Exploring the differentiation potential of Eomes^POS mouse trophoblast cells in mid-gestation

Mouse trophoblast stem (mTS) cells can be derived from the blastocyst or extraembryonic ectoderm as late as embryonic day (E) 6.5 and when cultured in vitro, can differentiate to all trophoblast subtypes of the mature placenta. Expression of the T-box transcription factor, Eomes, is required for the maintenance of, and used to identify mTS cells. During development, Eomes is restricted to the ExE and, by E7.5, to the chorion, after which its expression declines. The placental junctional zone and labyrinth layers are thought to develop exclusively from the ectoplacental cone and chorion, respectively. While it is well established that mTS cells express Eomes in vitro, it is unknown if Eomes-positive (Eomes^POS) trophoblast that reside in the chorion after E6.5 are restricted in their developmental potential to the labyrinth layer in vivo. This study utilized a lineage tracing technique to evaluate the in vivo differentiation of Eomes^POS trophoblast. Using an Ai6 reporter mouse crossed with a tamoxifen-inducible Eomes-Cre-ERT2 mouse, Cre was activated from E7.5 to E9.5, permanently marking all Eomes^POS trophoblast and daughter cells with the ZsGreen fluorescent protein. This approach was complemented with immunofluorescence staining to assess how the Eomes^POS trophoblast had contributed to the differentiated trophoblast population within the placenta by E17.5. Importantly, the results show that daughter cells of Eomes^POS trophoblast in which Cre was activated, contributed to both placental layers; specifically, spongiotrophoblast and glycogen trophoblast within the junctional zone and syncytiotrophoblast and sinusoidal trophoblast giant cells within the labyrinth. This confirms that Eomes^POS trophoblast maintain the capacity to contribute to both placental layers in vivo and do so after E7.5. This study expands our understanding of trophoblast differentiation in vivo and may prove useful in assessing how Eomes^POS trophoblast contribute placental development later in gestation and in the context of placental pathology, where Eomes expression has been reported.