Life Sciences Commons^™

Epigenomics Of Cell Fate In Development And Disease, Rebecca Faith Lowdon

Arts & Sciences Electronic Theses and Dissertations

Epigenetic features at regulatory elements provide instructive cues for transcriptional regulation during development. However, the particular epigenetic alterations necessary for proper cell fate acquisition and differentiation are not well understood. This dissertation explores the epigenetic dynamics of regulatory elements during development and uses epigenome annotations to document inappropriate transcriptional regulation in disease. First, I summarize my contributions to developing a new algorithm for detecting differential DNA methylation, M&M. I report the application of the M&M algorithm to identify distinct classes of DNA methylation dynamics in surface ectoderm (SE) progenitor cells and SE-derived lineages: epigenome alterations, and differential DNA methylation in …

Molecular Measurement Of Toxicity In Fish; Case Examples And Policy Implications, Jessica A. Freedman

Senior Honors Projects

Stormwater and oil are common urban contaminants that can be harmful to fish species. One way of recognizing exposed and impaired fish is by monitoring gene expression and gene induction. This study focused on the identification and validation of reference genes for measuring contaminant-induced changes in gene expression due to urban influence. In this study, reference genes (which are genes used to normalize data and remain consistent in varying exposures regardless of organism and tissue type) were established. Six genes were identified as reference genes (ef1a, wdtc1, mtm1, spop, rxrba and tuba1) from a longer list of potential …

Biochemical Changes In Animal Models Of Fetal Alcohol Spectrum Disorder, Christine E. M. Keller

Open Access Dissertations

Fetal alcohol spectrum disorder (FASD) is a completely preventable disease, that has profound effects on life-long health and function of the affected individual. Prevalence estimates of FASD in the United States indicate 33.5 per 1,000 live births are affected with this disorder (Roozen, 2016). FASD is caused by maternal ethanol intake during pregnancy. However, recommendations of the amounts of alcohol safe to drink during pregnancy are not established. Further, we lack a comprehensive understanding of the biochemical pathways modified in prenatal ethanol exposure. Biomarkers are also lacking. Our results demonstrate the vast array of biochemical pathways modified in the chronic …

The Effects Of Amphetamine Exposure On Neurodevelopment And Behavior In Early Life Stages Of Danio Rerio, Jennifer D. Bullard

Master of Science in Integrative Biology Theses

Amphetamines are frequently prescribed to young children for attention deficit hyperactivity disorder (ADHD). Due to the recent rise in ADHD diagnoses and the resulting increase in amphetamine intake in children, we investigated how amphetamine exposure impacts the development of stereotyped behaviors and the neural circuit that governs these behaviors. The zebrafish (Danio rerio) was used as our model organism because of the extensively studied developmental milestones these fish provide. Using escape responses as a behavior model, we exposed embryos to three doses of amphetamine (10 μg/ml, 20 μg/ml, and 30 μg/ml) environmentally during the first 48 hours of …

Gfap-Positive Radial Glial Cells Are An Essential Progenitor Population For Later-Born Neurons And Glia In The Zebrafish Spinal Cord, Kimberly Johnson, Jessica Barragan, Sarah Bashiruddin, Cody J. Smith, Chelsea Tyrrell, Michael J. Parsons, Rosemarie Doris, Sarah Kucenas, Gerald B. Downes, Carla M. Velez, Caitlin Schneider, Catalina Sakai, Narendra Pathak, Katrina Anderson, Rachael Stein, Stephen H. Devoto, Jeff S. Mumm, Michael J.F. Barresi

Biological Sciences: Faculty Publications

Radial glial cells are presumptive neural stem cells (NSCs) in the developing nervous system. The direct requirement of radial glia for the generation of a diverse array of neuronal and glial subtypes, however, has not been tested. We employed two novel transgenic zebrafish lines and endogenous markers of NSCs and radial glia to show for the first time that radial glia are essential for neurogenesis during development. By using the gfap promoter to drive expression of nuclear localized mCherry we discerned two distinct radial glial-derived cell types: a major nestin+/Sox2+ subtype with strong gfap promoter activity and a minor Sox2+ …

Radial Glia Inhibit Peripheral Glial Infiltration Into The Spinal Cord At Motor Exit Point Transition Zones, Cody J. Smith, Kimberly Johnson, Taylor G. Welsh, Michael J.F. Barresi, Sarah Kucenas

Biological Sciences: Faculty Publications

In the mature vertebrate nervous system, central and peripheral nervous system (CNS and PNS, respectively) GLIA myelinate distinct motor axon domains at the motor exit point transition zone (MEP TZ). How these cells preferentially associate with and myelinate discrete, non-overlapping CNS versus PNS axonal segments, is unknown. Using in vivo imaging and genetic cell ablation in zebrafish, we demonstrate that radial glia restrict migration of PNS glia into the spinal cord during development. Prior to development of radial glial endfeet, peripheral cells freely migrate back and forth across the MEP TZ. However, upon maturation, peripherally located cells never enter the …

