Abstract
This study aimed to characterize the molecules involved in osteogenesis in seabream and establish using in vitro/in vivo approaches the responsiveness of selected key genes to temperature. The impact of a temperature drop from 23 to 13 °C was evaluated in juvenile fish thermally imprinted during embryogenesis. Both, in vitro/in vivo, Fib1a, appeared important in the first stages of bone formation, and Col1A1, ON and OP, in regulating matrix production and mineralization. OCN mRNA levels were up-regulated in the final larval stages when mineralization was more intense. Moreover, temperature-dependent differential gene expression was observed, with lower transcript levels in the larvae at 18 °C relative to those at 22 °C, suggesting bone formation was enhanced in the latter group. Results revealed that thermal imprinting affected the long-term regulation of osteogenesis. Specifically, juveniles under the low and low-to-high-temperature regimes had reduced levels of OCN when challenged, indicative of impaired bone development. In contrast, gene expression in fish from the high and high-to-low-temperature treatments was unchanged, suggesting imprinting may have a protective effect. Overall, the present study revealed that thermal imprinting modulates bone development in seabream larvae, and demonstrated the utility of the in vitro MSC culture as a reliable tool to investigate fish osteogenesis.
Highlights
Gilthead sea bream is one of the most important farmed species in Spain, which is ranked fourth in total production volume in the Mediterranean area and third in the European Union[9]
A significant interaction between days in culture and media was observed for fibronectin 1a (Fib1a), Bone Morphogenetic Protein 2 (BMP2) and OP mRNA levels
In cells incubated in osteogenic medium (OM), the gene expression of Runx[2], a key transcription factor driving osteogenesis of MSCs, and of collagen type 1 alpha-1 (Col1A1), and tissue non-specific alkaline phosphatase (TNAP), did not vary significantly as differentiation progressed (Fig. 1A,D,H)
Summary
Gilthead sea bream is one of the most important farmed species in Spain, which is ranked fourth in total production volume in the Mediterranean area and third in the European Union[9]. In the Mediterranean Sea, gilthead sea bream is frequently exposed to severe temperature changes during winter, which is one of the causal factors of winter syndrome in reared populations[20] This pathology causes chronic mortality during the coldest months of the year and acute death episodes when the water temperature rises again[21,22]. Following on from our previous work, in the present study, we addressed the hypothesis that, modifications in the skeleton due to water temperature are linked to its effect on the gene expression of key osteogenic molecules during development To this end, we analysed in vivo how thermal regimes during development influenced the expression of genes involved in osteogenesis and used an in vitro primary culture of MSCs from juvenile gilthead sea bream vertebrae to investigate how temperature modified bone tissue specific gene expression. We assessed in vivo how thermal regimes experienced during early development influenced the expression of bone tissue specific genes in adults exposed to a cold challenge
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