Thesis (Ph.D.)--York University, 2009. Graduate Programme in Biology. / Typescript. Includes bibliographical references (leaves 275-298). Also available on the Internet. MODE OF ACCESS via web browser by entering the following URL: http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&res_dat=xri:pqdiss&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&rft_dat=xri:pqdiss:NR51670

La mise en place du système musculo-(exo)squelettique de la drosophile est un modèle d’organisation particulièrement propice à l’étude des interactions tissulaires au cours du développement.Notre étude vise à, d’une part, comprendre la myogenèse appendiculaire à travers l’étude des interactions précoces entre les précurseurs de tendon et les myoblastes, et d’autre part, étudier les mécanismes de différenciation des précurseurs de tendons associés au disque de patte. Dans ce contexte nous avons adapté la méthode GRASP (GFP Reconstitution Across Synaptic Partners) ainsi que l’imagerie en temps réel à notre modèle pour démontrer l’existence des interactions cellulaires entre les précurseurs de tendons et les myoblastes, nous avons aussi mis au point une approche cellule-spécifique afin de trier les précurseurs de tendons et les myoblastes associés au disque de patte, ce qui nous a permis d’obtenir dans un premier temps les données transcriptomiques des précurseurs de tendons. J’ai également étudié l’impact de l’altération des précurseurs de tendon sur le comportement des myoblastes associés et inversement. Nos résultats montrent que l’altération du développement des tendons entraîne une désorganisation spatiale des myoblastes environnants. Dans la seconde partie de mon projet, je me suis intéressée à l’implication de la voie Notch et des gènes de la famille odd-skipped dans la différenciation et la morphogenèse des précurseurs de tendon. J’ai ainsi démontré que Notch est nécessaire et localement suffisant pour induire l’expression de stripe et que les gènes odd-skipped et stripe coopèrent en aval cette voie pour permettre l’invagination et l’élongation sous forme de tube des longs tendons internes de la patte. / The formation of the musculo-(exo)skeletal system in drosophila is a remarkable example of tissue patterning making it a suitable model for studying multiple tissue interactions during development.The aim of our study is to better understand appendicular myogenesis through the identification of early interactions between tendon and muscle precursors, and by investigating the mechanisms governing the specification of tendon cell precursors of the leg disc. In order to characterize the interaction between these two tissues, we adapted the GRASP method (GFP Reconstitution Across Synaptic Partners) and set up live imaging experiments to reveal cellular interactions between tendon precursors and myoblasts. We have also conducted a genome wide cell-specific analysis using Fluorescence-activated cell sorting (FACS) on imaginal discs which allowed us to perform a tendon cell specific transcriptional analysis.To test whether reciprocal muscle-tendon interactions are necessary for correct muscle-tendon development, I performed experiments to specifically interfere with the development of tendon or muscle precursors. By altering tendon precursors formation during the early steps of leg development, we affect the spatial localization of the associated myoblasts. These findings provide the first evidence of the developmental impact of early interactions between muscle and tendon precursors in the leg disc.In the second part of my project, I investigated the role of Notch pathway and odd-skipped genes in the differentiation and morphogenesis of tendon precursors. Thus, I have demonstrated that Notch signalling pathway is necessary and locally sufficient for the initiation of stripe expression, and that both odd-skipped genes and stripe are required downstream of Notch to promote morphological changes associated with formation of long tubular tendons.

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.

The inhibitor of apoptosis (IAP) proteins traditionally regulate programmed cell death by binding to and inhibiting caspases. Recent studies have uncovered a variety of alternate cellular roles for several IAP family members. The cellular inhibitor of apoptosis 1 (cIAP1) protein, for instance, regulates different axes of the NF-κB signalling pathway. Given the extensive functions of NF-κB signalling in muscle differentiation and regeneration, I asked if cIAP1 also plays critical roles in skeletal muscle myogenesis. In a primary myoblast cell-culture system, genetic and pharmacological approaches revealed that loss of cIAP1 dramatically increases the fusion of myoblasts into myotubes. NF-κB signalling occurs along a classical and an alternative pathway, both of which are highly active in cIAP1-/- myoblasts. Suppression of the alternative pathway attenuates myotube fusion in wildtype and cIAP1-/- myoblasts. Conversely, constitutive activation of the alternative pathway increases myoblast fusion in wildtype myoblasts. cIAP1-/- mice have greater muscle weight and size than wildtypes, as well as an increased number of muscle stem cells. These results identify cIAP1 as a regulator of myogenesis through its modulation of classical and alternative NF-κB signalling pathways. Loss of the structural protein dystrophin in the mdx mouse model of Duchenne muscular dystrophy leads to chronic degeneration of skeletal muscle. The muscle pathology is strongly influenced by NF-κB signaling. Given the roles demonstrated for cIAP1 in cell culture and in vivo, I asked whether loss of cIAP1 would influence muscle pathology in the mdx mouse. To address this question, double-mutant mice were bred lacking both cIAP1 and dystrophin (cIAP1-/-;mdx). Histological analyses revealed that double-mutant mice exhibited reduced indications of damage on several measures, as compared to single-mutant (cIAP1+/+;mdx) controls. Unexpectedly, these reductions were seen in the “slow-twitch” soleus muscle but not in the “fast-twitch” extensor digitorum longus (EDL) muscle. The improvements in pathology of double-mutant solei were associated with reductions in muscle infiltration by CD68-expressing macrophages. Finally, the double-mutant mice exhibited improved endurance and resistance to damage during treadmill-running exercise. Taken together, these results suggest that loss of cIAP1, through its multiple regulatory functions, acts to improve myogenesis and increase muscle resistance to damage.

