Muscle development involves concerted action of a repertoire of mechanisms governing myoblast proliferation, migration, fusion and differentiation. Subsequently, there are cellular events administrating proper muscle function and maintenance of muscle integrity. Chapter 1 covers what is known about muscle development, building up of mass and maintenance in vertebrates and Drosophila, highlighting the myogenic programs and factors that play a role in them. The formation of vertebrate skeletal muscles can be recapitulated in Drosophila indirect flight muscles (IFMs), making IFMs an interesting model to dissect and understand the mechanisms of muscle development and maintenance. The cellular and developmental events that occur during IFM development have been discussed in detail along with their genetic control which encompasses both cell autonomous and cell non-autonomous mechanisms. The fly resources and tools used for experimentations have been described in Chapter 2. One of the hallmark events during muscle development is myoblast fusion. Myoblasts are kept in undifferentiated state until they fuse through a balanced action of anti-differentiation and pro-differentiation factors. The swarming myoblasts are in semi-differentiated state and just prior to fusion should exit cell cycle to achieve terminal differentiation. The mechanisms of cyclin/CDK complexes and their regulation via CKI (CDK inhibitor) are known in a cell. However, tissue specific factors exerting additional control on molecules that participate in cell cycle have been proposed but have not been shown in vivo. Chapter 3 uncovers a novel role played by the transcription factor, Erect wing (Ewg) in IFM development and patterning. Despite the fact that Ewg is known to express in fusing myoblasts and nuclei of developing IFMs and has long been used as a nuclear marker for IFMs, the mechanism(s) behind Ewg‟s function has remained enigmatic. Historical perspective of Ewg has been presented in Chapter 1. One set of IFMs; dorsal longitudinal muscles (DLMs) require larval templates for their formation and the other set; dorsal ventral muscles (DVMs) form de novo. Chapter 3 shows that Ewg is required in a spatio-temporal fashion to initiate myoblast fusion process. In the absence of Ewg, the number of fusion events in a given time is reduced. In addition de novo fusion is observed in the region of DLM development just like DVM and overall IFM development is delayed resulting in an aberrant adult IFM pattern. Genetic studies undertaken reveal a requirement for Ewg in exerting a temporal control on myoblast fusion. This is achieved by down-regulating Cyclin E levels, as a result of which the myoblasts exit cell cycle at G1/S stage. Through this study the proposal for DLM development and pattern has been put forth as follows: i) appropriate progression of DLM development commences on synchronous exit of myoblasts from cell cycle. This function is facilitated by Ewg expression in fusing myoblasts assisting symmetrical DLM formation in hemithoraces. ii) DLM pattern of six muscles in each hemithorax is dependent on template survival which requires fusion of enough myoblasts and further subsequent fusion events to support the splitting of three larval templates or presumptive DLM. The muscles that develop should preserve their structural integrity for efficient functional output. Muscles perform extensive activities warranting high energy requirements. IFMs are widely utilised for thorax movements that aid flight. IFMs are exclusively oxidative in nature with upto 40% mass contributed by large mitochondria themselves. Chapter 4 describes yet another novel finding for Ewg function in IFM maintenance. Vertebrate homolog of Ewg is nuclear respiratory factor 1 (NRF1) known for its role in mitochondrial biogenesis. This prompted an investigation on the role of Ewg, if any, in mitochondrial function and IFM maintenance. In this chapter, Ewg null adult IFMs are shown to undergo degeneration. Mitochondria in these muscles show rounder and smaller phenotype. Mitochondria morphology is traced throughout Drosophila pupal DLM development and extensive fusion is observed in last one-fourth of pupal phase. In Ewg null condition transcripts of Opa1-like required for inner mitochondrial membrane fusion is found to be absent, suggesting lack of mitochondrial fusion behind the smaller mitochondrial morphology. This emerged as an intriguing problem since Ewg expression follows until sarcomerogenesis (formation of sarcomeres) initiates at mid pupal stage. Developmentally extending Ewg‟s expression beyond mid pupal stage is not observed to increase Opa1-like levels pointing an indirect regulation by Ewg. However, Opa1-like knock-down beyond mid pupal stage is not observed to result in any muscle or flight defect. It is thus proposed that Ewg expression early during muscle development helps to up-regulate Opa1-like levels needed to support mitochondrial growth and fusion. In addition, this chapter provides additional data on requirement of Opa1-like for maintenance of mitochondrial as well as muscle integrity. This is the first ever report of tissue specific temporal regulation of Opa1-like by Ewg. Chapter 3 and Chapter 4 conclude spatially segregated functional requirements of Ewg which are also mechanistically distinct. Expression in fusing myoblasts channelizes fusing myoblasts to exit cell cycle and undergo timely fusion saving the larval template, subsequent fusion assists template splitting thus forming the appropriate adult DLM pattern. On the other hand expression until mid pupal stage up-regulates Opa1-like expression, facilitating mitochondrial fusion during the late pupal stage. This as a result helps maintain structural integrity of muscles in the adult. Vertebrate skeletal muscle contains multiple muscle fibres that provide appropriate mass and size to muscles. As DLM share similarity in development to that of the vertebrate skeletal muscle, DLM organisation is studied to get insights into the mechanisms which regulate the process. Chapter 5 discusses role of nuclear number and nuclear activity in determining the DLM organisation. In order to alter nuclear number, myoblast population is reduced to amounts lesser than that of the wild type and to alter nuclear activity, two nuclear encoded genes Opa1-like and Marf , involved in inner and outer mitochondrial membrane fusion respectively have been knocked down in IFMs. First, the DLM organisation is established by comparing it to the vertebrate skeletal muscle organisation. This organisation is affected on lowering the number of myoblasts destined to fuse and form IFMs, without affecting the differentiation. On the other hand, when nuclear encoded mitochondrial fusion genes are knocked down, not only DLM organisation but their differentiation is also affected. A proposal for achieving DLM organisation has been presented which should also apply to vertebrate skeletal muscle given their developmental similarity. In conclusion, the studies decipher a novel mechanism by which a transcription factor, Ewg exerts a temporal control on myoblast fusions directly influencing progression of DLM formation, and thereby, symmetry and pattern. Moreover, Ewg is also shown here to regulate mitochondrial fusion during later pupal stages helping muscles to attain greater function and maintain structural integrity. Discovery of such regulatory mechanisms controlling mitochondrial dynamics in vertebrates can open up new avenues to understand and design new therapeutic approaches to tackle mitochondrial diseases. Additionally, myoblast fusion and hence myonuclear number and their efficient functioning are shown to be important determinants of muscle organisation. This has further implications in understanding and using stem cell science in dystrophic or atrophic or ageing related muscle loss and therapy.

