Next Generation Sequencing Facilitates Quantitative Analysis of Zebrafish Osteoporosis Model Transcriptomes

The development of predictive markers to determine the tipping point prior to the onset of osteoporosis is essential to mitigate further irrevocable deterioration. In order to determine the early warning signs before osteoporosis, we used the dynamic network biomarker (DNB) approach to analyze the time series gene expression data in the zebrafish osteoporosis model and revealed that cyclin-dependent kinase inhibitor 1A (cdkn1a) is one core member of DNB. We demonstrated that cdkn1a negatively regulates osteogenesis, evidenced by the loss-of-function and gain-of-function studies. On one hand, CRISPR/Cas9-mediated cdkn1a knockout in the zebrafish significantly altered skeletal development and increased bone mineralization. On the other hand, inducible cdkn1a expression significantly contributed to the osteoclast differentiation. We also provide mechanistic clues that cdkn1a participates in osteoclast differentiation by regulating its upstream signaling cascades. Our data provided new insights into the dynamic nature of osteoporosis, and identified cdkn1a as the early-warning signals of osteoporosis onset. Overall design: Zebrafish samples treated with DEX for 3, 4, 5, 6 and 7 dpf (N=3) as well as those from the WT and cdkn1a Cas9/sgRNA crispants (N=5) were collected to perform RNA-seq.