Thesis (Ph. D.)--Case Western Reserve University, 2008. / [School of Medicine] Department of Biochemistry. Includes bibliographical references.

No description available.

Although much work has been carried out to identify the mechanisms by which muscle is formed, many of the regulatory pathways involved have yet to be fully elucidated. In creating perturbations during the embryonic period, either nutritionally (with a marginal vitamin A deficiency model) or pharmacologically (with the b₂-adrenerguic agonist clenbuterol), a comparison with 'normal' muscle development may be attained. Differences in the temporal expression of specific regulatory proteins may then enhance the existing knowledge of their function in regulating muscle development. Prior to studying changes in muscle regulatory proteins due to perturbations, it was first necessary to illustrate their temporal pattern in "normal" muscle development. The results indicated that a complex regulatory system operates in myogenesis with a number of proteins appearing to be involved in the process of muscle development. A marginal vitamin A deficiency model was established in which maternal retinol levels were clearly reduced in treatment animals in comparison with controls. This resulted in offspring that showed clearly symptoms of marginal vitamin A deficiency. Changes in the abundance of five proteins were observed in response to marginal vitamin A deficiency. Overall, these changes suggested a potential reduction in secondary myogenesis, based on reduced levels of MHCfast, associated with secondary fibres, following birth. Analysis of RNA, DNA and protein values suggested that neonates from clenbuterol fed dams may have reduced hyperplasia and/or increased hypertrophy. Biochemical analysis revealed that proteins such as GATA-2, PKC and Shh, which have previously been associated with hypertrophy, were altered in response to clenbuterol. Further evidence in support of hypertrophy was indicated in an apparent increase in fibre size of neonates detected by MHC immunolocalisation. In conclusion, it has been demonstrated that both nutritional and pharmacological manipulations throughout are gestation capable of altering myogeneiss <i>in utero</i> by two different mechanisms.

Thesis (Ph. D.)--University of Virginia, 1999. / Spine title: SNON modulates gene activity. Includes bibliographical references (p. 208-232). Also available online through Digital Dissertations.

In the UK annual lamb mortality rates range between 10-30% with the majority of deaths occurring within the first three days of life, however research has shown that lambs that stand and suck quickly are more likely to survive. Modern breeding strategies have led to breeds of sheep, such as the Suffolk, which despite greater lean muscle growth, show slower behavioural development and are less able to thermoregulate in the neonatal period than relatively unselected breeds, such as the Scottish Blackface. The reason for this is unclear however fast growing strains of pigs and cattle have been shown to have a greater proportion of fast-twitch fibres to slow fibres in their muscles, but it is still unknown as to whether these changes in fibre proportions affect muscle function and behaviour. Thus the aim of this project is to investigate whether selection for lean muscle growth in sheep has altered muscle development by affecting the proportions of different fibre types and determine what effect this may have on the animal’s ability to perform certain behaviours, such as neonatal progression to standing and sucking. As muscle fibre development occurs very early in gestation (starting around day 32) it was also hypothesised that there may also be a relationship between muscle fibre characteristics, and foetal behaviour and presentation at birth. A further hypothesis to be addressed was that maternal undernutrition of 75% of requirements for ewe maintenance and foetal growth for the first 90 days of gestation would have a greater negative effect on muscle fibre development in genotypes selected for lean muscle growth. It was found that Suffolk foetuses (genotype selected for lean growth) were significantly less active at days 56 and 77 of gestation than Blackface foetuses (genotype relatively unselected for growth) while nutritionally restricted foetuses were more active at day 56 than control foetuses. A subsequent study found that there was a negative relationship between foetal activity at day 56 and neonatal activity while activity at day 98 of gestation was positively associated with neonatal activity. A relationship was also found between foetal activity and presentation at birth with malpresented lambs being less active as foetuses at day 77 of gestation than normally presented lambs. Suffolk foetuses had lower proportions of slow twitch (SO) fibres and higher proportions of fast-oxidative-glycolytic (FOG) fibres in the soleus (postural muscle) than Blackface foetuses and SO fibre proportions were positively correlated with foetal activity at days 56 and 77 of gestation while fast twitch (FOG and FO) and transitional (Trans) fibre proportions were negatively correlated with foetal activity at each scanning period. Suffolk lambs showed significantly slower neonatal behavioural development than Blackface lambs and there was an interaction between breed and nutritional treatment with prenatally undernourished Suffolk lambs consistently being less active and prenatally undernourished Blackface lambs being more active than all other groups of lambs. At slaughter at 164 days old, Suffolk lambs had lower proportions of SO fibres and higher proportions of fastglycolytic (FG) fibres in the soleus muscle while also having a lower proportion of FG fibres in the plantaris (muscle involved in movement of the limb) than Blackface lambs. SO fibre proportions in the soleus muscle were found to be positively correlated with total duration standing and walking in the early neonatal period while proportion of FG fibres in the plantaris was negatively correlated with duration of lying laterally following birth. The results from this study indicate that divergent breeding strategies have led to differences in muscle fibre proportions within certain muscles in sheep and also that there may be a relationship between muscle development and both foetal and neonatal lamb behaviour. This research has added to our understanding of the consequences of selection for growth on the function of the animal and it is hoped that it will lead to the development of broader breeding goals which incorporate welfare characteristics.

