Due to their optical clarity and short development time, zebrafish are ideal for studying development. Through a BLAST search, we found that homologs of multiple autophagy-related genes, including ulk1/atg1, atg2, atg3, atg4, atg5, beclin 1/atg6, atg7, lc3/atg8, atg9, atg12
are present in the zebrafish genome, which suggests that this organism may be useful as a model system for studying the function and mechanism of autophagy. It has recently been reported that autophagy is required in mice for preimplantation embryonic development; autophagosomes are observed in GFP-LC3 mouse embryos at the one- to four-cell stage, and autophagy-defective embryos fail to develop beyond the four- and eight-cell stage.25
In contrast to zebrafish development that proceeds quickly after fertilization, mammalian development progresses relatively slowly. It is unknown if autophagy is required during the one- to four-cell stage in nonmammalian vertebrates such as fish, amphibians, or birds; however, it is unlikely that zebrafish undergo autophagy during the 1–4 cell stages as ulk1a, ulk1b, atg9a
(genes that are presumably required for autophagy as they are in other organisms) were not expressed in 0 hpf embryos (). However, by 24 hpf ulk1a, ulk1b, atg9b
and to a lesser extent atg9a
transcripts were easily detected. PE-conjugated Lc3 was not seen in 24 hpf embryos, but Lc3-II was clearly observed by 48 hpf. The appearance of transcripts for genes necessary for autophagy by 24 hpf, the subsequent appearance of PE-conjugated Lc3 by 48 hpf, and the high basal level of autophagy by the 48 hpf time point opens the possibility that autophagy may play a role in zebrafish development during later developmental stages.
In recent years autophagy has been shown to play multiple important roles in human health and pathology.4,5,31
Therefore, it is likely that chemical screens for compounds that regulate autophagy, and subsequent dissection of the mechanisms involved, will become an important area of research. Indeed a screen using a panel of 253 compounds was recently performed to identify drugs that induce autophagy.30
As a secondary screen these researchers used a zebrafish Huntington disease model to determine if these drugs caused clearance of aggregate-prone proteins, but they did not directly show autophagy induction in zebrafish.30
Here, we have shown for the first time that aqueous administration of rapamycin, calpeptin and 2’5’-ddA, and to a lesser extent verapamil, induce autophagy ( and ). Our findings not only provide the first instance of monitoring chemical upregulation of autophagy in zebrafish, but also corroborate the hypothesis that these drugs induce autophagy in this system. Because drug administration can be achieved simply by aqueous exposure, zebrafish would be an ideal organism for primary or secondary in vivo small molecule screens for compounds that regulate autophagy.32,33
The optical transparency of zebrafish combined with their external development make them an ideal organism for in vivo microscopy analysis, especially during the developmental period. We therefore have created transgenic GFP-Lc3 and GFP-Gabarap zebrafish lines. Both GFP-Lc3 and GFP-Gabarap accumulate in lysosomes in response to drug treatment, suggesting these GFP-tagged Atg8 homologs are incorporated into autophagosomes as found in other organisms. These fish will provide an essential tool for the study of autophagy by allowing monitoring of autophagosome formation in this model system as has been done in other organisms.