Differentiation is the process of regulated gene expression that gives rise to different phenotypes from a common genotype. Skeletal muscle differentiation, myogenesis, is a good example of this process. Skeletal muscle is susceptible to injury due to direct or indirect causes. If left unrepaired, these injuries may lead to a loss of muscle mass, locomotive deficiency, and even lethality. Thus, understanding the molecular mechanisms behind this process is an important first step in the design of treatment for muscle-related diseases. Once myogenesis is induced, the expression of MRF proteins, such as MyoD and myogenin, is maintained at high levels in myofibers without the need to increase their rates of transcription, suggesting a role for post-transcriptional regulatory mechanisms. HuR is a ubiquitously expressed member of the embryonic lethal, abnormal vision (ELAV) family of RNA binding proteins that is known to post-transcriptionally regulate its target messages. Here, I demonstrate that, in the C2C12 muscle cell line, HuR is a required factor for both the initiation and maintenance of the myogenic process. First in vitro RNA Electro-Mobility Shift Assays (REMSA) and immunoprecipitation experiments demonstrated that HuR specifically binds to the AU-rich elements (AREs) that are present in the 3' untranslated regions (3'UTRs) of the MyoD and myogenin mRNAs. In the absence of HuR at the time of differentiation induction, accomplished using the siRNA technology, the expression of the MyoD and myogenin messages is significantly reduced, leading to inhibition of myogenesis. At this early stage in the differentiation process, HuR, a shuttling protein, is predominantly nuclear; localization that is mediated by the import receptor Transportin2 (Trn2). Nuclear HuR was determined to be required for the negative regulation of nucleophosmin (NPM) translation. Forced overexpression of NPM, resulting in differentiation inhibition, shows that its downregulation is a requirement for induction of the differentiation process. Late in myogenesis, however, NPM RNA is no longer expressed, and HuR is seen to accumulate in the cytoplasm of myotubes. This cytoplasmic accumulation results from dissociation of HuR from Trn2 due to caspase-dependent cleavage within its HNS region. Specifically blocking HuR import through the use of cell-permeable peptides, as well as RNAi-mediated depletion of Trn2, leads to an increase in cytoplasmic HuR, as well as increased cytoplasmic localization and stabilization of the MyoD and myogenin messages, and a corresponding enhancement of differentiation. Overall, we conclude that HuR is required for myogenesis due to its ability to post-transcriptionally regulate genes required for the process, and that HuR itself is regulated at the level of its subcellular localization, mediated by the import receptor TRN2.

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.

Dissertation (Ph.D.) -- University of Texas Southwestern Medical Center at Dallas, 2006. / Vita. Bibliography: p. 96-98.

Thesis (Ph.D.)--Virginia Commonwealth University, 2005. / Prepared for: Dept. of Anatomy and Neurobiology. Bibliography: leaves 75-83. Also available online.

Thesis (Ph. D.) -- Virginia Commonwealth University, 2005. / Title from title-page of electronic thesis. Prepared for: Dept. of Anatomy and Neurobiology. Bibliography: leaves 75-83.

abstract: Skeletal muscles arise from the myotome compartment of the somites that form during vertebrate embryonic development. Somites are transient structures serve as the anlagen for the axial skeleton, skeletal muscle, tendons, and dermis, as well as imposing the metameric patterning of the axial musculoskeletal system, peripheral nerves, and vasculature. Classic studies have described the role of Notch, Wnt, and FGF signaling pathways in controlling somite formation and muscle formation. However, little is known about the transformation of myotome compartments into identifiable post-natal muscle groups. Using a mouse model, I have undertaken an evaluation of morphological events, including hypertrophy and hyperplasia, related to the formation of several muscles positioned along the dorsal surface of the vertebrae and ribs. Lunatic fringe (Lfng) deficient embryos and neonates were also examined to further understand the role of the Notch pathway in these processes as it is a modulator of the Notch receptor and plays an important role in defining somite borders and anterior-posterior patterning in many vertebrates. Lunatic fringe deficient embryos showed defects in muscle fiber hyperplasia and hypertrophy in the iliocostalis and longissimus muscles of the erector spinae group. This novel data suggests an additional role for Lfng and the Notch signaling pathway in embryonic and fetal muscle development. / Dissertation/Thesis / M.S. Biology 2012

Caffeine and retinoic acid were examined for effects upon limb morphogenesis and upon creatine kinase (CK) as a measure of limb myogenesis. Caffeine at 200 mg/kg, i.p., on E11 produced a low level of forelimb (1.2%) and hindlimb (2.0%) defects. Retinoic acid, at 50 mg/kg given orally as an oily suspension, induced a high level of reduction deformities. Hindlimbs (100%) were affected more than forelimbs (88%). Limbs (E16) were examined for CK isoenzymes using DEAE-Sephacel column chromatography. Untreated limbs had 88.04% skeletal muscle (MM), 6.98% hybrid (MB) and 5.08% brain (BB) CK isoenzyme. Caffeine had no effect. However, retinoic acid increased MM-CK to 92.67%, and decreased BB-CK to 2.24%. This is the first evidence that suggests that retinoic acid may modify the phenotypic expression of developing muscle.

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