Thesis (Ph. D.)--University of Washington, 2007. / Vita. Includes bibliographical references (leaves 188-204).

Thesis advisor: Eric S. Folker / Skeletal muscle is a syncytial cell type in which the multiple nuclei are evenly spaced along the cell periphery. During muscle development, the myonuclei undergo an elaborate set of movements to achieve this precise positioning throughout the muscle. The importance of proper nuclear positioning is highlighted by the correlation between mispositioned nuclei and muscle disease. However, the mechanisms that govern this energetically expensive process as well as the influence nuclear positioning has on muscle cell function remains to be elucidated. The goal of this thesis is to determine the molecular factors and subsequent mechanisms that regulate nuclear movement and how such pathways are disrupted in various muscle diseases. Since many of the key cellular features are conserved between Drosophila and mammalian muscles, we utilize Drosophila musculature as a model system to study myonuclear positioning during muscle development. In this thesis, we provide the first evidence that nuclei experience attractive and repulsive interactions with one another as they actively migrate. Furthermore, we demonstrate that these nucleus-nucleus interactions are critical for proper nuclear positioning, and that they are distinctly regulated by genes that are associated with two different muscle diseases, Emery-Dreifuss muscular dystrophy and Centronuclear myopathy (Chapter 2). We then elaborate upon the genetic mechanisms through which CNM-linked genes regulate nuclear positioning (Chapter 3). Finally, we show that proper nuclear movement requires both the separation of nuclei from their neighbors as well as the transmission of force, that is generated from the cytoskeleton, to move nuclei within the cell (Chapter 4). Together, the work presented in this thesis provides new perspective and mechanistic insights into the genetic factors and physical forces that regulate nuclear movement during muscle development and how such pathways are disrupted in disease, while emphasizing the importance of studying such dynamic processes within an in vivo system. / Thesis (PhD) — Boston College, 2020. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

