The Mechanism Study Of Mouse Abnormal Tongue And Mandible Activated By Spatio-temporal Specific Fgf8 Gene

Background and ObjectivesCraniofacial development depends on the migration and differentiation of ectodermal-derived Cranial Neural Crest Cells(CNCCs).The pluripotential CNCCs migrate into specific locations and differentiate into the most crucial connective tissues of the craniofacial region,including the upper and lower jaw,cartilage,palate,tongue(such as the tongue septum and the lingual tendon)and other tendons in the craniofacial region.The tongue and mandible have common origins and arise simultaneously from the mandibular arch.They are coordinated following strictly spatiotemporal development and growth,which are evident from several clinical conditions,such as Pierre Robin sequence(PRS).For example,human PRS formally comprises the triad of mandibular hypoplasia and glossoptosis causing respiratory obstruction,along with cleft palate.Micrognathia is considered to be the initiating factor leading to PRSTongue malformation and malposition are generally considered as the secondary defects caused by micrognathia.The tongue morphogenesis is not only regulated by molecular cues during the myogenesis,but also relies on the signaling emanated from the craniofacial CNCC-derived tongue mesenchymal cells which develop into the connective tissues,such as tendon and median septum.The tongue connective tissues not only provide a scaffold for the movements of tongue muscles,but also affect the migration,survival,proliferation and fusion of myoblasts in the embryonic tongue.A recent study revealed that multiple FGF ligands were involved in tongue development.As a typical paracrine factors,Fgf18,a member of Fgf8 sub-family,was activated in the lateral mesenchyme underlying tongue epithelium from E11.5 on.Although FGF18 is speculated to exert the same function as other FGFs,its exact role in the tongue development remains unknown.Enormous studies on limbs and trunk proved that musculoskeletal system including muscles and tendons,plays a pivotal role in skeletal development in recent years.Craniofacial morphology and function are possibly determined by the complex system constituted by tendons,muscles and skeletons.However,few studies have been reported so far about the interactions among tendons,muscles and skeletons during craniofacial development.Moreover,due to the lack of craniofacial tendon-specific Cre mouse line,almost all the studies about the tendon development are currently based on the trunk and limbs.There is a paucity of researches about craniofacial tendon development and molecular regulation mechanism.Whether the same pattern and regulation mechanism,such as FGF signaling,will be shared between craniofacial tendons and trunk-limb tendons is still to be demonstrated.Our team found that the transcription factor Osr2 encoded by the Odd-Skipped related 2 gene was specifically activated in the mouse masseter muscle tendon for the first time,but not in the mandible and masseter muscle.Our latest research showed that when Fgf8 was specifically activated in the developing masseter tendon of Osr2-cre;Rosa26R-Fgf8 mouse embryos,the tendon differentiation was impaired,which resulted in the regression of masseter muscle as well as the short mandibular bone secondarily.These findings strongly suggested that tendon development was indispensable for the normal morphology and function of craniofacial muscle and skeleton.We plan to investigate the normal process and the expression pattern of the key genes during craniofacial tendon development,and generate tendon-specific Cre mouse line for future study.Then,we will explore the role of FGF signaling in the development of craniofacial tendon by exploiting Osr2-cre;Rosa26R-Fgf8 mice.Finally,by applying the multiple conditional knock-in mice,we will address the molecular mechanisms through which the absence of masseter tendon leads to the regression of masseter and deformity of mandibular bone.Our study will disclose the pivotal role of the interactions among the tendons,muscles and skeleton in craniofacial development.Our work will provide novel ideas and research basis for the tendon regeneration,the diagnosis and therapy of craniofacial deformity.The study is