Meckel's cartilage and SOX9/Scx expression during morphogenesis of the mouse mylohyoid attachment site

Meckel's cartilage is a central structure in mandibular morphogenesis, but how developing muscles establish their attachment to the mandible in relation to Meckel's cartilage remains unclear. In this study, we investigated the fetal development of the mouse mylohyoid attachment site using histological analysis, immunohistochemistry, in situ hybridization, and three-dimensional reconstruction, with particular attention to SOX9 and Scleraxis (Scx), markers associated with cartilage and tendon development. At E13.5, the mylohyoid attachment site was located close to the inferior aspect of Meckel's cartilage, and SOX9-positive mesenchymal cells were observed between the mylohyoid muscle bundle and Meckel's cartilage. During subsequent development, the intramembranous mandibular bone extended into the space between Meckel's cartilage and the mylohyoid attachment site. By E17.5, the mylohyoid attachment site was closely associated with the mandibular bone. SOX9 expression was detected at the developing attachment site from E13.5 to E17.5, whereas Scx mRNA expression was detected transiently and became progressively reduced during this period. Three-dimensional reconstruction further supported the developmental shift in the spatial relationship among Meckel's cartilage, the mandibular bone, and the mylohyoid attachment site. These findings suggest that Meckel's cartilage may serve as a transient anatomical framework associated with early organization of the mylohyoid attachment site before definitive attachment to the mandibular bone is established.