Transgenic analysis has shown that the MyoD core enhancer and distal regulatory region (DRR) together recapitulate MyoD expression in postnatal skeletal muscle. Using mice with single-enhancer deletions, we have tested the requirement for each enhancer in directing MyoD expression in uninjured and regenerating skeletal muscle. In the absence of injury, the core enhancer is not required for normal MyoD expression, whereas DRR deletion reduces MyoD mRNA levels. Each enhancer is dispensable for normal MyoD expression early in regeneration, whereas at later stages, each enhancer directs approximately 50% of MyoD transcription. These are the first targeted deletion studies of postnatal MyoD transcriptional regulation. Neo-containing knockouts crossed into an X-linked muscular dystrophy (mdx) background exhibit a severe reduction in MyoD levels, yet the extent of regeneration is comparable to that of mdx mice. While an absence of MyoD expression is known to be detrimental, this work suggests that a low threshold level of MyoD suffices for normal regeneration. We have also tested the osteoprogenitor activity of three tissue types resident in limb musculature: skeletal muscle, vascular smooth muscle, and endothelium. Using Cre/lox labeling, we detect beta-galactosidase-labeled chondrocytes generated from skeletal muscle and Tie2-positive cells, but not from smooth muscle. Only mice with labeling of Tie2-positive precursors clearly generate labeled osteocytes. This is the first in vivo demonstration of the osteogenic capability of Tie2-positive cells. The osteogenic activity present in muscle is clinically relevant to human diseases of heterotopic ossification, such as fibrodysplasia ossificans progressiva (FOP). Another potential source of osteoprogenitors in FOP is the circulation. No labeled chondrocytes or osteocytes were detected in BMP-induced nodules of lethally irradiated hosts engrafted with ROSA26 bone marrow. Based on these data, Tie2-positive cells, most likely the resident vascular endothelium, are a source of ectopic ossification in skeletal muscle.
Adviser: David J. Goldhamer. Thesis (Ph.D. in Cell and Molecular Biology) -- University of Pennsylvania, 2002. Includes bibliographical references.