The Hedgehog (Hh) signalling pathway is one of the key signalling pathways orchestrating intricate organogenesis, including the development of neural tube, heart and skeletal muscle. Yet, insufficient mechanistic understanding of its diverse roles is available. Here, we show the molecular mechanisms regulating the neurogenic, cardiogenic and myogenic properties of Hh signalling, via effector protein Gli2, in embryonic and adult stem cells. In Chapter 2, we show that Gli2 induces neurogenesis, whereas a dominant-negative form of Gli2 delays neurogenesis in P19 embryonal carcinoma (EC) cells, a mouse embryonic stem (ES) cell model. Furthermore, we demonstrate that Gli2 associates with Ascl1/Mash1 gene elements in differentiating P19 cells and activates the Ascl1/Mash1 promoter in vitro. Thus, Gli2 mediates neurogenesis in P19 cells at least in part by directly regulating Ascl1/Mash1 expression. In Chapter 3, we demonstrate that Gli2 and MEF2C bind each other’s regulatory elements and regulate each other’s expression while enhancing cardiomyogenesis in P19 cells. Furthermore, dominant-negative Gli2 and MEF2C proteins downregulate each other’s expression while imparing cardiomyogenesis. Lastly, we show that Gli2 and MEF2C form a protein complex, which synergistically activates cardiac muscle related promoters. In Chapter 4, we illustrate that Gli2 associates with MyoD gene elements while enhancing skeletal myogenesis in P19 cells and activates the MyoD promoter in vitro. Furthermore, inhibition of Hh signalling in muscle satellite cells and in proliferating myoblasts leads to reduction in MyoD and MEF2C expression. Finally, we demonstrate that endogenous Hh signalling is important for MyoD transcriptional activity and that Gli2, MEF2C and MyoD form a protein complex capable of inducing skeletal muscle-specific gene expression. Thus, Gli2, MEF2C and MyoD participate in a regulatory loop and form a protein complex capable of inducing skeletal muscle-specific gene expression. Our results provide a link between the regulation of tissue-restricted factors like Mash1, MEF2C and MyoD, and a general signal-regulated Gli2 transcription factor. We therefore provide novel mechanistic insights into the neurogenic, cardiogenic and myogenic properties of Gli2 in vitro, and offer novel plausible explanations for its in vivo functions. These results may also be important for the development of stem cell therapy strategies.

One of the major drawbacks of using stem cell therapy to treat muscular dystrophies is the challenge of isolating sufficient numbers of suitable precursor cells for transplantation. As such, a deeper understanding of the molecular mechanisms involved during muscle development, which would increase the proportion of embryonic stem cells that can differentiate into skeletal myocytes, is essential. In conditional SOX7-/- mice, we observed that the loss of SOX7 in satellite cells resulted in poor differentiation and fusion. In vivo, we observed fewer Pax7+ satellite cells in the mice lacking SOX7 as well as smaller muscle fibers. RT-qPCR data also revealed that Pax7, MRF and MHC3 transcript levels were down-regulated in SOX7 knockdown mice. Surprisingly, when SOX7 was over-expressed in embryonic stem cells, we found that there was a defect in making muscle precursor cells, specifically a failure to activate Pax7 expression. Taken together, these results suggest that SOX7 expression is required for the proper regulation of skeletal myogenesis.

The age-related loss of skeletal muscle mass and function is accompanied by a decline in regenerative capacity. The processes that facilitate healthy muscle repair are complex, involving several phases of degradation and rebuilding of muscle tissue and the surrounding microenvironment. Specifically, myogenic progenitor cells known as satellite cells are the most influential in repairing damaged muscle tissue. Following injury, satellite cells become activated and migrate, proliferate and fuse with mature skeletal muscle fibers to restore homeostasis to the tissue. However, satellite cells do not act in isolation, a robust inflammatory response is necessary to facilitate successful and rapid healing. Macrophages are one of the first and most abundant immune cells to infiltrate damaged skeletal muscle tissue. Primarily, macrophages adapt to a proinflammatory state to clear the area of cellular debris, promote degradation of the extracellular matrix and stimulate satellite cell activation and proliferation. Afterwards, a timely transition to an anti-inflammatory state directs rebuilding of the extracellular matrix and terminal differentiation of satellite cells. Indeed, the inhibition of macrophage activity leads to impaired healing and loss of skeletal muscle function. Little is known regarding the behavior of macrophages in aged skeletal muscle following injury in humans. Thus, the objective of this dissertation is to investigate the age-related response of macrophages in human skeletal muscle, and their role in muscle repair.

