title: Dynamic visualization of transcription and RNA subcellular localization in zebrafish.
abstract: Live imaging of transcription and RNA dynamics has been successful in cultured cells and tissues of vertebrates but is challenging to accomplish in vivo. The zebrafish offers important advantages to study these processes--optical transparency during embryogenesis, genetic tractability and rapid development. Therefore, to study transcription and RNA dynamics in an intact vertebrate organism, we have adapted the MS2 RNA-labeling system to zebrafish. By using this binary system to coexpress a fluorescent MS2 bacteriophage coat protein (MCP) and an RNA of interest tagged with multiple copies of the RNA hairpin MS2-binding site (MBS), live-cell imaging of RNA dynamics at single RNA molecule resolution has been achieved in other organisms. Here, using a Gateway-compatible MS2 labeling system, we generated stable transgenic zebrafish lines expressing MCP, validated the MBS-MCP interaction and applied the system to investigate zygotic genome activation (ZGA) and RNA localization in primordial germ cells (PGCs) in zebrafish. Although cleavage stage cells are initially transcriptionally silent, we detect transcription of MS2-tagged transcripts driven by the βactin promoter at ∼ 3-3.5 h post-fertilization, consistent with the previously reported ZGA. Furthermore, we show that MS2-tagged nanos3 3'UTR transcripts localize to PGCs, where they are diffusely cytoplasmic and within larger cytoplasmic accumulations reminiscent of those displayed by endogenous nanos3. These tools provide a new avenue for live-cell imaging of RNA molecules in an intact vertebrate. Together with new techniques for targeted genome editing, this system will be a valuable tool to tag and study the dynamics of endogenous RNAs during zebrafish developmental processes.
authors.affiliation: Department of Developmental and Molecular Biology, Albert Einstein College of Medicine, Yeshiva University, 1300 Morris Park Av, Bronx, NY 10461, USA.
authors.forename: Philip D
authors.initials: PD
authors.lastname: Campbell
authors.affiliation: Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Yeshiva University, 1300 Morris Park Av, Bronx, NY 10461, USA Friedrich Meischer Institute for Biomedical Research, Maulbeerstrasse 66, Basel 4058, Switzerland.
authors.forename: Jeffrey A
authors.initials: JA
authors.lastname: Chao
authors.affiliation: Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Yeshiva University, 1300 Morris Park Av, Bronx, NY 10461, USA Department of Neuroscience, Albert Einstein College of Medicine, Yeshiva University, 1300 Morris Park Av, Bronx, NY 10461, USA Department of Cell Biology, Albert Einstein College of Medicine, Yeshiva University, 1300 Morris Park Av, Bronx, NY 10461, USA.
authors.forename: Robert H
authors.initials: RH
authors.lastname: Singer
authors.affiliation: Department of Developmental and Molecular Biology, Albert Einstein College of Medicine, Yeshiva University, 1300 Morris Park Av, Bronx, NY 10461, USA Department of Neuroscience, Albert Einstein College of Medicine, Yeshiva University, 1300 Morris Park Av, Bronx, NY 10461, USA florence.marlow@einstein.yu.edu.
authors.forename: Florence L
authors.initials: FL
authors.lastname: Marlow
mesh_terms: D020413:3' Untranslated Regions / Q000235:genetics; D000818:Animals; D030801:Animals, Genetically Modified; D001665:Binding Sites; D053487:DEAD-box RNA Helicases / Q000378:metabolism; D004625:Embryo, Nonmammalian / Q000378:metabolism; D016678:Genome; D005854:Germ Cells / Q000166:cytology / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism; D019913:Nuclear Localization Signals / Q000378:metabolism; D011485:Protein Binding; D055503:Protein Multimerization; D012313:RNA / Q000378:metabolism*; D015203:Reproducibility of Results; D013347:Subcellular Fractions / Q000378:metabolism; D014158:Transcription, Genetic; D014764:Viral Proteins / Q000378:metabolism; D015027:Zebrafish / Q000196:embryology / Q000235:genetics / Q000378:metabolism*; D029961:Zebrafish Proteins / Q000378:metabolism; D015053:Zygote / Q000378:metabolism
chemical_list: D020413:3' Untranslated Regions; D019913:Nuclear Localization Signals; D014764:Viral Proteins; D029961:Zebrafish Proteins; D049452:Green Fluorescent Proteins; D012313:RNA; C498289:ddx4 protein, zebrafish; D053487:DEAD-box RNA Helicases
keywords: In vivo RNA labeling; MS2; Transcription; Transgenic zebrafish