The temporal transcriptomic signature of cartilage formation

Abstract

ABSTRACTChondrogenesis is a multistep process, in which cartilage progenitor cells generate a tissue with distinct structural and functional properties. Although several approaches to cartilage regeneration rely on the differentiation of implanted progenitor cells, the temporal transcriptomic landscape ofin vitrochondrogenesis in different models has not been reported. Using RNA sequencing, we examined differences in gene expression patterns during cartilage formation in micromass cultures of embryonic limb bud-derived progenitors. Principal component and trajectory analyses revealed a progressively different and distinct transcriptome during chondrogenesis. Differentially expressed genes (DEGs), based on pairwise comparisons of samples from consecutive days were classified into clusters and analysed. We confirmed the involvement of the top DEGs in chondrogenic differentiation using pathway analysis and identified several chondrogenesis-associated transcription factors and collagen subtypes that were not previously linked to cartilage formation. Transient gene silencing ofATOH8orEBF1on day 0 attenuated chondrogenesis by deregulating the expression of key osteochondrogenic marker genes in micromass cultures. These results provide detailed insight into the molecular mechanism of chondrogenesis in primary micromass cultures and present a comprehensive dataset of the temporal transcriptomic landscape of chondrogenesis, which may serve as a platform for new molecular approaches in cartilage tissue engineering.GRAPHICAL ABSTRACTThis study provides insights into the mechanisms of cartilage formation. Gene expression patterns during cartilage formation in micromass cultures were analysed using RNA sequencing. SilencingATOH8orEBF1attenuates chondrogenesis.Key points•We examined the global gene expression patterns during in vitro chondrogenesis.•Using WGCNA, we created a module of genes with patterns similar to those of SOX9, ACAN, and COL2A1.•We identified ATOH8 and EBF1 transcription factors with a yet unexplored role in chondrogenesis.