abstract: Satellite cells are adult muscle stem cells that activate, proliferate, and differentiate into myofibers upon muscle damage. Satellite cells can be cultured and manipulated in vitro, and thus represent an accessible model for studying skeletal muscle biology, and a potential source of autologous stem cells for regenerative medicine. This work summarizes efforts to further understanding of satellite cell biology, using novel model organisms, bioengineering, and molecular and cellular approaches. Lizards are evolutionarily the closest vertebrates to humans that regenerate entire appendages. An analysis of lizard myoprogenitor cell transcriptome determined they were most transcriptionally similar to mammalian satellite cells. Further examination showed that among genes with the highest level of expression in lizard satellite cells were an increased number of regulators of chondrogenesis. In micromass culture, lizard satellite cells formed nodules that expressed chondrogenic regulatory genes, thus demonstrating increased musculoskeletal plasticity. However, to exploit satellite cells for therapeutics, development of an ex vivo culture is necessary. This work investigates whether substrates composed of extracellular matrix (ECM) proteins, as either coatings or hydrogels, can support expansion of this population whilst maintaining their myogenic potency. Stiffer substrates are necessary for in vitro proliferation and differentiation of satellite cells, while the ECM composition was not significantly important. Additionally, satellite cells on hydrogels entered a quiescent state that could be reversed when the cells were subsequently cultured on Matrigel. Proliferation and gene expression data further indicated that C2C12 cells are not a good proxy for satellite cells. To further understand how different signaling pathways control satellite cell behavior, an investigation of the Notch inhibitor protein Numb was carried out. Numb deficient satellite cells fail to activate, proliferate and participate in muscle repair. Examination of Numb isoform expression in satellite cells and embryonic tissues revealed that while developing limb bud, neural tube, and heart express the long and short isoforms of NUMB, satellite cells predominantly express the short isoforms. A preliminary immunoprecipitation- proteomics experiment suggested that the roles of NUMB in satellite cells are related to cell cycle modulation, cytoskeleton dynamics, and regulation of transcription factors necessary for satellite cell function. / Dissertation/Thesis / Doctoral Dissertation Molecular and Cellular Biology 2020

Very little is known about the development of cardiac parasympathetic ganglia and cholinergic innervation of the mouse heart. Accordingly, we evaluated the growth of cholinergic neurons and nerve fibers in mouse hearts from embryonic day 18.5 (E18.5) through postnatal day 21(P21). Cholinergic perikarya and varicose nerve fibers were identified in paraffin sections immunostained for the vesicular acetylcholine transporter (VAChT). Satellite cells and Schwann cells in adjacent sections were identified by immunostaining for S100β calcium binding protein (S100) and brain-fatty acid binding protein (B-FABP). We found that cardiac ganglia had formed in close association to the atria and cholinergic innervation of the atrioventricular junction had already begun by E18.5. However, most cholinergic innervation of the heart, including the sinoatrial node, developed postnatally (P0.5-P21) along with a doubling of the cross-sectional area of cholinergic perikarya. Satellite cells were present throughout neonatal cardiac ganglia and expressed primarily B-FABP. As they became more mature at P21, satellite cells stained strongly for both B-FABP and S100. Satellite cells appeared to surround most cardiac parasympathetic neurons, even in neonatal hearts. Mature Schwann cells, identified by morphology and strong staining for S100, were already present at E18.5 in atrial regions that receive cholinergic innervation at later developmental times. The abundance and distribution of S100-positive Schwann cells increased postnatally along with nerve density. While S100 staining of cardiac Schwann cells was maintained in P21 and older mice, Schwann cells did not show B-FABP staining at these times. Parallel development of satellite cells and cholinergic perikarya in the cardiac ganglia and the increase in abundance of Schwann cells and varicose cholinergic nerve fibers in the atria suggest that neuronal-glial interactions could be important for development of the parasympathetic nervous system in the heart.

The muscle hypertrophy of lambs expressing the Callipyge phenotype is possibly linked to characteristics of their muscle satellite cells. Therefore, characteristics (proliferation, fusion %, and protein accretion) of cultured satellite cells isolated from the longissimus muscle of Callipyge (n = 3) and normal (n = 3) lambs were compared in this study. In the first experiment, we tested whether or not the lll proliferation rates differ for satellite cells isolated from Callipyge or normal sheep when cultured in the presence of different serum types (horse, normal lamb, or Callipyge lamb). The average population doubling time (PDT, h) during log phase growth was calculated for cells from each animal grown in each serum type. Population doubling time was not affected (P > .1) by the interaction of satellite cell type with serum type, or by satellite cell type. Unexpectedly, PDT was longer (P < .05) for satellite cells grown in Callipyge serum (22 h) than for cells grown in normal sheep serum (20 h) or horse serum (18 h). These results suggest that muscle hypertrophy of Callipyge lambs is not linked to intrinsic differences in satellite cell proliferation, although hypertrophy may be associated with a decreased proliferation induced by a factor in Callipyge serum. In the second experiment, we tested whether cell fusion, or protein accretion differ for cultured satellite cells isolated from Callipyge or normal sheep. DNA and protein were determined at 24, 48, 72, and 96 h after satellite cell cultures were induced to differentiate. Fusion percentage was determined in a Giemsa stained plate after 72 h in differentiation medium (Dulbecco's Modified Eagle Medium containing 1.5% of horse serum). Callipyge cultures tended (P = .14) to have higher fusion% than normal cultures exhibited, suggesting that muscle hypertrophy of Callipyge lambs may be linked to an increased tendency of satellite cells to fuse. Protein content (μg/well) and protein:DNA ratio (ng of protein/ng of DNA) were not affected by satellite cell type (P = .80 and P = .79, respectively). Thus, there was no evidence for a link between increased protein accretion and Callipyge hypertrophy.