骨骼肌分化是由一个包括转录因子、表观遗传调控子和非编码RNA在内的复杂网络共同调控的。YY1能够通过募集PRC2抑制一系列肌肉结构基因的表达，进而抑制肌肉分化。miRNA是一组转录后调控基因表达的小片段非编码RNA，miRNA与转录因子的相互作用已经被广泛证实。在本次研究中，我们证实了一个YY1和肌肉特异性miRNA（miR-1,miR-133和miR-206）的调控回路。实验证实，YY1通过肌肉特异性miRNA增强子区域的YY1结合位点募集PRC2来抑制肌肉特异性miRNA的表达。YY1调控miR-1在体外和体内肌肉分化均被证实有重要意义。另外，我们还证实miR-1能够负反馈作用于YY1，抑制YY1的表达。 / 为了阐述YY1在基因组转录中的作用，我们做了肌肉中YY1的ChIP-seq。测序结果表明在C2C12肌肉母细胞中有1820个YY1结合位点，其中很大部分位于基因间的区域。进一步研究发现，基因间YY1的结合可能调控一些lincRNA，而这些lincRNA在肌肉发育的作用目前尚不清楚。进一步研究这些可能受YY1调节的lincRNA，我们证实了YY1能够正调控两个新的lincRNA，YAM-1和YAM-2。YAM-1在肌肉分化过程中逐渐下调，并且通过正调控他的临近基因miR-715，抑制肌肉分化，而YAM-2能够促进早期的肌肉分化。 / 总之，我们第一次在肌肉细胞中进行了YY1的ChIP-seq，并且证实在肌肉分化过程中转录因子和非编码RNA相互作用的重要性和普遍性。 / Skeletal muscle cell differentiation is a process orchestrated by a complex network of transcription factors, epigenetic regulators and non-coding RNAs. As a repressor of myogenesis, Yin Yang 1 (YY1) silences a number of muscle structural genes through recruiting Polycomb repressive complex2 (PRC2) in proliferating myoblasts. microRNAs (miRNAs) are small non-coding RNAs that regulate gene expression post-transcriptionally, and mounting evidences support the prevalence and functional significance of their interplay with transcription factors (TFs). Here we describe the identification of a regulatory circuit between muscle miRNAs (miR-1, miR-133 and miR-206) and Yin Yang 1 (YY1). The subsequent experimental results demonstrate that YY1 indeed represses muscle miRs expression in myoblasts and the repression is mediated through multiple enhancers and recruitment of Polycomb complex to several YY1 binding sites. YY1 regulating miR-1 is functionally important for both in vitro and in vivo myogenesis. Furthermore, we demonstrate that miR-1 in turn targets YY1, thus forming a negative feedback loop. / To elucidate its role on genome-wide regulation of transcription, here in the second part of this study we performed ChIP-Seq for YY1 in muscle cells. Our results revealed 1820 YY1 binding peaks genome-wide in myoblasts, with a large portion residing in the intergenic region. A close analysis of the intergenic region bound by YY1 uncovered that YY1 may regulate a large number of lincRNAs (Long Intergenic non-coding RNAs), whose roles in skeletal myogenesis have not been explored yet. As further elucidation of the functional roles of YY1-lincRNA regulation, we identified two novel lincRNAs, YAM-1 and YAM-2 as positively regulated by YY1. YAM-1 was found to be down-regulated upon myogenic differentiation and acts as an inhibitor of myoblast differentiation. We further demonstrated that YAM-1 functions by its in cis regulation on a downstream gene, miR-715 which promotes differentiation. YAM-2, on the other hand, appears to promote myogenesis. / Together, our studies not only provide the first genome-wide picture of YY1 association in muscle cells but also uncovered novel regulatory circuits required for skeletal myogenesis and reinforce the idea that regulatory circuitry involving non-coding RNAs and TFs is essential components of myogenic regulatory network. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Lu, Leina. