Live imaging of apoptotic cells in zebrafish
ABSTRACT Many debilitating diseases, including neurodegenerative diseases, involve apoptosis. Several methods have been developed for visualizing apoptotic cells in vitro or in fixed tissues, but few tools are available for visualizing apoptotic cells in live animals. Here we describe a genetically encoded fluorescent reporter protein that labels apoptotic cells in live zebrafish embryos. During apoptosis, the phospholipid phosphatidylserine (PS) is exposed on the outer leaflet of the plasma membrane. The calcium-dependent protein Annexin V (A5) binds PS with high affinity, and biochemically purified, fluorescently labeled A5 probes have been widely used to detect apoptosis in vitro. Here we show that secreted A5 fused to yellow fluorescent protein specifically labels apoptotic cells in living zebrafish. We use this fluorescent probe to characterize patterns of apoptosis in living zebrafish larvae and to visualize neuronal cell death at single-cell resolution in vivo.
- SourceAvailable from: Javier Terriente[Show abstract] [Hide abstract]
ABSTRACT: The introduction of mechanism-based targeted therapies to treat human cancers is fruit of decades of research into the molecular basis of cancer pathogenesis. Despite the growing knowledge about the molecular mechanisms governing its causes and progression, there is a lack of effective treatments for many types of cancer. The expensive and time-consuming preclinical pipeline for testing molecules slows the discovery of new therapies. Therefore, it is important to consider alternative methodologies both for accelerating therapeutic discovery and reducing costs. In that regard, zebrafish is becoming an attractive model for fast and efficient drug screening. Its use has expanded to many disease research areas, and the post-genomic era has led to the progression of functional studies and boosted the development of general databases, such as ZFIN, and the emergence of more specialized ones, including several catalogues of transgenic reporter screens. Taken together, they provide to the scientific community many tools that could be used for drug discovery. The use of zebrafish in cancer drug screenings could help to economize time and resources even more if we rationalize its use: we could use embryonic screens to identify drugs that address general hallmarks of cancer, and use adults for finding molecules that target specific cancer models. © 2012 Wiley Periodicals, Inc., a Wiley company.Developmental Dynamics 02/2013; 242(2). DOI:10.1002/dvdy.23912 · 2.67 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Vertebrate hematopoietic stem cells (HSCs) emerge in the aorta-gonad-mesonephros (AGM) region from "hemogenic" endothelium. Here we show that the proinflammatory cytokine interferon-γ (IFN-γ) and its receptor Crfb17 positively regulate HSC development in zebrafish. This regulation does not appear to modulate the proliferation or survival of HSCs or endothelial cells, but rather the endothelial-to-HSC transition. Notch signaling and blood flow positively regulate the expression of ifng and crfb17 in the AGM. Notably, IFN-γ overexpression partially rescues the HSC loss observed in the absence of blood flow or Notch signaling. Importantly, IFN-γ signaling acts cell autonomously to control the endothelial-to-HSC transition. IFN-γ activates Stat3, an atypical transducer of IFN-γ signaling, in the AGM, and Stat3 inhibition decreases HSC formation. Together, our findings uncover a developmental role for an inflammatory cytokine and place its action downstream of Notch signaling and blood flow to control Stat3 activation and HSC emergence. Copyright © 2014 Elsevier Inc. All rights reserved.
Article: Aspects of Larval Rearing[Show abstract] [Hide abstract]
ABSTRACT: Fish-and zebrafish (Danio rerio) in particular-are now the second-most used biomedical model in the United Kingdom. The use of fish in research rose by 23% in 2011, primarily reflecting a rise in the use of zebrafish. Despite the increasing importance of zebrafish as a biomedical model system, there are currently no legislative guidelines or requirements for larval husbandry in the United Kingdom, the European Union, or the United States. This has led to a variety of procedures and methods being developed for larval rearing, many of which are not derived from peer-reviewed protocols. This article reviews published work relating to larval rearing and some unpublished protocols to establish optimized and standardized husbandry procedures.ILAR journal / National Research Council, Institute of Laboratory Animal Resources 06/2012; 53(2):169-78. DOI:10.1093/ilar.53.2.169 · 1.05 Impact Factor