CCAAT/Enhancer Binding Proteins are a family of six bZIP transcription factors. C/EBPβ, the second member cloned, has been implicated in adipogenesis and osteogenesis, but the role of C/EBPβ in myogenesis remained undetermined. In adults, muscle-resident stem cells, called satellite cells (SCs), have the greatest propensity to regenerate the skeletal muscle. We found that C/EBPβ is expressed in SCs, and its expression progressively declines upon differentiation. Forcing the expression of C/EBPβ in myoblasts enhanced the expression of the SC marker Pax7, and repressed MyoD and the myogenic genes expression, resulting in the inhibition of myogenesis. Using a SC-specific conditional knockout (cKO) mouse model, we found that cKO myoblasts have decreased expression of Pax7, and we identified Pax7 as a direct target of C/EBPβ action. In vivo, excision of C/EBPβ resulted in muscle hypertrophy at the juvenile age, and adult cKO animals had enhanced muscle regeneration following BaCl2 muscle injury. Moreover, the number of Pax7+ cells in cKO animals decreased following BaCl2 injury. Upon performing a second injury into cKO animals, we demonstrate a decreased muscle fiber size and an exacerbation of the percentage number of SCs. While cKO animals repaired well a BaCl2 injury, regeneration failed in cKO animals following cardiotoxin (CTX) injury. We demonstrate that IL-1β expression is enhanced in muscle after CTX injury when compared to BaCl2, and we found that IL-1β can stimulate the expression of C/EBPβ in myoblasts. Ectopic C/EBPβ expression can protect myoblasts from apoptosis when triggered with thapsigargin, whereas cKO myoblasts are more sensitive to apoptosis. Using cancer cachexia as a model of chronic inflammation, we found that the expression of C/EBPβ is stimulated in the SCs of cachectic animals, and this correlated with a decrease in regenerative capacity. The severity of muscle wasting was not improved in cKO animals, but rather cKO SCs were lost to apoptosis. Together, this study establishes a protective role for C/EBPβ in muscle SCs in conditions of inflammation.

The transcription factor Six1 of the sine oculis homeobox family has been tied to skeletal muscle formation. Work completed thus far has allowed our research team to identify the precise mechanism by which Six1 regulates the expression of MyoD, a key myogenic gene, in muscle stem cells. Furthermore, loss-of-function of this protein, mediated by RNA interference, has implicated Six1 as essential towards normal myogenic differentiation. However, beyond Six1 and its involvement towards myogenesis, our data also suggests the transcription factor as a potential regulator of the cell cycle. Data from our lab shows that loss of Six1 expression significantly impairs primary myoblast proliferation and appears to impair satellite cell activation in response to muscle injury in vivo. Furthermore, loss of Six1 decreases the expression of key cell cycle genes. Combining functional genomics approaches such as ChIP-Seq and Gene Expression Profiling together with Gene Ontology Term Enrichment shows a significant representation for biological processes regarding the cell cycle and its regulation; these biological clusters contain a large subset of genes that are bound and modulated by Six1. In particular, Ccnd1 was found to display a similar expression pattern as Six1 in growing myoblasts and its expression was found to be directly controlled by Six1. Furthermore, Ccnd1 over-expression was sufficient to rescue the Six1-knockdown associated cell cycle phenotype. Together, these data suggest that in response to injury Six1 enhances the expression of the cell cycle gene Ccnd1 thus modulating myoblast proliferation for muscle regeneration.

