Chromatin dynamics in the regulation of cell fate allocation during early embryogenesis.

Nature Reviews Molecular Cell Biology | AOP, published online 10 October 2014; doi:10.1038/nrm3885

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Chromatin dynamics in the regulation of cell fate allocation during early embryogenesis Adam Burton and Maria-Elena Torres-Padilla

Abstract | Following fertilization, gametes undergo epigenetic reprogramming in order to revert to a totipotent state. How embryonic cells subsequently acquire their fate and the role of chromatin dynamics in this process are unknown. Genetic and experimental embryology approaches have identified some of the players and morphological changes that are involved in early mammalian development, but the exact events underlying cell fate allocation in single embryonic cells have remained elusive. Experimental and technological advances have recently provided novel insights into chromatin dynamics and nuclear architecture in single cells; these insights have reshaped our understanding of the mechanisms underlying cell fate allocation and plasticity in early mammalian development. Totipotent Totipotent cells are unique to the early embryo and have an unlimited potential to differentiate to the three germ layers of the embryo as well as to the extra-embryonic tissues.

Blastocyst The stage in mammalian development in which the embryo contains a fluid-filled cavity called the blastocoel.

Trophectoderm The first differentiated cell type that forms the outer layer of the blastocyst, which gives rise to extra-embryonic tissues that support the developing embryo.

Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM U964, Université de Strasbourg, F-67404 ILLKIRCH, Cité Universitaire de Strasbourg, France. Correspondence to M.E.T.-P. e-mail: [email protected] doi:10.1038/nrm3885 Published online 10 October 2014

The generation of an organism from a fertilized oocyte requires a complex interplay of events involving cell division, differentiation and cell death. In mammals, the genomes of the oocyte and sperm must be epigenetically reprogrammed to enable them to generate the multiple cell types in the organism and to support extraembryonic tissues. This reprogramming occurs immediately after fertilization and results in the formation of a totipotent zygote. The zygote first undergoes a series of divisions or cleavages, without significant increase in cell volume1, resulting in the formation of the blastocyst. By this point, the first differentiation event has occurred, segregating the outer trophectoderm, which is developmentally restricted to extra-embryonic tissues, from the inner cell mass (ICM), which comprises pluripotent embryonic cells that will develop into the embryo. In mice, one day later, at embryonic day 4.5 (E4.5), the embryo will undergo implantation. Thus, during pre-implantation development, the first two distinct cell lineages are generated: the outer cells support embryo implantation into the uterine wall and differentiate as a defined lineage first, whereas the inner cells retain pluripotency in order to enable the formation of the embryo. The acquisition of cellular identity or phenotype relies on the combined effects of the genetic and epigenetic information of a cell. Chromatin-mediated changes that regulate gene expression operate on multi ple levels, including DNA methylation (5meC), posttranslational histone modifications, the incorporation of specific histone variants, and chromatin remodelling

through nucleosome positioning2,3 (FIG. 1). These modifications, through the recruitment of effector proteins4,5 or through a direct effect on nucleosomal stability6,7, are key regulators of gene expression, although whether they are initiating or reinforcing factors for transcription is unclear. Nuclear organization can also have an impact on gene expression either globally or specifically in generating a local chromatin environment that is permissive or refractory to gene expression. Mammalian pre-implantation development is characterized by major dynamics in chromatin at all of these levels, with the possible exception of nucleosome positioning, which has not been addressed extensively during these developmental stages and awaits further investigation8–10. How embryonic cells adopt their fate after fertilization and whether the chromatin conformation within each cell gradually promotes its identity throughout development remain unknown. These questions are central for our understanding of cell plasticity, development and reprogramming. Here we discuss recent technical advances and new approaches to address the epigenome and nuclear architecture of single cells and the conclusions drawn from applying these techniques in the study of the early mammalian embryo. We discuss the characteristics of embryonic chromatin and the state of single-cell analyses of chromatin, and we postulate that chromatin structure can regulate cell fate by potentially operating at two levels: locally, by regulating the expression of specific genes (for example,

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REVIEWS Fertilization

Implantation

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OCT4 NANOG SOX2 ID2 CDX2 Zygote

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Morula

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Asymmetric divsions Totipotency

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H3.1/3.2 H3.3 H2A.Z mH2A H2A.X γH2A.X H4K20me3 H3K64me3 H3K9me3 H3K27me3 H3K4me3 DNAme Histone mobility Chromocentre formation

Figure 1 | Transcription factors and global chromatin changes in early embryonic Nature Reviews | Molecular Cell Biology development in the mouse. a | Depicted are the temporal dynamics of the expression of key transcription factors between fertilization and implantation. The onset of lineage allocation can be traced to the division between the 8‑cell stage and the 16‑cell stage that generates inner (green) and outer (purple) cells. Inner cells tend to become the pluripotent inner cell mass (ICM) cells in the early blastocyst, and outer cells become the trophectoderm. The transcription factors contributing to either lineage are colour coded accordingly. b | Global levels of histone modifications or chromatin features such as histone variants and histone mobility are depicted with their temporal level profile shown across pre-implantation development. Features showing an asymmetric distribution among individual cells of the same embryo at the 4‑cell stage are colour coded in pink; histone variants are shown in red; post-translational modifications characteristic of constitutive heterochromatin are shown in blue; facultative heterochromatin marks are shown in green; the histone modification of transcriptionally active chromatin is shown in yellow; DNA methylation levels are shown in purple; and nuclear architecture features are shown in orange. γH2A.X, phosphorylated H2A.X; CDX2, caudal type homeobox 2; DNAme, 5‑methyl cytosine DNA; H2A.Z, variant of H2A; H3, histone H3; H4, histone H4; ID2, inhibitor of DNA binding; me2, dimethylation; me3, trimethylation; mH2A, macro histone H2A; OCT4, octamer-binding transcription factor 4; PRDM14, PR domain containing 14; SOX2, sex determining region Y-box 2.

lineage-specific transcription factors), but also globally, by generating a chromatin environment that is permissive for cell fate allocation through global chromatin dynamics and nuclear organization (FIG. 2).

