| Background: The development of salivary glands is accomplished through the interaction between the epithelium and mesenchyme,and is regulated by a variety of growth factors and signaling pathways.As a paracrine factor,Fgf8 in the fibroblast growth factor family plays an important role in the development of salivary glands.The study found that when the Fgf8 gene was knocked out in mice,the number of epithelial branches of the salivary glands was reduced.In order to further explore the influence of the Fgf8 gene in the development of the salivary glands,it is necessary to overexpress Fgf8 in the salivary glands.And when the Fgf8 gene was knocked out in the whole body of the mice,it could not be clarified whether the deletion of Fgf8 in the epithelium or in the mesenchyme caused the reduction in the number of epithelial branches.Therefore,the differential effects in the salivary gland development were explored by separately activating Fgf8 in salivary gland epithelium and mesenchyme.Objective: By use Osr2-cre;Rosa26R-Fgf8 and Shh-cre;Rosa26R-Fgf8 mice to explore the differences in the regulation of salivary gland development when Fgf8 is overexpressed in the mesenchyme or in the epithelium of salivary glands.Methods:(1)Mate Rosa26R-Fgf8 male mice with Shh-cre and Osr2-cre female mice respectively to obtain Shh-cre;Rosa26R-Fgf8 and Osr2-cre;Rosa26R-Fgf8 mice models.The mice were used as the control group,and the mice were decapitated on E12.5,E13.5,E14.5 and E16.5 days,and the tissues were processed for paraffin-embedded sections.(2)Using frozen section technique,observe the expression patterns of Shh-cre and Osr2-cre in the salivary glands of Shh-cre;Rosa26R-m Tm G on E14.5 day and Osr2-cre;Rosa26R-m Tm G mice on E14.5 day.(3)The histological differences of salivary glands were observed by Masson staining.(4)α-SMA immunohistochemical staining was used to observe the differences in the distribution of myoepithelial cells in salivary glands.(5)Brd U assay and TUNEL assay were used to observe the differences in the proliferation and apoptosis levels of epithelial and mesenchymal cells in salivary glands.(6)Sox9 immunohistochemical staining was used to observe the differences of salivary gland epithelial progenitor cells.(7)In situ hybridization experiments were used to observe the differences in Shh expression pattern in the salivary glands of Shh-cre;Rosa26R-Fgf8 and Osr2-cre;Rosa26R-Fgf8 mice at E13.5.Results:(1)At E14.5,Osr2-cre was found to be expressed in the mesenchyme of the salivary gland of Osr2-cre;Rosa26R-m Tm G mice,while Shh-cre was expressed in the terminal buds and ducts of the salivary glands of Shh-cre;Rosa26R-m Tm G mice.(2)The results of Masson staining showed that at days E12.5,E14.5 and E16.5,Osr2-cre;Rosa26R-Fgf8 mice had not the obvious difference with the numbers of terminal buds and ducts in salivary gland compared with wild-type mice.At E13.5 and E14.5,Shh-cre;Rosa26R-Fgf8 mice had a sharp decrease in the number of salivary gland terminal buds and ducts compared with wild-type mice.And Shh-cre;Rosa26R-Fgf8 mice had less number of terminal buds and ducts than Osr2-cre;Rosa26R-Fgf8 mice.(3)The results of α-SMA immunohistochemical staining showed that myoepithelial cells of wild-type mice surrounded the inner periphery of terminal buds at E15.5.The myoepithelial cells of Osr2-cre;Rosa26R-Fgf8 mice also surround the inner periphery of terminal buds,while the myoepithelial cells of Shh-cre;Rosa26R-Fgf8 mice not only surround the inner periphery of terminal buds,but also exist inside of the bud at the same time.(4)The results of Brd U assey showed that: at E14.5,Osr2-cre;Rosa26R-Fgf8 mice had no significant difference in the proliferation of terminal buds and ducts compared with wild-type mice,but mesenchyme had increasing cell proliferation.At E13.5,Shh-cre;Rosa26R-Fgf8 mice showed reduced proliferation in both terminal bud and ductal cells as well as mesenchymal cells compared with wild-type mice.The results of TUNEL assay showed that compared with wild-type mice,the Osr2-cre;Rosa26R-Fgf8 mice at E14.5 and the Shh-cre;Rosa26R-Fgf8 mice at E13.5 showed no difference in apoptosis.(5)The results of Sox9 IHC experiment showed that compared with wild-type mice Osr2-cre;Rosa26R-Fgf8 mice and Shh-cre;Rosa26R-Fgf8 mice were found the expression of Sox9 in terminal buds and ducts,and there was no difference between Osr2-cre;Rosa26R-Fgf8 mice and Shh-cre;Rosa26R-Fgf8 mice.(6)The results of in situ hybridization showed that Shh was expressed in the terminal buds of wild-type mice and Osr2-cre;Rosa26R-Fgf8 mice,but in Shh-cre;Rosa26R-Fgf8 mice,it was not expressed in terminal buds.Conclusions:(1)The overexpression of Fgf8 in the epithelium of mouse salivary gland has a more serious effect on the branching morphogenesis of salivary gland epithelium than that in the mesenchyme.(2)The results of α-SMA IHC experiments showed that myoepithelial cells were not affected when Fgf8 was activated in the mesenchyme,but the activation of Fgf8 in the epithelium led to the transformation of epithelial cells into myoepithelial cells.(3)Brd U experiments showed that activation of Fgf8 in the mesenchyme of salivary gland promoted the proliferation of mesenchymal cells,while activation of Fgf8 in the epithelium of salivary gland decreased the proliferation of epithelial and mesenchymal cells.TUNEL experiments showed that the activation of Fgf8 in the epithelium and mesenchyme,respectively,resulted in the salivary gland epithelial and mesenchymal cell apoptosis no differcence.(4)The results of Sox9 IHC experiment showed that when Fgf8 was overexpressed in the epithelium and mesenchyme of salivary glands,the epithelial progenitor cells of salivary glands were not affected.(5)The results of in situ hybridization showed that because the activation of Fgf8 in the epithelium inhibited the expression of Shh in the epithelium,the effect on the development of salivary gland was more serious than the activation of Fgf8 in the mesenchyme. |
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