Adult skeletal muscles can regenerate after injury due to the presence of satellite cells, a quiescent population of myogenic progenitor cells characterized by expressing the transcription factor Pax7. Once activated, satellite cells repair the muscle damage and replenish the stem cell niche due to the coordinated function of several transcription factors including Pax7 and the myogenic regulatory factors (MRFs). MRFs are skeletal muscle-specific transcription factors that can convert non-muscle cells into the myogenic lineage. MRFs are known to cooperate with other transcription factors in regulating the complex transcriptional network driving myogenic differentiation of muscle progenitors. The Six4 transcription factor emerges as a strong candidate for cooperating with MRFs. Six4 is expressed in skeletal muscles; the lack of a muscle development phenotype in Six4-null mice has been attributed to compensation by other Six family members. However, this did not exclude a critical role for Six4 during muscle development as Six1;Six4 double mutant mice show a more severe muscle phenotype than Six1 mutant mice. Nevertheless, the role of Six4 during adult muscle regeneration has never been addressed. I combined a partial loss-of-function of Six4 with high-throughput approaches to address the role of Six4 during adult skeletal muscle regeneration. I observed an important function of Six4 during muscle regeneration in vivo and in in vitro cell models. Using RNA interference assays against Six4 in tibialis anterior muscle regeneration after cardiotoxin-induced muscle damage, I observed for the first time that Six4 plays a role in proper muscle regeneration. The ability of the MRF MyoD, a central regulator of skeletal myogenesis, to convert a non-muscle cell model into the myogenic lineage was impaired with attenuated Six4 expression. I employed genome-wide approaches by combining ChIP-sequencing with gene expression profiling and identified a set of muscle genes coordinately regulated by both Six4 and MyoD. Throughout the genome, the cooperation between Six4 and MyoD was associated with binding of the H3K27me3 demethylase Utx and depletion of the H3K27me3 repressive chromatin mark. Together, these results reveal an important role for Six4 during adult muscle regeneration, and suggest a widespread mechanism of cooperation between Six4 and MyoD that correlates with modifying the epigenetic landscape of the regulatory regions of a large set of genes needed for efficient myogenesis.

The post-natal regenerative capacity of skeletal muscle is attributed to myogenic satellite cells, which function as lineage-committed precursors to replace terminally differentiated muscle. The development and differentiation of the satellite cell lineage is regulated by Pax7 and the myogenic regulatory factors. While the expression of Pax7 is vital to the function of the satellite cell compartment, the paired domain alternative splicing events that regulate its DNA binding potential remain elusive. Interestingly, the generation of Pax7 splice variants differentially regulate Myf5 expression. We performed a global analysis of two Pax7 isoforms, which differ by a glycine-leucine dipeptide, to determine how paired domain splicing events modify the ability of Pax7 to regulate target genes. To this end, we observe that although the homeodomain is important for Pax7 binding, these isoform differences in the paired domain can regulate Pax7 targets during myogenesis. In addition to further examining the role of Pax7 during satellite cell proliferation and maintenance, it remains important to understand their downstream differentiation potential. Since activation of the canonical Wnt signalling pathway results in reduced regenerative efficiency in vivo, we undertook a global analysis of satellite cell derived myoblasts to examine their ability to respond to canonical Wnt signalling. We demonstrate that Wnt/β-Catenin signalling drives myogenic differentiation, via the myogenin-dependent control of follistatin expression, further fine-tuning the myogenic differentiation process. The effects of canonical Wnt signalling on myogenic differentiation complement our observations regarding Pax7 alternative splicing during myoblast proliferation and provide a greater comprehensive understanding of the molecular regulation of satellite cell development and differentiation during adult myogenesis.

Growth hormone (GH) has growth-stimulating effects on skeletal muscle and liver but a growth-inhibitory effect on adipose tissue. The mechanisms underlying these actions of GH are not fully understood. Two studies were conducted to achieve the following objectives: 1) to determine the cellular mechanism by which GH stimulates liver growth; 2) to determine the effects of GH on the commitment of mesenchymal stem cells (MSCs) to myogenic and adipogenic lineages. In the first study, the GH-deficient lit/lit male mice were injected (s.c.) daily with rbGH or vehicle for two weeks. GH-injected lit/lit mice tended to have a greater liver/body weight percentage than lit/lit control mice. GH injection did not alter the percentage of proliferating cells in the liver. However, GH-injected lit/lit mice had 18% larger hepatocytes and 16% less DNA per unit liver weight than those of lit/lit control mice. These data together indicate that GH stimulates liver growth in mice by increasing the size, not by increasing the number of hepatocytes. In the second study, we treated the MSC cell line C3H10T1/2 cells with or without 5'-azacytidine and rbGH for 4 days. We assessed the myogenic or adipogenic potential by determining the ability of these cells to differentiate into myotubes or adipocytes, respectively. C3H10T1/2 cells treated with 5'-azacytidine and GH formed more myotubes, myoblasts, and fewer adipocytes compared to cells treated with 5'-azacytidine alone. Taken together, these results suggest that GH enhances 5'-azacytidine-induced myogenic commitment but inhibits 5'-azacytidine-induced adipogenic commitment in C3H10T1/2 cells. / Master of Science

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