This Review does not include a discussion on the epigenetic features that are laid down before the zygote stage, during germline development, as this has been recently reviewed elsewhere11. Most of the work discussed below is based on the mouse embryo as a model system. Where pertinent, and when data are available, we have also discussed other mammalian species.

Characteristics of embryonic chromatin The development of the mammalian pre-implantation embryo is highly regulative in nature, meaning that cell fates are not pre-determined. This is in stark contrast to many non-mammalian species, in which fate decisions are determined as early as the zygote stage through polarization and unequal partitioning to daughter cells of certain determinants, such as the Par proteins in Drosophila melanogaster and Caenorhabditis elegans. These proteins, through their asymmetric cortical localization in the oocyte, determine the orientation of the future anterior–posterior axis of the embryo12–14. In contrast, up to the 8‑cell stage, mouse blastomeres remain morphologically identical, and individual blastomeres can give rise to all tissues of the embryo when transplanted into ‘carrier’ embryos14. Overall, it has therefore been difficult to ascertain, without experimental perturbation, whether cells prior to the 8‑cell stage consistently present any intra-embryonic differences, which could potentially indicate some degree of early cell fate allocation before blastocyst formation. The blastocyst asymmetry can be traced to the gener ation of the 16‑cell stage morula, whereby inner blasto meres arise that will later be enveloped by outer blastomeres12. Inner cells tend to form the ICM, whereas the outer blastomeres tend to form the trophectoderm (FIG. 1). However, cells at this point retain plasticity and are still not committed to their particular lineage15,16. Trophectoderm cells commit before the pluripotent ICM cells17. The ICM cells further differentiate into the true embryonic epiblast and extra-embryonic primitive endoderm lineages during the next cell cycle. Embryonic stem cells (ES cells) derived from the ICM also retain the ability to differentiate into trophectoderm, either through forced repression of octamer-binding transcription factor 4 (Oct4) or through overexpression of caudal type homeobox 2 (Cdx2) (REFS 18,19). Early lineage specification by transcription factors. How the transitions in cell potency and lineage allocation are regulated at the molecular level remains poorly understood. The control of gene expression by transcription factors and chromatin remodelling is critical for guiding developmental decisions. The trophectoderm expresses transcription factors, such as CDX2 and TEA domain family member 4 (TEAD4; also known as TEF3), that are restricted to, and required for, its formation or differentiation20,21. Likewise, the expression of transcription factors such as OCT4 and NANOG is maintained in the ICM only17,22 (FIG. 1). However, these factors may not be as important for lineage specification as previously thought; for example, NANOG and OCT4 are not required for the initial lineage establishment between inner and outer

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cells of the early blastocyst23,24 and undergo complete restriction only after the subsequent lineage segregation into epiblast and primitive endoderm17,25. In addition, CDX2 seems to be dispensable for trophectoderm specification as Cdx2–/– embryos form an expanded blastocyst that includes the trophectoderm, although they fail to maintain trophectoderm function21,26. How the expression of these lineage transcription factors is regulated in stem cell lines derived from the ICM (ES cells) and the trophectoderm (trophoblast stem cells) is well understood. However, although ES cells constitute a powerful system for studying pluripotency, the chromatin in ES cells differs significantly from that in preimplantation embryos in terms of — for example — its nuclear organization and the prevalence of constitutive heterochromatin, both of which are distinctive features of the embryo (see below). Therefore, much of our knowledge about chromatin regulation in ES cells cannot be directly extrapolated to early embryos, and the question of how the expression of pluripotency-associated transcription factors is regulated in vivo remains underinvestigated. This is mainly because biochemical approaches cannot be applied to pre-implantation embryos owing to the small amount of material that is available from each embryo. There is ample information about the steady state levels of mRNA and protein of lineage transcription factors throughout early embryogenesis, but how their expression is regulated by chromatin remodelling is unknown. A few studies have investigated nascent transcription in embryonic cells (for example, using RNA fluorescence in situ hybridization (RNA-FISH)), providing a first approximation of how embryonic gene expression is regulated27–30. RNA-FISH for Nanog in 2-cell, 4-cell and 8‑cell embryos revealed high heterogeneity in both the timing of Nanog transcription and number of cells that transcribe Nanog; this is in contrast to Oct4, which is transcribed more uniformly in the different cells within embryos28. Moreover, whereas Nanog seems to be transcribed primarily from a single allele27,28, other lineage transcription factors are transcribed from both alleles, which suggests that transcriptional regulation might be important in controlling the dose of such proteins during pre-implantation development. We are still far from understanding how transcription dynamics are achieved at the molecular level in vivo and how embryonic chromatin is made permissive or instructive for cell fate allocation: is it by promoting an open environment globally or exclusively at one or several ‘master’ genes? (FIG. 2). A thorough description of the composition of the embryonic chromatin, its modifications and their changes during cleavage is a prerequisite to understanding the potential role of chromatin dynamics in regulating cell fate allocation. Indeed, as we describe below, embryonic chromatin has several distinctive features that are different from those of the chromatin in somatic and stem cells.

