This application combines an embryological approach with a cellular analysis of events underlying cell commitment and stem cell maintenance of skeletal muscle. Past work by the applicant and others has yielded information regarding the identification and analysis of expression of myogenic factors that can initiate the muscle developmental program in a variety of cells following gene transfer. It is presently clear that these genes govern the final stages of myogenic commitment and are not necessary to establish stem cell identity. In the embryo, myoblasts originate during early somite formation and subsequently colonize various structures such as the limb bud. In the early limb, myogenic cells are present but do not express detectable levels of any identified myogenic factor. We present here a series of experiments designed to elucidate the role of growth factors (TGFbeta, bFGF, IGFI, II) during development and we propose to identify how they act in governing differentiation in the embryo and post- natal life. In addition, we propose to use cell lines grown in the presence of growth factors to uncover genes and their products that control early myogenic determination and mesodermal development. By combining in situ analysis during development, cDNA library screening, and functional analysis of novel genes, we will assemble a model for myogenesis in vivo. We will also examine the role of homeobox gene expression in relation to the regulation of the myogenic program. It is suggested that these genes may regulate the ability of a cell (e. g., myoblast) to respond to factors mediating cell growth. We have previously identified a homeobox gene, Hox- 7.1, which is expressed in the limb bud progress zone and is related to the drosophila muscle segmentation homeogene (msh). We will assess the action of this and other homeotic genes by gene transfer into a variety of cell lines. Preliminary results indicate that Hox-7 acts as a repressor of the myogenic program. We will analyze how this is achieved through a variety of techniques and subsequently isolate Hox target genes. It is predicted that these experiments will shed important new information regarding the actual biological roles of homeotic genes. This type of information may have implications for eventual in vitro maintenance of stem cells (important for gene therapy) and eventually help explain why growth control is sometimes lost such as the case in neoplastic cells. The fact that homeotic gene expression is almost unique to embryonic tissue, and that these genes are co-expressed in regions which are also responsive to characterized growth factors suggests a potential common pathway by which growth control is regulated in the embryo as compared to the adult. The embryological roles of the IGFs, FGFs, and TGFbeta related growth factors will be studied in vivo and in vitro and we will determine if they regulate muscle development via a homeobox gene(s) related pathway.
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