In vertebrate embryos, the first skeletal muscles are called myotome fibers and develop in ball-like clusters of cells called somites. Recent evidence from this laboratory suggests that nitric oxide (NO), which is a potent, ubiquitous, messenger molecule, plays an important "signaling" role in mediating the formation of myotome fibers in somites. Nitric oxide was found to be elevated and dynamically changing in the dorsal somite and in an adjacent tissue called the ectoderm layer. Furthermore, experimental treatments that stopped NO increases in the ectoderm layer resulted in a block of myotome formation. Therefore, the central problem in this proposal is to determine the role of NO signaling between ectoderm and dorsal somite for myotome formation. To investigate NO signaling, live tissue from early chicken embryos will be tagged with fluorescent molecules to monitor NO concentration changes and concurrent myotome formation will be detected by muscle protein antibody labeling. Confocal microscopy will be used to obtain clear images of fluorescent labeled cells that lie deep inside tissue. The effect of NO to induce follistatin gene expression in somites will also be studied. The follistatin protein is known to remove BMP4, a protein that inhibits myotome formation. It is expected that a pattern of NO signaling will be seen between the ectoderm and somite that correlates with the activation of the follistatin gene, which in turn will allow myotome to form. The outcome from this research will show for the first time that NO is a major regulator of myotome formation in somites which is not presently recognized in the vertebrate embryo.
