Endochondral ossification in the vertebrae is a process through which embryonic cartilage is replaced by bone. In this proposal, we intend to elucidate the mechanisms underlying endochondral ossification in the vertebrae through studies on the spatio-temporal patterns of three cartilage collagens, with a special emphasis on type X collagen; hypertrophic chondrocyte morphology; vascular invasion; and mineralization in the matrix. We will analyze these parameters in developing vertebrae of chick embryos subjected to the following treatments: (1) Decreased serum calcium levels in developing embryos (shell-less culture in vitro) and in adult animals (vitamin D-deficiency). (2) Interference with the synthesis of type X collagen in embryos (infection with retrovirus encoding truncated type X collagen chains). (3) Induction of morphogenesis or dysmorphogenesis in chick embryos (local application of retinoic acid). Endochondral ossification will be analyzed at various times after treatment using in situ hybridization to collagen mRNAs and immunohistochemistry for collagen proteins using serial sections from paraffin embedded tissues coupled with morphometric quantitation assisted by computer-based image analysis. Furthermore, we will investigate the presence of angiogenic activity of isolated hypertrophic chondrocytes using the chick embryo chorioallantoic membrane. In addition, we will try to correlate the unique spatio-temporal pattern of a mouse homeobox gene, Hox-3, in the spinal cord with other events in the vertebrae of mouse embryos. The results of these studies will provide a better understanding of the mechanisms underlying age dependent vertebral bone development and of its alterations produced by nutritional and genetic manipulations.
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