Identification of Mechanosensitive Genes during Embryonic Bone Formation

Although it is known that mechanical forces are needed for normal bone development, the current understanding of how biophysical stimuli are interpreted by and integrated with genetic regulatory mechanisms is limited. Mechanical forces are thought to be mediated in cells by “mechanosensitive” genes, but it is a challenge to demonstrate that the genetic regulation of the biological system is dependant on particular mechanical forces in vivo. We propose a new means of selecting candidate mechanosensitive genes by comparing in vivo gene expression patterns with patterns of biophysical stimuli, computed using finite element analysis. In this study, finite element analyses of the avian embryonic limb were performed using anatomically realistic rudiment and muscle morphologies, and patterns of biophysical stimuli were compared with the expression patterns of four candidate mechanosensitive genes integral to bone development. The expression patterns of two genes, Collagen X (ColX) and Indian hedgehog (Ihh), were shown to colocalise with biophysical stimuli induced by embryonic muscle contractions, identifying them as potentially being involved in the mechanoregulation of bone formation. An altered mechanical environment was induced in the embryonic chick, where a neuromuscular blocking agent was administered in ovo to modify skeletal muscle contractions. Finite element analyses predicted dramatic changes in levels and patterns of biophysical stimuli, and a number of immobilised specimens exhibited differences in ColX and Ihh expression. The results obtained indicate that computationally derived patterns of biophysical stimuli can be used to inform a directed search for genes that may play a mechanoregulatory role in particular in vivo events or processes. Furthermore, the experimental data demonstrate that ColX and Ihh are involved in mechanoregulatory pathways and may be key mediators in translating information from the mechanical environment to the molecular regulation of bone formation in the embryo. Author Summary: While mechanical forces are known to be critical to adult bone maintenance and repair, the importance of mechanobiology in embryonic bone formation is less widely accepted. The influence of mechanical forces on cells is thought to be mediated by “mechanosensitive genes,” genes which respond to mechanical stimulation. In this research, we examined the situation in the developing embryo. Using finite element analysis, we simulated the biophysical stimuli in the developing bone resulting from spontaneous muscle contractions, incorporating detailed morphology of the developing chick limb. We compared patterns of stimuli with expression patterns of a number of genes involved in bone formation and demonstrated a clear colocalisation in the case of two genes (Ihh and ColX). We then altered the mechanical environment of the growing chick embryo by blocking muscle contractions and demonstrated changes in the magnitudes and patterns of biophysical stimuli and in the expression patterns of both Ihh and ColX. We have demonstrated the value of combining computational techniques with in vivo gene expression analysis to identify genes that may play a mechanoregulatory role and have identified genes that respond to mechanical stimulation during bone formation in vivo.