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      Studying Autophagy in Zebrafish

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          Abstract

          Autophagy is an evolutionarily conserved catabolic process which allows lysosomal degradation of complex cytoplasmic components into basic biomolecules that are recycled for further cellular use. Autophagy is critical for cellular homeostasis and for degradation of misfolded proteins and damaged organelles as well as intracellular pathogens. The role of autophagy in protection against age-related diseases and a plethora of other diseases is now coming to light; assisted by several divergent eukaryotic model systems ranging from yeast to mice. We here give an overview of different methods used to analyse autophagy in zebrafish—a relatively new model for studying autophagy—and briefly discuss what has been done so far and possible future directions.

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          The zebrafish reference genome sequence and its relationship to the human genome.

          Kerstin Howe, Matthew D. Clark, Carlos F Torroja … (2013)
          Zebrafish have become a popular organism for the study of vertebrate gene function. The virtually transparent embryos of this species, and the ability to accelerate genetic studies by gene knockdown or overexpression, have led to the widespread use of zebrafish in the detailed investigation of vertebrate gene function and increasingly, the study of human genetic disease. However, for effective modelling of human genetic disease it is important to understand the extent to which zebrafish genes and gene structures are related to orthologous human genes. To examine this, we generated a high-quality sequence assembly of the zebrafish genome, made up of an overlapping set of completely sequenced large-insert clones that were ordered and oriented using a high-resolution high-density meiotic map. Detailed automatic and manual annotation provides evidence of more than 26,000 protein-coding genes, the largest gene set of any vertebrate so far sequenced. Comparison to the human reference genome shows that approximately 70% of human genes have at least one obvious zebrafish orthologue. In addition, the high quality of this genome assembly provides a clearer understanding of key genomic features such as a unique repeat content, a scarcity of pseudogenes, an enrichment of zebrafish-specific genes on chromosome 4 and chromosomal regions that influence sex determination.
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            The role of Atg proteins in autophagosome formation.

            Noboru Mizushima, Tamotsu Yoshimori, Yoshinori Ohsumi (2011)
            Macroautophagy is mediated by a unique organelle, the autophagosome, which encloses a portion of cytoplasm for delivery to the lysosome. Autophagosome formation is dynamically regulated by starvation and other stresses and involves complicated membrane reorganization. Since the discovery of yeast Atg-related proteins, autophagosome formation has been dissected at the molecular level. In this review we describe the molecular mechanism of autophagosome formation with particular focus on the function of Atg proteins and the long-standing discussion regarding the origin of the autophagosome membrane.
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              Efficient In Vivo Genome Editing Using RNA-Guided Nucleases

              Woong Hwang, Yanfang Fu, Deepak Reyon … (2013)
              Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems have evolved in bacteria and archaea as a defense mechanism to silence foreign nucleic acids of viruses and plasmids. Recent work has shown that bacterial type II CRISPR systems can be adapted to create guide RNAs (gRNAs) capable of directing site-specific DNA cleavage by the Cas9 nuclease in vitro. Here we show that this system can function in vivo to induce targeted genetic modifications in zebrafish embryos with efficiencies comparable to those obtained using ZFNs and TALENs for the same genes. RNA-guided nucleases robustly enabled genome editing at 9 of 11 different sites tested, including two for which TALENs previously failed to induce alterations. These results demonstrate that programmable CRISPR/Cas systems provide a simple, rapid, and highly scalable method for altering genes in vivo, opening the door to using RNA-guided nucleases for genome editing in a wide range of organisms.
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                Author and article information

                Journal
                Journal ID (nlm-ta): Cells
                Journal ID (iso-abbrev): Cells
                Journal ID (publisher-id): cells
                Title: Cells
                Publisher: MDPI
                ISSN (Electronic): 2073-4409
                Publication date (Electronic): 09 July 2017
                Publication date Collection: September 2017
                Volume: 6
                Issue: 3
                Electronic Location Identifier: 21
                Affiliations
                [1 ]Department of Molecular Medicine, Institute of Basic Medical Sciences, University of Oslo, Sognsvannsveien 9, 0317 Oslo, Norway; b.j.mathai@ 123456medisin.uio.no
                [2 ]Institute of Biology Leiden, Leiden University, Einsteinweg 55, 2333 CC Leiden, The Netherlands; a.h.meijer@ 123456biology.leidenuniv.nl
                Author notes
                Author information
                https://orcid.org/0000-0002-1325-0725
                Article
                Publisher ID: cells-06-00021
                DOI: 10.3390/cells6030021
                PMC ID: 5617967
                PubMed ID: 28698482
                SO-VID: 29f49be4-63d2-4762-8639-e160869a2f94
                Copyright © © 2017 by the authors.
                License:

                Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ( http://creativecommons.org/licenses/by/4.0/).

                History
                Date received : 07 June 2017
                Date accepted : 03 July 2017
                Categories
                Subject: Review

                Keywords: autophagy,zebrafish,gfp-lc3,confocal microscopy,mitophagy,aggrephagy,xenophagy
                Data availability:
                Keywords: autophagy, zebrafish, gfp-lc3, confocal microscopy, mitophagy, aggrephagy, xenophagy

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