composed of three parts to detect the mechanism of the malformed tongue and micrognathia.The first part will focus on the mechanism of tongue morphological malformation in Osr2-cre;Rosa26R-Fgf8 mice.The second part will analyze phenotype and mechanism of micrognathia during mandible developmental events in Osr2-cre;Rosa26R-Fgf8 mice in detail.The last part will address the molecular mechanisms through which the absence of masseter tendon leads to the regression of masseter and deformity of mandibular bone by applying the multiple conditional knock-in mice.Methods(1)Osr2-Cre mice and Osr2-cre;Rosa26R-mT/mG mice were crossed with Rosa-Fgf8 mice to generate Osr2-cre;Rosa26R-Fgf8 and Osr2-cre;Rosa26R-Fgf8;Rosa26R-m T/mG embryos,respectively.Osr2-Cre mice were mated with Rosa26R-DTA mice to produce Osr2-cre;Rosa26R-DTA embryos.Myf5-cre mice were crossed with Rosa26R-mT/mG and Rosa26R-Fgf8 mice to produce Myf5-cre;Rosa26R-mT/mG and Myf5-cre;Rosa26R-Fgf8 mice,respectively.(2)Osr2-expressing domain of Osr2-cre;Rosa26R-m T/m G,Osr2-cre;Rosa26R-Fgf8;Rosa26R-mT/mG and Myf5-cre;Rosa26R-mT/mG mice was detected by Cryostat section and fluorescence microscopy(3)Masson staining was performed for embryonic craniofacial histology.(4)Alizarin Red/Alcian Blue staining was applied to test the morphology of mandible bone and Meckel's cartilage.(5)Cell proliferation of tongue and mandible was assessed by BrdU labeling assay.(6)Apoptosis of tongue and mandible was detected by TUNEL assay.(7)In situ hybridizations were performed for the mRNA expression of Tnc,Colla1,Scx and Tnmd.(8)Immunohistochemistry assay was performed to detect the level distribution by using primary antibodies against Myosin,MyoD,Fibronectin,Osterix,p-Smad1/5/8,p-Erk 1/2,p-Akt,p-p38,Fgfrl and Lef1.(9)Western blot was used to confirm the protein level of RhoA in the tongue.(10)All measurements and counting were performed with Image-J 1.48 and GraphPad Prism 5 software.Data were assessed for statistical significance using Student's t-test by p<0.05(*),p<0.01(**)and p<0.001(***).Results1.Fgf8 activation resulted in the malformation of mice tongue by inhibiting mesenchymal cell differentiation.(1)Fgf8 was conditionally activated in the peripheral mesenchyme of mice tongue,causing the "protruding tongue" shape of Osr2-cre;Rosa26R-Fgf8 mice from E14.5 on.Although the gross view indicated that the total size of E16.5 Osr2-cre;Rosa26R-Fgf8 tongue was comparable to that of WT littermates,the width of the Osr2-cre;Rosa26R-Fgf8 mice anterior tongue was larger than that of WT mice,while the width of posterior tongue was smaller than that of control tongue.(2)The histological analysis showed that Osr2-cre;Rosa26R-Fgf8 mice had irregular changes in tongue morphology and the back of the tongue was high with the shape of "inverted V".The mesenchymal cells on the lingual side and among the inferior longitudinal muscles increased in number.There was no significant difference in the arrangement and distribution of the other tongue muscles,apart from the decrease in the density of the inferior longitudinal muscle bundle resulting from the increase in the space between the muscle bundles.(3)In addition,there was no significant difference in the expression of MyoD in tongue muscle of Osr2-cre;Rosa26R-Fgf8 mice and the decrease in the density of individual muscle fibers(labeled Myosin protein)in the inferior longitudinal muscle,suggesting that the tongue muscle development in Osr2-cre;Rosa26R-Fgf8 mice was incompatible,and the abnormal differentiation of tongue mesenchymal cells may contribute to the malformed tongue by destroying the integrity of tongue muscle structure and function.(4)Compared to the control group,the number of BrdU-labeled cells was higher in Osr2-cre;Rosa2 6R-Fgf8 tongue inferior longitudinal muscle,suggesting that Fgf8 activation promoted cellular proliferation in tongue mesenchyme.(5)The expression of Tenascin C as the extracellular matrix protein decreased significantly in Osr2-cre;Rosa26R-Fgf8 mice tongue,while that of Col1a1 and tendon differentiation markers,including Scx and Tnmd,showed no significant difference.This suggest that Fgf8 activation may influence tongue development by inhibiting Tenascin C.