Ambra1knockdown in zebrafish leads to incomplete development due to severe defects in organogenesis

Abstract

These first authors contributed equally to this work. ‡ These senior authors contributed equally to this work.Keywords: Ambra1, zebrafish, autophagy, development, morpholino Abbreviations: AMBRA1, autophagy/Beclin 1 regulator 1; ATG, autophagy-related proteins; ATG5, autophagy-related 5; ATG7, autophagy-related 7; ATG14, autophagy-related 14; ATGMOs, translation blocking morpholinos; Baf A1, bafilomycin A 1 ;BCL2, B-cell CLL/lymphoma 2; BECN1, Beclin 1, autophagy-related; cDNA, complementary DNA; chd, chordin; chrm4, cholinergic receptor, muscarinic 4; CNS, central nervous system; DIG, digoxigenin; dgkz, diacylglycerol kinase, zeta; DYNLL1, dynein, light chain, LC8-type 1; EGFP, enhanced green fluorescent protein; ER, endoplasmic reticulum; EST, expressed sequence tags; gsc, goosecoid homeobox; KIAA0226, RUBICON/RUN domain and cysteine-rich domain containing, Beclin 1-interacting protein; MAP1LC3/LC3, microtubule-associated protein 1A/1B-light chain 3; mdk, midkine (neurite growthpromoting factor 2); MISMMOs, five-nucleotide-mismatched MOs; MOs, morpholinos; mRNA, messenger RNA; NBT/BCIP, 5-nitro-blue tetrazolium chloride/bromo-4-chloro-3'-indolyl phosphate p-toluidine salt; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; PFA, paraformaldehyde; PIK3C3, phosphatidylinositol 3-kinase, catalytic subunit type 3; PIK3R4/Vps15/p150, phosphoinositide-3-kinase, regulatory subunit 4; phf21a, PHD finger protein 21A; qPCR, real timepolymerase chain reaction; RACE, rapid amplification of cDNA ends; RT-PCR, reverse transcriptase-polymerase chain reaction; SEM, scanning electron microscopy; S.E.M., standard error of the mean; shha, sonic hedgehog a; SH3GLB1, SH3-domain GRB2-like endophilin B1; SPLICMOs, splicing blocking morpholinos; TP53, tumor protein p53; TUNEL, terminal deoxynucleotidyl transferase-mediated fluorescein-dUTP nick-end labeling assay; UVRAG, UV radiation resistance-associated; WMISH, whole mount in situ hybridization AMBRA1 is a positive regulator of the BecN1-dependent program of autophagy recently identified in mouse. in this study, we cloned the full-length cDNAs of ambra1a and ambra1b zebrafish paralogous genes. As in mouse, both Ambra1 proteins contain the characteristic WD40 repeat region. The transcripts of both genes are present as maternal RNAs in the eggs and display a gradual decline until 8 hpf, being replaced by zygotic mRNAs from 12 hpf onwards. After 24 hpf, the transcripts are mainly localized in the head, suggesting a possible role in brain development. To check their developmental roles, we adopted morpholino knockdown to block either translation (ATGMOs) or splicing (sPLicMOs). Treatment with ATGMOs causes severe embryonic malformations, as prelarvae could survive for only 3 and 4 days in ambra1a and b morphants, respectively. Treatment with sPLicMOs led to developmental defects only at a late stage, indicating the importance of maternally supplied ambra1 transcripts. Analysis of the levels of Lc3-ii, an autophagosomespecific marker, in the presence of lysosome inhibitors e...

Autophagy is essential for cardiac morphogenesis during vertebrate development

“…Mice lacking Ambra1 are embryonic lethal at d E10-14 and show defective neural tube development and hyperproliferation of neural tissues. (In accord with this result, Benato and coworkers reported that knockdown of the zebrafish Ambra1 orthologs causes defects in body morphogenesis and brain development 20 ). rb1cc1 −/− mice are embryonic lethal at d E13.5-16.5, and display defects in heart and liver development.…”

“…The result is aberrant development of the cardiac chambers and the atrioventricular canal, as well as increased embryonic lethality. While previous studies have indicated that specific autophagy factors are required for development of the Drosophila eye, 12,43 the zebrafish brain, 19,20 and for mouse development (reviewed in ref. 7), our results provide the first evidence that autophagy contributes to the cardiac morphogenic program during vertebrate development.…”

“…atg5 and becn1 transcripts appeared to decrease at the early gastrulation (germ ring) stage, similar to what was reported for atg5, 19 ambra1a, and ambra1b. 20 He and coworkers previously reported that several transcripts including ulk1b are not expressed before 23 hpf. 26 The inconsistency with the previous report may be due to different RT-PCR conditions.…”

Beneficial Bacteria Affect Danio rerio Development by the Modulation of Maternal Factors Involved in Autophagic, Apoptotic and Dorsalizing Processes

“…A specific set of primers was designed to amplify all the isoforms of ambra1a [37], ambra1b, beclin1 and lc3 .…”

“…Previous experimental findings have asserted that probiotic administration is beneficial to the i) IGF system and the gonadal development [26], ii) body growth [19,20,21,22,38,39], iii) immune system [18,40,41] and iv) reproduction [31,32,37]. …”