Connexin31.1 (Gjb5) deficiency blocks trophoblast stem cell differentiation and delays placental development

The gap junction channel forming connexins (Cx) Cx31 (Gjb3) and Cx31.1 (Gjb5) are co-expressed in the mouse trophoblast lineage. Inactivation of either gene results in partial embryonic loss at mid gestation (60% and 30%, respectively, between embryonic days E10.5and E13.5) caused by placental phenotypes. Cx31 deficiency results in loss of stem cell potential and enhanced trophoblast giant cell (TGC) differentiation, whereas the molecular role of the co-expressed Cx31.1 remained unclear. It was assumed that both isoforms have overlapping functions and can compete for each loss in placentation as both knockout mice show similar survival rates, reduced placental weights, and growth restricted embryos. Instead, here we show that Cx31.1 has opposed functions in regulating trophoblast differentiation. Cx31.1 deficiency causes a shift in placental subpopulations, reduced area of fetal blood spaces, and a reduced number of secondary TGC in the junctional zone, as shown by stereology at E10.5. Cx31.1 is critical for terminal differentiation of trophoblast cells during placentation resulting in a delayed induction of marker genes Tpbpa, Prl3b1/Pl-2, and Ctsq in Cx31.1-deficient placentas. Derivation and analysis of Cx31.1-deficient trophoblast stem lines clearly indicates a delayed trophoblast differentiation manifested by repression of marker genes for placental subpopulations and continued expression of stem cell marker genes Id2 and Ascl2, which is correlated to enhanced proliferation capacity of differentiating stem cells These findings clarify the disparate actions of Cx31.1 and Cx31 that act in opposition to balance the fate of trophoblast cells during differentiation, with Cx31.1 promoting, and Cx31 delaying terminal differentiation.