A cell lineage derived from the inner cell mass that generates primarily extra-embryonic tissues, which will constitute the embryonic part of the placenta.

Starting development: parental epigenetic asymmetries. The two parental genomes remain physically segregated for about 24 hours after fertilization and exhibit remarkable asymmetry in several levels of chromatin

Inner cell mass (ICM). Cells in the interior of the blastocyst that give rise to all tissues of the embryo and are the source of embryonic stem cells. The ICM is completely surrounded by the trophectoderm cells.

Pluripotent Pluripotent cells have the potential to differentiate to the three germ layers of the embryo: the endoderm, ectoderm and mesoderm.

Histone variants Non-allelic variants of the canonical histone proteins that differ in their protein structure, possess 5′ and 3′ untranslated regions, and are not restricted in their expression and incorporation into chromatin to the S phase of the cell cycle.

Cell plasticity The ability of a cell to change state or fate, whether by differentiation, reprogramming or any other sort of transformation.

Regulative A term used to describe developmental progression in which cells remain plastic and their fates are not determined from an early stage.

Blastomeres Cells of the pre-implantation embryo.

Blastocyst asymmetry The two initial cell types that exist within the blastocyst: the trophectoderm cells forming the outer trophoblast layer that surrounds the cavity, and the inner cell mass.

Epiblast An embryonic compartment derived from the inner cell mass. It gives rise to the embryo proper and differentiates to form the three layers of the developing embryo: ectoderm, endoderm and mesoderm during gastrulation.

a Global chromatin-mediated regulation

b Local chromatin-mediated regulation

Gene X

Gene X Gene Y

Gene Y

Figure 2 | Model for global and local chromatin regulation of gene expression. Global and local Nature Reviews | Molecular Cell Biology chromatin can mediate intra-embryonic asymmetry between blastomeres. A 4‑cell stage embryo is shown as an example. a | In global chromatin asymmetry, the chromatin in some blastomeres is generally more compact, whereas in others it is generally more open, resulting in differential expression patterns. b | In local chromatin-mediated asymmetry, the chromatin in all nuclei contains both open and compacted regions; however, the chromatin state at particular loci differs between individual blastomeres at the same stage of development, enabling dissimilar gene expression programmes to be established. Both types of regulation could potentially contribute to generating chromatin-based asymmetries in gene expression.

organization. This has been reviewed elsewhere8–10, so we will only discuss the most recent data. It has long been known that the maternal and paternal genomes of most mammals31–33 undergo extensive DNA demethylation following fertilization, and recent technical advances, such as whole-genome bisulphite sequencing and reduced representation bisulphite sequencing (RRBS), have shown that the gametes differ in the extent and genomic localization of methylated cytosines34,35. The paternal genome seems to undergo demethylation much faster than the maternal genome31,36,37. However, the extent to which such data truly reflect DNA (de)methylation levels has recently been questioned, as bisulphite sequencing cannot distinguish between methylation and hydroxylation, and it was discovered that TET enzymes oxidize methylated DNA in the paternal pronucleus38,39. In addition, the DNA base-excision repair pathway has been implicated in reducing DNA methylation levels, and the precise contributions of each of these processes to overall paternal demethylation remains to be established40.



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Embryonic stem cells (ES cells). Pluripotent stem cells derived from the inner cell mass that can be cultured in vitro indefinitely and differentiated into all three germ layers.

Trophoblast stem cells Stem cells that are derived from the polar trophectoderm of pre-implantation embryos and retain the capacity to differentiate in vitro into all trophoblast derivatives of the placenta.

RNA fluorescence in situ hybridization (RNA-FISH). An approach for studying the localization of nascent transcripts. Single-molecule RNA-FISH is a quantitative adaptation of RNA-FISH.

Bisulphite sequencing A technique for analysing sequence-specific methylated cytosines, based on their specific resistance to bisulphite conversion.

Reduced representation bisulphite sequencing (RRBS). A variant of bisulphite sequencing that is used to analyse methylation patterns at specific loci with high CpG content.

TET enzymes Ten-eleven translocation methylcytosine dioxygenase enzymes that catalyse the conversion of 5‑methylcytosine to 5‑hydroxymethylcytosine by oxidation.

Protamines A group of small, highly basic proteins associated with the DNA, particularly in sperm, in place of histones.

Polycomb repressive complex 2 (PRC2). A di- and trimethyltransferase complex. Its substrate is Lys27 of histone H3, a mark of facultative heterochromatin.

Major satellite (Also known as a gamma satellite). An extensive region of tandem DNA repeats. Major satellites are normally found at pericentromeres and are mostly AT‑rich.

