Cooperative genetic networks drive embryonic stem cell transition from naïve to formative pluripotency

Abstract

In the mammalian embryo, epiblast cells must exit the naïve state and acquire formative pluripotency. This cell state transition is recapitulated by mouse embryonic stem cells (ESCs), which undergo pluripotency progression in defined conditions in vitro. However, our understanding of the molecular cascades and gene networks involved in the exit from naïve pluripotency remains fragmentary. Here, we employed a combination of genetic screens in haploid ESCs, CRISPR/Cas9 gene disruption, large‐scale transcriptomics and computational systems biology to delineate the regulatory circuits governing naïve state exit. Transcriptome profiles for 73 ESC lines deficient for regulators of the exit from naïve pluripotency predominantly manifest delays on the trajectory from naïve to formative epiblast. We find that gene networks operative in ESCs are also active during transition from pre‐ to post‐implantation epiblast in utero. We identified 496 naïve state‐associated genes tightly connected to the in vivo epiblast state transition and largely conserved in primate embryos. Integrated analysis of mutant transcriptomes revealed funnelling of multiple gene activities into discrete regulatory modules. Finally, we delineate how intersections with signalling pathways direct this pivotal mammalian cell state transition.

Genomic imprinting in mouse blastocysts is predominantly associated with H3K27me3

“…We next evaluated nBiX and nBsX gene expression by data mining, and in vitro in an ESC model. Both pubBsX and nBiX genes exhibited more variable transcriptional dynamics during the first 24 h of ESC differentiation 23 than other genes that were robustly expressed in blastocysts (Supplementary Fig. 2b).…”

“…We obtained allele-specific single-cell gene expression data for oocytes and preimplantation embryos 18 from GEO (GSE80810) and used these data to confirm parentally biased allele-specific expression of published, nBiX, nBsX, but not of published unconfirmed imprinted genes throughout preimplantation development. A comparison of absolute log2FCs between ESCs cultured in 2i and 24 h after induction of differentiation by 2i withdrawal 23 for different groups of genes was utilised to determine whether they exhibited dynamic gene-expression regulation during the exit from naïve pluripotency. Genes exhibiting parentally biased allele-specific expression, equal expression from both alleles, published imprinted genes and all genes, were used as gene groups to compare the dynamics in gene expression as stated in the text.…”

β-catenin links cell seeding density to global gene expression during mouse embryonic stem cell differentiation

“…To address whether seeding density impacts the mESC transcriptome, we plated mESCs in +LIF (leukemia inhibitory factor, for self-renewal) and -LIF (differentiation) medium starting at an initial seeding density of 5×10 5 cells (2.6×10 5 cells/cm 2 ) and serially diluting the cells 1:2 a total of six times, such that the lowest density was 4×10 3 cells/cm 2 . This represents two extremes, as previous mESC studies specify densities between these two values ( Liu et al., 2020a ; Lackner et al., 2021 ). After 96 h, we performed RNA sequencing (RNA-seq) and quantified expression using kallisto ( Bray et al., 2016 ).…”

Unraveling the Spatiotemporal Human Pluripotency in Embryonic Development

“…On the other hand, after 2i withdrawal, a gene-specific network (including Klf5, Nr0b1, Nr5a2, and Pdgfa) related to naïve pluripotency was detected in mESCs, which is necessary for exiting from the naïve state and transitioning to the formative stage. Interestingly, this network was found not only in mouse embryos in the implantation stage (E5.5) but also in macaque postEPI and capacitance hPSCs ( Lackner et al, 2021 ). These data suggest that conversion from the naïve to the formative phase is conserved in primates through a gene network mechanism.…”