the promoter region of the link protein gene. Gel retardation analysis suggested that the promoter and the enhancer of the collagen II gene could form a DNA-loop structure by interacting with multiple nuclear factors. Glucocorticoid responsive element was identified in the first intron of the link protein. A segment between -920 and -750 bp of the promoter of the link protein was found to increase transcriptional activity of the gene. This segment contained a sequence homologous to a portion of the enhancer of the collagen II gene suggesting that a common nuclear factor was involved in the coordinate regulation of the collagen II and link, protein genes in chondrocytes. We have initiated a genome project to identify novel genes which regulate craniofacial and tooth development. Two cDNA libraries were constructed using mRNA from mouse embryo maxillofacial tissues and rat incisor pulp tissues. About 400 cDNA clones from each library were sequenced. We have been characterizing these clones by examining their stage- and tissue-specific expression.
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