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      The zebrafish lysozyme C promoter drives myeloid-specific expression in transgenic fish

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          Abstract

          Background

          How different immune cell compartments contribute to a successful immune response is central to fully understanding the mechanisms behind normal processes such as tissue repair and the pathology of inflammatory diseases. However, the ability to observe and characterize such interactions, in real-time, within a living vertebrate has proved elusive. Recently, the zebrafish has been exploited to model aspects of human disease and to study specific immune cell compartments using fluorescent reporter transgenic lines. A number of blood-specific lines have provided a means to exploit the exquisite optical clarity that this vertebrate system offers and provide a level of insight into dynamic inflammatory processes previously unavailable.

          Results

          We used regulatory regions of the zebrafish lysozyme C ( lysC) gene to drive enhanced green fluorescent protein (EGFP) and DsRED2 expression in a manner that completely recapitulated the endogenous expression profile of lysC. Labeled cells were shown by co-expression studies and FACS analysis to represent a subset of macrophages and likely also granulocytes. Functional assays within transgenic larvae proved that these marked cells possess hallmark traits of myelomonocytic cells, including the ability to migrate to inflammatory sources and phagocytose bacteria.

          Conclusion

          These reporter lines will have utility in dissecting the genetic determinants of commitment to the myeloid lineage and in further defining how lysozyme-expressing cells participate during inflammation.

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          Most cited references61

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          A transposon-mediated gene trap approach identifies developmentally regulated genes in zebrafish.

          We report here development of a novel gene trap method in zebrafish using the Tol2 transposon system. First, we established a highly efficient transgenesis method in which a plasmid DNA containing the Tol2 transposon vector and the transposase mRNA synthesized in vitro were coinjected into one-cell stage embryos. The transposon vector inserted in the genome could be transmitted to the F1 progeny at high frequencies, and regulated gene expression by a specific promoter could be recapitulated in transgenic fish. Then we constructed a transposon-based gene trap vector containing a splice acceptor and the GFP gene, performed a pilot screen for gene trapping, and obtained fish expressing GFP in temporally and spatially restricted patterns. We confirmed the endogenous transcripts were indeed trapped by the insertions, and the insertion could interfere with expression of the trapped gene. We propose our gene trap approach should facilitate studies of vertebrate development and organogenesis.
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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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              The vascular anatomy of the developing zebrafish: an atlas of embryonic and early larval development.

              We have used confocal microangiography to examine and describe the vascular anatomy of the developing zebrafish, Danio rerio. This method and the profound optical clarity of zebrafish embryos make it possible to view the entire developing vasculature with unprecedented resolution. A staged series of three-dimensional images of the vascular system were collected beginning shortly after the onset of circulation at 1 day postfertilization through early- to midlarval stages at approximately 7 days postfertilization. Blood vessels in every region of the animal were imaged at each stage, and detailed "wiring patterns" were derived describing the interconnections between every major vessel. We present an overview of these data here in this paper and in an accompanying Web site "The interactive atlas of zebrafish vascular anatomy" online at (http://eclipse.nichd.nih.gov/nichd/lmg/redirect.html). We find a highly dynamic but also highly stereotypic pattern of vascular connections, with different sets of primitive embryonic vessels severing connections and rewiring in new configurations according to a reproducible plan. We also find that despite variation in the details of the vascular anatomy, the basic vascular plan of the developing zebrafish shows strong similarity to that of other vertebrates. This atlas will provide an invaluable foundation for future genetic and experimental studies of vascular development in the zebrafish.
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                Author and article information

                Journal
                BMC Dev Biol
                BMC Developmental Biology
                BioMed Central (London )
                1471-213X
                2007
                4 May 2007
                : 7
                : 42
                Affiliations
                [1 ]Department of Molecular Medicine and Pathology, School of Medical Sciences, The University of Auckland, Auckland, New Zealand
                Article
                1471-213X-7-42
                10.1186/1471-213X-7-42
                1877083
                17477879
                26150359-2ce8-4ca8-aab1-aee06bf7a8fe
                Copyright © 2007 Hall et al; licensee BioMed Central Ltd.

                This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

                History
                : 8 November 2006
                : 4 May 2007
                Categories
                Research Article

                Developmental biology
                Developmental biology

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