Abstract
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.
Highlights
Osteoporosis and osteopenia are common bone diseases affecting millions of people worldwide (Pisani et al, 2013) and are characterized by both reduced mass and the structural deterioration of bone, causing its fragility and increased susceptibility to fractures
While both the area of the iris and the eye and the length between snout and cleithrum exhibited a good correlation with the area of the operculum (R2 = 0.88, 0.90 and 0.92, respectively), the area of the head appeared to be the most accurate parameter to correct for inter-specimen variability of the operculum area (R2 = 0.94), which showed the lowest coefficient of variance (CV) (27.0 ± 9.9 %) (Supplementary figure 1)
We propose that the screening or study of osteogenic bioactivities in zebrafish larvae should be initiated at 3 dpf and last for 3 days (Figure 6)
Summary
Osteoporosis and osteopenia are common bone diseases affecting millions of people worldwide (Pisani et al, 2013) and are characterized by both reduced mass and the structural deterioration of bone, causing its fragility and increased susceptibility to fractures. Various in vivo tools exist to study how drugs affect the skeleton (osteogenic or anti-osteogenic activities) (Laizé et al, 2014; Cardeira et al, 2016), including zebrafish mutant lines mimicking human skeletal disorders (Barrett et al, 2006; de Vrieze et al, 2014) and transgenic lines able to express fluorescent markers at sites of bone-related gene expression (Knopf et al, 2011a) These are valuable, both for the discovery of therapeutic molecules capable of rescuing skeletal pathologies and in studying their mechanisms of action. This study aims to establish and optimize a methodology to screen for potential bioactives in laboratories using zebrafish This novel method should overcome limitations of current systems used to assess osteogenic effects in zebrafish, such as long exposure time, late assessment, no correction for inter-specimen variability, and long image acquisition and analysis. The suitability of a double transgenic line, expressing fluorescence proteins under the control of bone marker gene promoters, will be tested to gain insights into the mechanisms underlying the osteogenic effects of calcitriol and cobalt
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