In any case, the function of changes in DNA methylation and the potential functional consequences of parental asymmetry, if any, remain unknown. Conceivably, it could be an evolutionary by‑product or a consequence of the genome-wide exchange of protamines with histones in the paternal genome. Subsequently, throughout the course of the following cell divisions, passive demethylation occurs in both parental genomes until the blastocyst stage31,32,41, when methylation patterns are re‑established in a lineage-specific manner. The maternal and paternal genomes possess strikingly different patterns of histone modifications42–49. Whereas the maternal genome has a global pattern of histone marks typically resembling that of somatic cells, the paternal genome is markedly atypical and seems to be devoid of trimethylation marks and of constitutive heterochromatin features in particular9. Histone methylation occurs in a stepwise manner and, in general, monomethylation is functionally uncoupled from dimethylation and trimethylation and is established by different enzyme complexes, which results in a delay in the appearance of trimethylated histone marks in early development. Indeed, monomethylation of histone H3 at Lys4 (H3K4me), H3K9me, H3K27me and H4K20me are observed in the paternal pronucleus at the early zygotic stages, whereas the corresponding trimethylated (me3) states are absent. Although the responsible methyltransferases have been relatively well studied in vitro, they have not been well characterized in the pre-implantation embryo. The delayed conversion from H3K27me to H3K27me3 in the paternal pronucleus is concomitant with a delayed localization of Polycomb repressive complex 2 (PRC2) to the paternal pronucleus44,45. The changes in chromatin modifications and the asymmetry of the two parental nuclei are thought to be necessary for epigenetic reprogramming. An attractive hypothesis is that the initial parental asymmetry is involved in setting a lineage cue, but this has not been addressed experimentally. This is because, although cell fate regulation and epigenetic reprogramming are two conceptually different processes, they are difficult to discern experimentally, as they temporally coincide. Histone variant incorporation into embryonic chromatin. De novo chromatin assembly is necessary following the unpackaging of the paternal genome from protamines. Although some histones remain at certain loci in the paternal genome in human, mouse and zebrafish50–53, most of the genome acquires new histones, which are derived from the oocyte. The newly incorporated histones are hyperacetylated and hypomethylated, and the resulting genome is devoid of heterochromatin in the traditional sense9. Chromatin structure and function are also regulated by the incorporation of histone variants, and the two parental genomes also differ in this regard. Notably, the histone H3 variant H3.3, a replacement variant that differs from canonical H3.1 and H3.2 by four and five amino acids, respectively, is rapidly incorporated into the paternal genome, in a replication-independent manner, specifically after fertilization45,54,55. It remains to be ascertained whether the

selective incorporation of H3.3 into the decondensing paternal pronucleus is merely a stop-gap until canonical H3 incorporation can occur during replication or whether H3.3 incorporation has a role in facilitating the open chromatin configuration observed particularly in the paternal pronucleus. H3.3 is important for the initial establishment of paternal pericentric heterochromatin: H3.3 localizes to the paternal pericentric chromatin, and the mutation of H3.3K27 — but not of H3.1K27 — leads to developmental arrest45. This phenotype is rescued by the injection of major satellite transcripts, which suggests a role for H3.3 in facilitating transcription from these regions45, an event that is specific to the stages immediately following fertilization and that may be required for the subsequent establishment of paternal pericentric heterochromatin. Histone H2A variants are also used during preimplantation development. Foci of phosphorylated H2A.X, which normally mark sites of DNA doublestrand breaks, are abundant throughout pre-implantation development and enriched in the early paternal pro nucleus38,56. This may be independent of DNA damage, as tumour suppressor p53-binding protein 1 (TP53BP1), which is also normally recruited to damage sites, does not colocalize with these foci. Although H2A.Z is expressed at very low levels in zygotes, it is incorporated from the late 2‑cell stage in euchromatin57,58. Absence of H2A.Z results in a failure of development at around the time of implantation59. This is possibly due to a specialized function of H2A.Z in trophectoderm differentiation, as this histone variant is enriched in the trophectoderm60 (although it is also expressed in the ICM57). Acetylation of H2A.Z, which occurs on amino-terminal Lys residues, also occurs in pre-implantation embryos, following similar dynamics to the incorporation of H2A.Z itself. As a marker of active chromatin, the absence of acetyl‑H2A.Z from the 2‑cell stage in mice, when major embryonic genome activation (EGA) occurs, is intriguing. Remarkably, the appearance of active marks of transcription does not necessarily correlate with the timing of EGA across species; for example, H3K36me3 is undetectable at the 2‑cell stage in mice but appears before EGA in bovine embryos at the 4-cell to 8-cell stage, which suggests that it has an alternative function than facilitating transcription in early embryogenesis57. Thus, the chromatin in the period immediately following fertilization is markedly atypical in its composition, and there are several epigenetic asymmetries between the parental genomes. The atypical organization of embryonic chromatin. In the hours following fertilization, heterochromatic features are removed from the maternal chromatin. For example, the maternal genome rapidly loses two histone methylation marks that are typical of heterochromatin — H4K20me3 and H3K64me3 — as early as the 2‑cell stage 46,48. H3K9me3, which is another classic mark of constitutive heterochromatin, becomes diluted, apparently passively; the de novo establishment of H3K9me3 is prevented for at least two or three cell divisions42,44 (FIG. 1). The molecular basis of this decrease in constitutive heterochromatin marks is unknown, but



REVIEWS Germinal vesicle oocyte

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Chromocentres

Figure 3 | Global chromatin reorganization during early mouse development. Nature Reviews | Molecular Biology Confocal sections of DAPI (4ʹ,6‑diamidino-2‑phenylindole)-stained nuclei of Cell a germinal vesicle oocyte, zygote, early 2‑cell stage and 4-cell stage mouse embryos. AT‑rich sequences from the major satellites localize sharply in rings around the nucleolar-like bodies (black holes on the figure). These images show progressive relocalization into chromocentres, which are neatly visible by the 4‑cell stage. The scale bar represents 10 µm. Images courtesy of J. Jackowicz, Institut de Génétique et de Biologie Moléculaire et Cellulaire, Strasbourg, France.

