The mouse preimplantation blastocyst contains a population of pluri- potential inner cell mass cells. Embryonic stem (ES) cell lines which represent these cells have potent proliferative and differentiative capacities and are capable of forming endodermal, mesodermal and ectodermal derivatives when allowed to differentiate in vitro. The mesodermal derivatives formed in culture include beating heart cells and skeletal myocytes. ES cells transfected with gene constructs can be transferred into host blastocyst stage embryos, and the genetically altered ES cells will contribute with a high efficiency to each of the three germ layers as well as the germ line in chimeric embryos. ES cells offer a unique opportunity to examine and manipulate a developmental window on early differentiation events in the embryo. The goal of the proposed research is to identify and isolate new genes which are expressed at the initial stages of myogenic determination and differentiation. To accomplish this goal, the following specific aims are proposed: (1) to isolate mouse ES cell clones which express a transfected gene-trap construct, containing a lacZ marker gene, specifically in cardiac and/or in skeletal myocytes during in vitro differentiation; (2) to inject these undifferentiated ES cell clones into blastocysts and study the expression of the lacZ gene in chimeric embryos, fetuses and postnatal animals in vivo; (3) to isolate by cDNA cloning previously unidentified genes which are turned on early in myogenesis; (4) to sequence these new cDNAs and to begin to characterize their roles in myogenesis; (5) to identify mutations in muscle development which may result when insertion of the lacZ gene disrupts the normal function of the protein encoded by that muscle gene. Accomplishment of these goals will be important in the future understanding of events involved in muscle cell determination and differentiation, and how these events may go awry in neuromuscular diseases.
