Modified Cryosectioning And Sox2 Cell Localization And Spatiotemporal Expression In Chicken Brain,Lung And Testis

Sox2,a member of the Sox gene family,contains a DNA binding HMG domain that interacts with different transcription factors,multiple DNA promoters or enhancers,and Micro RNAs,exerting a variety of biological functions in the development of oosperm,embryonic development,maintenance of stem cell pluripotency,aging and disease occurrence.However,little is known about the tissue distribution and localization of Sox2 and its temporal and spatial expression patterns during Chick embryo development.The aim of this study was to improve the cryosectioning procedure,and through immunohistochemical(IHC)staining to investigate the distribution and localization of Sox2 in cerebrum,midbrain,cerebellum,lung and testis of "Guangxi three yellow chicken" embryos at different time points during the development.At the same time,the spatiotemporal expression pattern of Sox2 in these tissues was detected by q-PCR and Western Blot.1.Improvement of cryosectioning method and its application in Sox2 cell localization in chickenThe tissues of brain,lung and testis were fixed with Paraformaldehyde and dehydrated by sucrose,and then embedded with OCT embedding reagent before sectioning,the differences of HE staining and immunohistochemical staining from the modified cryosectioning,the conventional cryosectioning and the paraffin sectioning were observed under the microscope,using Image J and Graph Pad software,the quantity of ice crystals in cryosections made by different methods was quantified and analyzed statistically.The localization of Sox2 immunopositive cells in the brain,lungs and testis of chicken embryos was subsequently detected as previously reporte method with little modification.The results showed that the quality of cryosections could be significantly improved by fully infiltrating the tissues with Oct embedding reagent for 6 hours before slicing,and the structure of cryosections of testicular and brain tissues prepared by the modified cryosectioning is clearer than that by the conventional cryosectioning method;compared with the traditional paraffin section,the quality of the cryosections of lung tissue derived from the modified method is more better.The cerebrum,midbrain,cerebellum,lung and testis of chicken embryo are all positive to Sox2 IHC staining.The Sox2-positive cells are found in every tissue layer of Cerebellum,and ome scattered ones are found in the stratum griseum et fibrosum superficiale(SGFS)and the stratum album centrale(SAC)of optic tectum;the Sox2-positive cells are also located in the cerebral cortex and the neuroepithelium of lateral ventricle;Sox2-positive cells are distributed in primary bronchial mucosa epithelium,secondary bronchial epithelium and glands in submucosa of lung.The spermatogenic cells in spermatogenic tubules and a small number of stromal cells in the interstitium of testis are positive to Sox2 IHC staining.2.Spatiotemporal expression of Sox2 during chick embryo developmentChicken embryos were collected at four different time points(7,11,15 and 20 day)after incubation.Firstly,the embryos were sexed by PCR amplification of CDH1 gene,then the cerebrum,mesencephalon,cerebellum,lung and testis tissues were removed from the male embryos,quantitative Real-time PCR and Western Blotting were used to detect the spatio-temporal expression of Sox2 in these tissues.The results showed that the results showed that in all brain tissues,Sox2 mRNA reached its highest level at 15 days of development,and the expression pattern of Sox2 protein at different development time points was different from Sox2 mRNA.In the lungs,Sox2 mRNA and protein are gradually reduced as development progresses.Sox2 mRNA and protein decreases gradually in testis with the progress of development,Sox2 mRNA decreases at 11 d,then increases and remains stable,but Sox2 protein expression in testis is stable at all stages of development.In conclusion,this study provides a new Cryosectioning method for immunohistochemical study.Sox2 protein is localized in the testis and lungs of chickens in a manner similar to that of mammals,and is more widely distributed in brain tissue,especially in the optic tectum.Throughout embryonic development,Sox2 mRNA and protein were continuously expressed in brain,lung and testis,indicating that Sox2 may be involved in the development of central nervous system,lung formation,airway branching and spermatogenesis.This study has provided basic data for understanding the role of Sox2 in chick embryo development.