title: Imaging Subcellular Structures in the Living Zebrafish Embryo.
abstract: In vivo imaging provides unprecedented access to the dynamic behavior of cellular and subcellular structures in their natural context. Performing such imaging experiments in higher vertebrates such as mammals generally requires surgical access to the system under study. The optical accessibility of embryonic and larval zebrafish allows such invasive procedures to be circumvented and permits imaging in the intact organism. Indeed the zebrafish is now a well-established model to visualize dynamic cellular behaviors using in vivo microscopy in a wide range of developmental contexts from proliferation to migration and differentiation. A more recent development is the increasing use of zebrafish to study subcellular events including mitochondrial trafficking and centrosome dynamics. The relative ease with which these subcellular structures can be genetically labeled by fluorescent proteins and the use of light microscopy techniques to image them is transforming the zebrafish into an in vivo model of cell biology. Here we describe methods to generate genetic constructs that fluorescently label organelles, highlighting mitochondria and centrosomes as specific examples. We use the bipartite Gal4-UAS system in multiple configurations to restrict expression to specific cell-types and provide protocols to generate transiently expressing and stable transgenic fish. Finally, we provide guidelines for choosing light microscopy methods that are most suitable for imaging subcellular dynamics.
authors.affiliation: Institute of Neuronal Cell Biology, Technische Universität München; Peter.Engerer@tum.de.
authors.forename: Peter
authors.initials: P
authors.lastname: Engerer
authors.affiliation: Institute of Neuronal Cell Biology, Technische Universität München; Cell Biology, Department of Biology, Faculty of Science, Utrecht University.
authors.forename: Gabriela
authors.initials: G
authors.lastname: Plucinska
authors.affiliation: Institute of Neuronal Cell Biology, Technische Universität München; Faculty of Biology, Ludwig-Maximilians-Universität-München.
authors.forename: Rachel
authors.initials: R
authors.lastname: Thong
authors.affiliation: Institute of Neuronal Cell Biology, Technische Universität München.
authors.forename: Laura
authors.initials: L
authors.lastname: Trovò
authors.affiliation: Adolf-Butenandt-Institute, Biochemistry, Ludwig-Maximilians-Universität-München; German Center for Neurodegenerative Diseases; Laboratory of Brain Development and Repair, The Rockefeller University.
authors.forename: Dominik
authors.initials: D
authors.lastname: Paquet
authors.affiliation: Institute of Neuronal Cell Biology, Technische Universität München; Leanne.Godinho@tum.de.
authors.forename: Leanne
authors.initials: L
authors.lastname: Godinho
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D018385:Centrosome / Q000502:physiology*; D004625:Embryo, Nonmammalian / Q000166:cytology*; D005260:Female; D005456:Fluorescent Dyes / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008297:Male; D018613:Microscopy, Confocal; D008928:Mitochondria / Q000502:physiology*; D013194:Staining and Labeling / Q000379:methods; D015027:Zebrafish / Q000196:embryology*
chemical_list: D001426:Bacterial Proteins; C485182:Cyan Fluorescent Protein; D005456:Fluorescent Dyes; D008164:Luminescent Proteins; C054562:yellow fluorescent protein, Bacteria; D049452:Green Fluorescent Proteins