Date of Award

2007

Level of Access

Campus-Only Thesis

Degree Name

Master of Science (MS)

Department

Zoology

Advisor

Clarissa A. Henry

Second Committee Member

Sharon L. Ashworth

Third Committee Member

Mary S. Tyler

Abstract

Muscle development involves the specification and morphogenesis of muscle fibers that attach to tendons. Skeletal muscle is derived from somites, transient segmental structures that form during early development. Subsequent to somite formation, initially short somite cells form long multinucleate myotubes that orient along the anterior-posterior axis and attach to tendons. This attachment site is the primary site of force transmission between musculature and the skeletal system. Although a great deal is known about muscle and tendon specification during early development, less is known about morphogenesis of these structures. Specifically, the cellular and molecular mechanisms that underlie myotube morphogenesis in vertebrates in vivo are as yet unknown. Time-lapse confocal microscopy indicates that myotube formation proceeds via intercalation: initially short muscle precursor cells extend protrusions and intercalate between other muscle precursor cells. This cellular behavior generates long muscle fibers that then fuse to become multinucleate myotubes. Morpholino-mediated inhibition of the extracellular matrix protein laminin disrupts fast muscle fiber morphogenesis as well as normal expression of myogenic regulatory factors, a set of transcription factors necessary for muscle development. Laminin deficiency results in an increase in the number of muscle stem cells, revealing a role for laminin in specification. Integrin alpha 6 is the relevant receptor for laminin in muscle morphogenesis and is required for timely fast muscle cell elongation. The subsequent attachment of fast-twitch myofibers to the nascent myoseptal tendon correlates with the formation of novel microenvironments within the myoseptal tendon. The expression or activation of two proteins involved in anchoring the intracellular cytoskeleton to the extracellular matrix, Focal Adhesion Kinase and p-Dystroglycan, is upregulated. Conversely, the extracellular matrix protein Fibronectin (Fn) is downregulated. Laminin expression within the developing myoseptal tendon does not change. The downregulation of Fn as fast-twitch fibers attach to the myoseptal tendon results in Fibronectin protein defining a novel microenvironment within the myoseptal tendon adjacent to slow-twitch, but not fast-twitch, muscle. Taken together, these data indicate that multiple dynamic interactions between developing muscle fibers, the extracellular matrix (comprising the myoseptal tendon), and the receptors binding them, mediate muscle morphogenesis.

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