its timing indicates that they are actively removed, at least in the case of H4K20me3 and H3K64me3, which suggests an important role for the loss of constitutive heterochromatin in embryonic chromatin reprogramming. The absence of typical heterochromatic features is in line with a more open chromatin configuration characterizing pre-implantation development61. Major changes in global chromatin organization occur during pre-implantation development, and many of these changes are unique to the period of time following fertilization. In somatic cells, AT‑rich genomic regions, which in the mouse are mostly the major satellites and the minor satellites surrounding the centro meres, typically associate to form chromocentres , visualized as DAPI-rich foci. However, in mature oocytes and fertilized embryos, these centromeric and pericentromeric regions are neatly arranged in rings surrounding nucleolar-like bodies (NLBs), as determined by DNA-FISH62–64. This organization persists until the

late 2‑cell stage, although the NLBs themselves remain for another cell division (FIG. 3). The purpose of this perinucleolar organization is unknown; however, forcing major satellite regions to relocate away from the NLBs (by the expression of zinc-finger targeting major satellites fused to emerin) impedes pre-implantation development, even though it has no effect on gene expression65. This suggests that the atypical nuclear organization is important and may be required for the consolidation of the two parental genomes after fertilization. Alternatively, it may be necessary for epigenetic reprogramming by promoting an open chromatin environment that is permissive for the removal of gametespecific epigenetic modifications and the subsequent re‑establishment of new epigenetic marks. Indeed, during somatic cell nuclear transfer (SCNT; BOX 1), formation of NLBs in bovine and mouse embryos is observed66–68. Reprogramming by transcription factors does not support such a reorganization, perhaps as this results in reprogramming to pluripotency, and not to totipotency as in SCNT. Thus, the analysis of the chromatin-based organizational changes that accompany natural reprogramming is likely to assist in the development of artificial reprogramming strategies. In combination with the atypical organization of satellite repeats that normally form constitutive heterochromatin, the pericentromeric regions acquire marks of facultative heterochromatin after fertilization, such as H3K27me3 (REF. 43), instead of marks of constitutive heterochromatin (FIG. 1). This is possibly because the more resilient compaction of constitutive chromatin is unfavourable for the execution of major epigenetic reprogramming. Studying the function and mechanisms of heterochromatin formation in the pre-implantation embryo is therefore likely to yield unique insights into how different states of chromatin may contribute to sustaining cell plasticity and cell fate allocation.

Box 1 | Somatic cell nuclear transfer

Embryonic genome activation (EGA). The process by which the embryonic genome begins to transcribe the major portion of its genome.

Chromocentres Irregular, densely stained aggregations of DNA, consisting of heterochromatic, centromeric and pericentromeric regions.

Nucleolar-like bodies (NLBs). Spherical bodies of uncertain structure and function that are unique to the pre-implantation embryo of mammals and are thought to be the non-functional precursors of nucleoli.

Somatic cell nuclear transfer (SCNT) consists of transferring a nucleus from a (relatively) differentiated cell into a de‑differentiated cell to induce cellular reprogramming of the donor nucleus to a more plastic state. SCNT enables the creation of an embryo from the nucleus of an adult cell, demonstrating that the potential for totipotency is not lost in most adult cell types122. SCNT exploits the inherent ability of the oocyte (or less frequently, the zygote) cytoplasm to reprogramme a more differentiated cell nucleus and enables the study of the mechanisms regulating reprogramming to totipotency. Better outcomes and more typical epigenetic resetting are achieved with reprogramming through nuclear transfer to oocytes than with other techniques, such as the reprogramming of induced pluripotent stem cells with transcription factors. This shows the advantage of passing through an early embryonic-like state, which has been the preferred configuration throughout mammalian evolution. However, SCNT is inefficient, particularly when more differentiated cell types are used as nuclear donors, which indicates the existence of an inherent resistance to epigenetic reprogramming following differentiation123. Several recurrent defects in SCNT-derived embryos have been identified; these have been targeted to improve the success rates of embryos developed to term124. For example, incomplete chromatin remodelling and DNA demethylation occurs during SCNT, and therefore the inhibition of DNA methyltransferase and histone deacetylases improves SCNT efficiency125. Furthermore heterochromatin marks seem to be inhibitory towards reprogramming126,127, and their maintenance correlates with poor SCNT efficiency128. In Xenopus laevis, several other chromatin constituent factors regulate reprogramming efficiency both positively (histone B4 and nucleophosmin) and negatively (macro histone 2A; for a review, see REF. 123). H3.3 seems also to be required for efficient SCNT reprogramming in frogs129 and mice130. An alternative method for analysing mechanisms of reprogramming is to use the germinal-vesicle stage oocyte of X. laevis as a recipient cell, into which several donor nuclei are injected. The nuclei do not replicate but rapidly change their transcriptional programme, which enables one to distinguish mechanisms driving transcriptional reprogramming from those driving developmental effects (such as those relating to DNA replication).



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4-cell stage

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CARM1 H3R26me2 levels

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Figure 4 | Asymmetries between cells of 4‑cell and 8‑cell mouse embryos. a | A gradient of dimethylated histone 3 Arg26 (H3R26me2) levels is found across the four blastomeres of some four‑cell stage embryos. b | |PR domain Cell Biology Nature Reviews Molecular containing 14 (Prdm14) is asymmetrically expressed in four‑cell stage embryos: mRNA and protein levels are high in two cells (green nuclei) and low in two cells (purple nuclei). Overexpression of Prdm14 in single blastomeres of 2‑cell stage embryos results in increased H3R26me2 levels in 4‑cell stage embryos and drives these cells towards the inner cell mass lineage. Coactivator-associated arginine methyltransferase 1 (CARM1) and PRDM14 interact physically, which suggests a model whereby PRDM14 might promote CARM1 binding to its target genes. c | Blastomeres of 8‑cell stage embryos show consistent intra-embryonic differences in OCT4 kinetics. Two distinct OCT4 pools — one with higher mobility, the other with lower mobility — are observed in individual cells, pointing to potential differences in OCT4 association to the chromatin.

