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.
"Van Ham et al. (2010) recently introduced a new transgenic fluorescent marker allowing in vivo imaging of apoptotic cells to understand their dynamics. They fused secreted A5 (secA5) protein to yellow fluorescent protein (YFP) (secA5-YFP) and showed that this fusion product specifically labels apoptotic cells in living ZF; the fluorescent probe can characterize patterns of apoptosis in living ZF larvae and visualize cell death at single-cell resolution in vivo. "
[Show abstract][Hide abstract] ABSTRACT: Zebrafish (ZF, Danio rerio) has emerged as an important and popular model species to study different human diseases. Key regulators of skeletal development and calcium metabolism are highly conserved between mammals and zebrafish. The corresponding orthologs share significant sequence similarities and an overlap in expression patterns when compared to mammals, making ZF a potential model for the study of mineralization related disorders and soft tissue mineralization. To characterize the function of early mineralization-related genes in ZF, these genes can be knocked down by injecting morpholinos (MOs) into early stage embryos. Validation of the MO needs to be performed and the concern of aspecific effects can be addressed by applying one or more independent techniques to knock down the gene of interest. Post-injection assessment of early mineralization defects can be done using general light microscopy, calcein staining, Alizarin red staining, Alizarin red-Alcian blue double staining and by the use of transgenic lines. Examination of general molecular defects can be done by performing protein and gene expression analysis, and more specific processes can be explored by investigating ectopic mineralization related mechanisms such as apoptosis and mitochondrial dysfunction. In this paper, we will discuss all details about the aforementioned techniques; shared knowledge will be very useful for the future investigation of zebrafish models for ectopic mineralization disorders and to understand the underlying pathways involved in soft tissue calcification.
Frontiers in Genetics 04/2013; 4(74):1-17. DOI:10.3389/fgene.2013.00074
"TABLE 1. Cancer-Related Drug Screenings Using Zebrafish Embryos Targeted molecules Detection method Alternative method References Cytotoxic molecules TUNNEL assay/p21 and mdm2 in situ hybridization Ubiquitous SecA5-YFP (Langheinrich et al., 2002; van Ham et al., 2010 "
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