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 144-167). / Abstract also in Chinese. / Abstract / 摘要 / Acknowledgement / Publications / List of figures / List of tables / Abbreviations / Table of content / Chapter Chapter 1: --- INTRODUCTION / Chapter 1.1 --- Skeletal Myogenesis --- p.1 / Chapter 1.2 --- Transcriptional Regulation of myogenic differentiation --- p.3 / Chapter 1.2.1 --- Transcriptional regulatory network in myogenic differentiation --- p.3 / Chapter 1.2.2 --- YY1 as a transcription factor in myogenic differentiation --- p.5 / Chapter 1.3 --- Epigenetic Regulation during skeletal muscle differentiation --- p.6 / Chapter 1.4 --- microRNA: Post-transcriptional regulation on myogenic differentiation --- p.11 / Chapter 1.4.1 --- Muscle specific miRNAs in skeletal myogenic differentiation --- p.15 / Chapter 1.4.2 --- Non-muscle specific miRNAs in skeletal myogenic differentiation --- p.20 / Chapter 1.4.3 --- miRNAs and skeletal muscle diseases --- p.23 / Chapter 1.5 --- Long Non-coding RNAs --- p.26 / Chapter 1.5.1 --- Long Non-coding RNAs and lincRNAs --- p.26 / Chapter 1.5.2 --- LincRNAs in muscles --- p.30 / Chapter Chapter 2: --- MATERIALS AND METHODS / Chapter 2.1 --- C2C12 cell line --- p.32 / Chapter 2.2 --- Primary Myoblast isolation and in vitro culture --- p.32 / Chapter 2.3 --- Animal studies --- p.33 / Chapter 2.4 --- RNA extraction --- p.34 / Chapter 2.5 --- RT-PCR and Real-Time RT-PCR --- p.35 / Chapter 2.6 --- Transfection and infection --- p.37 / Chapter 2.7 --- Oligonucleotides --- p.38 / Chapter 2.8 --- Dual-luciferase reporter assay --- p.43 / Chapter 2.9 --- Immunofluorencence staining --- p.44 / Chapter 2.10 --- Antibodies --- p.45 / Chapter 2.11 --- Protein extraction and Western blotting --- p.46 / Chapter 2.12 --- DNA constructs --- p.48 / Chapter 2.13 --- Mutagenesis --- p.49 / Chapter 2.14 --- RNA-Fluorescence In Situ Hybridization (RNA-FISH) --- p.51 / Chapter 2.15 --- C2C12 cells with YY1-stably knocked down --- p.52 / Chapter 2.16 --- Rapid Amplification of cDNA Ends (RACE) --- p.53 / Chapter 2.17 --- Chromatin Immunoprecipitation (ChIP) --- p.55 / Chapter 2.18 --- ChIP-PCR --- p.58 / Chapter 2.19 --- ChIP-sequencing --- p.58 / Chapter 2.20 --- Northern blotting --- p.59 / Chapter 2.21 --- Prediction of miRNA targets --- p.60 / Chapter 2.22 --- Statistical analysis --- p.60 / Chapter Chapter 3: --- Results / Chapter 3.1 --- YY1-miR-1/133 regulatory circuitry in skeletal myogenesis --- p.61 / Chapter 3.1.1 --- YY1 decreases miR-1/133 during skeletal muscle differentiation --- p.61 / Chapter 3.1.1.1 --- Negative correlation between YY1 and miR-1/133 during C2C12 differentiation --- p.61 / Chapter 3.1.1.2 --- Negative correlation between YY1 and miR-1/133 in primary cell differentiation --- p.63 / Chapter 3.1.1.3 --- Negative correlation between YY1 and miR-1/133 in postnatal muscle development and mdx mouse model --- p.65 / Chapter 3.1.1.4 --- Deletion of YY1 upregulates miR-1/133 both in C1C12 and primary myoblast --- p.68 / Chapter 3.1.1.5 --- Deletion of YY1 upregulates miR-1/133 at the transcriptional level --- p.70 / Chapter 3.1.2 --- YY1 represses miR-1/133 by binding to 4 enhancers --- p.72 / Chapter 3.1.2.1 --- Four enhancers of miR-1/133 with potential YY1 targeting sites --- p.72 / Chapter 3.1.2.2 --- YY1 represses the four enhancers’ activities --- p.75 / Chapter 3.1.2.3 --- Depletion of YY1 up-regulates the four enhancers’ activities --- p.77 / Chapter 3.1.2.4 --- YY1 