• DEVELOPMENT OF TRANSGENIC ZEBRAFISH MODEL FOR INVESTIGATION OF THE FUNCTION OF MICROGLIA

      Sura, Survasha; Department of Biological Sciences; Department of Biochemical and Molecular Biology; Georgia Cancer Center; Rajpurohit, Surendra K; Augusta University (2019-02-13)
      Zebrafish have emerged as a powerful model organism for elucidating the development and function of microglia. Generation of new transgenic reporter lines and imaging tools strengthen the zebrafish model in microglia study�in-vivo. The aim is to develop a novel compound transgenic line to study the inflammatory process mediated by NF-kB in microglia cells. This novel compound transgenic line will establish a new model for microglia study. To generate the novel compound zebrafish transgenic model for microglia, we are crossbreeding microglia transgenic line zebrafish (Tg(mpeg1:mCherry) with the NF-kB Tg(6xNFkB:EGFP) transgenic progeny. We first generate a heterozygous F1 progeny which will be bred to generate an F2 homozygous progeny. Once the F1 progeny of the Microglia-NfkB transgenic line is developed, they will be crossbred to develop the Homozygous compound transgenic line. Fluorescent Microscopy will be used to screen the larvae generated from the breeding events. By developing the compound transgenic line, we are optimizing microglia isolation and sorting methodology by using the related antibodies as the marker. The NF-kB microglia transgenic line will provide a unique platform for drug screening to address microglial based ailments, thus furthering the understanding and treatment of human disease.
    • Developmental Biology of Zebrafish and Integration of Transgenic Lines to Study Microglia in Perspective of Glioblastoma

      O'Keefe, Anabelle; McCartney, Katherine; Kandepu, Umasai; Rajpurohit, Surendra; Biological Sciences; Department of Biochemistry and Molecular Biology; Rajpurohit, Surendra; Augusta University (1/31/2020)
      Glioblastoma is a vicious cancer of the brain that is extremely invasive. Our innovative approach to studying glioblastoma utilizes zebrafish as model for scientific study because of their affordable maintenance, transparent body plan during embryo and larval stages, and genomic accessibility. We aim to use zebrafish as an organismal model to study how glioblastoma and microglial cells interact in the neural region. To achieve this, we are developing an all-encompassing in-vivo transgenic and transparent zebrafish modeling system to study microglia function and manipulation in the context of adverse conditions such as glioblastoma and inflammation. Microglia are the resident macrophages found in zebrafish and humans located along the central nervous system in the brain and spinal cord. These cells support the immune system by cleaning any foreign debris. The model will integrate Microglia, NF-kB, and Annexin-5 transgenic lines displaying which genes in the brain are activated via their corresponding fluorescent protein upon the introduction of glioblastoma. Furthermore, a mutant Casper line of zebrafish will introduce a transparent characteristic in adult zebrafish that allows for simpler visualization and observation in the final model. Ultimately, the transgenic model will utilize microglia cells as a mechanism to approach glioblastomas.
    • A Prospective Dominant Negative Mutant of Wnt Signaling In Zebrafish Causes Craniofacial Asymmetry with Low Penetrance

      Ravilla, Dheeraj; Neiswender, Hannah; Department of Biological Sciences; Department of Cellular Biology and Anatomy (2016-03)
      Wnt gene signaling pathways have been implicated in development, cell behavior, and diseases, including craniofacial abnormalities. We created mutant complementary DNA constructs using QuikChange mutagenesis and then compared them to wild-type cDNA for effects on zebrafish development following injection into one-cell embryos. We hypothesized that disrupting a putative Wnt-binding lipocalin motif would allow mutant tinagl1 mRNAs to induce a dominant negative phenotype, similar to tinagl1 gene knockdown. Mutant LCN-W2 but not WT mRNA preferentially gave small eyes and ventral body curvature similar to the gene knockdown. Our main focus was on craniofacial development using Alcian blue staining of cartilage elements in 5 day old zebrafish. Abnormalities were seen at low penetrance in high-survival (high-quality) clutches with the highest injected dose of TIN LCN-W2,150 pg. These included smaller head and asymmetric head skeleton with one smaller eye on side of variable cartilage defects. Craniofacial defects, especially asymmetry, were more prevalent in clutches with lower survival rates. These asymmetric defects had not been seen in the gene knockdown. In summary, the phenotypes of LCN-W2 partially support similarity to a dominant negative phenotype with cartilage defects, small eyes, and ventrally curved body, but the craniofacial asymmetry appears novel. More research is needed for further understanding.
    • Role of the Aryl Hydrocarbon Receptor (Ahr) in Skeletal Muscle

      Bowles, Jessica; Lambert, Andrea; Dukes, Amy; Mendhe, Bharati; Department of Biological Sciences (2016-03)
      The AhR is a ligand-activated transcription factor known to mediate the negative effects of environmental contaminants such as dioxin. Inactivation of AhR in skeletal muscle appears to be a response to both resistance exercise training and endurance exercise training, whereas activation of the receptor impairs tissue regeneration in zebrafish. AhR is also a receptor for kynurenine, an oxidized metabolite of the aromatic amino acid tryptophan. We have found that while tryptophan can preserve lean mass and stimulate muscle-derived IGF-1 in the setting of dietary protein deficiency, kynurenine decreases both muscle mass and IGF-1. Aside from these few studies, very little is known about the role of AhR in muscle wasting in catabolic settings such as aging or disease, or how it mediates the response to exercise. We have identified expression of the AhR in skeletal muscle using immunostaining and gene expression (PCR) of mouse hindlimb muscles (tibialis anterior). We are currently working to determine whether AhR expression levels change with age, or differ between males and females. The ultimate goal of this research is to develop novel therapeutic approaches, perhaps targeting AhR, to prevent muscle loss with aging and disuse. Funding Source: National Institute on Aging