The Study Of PTEN On Cranil Neural Crest Cell Emigration In Early Chick Embryo

ObjectiveTo provide the clinical therapy of neural crest cell migration related diseases withmuch more theory foundation through investigating the effects of tumor suppressor genePTEN on the migration and differentiation of neural crest cell in early chick embryo.MethodWhole mount in situ hybridization, PTEN was detected in the early embryo,especially the situ expression of PTEN in neural crest cells and neural crest precursorcells using chick PTEN RNA probes synthesised by molecular biology methods.Electroporation, the neural crest cell migration was detected using this technologyafter the overexpression of PTEN and PTEN mutant.Immunofluorescence, the neural crest cells were specifically markered by HNK1and Pax7, in order to show the migration of them.The primary culture of neural crest cells, the area concerning the migration of neuralcrest cells and the amount of it could be statistically analyzed using primary culturemethod, and so the neural crest cells could be studied and quantified better todemonstrate the effect of PTEN on them.ResultChick PTEN begins to express in the endoderm and mesoderm in HH4chickembryos, and continues to express in the dark area and the embryo body as the embryodevelops. And it expresses majorly in the neural tube and neural crest cells in HH9chickembryos. There was no change of the migration of neural crest cells and the number ofthem been found after the over-expression of PTEN C124S detected by the neural crestcell-specfic marker. However, the inhibition of neural crest cell migration could bedetected after the over-expression of PTEN WT, as manifested by a significance decreaseof migration longitudinal and horizontal distance compared to control (p<0.05), and aremarkable decrease in the number of neural crest cells migrating from the dorsal neuraltube compared to control (p<0.05). A similar result could be obtained through theover-expression of PTEN Y138L which only possesses lipid phosphatase activity.However, the over-expression of PTEN G129E that only possesses protein phosphatase activity, did not have an impact on neural crest cell migration and the number of them.The results obtained from primary neural crest cell culture showed that LY294002couldinhibite the migration of neural crest cell which indicated the involvement of PI3Ksignaling pathway.ConclusionAfter over-expression of PTEN WT, adhesion molecule of neural crest precursorcells, E-cadherin was up-regulated which could subsequently inhibite the migration ofneural crest cell. And PTEN functioned through its lipid phosphatase activity.