Identification of mechanosensitive genes during skeletal development: alteration of genes associated with cytoskeletal rearrangement and cell signalling pathways

Abstract

BackgroundMechanical stimulation is necessary for regulating correct formation of the skeleton. Here we test the hypothesis that mechanical stimulation of the embryonic skeletal system impacts expression levels of genes implicated in developmentally important signalling pathways in a genome wide approach. We use a mutant mouse model with altered mechanical stimulation due to the absence of limb skeletal muscle (Splotch-delayed) where muscle-less embryos show specific defects in skeletal elements including delayed ossification, changes in the size and shape of cartilage rudiments and joint fusion. We used Microarray and RNA sequencing analysis tools to identify differentially expressed genes between muscle-less and control embryonic (TS23) humerus tissue.ResultsWe found that 680 independent genes were down-regulated and 452 genes up-regulated in humeri from muscle-less Spd embryos compared to littermate controls (at least 2-fold; corrected p-value ≤0.05). We analysed the resulting differentially expressed gene sets using Gene Ontology annotations to identify significant enrichment of genes associated with particular biological processes, showing that removal of mechanical stimuli from muscle contractions affected genes associated with development and differentiation, cytoskeletal architecture and cell signalling. Among cell signalling pathways, the most strongly disturbed was Wnt signalling, with 34 genes including 19 pathway target genes affected. Spatial gene expression analysis showed that both a Wnt ligand encoding gene (Wnt4) and a pathway antagonist (Sfrp2) are up-regulated specifically in the developing joint line, while the expression of a Wnt target gene, Cd44, is no longer detectable in muscle-less embryos. The identification of 84 genes associated with the cytoskeleton that are down-regulated in the absence of muscle indicates a number of candidate genes that are both mechanoresponsive and potentially involved in mechanotransduction, converting a mechanical stimulus into a transcriptional response.ConclusionsThis work identifies key developmental regulatory genes impacted by altered mechanical stimulation, sheds light on the molecular mechanisms that interpret mechanical stimulation during skeletal development and provides valuable resources for further investigation of the mechanistic basis of mechanoregulation. In particular it highlights the Wnt signalling pathway as a potential point of integration of mechanical and molecular signalling and cytoskeletal components as mediators of the response.

Relationships between bone mineral density and new indices of body composition in young, sedentary men and women

“…On the other hand, it should be stressed that the mechanism of associations between body composition and BMD is far from being fully elucidated [23,24].…”

TGFβ and FGF promote tendon progenitor fate and act downstream of muscle contraction to regulate tendon differentiation during chick limb development

“…These pathways are known to be sensitive to mechanical forces in other biological systems (Humphrey et al, 2014). Moreover, transcriptome profiling analyses have identified FGF and TGFβ signalling as being downregulated in developing humerus (limb bone) of immobilised mouse foetuses (Rolfe et al, 2014). In addition to being downregulated in tendons of immobilised embryos, both FGF4 and TGFβ2 prevent the decrease of SCX expression in chick limbs in immobilisation conditions.…”

Validating a Noninvasive Technique for Monitoring Embryo Movement In Ovo

“…Whether this impact on embryo behavior extends to very early stages or how such alterations in behavior may influence cellular activity and gene expression patterns remains to be defined. Given the demonstrated role of movement in regulating gene expression (Dowthwaite et al 1999;Groenendijk et al 2005;Rolfe et al 2014), we suggest that care should be taken when interpreting data gathered solely from windowed eggs.…”

“…There is abundant evidence that musculoskeletal development is impaired, blood flow altered, and gene expression modified in the absence of normal movement (Hosseini and Hogg 1991;Pitsillides 2006;Nowlan et al 2010;Rolfe et al 2014). Moreover, embryo movement can be influenced by many factors, including pharmacological agents (Fanconi et al 1995;Heywood et al 2005;Jensen et al 2014) and external cues such as temperature and circadian rhythms (Hammond et al 2007;Ozkaya et al 2012).…”