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Mouse embryonic stem cells (ESCs) display pluripotency features characteristic of the inner cell mass of the blastocyst. Mouse embryonic stem cell cultures are highly heterogeneous and include a rare population of cells, which recapitulate characteristics of the 2‐cell embryo, referred to as 2‐cell‐like cells (2CLCs). Whether and how ESC and 2CLC respond to environmental cues has not been fully elucidated. Here, we investigate the impact of mechanical stress on the reprogramming of ESC to 2CLC. We show that hyperosmotic stress induces 2CLC and that this induction can occur even after a recovery time from hyperosmotic stress, suggesting a memory response. Hyperosmotic stress in ESCs leads to accumulation of reactive‐oxygen species (ROS) and ATR checkpoint activation. Importantly, preventing either elevated ROS levels or ATR activation impairs hyperosmotic‐mediated 2CLC induction. We further show that ROS generation and the ATR checkpoint act within the same molecular pathway in response to hyperosmotic stress to induce 2CLCs. Altogether, these results shed light on the response of ESC to mechanical stress and on our understanding of 2CLC reprogramming. Synopsis: Hyperosmotic stress induces embryonic stem cell reprogramming to 2‐cell‐like cells through ROS elevation and ATR checkpoint activation. Hyperosmotic stress induces 2CLCs.Hyperosmotic‐mediated 2CLC induction is mediated through ROS generation and ATR activation.ROS generation and the ATR pathway act in the same molecular pathway in response to hyperosmotic stress. [ABSTRACT FROM AUTHOR]

After fertilization, the zygote genome undergoes dramatic structural reorganization to ensure the establishment of totipotency, and then the totipotent potential of the zygote or 2-cell-stage embryo progressively declines. However, cellular potency is not always a one-way street. Specifically, a small number of embryonic stem cells (ESCs) occasionally overcome epigenetic barriers and transiently convert to a totipotent status. Despite the significant potential of the somatic cell nuclear transfer (SCNT) technique, the establishment of totipotency is often deficient in cloned embryos. Because of this phenomenon, the question arises as to whether strategies attempting to induce 2-cell-like cells (2CLCs) can provide practical applications, such as reprogramming of somatic cell nuclei. Inspired by strategies that convert ESCs into 2CLCs, we hypothesized that there will be a similar pathway by which cloned embryos can establish totipotent status after SCNT. In this review, we provide a snapshot of the practical strategies utilized to induce 2CLCs during investigations of the development of cloned embryos. The 2CLCs have similar transcriptome and chromatin features to that of 2-cell-stage embryos, and we propose that 2CLCs, already a valuable in vitro model for dissecting totipotency, will provide new opportunities to improve SCNT efficiency. [ABSTRACT FROM AUTHOR]

Repeat element transcription plays a vital role in early embryonic development. The expression of repeats such as MERVL characterises mouse embryos at the 2‐cell stage and defines a 2‐cell‐like cell (2CLC) population in a mouse embryonic stem cell culture. Repeat element sequences contain binding sites for numerous transcription factors. We identify the forkhead domain transcription factor FOXD3 as a regulator of major satellite repeats and MERVL transcription in mouse embryonic stem cells. FOXD3 binds to and recruits the histone methyltransferase SUV39H1 to MERVL and major satellite repeats, consequentially repressing the transcription of these repeats by the establishment of the H3K9me3 heterochromatin modification. Notably, depletion of FOXD3 leads to the de‐repression of MERVL and major satellite repeats as well as a subset of genes expressed in the 2‐cell state, shifting the balance between the stem cell and 2‐cell‐like population in culture. Thus, FOXD3 acts as a negative regulator of repeat transcription, ascribing a novel function to this transcription factor. SYNOPSIS: The forkhead domain transcription factor FOXD3 is involved in the heterochromatin‐mediated repression of major satellite repeats and MERVL, thereby counteracting the transcription of 2‐cell state promoting genes in mouse embryonic stem cells. FOXD3 binds to repeat elements, especially major satellite repeats (MSRs) and MERVL in mouse embryonic stem cells.FOXD3 depletion leads to an upregulation of MSRs, MERVL and a subset of 2‐cell state genes.FOXD3 regulates repeat transcription by recruiting the heterochromatin histone methyltransferase SUV39H1.SUV39H1 recruitment establishes repressive H3K9me3 marks on MSRs and MERVL. [ABSTRACT FROM AUTHOR]

