Myocardial patterning--the specification of the correct number of myocardial precursor cells from the multipotential heart field--is critical for achieving normal heart development. Little is known about factors that limit myocardial differentiation. Two zebrafish mutations, neckless (nls) and untamed heart (unt), result in abnormally expanded myocardial precursor populations. As the nls mutation inactivates the rate-limiting enzyme for retinoic acid (RA) synthesis, RA signaling may refine the heart field by restricting myocardial specification. This proposal focuses on using the nls and unt mutants as tools to clarify this role for RA signaling and to identify additional players that cooperate in the process of myocardial fate determination. A three-fold approach will be used to determine when and where RA signaling normally occurs during heart development: pharmacological RA signaling inhibition to identify the critical RA patterning period, examination of RA-responsive transgene expression to locate cells receiving the RA signal, and double mutant analysis to determine epistasis relationships between nls and factors that promote myocardial differentiation. As unt likely cooperates with RA signaling, unt will be cloned as a first step toward its biochemical characterization and testing of its role in myocardial patterning. A screen to identify additional factors that influence the formation of myocardial precursors will also be conducted, incorporating a novel myocardium-specific transgene. The long-term objective of this research is to increase our understanding of genetic networks controlling myocardial patterning, which will help to elucidate mechanisms underlying human congenital heart defects.