Mechanism Of Inhibiting BMP Signal Pathway In The Palate Mesenchymal Leading To Abmormal Development Of Soft Palate Connective Tissue

Background:The soft palate is located at the back of the palate and affected during development causing a cleft soft palate.The development of the soft palate is not only afftected by different genes but also regulated by a varietu of signalinng pathways,among which the BMP signaling pathway is one of the pathways regulate the development of the soft palate.The mouse model of Noggin,an antagonist of the BMP signaling pathway overexpressed in the palate mesenchyme,inhibited the BMP signaling pathway and found that Osr2-Cre ^KI;p Mes-Noggin mice exhibited soft palate cleft,affected soft palate muscle development,and inhibited tendon differentiation.However,studies have shown that soft palate muscle development has little to do with soft palate clefts,So we want to understand the influence of connective tissue on soft palate development.Previously,a mouse model using inactived typeⅠBMP receptors in the palatal mesenchyme to inhibit the BMP signaling pathway was studied,and it was found that Osr2-Cre;Bmpr1a^f/f mice had submucosal clefts in the anterior hard palate,but had no effect on the soft palate,what are the effects on development not yet mentioned.Therefore,we use a mouse model in which in activation of typeⅠBMP receptors inhibits BMP signaling in the palate mesenchyme to investigate changes in connective tissue and its impact on soft palate development.Objective:In this study,the Osr2-Cre^KI mouse tool was used to inactivate Bmpr1a at the time of palate development and the specific position of palate mesenchyme,that is to use Osr2-Cre;Bmpr1a^f/f mice to study the inhibition of BMP signaling pathway in palate mesenchyme effects on the development of soft palate connective tissue.we use the Osr2-Cre;Bmpr1a^f/ftool mouse research inhibit BMP signaling pathway effect on connective tissue of soft palate in palatal mesenchyme.Methods:（1）Osr2-Cre;Bmpr1a^f/+mice were crossbred with Bmpr1a^f/+mice to obtain the Osr2-Cre;Bmpr1a^f/f mouse model,and their normal phenotype mice in the same litter were used as the control group.tissue fixation,delydation,embedding and histological sectioning.（2）Using Masson staining to observe the changes of connective tissue in the soft palate.（3）Detection of expression level of mouse soft palate cartilage marker Sox9by immunohistochemical staining.（4）The expression level of myosin,CD3 andβ-tubulin were detected by immunohistochemical staining,and the differentiation of soft palate nerves,blood vessels,and soft palate muscles were analyzed.（5）The expression of Scx,corresponding indicators of early differentiation of soft palate tendon and extracellular matrix Tnc,were detected by in situ hybridization.Results:（1）Masson staining showed that the palatine aponeurrosis in the soft palate of Osr2-Cre;Bmpr1a^f/fmice was not connected at the midline compared with WT mice at E18.5 days,and the levator veli palati muscle is not attached to the palatel aponeurosis and palatopharyngeal muscle is disconnection at the midline.（2）Immunohistochemical staining showed that the expression level of Sox9 in the soft palate aponeurosis of Osr2-Cre;Bmpr1a^f/f mice was lower than that of WT mice.（3）In the soft palate of Osr2-Cre;Bmpr1a^f/f mice,the expression of Myosin,a marker of mature muscle fibers,could be detected at E16.5 and E18.5 days,suggesting that the inhibition of BMP signaling pathway in the palate mesenchyme has a positive effect on the soft palate.muscle development is not significantly affected.（4）Immunohistochemical stainingd showed that the nerve and blood vessel differentiation of soft palate of Osr2-Cre;Bmpr1a^f/f mice were inhibited.（5）In situ hybridization show that expression of Scx,an early tendon differentiation index of soft palate tendon and extracellular matrix Tnc was down-regulated in Osr2-Cre;Bmpr1a^f/f mice,suggesting that inhibit of BMP signaling pathway in palate mesenchyme can inhibit the differentiation of soft palate tendon..Conclusion:（1）Knockout of the soft palate mesenchymal BMP typeⅠreceptor in mice lead to hard palate submucosa and soft palate aponeurotic dysplasia.（2）Knockout of palate mesenchymal typeⅠBMP receptor does not affectthe development of soft palate muscle,but affects tendon differentiation,resulting in discountinuity of the palatine aponeurosis in the midline of the soft palate.