ICM-autonomous regulation of mouse blastocyst primitive endoderm formation by p38-MAPK is independent of cavity expansion

Abstract

During early mouse blastocyst ICM maturation, we previously described that pharmacological inhibition of p38 mitogen-activated-kinase (p38-MAPKi) significantly impairs primitive endoderm (PrE) differentiation from an initially uncommitted population of inner cell mass (ICM) cells but does not affect pluripotent epiblast (EPI) specification. A recent report details a positive role for blastocyst cavity expansion in assisting ICM lineage formation and marker gene expression. As p38-MAPKi also results in smaller cavity volumes, we addressed to what extent p38-MAPKi-mediated impaired PrE differentiation is driven by ICM autonomous or cavity expansion mechanisms. We compared ICM differentiation phenotypes associated with either chemically inhibited cavity volume expansion or p38-MAPKi on individual cell and ICM lineage population levels. Whilst recapitulating previously observed decreases in expression of both EPI and PrE markers, we discovered that cavity expansion phenotypes are manifested in impaired numbers of specified EPI and increased numbers of uncommitted cells, rather than impaired PrE differentiation, as observed after p38-MAPKi. Moreover, using both 2D ES-cell and 3D ICM organoid models, we show PrE differentiation is also significantly impaired by p38-MAPKi in the absence of a blastocyst cavity; a result recapitulated in cultured immuno-surgically (IS) isolated early blastocyst ICMs, in which an outer PrE and inner EPI population are ordinarily formed. These data confirm that the early blastocyst requirement for p38-MAPK activity to permit PrE differentiation from uncommitted ICM progenitors is primarily ICM autonomous rather than caused by impaired cavity expansion.