Ubr3, A Novel Modulator Of Hh Signaling Affects The Degradation Of Costal-2 And Kif7 Through Poly-Ubiquitination, Tongchao Li, Junkai Fan, Bernardo Blanco-Sánchez, Nikolaos Giagtzoglou, Guang Lin, Shinya Yamamoto, Manish Jaiswal, Kuchuan Chen, Jie Zhang, Wei Wei, Michael T. Lewis, Andrew K. Groves, Monte Westerfield, Jianhang Jia, Hugo J. Bellen

Markey Cancer Center Faculty Publications

Hedgehog (Hh) signaling regulates multiple aspects of metazoan development and tissue homeostasis, and is constitutively active in numerous cancers. We identified Ubr3, an E3 ubiquitin ligase, as a novel, positive regulator of Hh signaling in Drosophila and vertebrates. Hh signaling regulates the Ubr3-mediated poly-ubiquitination and degradation of Cos2, a central component of Hh signaling. In developing Drosophila eye discs, loss of ubr3 leads to a delayed differentiation of photoreceptors and a reduction in Hh signaling. In zebrafish, loss of Ubr3 causes a decrease in Shh signaling in the developing eyes, somites, and sensory neurons. However, not all tissues that require …

Single-Cell Transcriptional Analysis Of Normal, Aberrant, And Malignant Hematopoiesis In Zebrafish, Finola E. Moore, Elaine G. Garcia, Riadh Lobbardi, Esha Jain, Qin Tang, John C. Moore, Mauricio Cortes, Aleksey Molodtsov, Melissa Kasheta, Christina C. Luo, Amaris J. Garcia, Ravi Mylvaganam, Jeffrey A. Yoder, Jessica S. Blackburn, Ruslan I. Sadreyev, Craig J. Ceol, Trista E. North, David M. Langenau

Molecular and Cellular Biochemistry Faculty Publications

Hematopoiesis culminates in the production of functionally heterogeneous blood cell types. In zebrafish, the lack of cell surface antibodies has compelled researchers to use fluorescent transgenic reporter lines to label specific blood cell fractions. However, these approaches are limited by the availability of transgenic lines and fluorescent protein combinations that can be distinguished. Here, we have transcriptionally profiled single hematopoietic cells from zebrafish to define erythroid, myeloid, B, and T cell lineages. We also used our approach to identify hematopoietic stem and progenitor cells and a novel NK-lysin 4⁺ cell type, representing a putative cytotoxic T/NK cell. Our platform …

Characterization Of Stem Cell Turnover In A Living Epithelial Bilayer, Elizabeth Sumner

Dissertations & Theses (Open Access)

Homeostatic maintenance of epithelia requires the renewal and replacement of old or dying cells while sustaining a functional barrier. Imbalance between cell production and elimination are hypothesized to underlie many pathological conditions. However, our knowledge of cell turnover within living tissues remains largely restricted to static images due to the limited ability to study epithelia in their native context. Here we report that clearance of damaged basal stem cells promotes compensatory proliferation of neighboring stem cells to maintain overall population numbers in a bilayered epithelium. Time-lapse imaging and electron microscopy experiments reveal that dying cells are rapidly cleared as nearby …

Diversity Of Small Rna Expression During Zebrafish Caudal Fin Regeneration, Jefferson Adams

Honors College

Over the years microRNA have been shown to play a role in the regulation of genes involved in regeneration of zebrafish (Danio rerio) tissues. However, recent research suggest that there may be other types of small RNA that play a regulatory role in these regenerative processes. For the most part these other small RNA (sRNA), such as transfer RNA (tRNA) derived fragments, ribosomal RNA (rRNA) derived fragments, and small nucleolar RNA, are disregarded. Here I analyzed the expression pattern of these sRNA during the regeneration of the zebrafish caudal fin. High-throughput sequencing was used to characterize the expression of small …

Preliminary Analysis Of Β-Methylamino-L-Alanine Interactions With Cu/Zn Superoxide Dismutase In Amyotrophic Lateral Sclerosis Zebrafish Models, Elizabeth Pflugradt

Honors College

Amyotrophic Lateral Sclerosis (ALS) is a terminal neurodegenerative disease

with symptoms including limb-onset muscle wasting, difficulties swallowing and breathing, leading to death. Sporadic ALS occurs in 90% of patients, while 10% of cases are familial (FALS). Twenty percent of FALS cases are a result of mutation in the copper-zinc superoxide dismutase (SOD1) gene, leading to the activation of the mitochondrial apoptosis pathway. Meanwhile, a prominent cause of sporadic ALS is exposure to neurotoxins, such as β-methylamino-L-alanine (BMAA). BMAA has been suggested to induce selective motor neuron death, which is observed in ALS patients. While research has been done to how …

Innate Immune Recognition Of Candida Albicans In Zebrafish, Monique Elaine Theriault

Honors College

Candida albicans is an opportunistic fungal pathogen that has the capability to switch from commensal to pathogen in immunocompromised individuals. Recognition of pathogens, like C. albicans, during infection is poorly characterized primarily due to the difficulties in visualizing the host/pathogen interaction without killing the host. Transparent animal hosts, such as Danio rerio (zebrafish), enable imaging of pathogen recognition while maintaining host viability. For pathogen recognition, zebrafish likely use immune receptors similar to mammalian receptors including C-type lectin receptors. Human C-type lectin receptors have already been shown to be crucial in recognition of fungal pathogens like C. albicans, and our goal …

