Our work is directed toward understanding the cellular and molecular mechanisms for regulation of diversification cells as vertebrate organisms develop and age. We are concerned with the functional and structural importance of proteins that undergo isoform switching in embryonic, fetal, and adult development. We study the differentiation of avian skeletal muscle cells and the structural and functional characteristics of the myosin isoforms expressed as myogenic cells differentiate. Myosin heavy chain is both a structural and a functional (enzyme) protein within the sarcomeres of developing muscle fibers. Expression of multiple isoforms of the myosin heavy chain family within developing muscle fibers is regulated temporally and anatomically, suggesting that at different times during development, the importance of either structure or functional properties of particular myosin heavy chains may predominate. By mapping the structural and functional domains of myosin heavy chain isoforms, we will examine the role of isoform switching in normal development and aging. We also will examine the relative importance of commitment and modulation (switching) to expression of myosin heavy and light chains and relate changes in myosin heavy chain isoform expression to these two cellular processes. Having shown that early in development myogenic precursor cells become committed to several specific fates (each synthesizes only certain isoforms of myosin heavy chain when they differentiate), we have developed in vitro cell culture systems for studies on the mechanisms that regulate restriction of myogenic cells to the expression of only certain members of the myosin heavy chain gene family as muscle fibers form and mature. Using monoclonal antibodies, peptide mapping, immunoblotting, immunohistochemistry, cell cloning techniques, and molecular genetic approaches, we propose to examine the molecular and cellular basis for the regulation of myosin expression in skeletal muscle with the following specific aims: 1) To determine the structural features that account for differences between the fast and slow myosin heavy chain classes and to analyze differences in location of these isoforms within the sarcomeres of developing myotubes. 2) To determine the molecular basis for developmental regulation of fast myosin light chain LC1f and LC3f expression in systems where differences in the structure of the promoters of the genes for these proteins appear to regulate expression of these isoforms. 3) To develop new immunological and nucleic acid probes for slow myosin heavy chains and for the myoDl gene. 4) To determine the cellular and molecular events in the expression of fast and slow isoforms of myosin heavy chains in myogenic cell culture models in which isoform switching is subject to experimental control. 5) To determine where and when the myoDl gene, the commitment gene, is first transcribed and expressed in the early avian embryo and how this relates to expression of fast and slow myosin heavy chains within myogenic cells and their precursors.

National Institute of Health (NIH)
National Institute on Aging (NIA)
Research Project (R01)
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Molecular Cytology Study Section (CTY)
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Stanford University
Schools of Medicine
United States
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Stockdale, F E (1997) Mechanisms of formation of muscle fiber types. Cell Struct Funct 22:37-43
Nikovits Jr, W; Wang, G F; Feldman, J L et al. (1996) Isolation and characterization of an avian slow myosin heavy chain gene expressed during embryonic skeletal muscle fiber formation. J Biol Chem 271:17047-56
Wang, G F; Nikovits, W; Schleinitz, M et al. (1996) Atrial chamber-specific expression of the slow myosin heavy chain 3 gene in the embryonic heart. J Biol Chem 271:19836-45
Schafer, D A; Miller, J B; Stockdale, F E (1987) Cell diversification within the myogenic lineage: in vitro generation of two types of myoblasts from a single myogenic progenitor cell. Cell 48:659-70
Schafer, D A; Stockdale, F E (1987) Identification of sarcolemma-associated antigens with differential distributions on fast and slow skeletal muscle fibers. J Cell Biol 104:967-79
Miller, J B; Stockdale, F E (1986) Developmental regulation of the multiple myogenic cell lineages of the avian embryo. J Cell Biol 103:2197-208
Crow, M T; Stockdale, F E (1986) Myosin expression and specialization among the earliest muscle fibers of the developing avian limb. Dev Biol 113:238-54
Miller, J B; Stockdale, F E (1986) Developmental origins of skeletal muscle fibers: clonal analysis of myogenic cell lineages based on expression of fast and slow myosin heavy chains. Proc Natl Acad Sci U S A 83:3860-4
Crow, M T; Stockdale, F E (1986) The developmental program of fast myosin heavy chain expression in avian skeletal muscles. Dev Biol 118:333-42
Miller, J B; Crow, M T; Stockdale, F E (1985) Slow and fast myosin heavy chain content defines three types of myotubes in early muscle cell cultures. J Cell Biol 101:1643-50