Combinatorial Smad2/3 Activities Downstream of Nodal Signaling Maintain Embryonic/Extra-Embryonic Cell Identities during Lineage Priming [array]

Epiblast cells in the early post-implantation stage mammalian embryo undergo a transition described as lineage priming before cell fate allocation, but signaling pathways acting upstream remain ill defined. Genetic studies demonstrate that Smad2/3 double-mutant mouse embryos die shortly after implantation. To learn more about the molecular disturbances underlying this abrupt failure, here we characterised Smad2/3-deificient embryonic stem cells (ESCs). We found that Smad2/3 double-knockout ESCs induced to form epiblast-like cells (EpiLCs) display changes in naïve and primed pluripotency marker gene expression, associated with the disruption of Oct4-bound distal regulatory element. In the absence of Smad2/3, we observed enhanced Bmp target gene expression and de-repression of extra-embryonic gene expression. Cell fate allocation into all three embryonic germ lakers is disrupted. Collectively, these experiments demonstrate that combinatorial Smad2/3 functional activities are required to maintain distinct embryonic and/or extra-embryonic cell identity during lineage priming in the epiblast before gastrulation. Total RNA was obtained from wild type, Smad2 KO, Smad3 KO or Smad2/3 DKO undifferentiated embryonic stem cells (ESCs) and embryoid bodies (EBs) differentiated from 100 ESCs each in the absence of LIF for two or three days (d2 and d3 EBs). Biotyinylated cRNA (1500 ng) was randomly hybridised to Illumina WG6_V2 arrays.