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      Automated Reporter Quantification In Vivo: High-Throughput Screening Method for Reporter-Based Assays in Zebrafish

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          Abstract

          Reporter-based assays underlie many high-throughput screening (HTS) platforms, but most are limited to in vitro applications. Here, we report a simple whole-organism HTS method for quantifying changes in reporter intensity in individual zebrafish over time termed, Automated Reporter Quantification in vivo (ARQiv). ARQiv differs from current “high-content” (e.g., confocal imaging-based) whole-organism screening technologies by providing a purely quantitative data acquisition approach that affords marked improvements in throughput. ARQiv uses a fluorescence microplate reader with specific detection functionalities necessary for robust quantification of reporter signals in vivo. This approach is: 1) Rapid; achieving true HTS capacities (i.e., >50,000 units per day), 2) Reproducible; attaining HTS-compatible assay quality (i.e., Z'-factors of ≥0.5), and 3) Flexible; amenable to nearly any reporter-based assay in zebrafish embryos, larvae, or juveniles. ARQiv is used here to quantify changes in: 1) Cell number; loss and regeneration of two different fluorescently tagged cell types (pancreatic beta cells and rod photoreceptors), 2) Cell signaling; relative activity of a transgenic Notch-signaling reporter, and 3) Cell metabolism; accumulation of reactive oxygen species. In summary, ARQiv is a versatile and readily accessible approach facilitating evaluation of genetic and/or chemical manipulations in living zebrafish that complements current “high-content” whole-organism screening methods by providing a first-tier i n vivo HTS drug discovery platform.

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          Most cited references 45

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          Genetic interaction of PGE2 and Wnt signaling regulates developmental specification of stem cells and regeneration.

          Interactions between developmental signaling pathways govern the formation and function of stem cells. Prostaglandin (PG) E2 regulates vertebrate hematopoietic stem cells (HSC). Similarly, the Wnt signaling pathway controls HSC self-renewal and bone marrow repopulation. Here, we show that wnt reporter activity in zebrafish HSCs is responsive to PGE2 modulation, demonstrating a direct interaction in vivo. Inhibition of PGE2 synthesis blocked wnt-induced alterations in HSC formation. PGE2 modified the wnt signaling cascade at the level of beta-catenin degradation through cAMP/PKA-mediated stabilizing phosphorylation events. The PGE2/Wnt interaction regulated murine stem and progenitor populations in vitro in hematopoietic ES cell assays and in vivo following transplantation. The relationship between PGE2 and Wnt was also conserved during regeneration of other organ systems. Our work provides in vivo evidence that Wnt activation in stem cells requires PGE2, and suggests the PGE2/Wnt interaction is a master regulator of vertebrate regeneration and recovery.
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            Notch signaling: from the outside in.

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              Transactivation from Gal4-VP16 transgenic insertions for tissue-specific cell labeling and ablation in zebrafish.

              Prior studies with transgenic zebrafish confirmed the functionality of the transcription factor Gal4 to drive expression of other genes under the regulation of upstream activator sequences (UAS). However, widespread application of this powerful binary system has been limited, in part, by relatively inefficient techniques for establishing transgenic zebrafish and by the inadequacy of Gal4 to effect high levels of expression from UAS-regulated genes. We have used the Tol2 transposition system to distribute a self-reporting gene/enhancer trap vector efficiently throughout the zebrafish genome. The vector uses the potent, hybrid transcription factor Gal4-VP16 to activate expression from a UAS:eGFP reporter cassette. In a pilot screen, stable transgenic lines were established that express eGFP in reproducible patterns encompassing a wide variety of tissues, including the brain, spinal cord, retina, notochord, cranial skeleton and muscle, and can transactivate other UAS-regulated genes. We demonstrate the utility of this approach to track Gal4-VP16 expressing migratory cells in UAS:Kaede transgenic fish, and to induce tissue-specific cell death using a bacterial nitroreductase gene under UAS control. The Tol2-mediated gene/enhancer trapping system together with UAS transgenic lines provides valuable tools for regulated gene expression and for targeted labeling and ablation of specific cell types and tissues during early zebrafish development.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, USA )
                1932-6203
                2012
                4 January 2012
                : 7
                : 1
                Affiliations
                [1 ]Department of Cellular Biology and Anatomy, Georgia Health Sciences University, Augusta, Georgia, United States of America
                [2 ]Luminomics, Inc., Augusta, Georgia, United States of America
                [3 ]Cancer Center, Georgia Health Sciences University, Augusta, Georgia, United States of America
                [4 ]Institute of Developmental Genetics, Helmholtz Zentrum München, Neuherberg, Germany
                [5 ]Department of Surgery, Johns Hopkins University, Baltimore, Maryland, United States of America
                Center for Regenerative Therapies Dresden Group Leader CRTD, Germany
                Author notes

                Conceived and designed the experiments: SLW JA JRM VC KNB MJP MTS JSM. Performed the experiments: SLW JA JRM VC. Analyzed the data: SLW JA JRM VC JSM. Contributed reagents/materials/analysis tools: JA XX MD RWK JRM MJP. Wrote the paper: JSM.

                [¤a]

                Current address: Department of Cellular and Molecular Medicine, University of California San Diego, La Jolla, California, United States of America

                [¤b]

                Current address: Zoological Institute, Technical University of Braunschweig, Braunschweig, Germany

                [¤c]

                Current address: University of Kansas Cancer Center, Kansas City, Kansas, United States of America

                Article
                PONE-D-11-15816
                10.1371/journal.pone.0029916
                3251595
                22238673
                Walker et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
                Page count
                Pages: 14
                Categories
                Research Article
                Biology
                Biotechnology
                Genetic Engineering
                Developmental Biology
                Morphogenesis
                Organism Development
                Stem Cells
                Genetics
                Model Organisms
                Animal Models
                Molecular Cell Biology
                Signal Transduction
                Signaling in Selected Disciplines
                Neuroscience
                Sensory Systems

                Uncategorized

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