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      Developmental Expression of the Nfe2-Related Factor (Nrf) Transcription Factor Family in the Zebrafish, Danio rerio

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          Abstract

          Transcription factors in the CNC-bZIP family (NFE2, NRF1, NRF2 and NRF3) regulate genes with a wide range of functions in response to both physiological and exogenous signals, including those indicating changes in cellular redox status. Given their role in helping to maintain cellular homeostasis, it is imperative to understand the expression, regulation, and function of CNC-bZIP genes during embryonic development. We explored the expression and function of six nrf genes ( nfe2, nrf1a, nrf1b, nrf2a, nrf2b, and nrf3) using zebrafish embryos as a model system. Analysis by microarray and quantitative RT-PCR showed that genes in the nrf family were expressed throughout development from oocytes to larvae. The spatial expression of nrf3 suggested a role in regulating the development of the brain, brachia and pectoral fins. Knock-down by morpholino anti-sense oligonucleotides suggested that none of the genes were necessary for embryonic viability, but nfe2 was required for proper cellular organization in the pneumatic duct and subsequent swim bladder function, as well as for proper formation of the otic vesicles. nrf genes were induced by the oxidant tert-butylhydroperoxide, and some of this response was regulated through family members Nrf2a and Nrf2b. Our results provide a foundation for understanding the role of nrf genes in normal development and in regulating the response to oxidative stress in vertebrate embryos.

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          Zebrafish hox clusters and vertebrate genome evolution.

          HOX genes specify cell fate in the anterior-posterior axis of animal embryos. Invertebrate chordates have one HOX cluster, but mammals have four, suggesting that cluster duplication facilitated the evolution of vertebrate body plans. This report shows that zebrafish have seven hox clusters. Phylogenetic analysis and genetic mapping suggest a chromosome doubling event, probably by whole genome duplication, after the divergence of ray-finned and lobe-finned fishes but before the teleost radiation. Thus, teleosts, the most species-rich group of vertebrates, appear to have more copies of these developmental regulatory genes than do mammals, despite less complexity in the anterior-posterior axis.
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            Transplantation and in vivo imaging of multilineage engraftment in zebrafish bloodless mutants.

            The zebrafish is firmly established as a genetic model for the study of vertebrate blood development. Here we have characterized the blood-forming system of adult zebrafish. Each major blood lineage can be isolated by flow cytometry, and with these lineal profiles, defects in zebrafish blood mutants can be quantified. We developed hematopoietic cell transplantation to study cell autonomy of mutant gene function and to establish a hematopoietic stem cell assay. Hematopoietic cell transplantation can rescue multilineage hematopoiesis in embryonic lethal gata1-/- mutants for over 6 months. Direct visualization of fluorescent donor cells in embryonic recipients allows engraftment and homing events to be imaged in real time. These results provide a cellular context in which to study the genetics of hematopoiesis.
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              Characterization of housekeeping genes in zebrafish: male-female differences and effects of tissue type, developmental stage and chemical treatment

              Background Research using the zebrafish model has experienced a rapid growth in recent years. Although real-time reverse transcription PCR (QPCR), normalized to an internal reference ("housekeeping") gene, is a frequently used method for quantifying gene expression changes in zebrafish, many commonly used housekeeping genes are known to vary with experimental conditions. To identify housekeeping genes that are stably expressed under different experimental conditions, and thus suitable as normalizers for QPCR in zebrafish, the present study evaluated the expression of eight commonly used housekeeping genes as a function of stage and hormone/toxicant exposure during development, and by tissue type and sex in adult fish. Results QPCR analysis was used to quantify mRNA levels of bactin1, tubulin alpha 1(tuba1), glyceraldehyde-3-phosphate dehydrogenase (gapdh), glucose-6-phosphate dehydrogenase (g6pd), TATA-box binding protein (tbp), beta-2-microglobulin (b2m), elongation factor 1 alpha (elfa), and 18s ribosomal RNA (18s) during development (2 – 120 hr postfertilization, hpf); in different tissue types (brain, eye, liver, heart, muscle, gonads) of adult males and females; and after treatment of embryos/larvae (24 – 96 hpf) with commonly used vehicles for administration and agents that represent known environmental endocrine disruptors. All genes were found to have some degree of variability under the conditions tested here. Rank ordering of expression stability using geNorm analysis identified 18s, b2m, and elfa as most stable during development and across tissue types, while gapdh, tuba1, and tpb were the most variable. Following chemical treatment, tuba1, bactin1, and elfa were the most stably expressed whereas tbp, 18s, and b2m were the least stable. Data also revealed sex differences that are gene- and tissue-specific, and treatment effects that are gene-, vehicle- and ligand-specific. When the accuracy of QPCR analysis was tested using different reference genes to measure suppression of cyp19a1b by an estrogen receptor antagonist and induction of cyp1a by an arylhydrocarbon receptor agonist, the direction and magnitude of effects with stable and unstable genes differed. Conclusion This study provides data that can be expected to aid zebrafish researchers in their initial choice of housekeeping genes for future studies, but underlines the importance of further validating housekeeping genes for each new experimental paradigm and fish species.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, USA )
                1932-6203
                2013
                24 October 2013
                25 November 2013
                : 8
                : 10
                : e79574
                Affiliations
                [1 ]Biology Department, Woods Hole Oceanographic Institution, Woods Hole, Massachusetts, United States of America
                [2 ]Biology Department, Bates College, Lewiston, Maine, United States of America
                [3 ]Andrew McArthur Consulting, Hamilton, Ontario, Canada
                [4 ]Department of Biology and Marine Biology, Roger Williams University, Bristol, Rhode Island, United States of America
                Deakin School of Medicine, Australia
                Author notes

                Competing Interests: Dr. McArthur is the sole employee of Andrew McArthur Consulting. Dr. McArthur, a long-time colleague formerly at the Marine Biological Laboratory in Woods Hole, is a collaborator whose effort is paid for (by the grant to M.E.H.) as a consultant. The fact that Dr. McArthur collaborates with the authors as a private consultant does not alter their adherence to all the PLOS ONE policies on sharing data and materials.

                Conceived and designed the experiments: LMW ARTL AGM JJS MEH. Performed the experiments: LMW ARTL JVG. Analyzed the data: LMW ARTL AGM JVG RMS. Contributed reagents/materials/analysis tools: JJS RMS MEH. Wrote the manuscript: LMW ARTL AGM JVG JJS RMS MEH.

                Article
                PONE-D-13-12814
                10.1371/journal.pone.0079574
                3840143
                24298298
                dd808334-736c-497d-9a7d-188ccaf152e5
                Copyright @ 2013

                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.

                History
                : 27 March 2013
                : 24 September 2013
                Funding
                This work was supported, in whole or in part, by National Institutes of Health grants F32ES019832 (to L.M.W.), F32ES017585 (to A.R.T.-L.), R01ES015912 (to J.J.S.), and R01ES016366 (to M.E.H.). This work was also supported by Walter A. and Hope Noyes Smith and the J. Seward Johnson Fund. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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