After fertilization, the zygote genome undergoes dramatic structural reorganization to ensure the establishment of totipotency, and then the totipotent potential of the zygote or 2-cell-stage embryo progressively declines. However, cellular potency is not always a one-way street. Specifically, a small number of embryonic stem cells (ESCs) occasionally overcome epigenetic barriers and transiently convert to a totipotent status. Despite the significant potential of the somatic cell nuclear transfer (SCNT) technique, the establishment of totipotency is often deficient in cloned embryos. Because of this phenomenon, the question arises as to whether strategies attempting to induce 2-cell-like cells (2CLCs) can provide practical applications, such as reprogramming of somatic cell nuclei. Inspired by strategies that convert ESCs into 2CLCs, we hypothesized that there will be a similar pathway by which cloned embryos can establish totipotent status after SCNT. In this review, we provide a snapshot of the practical strategies utilized to induce 2CLCs during investigations of the development of cloned embryos. The 2CLCs have similar transcriptome and chromatin features to that of 2-cell-stage embryos, and we propose that 2CLCs, already a valuable in vitro model for dissecting totipotency, will provide new opportunities to improve SCNT efficiency.

Embryonic stem (ES) cells, derived from the inner cell mass of a blastocyst, are believed to pluripotent cells and give rise to embryonic, but not extraembryonic, tissues. In mice, totipotent 2-cell stage embryo-like (2-cell-like) cells, which are identified by reactivation of murine endogenous retrovirus with leucin transfer RNA primer (MuERV-L), arise at a very few frequencies in ES cell cultures. Here, we found that a lipid droplet forms during the transition from ES cells to 2-cell-like cells, and we propose that 2-cell-like cells utilize a unique energy storage and production pathway.

The double homeobox (Dux) gene, encoding a double homeobox transcription factor, is one of the key drivers of totipotency in mice. Recent studies showed Dux was temporally expressed at the 2-cell stage and acted as a transcriptional activator during zygotic genome activation (ZGA) in embryos. A similar activation occurs in mouse embryonic stem cells, giving rise to 2-cell-like cells (2CLCs). Though the molecular mechanism underlying this expanded 2CLC potency caused by Dux activation has been partially revealed, the regulation mechanisms controlling Dux expression remain elusive. Here, we discuss the latest advancements in the multiple levels of regulation of Dux expression, as well as Dux function in 2CLCs transition, aiming to provide a theoretical framework for understanding the mechanisms that regulate totipotency.

Currently, two main cell culture models predominate pluripotent stem cell research: embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). Thanks to their ability to contribute to and form all tissues within the body, ESCs and iPSCs have proven invaluable in understanding pluripotent states, early embryonic development and cell differentiation, as well as in devising strategies for regenerative medicine. Comparatively little is known about totipotency -- a cellular state with greater developmental potential. In mice, only the zygote and the blastomeres of the 2-cell-stage embryo are truly totipotent, as they alone can develop to form the embryo and all of its supportive extra-embryonic tissues. However, the discovery of a rare subpopulation of cells in murine ESC cultures, possessing features of 2-cell embryo blastomeres and expanded cell fate potential, has provided a biochemically tractable model to enable the in vitro study of totipotency. Here, we summarize current known features of these 2-cell-like cells (2CLCs) in an effort to provide a reference for the community, and to clarify what we know about their identity so far. [ABSTRACT FROM AUTHOR]

