Biological Function Of Transcription Factor Sox2 During Mouse Secondary Palate Development

Objective:The transcription factor Sox2 plays important roles in maintaining the pluripotency of embr yonic stem cells and self-renewal,and take part in the formation of various tissues and organs.Whether Sox2 participates in the development of the palate and its mechanism remains unclear.Aims of this study:(1)to detect the spatio-temporal expression characteristics of the transcription factor Sox2 in the palatal development in mice;(2)to study the biological function of Sox2 during mouse palatal development by constructing the Shh-cre,Sox2fl/fl conditional knockout mice.Material and methods:(1)Immunohistochemistry(IHC)techniques were used for examining the different development stages of mice palate,further to observe and analyze the expression pattern of Sox2 during mice palatal development;(2)Building Shh-cre,Sox2fl/fl conditional knockout mice as the research model,the stereoscopic microscope,SEM,HE staining,IHC,EdU staining,TUNEL apoptosis staining and RT-PCR were used for exploring the biological function and molecular mechanism of Sox2 during palatal development.Results:(1)Immunohistochemical staining was performed to ensure the expression pattern of Sox2 during palate development in mice.As shown in the results,Sox2 showed similarly expression between E11.5 and PNO(2 positions along the anterior-posterior axis of the palate).Sox2 protein expression was detected in the developing oral epithelium,and showed strong expression in the palatal epithelium and tongue epithelium.At E14.5,the palatal shelves elevated to a horizontal position above the tongue,and Sox2 expression was reduced in the front of palatal shelves.At E14.75,Sox2 protein in the MES continue reducing.By E15.5,when the palatal shelves fused and the MES disappeared,Sox2 was not expressed in the middle.The expression of Sox2 in the tongue epithelium was always present.(2)Compared with wild type mice which bom in the same litter,immunohistochemistry in Shh-cre,Sox2fl/fl mice demonstrated that Sox2 expression was almost inactivated in the palatal epithelium.Some remaining Sox2 protein was still detected in the palatal epithelium.But there was no expression of Sox2 protein in the tongue epithelium.The Shh-cre,Sox2fl/fl mice showed severe cleft palate,and the embryos died at PNO.Furthermore,Shh-cre,Sox2fl/fl mice had a similar remarkable feature that the tongue was attached to mandible and all tongues had an obvious abnormal morphology.However,the Shh-cre,Sox2fl/fl mice exhibited no difference in the presence of frontal and lateral views compared with wild type.(3)At E14.5,the palatal shelves had already completed their elevation and formed the palatal midline at E14.75.And the palate shelves fused with opposite side and formed a complete palate in the wild type at E15.5.Nevertheless,from E14.5 to E15.5,Shh-cre,Sox2fl/fl mice demonstrated a wide crack across the second palate,and one of the palatal shelves still grew vertically along the developmenting tongue,while the other elevated in the horizontal position.By EdU staining,in the elevated palatal shelves of Shh-cre,Sox2fl/fl mice,the proliferation rate had no obvious difference compared with the wild type,not only the epithelium but also the mesenchyme.But both the anterior and posterior level of sections,the non-elevated palatal shelves of Shh-cre,Sox2fl/fl mice had fewer EdU positive cells in the epithelium than wild type,and the rate means were significantly different(P<0.05).However,the reduction of mesenchymal proliferation had no significant difference.TUNEL staining indicated that there were no obvious difference between the wild type and Shh-cre,Sox2fl/fl mice in the palatal epithelium and mesenchyme,not only the anterior but also the posterior sections.(4)At E14.5,the expression of Fgf10,Shh,Fgf8,Pax9,Osr2 in elevated palatal shelves showed down-regulation,but the expression of Bmp4,Msxl exhibited up-regulation;the quantity of Bmp4,Fgf10,Shh,Fgf8,Pax9,Osr2 in non-elevated palatal shelves exhibited down-regulation,the expression of Msrx1 showed up-regulation.Condusions:(1)The expression pattern(temporal and spatial)of Sox2 during mice palatal development indicating that Sox2 participates in the palatal development in mice;(2)The result that Shh-cre,Sox2fl/fl conditional knockout mice showed severe secondary palate cleft palate,prompt that Sox2 plays an important biological role in the process of palate development;(3)The reasons of Shh-cre,Sox2fl/fl conditional knockout mice showed severe secondary palate cleft palate maybe as followed:A.the reduction of palatal epithelial proliferation B.the abnormal tongue morphology(the tongue was attached to the mandible).(4)The results of RT-PCR showed that not only the elevated shelves,but also the non-elevated shelves,the genes involved were all affected.