Molecular mechanism of placental development: The role of TMED2, a vesicular transport protein during syncytiotrophoblast formation

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Our group investigates the genetic and cellular basis of placental development in the mouse model. We discovered that TMED2, a transmembrane protein involved in anterograde transport of proteins between the ER and Golgi, was expressed and required for normal placental development. In Tmed2 homozygous mutant (Tmed2-/-) embryos the functional labyrinth layer fails to form. Furthermore, syncytiotrophoblast cells required for function and differentiation of the labyrinth layer are not found in Tmed2-/- embryos. TMED2 is conserved in human and is expressed in both cytotrophoblast cells and in syncytiotrophoblast. To characterize the role of TMED2 in syncytiotrophoblast differentiation, we turned to in vivo human trophoblast cell culture models. We found that TMED2 mRNA and protein levels were higher in the fusion competent BeWo cell line when compared to the non-fusing Jeg-3 cell line. We generated stable BeWo cells with knockdown of TMED2, and stable JEG-3 cells with inducible over-expression of TMED2. We found significantly reduced syncytiotrophoblast formation in BeWo cells with knockdown of TMED2, and syncytiotrophoblast formation in JEG-3 cells expressing TMED2. We hypothesise that TMED2 is a key factor that transports one or more proteins required for syncytiotrophoblast formation. The Aims of this proposal are:******1. To determine the tissue and cell type-specific requirement for Tmed2 in syncytiotrophoblast differentiation - TMED2 is expressed in both the chorion and allantois, which attach to form the labyrinth layer. We will generate mice with conditional mutation in Tmed2 in order to inactivate it in the allantois or chorion. Placental abnormalities in tissue-specific knockouts will be characterized by histology, immunohistochemistry and in situ hybridization.******2. To identify and characterize the TMED2 cargo(es) involved in syncytiotrophoblast formation - Candidate approaches and unbiased proteomic screens will be used to identify TMED2 cargoes in JEG 3 cells with induced expression of TMED2, in BeWo cells with knockdown of TMED2, and in Tmed2 mutant tissues. Expression and localization of TMED2 cargoes will be examined by western blot analysis, immunofluorescence, and co-immunoprecipitation. Proteomics of ER and Golgi fractions will be used to identify abnormally transported proteins.******3. To identify TMED2 cargo proteins that regulate syncytiotrophoblast formation -Validated novel TMED2-cargo proteins conserved in human and mouse will be studied in trophoblast cells and mouse mutant embryos. shRNA will be use to knockdown the specific cargo in trophoblast cells. Placentas of embryos with mutation in TMED2 cargo proteins will be analyzed by transmission electron microscopy and histology for abnormalities in the labyrinth layer. ***The proposed studies will provide novel insights into the role of TMED2 in syncytiotrophoblast formation.**