Skeletal muscle development and growth involve significant changes in gene expression. The overall objective of this dissertation project was to identify transcription factors, enhancers, and mechanisms that control gene expression during skeletal muscle development and growth on a genome-wide scale. Three independent studies were conducted in this project. The objective of the first study was to identify potentially novel mechanisms that mediate myoblast differentiation, a process whereby the mononuclear muscle precursor cells myoblasts express skeletal muscle-specific genes and fuse with each other to form multinucleated myotubes. Comparing gene expression profiles in C2C12 cells, a widely used model of myoblasts, before and 6 days after induced myogenic differentiation by RNA sequencing (RNA-seq) revealed 11,046 differentially expressed genes, of which 5,615 and 5,431 were upregulated and downregulated, respectively. Functional enrichment analyses revealed that the upregulated genes were associated with biological processes or cellular components such as skeletal muscle contraction, autophagy, and sarcomere. In contrast, the downregulated genes were associated with biological processes or cellular components such as ribonucleoprotein complex biogenesis, mRNA processing, and ribosome. Western blot analyses showed an increased conversion of LC3-I to LC3-II protein during myoblast differentiation, further demonstrating the upregulation of autophagy during myoblast differentiation. Blocking the autophagic flux in C2C12 cells with chloroquine inhibited the expression of skeletal muscle-specific genes and the formation of myotubes, confirming a positive role of autophagy in myoblast differentiation and fusion. The aim of the second study was to identify enhancers and transcription factors that regulate gene expression during the differentiation of bovine satellite cells, which are the myogenic precursor cells in adult skeletal muscle, into myotubes. In this study chromatin immunoprecipitation followed by sequencing (ChIP-seq) was used to identify active enhancers, i.e., genomic regions marked with histone modification H3K27ac (acetylation of lysine 27 of H3 histone protein). 19,027 and 47,669 H3K27ac-marked enhancers were identified from undifferentiated and differentiating bovine satellite cells, respectively. Of these enhancers, 5,882 and 35,723were specific to undifferentiated and differentiating bovine satellite cells, respectively while 13,199 were shared by both undifferentiated and differentiating bovine satellite cells. Many of the H3K27ac-marked enhancers specific to differentiating bovine satellite cells were associated with muscle structure and development genes and were enriched with binding sites for MyoD, AP-1, AP-4, KLF, TEAD, and MEF2 transcription factors. Through siRNA-mediated knockdown, AP-4 was found to be essential for differentiation of bovine satellite cells into myotubes. The objective of the third study was to identify enhancers and transcription factors that control differential gene expression in skeletal muscle between neonatal and adult cattle. First, RNA-seq was performed to compare gene expression profiles in skeletal muscle between neonatal calves and adult steers. This analysis identified 924 genes downregulated and 1,021 upregulated from calf to steer muscle. Among genes downregulated in steer muscle were myosin heavy chain3 (MYH3) and MYH8, and among genes upregulated in steer muscle were MYH7 and myoglobin. Surprisingly, many so-called adult muscle genes, such as MYH1 and MYH2, were not differentially expressed between calf and steer muscle. Gene ontology analyses showed that many genes downregulated in steer muscle are involved in protein synthesis and glycolysis and that many genes upregulated in steer muscle function in blood vessel development and immune cell activation. Next, ChIP-seq was performed to identify genomic regions marked with H3K27ac, i.e., active enhancers, in the skeletal muscle of neonatal calves and adult steers. This experiment led to the finding of 20,163 enhancers specifically active in the calf muscle, 14,909 enhancers specifically active in the steer muscle, and 27,002 enhancers active in both the calf and steer muscle. Motif enrichment analyses revealed the enrichment of binding sites for the KLF family and TEAD family transcription factors in enhancers active specifically in the calf muscle, the enrichment of binding sites for the FOXO family and the SMAD family transcription factors in enhancers specifically active in the steer muscle, and the enrichment of binding sites for the MRF family and MEF2 family transcription factors in enhancers active in both the calf and steer muscle. . These results shed light on the differences in gene expression and biology between newborn calf and adult steer skeletal muscle. These results also shed light on the enhancers and transcription factors that control these differences. / Doctor of Philosophy / Muscle is the central part of meat. So, to improve meat yield, it is essential to know how muscle development is controlled. Muscle development, also called myogenesis, starts with muscle progenitor cells developing into myoblasts. Myoblasts then differentiate and fuse with each other to form myotubes. Myotubes undergo hypertrophy and form functional muscle fibers. During myogenesis, each step involves significant changes in gene expression. Gene expression is controlled mainly by proteins called transcription factors. The overall goal of this project was to identify transcription factors and DNA sequences bound by these factors that control gene expression during muscle development. This project consisted of three studies. In the first study, we used the RNA sequencing (RNA-seq) technique to find genes differentially expressed in myoblasts between before and after terminal differentiation. Analyzing the RNA-seq data led to the discovery that autophagy, a 'self-eating' biological process, is required for myoblast differentiation. In the second study, we used a technique called chromatin immunoprecipitation followed by sequencing (ChIP-seq) to identify genomic regions called active enhancers in differentiating bovine myoblasts. This work led to the identification of thousands of active enhancers and dozens of transcription factors binding to these genomic regions that control the differentiation of bovine myoblasts. In the third study, we combined RNA-seq and ChIP-seq to explore the genes and genomic regions controlling muscle transition from newborn calves to adult cattle. This part of the project led to the finding of thousands of genes differentially expressed and thousands of genomic regions differentially activated between newborn calf and adult steer muscle.