directly binds to the putative binding sites and mediates the repression on miR-1/133 --- p.79 / Chapter 3.1.2.5 --- YY1 recruits Ezh2 to the enhancers which subsequently causes histone modification --- p.82 / Chapter 3.1.3 --- YY1 repressing miR-1/133 is functionally significant in myogenesis --- p.84 / Chapter 3.1.3.1 --- Negative correlation between YY1 and miR-1/133 in CTX induced muscle regeneration model --- p.84 / Chapter 3.1.3.2 --- Depletion of YY1 in CTX induced muscle regeneration model promotes miR-1/133 expression --- p.87 / Chapter 3.1.3.3 --- Depletion of YY1 in CTX induced muscle regeneration model promotes muscle differentiation --- p.89 / Chapter 3.1.4 --- miR-1 can target YY1 forming a feedback loop --- p.92 / Chapter 3.1.5 --- miR-1 can repress Pax7 by targeting two binding sites on 3’UTR --- p.95 / Chapter 3.1.5.1 --- miR-1 targets Pax7 by binding to two target sites --- p.95 / Chapter 3.1.5.2 --- miR-1 represses Pax7 forming an YY1-miR-1-Pax7 regulating circuitry in skeletal myogenesis --- p.98 / Chapter 3.1.6 --- Conclusion: YY1-miR-1-Pax7 regulatory circuitry in skeletal myogenesis --- p.100 / Chapter 3.2 --- ChIP-seq reveals YY1-lincRNA regulation in skeletal myogenesis --- p.102 / Chapter 3.2.1 --- ChIP-seq uncovered a large number of genes under YY1 regulation --- p.102 / Chapter 3.2.2 --- ChIP-seq reveals that YY1 associates with lincRNA loci --- p.105 / Chapter 3.2.2.1 --- YY1 associates with lincRNA-YAM loci --- p.105 / Chapter 3.2.2.2 --- YY1 positively regulates YAM-1 and YAM-2 both in vitro and in vivo --- p.107 / Chapter 3.2.3 --- YY1-YAM-1-miR-715 regulatory pathway in muscle differentiation --- p.109 / Chapter 3.2.3.1 --- Genomic organization and cellular localization of YAM-1 --- p.109 / Chapter 3.2.3.2 --- Expression of YAM-1 decreases during myogenic differentiation --- p.112 / Chapter 3.2.3.3 --- YAM-1 represses myogenic differentiation both in vitro and in vivo --- p.115 / Chapter 3.2.3.3.1 --- YAM-1 inhibits C2C12 differentiation --- p.115 / Chapter 3.2.3.3.2 --- YAM-1 inhibits muscle differentiation in vivo --- p.117 / Chapter 3.2.3.4 --- A functional YY1-YAM-1-miR-715 regulatory axis in skeletal myogenic differentiation --- p.119 / Chapter 3.2.3.4.1 --- miR-715 is down-regulated during muscle differentiation --- p.119 / Chapter 3.2.3.4.2 --- miR-715 is under the regulation of YY1-YAM-1 --- p.122 / Chapter 3.2.3.4.3 --- miR-715 represses muscle differentiation forming a YAM-1-miR-715 regulatory axis during muscle differentiation --- p.124 / Chapter 3.2.4 --- YAM-2 promotes early myogenic differentiation --- p.126 / Chapter 3.2.4.1 --- Genomic organization and cellular localization of YAM-2 --- p.126 / Chapter 3.2.4.2 --- YAM-2 is regulated during myogenic differentiation --- p.129 / Chapter 3.2.4.3 --- YAM-2 promotes early myogenic differentiation --- p.131 / Chapter Chapter 4: --- DISCUSSION / Chapter 4.1. --- YY1-miRNA regulatory circuit in skeletal myogenesis --- p.133 / Chapter 4.2 --- YY1 mediates epigenetic modification in skeletal myogenesis --- p.135 / Chapter 4.3 --- miRNAs in skeletal myogenesis --- p.136 / Chapter 4.4 --- YY1 regulates long intergenic non-coding RNAs in skeletal myogenesis --- p.138 / Chapter Chapter --- 5: SUMMARY AND FUTURE WORK --- p.142 / REFERENCE --- p.144