Bivalent domains Regions of chromatin that contain both activating and repressive chromatin marks coincidently, notably trimethylated histone H3 Lys4 (H3K4me3) and H3K27me3.

Asymmetry in histone modifications in the blastocyst. Asymmetries in the global levels of some histone modifications, although not necessarily an indicator of a role of chromatin structure and nuclear architecture in cell fate allocation, have been observed in the blastocyst. Compared with the trophectoderm, the cells of the ICM have globally higher levels of DNA methylation and H3K27 methylation, as well as lower levels of histone H2A and/‌or H4 phosphorylation37,69–71. Bivalent domains are also enriched in the ICM relative to the trophectoderm, owing to lower levels of H3K27me3 in the trophectoderm72. Furthermore, mouse embryos with knockouts of methyltransferases, such as enhancer of zeste homologue 2 (Ezh2), which are responsible for the DNA and histone methylations that are enriched in the ICM are often embryonic lethal at early stages, even when the maternal contribution is intact74,75. These knockouts are more severely affected in embryonic tissues than in trophectoderm tissues during development, which suggests that the asymmetries in the epigenetic landscape in the blastocyst and potentially in earlier stages are functionally important70,76–79. However, these epi genetic differences might not be reflected in differences in gene expression. Few epigenetic pathways have been shown to regulate lineage allocation in the early embryo by affecting the expression of specific genes or by promoting a global chromatin environment that supports cell fate determination. For example, DNA methylation in the promoter of the ETS-related transcription factor E74-like factor 5 (ELF5) was shown to be required for its repression in ES cells and consequently for its restricted expression in the trophoblast lineage80. The epigenetic asymmetries of the two blastocyst lineages are more evident after their fate has been allocated, but it is not known whether epigenetic asymmetry can

also function as a driver for lineage allocation or whether it is merely a locking mechanism to enforce cell fate, as in the case of ELF5. In a proportion of 4‑cell embryos, one blastomere of four has lower levels of H3R26me2 and is more likely to develop into trophectoderm derivatives81,82 (FIG. 4a). Increasing the levels of H3R26me2 by overexpressing coactivator-associated arginine methyltransferase 1 (CARM1) in individual blastomeres at the late 2‑cell stage resulted in the allocation of their progeny to the ICM lineage, providing evidence of a driver role for an epigenetic modification of chromatin during early embryo development81. Using single-cell gene expression profiling, the levels of chromatin modifiers were shown to distinguish the ICM and trophectoderm lineages83. This was mostly based on the enrichment of DNA methyltransferase 3β (Dnmt3b) and DNA methyltransferase 3-like (Dnmt3l) in the trophectoderm and of PR domain containing 14 (Prdm14) in the ICM, which confirms previous findings that Prdm14 is enriched in the ICM in the early blastocyst84,85. Single-cell transcriptomics also revealed that Prdm14 is expressed at the 4‑cell stage in an interesting heterogeneous pattern: two of the blastomeres in each embryo express high levels of Prdm14 mRNA and protein, whereas the other two blastomeres express no, or very low levels of, Prdm14 (FIG. 4b). This pattern emerged only by the late 4‑cell stage, suggesting that the 4‑cell stage might be a transitional stage in which potential epigenetic asymmetries arise. Furthermore, similarly to CARM1 (albeit to a lesser extent) PRDM14 was found to promote lineage allocation to the ICM from the 4‑cell stage. A potential functional link with CARM1 was also implicated by the finding that PRDM14 overexpression increased levels of the CARM1 substrate H3R26me2, and both proteins were shown to be able to interact83 (FIG. 4).



REVIEWS a Zygotic knockout (heterozygous crosses) Sperm Knockout allele Germinal vesicle oocyte Zygote

Maternal and zygotic knockout Sperm Knockout allele Germinal vesicle oocyte Zygote

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Fertilization

Wild-type allele Knockout allele Maternally provided protein and/or mRNA

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2-cell stage

4-cell stage Blastocyst

Figure 5 | Genetic approaches to study gene function in pre-implantation embryos. Nature Reviews | Molecular Cell Biology a | A comparison between zygotic and maternal knockout strategies is shown. To reveal gene function in the earliest stages of development, it is necessary to deplete the maternal contribution of its mRNA and/or protein. In a zygotic knockout (left), pre-implantation embryos are generated from crosses of heterozygous females, where diploid oocytes harbour a wild-type allele and a knockout allele. These oocytes therefore contain maternally inherited, persisting, functional transcripts and/or proteins of the gene of interest. Thus, these strategies cannot be used to elucidate the function of chromatin modifiers in pre-implantation embryos. Instead, maternal knockout strategies must be carried out (right), in which knockouts of both alleles are generated in the growing oocyte using a Cre recombinase expressed early during germline development, thereby preventing the expression of maternal mRNA and protein. The general lack of maternal germline knockouts has hampered our understanding of chromatin function during pre-implantation development, and alternative approaches are needed to study chromatin function in the early embryo. b | A strategy for addressing the function of a given gene in individual cells of the early embryo. Expression of a dominant negative or mutant protein, or of siRNAs targeting the endogenous transcripts, can be achieved in individual cells at the 2‑cell stage using RNA microinjection. Co‑injection of a lineage tracer, such as GFP, is carried out in parallel. The effect of these manipulations in development can then be assessed in vitro, and potential effects can be followed at the blastocyst stage based on lineage tracing of the GFP-positive cells, representing the progeny of the injected cells. Embryos can then be reconstructed in three dimensions to determine cell lineages based on inner and outer positions within the blastocyst.

