Secondary Chondrogenesis on The Coronoid Process of The Mandible Requires Both FGF And TGFβ Signaling
Authors: Woronowicz Katherine C , Jaul Danielle Z , Herfat Safa T , Fields Aaron J , Lotz Jeffrey C , Schneider Richard A 
Published: November 2023

The musculoskeleton senses and adapts to its mechanical environment. This relationship between biomechanical forces and skeletal form begins in the embryo. Muscle contractions influence the embryonic skeleton in ways that presage adult function. Secondary cartilage, which forms on the surface of bone within certain muscle insertions and articulations, requires proper mechanical stimulation for its induction and maintenance. As in humans, the coronoid process on the duck mandible develops via a secondary cartilage. This secondary cartilage arises within the enthesis of the mandibular adductor muscle and provides a robust insertion site on the surangular bone. During development, mechanical cues induce enthesis secondary chondrogenesis seemingly in anticipation of the high magnitude forces that the duck‐specific feeding behaviors of levered straining and suction pump movement will exert upon the jaw. In quail, which feed by pecking, the coronoid process also serves as the insertion site for the mandibular adductor, but no secondary cartilage is present. This species‐specific difference occurs even though we find that the onset and frequency of embryonic jaw movement is the same in quail and duck, suggesting that the quality of mechanical stimulation, rather than quantity, is critical. Indeed, our 3D reconstructions of duck and quail jaw complexes prior to secondary chondrogenesis reveal a key difference. In duck, the mandibular adductor muscle inserts laterally on the surangular bone. In quail, this muscle inserts dorsally. Using finite element modeling, we find that this difference in position creates distinct mechanical environments in duck versus quail.The species‐specific mechanical forces present at the duck mandibular adductor insertion likely activate pathways that promote enthesis secondary chondrogenesis. To identify mechanically responsive genes, we assayed for changes in expression in paralyzed duck embryos. We found that members and targets of the FGF and TGFβ pathways are differentially affected. We then tested the extent to which FGF and TGFβ signaling are required for secondary chondrogenesis. Surgically implanting beads soaked in small molecule inhibitors of FGF (10mM SU5402) or TGFβ (100mM SB431542) signaling results in absent or reduced enthesis secondary cartilage in 48% (n=27) and 39% (n=66) of embryos respectively. Treating with TGFβ inhibitor one stage earlier produces a phenotype in 37% of cases, but the frequency of absent secondary cartilage increases from 3.1% (n=66) to 13.5% (n=37). This suggests that TGFβ signaling is important early in the process, and that the TGFβ and FGF pathways may work in tandem or they may operate in succession to induce secondary cartilage. We are now conducting qPCR and in‐situ hybridization analyses to determine the roles of individual pathway members and to understand the ways in which paralysis alters signaling. Taken together, our data support a model whereby the mechanical environment interacts with FGF and TGFβ signaling to induce secondary chondrogenesis at the duck coronoid process.Support or Funding InformationR01 DE016407F31 DE024405