Circadian BMAL1 Regulates Mandibular Condyle Development By Hedgehog Pathway

Background:Chondrogenesis and endochondral ossification in mandibular condyle play crucial roles in maxillofacial morphogenesis and function.Deviations in the growth of mandibular condylar cartilages(MCC)affect the function of occlusion,leading to severe facial dysmorphism.Circadian regulator brain and muscle arnt-like 1(BMAL1)is proven to be essential for embryonic and postnatal development.Objective:The goal of this study was to define the functions of BMAL1 in the embryonic and postnatal growth of MCC.Methods:In order to explore the critical role of circadian clock in endochondral ossification of MCC,micro-CT,H&E,Safranin O-green and immunohistochemistry assays were performed using BMAL1-deficiency(Bmal1^-/-)mice and conditional Bmal1knockout mice in mesenchymal stem cells(MSCs)(Bmal1^fl/fl;Twist2-Cre)model.In vitro experiments were performed using rat chondrocytes isolated from mandibular condylar cartilages.RNA-sequencing of Bmal1^-/-mice and wild-type mice was used for transcriptional profiling.Chromatin immuneprecipitation and dual-luciferase assays illuminated the molecular mechanism of BMAL1 in regulating MCC development.Results:We report that the mandibular condylar cartilages contain a functional circadian clock.Meanwhile,we detected that the expression levels of circadian regulator BMAL1decreased gradually in MCC.BMAL1 is proved to regulate sequential chondrocyte differentiation,and its deficiency can result in the impairment of chondrogenesis and endochondral ossification of MCC.Morphological evaluation of MCC further verified that the height of PZ and HZ were diminished obviously,and the number of chondrocytes per column in PZ was decreased significantly compared to the age matched wild-type mice.The expression of basic components that are cartilage matrix type II collagen(COL2?1),Aggrecan(ACAN)and type X collagen(COL10?1)were dramatically less in Bmal1^-/-mice at prenatal and postnatal development stage.Immunohistochemical staining for Ki67 revealed that chondrocyte proliferation was significantly attenuated in PZ.Moreover,TUNEL staining showed that cell apoptosis of chondrocyte was increased in MCC of Bmal1^-/-mice compared to the age matched wild-type mice.RNA-sequencing reveals that the transcriptional effects of BMAL1 in MCC are gradually decreased along with the development of mandibular condyle and hedgehog signaling pathway is the potential target of BMAL1.Chromatin immuneprecipitation and dual-luciferase assays illuminated that BMAL1 regulates hedgehog signaling and affects its downstream cascades through directly binding to the promoters of Ptch1 and Ihh,modulating targets of hedgehog signaling which is indispensable for endochondral ossification.Importantly,the short stature phenotypes caused by BMAL1-deficiency can be rescued by hedgehog signaling activator,SAG.Conclusion:We concluded that the BMAL1 is a critical component of chondrogenesis and endochondral ossification of MCC.We also verified that BMAL1-deficiency inhibits chondrogenesis and endochondral ossification in mandibular condyle by reducing sequential chondrocyte differentiation.Hh pathway is an essential factor for BMAL1regulating MCC development and presents potential therapeutic strategies for skeletal dysplasia.