Temperature and the expression of myogenic regulatory factors (MRFs) and myosin heavy chain isoforms during embryogenesis in the common carpCyprinus carpioL.

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Abstract
SUMMARY: Embryos of the common carp, Cyprinus carpio L., were reared from fertilization of the eggs to inflation of the swim bladder in the larval stage at 18 and 25°C. cRNA probes were used to detect transcripts of the myogenic regulatory factors MyoD, Myf-5 and myogenin, and five myosin heavy chain (MyHC) isoforms during development. The genes encoding Myf-5 and MyoD were switched on first in the unsegmented mesoderm, followed by myogenin as the somites developed. Myf-5 and MyoD transcripts were initially limited to the adaxial cells, but Myf-5 expression spread laterally into the presomitic mesoderm before somite formation. Two distinct bands of staining could be seen corresponding to the cellular fields of the forming somites, but as each furrow delineated, Myf-5 mRNA levels declined. Upon somite formation, MyoD expression spread laterally to encompass the full somite width. Expression of the myogenin gene was also switched on during somite formation, and expression of both transcripts persisted until the somites became chevron-shaped. Expression of MyoDwas then downregulated shortly before myogenin. The expression patterns of the carp myogenic regulatory factor (MRF) genes most-closely resembled that seen in the zebrafish rather than the rainbow trout (where expression of MyoD remains restricted to the adaxial domain of the somite for a prolonged period) or the herring (where expression of MyoD persists longer than that of myogenin). Expression of two embryonic forms of MyHC began simultaneously at the 25-30 somite stage and continued until approximately two weeks post-hatch. However, the three adult isoforms of fast muscle MyHC were not detected in any stage examined, emphasizing a developmental gap that must be filled by other, as yet uncharacterised, MyHC isoform(s). No differences in the timing of expression of any mRNA transcripts were seen between temperature groups. A phylogenetic analysis of the MRFs was conducted using all available full-length amino acid sequences. A neighbour-joining tree indicated that all four members evolved from a common ancestral gene, which first duplicated into two lineages, each of which underwent a further duplication to produce Myf-5 and MyoD, and myogenin and MRF4. Parologous copies of MyoD from trout and Xenopus clustered closely together within clades, indicating recent duplications. By contrast, MyoDparalogues from gilthead seabream were more divergent, indicating a more-ancient duplication.