(Taken directly from the application) Secreted proteins direct the initiation, growth and patterning of the developing skeletal elements. This project will utilize the powerful advantages of retroviral gene transfer into developing chick limb buds to address aspects of each of these steps, and will take advantage of the Transgenic Core facility provided by this Program to carry out complementary genetic experiments in mice. Autotaxin (Atx) is a secreted glycoprotein expressed extremely early in the chondrogenic centers. Atx has been previously studied in the context of human melanoma cells where it is a potent inducer of chemotaxis. Misexpression of Atx in the developing chick limb bud and in micromass culture will allow its ability to affect chondrogenesis to be assessed. Its potential effect on limb mesenchyme migration will be examined in a two-chamber chemotaxis apparatus. The requirement for Atx in mesenchymal condensation will be tested by targeted deletion in mice, utilizing the Transgenic Core. Once condensations have formed their growth is regulated by a number of secreted proteins. The role of Wnt5b, a signal produced in the hypertrophic chondrocytes, and Wnt14, produced in the forming joints, will be examined by retroviral misexpression in developing chick bones. Targeted deletions of Wnt5b and Wnt14 will also be generated in conjunction with our Transgenic Core to determine the requirement for these factors. Epistasis relationships to Ihh and PTHrP will be determined by examining gene expression in the gain and loss of functional Wnt5b alleles. Wnt5b will also be over-expressed by directing its transcription from a ColII promoter in mice in our Transgenic Core. These mice will be crossed with mice carrying mutations in Ihh and PTHrP, studied in other projects in this Program, to further explore the functional relationships between these signals. Wnt signal transduction will be studied during cartilage formation by misexpression of activated forms of Fz receptors and dominant-negative form of beta-catenin. While all the skeletal elements express the same signals, they grow to distinct shapes and sizes. This is, at least in part, mediated by differential expression of Hox genes. Mice carrying targeted deletions of both Hoxa-11 and Hoxd-11, or harboring the phenocopy dominant mutation ulnaless, form skeletal limb patterns where the zeugopod forelimb elements, the radius and ulna, fail to grow. Changes in regulation of various signaling molecules will be assessed in these mutants. The ability of these purified factors to complement the Hox and ulnaless defects will be assessed in an in vitro culture system.
Showing the most recent 10 out of 79 publications
