Postnatal Craniofacial Skeletal Development In C57BL/6NCrl Mice And Effect And Mechanism Of Tsc1 Deletion On The Craniofacial Bone Development

The craniofacial skeleton,which can be regarded as an independent structure-functional complex,differs from the body skeleton in the complexity of organization,embryonic origin,and the molecular mechanisms inducing skeletogenesis.The perturbation of craniofacial bone development compromises not only esthetics and function,but also individual's mental well-being.Therefore,a thorough knowledge of physiological craniofacial skeletal development and regulatory mechanisms is required for a better understanding and effective treatment of defective craniofacial skeletal development.The growth and development of craniofacial skeleton is regulated by multiple signaling pathways.Among them,the mammalian target of rapamycin(mTOR)signaling integrates intra-and extracellular signals and plays critical roles in determining cell growth and size,proliferation and differentiation,protein synthesis and autophagy,etc.However,the recent results of regulatory effect of mTOR on osteogenesis are controversial.In addition,most of previous studies focus on the effect of mTOR signaling in long bones,but not in the development of craniofacial skeleton,which is unique and distinct from long bones.In this study,we analyzed the postnatal craniofacial skeletal development of C57BL/6NCrl female mice,as well as the effect of Tscl deletion on craniofacial bone development.The objectives are to(1)enhance the understanding of the physiological development of craniofacial skeleton in mice,including morphological assessment,dynamic analysis of bone quantity and quality parameters;(2)determine the effect and mechanism of upregulation of mTORC1 signaling on craniofacial bone development.In Chapter 1,we reviewed and summarized the knowledge about growth and development of craniofacial skeleton in human,as well as cranial base development and related diseases of craniofacial anomalies.Additionally,we introduced the constitution and biological functions of mTOR signaling,TSC1/TSC2 complex,interactions between mTOR signaling and other signaling pathways and regulatory effect of mTOR in bone remodeling.In Chapter 2,we employed high-resolution micro-computed tomography(?CT)in combination with morphometric measurements to analyze the postnatal craniofacial skeletal development from postnatal day 7(P7)through day 390(P390)of female C57BL/6NCrl mice,a widely used mouse strain.We tracked 47 skeletal landmarks and measured 44 linear and angular measurement,as well as craniofacial proportional change and coordinated growth of jaws.Our data demonstrated a unique craniofacial skeletal development pattern in female C57BL/6NCrl mice and differentiates the early vs.late craniofacial growth patterns.Additionally,our data documents the complex and differential changes in bone parameters(thickness,bone volume,bone volume/tissue volume,bone mineral density and tissue mineral density)of various craniofacial bones with different embryonic origins and ossification mechanisms during postnatal growth,which underscores the complexity of craniofacial bone development and provides a reference standard for future quantitative analysis of craniofacial bones.In Chapter 3,we assessed the craniofacial anomalies of a specific transgenic mouse model with Tscl conditional deletion,aiming to reveal the effect of dysregulated mTORC1 signaling on the craniofacial morphology in mice.Our data demonstrated a series of consistently inherited phenotypes in Tsc1 conditional knockout(CKO)mice,including shorter wider and higher head,with orbital hypertelorism and bowed forehead.These manifestations were due to premature fusion of inter-sphenoid synchondrosis(ISS)at early developmental stage,which subsequently affected craniofacial morphology.Micro-CT showed 60%(6/10)and 100%(10/10)of CKO mice who have ISS fusion at postnatal day 7 and 2 month,respectively.In CKO,ISS fusion followed a unique direction in which start ventrally,and end up dorsally.The underlying mechanism may due to the early disorganization of Golgi apparatus in resting zone chondrocytes,thus disturbed the cell orientation/polarity in ISS.In Chapter 4,we investigated the effect of Tscl deletion on craniofacial bone mass and bone quality.Herein we showed that Tscl deletion in neural crest derived cells cause progressive increase in bone mass,mineral density and extracellular matrix(ECM)formation in both intramembranous and endochondral ossified craniofacial bones.RNA-seq data indicated enhanced osteoblast differentiation and mineralization in 2-month-old frontal bone of CKO.Hyperexpression of collagen genes and non-collagen ECM genes as well as ECM receptors confirmed intensive ECM formation.Focal adhesion signaling integrates signals from ECM/integrin and induces multiple intracellular biological functions.Our data showed that FAK was activated in CKO frontal bone.However,preliminary data showed treatment with FAK inhibitor cannot prevent craniofacial bone from thickening in CKO.Rapamycin,an inhibitor of mTORCl signaling,can mostly rescue the bone mass and mineral density increase in CKO.These data unfolded the physiological growth and development of craniofacial skeleton of C57BL/6NCr1 female mice,and indicated the critical role of mTORC1 in regulating craniofacial bone development,enlightening the potential therapies for the patients with tuberous sclerosis or osteoporosis.