The study of vertebrate muscle development presents exceptional opportunities for the investigation of gene regulation during complex ontogenetic processes. During myogenesis both tissue- specific and developmental stage-specific gene switching occur, culminating in the formation of the two major types of skeletal muscle: fast-twitch and slow-twitch myofibers. Each of the muscle fiber types is characterized by the expression of unique combinations is contractile protein isoforms. The establishment of muscle fiber type arises from the interplay of factors such as cell lineage, type of innervation the myofiber receives, and the level of circulating hormones. Whereas much progress has been made in understanding the molecular mechanisms responsible for muscle-specific gene expression, almost nothing is known about differential myofiber-type-specific gene expression. Dr Wade proposes to investigate the mechanisms regulating myofiber-type specific gene expression through the study of the human troponin I gene family. The adult slow-fiber TnI gene is differentially expressed during embryonic muscle fiber formation, and can serve as a model for the developmental regulation of fiber-type- specific gene expression. He has cloned the human skeletal muscle TnI slow gene, and has identified multiple cis-acting sequences required for its muscle-specific expression by the use of in vitro transient expression assays. These studies have identified a muscle specific enhancer which is not trans- activated by the myogenic regulatory factor MyoD1. He will further characterize the muscle-specific regulatory elements and extend these studies to examine muscle fiber-type-specific gene activity. The mechanisms underlying fiber-type-specific gene expression will be investigated in vivo by the novel approach of directly introducing TnI gene constructs into specific fast and slow twitch muscles in mice and rats, and by the development of transgenic mice carrying hybrid TnI gene constructs. The in vivo expression of these TnI gene constructs will be monitored by CAT activity assays, RNase protection assay,and by in situ hybridization of gene specific probes to cross sections of muscle tissue. These in vitro and in vivo expression studies will assess whether specific cis-acting sequences can confer muscle fiber-type-specific gene expression and evaluate the role of trans-acting factors in the process. The putative trans-acting factors regulating fiber-type-specific gene expression will be characterized by gel mobility shift assays, and DNA footprinting techniques. If such factors are found, he will clone representative cDNAs from expression libraries. These studies will be among the first to examine the molecular mechanisms regulating muscle fiber type development and maintenance.