Carrier-chromatin immunoprecipitation (CChIP). A chromatin immunoprecipitation approach of native chromatin (prepared by nuclease digestion as opposed to crosslinking) modified for low sample quantities.

Many questions remain open. If epigenetic asymmetries arise before lineage allocation, do they act through a global change of chromatin structure or rather by the specific regulation of key factors (FIG. 2)? Do these asymmetries arise stochastically? Are they reinforced in later stages by additional epigenetic asymmetries? Genetic approaches have contributed considerably to the identification of the players involved in early developmental progression. However, care should be taken when drawing conclusions about the roles of specific genes in pre-implantation embryos on the basis of these genetic approaches alone, as maternal proteins and mRNAs from the mature oocyte frequently persist in zygotic knockouts (FIG. 5a). Indeed, in the few cases in which chromatin modifiers such as Ezh2, E3 ubiquitin-protein ligase RING2 (Ring1b), Ring1a and Bhrama-related gene 1 (Brg1) were deleted in the maternal germ line, early preimplantation lethality was observed70,86,87.

Owing to the limitations of such traditional method ologies, there has been an increasing need to develop and adapt alternatives — mainly based on experimental embryology (such as lineage tracing) and, more recently, on single-cell analyses and imaging — to help to discern whether chromatin modifiers have a general role in developmental progression or a more specific role in lineage allocation of individual cells during development. Although, as we discuss below, there are only a few examples of experimental manipulation of chromatin modifiers (FIG. 5b), such as Carm1, Prdm14 and Tet2 (REFS 81,83,88), in single cells, the results support the hypothesis that the manipulation of epigenetic information in the early embryo can regulate cell fate allocation. Studying the chromatin of early embryos. When studying pre-implantation development, one can access between 10 and 1,500 cells. This implies that large-scale chromatin preparations cannot be carried out using biochemical approaches, which are rarely amenable to low cell numbers. Histone radiolabelling and western-blot analyses, although rarely carried out, have shed light on the composition of embryonic chromatin up to the morula stage89,90 and have revealed major changes in histone H1 composition between the oocyte and blastocyst. However, no quantitative information is available regarding the abundance of other histones and their modifications. One of the first breakthroughs that enabled the investigation of native chromatin in the early embryo was carrier-chromatin immunoprecipitation (CChIP), which is a protocol that is amenable to low cell numbers91. CChIP on isolated ICM cells revealed a high and moderate enrichment of the active marks H4K16ac and H3K4me3, respectively, on the Nanog and Oct4 promoters, but not on that of Cdx2. Conversely, the repressive mark H3K9me2 was found to be enriched at the Cdx2 promoter but not on Nanog or Oct4, which shows a correlation between histone modifications and gene expression in the two blastocyst lineages. H4K8ac and H3K4me3 were also found to correlate with transcriptional activity in 8‑cell and morula-stage mouse embryos73,92. In ICM cells, H3K27me3 was also found to be enriched at promoters of genes that are bivalent in ES cells, which suggests that Polycomb complexes might help to delineate lineage commitment in the blastocyst73. The quantification of enrichment of chromatin marks in embryos was enabled by the development of ‘microChIP’ (REF. 93) and this was re‑adapted to determine the levels of H3K4me3 and H3K9me3 on repetitive elements in 2‑cell and 8‑cell embryos, and in isolated epiblasts of E6.5 embryos. This revealed that long interspersed element 1 (LINE-1), short interspersed nuclear element (SINE) B2 and intracisternal A-particle (IAP) retrotransposons, which are typically silenced in somatic cells, are strongly enriched in H3K4me3 at the beginning of development at the 2‑cell stage, which correlates with the timing of their transcriptional reactivation after fertilization94. This led to the suggestion that retrotransposons adopt an atypical chromatin signature in pre-implantation embryos. Only one genome-wide study of histone



REVIEWS modifications in embryos is currently available; in this study, the H3K4me3 in growing oocytes and blastocysts was found to generally anticorrelate with CpG islands34. The improvement of ChIP approaches and genome amplification procedures in recent years has provided initial observations of the presence of a few histone modifications on some isolated genes, but we know little about the chromatin composition at other genomic loci. In contrast to the limited data on histone modifications, we have high-resolution information on genome-wide DNA methylation in the oocyte and in the early embryo. This information has challenged the view that passive, genome-wide DNA demethylation occurs after fertilization and continues throughout pre-implantation development. Instead, RRBS revealed waves of remethylation at specific gene promoters before blastocyst formation34,41,95. Moreover, it seems that gamete DNA methylation predisposes for DNA methylation patterns in pre-implantation embryos, but it is not their main determinant34. Importantly, methylated DNA immunoprecipitation (MeDIP) analysis of promoter DNA methylation identified some non-imprinted sequences that resist demethylation during pre-implantation development95, contrary to the previously accepted view that only imprinted sequences resist DNA demethylation. Analysis of the DNA methyl ation status of ~1 million CpG dinucleotides in the different pre-implantation stages41 confirmed that the most dramatic changes in DNA methylation occur during two transitions: one between the sperm and the zygote and one between the ICM of the early blastocyst and the postimplantation embryo, with more subtle global changes from the zygote through to the 8‑cell stage. CpG islands Regions in the genome that contain a high frequency of CpG dinucleotides. They are often found in gene promoters.

