Doctor of Philosophy, Case Western Reserve University, 2021, Biology

Regulation of Cellular Differentiation by Ezh2 during Skin and Muscle Development Abstract by VENKATA REVANTH SAI KUMAR THULABANDU The dermis of the skin and muscle are vital tissues that provide protection and movement to the body, respectively. To derive these specialized tissues from mesenchymal stem cells, a controlled sequential differentiation process is crucial. The lineage-guiding transcription and secreted factors, expressed during dermal and muscle development, are known targets of epigenetic pathways, primarily governed by Polycomb Repressive Complex 2 (PRC2). However, it is unknown whether and how PRC2 coordinates lineage selection and differentiation during dermal and muscle development. Here we examined the role of PRC2 in dermal and muscle development using murine in vivo conditional knockout of PRC2's catalytic component, Ezh2. We found that PRC2 is active throughout dermal and muscle development, as seen from its histone modification, H3K27me3, staining. The Ezh2 mutants exhibited ectopic specification of lower dermal mesenchymal cells to dermal progenitor fate due to upregulation in Wnt/β-catenin signaling. The Ezh2 mutants also exhibited delayed secondary hair follicle initiation, perhaps via Noggin. Additionally, the Ezh2 mutants showed non-cell autonomous epidermal hyperplasia that can be phenocopied by a single dose of retinoic acid administration to the embryos. The Ezh2 mutants had a complete loss of muscle due to hindered myocytes' differentiation into myotubes. Both epidermal hyperplasia and myocyte differentiation were rescued by the administration of retinoic acid signaling antagonist BMS453. These results demonstrate a complex circuitry at play wherein PRC2 regulates (i) Wnt/β-catenin to regulate differentiation in dermal fibroblasts and (ii) retinoic acid signaling to regulate proliferation in epidermal keratinocytes and differentiation in myocytes. The insights drawn from this project have implications in improving current tiss (open full item for complete abstract)

Committee: Radhika Atit Dr. (Advisor); Abbott Karen Dr. (Committee Chair); Basch Martin Dr. (Committee Member); Mizutani Claudia Dr. (Committee Member); Ezhkova Elena Dr. (Committee Member) Subjects: Biology; Cellular Biology; Developmental Biology; Molecular Biology

Doctor of Philosophy (PhD), Ohio University, 2021, Translational Biomedical Sciences

Age-related declines in physical function and mobility can be attenuated by routine physical activity, however, less than 50% of adults over the age of 65 exercise regularly. As such, time-efficient exercise strategies are being investigated as a means of improving and/or maintaining physical function. If these strategies are to be systematically evaluated for effectiveness, it is essential that the techniques employed to quantify exercise-induced changes have been validated to assess the outcome of interest. The widespread use of standardized measurement techniques allows for more accurate cross-investigation analyses (i.e., meta-analysis), while simultaneously making it easier to assess reproducibility. However, our search for knowledge is hampered when inappropriate techniques are employed, or when appropriate techniques are employed in a manner for which they have not been validated (e.g., cross-sectional vs. longitudinal). These potentially erroneous reports result in inconsistent messages, making it difficult to determine optimal treatment strategies for unique clinical populations (e.g., older adults). Thus, the global aim of this dissertation was to 1) systematically examine tools for assessing physiological, morphological, and functional adaptations where exercise served as a stimulus for change, and 2) employ said tools in the development and execution of a proof-of-concept, proof-of-mechanism clinical trial investigating the effectiveness of a novel exercise strategy against existing strategies. Experiment 1 challenged the recommendation that dual-energy X-ray absorptiometry (DXA) should be considered the reference standard for the assessment of muscle mass, particularly as it relates to exercise- and/or disease-induced changes in muscle mass. Thigh muscle size was quantified with DXA and magnetic resonance imaging (MRI) in 26 adults (29.2 ﰀ 9.5 years) before and after 10 weeks of low-load resistance exercise, and relationships between the two measures (open full item for complete abstract)

