WNT3 and membrane-associated β-catenin regulate trophectoderm lineage differentiation in human blastocysts

Abstract

WNT/β-catenin signaling has been described as a crucial regulator of embryonic stem cells and embryogenesis. However, little is known on its role during human preimplantation embryo development, besides the RNA expression of its multiple players. In this study, we performed b-catenin loss- and gain-of-function studies on human preimplantation embryos by adding either Cardamonin or GSK3 inhibitor, 1-Azakenpaullone, to the embryo culture medium from the cleavage until blastocyst stages (Days 3-5/6). β-Cateninwas displayed in the cortical region underneath the membrane during all stages, but it only showed nuclear localization at cleavage stages after stabilization with 1-Azakenpaullone. We did not observe any effects on the inner cell mass markersNANOG, POU5F1,SOX2 and SALL4 in these functional experiments.However, both β-catenin degradation and stabilization caused inhibition of the trophectoderm (TE) fate, illustrated by KRT18 and GATA3 RNA, and CDX2 protein expression. Based on the TE-specificWNT3protein expression in blastocysts, we postulated that this protein may be an upstream regulator for the observed membrane β-catenin function. The addition of either WNT3 or 1-Azakenpaullone to the culture medium promoted EOMESexpression specific for trophoblast development. In both studies, the canonicalWNTpathway target gene, TCF1,was not affected. Therefore, we conclude thatWNT3 and membrane-associated b-catenin promote progenitor trophoblast development in human blastocysts. These results have important implications in assisted reproduction and stem cell biology.