Glyphosate Induces Neurotoxicity In Zebrafish, Nicole M. Roy, Bruno Carneiro, Jeremy Ochs

Biology Faculty Publications

Glyphosate based herbicides (GBH) like Roundup^® are used extensively in agriculture as well as in urban and rural settings as a broad spectrum herbicide. Its mechanism of action was thought to be specific only to plants and thus considered safe and non-toxic. However, mounting evidence suggests that GBHs may not be as safe as once thought as initial studies in frogs suggest that GBHs may be teratogenic. Here we utilize the zebrafish vertebrate model system to study early effects of glyphosate exposure using technical grade glyphosate and the Roundup^® Classic formulation. We find morphological abnormalities including cephalic and …

Imaging Tumour Cell Heterogeneity Following Cell Transplantation Into Optically Clear Immune-Deficient Zebrafish, Qin Tang, John C. Moore, Myron S. Ignatius, Inês M. Tenente, Madeline N. Hayes, Elaine G. Garcia, Nora Torres Yordán, Caitlin Bourque, Shuning He, Jessica S. Blackburn, A. Thomas Look, Yariv Houvras, David M. Langenau

Molecular and Cellular Biochemistry Faculty Publications

Cancers contain a wide diversity of cell types that are defined by differentiation states, genetic mutations and altered epigenetic programmes that impart functional diversity to individual cells. Elevated tumour cell heterogeneity is linked with progression, therapy resistance and relapse. Yet, imaging of tumour cell heterogeneity and the hallmarks of cancer has been a technical and biological challenge. Here we develop optically clear immune-compromised rag2^E450fs (casper) zebrafish for optimized cell transplantation and direct visualization of fluorescently labelled cancer cells at single-cell resolution. Tumour engraftment permits dynamic imaging of neovascularization, niche partitioning of tumour-propagating cells in embryonal rhabdomyosarcoma, emergence of clonal …

Characterization Of The Three Zebrafish Orthologs Of The Mitochondrial Gtpase Miro/Rhot, Brittany M. Hollister, Kelsey A. Oonk, Douglas C. Weiser, Susan Walsh

Faculty Publications

Mitochondria exhibit dynamic locomotion and spatial rearrangement. This movement is necessary for a cell to maintain basic metabolic functions, and disruption of motility often results in cell death. Miro is a mitochondrial outer membrane Rho GTPase essential for mitochondrial movement and distribution in diverse systems, including yeast, animals, and plants. We sought to study the previously uncharacterized Miro protein family in zebrafish. We confirmed that, like human Miro, the zebrafish Miro proteins (Rhot1a, Rhot1b, and Rhot2) localize to mitochondria in mammalian tissue culture cells by both biochemical fractionation and immunofluorescent colocalization. In addition, using whole mount in situ hybridization, we …

Role Of Hairy-Related (Her) Genes During Vertebrate Retinal Development And Regeneration, Stephen G. Wilson

Theses and Dissertations--Biology

Development and regeneration of the vertebrate eye are the result of complex interactions of regulatory networks and spatiotemporally controlled gene expression events. During embryonic retinal development, the coordination of cell signaling and transcriptional regulation allows for a relatively homogenous sheet of neuroepithelial cells to proliferate and differentiate in-to a multilayered, light sensitive retinal tissue. Following injury, the retinas of many cold-blooded vertebrates, such as the zebrafish, undergo a proliferative response that results not only in new retinal cells of the correct type in the correct location, but also functional integration of these cells and restoration of vision. In order for …

The Role Of Integrin Alpha 6 On Tumor Metastasis, Shauntell N. Luke Ms.

Honors College Theses

Cancer research is a large topic in science, because over 14 million people are diagnosed worldwide, and of those, 8 million will die annually. Traditional therapies are the mainstays of treatment strategies, but a cure less invasive or with less side effects have not yet been identified. In order to develop a better cure, we need to understand the tumor and molecules present in its tissues. Integrins, cell surface proteins that aid in cell communication, have been found in the tumor tissues of several types of cancers, including prostate, lung and bone. Integrin alpha-6 (ITGA6), in particular, has been found …

The Behavioral, Biochemical And Genetic Effects Of Sleep Deprivation In Zebrafish (Danio Rerio), Jade Catherine Boykin

Electronic Theses and Dissertations

Vertebrate sleep is a universal phenomenon and encompasses an array of conserved behavioral and physiological characteristics. Studies of sleep in humans have shown that sleep deprivation leads to adverse effects to human health and additional stress. Teleosts have emerged as a useful model species to study the vertebrate stress response, given its stress axis has high levels of homology with the mammals, including humans. For this study, the behavior of zebrafish (Danio rerio) subjected to acute (24 hours) and chronic (20 days) extended light exposure was monitored and whole-body cortisol was analyzed as an indication of activation of …