Committee: Brian Clark Ph.D. (Advisor); David Russ Ph.D. (Advisor); Janet Simon Ph.D. (Committee Chair); Jeff Russell Ph.D. (Committee Member); Leslie Consitt Ph.D. (Committee Member); Nathan Wages Ph.D. (Committee Member) Subjects: Biomedical Research; Gerontology; Health Sciences; Medical Imaging; Neurosciences; Physiology

Master of Science, The Ohio State University, 2019, Animal Sciences

In recent years, a variety of muscle myopathies, specifically wooden breast, white striping, and deep pectoralis myopathy, have severely impacted the poultry industry. Although broilers have become more efficient at producing muscle mass, the rapid increase in growth has exceeded the structural limits of the muscle. As a result, myopathies such as wooden breast are commonly associated with fast-growing, heavy weight broilers. However, there is little genetic correlation with breast muscle yield, indicating that environmental factors likely play a vital role. Environmental factors such as nutrient restriction and thermal stress can alter satellite cell populations, which are a type of stem cell responsible for all post-hatch muscle growth. Changes in the satellite cells will have long-lasting impacts on muscle development and meat quality. Although there have been studies that have elucidated the independent effects of nutrient restriction and thermal stress, there is little information regarding the effect of a simultaneous temperature and nutritional restriction on satellite cells, specifically, immediately after hatch, when satellite cells are the most susceptible to environmental stressors. Since chicks are rarely exposed to a single stressor, it is important to understand how simultaneous environmental factors will impact long-term muscle growth. The effect of a simultaneous post-hatch feed restriction and thermal stress on skeletal muscle growth and meat quality was studied by applying a 20% feed restriction during the first week post-hatch. At the time of hatch broiler chicks were divided into either a 20% feed restriction group, or given ad libitum access to feed and held at an ambient temperature of 31°C, 35°C, or 39°C. Gene expression for satellite cell genes Paired Box Protein 7 (PAX7), Myogenic Factor 5 (MYF5), Myogenic Differentiation 1 (MYOD1), and Myogenin (MYOG), were measured at d 7, and no significant differences were detected (P > 0.25). Muscle fib (open full item for complete abstract)

Committee: Daniel Clark PhD (Advisor); Sandra Velleman PhD (Committee Member); Michael Cressman PhD (Committee Member); Sheila Jacobi PhD (Committee Member) Subjects: Agriculture; Animal Sciences

MS, University of Cincinnati, 2017, Medicine: Molecular and Developmental Biology

The formation of skeletal muscle begins during embryogenesis, when muscle stem cells (MuSCs) differentiate into myoblasts and fuse to form multinucleated myofibers. This process continues into the early postnatal period, until skeletal muscle development is complete by P21. Once development is complete, MuSCs continue to contribute to the adult myofiber for the duration of an animal's life. While it is well established that skeletal muscle development occurs during the first three weeks of life, the necessity of myoblast fusion during the neonatal period for normal muscle growth and function is poorly understood. In addition, whether myoblast fusion during homeostasis in the adult is physiologically relevant is yet to be determined. Here we show that addition of new myonuclei through fusion is required during the first two weeks of development for myofiber cross sectional area (CSA). Genetic deletion of myomaker in Pax7+ MuSCs at either P2 or P8 leads to decreases in muscle weight and myonuclear number per myofiber four weeks later. Further, we demonstrate that myoblast fusion is not necessary during homeostasis to maintain myofiber size. Our findings indicate that myoblast fusion during development is essential to establish the number of myonuclei needed to support normal myofiber size in the adult.

