Assaying autophagic activity in transgenic GFP-Lc3 and GFP-Gabarap zebrafish embryos

Life Sciences Institute and Departments of Molecular, Cellular and Developmental Biology, and Biological Chemistry, University of Michigan, Ann Arbor, MI 48109, USA.
Autophagy (Impact Factor: 11.75). 06/2009; 5(4):520-6. DOI: 10.4161/auto.5.4.7768
Source: PubMed


Autophagy mediates the bulk turnover of cytoplasmic constituents in lysosomes. During embryonic development in animals, a dramatic degradation of yolk proteins and synthesis of zygotic proteins takes place, leading to intracellular remodeling and cellular differentiation. Zebrafish represents a unique system to study autophagy due in part to its rapid embryonic development relative to other vertebrates. The technical advantages of this organism make it uniquely suited to various studies including high-throughput drug screens. To study autophagy in zebrafish, we identified two zebrafish Atg8 homologs, lc3 and gabarap, and generated two transgenic zebrafish lines expressing GFP-tagged versions of the corresponding proteins. Similar to yeast Atg8 and mammalian LC3, zebrafish Lc3 undergoes post-translational modification starting at the pharyngula stage during embryonic development. We observed a high level of autophagy activity in zebrafish embryos, which can be further upregulated by the TOR inhibitor rapamycin or the calpain inhibitor calpeptin. In addition, zebrafish Gabarap accumulates within lysosomes upon autophagy induction. Thus, we established a convenient zebrafish tool to assay autophagic activity during embryogenesis in vivo.

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    • "Wild-type embryos were collected and treated with the following drugs dissolved in DMSO. Rapamycin, which is a known inducer of autophagy, was added to a final concentration of 1 mM to 48 hpf embryos for 24 h [28] [29]. Chloroquine, a chemical that increases lysosomal pH thus inhibiting autophagy was added at a concentration of 50 mM to 56 hpf embryos for 16 h [25,30]. "
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    ABSTRACT: Autophagy is the principle pathway in a cell involved in clearing damaged proteins and organelles. Therefore autophagy is necessary to maintain turnover balance of peptides and homeostasis. Autophagy occurs at basal levels under normal conditions but can be upregulated by chemical inducers or stress conditions. The zebrafish (Danio rerio) serves as a versatile tool to understand the function of genes implicated in autophagy. We report the identification of the zebrafish orthologue of mammalian genes MAP1LC3A (map1lc3a) and MAP1LC3B (map1lc3b) by phylogenetic and conserved synteny analysis and examine their expression during embryonic development. Both the zebrafish map1lc3a and map1lc3b genes show maternally contributed expression in early embryogenesis. However, levels of map1lc3a transcript steadily increase until at least 120 hours post fertilisation (hpf) while the levels of map1lc3b show a more variable pattern across developmental time. We have also validated the LC3II/LC3I immunoblot assay in the presence of chloroquine (a lysosomal proteolysis inhibitor). We found that the LC3II/LC3I ratio is significantly increased in the presence of sodium azide treatment supporting that hypoxia induces autophagy in zebrafish. This was supported by our qPCR assay that showed an increase in map1lc3a transcript levels in the presence of sodium azide. In contrast levels of map1lc3b transcripts were reduced in the presence of rapamycin but showed no significant difference in the presence of sodium azide. Our study thus identifies the zebrafish orthologues of MAP1LC3A & MAP1LC3B and supports the use of zebrafish for the interaction between hypoxia, development and autophagy.
    Experimental Cell Research 10/2014; 328(1). DOI:10.1016/j.yexcr.2014.07.014 · 3.25 Impact Factor
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    • "Autophagy can be analyzed in zebrafish by observing induction of the LC3-II protein by western immunoblotting using a human antibody against LC3 that cross-reacts with zebrafish Lc3 (He et al., 2009; He and Klionsky, 2010). Transgenic zebrafish have also been developed that express GFP fused to Lc3 (He et al., 2009). "
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    ABSTRACT: Rodent models have been extensively used to investigate the cause and mechanisms behind Alzheimer's disease. Despite many years of intensive research using these models we still lack a detailed understanding of the molecular events that lead to neurodegeneration. Although zebrafish lack the complexity of advanced cognitive behaviors evident in rodent models they have proven to be a very informative model for the study of human diseases. In this review we give an overview of how the zebrafish has been used to study Alzheimer's disease. Zebrafish possess genes orthologous to those mutated in familial Alzheimer's disease and research using zebrafish has revealed unique characteristics of these genes that have been difficult to observe in rodent models. The zebrafish is becoming an increasingly popular model for the investigation of Alzheimer's disease and will complement studies using other models to help complete our understanding of this disease.
    Frontiers in Genetics 06/2014; 5:189. DOI:10.3389/fgene.2014.00189
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    • "The changes in myoD1 and shh expression, together with the reduced locomotor activity, observed in ambra1 morphants occur at very early developmental stages, when embryonic cells are likely not yet competent for autophagy, e.g. the biological process in which Ambra1 has a well-established role. Actually, autophagy can be observed in zebrafish embryos starting from 32 hpf [29], and thus relatively late compared with the onset of the muscle developmental defects. Interestingly, ambra1 depletion seems to interfere with the gene expression program responsible for correct muscle development, as suggested by the displaced expression of shh [14] and of myoD1, however it is still unclear whether these effects are related to Ambra1 pro-autophagic roles. "
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    ABSTRACT: The essential role of autophagy in muscle homeostasis has been clearly demonstrated by phenotype analysis of mice with muscle-specific inactivation of genes encoding autophagy-related proteins. Ambra1 is a key component of the Beclin 1 complex and, in zebrafish, it is encoded by two paralogous genes, ambra1a and ambra1b, both required for normal embryogenesis and larval development. In this study we focused on the function of Ambra1, a positive regulator of the autophagic process, during skeletal muscle development by means of morpholino (MO)-mediated knockdown and compared the phenotype of zebrafish Ambra1-depleted embryos with that of Ambra1gt/gt mouse embryos. Morphological analysis of zebrafish morphant embryos revealed that silencing of ambra1 impairs locomotor activity and muscle development, as well as myoD1 expression. Skeletal muscles in ATG-morphant embryos displayed severe histopathological changes and contained only small areas of organized myofibrils that were widely dispersed throughout the cell. Double knockdown of ambra1a and ambra1b resulted in a more severe phenotype whereas defects were much less evident in splice-morphants. The morphants phenotypes were effectively rescued by co-injection with human AMBRA1 mRNA. Together, these results indicate that ambra1a and ambra1b are required for the correct development and morphogenesis of skeletal muscle.
    PLoS ONE 06/2014; 9(6):e99210. DOI:10.1371/journal.pone.0099210 · 3.23 Impact Factor
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