Identification and expression analysis of the zebrafish orthologues of the mammalian MAP1LC3 gene family

Abstract

Elsevier's AAM Policy: Authors retain the right to use the accepted author manuscript for personal use, internal institutional use and for permitted scholarly posting provided that these are not for purposes of commercial use or systematic distribution. Permitted scholarly postingVoluntary posting by an author on open websites operated by the author or the author's institution for scholarly purposes, as determined by the author, or (in connection with preprints) on preprint servers. 7th October, 2014Identification and expression analysis of the zebrafish orthologues of mammalian MAP1LC3 gene family AbstractAutophagy 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.

HS1BP3 negatively regulates autophagy by modulation of phosphatidic acid levels

“…At 2 days post fertilization (dpf), abundant GFP-LC3 puncta are present in the trunk region of the morphant compared with the control embryos (Fig. 2a–d) and this difference is even more pronounced after chloroquine treatment, known to block autophagosome degradation in zebrafish3435. On injection of in vitro -transcribed-capped human Hs1bp3 mRNA alongside the Hs1bp3 morpholino, we observe a partial rescue of the phenotype at 2 dpf both with and without chloroquine treatment (Fig.…”

Functionally conserved effects of rapamycin exposure on zebrafish

“…In vivo studies indicate that rapamycin results in developmental delay in different organisms including Drosophila melanogaster, zebrafish and mice (6,13,14). Previous studies using zebrafish have identified significant effects of rapamycin on autophagy (15), prevention of hepatic steatosis (16), heart development (17) and demonstrated the importance of zebrafish as a mitochondrial and ribosomal disease model (18,19). Although the zebrafish is an emerging model in drug-screening and in vivo disease models (20), evolutionarily conserved effects of rapamycin on the zebrafish transcriptome in addition to the dose-dependency in embryonic/larval size and pigmentation have not previously been studied.…”

Ratiometric assays of autophagic flux in zebrafish for analysis of familial Alzheimer’s disease-like mutations

“…48 and 52 hpf-old embryos were firstly dechorionated and deyolked and then placed in sample buffer (2% sodium dodecyl sulphate (SDS), 5% β-mercaptoethanol, 25% v/v glycerol, 0.0625 M Tris–HCl [pH 6.8], and bromophenol blue) (16), heated immediately to 95°C for 10 min and then sonicated (Diagenode Bioruptor UCD-200) in an ice-water bath at high power mode for 10 min before loading onto polyacrylamide gels for electrophoresis (see below). The 96 hpf-old larvae were not deyolked before lysis in sample buffer.…”