Systematic Reconstruction of Molecular Cascades Regulating GP Development Using Single-Cell RNA-Seq

•Unbiased clustering of single cells from GPs identifying stage transitions•Temporal and spatial reconstruction of GP maturation•Prediction of the TF portfolio directing GP maturation•Recapitulation of TF portfolio using an in vitro screening system

SummaryThe growth plate (GP) comprising sequentially differentiated cell layers is a critical structure for bone elongation and regeneration. Although several key regulators in GP development have been identified using genetic perturbation, systematic understanding is still limited. Here, we used single-cell RNA-sequencing (RNA-seq) to determine the gene expression profiles of 217 single cells from GPs and developed a bioinformatics pipeline named Sinova to de novo reconstruct physiological GP development in both temporal and spatial high resolution. Our unsupervised model not only confirmed prior knowledge, but also enabled the systematic discovery of genes, potential signal pathways, and surface markers CD9/CD200 to precisely depict development. Sinova further identified the effective combination of transcriptional factors (TFs) that regulates GP maturation, and the result was validated using an in vitro EGFP-Col10a screening system. Our case systematically reconstructed molecular cascades in GP development through single-cell profiling, and the bioinformatics pipeline is applicable to other developmental processes.Video Abstract To view the video inline, enable JavaScript on your browser. However, you can download and view the video by clicking on the icon belowHelp with MP4 filesOptionsDownload video (77632 K)

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