Hedgehog (Hh) signalling from the notochord induces the slow muscle cell fate in the adaxial cells of the developing zebrafish embryo. Slow muscle formation is disrupted in zebrafish ??you-type?? mutants resulting in U-shaped somites. In many you-type mutants, genes encoding components of the Hh signalling pathway are mutated. scube2, a gene not previously known to be involved in Hh signalling, is disrupted in the you-type mutant ??you??. you mutants are deficient in several Hh dependent cell types and show decreased expression of Hh target genes. The Scube (signal peptide-CUB domain-EGF-related) family of proteins act as secreted glycoproteins or cell-surface proteins and are thought to be involved in protein-protein interactions and ligand binding. At the protein level, the Scube family resembles the endocytic receptor Cubilin. Cubilin is known to interact with another endocytic receptor Megalin, which can function as an endocytic receptor for Sonic Hedgehog (SHH) in vitro. Megalin endocytosis of Shh may be an important part of the Hh signal transduction pathway. An anti-Scube2 antibody was developed during this work to investigate the intracellular localization pattern of Scube2 and facilitate the identification of potential Scube2 binding partner(s). In addition, this work identified and characterized two homologs of scube2 in zebrafish, scube 1 and scube 3. The high level of similarity amongst the Scube family of proteins and the weak phenotype of the you mutant suggested scube1 and scube3 might also be involved in slow muscle development. Loss of function experiments performed by antisense morpholino knockdown of scube1 and scube3 in the you mutant decreases the expression of Hh target genes to levels seen in embryos lacking Hh signalling and dramatically enhances the loss of slow muscle fibres compared to you mutants alone. Thus, injecting both scube1 and scube3 morpholinos into you blocks Hh signalling and these embryos fail to develop slow muscle. Inhibition of the three partially redundant scube genes inhibits Hh signalling in zebrafish embryos, thereby demonstrating the essential requirement for scube gene function in the Hh signalling pathway.

Attaining an understanding of the mechanisms underpinning development has been amongst the cardinal scientific challenges of our age. The transition from a single cell organism to the level of complexity evidenced in higher eukaryotes has been facilitated by the advent of intricate developmental networks involving a plethora of factors that synergise to allow for precise spatio-temporal expression of the proteins present in higher organisms. Development is often portrayed as a domino like cascade of events stemming from relatively uncomplicated origins that go on to branch out and form associations and interactions amongst multitudinous actors that will inexorably lead towards a higher state of order. Transcription factors occupy a central position within this tapestry of interactions. They regulate expression of the various required proteins and they provide the cues for the developmental events that will eventually shape an organism. These factors frequently remain unknown until some occurrence causes developmental processes to fail and inadvertently focus attention on the factors that facilitate development. Myotonic dystrophy is a useful paradigm of such a developmental dysfunction that has led to the discovery of a transcription factor integral to both muscle development and gonadogenesis in both Drosophila and higher eukaryotes.