The zebrafish operculum: A powerful system to assess osteogenic bioactivities of molecules with pharmacological and toxicological relevance.

Bone 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 3days initiated at 3days 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.

Development of an in vitro cell system from zebrafish suitable to study bone cell differentiation and extracellular matrix mineralization.

Mechanisms of bone formation and skeletal development have been successfully investigated in zebrafish using a variety of in vivo approaches, but in vitro studies have been hindered due to a lack of homologous cell lines capable of producing an extracellular matrix (ECM) suitable for mineral deposition. Here we describe the development and characterization of a new cell line termed ZFB1, derived from zebrafish calcified tissues. ZFB1 cells have an epithelium-like phenotype, grow at 28°C in a regular L-15 medium supplemented with 15% of fetal bovine serum, and are maintained and manipulated using standard methods (e.g., trypsinization, cryopreservation, and transfection). They can therefore be propagated and maintained easily in most cell culture facilities. ZFB1 cells show aneuploidy with 2n=78 chromosomes, indicative of cell transformation. Furthermore, because DNA can be efficiently delivered into their intracellular space by nucleofection, ZFB1 cells are suitable for gene targeting approaches and for assessing gene promoter activity. ZFB1 cells can also differentiate toward osteoblast or chondroblast lineages, as demonstrated by expression of osteoblast- and chondrocyte-specific markers, they exhibit an alkaline phosphatase activity, a marker of bone formation in vivo, and they can mineralize their ECM. Therefore, they represent a valuable zebrafish-derived in vitro system for investigating bone cell differentiation and extracellular matrix mineralization.