MOLECULAR MECHANISMS OF CRANIOFACIAL SKELETAL MUSCLE REGENERATION

Limited therapeutic options exist to rebuild functional craniofacial skeletal muscle in individuals suffering from orofacial muscle diseases, trauma, and oncologic surgical defects. The lack of a comprehensive investigation of the craniofacial musculature and its stem population has resulted in a poor understanding of the regenerative capacity of this tissue when faced with disease or trauma. Our current knowledge of skeletal muscles and their stem cell populations comes from the limb muscles; however, it is known that distinct differences in embryological origin exist between muscles of the limb and those of the craniofacial region.The objective of this study is to characterize the molecular mechanisms surrounding the regenerative program of craniofacial skeletal muscle and its resident stem cells, satellite cells in order to identify therapeutic targets for craniofacial muscle regeneration.The first aim was to assess the regenerative capacity of craniofacial muscle in vivo and the function of satellite cells, termed muscle stem cells (MuSCs) in vitro. To do this, I characterized the morphology of regenerating craniofacial muscle following injury using in vivomodels of muscle regeneration. To characterize the functional capacity of craniofacial MuSCs, I isolated MuSCs from limb and craniofacial muscle using FACS and determined MuSC number and used in vitro cellular assays to measure the ability of MuSCs to proliferate and differentiate.The second aim was to identify unique transcriptional signatures between limb and craniofacial muscle satellite cells. To do this, I used single-cell RNA sequencing (scRNA-seq) on FACS-isolated satellite cells to identify differential gene expression between craniofacial and limb satellite cells throughout a time course of muscle regeneration.ivThe final aim was to determine the roles of identified genes of interest, Ebf1 and Postn, in the molecular regulation of satellite cells during craniofacial muscle regeneration. To do this, I used quantitative real-time PCR (qPCR) to determine gene expression over a time course of myogenesis as well as viral-mediated gene manipulation to knock down the genes of interest in vitro.Doctor of Philosoph