The Role And Mechanism Of Gcml In Mouse Syncytiotrophoblast Cell Formation

A functional placenta with a complete structure is essential for a successful pregnancy.The formation of the placental labyrinth layer,an exchange interface of the maternal-fetal nutrient and gas,is essential for placental development.Glial cells missing-1(Gcml)is a key transcription factor regulating the development of the placental labyrinth layer.As a transcriptional factor,Gcm1 exerts its biological function via the special DNA binding to regulate gene expression.The lack of Gcm1 results in branch failure during the development of the labyrinth layer in the mouse placenta,and failed fusion of trophoblast cells to form syncytial trophoblast cells,ultimately leading to embryonic leathal.Previous studies have shown that Gcm1 promotes the withdrawal of trophoblast cells from the cell cycle and syncytosis of trophoblast cells.However,the potential molecular mechanism underlying the differentiation of trophoblast cells into syncytial cells through Gcml still needs to be explored.In addition,there is no faithful in vitro differentiation system for trophoblast stem cells to differentiate into syncytial trophoblast cells,which further hinders the research on the molecular mechanism of Gcml regulating trophoblast syncytialization.Here,we constructed a trophoblast cell line with induced expression of Gcm1 using the Piggybac transposon system.A mouse placental trophoblast cell line with uniform expression of Gcml was obtained by sorting monoclonal cell.By stimulating cell differentiation in different systems,we found that the differentiation culture system is more suitable for studying the mechanism of Gcml regulating the differentiation from trophoblast cells to syncytial cells compared with proliferation conditions.In order to further improve the efficiency of inducing syncytiotrophoblast layer Ⅱ in the differentiation culture system,we optimize a method that is suitable for inducing Gcm l expression,cell fusion and formation of syncytiotrophoblast layer Ⅱ(SynT Ⅱ).Trophoblast cells are treated with proliferation medium(TSCM)for one day before treated with differentiation medium(TSM)for one day,and then induced to express Gcml with Dox.Some studies have reported that inhibition of Mek/Erk pathway can impair the stem cell characteristics of trophoblast cells and promote the differentiation of trophoblast cells.By adding different Mek/Erk signaling pathway inhibitors to our established trophoblast cell differentiation system,we found that inhibition of Mek/Erk signaling can promote the expression of syncytial trophoblast cell related genes and the differentiation of trophoblast cells into syncytial trophoblast cells.By exploring the single-cell sequencing data of the labyrinth layer from a recent published paper and searching for the unique DNA binding motif of Gcml,we got several potential downstream target genes of Gcm1.A number of potential downstream target genes of Gcm1 were predicted,and three candidate molecules,Apela,Igf1r and Itga6,were focused.Apela and Igf1r were found to be highly likely downstream target genes of Gcm1,which jointly regulate the process of trophoblast stem cells to syncyte cells.Immunohistochemical and in situ hybridization experiments showed that Apela and Igf1r expressing in the syncytial trophoblast Ⅱ togethter with Gcm1.ChIP-PCR and luciferase reportor assays showed that Gcm1 binds to the promoter regions of Apela to regulate its expression.In summary,this study established an in vitro cell model for the differentiation of trophoblast cells into syncytial trophoblast cells,and a new direct target gene of Gcml was found.Our research not only explain Gcml mediated regulatory mechanism of trophoblast cell syncytialization,but also provide a reliable in vitro model for studying the molecular mechanisms of the fate determination and differentiation of syncytial trophoblast cells.