Doctor of Philosophy

dissertationSkeletal muscle is among the few adult tissues with the capacity to regenerate after injury. This is due to the resident stem cells within the muscle, the satellite cells. In the absence of injury, these stem cells remain quiescent and reside within their niche beneath the basement membrane adjacent to the myofiber. Upon muscle injury, satellite cells are activated and will proliferate, self renew, and differentiate into transiently amplifying myoblasts, which also self renew and give rise to differentiating myocytes. These myocytes will fuse to themselves and to the injured myofibers to repair muscle damage. While the cellular processes of muscle regeneration are understood, many questions remain. Despite similarities in their expression patterns and function, many characteristics are dissimilar between developmental myogenic precursors and satellite cells. Chapter 2 of this dissertation reviews what is known about the unique properties of these closely related cells, and Chapter 3 of this dissertation directly tests the requirement of satellite cells during muscle regeneration. In addition to the myogenic cells, many nonmuscle cells are involved in the process of muscle regeneration. Chapter 3 of this dissertation also shows that connective tissue fibroblasts prevent premature differentiation of satellite cells and are an important component of the satellite cell niche. One signaling pathway shown to regulate stem cells in other tissue contexts is the Wnt/beta-catenin pathway. Multiple studies describe the role of Wnt/beta-catenin signaling in muscle regeneration; however, there is no consensus as to the functional role of this signaling pathway in adult myogenesis. In Chapter 4 of this dissertation, the requirement for Wnt/beta-catenin in the satellite cells and their progeny is tested in vivo. Surprisingly, despite evidence that the Wnt/beta-catenin signaling pathway is active in myogenic cells during regeneration, satellite cells and their progeny do not require beta-catenin. Chapter 4 also discusses our results that show that extension of Wnt/beta-catenin signaling prolongs the time myogenic cells spend in the myoblast phase of regeneration. This dissertation demonstrates the importance of the connective tissue fibroblasts and also critically tests the function of the Wnt/beta-catenin signaling pathway in muscle regeneration

Leptin-Receptor-Expressing Mesenchymal Stromal Cells Represent the Main Source of Bone Formed by Adult Bone Marrow

SummaryStudies of the identity and physiological function of mesenchymal stromal cells (MSCs) have been hampered by a lack of markers that permit both prospective identification and fate mapping in vivo. We found that Leptin Receptor (LepR) is a marker that highly enriches bone marrow MSCs. Approximately 0.3% of bone marrow cells were LepR+, 10% of which were CFU-Fs, accounting for 94% of bone marrow CFU-Fs. LepR+ cells formed bone, cartilage, and adipocytes in culture and upon transplantation in vivo. LepR+ cells were Scf-GFP+, Cxcl12-DsRedhigh, and Nestin-GFPlow, markers which also highly enriched CFU-Fs, but negative for Nestin-CreER and NG2-CreER, markers which were unlikely to be found in CFU-Fs. Fate-mapping showed that LepR+ cells arose postnatally and gave rise to most bone and adipocytes formed in adult bone marrow, including bone regenerated after irradiation or fracture. LepR+ cells were quiescent, but they proliferated after injury. Therefore, LepR+ cells are the major source of bone and adipocytes in adult bone marrow

Transiently Active Wnt/β-Catenin Signaling Is Not Required but Must Be Silenced for Stem Cell Function during Muscle Regeneration

Adult muscle’s exceptional capacity for regeneration is mediated by muscle stem cells, termed satellite cells. As with many stem cells, Wnt/β-catenin signaling has been proposed to be critical in satellite cells during regeneration. Using new genetic reagents, we explicitly test in vivo whether Wnt/β-catenin signaling is necessary and sufficient within satellite cells and their derivatives for regeneration. We find that signaling is transiently active in transit-amplifying myoblasts, but is not required for regeneration or satellite cell self-renewal. Instead, downregulation of transiently activated β-catenin is important to limit the regenerative response, as continuous regeneration is deleterious. Wnt/β-catenin activation in adult satellite cells may simply be a vestige of their developmental lineage, in which β-catenin signaling is critical for fetal myogenesis. In the adult, surprisingly, we show that it is not activation but rather silencing of Wnt/β-catenin signaling that is important for muscle regeneration