| Background: During embryonic development,the tendons and muscles of the limbs and trunk(all derived from the mesoderm)can promote their own development and maturity through inter-tissue interactions,but whether the relationship between the maxillofacial tendons(derived from the ectoderm)and muscles(mostly derived from the unsegmented branchial arch mesoderm)during development is similar to the trunk and limbs is still unknown.The previous research of our research group found that we could specifically and continuously activate FGF family member Fgf8 in the maxillofacial mandible-masseter tendon by utilizaing Osr2-Cre;Rosa26R-Fgf8 gene knock-in mouse model.Then,we found not only the masseter tendon of Osr2-Cre;Rosa26R-Fgf8 mice was lost,but also its masseter muscle was missing.Therefore,our group analyzed the phenotype of the mouse masseter muscle loss,and found that the number and proliferation of masseter muscle myoblasts were significantly reduced,resulting in the masseter muscle development disorders.In order to further explore the mechanism of the mouse masseter muscle loss,two conjectures were made about the cause of the mouse masseter muscle loss in this study: First,Fgf8 activated in the maxillofacial mandible-masseter tendon directly acted on the adjacent masseter muscle through the paracrine pathway,and then caused the masseter muscle loss through an unknown mechanism.Second,the lack of tendon led to the lack of signal molecules secreted by the tendon and the inter-tissue interaction during the muscle development,which finally led to the failure of the mouse masseter muscle to form.Objective: Myf5 is the earliest expressed myogenic regulatory factor during embryonic development.In order to explore whether the masseter muscle loss in Osr2-Cre;Rosa26R-Fgf8 mice is related to the paracrine effect of Fgf8,this study used Myf5-Cre;Rosa26R-Fgf8 gene knock-in mouse model to observe the effect of Fgf8 activation in myogenic progenitor cells on masseter muscle development.At the same time,DTA can cause apoptosis of cells specifically expressing Cre.In order to verify whether the masseter muscle loss in Osr2-Cre;Rosa26R-Fgf8 mice is caused by its tendon loss,this study observed the effect of tendon precursor cell apoptosis on masseter muscle development through Osr2-Cre;Rosa26R-DTA gene knock-in mouse model.In addition,this study also used Myf5-Cre;Rosa26R-DTA gene knock-in mouse model to observe the influence of masseter precursor cell apoptosis on tendon development,so as to explore the importance of the interaction between maxillofacial skeletal muscle and tendon during embryonic development.Materials and Methods: Myf5-Cre mice was mated with Rosa26R-Fgf8 mice to obtain Myf5-Cre;Rosa26R-Fgf8 embryos(specific and continuous activation of Fgf8 in myogenic progenitor cells).Osr2-Cre mice and Myf5-Cre mice were crossed with Rosa26R-DTA mice to generate Osr2-Cre;Rosa26R-DTA(specific apoptosis of masseter tendon precursor cells)and Myf5-Cre;Rosa26R-DTA(specific apoptosis of masseter precursor cells)embryos,respectively.The mouse embryos were selected according to the requirement of the experiments.Masson staining was used to observe the morphology of mouse masseter muscle and tendon tissue,Brd U labeling was used to detect cell proliferation level.Immunohistochemistry was used to detect muscle differentiation indicators such as Myosin and Myo D,and proliferation marker protein Ki67,as well as p-Smad3 and other signaling pathway molecules.Results:(1)The masseter muscle of Myf5-Cre;Rosa26R-Fgf8 mice still existed,but the area occupied by the anterior and posterior masseter muscles and the number of muscle fibers per unit area in the posterior masseter muscle were significantly lower than that of WT mice.This result indicates that continuous activation of Fgf8 in myogenic progenitor cells can inhibit the development of mouse masseter muscle,resulting in the decrease in the number of mature masseter muscle fibers.(2)The area occupied by Myo D-positive cells in the anterior masseter muscles and the number of Myo D-positive cells per unit area in the anterior and posterior masseter muscles of Myf5-Cre;Rosa26R-Fgf8 mice were significantly higher than that of WT mice,indicating that continuous activation of Fgf8 in myogenic progenitor cells can lead to a significant increase in the number of mouse masseter muscle myoblasts.Besides,the number of Brd U-positive cells(proliferating cells)per unit area in its masseter muscle was also higher than that of WT mice,which indicates that continuous activation of Fgf8 in myogenic progenitor cells can also promote the proliferation of mouse masseter muscle myoblasts.(3)The area occupied by the masseter muscle and the number of mature masseter muscle fibers of Osr2-Cre;Rosa26R-DTA mice were dramatically less than that of WT mice.The changes in its posterior masseter muscle were particularly significant.This result indicates that the tendon loss can severely inhibits mouse masseter muscle development,even resulting in the loss of mouse posterior masseter muscle.(4)The area occupied by Myo D-positive cells in the posterior masseter muscle and the number of myoblasts per unit area in the masseter muscle of Osr2-Cre;Rosa26R-DTA mice were significantly lower than that of WT mice,indicating that the tendon loss can cause a significant decrease in the number of mouse masseter myoblasts.Besides,the number of Ki67-positive cells(proliferative cells)per unit area in its masseter muscle was also lower than that of WT mice,indicating that the tendon loss can decrease the proliferation level of mouse masseter muscle myoblasts.(5)p-Smad3(the intracellular regulatory factor of the classical TGF-β signaling pathway)was widely expressed in the anterior and posterior masseter muscles of WT mice,and was strongly expressed in the masseter muscles adjacent to the myotendinous junction(MTJ).However,the expression position of p-Smad3 in the anterior masseter muscle of Osr2-Cre;Rosa26R-DTA mice and Osr2-Cre;Rosa26R-Fgf8 mice all changed,and there was almost no expression of p-Smad3 in their posterior masseter muscles.This result indicates that the tendon loss can obviously decrease the p-Smad3 level in mouse masseter muscle.(6)The tendon of masseter muscle of Myf5-Cre;Rosa26R-DTA mice was lost,indicating that the masseter muscle loss can also inhibit the development of tendon.Conclusion:(1)Continuous activation of Fgf8 can inhibit the development of masseter muscle in mice,but the determinant of masseter muscle loss in Osr2-Cre;Rosa26R-Fgf8 mice is not the paracrine effect of Fgf8,but the loss of masseter muscle tendon.(2)Loss of tendon may inhibit the transmission of mechanical force and inhibit TGF-β/Smad3 signaling,thereby inhibiting the proliferation of masseter muscle myoblasts,leading to abnormal masseter muscle development in mice. |
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