Cloning and characterization of the executioner caspases 3, 6, 7 and Hsp70 in hyperthermic Atlantic salmon (Salmo salar) embryos.
Hyperthermia during embryogenesis has been reported to induce deformities in Atlantic salmon (Salmo salar). To examine the involvement of executioner caspases in hyperthermia-induced cell-death in a poikilotherm vertebrate species, five genes encoding caspase-3,-6, and -7 were cloned from Atlantic salmon, and the expression was studied in thermal stressed salmon embryos. The salmon genome contained two genetically distinct variants of both salmon caspase-3 and caspase-6 that is likely the result of two independent chromosome or genome duplications. Whereas only partial caspase-3A encoding sequences were isolated, the full-length caspase-3B cDNA encodes the inactive proenzyme of 279 amino acids (aa) consisting of an N-terminal prodomain and the large and the small subunit. The salmon caspase-6A and caspase-6B proenzymes include an additional linker region between the two subunits. The deduced salmon caspase-7 consists of only 245 aa and lacks the prodomain and part of the large subunit similar to the predicted caspase-7 of the puffer fish Tetraodon sp.. Increased apoptotic activity as evidenced by cleavage of nuclear DNA was demonstrated in salmon embryos incubated at 18-20 degrees C for 84 h after acclimatization at 8 degrees C. Hyperthermia-induced activation of the executioner caspases was indicated by the increased mRNA levels of caspase-3B, caspase-6A/B and caspase-7 after 54 h heat exposure as quantified by real-time RT-PCR. The 2-2.5 fold increase in the mRNA expression of the heat shock protein Hsp70 gene coincided with the peak mRNA values of the executioner caspases. Whole-mount in situ hybridization of the salmon embryo identified caspase-7 mRNA in the lens exclusively, while caspase-3B and caspase-6A/B were expressed in multiple tissues of exposed and control embryos. Interestingly, cardiac expression of caspase-6A/B was only identified in heat stressed embryos. Altogether, these results shed light on evolutionary aspects of the executioner caspases in vertebrates and their expression in salmon embryos exposed to hyperthermia. In particular, the heat sensitive caspase-6 expression in the embryonic heart is of interest since cardiac malformations are an emergent problem in salmon aquaculture.
Takle
Harald
H
AKVAFORSK (Institute of Aquaculture Research), Norwegian University of Life Sciences, P.O. Box 5010, N-1432 Aas, Norway. harald.takle@akvaforsk.no
McLeod
Anette
A
Andersen
Oivind
O
D000595:Amino Acid Sequence; D000818:Animals; D017209:Apoptosis; D020169:Caspases / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D004625:Embryo, Nonmammalian / Q000033:anatomy & histology; D005260:Female; D020440:Gene Duplication; D015870:Gene Expression; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics* / Q000378:metabolism; D006358:Hot Temperature / Q000009:adverse effects; D006979:Hyperthermia, Induced / Q000009:adverse effects; D008297:Male; D008969:Molecular Sequence Data; D012474:Salmon / Q000196:embryology* / Q000235:genetics*; D017386:Sequence Homology, Amino Acid; D014018:Tissue Distribution / Q000235:genetics
D018840:HSP70 Heat-Shock Proteins; D020169:Caspases