Live Fluorescent Staining Platform for Drug-Screening and Mechanism-Analysis in Zebrafish for Bone Mineralization

Abstract

Currently, drug screening relies on cell-based experiments or on animal models to confirm biological effects. The mammalian system is considered too time-consuming, expensive and complex to perform high-throughput drug screening. There is a gap between in vitro cell-based models and the in vivo mammalian models. The zebrafish is an ideal model that could link preclinical toxicity screening with the drug development pipeline. Taking advantage of a highly conservative genomic, rapid development, large number of offspring, low cost and easy manipulation, zebrafish has been considered an excellent animal model for disease-based drug screening. In this study, zebrafish embryos were incubated with small molecular compounds that potentially affected bone mineralization in microplates. Two compounds of alendronate and dorsomorphin were used as positive and negative controls, respectively. The level of osteogenic mineralization was measured and quantified by using ImageJ software with fluorescent calcein-staining images. Among twenty-four tested compounds from the kinase inhibitor library, we identified two compounds, pentamidine and BML-267, which showed increased embryonic mineralization; while six compounds, RWJ-60475, levamisole HCL, tetramisole HCL, fenvalerate, NSC-663284, and BML-267ester, were inhibitory to bone mineralization. In addition, real time quantitative PCR (RT-qPCR) was performed to evaluate the biological pathways involved in bone metabolism at the molecular level. We confirmed that alendronate enhanced the level of bone mineralization by inhibiting osteoclast-related genes. In summary, our research established a simple method to screen potential bone metabolic drugs and to perform mechanism analysis for bone mineralization in vivo.

Keywords

• drug screening
• bone mineralization
• osteoclast
• calcein
• zebrafish