Formation of the skeletal system in a developing embryo is dependent on mechanical movement. A recent study published in the journal Development has narrowed down the genes involved in this pathway. This was a joint study by researchers led by Amitabha Bandyopadhyay from Department of Biological Sciences and Bioengineering, Indian Institute of Technology, Kanpur (IIT-K) and scientists from the laboratory of Paula Murphy from School of Natural Science, Trinity College, Dublin (TCD) in Ireland.
Mechanical forces due to muscle contractions in an embryo act as biophysical stimuli for development of its skeletal system. Proof of this can be found in cases of congenital disorders that restrict fetal movement, wherein the effects can range from thinner and weaker long bones to delayed skeletal maturation.
In a developing vertebrate embryo, the majority of skeleton is initially formed from cartilage. These cartilage cells are bi-potential, i.e., they can differentiate into two types, transient as well as permanent cartilage. Further development replaces cartilage tissue all over the skeleton with bone tissue [due to Bone Morphogenetic Protein (BMP) signaling], except at joints, where permanent or articular cartilage can be found (due to signaling by the Wnt pathway). An older study by Murphy’s team showed that fetal movements are vital for normal development of the joints. Muscle development in the embryo occurs almost in tandem with that of the skeletal system; therefore, these groups of tissue can influence each other through paracrine signaling.
Bandyopadhyay explains the rationale behind this study, “The fact that immobilisation causes differentiation of transient cartilage at the expense of joint cartilage immediately suggested to me that there must be dysregulation of BMP signalling. Thus, we started investigating this observation, for which no molecular explanation existed, due to curiosity and as a validation of the model that we proposed in 2015”.
The researchers used immobilised chick embryos and transgenic muscle-less mouse models for this study. This was a huge challenge for the IIT-K scientists since they did not have the transgenic mouse strains neither did they have the protocol for immobilising chick embryos. This resulted in collaboration between the IIT-K researchers and Paula Murphy’s team in TCD. “These mice only lack limb muscles; other muscles are fine, telling us that it is only limb muscles (and presumably their contractions) that are responsible for this phenotype,” says Raj Ladher, developmental biologist and associate professor at National Centre for Biological Sciences.
In the knee joint of the immobilised embryos, Wnt signaling (that promotes permanent cartilage formation) was seen to be down regulated while BMP signaling was turned on. However, expression of the signaling molecule Noggin was found to be high in the joint line of both chick and mouse joints. This did not make sense since Noggin is an inhibitor of BMP, and should have been at lower than normal levels. The breakthrough came with a suggestion from a colleague, Sandeep Gupta, to investigate Smad ubiquitination regulatory factor 1(Smurf1), an intracellular inhibitor of BMP signaling at a laboratory meeting.
Bandyopadhyay’s team then got down to examine the expression of Smurf 1 and 2 in immobilised chick knee joints. They found that both Smurf 1 and 2 mRNAs were down regulated in immobilised embryos as compared to control embryos. This caused ectopic activation of BMP signaling, thus not allowing development of articular cartilage. The team also showed that Sfrp2 (an inhibitor of Wnt signaling) was up regulated in the immobilised chick knee joints.
Raj Ladher highlights the importance of this study, “The study beautifully shows how mechanical stimulation modifies the response to genetic cues. More precisely, they show that mechanical stimulation of the cartilage induces an inhibitor of a bone-inducing signal keeping this part of the skeletal element free of bone. The study is the first to integrate mechanical stimulation with the genetic program that induces articular cartilage.”
This team’s findings may also help in understanding molecular mechanisms of Osteoarthritis, where permanent cartilage tissue breaks down and begins resembling transient cartilage tissue. Bandyopadhyay charts out the future course for this team, “Mechanical movement favours joint cartilage formation by keeping high Wnt and low BMP (low Wnt inhibitor and high BMP inhibitor) levels whereas immobilisation causes the opposite. I would like to understand the molecular nature of this toggle switch i.e. transcriptional regulation of Sfrp1 and Smurf1. I would also like to understand how mechanical forces impinge on this transcriptional regulation.”