Ex vivo culture of pre-placental tissues reveals that the allantois is required for maintained expression of Gcm1 and Tpbpα

Abstract

IntroductionChorioallantoic fusion is essential for development of the labyrinth layer of the mouse placenta. However, events that occur after chorioallantoic attachment remain poorly described, partly due to difficulties of conducting ex vivo analysis of the placenta. Herein, we report conditions for ex vivo culture of the developing murine placenta. MethodsMesometrial halves of decidua containing pre-attachment chorions were cultured alone or with explants of allantoides from stage-matched controls and analyzed by confocal and immunofluorescence microscopy. Expression and levels of marker genes associated with specific placental cell types were measured by in situ hybridization and qRT-PCR, respectively. ResultsAfter 24 h (hr) of co-culture, a mosaic pattern of eGFP+ and eGFP− cells were found when explants of pre-attachment chorions from eGFP+ embryos were co-cultured with stage-matched allantoides from eGFP− embryos or vice versa. In addition, proliferation increased in the allantoic region and folds formed on the chorionic plate. PECAM positive cells derived from the allantois were found in the chorionic region. Levels of the SynT-II marker, Gcm1, significantly increased at 24 h, although expression of Gcm1, was only found in explants co-cultured with an allantois at 12 h and 24 h. In addition, though levels of Tpbpα was not altered by co-culture with an allantois, Tpbpα was only detected in explants co-cultured with an allantois for 24 h. DiscussionOur data show that chorioallantoic fusion and events associated with initiation of labyrinth layer formation can be modeled ex vivo, and reveal a previously unsuspected requirement of chorioallantoic fusion for Tpbpα expression.