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

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. Tn5-accessible chromatin profiles of wild type and Smad2/3 DKO day 2 epiblast-like cells (EpiLCs) were generated by deep sequencing in biological triplicate using Illumina HiSeq4000.

哺乳动物着床后早期胚胎的上胚层细胞在细胞命运决定前会经历被称为谱系预置（lineage priming）的转变过程，但其上游调控的信号通路仍未明确。遗传学研究显示，Smad2/3双突变小鼠胚胎会在着床后不久死亡。为深入探究该致死现象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）进行了表征分析。研究发现，被诱导形成上胚层样细胞（epiblast-like cells, EpiLCs）的Smad2/3双敲除ESCs，其初始态多能性（naïve pluripotency）与始发态多能性（primed pluripotency）标记基因的表达发生改变，且该改变与Oct4结合的远端调控元件的破坏相关。在Smad2/3缺失的情况下，可观察到骨形态发生蛋白（Bone morphogenetic protein, Bmp）靶基因的表达增强，同时胚外基因的表达出现去抑制现象。所有三种胚胎胚层的细胞命运分配过程均受到干扰。综上，本实验证明，在原肠胚形成（gastrulation）前的上胚层谱系预置过程中，需要Smad2/3的协同功能活性，以维持不同的胚胎和/或胚外细胞身份。本研究采用Illumina HiSeq4000测序平台，通过三次生物学重复，对野生型与Smad2/3双敲除（DKO）的第2天上胚层样细胞（EpiLCs）的Tn5转座酶可及性染色质图谱（Tn5-accessible chromatin profiles）进行了深度测序。