Intermuscular bones are found in the myosepta in teleosts. However, there is very little information on the development and ossification of these intermuscular bones. In this study, we performed an in-depth investigation of the ossification process during development in zebrafish (Danio rerio) and Japanese eel (Anguilla japonica). In Japanese eel, a typical anguilliform swimmer, the intermuscular bones ossified predominantly from the anterior to the posterior. By contrast, in the zebrafish, a sub-carangiform or carangiform swimmer, the intermuscular bones ossified predominantly from the posterior to the anterior regions of the fish. Furthermore, tail amputation affected the ossification of the intermuscular bones. The length of the intermuscular bones in the posterior area became significantly shorter in tail-amputated zebrafish and Japanese eels, and both had less active and lower swimming speeds; this indicates that swimming might induce the ossification of the intermuscular bones. Moreover, when a greater length of tail was amputated in the zebrafish, the intermuscular bones became even shorter. Tail amputation affected the length and ossification of intermuscular bones in the anterior part of the fish, close to the head, differently between the two fish: they became significantly shorter in the zebrafish, but did not in the Japanese eel. This might be because tail amputation did not significantly affect the undulations in the anterior of the Japanese eel, especially near the head. This study shows that the ossification of intermuscular bones might be induced through mechanical force loadings that are produced by swimming.

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ABSTRACT. Intermuscular bones, ossified from tendons within the myosepta, occur only in teleost fish. Current understanding of the homology and origins of intermuscular bones in fishes is based mainly on morphological data. To date, there is no published data regarding molecular mechanisms of intermuscular bone formation. In this study, we cloned the gene muscle segment homeobox C (MsxC). MsxC potentially plays a role in intermuscular bone development of Hemibarbus labeo, an important species of cyprinid fish in the Chinese aquaculture industry. Sequence analysis of MsxC revealed motifs characteristic of the homeobox domain family. Whole-mount in situ hybridization showed that MsxC was primarily expressed in the myosepta and brain. MsxC was expressed in the myosepta from 26 to 41 days after hatching (DAH); this coincided with the onset of intermuscular bone ossification, which occurred between 35 and 62 DAH. Evidence for localization of MsxC expression by in situ hybridization correlated 11325 MsxC involved in intermuscular bone development ©FUNPEC-RP www.funpecrp.com.br Genetics and Molecular Research 14 (3): 11324-11334 (2015) with its detection by quantitative real-time PCR. In vertebrates, MsxC plays a role in the regulation of mesenchymal cell differentiation during bone formation. We therefore conclude that MsxC may have a role in epithelium-mesenchyme interactions during intermuscular bone formation in H. labeo.

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