Committee: Rashmi Hegde Ph.D. (Committee Chair); Samantha Brugmann Ph.D. (Committee Member); Douglas Millay Ph.D. (Committee Member); Kaushik Roychoudhury Ph.D. (Committee Member) Subjects: Developmental Biology

Doctor of Philosophy, The Ohio State University, 2017, Animal Sciences

The overarching objective of the current research was to investigate morphological (growth) and biochemical (transcription) events underlying skeletal muscle growth and developmental in an ontogeny study using the P. major breast muscle of broiler chickens, from days 2 through 46. Another main objective was to use this time course approach to investigate growth/age dependent nutritional influences, using 3 dietary treatments; a control and two treatment groups containing differential decreases in percent digestible amino acid (NC1 -5% and NC2 -10%). This approach allowed the current research to address a wide-range of diversely relevant research areas, pertaining to both fundamental biology and industry implications in raising broiler chickens for the production of meat. Through quantitative gene transcriptional analysis we were able to investigate the transcriptional events underlying post-hatch skeletal muscle growth and development. Of particular interest was the temporal transcription of developmental MyHC isoforms, as the functional diversity driving their transitions remains unknown. In an effort to further our knowledge in trying to understand their functional diversity, we characterized their temporal transcription patterns under differential dietary treatments during post-hatch growth period using the P. major breast muscle. From which, we report an unexpected post-hatch increase in the transcription of embryonic MyHC isoform, Cemb3. For which, cemb3 transcription was markedly higher in the control diet. Alternatively, the second MyHC isoform for which dietary differences were observed, Cadult, showed the opposite transcription pattern, where an increase in transcription coincided with a further decrease in digestible amino acid levels (Control < NC1 < NC2). The overarching goal of maximizing growth in broiler chickens, for the production of meat, has perpetuated two areas of research that remain a central area of interest in both industry and academ (open full item for complete abstract)

Committee: Michael Lilburn PhD (Advisor); Macdonald Wick PhD (Committee Member); William Pope PhD (Committee Member); Christine Alvarado PhD (Committee Member) Subjects: Animal Sciences; Morphology

Doctor of Philosophy, The Ohio State University, 2009, Integrated Biomedical Sciences

The NF-kappa B family of transcription factors has been implicated in regulating cellular processes such as immune response, cell survival, cellular proliferation, and differentiation. This dissertation will focus on the involvement of NF-kappa B in cellular proliferation and differentiation. Studies support that NF-kappa B functions in cellular proliferation through the transcriptional regulation of cyclin D1, but whether such regulation is attributed to a single NF-kappa B subunit remains unclear. In chapter two we examine endogenous cyclin D1 levels during cell cycle re-entry in mouse embryonic fibroblasts (MEFs) lacking specific NF-kappa B signaling subunits. We demonstrate that each of these subunits is dispensable for regulating cyclin D1 transcription. However, we found that resulting cyclin D1 protein was severely reduced in MEFs lacking only RelA/p65. Cycloheximide treatment revealed that this regulation was due to an increase in protein turnover. Similarly downregulation of cyclin D1 protein, but not RNA, was observed in vivo in multiple tissues lacking p65. Co-immunoprecipitation analysis also showed p65 and cyclin D1 were capable of interacting, thus providing a possible explanation for cyclin D1 protein stability. In addition, although the decrease in cyclin D1 in p65-/- MEFs was concomitant with lower CDK4 activity during cell cycle re-entry, this was not sufficient to affect S phase progression. Nevertheless, similar decreases in cyclin D1 protein in primary p65-/- myoblasts was adequate to accelerate cell cycle exit and myogenesis in these cells. A number of studies have identified classical NF-kappa B activity as an inhibitor of the myogenic program. Recent findings reveal that in newborn p65-/- mice, myofiber numbers are increased over that of wild type mice, suggesting that NF-kappa B may be a contributing factor in early postnatal skeletal muscle development. In chapter 3 we show that in addition to p65 deficiency, repression of NF-kappa B w (open full item for complete abstract)

Committee: Denis Guttridge Ph.D. (Advisor); Kay Huebner Ph.D. (Committee Member); Lawrence Kirschner MD./Ph.D. (Committee Member); Matthew Ringel MD. (Committee Member) Subjects: Biology; Biomedical Research; Cellular Biology; Molecular Biology