Thesis (Ph.D.)--University of Alabama at Birmingham, 2007. / Title from PDF title page (viewed on Feb. 18, 2010). Includes bibliographical references.

Having an in vivo model of development that develops quickly and efficiently is important for investigators to elucidate the critical steps, components and signaling pathways involved in building a myofibril; hence a compliant in vivo model would provide a pivotal foundation for deciphering muscle disease mechanisms as well as the development of myopathy-related therapeutics. Here, we take advantage of a relatively quick, cost effective, and molecularly pliable developmental model system in the Xenopus laevis (frog) embryo and establish it as an in vivo model to study the roles of sarcomeric proteins during de novo myofibrillogenesis.Using the Xenopus model, we elucidated the functional roles of Leiomodin3 (Lmod3) and Tropomodulin 4 (Tmod4) during de novo skeletal myofibrillogenesis. Tmods have been demonstrated to contribute to thin filament length uniformity by regulating both elongation and depolymerization of actin-thin filaments' pointed-ends. Lmods, which are structurally related to Tmod proteins also localize to actin filament pointed-ends. In situ hybridization studies demonstrated that of their respective families, only tmod4 and lmod3 transcripts were expressed at high levels in skeletal muscle from the earliest stages of development. When reducing their protein levels via morpholino (MO) treatment, thin filament regulation and sarcomere assembly were compromised. Surprisingly, alternate rescues (i.e., lmod3 mRNA co-injected with Tmod4 MO and vice versa) partially restored myofibril structure and actin-thin filament organization. Thus, our results not only indicate that both Tmod4 and Lmod3 are critical for myofibrillogenesis during Xenopus skeletal muscle development, but also revealed that they may share redundant functions during skeletal muscle thin filament assembly.

Thesis (Ph. D.)--Cornell University, August, 2008. / Vita. Includes bibliographical references (leaves 290-306).

Thesis (Ph. D.)--University of Washington, 2004. / Vita. Includes bibliographical references (leaves 84-92).

Thesis (Ph. D.) -- University of Texas Southwestern Medical Center at Dallas, 2001. / Vita. Bibliography: 91-102.

Thesis (Ph. D.)--University of Washington, 2004. / Vita. Includes bibliographical references (leaves 70-79).

Thesis (Ph.D.) -- University of Texas Southwestern Medical Center at Dallas, 2005. / Embargoed. Vita. Bibliography: 170-172.

Thesis (Ph.D.) -- University of Texas Southwestern Medical Center at Dallas, 2005. / Embargoed. Vita. Bibliography: References located at the end of each chapter.