Zakany, R. | bioRxiv Science

bioRxiv2022

The temporal transcriptomic signature of cartilage formation

Chondrogenesis 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 of in vitro chondrogenesis 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 of ATOH8 or EBF1 on 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 ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=181 SRC="FIGDIR/small/486765v2_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@1b599d5org.highwire.dtl.DTLVardef@18670daorg.highwire.dtl.DTLVardef@15afc12org.highwire.dtl.DTLVardef@5b0791_HPS_FORMAT_FIGEXP M_FIG This study provides insights into the mechanisms of cartilage formation. Gene expression patterns during cartilage formation in micromass cultures were analysed using RNA sequencing. Silencing ATOH8 or EBF1 attenuates chondrogenesis. C_FIG 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.

molecular biology↗

bioRxiv2021

Cyclic uniaxial mechanical load enhances chondrogenesis through entraining the molecular circadian clock

The biomechanical environment plays a key role in regulating cartilage formation, but current understanding of mechanotransduction pathways in chondrogenic cells is incomplete. Amongst the combination of external factors that control chondrogenesis are temporal cues that are governed by the cell-autonomous circadian clock. However, mechanical stimulation has not yet directly been proven to modulate chondrogenesis via entraining the circadian clock in chondroprogenitor cells. The purpose of this study was to establish whether mechanical stimuli entrain the core clock in chondrogenic cells, and whether augmented chondrogenesis caused by mechanical loading was at least partially mediated by the synchronised, rhythmic expression of the core circadian clock genes, chondrogenic transcription factors, and cartilage matrix constituents at both transcript and protein levels. We report here, for the first time, that cyclic uniaxial mechanical load applied for 1 hour for a period of 6 days entrains the molecular clockwork in chondroprogenitor cells during chondrogenesis in limb bud-derived micromass cultures. In addition to the several core clock genes and proteins, the chondrogenic markers SOX9 and ACAN also followed a robust sinusoidal rhythmic expression pattern. These rhythmic conditions significantly enhanced cartilage matrix production and upregulated marker gene expression. The observed chondrogenesis-promoting effect of the mechanical environment was at least partially attributable to its entraining effect on the molecular clockwork, as co-application of the small molecule clock modulator longdaysin attenuated the stimulatory effects of mechanical load. This study suggest that an optimal biomechanical environment enhances tissue homeostasis and histogenesis during chondrogenesis at least partially through entraining the molecular clockwork.

cell biology↗