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.