No description available.

Previous work from our laboratory in the mdx mouse model of Duchenne muscular dystrophy (DMD) demonstrated that a fraction of muscle stem cells (i.e., satellite cells) (MuSCs) progressively lose the expression of myogenic markers during the progression of the disease. In the process of characterizing this aberrant behaviour, we serendipitously discovered that MuSCs might be separated into two distinct subpopulations based on the expression of the GPI-anchored surface protein CD90. Crucially, this separation does not correlate with a divergence from the myogenic lineage; instead, it separates the pool of MuSCs into two subpopulations, both maintaining myogenic properties in healthy muscles. These two newly identified subpopulations do not overlap with any previously reported subpopulation and may be prospectively isolated; present a different response in terms of kinetics of activation and differentiation during the regenerative process induced by acute muscle damage; show a different propensity to enter in GAlert state upon distal injury; display dissimilar pAMPK activity and contain a different amount of mitochondria; are present in different proportions in distinct muscle groups; differentially express ECM encoding genes during quiescence. Moreover, one of the two subpopulations can give rise to the other and therefore appears to be upstream in the lineage hierarchy. Notably, loss of function experiments, in which CD90 was downregulated in MuSCs, diminish the difference in activation displayed by the two subpopulations. This demonstrates that CD90 is a molecular determinant of MuSCs functional diversification. Importantly, although the two subpopulations of MuSCs are numerically similar in healthy limb muscles, one of the two subpopulations is lost with time in dystrophic mdx mice. Based on these data, we are hypothesizing that an imbalance between the two newly identified subpopulations may impair regeneration in the dystrophic muscles. These observations not only increase our knowledge of the molecular and cellular dynamics that are controlling normal and pathological muscle homeostasis but also open the possibility that restoring the proper functional equilibrium between subpopulations of MuSCs may counteract the progression of the dystrophic disease.

Myotonic dystrophy type 1 (DM1) is an autosomal dominant and progressive neuromuscular disorder caused by the CTG trinucleotide repeat expansion in the 3’ untranslated region of the DMPK gene. Clinical manifestations include extensive atrophy of skeletal muscle (SkM) concomitant with muscle weakness, that develops in a distal to proximal fashion. Central to muscle plasticity is the satellite cell (SC), a muscle specific stem cell that, upon activation, facilitates muscle repair and regeneration. To date, SCs have yet to be elucidated in DM1; therefore, the aim of the present study was to extensively characterize the PAX7+ SC population, along with other indices of muscle quality in SkM. DM1 patients (6 women, 5 men) performed stationary cycling 3 times per week for 12wks, with biopsies taken from the Vastus lateralis pre- (PRE) and post-endurance exercise intervention (POST). Age-matched, healthy controls (CTRL) were used for comparison of baseline measures. Type 1 and 2 myofiber-specific PAX7+ cells were significantly greater in DM1 patients (PRE), in comparison to CTRL (2.24- and 1.84-fold, respectively), with type 2 SC content further increasing following training (p<0.05). In addition, protein expression of myogenic regulatory factors PAX7 and myogenin were significantly higher in DM1 compared to CTRL, with no training effects observed. Both immunohistochemical and immunoblotting analysis showed that activated MYOD+/PAX7+ cells did not significantly differ in DM1 vs. CTRL. FISH- IF analysis of CUG repeats show that 30% of SCs in DM1 were positive for these inclusions. Muscle capillarization was significantly lower in type 2 fibers in DM1 vs CTRL, which was fully rescued with training (p<0.05). At baseline, DM1 muscle showed the presence of de novo and fat infiltrated fibres, as well as fibrosis, that were relatively non-existent in the CTRL. In vitro results show patient-derived myoblasts exhibit a proliferation defect. Furthermore, myoblasts showed impairments in both glycolysis and mitochondrial respiration, with the latter being completely normalized to CTRL in myotubes. Our novel findings display an increased, albeit non-functional, SC pool in DM1 SkM indicated by disturbances in the myogenic program and overall poor muscle quality. We show that both SCs and SkM remain responsive to exercise training, suggesting therapeutic potential. We also suggest that mitochondrial dysfunction may underpin these impairments in the myogenic program. / Thesis / Master of Science (MSc) / Myotonic dystrophy type 1 (DM1) is the most common muscular dystrophy in adults worldwide affecting 1:8000 individuals, with certain areas in northeastern Quebec having a higher prevalence of 1:600 individuals. DM1 is caused by an autosomal dominant genetic mutation that leads to muscle weakness, respiratory insufficiency, cataracts and cardiac conduction block, ultimately resulting in poor quality of life and shortened lifespan. Preliminary evidence suggests that the maintenance of muscle health can greatly improve quality of life and life-span of these individuals, making an in-depth research focus on this therapeutic intervention extremely important. Optimal muscle health is maintained by the functionality of muscle stem cells, that aid in muscle repair and facilitate adaptations in muscle following exercise interventions. These cells are shown to be dys- or non-functional in various muscular dystrophies which coincide with the observation of poor muscle health. Therefore, the aim of this study was to examine the number and functionality of muscle stem cells, and physiological factors of muscle health in DM1. In addition, we also aimed to explore whether exercise has therapeutic potential to alleviate poor muscle quality in DM1. In general, we found that DM1 patients have a higher proportion of muscle stem cells; however, they are inherently dysfunctional but did respond to exercise. Consistent with the latter observation, we found poor muscle quality metrics in DM1 patients, with aerobic training leading to improvements in muscle health. Altogether, our results provide in-depth analysis that underscores muscle dysfunction observed in DM1 and the benefits of exercise interventions.

