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The transcription factors SMAD2 and SMAD3 are the effectors of classical transforming growth factor beta (TGFβ) signalling. This signalling cascade is involved in many cellular processes including proliferation and differentiation and is known to be a potent inhibitor of myogenic differentiation through SMAD3. We have previously shown that retinoic acid (RA) can upregulate SMAD3 in models of adipogenesis and mesenchymal stem cells and that SMAD3 can interact with the bZIP transcription factor C/EBPβ to disrupt its DNA binding. Forced expression of C/EBPβ inhibits myogenic differentiation but the mechanism has not been fully elucidated. Herein we show that RA increases Smad3 expression in myoblasts and that RA treatment antagonizes TGFβ-mediated inhibition of myogenic differentiation. TGFβ treatment increased C/EBPβ expression which was reversed by RA treatment. Further, RA was able to disrupt C/EBPβ occupancy of the Pax7 and Smad2 promoters in myoblasts. Loss of C/EBPβ in primary myoblasts using a conditional knockout model partially protected these cells from the anti-myogenic effects of TGFβ treatment. The TGFβ effector protein SMAD2 is expressed in myoblasts but its specific function in myogenesis has not been determined, as Smad2 knockout models are embryonic lethal. Thus, we created a novel Smad2 conditional knockout model where Smad2 is excised in PAX7-expressing muscle satellite cells. Herein we demonstrate a role for SMAD2 specifically in myogenic fusion. We describe a regeneration defect after acute injury and decreased fiber cross-sectional area at P21 (post-natal day 21) in Smad2cKO muscle, without affecting the numbers of PAX7-positive cells. Further, we reveal a mechanism whereby SMAD2 regulates KLF4 expression and mediates the KLF4-induced increased of the fusion gene Npnt. This work describes the pro-myogenic actions of RA-induced SMAD3 and the novel function of SMAD2 in terminal myogenic differentiation and fusion. This work also discusses future directions, implications and new insights into non-canonical SMAD actions.

Thesis (M.S.)--University of Wyoming, 2008. / Title from PDF title page (viewed on Mar. 8, 2010). Includes bibliographical references.

Thesis (M. Phil.)--Hong Kong University of Science and Technology, 2004. / Includes bibliographical references (leaves 102-115). Also available in electronic version. Access restricted to campus users.

Thesis (Ph. D.)--York University, 2002. Graduate Programme in Biology. / Typescript. Includes bibliographical references (leaves 160-183). Also available on the Internet. MODE OF ACCESS via web browser by entering the following URL: http://wwwlib.umi.com/cr/yorku/fullcit?pNQ82779.

Thesis (doctoral)--Ludwig-Maximilians-Universität München, 2001. / Title from PDF t.p. (viewed on July 15, 2006). Includes bibliographical references.

Rhabdomyosarcomas (RMS) are the most common soft tissue cancer among children and are characterized by their expression of the myogenic regulatory factors MyoD and myogenin. Yet RMS cells cannot undergo normal myogenesis and are caught between the proliferation program and the terminal differentiation program. Many questions still remain about the defects present in rhabdomyosarcoma cells. In this work, we set out to understand the role of myogenin in these cells. To begin, we found that myogenin and its co-factors were present in rhabdomyosarcoma cells at levels that should support terminal differentiation. We examined the expression profile of several myogenin target genes in rhabdomyosarcoma cells and then assayed for myogenin activity using luciferase reporter constructs that contain myogenin dependent promoters to test for myogenin function. Many myogenin target genes were down regulated in RMS cells but that the target promoters on the luciferase constructs were activated. Terminal differentiation is a complicated process that involves many proteins. In cancer cells, it is important to compare the levels proteins with known functions to those levels in wild-type cells at the protein and RNA levels. Establishing the defect of rhabdomyosarcoma cells can lead to further insights into normal myogenesis, and may also lead to new therapeutic approaches in the treatment of this childhood cancer.

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ARS2 is a member of the nuclear cap-binding complex (CBC) that is critical for a number of RNA processing pathways. The emerging model is that ARS2 acts as a master regulator of RNAPII transcript maturation by bringing capped RNA substrates together with the appropriate processing machinery. ARS2 is essential for early mammalian development but it remains unclear precisely how ARS2 functions in stem and progenitor cell maintenance and differentiation. The purpose of this study was to answer basic questions about the localization and function of ARS2 in muscle progenitor cells. Here I describe the localization of ARS2 in proliferating myoblasts and post-mitotic differentiating myotubes and show that disruption of ARS2 expression levels by knockdown or overexpression results in impaired myogenic differentiation. I also discovered a new isoform of ARS2 that is localized exclusively in the cytoplasm and found preliminary evidence that ARS2 is required for nonsense-mediated decay (NMD). This study includes the first evidence that an ARS2 isoform is expressed in the cytoplasm and opens the door for the discovery of new ARS2 functions beyond its reported roles in the nucleus. / Graduate

Embryonic stem cells (ESCs) represent a promising source of cells for cell replacement therapy in the context of muscle diseases; however, before ESC-based cell therapy can be translated to the clinic, we must learn to modulate cell-fate decisions in order to maximize the yield of myocytes from this systems. In order to gain a better understanding of the myogenic cell fate, we sought to define the molecular mechanisms underlying the specification and differentiation of ESCs into cardiac and skeletal muscle. More specifically, the central hypothesis of the thesis is that myogenic signalling cascades modulate cell fate via regulation of transcription factors. Retinoic acid (RA) is known to promote skeletal myogenesis, however the molecular basis for this remains unknown. We showed that RA expands the premyogenic progenitor population in mouse stem cells by directly activating pro-myogenic transcription factors such as Pax3 and Meox1. RA also acts indirectly by activating the pro-myogenic Wnt signalling cascade while simultaneously inhibiting the anti-myogenic influence of BMP4. This ultimately resulted in a significant enhancement of skeletal myogenesis. Furthermore, we showed that this effect was conserved in human embryonic stem cells, with implications for directed differentiation and cell therapy. The regulation of cardiomyogenesis by the Wnt pathway was also investigated. We identified a novel interaction between the cardiomyogenic transcription factor Nkx2.5 and the myosin phosphatase (MP) enzyme complex. Interaction with MP resulted in exclusion of Nkx2.5 from the nucleus and inhibition of its transcriptional activity. Finally, we showed that this interaction was modulated by phosphorylation of the Mypt1 subunit of MP by ROCK, downstream of Wnt3a. Treatment of differentiating mouse ESCs with Wnt3a resulted in exclusion of Nkx2.5 from the nucleus and a subsequent failure to undergo terminal differentiation into cardiomyocytes. This likely represents part of the molecular basis for Wnt-mediated inhibition of terminal differentiation of cardiomyocytes. Taken together, our results provide novel insight into the relationship between myogenic signalling cascades and downstream transcription factors and into how they function together to orchestrate the myogenic cell fate in stem cells.