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      Nfatc1 Promotes Interstitial Cell Formation During Cardiac Valve Development in Zebrafish

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          Abstract

          <p class="first" id="d3304694e101">The transcription factor NFATC1 (nuclear factor of activated T-cell 1) has been implicated in cardiac valve formation in humans and mice, but we know little about the underlying mechanisms. To gain mechanistic understanding of cardiac valve formation at single-cell resolution and insights into the role of NFATC1 in this process, we used the zebrafish model as it offers unique attributes for live imaging and facile genetics. </p>

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          Efficient multiplex biallelic zebrafish genome editing using a CRISPR nuclease system.

          Li-En Jao, Susan Wente, Wenbiao Chen (2013)
          A simple and robust method for targeted mutagenesis in zebrafish has long been sought. Previous methods generate monoallelic mutations in the germ line of F0 animals, usually delaying homozygosity for the mutation to the F2 generation. Generation of robust biallelic mutations in the F0 would allow for phenotypic analysis directly in injected animals. Recently the type II prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated proteins (Cas) system has been adapted to serve as a targeted genome mutagenesis tool. Here we report an improved CRISPR/Cas system in zebrafish with custom guide RNAs and a zebrafish codon-optimized Cas9 protein that efficiently targeted a reporter transgene Tg(-5.1mnx1:egfp) and four endogenous loci (tyr, golden, mitfa, and ddx19). Mutagenesis rates reached 75-99%, indicating that most cells contained biallelic mutations. Recessive null-like phenotypes were observed in four of the five targeting cases, supporting high rates of biallelic gene disruption. We also observed efficient germ-line transmission of the Cas9-induced mutations. Finally, five genomic loci can be targeted simultaneously, resulting in multiple loss-of-function phenotypes in the same injected fish. This CRISPR/Cas9 system represents a highly effective and scalable gene knockout method in zebrafish and has the potential for applications in other model organisms.
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            Hematopoietic stem cells derive directly from aortic endothelium during development

            Julien Y. Bertrand, Neil Chi, Buyung Santoso … (2010)
            A major goal of regenerative medicine is to instruct formation of multipotent, tissue-specific stem cells from induced pluripotent stem cells (iPSCs) for cell replacement therapies. Generation of hematopoietic stem cells (HSCs) from iPSCs or embryonic stem cells (ESCs) is not currently possible, however, necessitating a better understanding of how HSCs normally arise during embryonic development. We previously showed that hematopoiesis occurs through four distinct waves during zebrafish development, with HSCs arising in the final wave in close association with the dorsal aorta. Recent reports have suggested that murine HSCs derive from hemogenic endothelial cells (ECs) lining the aortic floor1,2. Additional in vitro studies have similarly suggested that the hematopoietic progeny of ESCs arise through intermediates with endothelial potential3,4. In this report, we have utilized the unique strengths of the zebrafish embryo to image directly the birth of HSCs from the ventral wall of the dorsal aorta. Utilizing combinations of fluorescent reporter transgenes, confocal timelapse microscopy and flow cytometry, we have identified and isolated the stepwise intermediates as aortic hemogenic endothelium transitions to nascent HSCs. Finally, using a permanent lineage tracing strategy, we demonstrate that the HSCs generated from hemogenic endothelium are the lineal founders of the adult hematopoietic system.
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              Ubiquitous transgene expression and Cre-based recombination driven by the ubiquitin promoter in zebrafish.

              Megan K. Pugach, Leonard Zon, Christian Mosimann … (2010)
              Molecular genetics approaches in zebrafish research are hampered by the lack of a ubiquitous transgene driver element that is active at all developmental stages. Here, we report the isolation and characterization of the zebrafish ubiquitin (ubi) promoter, which drives constitutive transgene expression during all developmental stages and analyzed adult organs. Notably, ubi expresses in all blood cell lineages, and we demonstrate the application of ubi-driven fluorophore transgenics in hematopoietic transplantation experiments to assess true multilineage potential of engrafted cells. We further generated transgenic zebrafish that express ubiquitous 4-hydroxytamoxifen-controlled Cre recombinase activity from a ubi:cre(ERt2) transgene, as well as ubi:loxP-EGFP-loxP-mCherry (ubi:Switch) transgenics and show their use as a constitutive fluorescent lineage tracing reagent. The ubi promoter and the transgenic lines presented here thus provide a broad resource and important advancement for transgenic applications in zebrafish.
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                Author and article information

                Journal
                Title: Circulation Research
                Abbreviated Title: Circ Res
                Publisher: Ovid Technologies (Wolters Kluwer Health)
                ISSN (Print): 0009-7330
                ISSN (Electronic): 1524-4571
                Publication date Created: April 10 2020
                Publication date (Print): April 10 2020
                Volume: 126
                Issue: 8
                Pages: 968-984
                Affiliations
                [1 ]From the Department of Developmental Genetics, Max Planck Institute for Heart and Lung Research, Bad Nauheim, Germany (F.G., A.G., S.G., D.Y.R.S., A.B.-B.)
                [2 ]German Centre for Cardiovascular Research (DZHK), Partner Site Rhine-Main, Bad Nauheim (F.G., S.G., D.Y.R.S., A.B.-B.)
                Article
                DOI: 10.1161/CIRCRESAHA.119.315992
                PubMed ID: 32070236
                SO-VID: 26e82c22-601a-4563-b53e-b7d3d2c4d8db
                Copyright © © 2020
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