Pax7 is a paired box transcription factor expressed by all satellite cells which are critically required for muscle regeneration and growth. The absolute requirements of Pax7 in the maintenance of the satellite cell pool are widely acknowledged. However the mechanisms by which Pax7 executes muscle regeneration or contributes to satellite cell homeostasis remain elusive. We performed cell and molecular analysis of Pax7 null satellite cells to investigate muscle stem cell function. Through genome wide studies, we found that genes involved in cell cell interactions, regulation of migration, control of lipid metabolism and inhibition of myogenic differentiation were significantly perturbed in Pax7 null satellite cells. Analysis of satellite cells in vitro showed that Pax7 null satellite cells undergo precocious myogenic differentiation and have perturbed expression of genes involved in the Notch signaling pathway. We showed that Notch 1 is a novel Pax7 target gene and by using a genetic approach we demonstrated that ectopic expression of the constitutively active intracellular domain of Notch1 (NICD1) in Pax7 null satellite cells is sufficient to maintain the satellite cell pool as well as to restore their proliferation. Instead of differentiating into myogenic cells and in the absence of a myogenic cue, NICD1 Pax7 null satellite cells become a source of ectopic brown fat within muscles and give rise to brown adipocytes both in vivo and in vitro. In conclusion we showed that Notch 1 partially rescues Pax7 deficient satellite cells loss and proliferation. Additionally we provide the first evidence that Notch signalling contributes to satellite cell fate by inhibiting terminal myogenic differentiation and inducing brown adipogenesis.

Muscle satellite cells are a distinct population of myogenic progenitors that mediate the postnatal growth and regeneration of skeletal muscle. To gain insight into the genetic regulation of satellite cell function during muscle regeneration, genes expressed specifically in these cells were identified by representational difference analysis of cDNAs. Notably, the paired-box transcription factor Pax7 was isolated as a gene specifically expressed in quiescent and activated satellite cells. Cell culture and histological analysis of PaxZ-deficient muscle revealed a complete absence of satellite cells. This result demonstrates a requirement for Pax7 upstream of MyoD and Myf5 in the specification of muscle satellite cells. Consistent with their lack of satellite cells, adult PaxT mice displayed an aggravated muscle wasting phenotype characterized by spinal kyphosis and reduced muscle mass. Acute muscle damage led to extensive calcification and deposition of adipose and fibrotic tissues with the appearance of rare regenerated myofibers. Importantly, analysis of Pax7 muscle suspensions indicated that myogenic cells expressing Pax3 and MyoD were responsible for this low level of regeneration. To characterize the role of adult stem cells in skeletal muscle, we investigated the myogenic potential of muscle-derived CD45+:Sca1+ cells in vivo during regeneration and in vitro using coculture assays. CD45+ and Sca1+ cells isolated from uninjured muscle were uniformly non-myogenic. Strikingly, 7-10% of CD45+:Sca1+ cells purified from regenerating muscle activated the myogenic program by a Pax7-dependent mechanism in response to activation of the Wnt signaling pathway. Furthermore, expression of Pax7 was sufficient to induce myogenic commitment in CD45f+Scal cells from uninjured muscle. This result demonstrates that non-satellite cell derived myogenic progenitors possess a physiological role in muscle regeneration and tissue homeostasis. Taken together, this work establishes a requirement for Pax7 in the specification of muscle satellite cells and for the myogenic recruitment of adult stem cells populations during tissue repair. Importantly, these studies also suggest that targeted therapies to activate Wnt signaling and Pax7 expression in adult stem cells will be effective for promoting muscle regeneration in patients with degenerative neuromuscular diseases or muscular dystrophies. / Thesis / Doctor of Philosophy (PhD)

