Biological Signaling Pathways Associated with Late Tendon Development in Chicken Embryos and Their Dependence on Mechanical Stimulation

During embryonic and neonatal development, tendons exhibit rapid changes in mechanics and structure, including increases in collagen content, crosslinking, and fibril length. These functional and structural changes are partly dependent on mechanical stimulation. However, the specific mechanobiological mechanisms driving these changes remain poorly understood. The objective of this study was to identify the potential biological mechanisms and pathways driving tendon maturation into load-bearing materials using an embryonic chick model. Using bulk RNA sequencing, gene set enrichment analysis, and transcription factor analysis, we produced a transcriptomic profile of chick hindlimb tendons during late embryonic development (E16, E18, and E20) and tendons from rigid and flaccid paralysis-treated embryos. Additionally, we assessed the results from two differential expression analysis models to improve the replicability of our findings. Our results suggest that the largest transcriptomic response occurs between embryonic day 16 and 20, and that among paralysis conditions, rigid paralysis produces the greatest effect relative to controls. Across development, we found positive enrichment in processes related to mechanotransduction, extracellular matrix remodeling, cell adhesion, and contractility, and identified candidate pathways upregulated with development, including TGF-β, PI3K-AKT-mTOR, Hippo, circadian rhythm, and focal adhesion signaling. In paralysis-treated embryos, our data suggest impairment in extracellular matrix remodeling and mechanotransduction events. Specifically, we found that both modes of paralysis are associated with a cellular stress response and downregulation of WNT signaling. Together, our findings identify candidate mechanotransduction pathways that are associated with late tendon maturation and altered by paralysis, which may inform tendon tissue engineering and regenerative approaches.