Background: Rhabdomyosarcoma (RMS) is a soft tissue malignancy of the skeletal muscle that occurs primarily in pediatric populations. The prevailing treatment for RMS is a combination of chemoradiation therapy and surgery which has contributed to its 5-year survival rate of 75%. However, the combination of RMS and chemoradiation therapy can lead to impaired muscle growth and development which results in life-long skeletal muscle atrophy and weakness for RMS survivors. Skeletal muscle is a highly plastic tissue due, in part, to dynamic interactions between muscle-resident stem and progenitor cells (i.e., satellite cells (SCs) and fibro/adipogenic progenitors (FAPs)), which are necessary for muscle maintenance, growth, and adaptation to anabolic stimuli such as resistance exercise training. There is a clear gap in research investigating whether resistance endurance training (RET) stimulates muscle growth and preserves muscle function after juvenile chemoradiation therapy. Purpose: To determine the extent to which RET ameliorates the skeletal muscle defects in a preclinical model of RMS survivorship. Hypothesis: RET will improve physical performance, muscle cross-sectional area (CSA), and stem/progenitor cell populations compared to sedentary mice following RMS and chemoradiation therapy. Methods: RMS (M3-9-M cells) was injected into a single hindlimb of juvenile (4 week) C57Bl/6 mice that underwent systemic chemotherapy followed by targeted, fractionated radiation therapy to the RMS-injected limb. Following therapy, mice underwent RET (RET; n=10) or remained sedentary (SED; n=10) for 8 weeks. Body composition and performance tests were completed pre- and post-therapy and throughout the exercise intervention. Fibre typing, cross-sectional area, myonuclear characteristics and trichrome staining were evaluated following muscle harvest and progenitor cell populations were assessed using flow cytometry. Results: RET led to increased endurance performance (p<0.0001) as well as a reduction in body fat percentage (p=0.0004). RET rescued atrophy induced by RMS+therapy as evidenced by a significant increase in gastrocnemius/soleus to body weight ratio for the RET group compared to the SED group (p=0.0303), despite the decrease in muscle weight observed in the treated limb compared to the control limb (p=0.015). A distinct increase in intramuscular fibrosis was noted in the treated limb compared to the control limb in both groups (p=0.0283). Furthermore, RET resulted in larger myofibre cross-sectional area (p<0.05) and a shift from Type IIX to IIA fibres (p<0.05). We also noted a greater Type IIA myonuclear domain in the RET group compared to the SED group (p=0.0015) and an overall decrease in myonuclear domain (the cytoplasmic volume controlled by each myonucleus) for the treated limb compared to the control limb (p<0.05). Interestingly, we noticed overall cell death and an increase in immune cells in the RMS treated limb, while exercise resulted in increased endothelial and mesenchymal stromal cells. Significance: These preclinical findings will provide the rationale for further investigation of the mechanisms responsible for the beneficial effects of RET as well as optimizing the RET protocol in this juvenile cancer survivorship model.