INTRODUCTION: Damage to the musculoskeletal system through disease, injury, or ageing can have long-lasting, and detrimental effects on one’s overall well-being. By understanding the processes by which the different tissues of the musculoskeletal system function and communicate, we can apply it to a variety of medical interventions that will benefit the patient population. These include reducing the prevalence of injury-inducing ectopic bone formation in muscle and slowing the degeneration of muscle and bone tissue associated with aging. A major focus is the relationship between muscle and bone tissue, specifically the stem cell populations found in each tissue type. Two genes that are thought to mark stem cell populations associated with muscle and bone tissue are Pax7 and Prx1, respectively. OBJECTIVES: Establish the capability and define optimal conditions to culture primary stem cells isolated from the muscle tissue of the reporter animals that fluorescently tag the Pax7 and Prx1 cell populations. Manipulate culture medium conditions to characterize the differentiation potential for multiple lineages, osteogenic, adipogenic, and myogenic. Lastly, assess whether there is more adipogenic cell differentiation in older animal cell cultures. METHODS: The tamoxifen inducible Pax7^tm1(cre/ER2)Gaka/J and Prx1^CreER-GFP were both crossed with B6.Cg-Gt(ROSA)26sor<tm14(CAG-tdTomato)Hze>/J to create the tamoxifen inducible Pax7/Ai14 and Prx1/Ai14 reporter mice. These animals were then crossed to the B6,129S7-Rag1^tm1Mom/J , creating the Pax7/Ai14/Rag and Prx1/Ai14/Rag reporter mice strains. This transgenic mouse model made it possible to fluorescently identify the Pax7 and Prx1 population of cells isolated from the muscle tissue and characterize the differentiation potential to different cell lineages in vitro. Cells were harvested from both male and female mice that received two tamoxifen injections. Cells were then cultured in various culture media conditions. Determination of specific cell densities, culture conditions, and differentiation time points were determined by manipulating these variables, and assessing the levels of proliferation and differentiation. Multiple assays were run in order to quantify and identify the different cell lineages that were grown in culture under different culture conditions. RESULTS: Cells grown on gelatin coated dishes at densities of 2.2x10^4 to 2.2x10^5 showed optimal performance in proliferation and differentiation. Cells grown in Growth Media containing Chick Embryo Extract (GM) and without (GM-) produced myogenic and adipogenic cell types that were positive for Prx1 expression. Prx1 positive cells grown in the Osteo-Inductive Media (GMOI) produced osteogenic cell types validated through tetracycline uptake. Pax7 expression was low in all culture media conditions. Finally, adipogenic cells were present in both younger and older animals. The adipogenic cells for both populations showed positive Prx1 expression. Younger animals showed a larger relative expression of Plin1 in qRT-PCR analysis. CONCLUSIONS: Although Prx1 is thought to be associated with bone tissue, Prx1 positive cells are located within the muscle and able to be cultured. This muscle derived Prx1 cell population is able to differentiate toward the myogenic, adipogenic, and osteogenic cell lineages. By altering the specific components of culture conditions such as extracellular matrices, seeding density, and media constituents, it is possible to force a particular lineage differentiation for Pax7 and Prx1 muscle derived stem cells. Further studies are needed to elucidate the differentiation potential of Prx1 cells isolated from animals of various ages. Additional in vivo studies are needed to understand the mechanisms surrounding the Prx1 and Pax7 population of cells with their roles in healing and regeneration in response to degeneration and trauma.

Epigenetic mechanisms are of fundamental importance for resolving and maintaining cellular identity. The mechanisms regulating muscle stem and progenitor cell identity have ramifications for understanding all aspects of myogenesis. The epigenetic mechanisms regulating muscle stem cells are therefore important aspects for understanding the regulation of muscle regeneration and maintenance. Important roles for the trithorax H3K4 histone methyltransferase (HMT) MLL1 have been established for early embryogenesis, and for hematopoietic and neural identity. Here, using a conditional Mll1 knockout (KO), we find that in vivo, MLL1 is necessary for efficient muscle regeneration, and for maintenance and proliferation of muscle stem and progenitor cells. Loss of Mll1 in cultured myoblasts reveals an essential role for expression of the myogenic specification gene Pax7. Mll1 KO results in a minor decrease in Pax7 mRNA and a strong decrease of Pax7 protein. While MLL1 was found to bind the Pax7 promoter, Mll1 KO results in a minor decrease of H3K4me3 at Pax7, supporting a recognized non-HMT role for Mll1 at Pax7. Microarray analysis of mRNA expression in Mll1 KO myoblasts finds that Myf5 is the most strongly downregulated of all genes, unexpectedly, mRNA expression of previously identified MLL1 targets are unaffected by loss of MLL1 in myoblasts. Pax7 activates Myf5 expression through recruitment of a H3K4 HMT, and in Mll1 KO myoblasts expression of, and H3K4me3 at Myf5 is lost. Exogenous Pax7 rescues Myf5 expression and H3K4me3 at Myf5 in the absence of MLL1, indicating that Myf5 expression is conditional on Pax7, but not MLL1. We also show that Myf5 DNA is methylated in non-myogenic cells, and in satellite stem cells that have never expressed Myf5, but is not methylated in satellite cells that are committed to the myogenic lineage, indicating that demethylation of Myf5 may be a fundamental step in myogenic commitment. Intriguingly, Myf5 promoter DNA becomes remethylated in Mll1 KO myoblasts. This work finds that Pax7 expression and myogenic identity is partly dependent on MLL1 expression. Further, evidence is uncovered that myogenic commitment is initiated by demethylation of Myf5. These findings add to the understanding of the epigenetic mechanisms that regulate and define muscle stem cells.

