Emerging cooperativity between Oct4 and Sox2 governs the pluripotency network in mouse early embryos [RNA-seq]

During the first lineage segregation, mammalian embryos generate the inner cell mass (ICM) and trophectoderm (TE). ICM gives rise to the epiblast (EPI) that forms all cell types of the body, an ability referred to as pluripotency. The molecular mechanisms that induce pluripotency in embryos remain incompletely elucidated. Using knockout (KO) mouse models in conjunction with low-input ATAC-seq and RNA-seq, we found that Oct4 and Sox2 gradually come into play in the early ICM, coinciding with the initiation of Sox2 expression. Oct4 and Sox2 directly activate the pluripotency-related genes through the corresponding OCT-SOX enhancers in the early ICM. Furthermore, we observed a substantial reorganization of chromatin landscape and transcriptome from the morula to the early ICM stages, which was partially driven by Oct4 and Sox2, highlighting their pivotal role in promoting the developmental trajectory towards the ICM. Our study provides new insights into the establishment of the pluripotency network in mouse preimplantation embryos. To investigate the role of Oct4 and Sox2 in the development of preimplantation embryos, we produced four transgenic mouse lines: Oct4 KO labeled with mKO2 (monomeric kusabira-orange 2, Oct4mKO2), Sox2 KO labeled with EGFP (Sox2EGFP), floxed Oct4 (Oct4flox) and floxed Sox2 (Sox2flox). Oct4 +/mKO2 and Sox2 +/EGFP heterozygous male mice were mated with Oct4 flox/flox; ZP3-Cre and Sox2 flox/flox; ZP3-Cre maternal KO female mice, respectively, to generate fluorescently labeled maternal-zygotic KO and unlabeled maternal KO (herein called control, Ctrl) embryos. To assess the genome-wide molecular impact of the loss of Oct4 and Sox2, we performed low-input ATAC-seq and RNA-seq using early (E2.75) and late (E3.25) compacted morulae, as well as early (E3.75) and late (E4.5) ICMs. For ATAC-seq, we pooled embryos based on our reporter systems because single-embryo samples yielded sparse signals and too few peaks. We opted to collect single morulae or ICMs for RNA-seq, as this approach enabled us to account for embryo-to-embryo variability and detect transcripts with greater sensitivity compared to scRNA-seq. Given that the ICM consists of the progenitors of EPI and PE cells, we treated the embryos with the MEKi (PD0325901) from E2.5 to suppress PE development and specify the entire ICM to the EPI.在第一次谱系分化阶段，哺乳动物胚胎会生成内细胞团（inner cell mass, ICM）和滋养外胚层（trophectoderm, TE）。内细胞团可分化为上胚层（epiblast, EPI），后者可发育为机体所有细胞类型，这种能力被称为多能性（pluripotency）。目前，胚胎中诱导多能性的分子机制仍未完全阐明。本研究结合敲除（knockout, KO）小鼠模型与低起始量ATAC-seq及RNA-seq技术，发现Oct4与Sox2会在早期内细胞团中逐步发挥功能，这一过程与Sox2表达的启动相吻合。Oct4与Sox2可通过早期内细胞团中对应的OCT-SOX增强子，直接激活多能性相关基因。此外，我们观察到从桑葚胚到早期内细胞团阶段，染色质开放图谱与转录组发生了显著重编程，这一过程部分由Oct4与Sox2驱动，凸显了二者在推动胚胎向内部细胞团发育轨迹中的关键作用。本研究为小鼠植入前胚胎多能性网络的建立提供了新的见解。为探究Oct4与Sox2在植入前胚胎发育中的功能，我们构建了四种转基因小鼠品系：分别用mKO2（单体kusabira-orange 2，即Oct4mKO2）标记的Oct4敲除小鼠、用EGFP标记的Sox2敲除（Sox2EGFP）小鼠、条件性敲除Oct4（Oct4flox）小鼠以及条件性敲除Sox2（Sox2flox）小鼠。将Oct4+/mKO2与Sox2+/EGFP杂合雄性小鼠分别与Oct4 flox/flox; ZP3-Cre及Sox2 flox/flox; ZP3-Cre母源敲除雌性小鼠交配，分别获得荧光标记的母源-合子敲除胚胎，以及未标记的母源敲除胚胎（以下称为对照组，Ctrl）。为评估Oct4与Sox2缺失对全基因组分子水平的影响，我们分别对早期（E2.75）与晚期（E3.25）致密化桑葚胚，以及早期（E3.75）与晚期（E4.5）内细胞团进行了低起始量ATAC-seq与RNA-seq。对于ATAC-seq，由于单胚胎样本的信号稀疏且峰数过少，我们根据报告基因系统对胚胎进行混合处理。而对于RNA-seq，我们选择收集单个桑葚胚或内细胞团，相较于单细胞RNA-seq（scRNA-seq），该方法可更好地反映胚胎间的异质性，并能更灵敏地检测转录本。考虑到内细胞团包含上胚层与原内胚层（primitive endoderm, PE）的祖细胞，我们从E2.5起使用MEKi（PD0325901）处理胚胎，以抑制原内胚层发育，将全部内细胞团定向为上胚层。