• Artificial Chromosome Transgenesis Reveals Long-Distance Negative Regulation of ragl in Zebrafish

      Jessen, Jason R.; Department of Biochemistry and Molecular Biology (1999-11)
      Despite the essential roles played by the recombination activating genes (ragl and rag2) during V(D)J recombination, the mechanisms that restrict their expression to lymphoid cells are undefined. Using a novel approach to achieve artificial chromosome transgenesis in zebrafish, we demonstrate that distal regulatory elements are critical to suppress ragl expression in inappropriate tissues. In contrast to smaller reporter gene constructs, 125 and 75 kb artificial chromosomes containing the zebrafish rag genomic locus directed GFP expression in a pattern reflective of endogenous rag 1. Mapping experiments identified a positive element 5' of ragl that enhances GFP expression in both lymphoid and non-lymphoid tissues and a negative element 5' of ra g l that specifically suppresses GFP expression in the skeletal muscle. Our transgenic zebrafish also express GFP in olfactory neurons which we show represent an authentic ra g l expression site in zebrafish.
    • Characterization o f Zebrafish Mutant m erlot as a Non-Mammalian Vertebrate Model for Congenital Anemia Due to Protein 4.1 Deficiency

      Shafizadeh, Ebrahim; Department of Biochemistry and Molecular Biology (2002-08)
      The zebrafish mutant merlot (mot) is characterized by onset o f a severe anemia at 96 hours post fertilization. We performed whole mount RNA in situ hybridization and showed that the process o f primitive erythropoiesis is not interrupted in the mot embryos. Blood analysis demonstrated that mot suffers from a severe congenital hemolytic anemia. Using the TU N E L assay, we detected apoptotic erythroid progenitors in the kidneys. We performed electron microscopic analysis and detected membrane abnormalities and a loss o f the cortical membrane organization in the mot cells. We used positional cloning techniques w ith a candidate gene approach to demonstrate that mot encodes the erythroid specific isoform o f protein 4.1R, a critical component o f the red blood cell membrane skeleton. Sequence analysis o f 4.IR cD N A detected nonsense point mutations in both alleles o f mot resulting in premature stop codons. We performed linkage analysis and transgenic rescue experiments to provide further confirmation that the molecular defect in the protein 4 .1R is the underlying cause o f anemic phenotype in mot fish. This study presents the zebrafish mutant merlot as the first characterized non-mammalian vertebrate model o f congenital anemia due to a defect in protein 4.1R integrity.
    • 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 and Behavioral Analyses of clarin-2: A Novel Somatosensory Neuron Subtype-Enriched Gene

      Roberts, Rachel; Department of Neuroscience and Regenerative Medicine (2017-06)
      The trigeminal ganglion (TG) is a somatosensory organ that relays stimuli in the head to the hindbrain and spinal cord, and it comprises multiple subtypes of sensory neurons that respond to different somatosensory stimuli and establish distinct neuronal circuits. The Trpa1b subtype of TG sensory neurons (TGSNs) are responsible for sensing noxious chemicals, but the molecular cues that specify the development of this neuronal subtype remain poorly understood. Zebrafish were previously established as a robust model for studying the development of TGSNs due to its small size, translucency, and robust somatosensory behaviors. A previous microarray study in zebrafish found a novel four transmembrane-domain protein, clarin-2, to be enriched in Trpa1b-expressing cells. Nothing is known about the function of clarin-2, but a close homolog, clarin-1, is one of the causative genes for Usher Syndrome Type 3, a disorder characterized by progressive hearing and vision loss. We hypothesize that clarin-2 may play a role in the development and sensory function of TGSNs. To test this hypothesis, we examined the expression of clarin-2 within the TG during development and used clarin-2 knockout (KO) fish to study the genesis and neurite outgrowth of Trpa1b TGSNs. We found that clarin-2 is indeed enriched in a subset of TGSNs but is not required for the morphogenesis of the TG or the specification of nociceptive sensory neurons. Furthermore, axon projections from Trpa1b neurons were normal in clarin-2 KO fish, compared to control siblings. To test whether clarin-2 is required for the function of TGSNs, we tested somatosensory behaviors in larval zebrafish, including chemo-, thermo-, and mechanosensation. Behavioral analyses showed that clarin-2 is not required for the ability of Trpa1b neurons to detect the chemical irritant mustard oil. Additionally, the detection of heat or vibration was not affected in clarin-2 KO fish. Together, these results suggest that although clarin-2 is enriched in a subset of TGSNs, it is not required for the general morphogenesis of TGSNs or for somatosensation.
    • Multicolor Time-lapse Imaging of Transgenic Zebrafish: Visualizing Retinal Stem Cells Activated by Targeted Neuronal Cell Ablation

      Ariga, Junko; Walker, Steven L.; Mumm, Jeff S.; Department of Cellular Biology and Anatomy (2010-09-20)
      High-resolution time-lapse imaging of living zebrafish larvae can be utilized to visualize how biological processes unfold (for review see 1). Compound transgenic fish which express different fluorescent reporters in neighboring cell types provide a means of following cellular interactions 2 and/or tissue-level responses to experimental manipulations over time. In this video, we demonstrate methods that can be used for imaging multiple transgenically labeled cell types serially in individual fish over time courses that can span from minutes to several days. The techniques described are applicable to any study seeking to correlate the "behavior" of neighboring cells types over time, including: 1) serial 'catch and release' methods for imaging a large number of fish over successive days, 2) simplified approaches for separating fluorophores with overlapping excitation/emission profiles (e.g., GFP and YFP), 3) use of hypopigmented mutant lines to extend the time window available for high-resolution imaging into late larval stages of development, 4) use of membrane targeted fluorescent reporters to reveal fine morphological detail of individual cells as well as cellular details in larger populations of cells, and 5) a previously described method for chemically-induced ablation of transgenically targeted cell types; i.e., nitroreductase (NTR) mediated conversion of prodrug substrates, such as metronidazole (MTZ), to cytotoxic derivatives 3,5.
    • 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