Ambra1 knockdown in zebrafish leads to incomplete development due to severe defects in organogenesis.
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 autophagosome-specific marker, in the presence of lysosome inhibitors evidenced a reduction in the rate of autophagosome formation in both MOs-injected embryos at 48 hpf, more pronounced in the case of ambra1a gene. Although some defects, such as body growth delay, curved shape and hemorrhagic pericardial cavity were present in both morphants, the occurrence of specific phenotypes, such as major abnormalities of brain development in ambra1a morphants, suggests the possible acquisition of specific functions by the two paralogous genes that are both required during development and do not compensate each other following knockdown.
Department of Biology, University of Padova, Padova, Italy.
Francesca
F
Benato
Tatjana
T
Skobo
Giorgia
G
Gioacchini
Isabella
I
Moro
Fabiola
F
Ciccosanti
Mauro
M
Piacentini
Gian Maria
GM
Fimia
Oliana
O
Carnevali
Luisa
L
Dalla Valle
D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000235:genetics; D001343:Autophagy / Q000187:drug effects / Q000235:genetics; D017124:Conserved Sequence / Q000235:genetics; D004625:Embryo, Nonmammalian / Q000187:drug effects / Q000378:metabolism / Q000473:pathology / Q000648:ultrastructure; D047108:Embryonic Development / Q000187:drug effects / Q000235:genetics; D019143:Evolution, Molecular; D018507:Gene Expression Regulation, Developmental / Q000187:drug effects; D055785:Gene Knockdown Techniques; D056426:Genetic Loci; D016678:Genome / Q000235:genetics; D006801:Humans; D007814:Larva / Q000187:drug effects / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D060172:Morpholinos / Q000494:pharmacology; D038081:Organogenesis / Q000187:drug effects / Q000235:genetics; D010641:Phenotype; D010802:Phylogeny; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D026801:Synteny / Q000235:genetics; D013997:Time Factors; D015027:Zebrafish / Q000196:embryology* / Q000235:genetics; D029961:Zebrafish Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D048868:Adaptor Proteins, Signal Transducing; C583440:Ambra1a protein, zebrafish; C583441:Ambra1b protein, zebrafish; D060172:Morpholinos; D012333:RNA, Messenger; D029961:Zebrafish Proteins
Ambra1; autophagy; development; morpholino; zebrafish