Cell-cell adhesion is likely to be an important developmental interaction responsible for the correct formation of the vertebrate central nervous system from a embryonic neuroepithelium. Neuroblast migration, cell death, neurite extension and synaptogenesis all may be influenced, in some degree, by adhesion properties of specific cell-surface glycoproteins expressed in a spacially and temporally precise pattern. In the initial period supported by this grant, we used the gene encoding the neural antigen Thy-1 as a model for the regulation of gene expression in the rodent nervous system and localized novel promoters and intragenic regulatory sequences which are important for tissue-specific expression and developmental regulation. We also demonstrated that a relatively small fragments of DNA containing the Thy-1.2 gene could be expressed in a cell-specific and temporally correct way after injection into mouse embryos to establish transgenic strains of mice. Modified Thy-1.2 genes were used to demonstrate that the expression of this gene could be redirected to other tissues and cell-types. In this competitive renewal application, we propose to use the molecular tools developed during the first grant period to ask questions about the effects of cell surface glycoproteins with cell-cell and cell-substrate adhesion properties on the developing mouse nervous system in vivo. Specifically, we will construct a series of transgenic expression vectors (TEV), using portions of the Thy-1 gene and other tissue-specific genes, to direct neuron- specific gene expression in transgenic mice. Then, cDNA or genomic fragments of the gene encoding the mouse and human neural cell adhesion molecule, N-CAM, will be introduced into the appropriate TEV, introduced into the germline of transgenic mice, and the expression charcteristics and morphological phenotype of the resulting strain analyzed. By creating gene fusions with genes expressed early or late in neural development, it may be possible to regulate the temporal pattern of transgene expression. Similarly, other gene products with similar adhesion properties, particularly members of the fibronectin/vitronectin gene family, may be expressed using a vector for directed expression. Finally, we will attempt to develop methods for the creation of pseudo- recessive mutatins using anti-sense RNA expression or artificial gene products competing for post-translational processing machinery. These studies will use molecular and cellular approaches applied in vivo to refine our understanding of the role of cell-cell and cell-substrate adhesion on nervous system development.
Showing the most recent 10 out of 20 publications
