EXPRESSION AND ROLE OF T-BOX GENES IN MOUSE DEVELOPMENT

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The long-term objectives of the proposed research are to characterize the members of a newly discovered, ancient family of embryonically-expressed mouse genes, the T-box genes, and to determine the functional roles they play in embryonic development. These genes share a conserved proton motif, the T-box, which co-localizes with the DNA binding activity of the prototypical T locus of the mouse, a locus which regulates mesoderm formation. Because of the evolutionary conservation between widely divergent species from nematode to mouse, these genes are likely to play a role in the development of all vertebrates. Our preliminary results with 6 newly discovered T-box genes reveal temporal and spatial expression patterns during embryogenesis, some unique and some overlapping that suggest interactive roles for these genes in different aspects of development. We will detail the expression of these genes and critically test their functional roles using mutational analysis. Although the research has specific goals that will be accomplished for the individual genes already identified, the overall objective is much broader in scope, and encompasses an attempt to understand the relationship between the various members of the gene families, and the basis of the evolutionary conservation of the shared motif. It is our belief, based our expression data and by analogy with other gene families with conserved, shared motifs such as Hox and Pax, that the T-box family of genes encodes a set of DNA binding proteins that each play a distinctive role in developmental signaling. our intention is to add new T-box genes to our investigation as they are identified and to concentrate efforts on individual genes or groups of related genes that appear most informative in elucidating developmental mechanisms. In order to accomplish this larger aim, this proposal is one component of a two-part Investigator-Initiated Interactive Research Project Grant (IRPG). Our collaborator, Dr. Lee Silver of Princeton University has been funded to pursue complementary work in his laboratory. The submission of this proposal as an IRPG reflects our shared objectives, complementary expertise and intention of continuing our existing collaborative arrangement to investigate this ancient family of mouse genes. Specific Aim 1. Determine the expression pattern of mouse T-box genes, 1- 6, during embryogenesis, organogenesis and in the adult. We will use Northern blot analysis, in situ hybridization of tissue sections and whole mounts, and gene targeting constructs that include a marker gene driven by the endogenous promoter. Specific Aim 2. To determine the function of T-box genes by creating null mutations using homologous recombination in embryonic stem cells. The phenotype of resulting mutant animals will be analyzed and the mutants will be mated together to study possible interactions between different genes.