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

哺乳动物着床后早期胚胎的上胚层细胞在细胞命运分配前会经历被称为谱系预启动（lineage priming）的转变，但上游调控的信号通路仍未明确。已有遗传研究表明，Smad2/3双突变小鼠胚胎在着床后不久即发生死亡。为深入探究这种突发性胚胎发育失败背后的分子紊乱机制，本研究对Smad2/3缺陷型胚胎干细胞（embryonic stem cells (ESCs)）进行了系统表征。我们发现，被诱导形成上胚层样细胞（epiblast-like cells (EpiLCs)）的Smad2/3双敲除胚胎干细胞，其初始态（naïve）与预启动态（primed）多能性标志物的基因表达发生显著改变，该改变与Oct4结合的远端调控元件的破坏密切相关。在缺失Smad2/3的条件下，我们观察到骨形态发生蛋白（BMP）靶基因的表达水平显著增强，同时胚外基因表达的去抑制现象。向三个胚胎生殖层的细胞命运分配均受到破坏。综上，本实验证实：在原肠胚形成前的上胚层谱系预启动过程中，Smad2/3的协同功能活性是维持胚胎和/或胚外细胞身份所必需。实验整体设计：从野生型、Smad2敲除（Smad2 KO）、Smad3敲除（Smad3 KO）或Smad2/3双敲除（Smad2/3 DKO）的未分化胚胎干细胞（ESCs），以及由每100个ESCs在无白血病抑制因子（leukemia inhibitory factor (LIF)）条件下分别分化2天（d2）与3天（d3）得到的胚状体（embryoid bodies (EBs)）中提取总RNA。取1500 ng生物素标记的cRNA随机杂交至Illumina WG6_V2基因芯片。