Doctor of Philosophy, The Ohio State University, 2009, OSU Nutrition

The role of Delta-like protein 1 (DLK1) has been implicated in the muscle hypertrophy observed in DLK1 transgenic mice, callipyge sheep, mouse paternal uniparental disomy (pUPD) 12 and human pUPD14 syndromes. However, no study on DLK1 has been conducted in the avian species. The ultimate goal of this dissertation is to identify the role of chicken delta-like protein 1 (gDLK1) in skelelal muscle development, growth, and regeneration in poultry. The objectives of the first study were to clone and sequence a full length of chicken DLK1 cDNA, and to investigate the developmental regulation of gDLK1 during embryonic muscle development and postnatal muscle growth. The cloning and sequencing data revealed that gDLK1 contains a total of 1,161 base pairs, encoding 386 amino acids. The similarities of gDLK1 nucleotide and protein sequences were over 50% compared to mammalian species. In addition, chickens only express a full-length gDLK1 in various tissues at different ages without the alternative splicing variants. The expression of gDLK1 gene in pectoralis major muscle was significantly higher in embryonic stages rather than posthatching stages during muscle development (P < 0.05). The expression pattern of gDLK1 was very similar to the expression patterns of myogenin and Pax7 genes, suggesting a close association with myogenic activities. The objectives of the second study were to determine gDLK1 mRNA expression during primary muscle cell differentiation and during muscle regeneration after cold injury, as well as to compare gDLK1mRNA expression during skeletal muscle development in layers and broilers. In the chicken primary muscle cell culture, the peak expression of gDLK1 mRNA was highly associated with late stages of myogenesis after primary myoblast differentiation. During muscle regeneration, the induction of the gDLK1 gene consistently appeared at late stages of regenerative myogenesis. The expression data of gDLK1 associated with myogenic markers (Pax7, MyoD, and m (open full item for complete abstract)

Committee: Kichoon Lee PhD (Advisor); Earl Harrison PhD (Committee Member); Martha A. Belury PhD (Committee Member); Sandra G. Velleman PhD (Committee Member) Subjects: Biology

Master of Sciences, Case Western Reserve University, 2011, Biology

In Drosophila, muscle cells develop from the mesoderm, the middle germ layer of the embryo. During myogenesis each muscle fiber in the pattern is prefigured by a single founder cell, which seeds muscle formation by fusing with surrounding fusion-competent myoblasts. In this work, we have examined the fate of mesodermal precursors in four Drosophila species that display variations in mesoderm size. We characterized a newly discovered morphological difference in muscle size and locomotion patterns between these species. First, our comparison indicates that the overall muscle pattern is conserved while the total number of myoblasts within a fiber is species- specific and depends on the mesoderm width. This suggests that the number of founder cells has not changed during evolution, while the cell-counting mechanism that regulates myoblast fusion is differently regulated. Second, we find that each species display characteristic NMJs and locomotion patterns which are not depended exclusively on muscle size. Finally, we provide evidence for a maternal inheritance of both muscle size and locomotion behavior in third instar larvae.

Committee: Jane Burns PhD (Committee Chair); Jocelyn McDonald PhD (Committee Member); Claudia M. Mizutani PhD (Advisor); Rui Sousa- Neves PhD (Committee Member) Subjects: Biology