Our perception of the role of the previously considered 'selfish' or 'junk'DNA has been dramatically altered in the past 20 years or so. A large proportion of this non-coding part of mammalian genomes is repetitive in nature, classified as either satellites or transposons. While repetitive elements can be termed selfish in terms of their amplification, such events have surely been coopted by the host, suggesting by itself a likely altruistic function for the organism at the subject of such natural selection. Indeed numerous examples of transposons regulating the functional output of the host genome have been documented. Transposons provide a powerful framework for large-scale relatively rapid concerted regulatory activities with the ability to drive evolution. Mammalian totipotency has emerged as one key stage of development in which transposon-mediated regulation of gene expression has taken centre stage in the past few years. During this period, large-scale (epigenetic) reprogramming must be accomplished in order to activate the host genome. In mice and men, one particular element murine endogenous retrovirus with leucine tRNA primer (MERVL) (and its counterpart human ERVL (HERVL)) appears to have acquired roles as a key driving force in this process. Here, I will discuss and interpret the current knowledge and its implications regarding the role of transposons, particularly of long interspersed nuclear elements (LINE-1s) and endogenous retroviruses (ERVs), in the regulation of totipotency. [ABSTRACT FROM AUTHOR]

The double homeobox (Dux) gene, encoding a double homeobox transcription factor, is one of the key drivers of totipotency in mice. Recent studies showed Dux was temporally expressed at the 2-cell stage and acted as a transcriptional activator during zygotic genome activation (ZGA) in embryos. A similar activation occurs in mouse embryonic stem cells, giving rise to 2-cell-like cells (2CLCs). Though the molecular mechanism underlying this expanded 2CLC potency caused by Dux activation has been partially revealed, the regulation mechanisms controlling Dux expression remain elusive. Here, we discuss the latest advancements in the multiple levels of regulation of Dux expression, as well as Dux function in 2CLCs transition, aiming to provide a theoretical framework for understanding the mechanisms that regulate totipotency. [ABSTRACT FROM AUTHOR]

Pluripotent stem cells are thought of as a surrogate of early developmental stages that sustain the capacity to generate all cell types in the body, thereby constituting an invaluable tool to address the mechanisms underlying cellular plasticity. In the mouse, cells resembling totipotent 2‐cell‐stage embryos (2‐cell‐like cells) arise at a very low frequency in embryonic stem cell (ESC) cultures. However, the extent to which these early‐embryonic‐like cells recapitulate the molecular features of the early embryo is unclear. Here, we have undertaken a characterization of some of the metabolic features of early‐embryonic‐like cells in culture. Our data indicate that early‐embryonic‐like cells exhibit decreased glycolytic and respiratory activity, lower levels of reactive oxygen species and increased glucose uptake, suggesting a shift of the metabolic programme during 2‐cell‐like cell reprogramming. Accordingly, we find that 2‐cell‐like cells can be induced by defined metabolites. Thus, in addition to their transcriptional and chromatin features, 2‐cell‐like cells recapitulate some of the metabolic features of their in vivo counterpart. Altogether, our work underscores a distinct metabolic state of early‐embryonic‐like cells and identifies compounds that can induce their emergence in vitro. Synopsis: 2‐cell‐like cells share some metabolic features with the early mouse embryo, indicating a shift of the metabolic programme during 2‐cell‐like cell reprogramming. 2‐cell‐like cells exhibit decreased glycolytic and respiratory activity compared with ES cells.2‐cell‐like cells do not upregulate oxygen consumption upon acute supplementation of pyruvate or of respiration‐uncoupling agents, suggesting diminished respiratory capacity.Metabolites such as sodium acetate, sodium l‐lactate or d‐ribose can induce the emergence of Zscan4+ and 2‐cell‐like cells in culture. [ABSTRACT FROM AUTHOR]

Embryonic stem (ES) cells, derived from the inner cell mass of a blastocyst, are believed to pluripotent cells and give rise to embryonic, but not extraembryonic, tissues. In mice, totipotent 2-cell stage embryo-like (2-cell-like) cells, which are identified by reactivation of murine endogenous retrovirus with leucin transfer RNA primer (MuERV-L), arise at a very few frequencies in ES cell cultures. Here, we found that a lipid droplet forms during the transition from ES cells to 2-cell-like cells, and we propose that 2-cell-like cells utilize a unique energy storage and production pathway.