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- The zebrafish operculum: a powerful system to assess osteogenic bioactivities of molecules with pharmacological and toxicological relevancePublication . Tarasco, Marco; Laizé, Vincent; Cardeira, João; Leonor Cancela, M.; Gavaia, PauloBone disorders affect millions of people worldwide and available therapeutics have a limited efficacy, often presenting undesirable side effects. As such, there is a need for novel molecules with bone anabolic properties. The aim of this work was to establish a rapid, reliable and reproducible method to screen for molecules with osteogenic activities, using the zebrafish operculum to assess bone formation. Exposure parameters were optimized through morphological analysis of the developing operculum of larvae exposed to calcitriol, a molecule with known pro-osteogenic properties. An exposure of 3 days initiated at 3 days post-fertilization was sufficient to stimulate operculum formation, while not affecting survival or development of the larvae. Dose dependent pro- and anti-osteogenic effects of calcitriol and cobalt chloride, respectively, demonstrated the sensitivity of the method and the suitability of the operculum system. A double transgenic reporter line expressing fluorescent markers for early and mature osteoblasts was used to gain insights into the effects of calcitriol and cobalt at the cellular level, with osteoblast maturation shown to be stimulated and inhibited, respectively, in the operculum of exposed fish. The zebrafish operculum represents a consistent, robust and rapid screening system for the discovery of novel molecules with osteogenic, anti-osteoporotic or osteotoxic activity.
- Anti-osteogenic activity of cadmium in zebrafishPublication . Tarasco, Marco; Cardeira Da Silva, João; Viegas, Michael; Caria, Joana; Martins, Gil; Gavaia, Paulo; Cancela, M. Leonor; Laizé, VincentAmong the many anthropogenic chemicals that end up in the aquatic ecosystem, heavy metals, in particular cadmium, are hazardous compounds that have been shown to affect developmental, reproductive, hepatic, hematological, and immunological functions in teleost fish. There is also evidence that cadmium disturbs bone formation and skeletal development, but data is scarce. In this work, zebrafish was used to further characterize the anti-osteogenic/osteotoxic effects of cadmium and gain insights into underlying mechanisms. Upon exposure to cadmium, a reduction of the opercular bone growth was observed in 6-days post-fertilization (dpf) larvae and an increase in the incidence of skeletal deformities was evidenced in 20-dpf post-larvae. The extent and stiffness of newly formed bone was also affected in adult zebrafish exposed to cadmium while regenerating their caudal fin. A pathway reporter assay revealed a possible role of the MTF-1 and cAMP/PKA signaling pathways in mechanisms of cadmium osteotoxicity, while the expression of genes involved in osteoblast differentiation and matrix production was strongly reduced in cadmium-exposed post-larvae. This work not only confirmed cadmium anti-osteogenic activity and identified targeted pathways and genes, but it also suggested that cadmium may affect biomechanical properties of bone.