4B)

4B). system promotes the C-terminal processing of the ubiquitin-like protein Atg8 and its eventual attachment to phosphatidylethanolamine (PE). Atg8 processing occurs by means of its initial cleavage by Atg4, activation by the E1 enzyme Atg7 and transfer to the E2 enzyme Atg3, which lipidates Atg8.7,8 Unlike most of the other known autophagy gene products, a population of Atg8 remains associated with the autophagosome and is degraded in the lysosome.9 This unique localization pattern of Atg8 has made it an excellent tool for monitoring the formation of autophagosomes in multiple organisms including (yeast), (zebrafish), (rat) and (human) were aligned using the ClustalW program. Amino acid identities, and high and low similarities are highlighted in black, dark gray and light gray, respectively. Arrows indicate the potential cleavage and lipidation site. (C) Phylogenetic tree of Atg8 homologs in yeast, zebrafish and mammals. To determine when autophagy might be induced in embryos during development, we first analyzed the temporal expression pattern of Lc3 and Gabarap by RT-PCR. and transcripts were detected at the early cleavage stage (1C2 cell, 0 hours post-fertilization (hpf)) (Fig. 2A), indicating that both and mRNA are maternally deposited, since transcription of zebrafish zygotic DNA does not start until 3 hpf.24 To investigate whether the presence of transcripts corresponded to expression of the protein and if conjugation of Lc3 occurred, we examined the BD-AcAc 2 appearance of Lc3 with an antimammalian LC3 antibody that BD-AcAc 2 cross-reacts with the zebrafish homolog. We were able to detect a 16 kDa band that presumably corresponds to the Lc3-I form and a 14 kDa band that is equivalent to Lc3-II (Fig. 2B). In contrast to mouse embryos, in which LC3 conversion is usually observed even in metaphase II oocytes,25 the conversion of Lc3-I to Lc3-II was evident at BD-AcAc 2 48 hpf but not at 24 hpf. In addition, the total expression level of Lc3 increased in 48 hpf embryos compared to 24 hpf. The earliest time point at which we observed conversion to the Lc3-II form was at 32 hpf (data not shown), suggesting that autophagy is usually upregulated in zebrafish embryos at the pharyngula BD-AcAc 2 period. One possible explanation for the delay in autophagy induction is usually that other proteins essential for autophagy are not yet produced. In support of this hypothesis we found that although and are maternally deposited, some other predicted autophagy-related genes in zebrafish start transcription at, or after, approximately 24 hpf (Fig. 2C). In particular, mRNA transcripts for the zebrafish homologs of and were not detected at 0 hpf, indicating that they were not maternally deposited, whereas transcripts were detected at 0 hpf similar to and mRNAs are maternally deposited in zebrafish embryos. RT-PCR was performed with RNA isolated from 0, 23 and 42 hpf wild-type embryos using gene-specific primers. (B) Lc3-I converts to Lc3-II after 24 hpf. Protein extracts were isolated from 24 and 48 hpf wild-type embryos, analyzed by SDS-PAGE and detected using anti-LC3 antibody. (C) Some autophagy-related genes start transcription at or after 23 hpf. RT-PCR was performed with RNA isolated from 0, 23, and 42 hpf wild-type embryos using specific primers to the zebrafish and genes. Lc3-I to Lc3-II conversion CD79B is enhanced in the presence of lysosomal inhibitors Having established that this Lc3 protein is expressed and that it undergoes post-translational modification similar to its mammalian and yeast homologs during zebrafish embryonic development, we decided to study the extent of autophagy during embryogenesis by analyzing Lc3-II conversion. To evaluate the basal level of autophagy, we utilized two lysosomal protease inhibitors, pepstatin A, an inhibitor of cathepsins D and E, and E64d, an inhibitor of cathepsins B, H and L. 26 At the concentration and time used in this analysis, these inhibitors imposed no readily observable effects on embryo viability when applied in the embryo water (data not shown). We treated two days postfertilization (dpf) embryos with the above drugs for 24.