Doctor of Philosophy, Case Western Reserve University, 2009, Biochemistry

Ski is the most studied member of a family of proteins all sharing a conserved Dachshund homology domain. It has been implicated in oncogenic transformation, myogenic conversion of avian embryo fibroblasts and also many aspects of vertebrate development, especially myogenesis. Ski-/- mice exhibit severe defects in skeletal muscle and die at birth, yet little is know about either the underlying mechanisms or the role of Ski in adult muscle regeneration. In these studies, I used Ski knockout mice and C2C12 myoblast cultures to address these issues, respectively. Detailed analysis of Ski-/- embryos revealed dramatically reduced hypaxial muscles but less affected epaxial muscles. The reduced number of myogenic regulatory factor positive cells in Ski-/- mice suggested an insufficient myogenic cell pool to support muscle formation. However, both the dermomyotomal hypaxial progenitors and myotomal epaxial progenitors formed and committed to myogenic fate appropriately. The hypaxial muscle defect in Ski-/- mice was not caused by abnormal proliferation, terminal differentiation or apoptosis of the myogenic cells either, but due to impaired migration of embryonic hypaxial progenitors. Surprisingly, the normal distribution of fetal/postnatal myogenic progenitors in Ski-/- mice suggested different effects of Ski on the behaviors of embryonic and fetal/postnatal myogenic progenitors. In addition, although not affecting the terminal differentiation of embryonic myogenic cells, Ski was necessary for that of adult satellite-cell derived C2C12 myoblasts as evidenced by impaired myotube formation and reduced induction of genes essential for myogenic differentiation in the absence of Ski. This function was mainly mediated by Ski's ability to form a complex with Six1 and Eya3 and activate Myog transcription through a MEF3 site. It is important in the future to further study mechanisms underlying the contrasting effects of Ski on embryonic, fetal and adult muscle development, to investi (open full item for complete abstract)

Committee: David Samols PhD (Committee Chair); Ed Stavnezer PhD (Advisor); Clemencia Colmenares PhD (Committee Member); Nikki Harter PhD (Committee Member); Lynn Landmesser PhD (Committee Member) Subjects: Biomedical Research

PhD, University of Cincinnati, 0, Medicine: Molecular and Developmental Biology

Congenital cardiac malformations are the most common birth defects in the United States, and cardiovascular disease (CVD) is the leading cause of death in the U.S. and worldwide. Therefore, identifying conserved molecular mechanisms that regulate cell fate in cardiac development and disease is of great clinical significance. During embryonic heart development, epicardium-derived cells (EPDCs) invade the myocardium and differentiate into fibroblasts and vascular smooth muscle (SM) cells, which support the coronary vessels. Work described in this dissertation demonstrates that the transcription factors Tcf21/Pod1, WT1, NFATC1, and Tbx18 are expressed in overlapping and distinct epicardial and EPDC populations. Retinoic acid (RA) signaling promotes Tcf21 and WT1 expression, while inhibiting EPDC differentiation into SM. Loss of Tcf21 in mice leads to epicardial blistering, increased SM differentiation on the surface of the heart, and a paucity of interstitial fibroblasts, with neonatal lethality. On the surface of the myocardium, SM marker expression is increased in Tcf21-deficient EPDCs, demonstrating premature SM differentiation. Increased SM differentiation also is observed in Tcf21-deficient lung and kidney mesenchyme. Together, these data demonstrate a critical role for Tcf21 in controlling mesenchymal progenitor cell differentiation into SM and fibroblast lineages during cardiac development. The role of Tcf21 in adult cardiac homeostasis or disease, however, is unknown. In order to define the role of Tcf21 in cardiac fibrosis, its expression was examined in mouse models of heart disease as well as in human congestive heart failure (CHF) tissue. Mouse models of ischemia and pressure overload were examined. Studies detailed in this dissertation demonstrate that ischemic injury leads to increased subepicardial cells positive for WT1, Tbx18, and Tcf21 in mice and human hearts. Mice subjected to pressure overload have extensive coronary perivascular fi (open full item for complete abstract)

Committee: Katherine Yutzey Ph.D. (Committee Chair); Jo El Schultz Ph.D. (Committee Member); John Shannon Ph.D. (Committee Member); Stephanie Ware M.D. Ph.D. (Committee Member); Aaron Zorn Ph.D. (Committee Member) Subjects: Developmental Biology