Pax3 and Pax7 belong to a family of conserved transcription factors that play important and diverse roles in development. In the embryo, they carry out similar roles in neural and somite development, but Pax7 fails to compensate for critical functions of Pax3 in the development of limb musculature. Conversely, in the adult, Pax7 is necessary for the maintenance and survival of muscle satellite cells, whereas Pax3 cannot effectively fulfill these roles in the absence of Pax7. To identify the unique roles of Pax7 in adult muscle cells, we have analyzed global binding of Pax3 and Pax7 by ChIP-Seq. Here, we show that despite highly homologous DNA-binding domains, the majority of binding sites are uniquely recognized by Pax7 and are enriched for homeobox motifs. Genes proximal to conserved, unique Pax7 binding sites cluster into specific functional groups which may reflect the unique biological roles of Pax7. Combining Pax7 binding sites with gene expression data, we describe the regulatory networks directed by Pax7 and show that Pax7 binding is associated with positive gene regulation. Moreover, we show Myf5 is a direct target of Pax7 and identify a novel binding site in the satellite cell control region upstream of Myf5.

Pax3 and Pax7 belong to a family of conserved transcription factors that play important and diverse roles in development. In the embryo, they carry out similar roles in neural and somite development, but Pax7 fails to compensate for critical functions of Pax3 in the development of limb musculature. Conversely, in the adult, Pax7 is necessary for the maintenance and survival of muscle satellite cells, whereas Pax3 cannot effectively fulfill these roles in the absence of Pax7. To identify the unique roles of Pax7 in adult muscle cells, we have analyzed global binding of Pax3 and Pax7 by ChIP-Seq. Here, we show that despite highly homologous DNA-binding domains, the majority of binding sites are uniquely recognized by Pax7 and are enriched for homeobox motifs. Genes proximal to conserved, unique Pax7 binding sites cluster into specific functional groups which may reflect the unique biological roles of Pax7. Combining Pax7 binding sites with gene expression data, we describe the regulatory networks directed by Pax7 and show that Pax7 binding is associated with positive gene regulation. Moreover, we show Myf5 is a direct target of Pax7 and identify a novel binding site in the satellite cell control region upstream of Myf5.

Myogenesis is the process of skeletal muscle tissue formation where committed muscle progenitor cells differentiate into skeletal muscle fibres. Depending on the instructive cues the muscle progenitor cells receive they will differentiate into specific fibre types with different properties. The skeletal muscle fibres can be broadly classified as fast-twitch fibres or slow-twitch fibres, based on their contractile speed. However, subgroups of fast- and slow-twitch fibres with different metabolic properties, endurance and different isoforms of sarcomeric components have also been identified, adding complexity to the process of muscle tissue patterning. The skeletal muscle tissue has the capacity to regenerate throughout life. Upon muscle tissue damage muscle satellite cells are recruited to the area of injury where they proliferate and either form new fibres similar to those damaged, or fuse with existing fibres. This thesis aims to investigate the process of muscle progenitor cell proliferation and differentiation, as well as the fast-twitch fibre formation and muscle tissue patterning in the zebrafish embryo. I present results identifying the previously uncharacterised gene myl1, encoding an alkali-like myosin light chain, which is specifically expressed in fast-twitch muscle progenitors before fibre formation. Furthermore, I introduce data showing that the transcription factor six1 is expressed in Pax7+ muscle progenitor cells, which has been reported to contribute to part of the fast-twitch muscle tissue as well as to a pool of quiescent muscle satellite cells. With support from the presented data, I hypothesise that six1 keeps the Pax7+ muscle progenitor cells in a proliferative state and consequently prevents them from differentiating into muscle fibres. In addition, I demonstrate that the zebrafish fast-twitch muscle fibres can be divided into different subgroups that express unique forms of fast myosin heavy chain genes along the anterior-posterior (head-tail) axis, and that this subspecification depends on a balance between RA and Wnt signalling. Collectively I propose a previously unknown role for Six1 in zebrafish Pax7+ muscle progenitor cell proliferation and differentiation. Furthermore, I present novel data suggesting that distinct regions of the zebrafish body musculature are composed of different fast-twitch fibre types, and that this regionalisation is conserved in adult zebrafish.

