Role of Indian Hedgehog in Mandibular Symphyseal Cartilage Development

Objectives: Symphyseal cartilage and other secondary cartilages are important components of the developing mandible, but the mechanisms underlying their formation remain largely unclear. Intriguingly, these cartilages form in close association with developing mandibular intramembranous bones, but undergo endochondral ossification instead. Indian Hedgehog (IHH) is a key signaling protein previously shown to regulate skeletogenesis in limbs and other sites. Thus, we asked here whether IHH regulates symphyseal cartilage development as well. Methods: IHH-null mice (kindly provided by Dr. A. McMahon) and wild type littermates from embryonic day 15 (E15) to neonatal stage were used to analyze and test formation of symphyseal cartilage and mandibular bone by histochemical and in situ hybridization procedures. Results: Symphyseal cartilage formation first became apparent by E16 as a mass of condensed TGF beta-positive mesenchymal cells. The cells differentiated into chondrocytes by E17 and the resulting cartilaginous tissue filled the space between Meckel's cartilage and developing mandibular bone. Histological and gene expression analyses in wild type mice indeed revealed that IHH was expressed starting on E18. The IHH-expressing chondrocytes resembled pre-hypertrophic chondrocytes in developing long bone growth plates. They were flanked by Histone H4C-positive proliferating chondrocytes and type X collagen- and osteopontin-positive hypertrophic chondrocytes. Interestingly, IHH-null mice exhibited markedly less symphyseal cartilage that was totally disorganized and contained few proliferating chondrocytes. Rostral mandibular cartilage shape was suboptimal and medial symphysis formation was incomplete, leaving the latter widely open at the midline. Treatment of IHH-null symphyseal chondrocytes in culture with recombinant IHH led to increased expression of hedgehog target molecules Patched and GLIs and stimulated chondrocyte proliferation. Conclusion: Our data show for the first time that IHH is involved in symphyseal cartilage development. IHH appears to orchestrate distinct events that lead to, and are necessary for, formation of the rostral mandible. This study is supported by NIH R01AR47543.