(6)There was no obvious difference in Fgfrl and the downstream intracellular signaling proteins of the FGF signaling pathway including p-Erk 1/2,p-Akt,and p-P38 in Osr2-cre;Rosa26R-Fgf8 mice,suggesting that Fgf8 activation did not affect tongue development through the above signaling pathway.(7)Fibronectin,as an important receptor on the cell surface was significantly decreased in Osr2-cre;Rosa26R-Fgf8 mice tongue,especially in the lingual mesenchyme.Moreover,the level of RhoA signal protein descended in Osr2-cre;Rosa26R-Fgf8 mice tongue2.Micrognathia phenotype analysis in Osr2-cre;Rosa26R-Fgf8 mice(1)Osr2-cre was not activated in Osr2-cre;mT/mG mice mandibular bone,but specifically activated in the masseter tendon.However,the Osr2-cre;Rosa26R-Fgf8 mice also exhibited a shorten mandible and mandibular bone from E14.5 on,namely micrognathia.(2)Osr2-cre;Rosa26R-Fgf8 embryos showed a reduced condensation of the mesenchymal cells in the presumptive mandibular primordia and a decreased cellular proliferation in the superior mandibular primordia as well as osteogenic differentiation through a series of analysis about important events during mandibular bone development.(3)The level of p-Erk1/2 signal in the osteoprogenitor cells outside the early mandibular primordia and the canonical BMP signal controlling the osteogenic differentiation process were both significantly decreased.This indicates that Fgf8 activated in the Osr2-cre;Rosa26R-Fgf8 mice masseter tendon may suppress the osteoprogenitors proliferation and osteogenic differentiation by inhibiting ERK signaling and canonical BMP/Smad signaling in the mandible,which in turn affects the growth and mineralization of the mandibular surface structure and eventually leads to the micrognathia in Osr2-cre;Rosa26R-Fgf8 mice3.Mechanism of specific Fgf8 activation in masseter muscle tendon to cause micrognathia by suppressing the differentiation of tendon-masseter complex.(1)Osr2-cre was specifically activated in Osr2-cre;Rosa26R-mT/mG mice masseter tendon attaching to the buccal side of the mandibular bone,while the green fluorescence boundary of the similar region in Osr2-cre;Rosa26R-Fgt8;Rosa26R-mT/mG mice was significantly enlarged.Also,the structure of tendon-masseter complex was not clear.(2)Osr2-cre;Rosa-Fgf8 mouse embryos showed impaired tendon differentiation,including the decreased expression of Scx and Tnmd as the tenocyte differentiation and maturation specific markers,along with the regression of masseter muscle.Myosin and MyoD level were both declined in the Osr2-cre;Rosa26R-Fgf8 mice masseter region,particularly in the posterior region where Myosin and MyoD were almost lost.The cellular proliferation in the Osr2-cre;Rosa26R-Fgf8 mice masseter region was declined significantly as well.(3)Fgfr1 and p-Erk1/2 as important proteins of FGF/ERK-MAPK signaling pathway were both activated extraordinarily in Osr2-cre;Rosa26R-Fgf8 mice tendon-masseter complex region,as well as Lef1 of canonical Wnt/?-catenin signaling pathway.(4)Myf5-cre;Rosa26R-Fgf8 mice showed a relatively normal tendon-masseter complex,except for the slightly decreased density of the muscle fibers.It is noticeable that no micrognathia was present in Osr2-cre;Rosa26R-Fgf8 mice.(5)Osr2-cre;Rosa26R-DTA mice showed the phenotype similar to that of Osr2-cre;Rosa26R-Fgf8 mice,such as invisible tendon,regression of masseter muscle and abnormal mandibular primordia.Conclusion(1)The activation of Fgf8 in the tongue mesenchyme of Osr2-cre;Rosa26R-Fgf8 mice may lead to tongue malformation by suppressing the level of Fibronectin and RhoA signaling,inhibiting the extracellular matrix Tenascin C expression in the tongue and destroying the balance of peripheral surface tension of the tongue.(2)Specific and sustained Fgf8 activation in masseter tendon may inhibit the tendon cell fate as well as its differentiation and maturation by activating the Fgfrl/Erk/Wnt signaling pathway,leading to regression of the tendon.At the same time,the development of adjacent masseter may be inhibited by Wnt signaling activated in the tendon.(3)Fgfr1/Erk/Wnt signaling activated by Fgf8 in the tendon may inhibit the osteoprogenitors proliferation and differentiation in the mandibular bone by suppressing ERK signaling and canonical BMP signaling though unknown signalings.