Abstract
SummaryEpiblast 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 characterized Smad2/3-deficient embryonic stem cells (ESCs). We found that Smad2/3 double-knockout ESCs induced to form epiblast-like cells (EpiLCs) display changes in naive and primed pluripotency marker gene expression, associated with the disruption of Oct4-bound distal regulatory elements. 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 layers 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.
Highlights
The strict segregation of embryonic and extra-embryonic tissues constitutes the earliest cell fate decision in the pre-implantation mammalian embryo
WT, Smad2 KO, Smad3 KO, and Smad2/3 DKO embryonic stem cells (ESCs) cultured under 2i + LIF (2iL) or serum + LIF (SL) conditions were morphologically indistinguishable (Figure S1D)
We identified 422 genes with significantly changed expression levels (209 down and 213 up) in Smad2/3 DKO ESCs cultured under SL conditions compared to WT ESCs (Figure 1B; Table S1)
Summary
The strict segregation of embryonic and extra-embryonic tissues constitutes the earliest cell fate decision in the pre-implantation mammalian embryo. Studies demonstrate that epiblast cells acquire competence to differentiate in response to inductive signaling cues at earlier stages (Smith, 2017). This cellular transition, designated as lineage priming or epiblast maturation and characterized in cultured epiblast-like cells (EpiLCs) (Buecker et al, 2014; Hayashi et al, 2011), is associated with genome-wide reorganization of active enhancers, resulting in decreased expression of naive pluripotency genes, activation of primed and early differentiation genes, and importantly, stable repression of extra-embryonic gene expression (Morgani et al, 2017; Murakami et al, 2016). Double-mutant embryos lacking both Smad2/3 abruptly arrest shortly after implantation and are severely disorganized (Dunn et al, 2004)
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