The goal of this proposal is to understand the molecular mechanisms that regulate the formation of the notochord in the vertebrate embryo. This is relevant to the understanding of the causes of axial birth defects. Although a number of genes that are expressed in the notochord have been described, little is known about the molecular processes that regulate the formation of the notochord. An important step forward has occurred with the isolation of the zebrafish mutation named floating head (flh) that does not form notochord precursor cells, and the identification of the gene that is defective in flh embryos. The hypothesis is that the flh gene functions at the junction between early signaling factors that specify the notochord from a subset of the mesodermal tissues in the embryo, and downstream genes involved in the differentiation of the notochord. The flh gene will serve as a starting point to study the molecular hierarchy that leads to the establishment of the notochord. The specific aims are: l) To determine which factors activate flh expression by injecting wild type and mutant zebrafish embryos with RNA encoding different candidate transcription and intercellular signaling factors, and monitoring the effect on flh gene expression using whole mount in situ hybridization on whole embryos, and rtPCR on embryo animal cap explants dissected from the injected embryos. 2) To characterize the cis-acting sequences that mediate flh transcriptional regulation by upstream regulatory factors, using the flh promoter in luciferase reporter transgene injections, combined with Southwestern and gel retardation techniques. 3) To isolate and characterize the transcription factors that bind to the flh promoter in response to intercellular signaling factors by screening an expression library with the target binding sequence in the flh promoter.