Pax3 and Pax7 belong to a family of conserved transcription factors that play important and diverse roles in development. In the embryo, they carry out similar roles in neural and somite development, but Pax7 fails to compensate for critical functions of Pax3 in the development of limb musculature. Conversely, in the adult, Pax7 is necessary for the maintenance and survival of muscle satellite cells, whereas Pax3 cannot effectively fulfill these roles in the absence of Pax7. To identify the unique roles of Pax7 in adult muscle cells, we have analyzed global binding of Pax3 and Pax7 by ChIP-Seq. Here, we show that despite highly homologous DNA-binding domains, the majority of binding sites are uniquely recognized by Pax7 and are enriched for homeobox motifs. Genes proximal to conserved, unique Pax7 binding sites cluster into specific functional groups which may reflect the unique biological roles of Pax7. Combining Pax7 binding sites with gene expression data, we describe the regulatory networks directed by Pax7 and show that Pax7 binding is associated with positive gene regulation. Moreover, we show Myf5 is a direct target of Pax7 and identify a novel binding site in the satellite cell control region upstream of Myf5.

The long-term treatment of injured, aging, or pathological skeletal muscle using stem cell therapy requires an abundant source of skeletal muscle progenitors (SMP) that are capable of self-replenishment. While adult SMPs—known as satellite cells and marked by PAX7 expression—can be collected from healthy donors, these satellite cells have limited replication potential once extracted, and may have difficulties providing sufficient numbers for therapy. Therefore, we sought to utilize the near-unlimited replication potential of human embryonic stem cells (hESC) to generate large quantities of SMPs in vitro. We developed a 50-day directed hESC differentiation that produced cultures with up to 90% myogenic identity; roughly 43 ± 4% become PAX7+ SMPs, and 47 ± 3% of cells become skeletal myocytes. We also performed gene expression profiling on our differentiating cultures to better understand in vitro skeletal myogenesis, and to better characterize in vitro hESC-derived SMPs, which remain poorly understood relative to adult satellite cells. 50-day cultures shared gene expression profiles more similar to quiescent rather than activated satellite cells, featuring a number of genes related to FOS/JUN, NOTCH, and TGFB-signaling. Day 50 cultures also expressed surface proteins known to mark adult or embryonic SMPs: CD82, CXCR4, ERBB3, NGFR, and PDGFRA. Transplanting 50-day cultures into cardiotoxin or BaCl2 injured immunodeficient murine muscle showed donor human cells persisted within the host muscle for 1 – 2 months post-injection; however, donor cells were confined to the interstitial space and did not contribute to host myofibers or the satellite cell niche. Together, these studies provide a tool for generating large quantities of embryonic skeletal muscle, and a gene expression resource that can provide insight into signaling factors that might improve or accelerate SMP development, or provide putative new surface receptors that may isolate embryonic SMPs better suited for in vivo transplantation.

Pax3 and Pax7 belong to a family of conserved transcription factors that play important and diverse roles in development. In the embryo, they carry out similar roles in neural and somite development, but Pax7 fails to compensate for critical functions of Pax3 in the development of limb musculature. Conversely, in the adult, Pax7 is necessary for the maintenance and survival of muscle satellite cells, whereas Pax3 cannot effectively fulfill these roles in the absence of Pax7. To identify the unique roles of Pax7 in adult muscle cells, we have analyzed global binding of Pax3 and Pax7 by ChIP-Seq. Here, we show that despite highly homologous DNA-binding domains, the majority of binding sites are uniquely recognized by Pax7 and are enriched for homeobox motifs. Genes proximal to conserved, unique Pax7 binding sites cluster into specific functional groups which may reflect the unique biological roles of Pax7. Combining Pax7 binding sites with gene expression data, we describe the regulatory networks directed by Pax7 and show that Pax7 binding is associated with positive gene regulation. Moreover, we show Myf5 is a direct target of Pax7 and identify a novel binding site in the satellite cell control region upstream of Myf5.