Methylated DNA immunoprecipitation (MeDIP). A genome-wide, high-resolution approach to quantifying DNA methylation. The antibody used for the precipitation recognizes 5‑methylcytosine.

Microfluidics A technology that enables the analysis of a set of transcripts (typically 48 or 96) in single cells by quantitative PCR in nanolitre volumes, allowing for truly quantitative information on gene expression to be extracted.

Cap analysis of gene expression (CAGE). A technique for capturing mRNAs by the addition of linkers at their 5′ end. It provides information on the 5′ end of a transcript and therefore on its transcription start sites.

Single-cell analyses of chromatin Phenotypic changes in individual cells are regulated by epigenetic mechanisms, as they occur without changes in their genetic composition96. Therefore assessing the chromatin features of individual cells across development is essential for understanding the molecular mechanisms underlying lineage allocation. Such features can provide information about the molecular characteristics of a given cell (static information), as well as the changes that take place in these cells during development (dynamic information). The analysis of single cells in stem cell populations and embryos has revealed the high degree of heterogeneity found both in stem cell populations and during pattern formation in development. The idea of heterogeneity in mRNA and protein expression between individual cells in a given population, often of particular lineage transcription factors, has shaped the way in which the process of cell fate allocation is perceived and has led to the understanding that pluripotency and cell fate allocation are dynamic, stochastic processes that impart robustness to a population of cells85,97–99. Many of the single-cell experimental approaches applied to the early embryo have relied on the development of imaging techniques. The continuing improvement of imaging techniques and the increasing sensitivity and coverage of genome-wide analyses in single cells should together help to establish a mechanistic link between dynamic changes in individual cells and

specific cell fates. If changes between cells are important for establishing cell fate, approaches for studying chromatin function based on pooled embryos will not provide sufficient information to understand cell fate allocation. Single-cell analyses could also be a means to portray multiple ‘snapshots’ of a cell over time on its way to fate allocation. It is possible to assess the global transcriptional profiles of individual embryonic cells using microarrays and RNA sequencing (RNA-seq)85,98,100, and these techniques have recently been complemented by more quantitative approaches based on microfluidics25,83,101. Below, we first discuss some of the main results recently obtained using these approaches in terms of cell plasticity and cell fate allocation, before discussing the live-cell imaging approaches that have substantially improved our understanding of how chromatin dynamics and function are shaped throughout early development. Quantitative single-cell expression analysis. The genomewide heterogeneity of the ICM became apparent following the first systematic analysis of single-cell transcriptomes in the pre-implantation mouse embryo. The profiling of individual cells of the ICM using microarrays85 showed that cells within the ICM are highly heterogeneous in the genes that they express. This finding supported a salt-andpepper model of lineage specification of the ICM into epiblast and primitive endoderm, whereby rather than arising from a coherent group of cells within the ICM, the precursors of the epiblast and primitive endoderm are heterogeneously distributed across the ICM102,103. Indeed, it is now established that stochastic cell‑to‑cell variability with subsequent reinforcement by antagonistic signalling underlies ICM lineage segregation into epiblast and primitive endoderm12,98. Following this work, a single-cell RNA-seq analysis of earlier developmental stages in the mouse based on polymorphisms indicated that more that one-half of the transcripts analysed were subject to random, allelespecific gene expression100. The significance of this observation is unclear, but using the same technique in hybrid pre-implantation embryos, it was recently shown that the proportion and type of genes that are subject to allelic expression varies depending on the developmental stage analysed27. Moreover, the gene ontology terms of the expressed genes in the pre-implantation embryo seem to be conserved between mouse and human: RNAseq analyses of individual blastomeres of the human embryo found overall similarities in the categories of genes expressed, albeit with differences in timing of activation104,105. Thus, we currently have an exhaustive data set of single-copy genes that are expressed in individual cells in the mouse and human pre-implantation embryo. However, all of these transcriptomic approaches are based on the amplification of polyadenylated mRNAs, and as it is uncertain whether all repetitive elements are polyadenylated, alternative approaches such as cap analysis of gene expression (CAGE)94,106 should be explored to identify different types of transcripts (for example, retrotransposons) that can potentially regulate chromatin function.



REVIEWS

Fluorescence recovery after photobleaching (FRAP). This technique uses fluorescently tagged proteins of interest. The recovery of the fluorescent signal after bleaching can provide information on the mobility of the protein of interest.

Fluorescence decay after photoactivation (FDAP). This technique uses a photoactivatable fluorescent molecule tagged to the protein of interest. The loss of fluorescence after photoactivation is then measured over time to determine protein mobility parameters.

Fluorescence correlation spectroscopy (FCS). This is a correlation analysis of fluorescence fluctuations to study the concentration and dynamics of a fluorescently tagged molecule. In combination with photoactivation (paFCS) it provides information on the number of molecules analysed.

Heterogeneity across the ICM of the early blastocyst has been undoubtedly demonstrated. However, whether individual cells exhibit different transcription profiles within the same embryo at stages earlier than the blastocyst has been difficult to establish using single-cell RNAseq data, owing to the inability to distinguish biological variability from technical variability. By contrast, the use of microfluidics results in fewer technical errors25, and a study that used a microfluidic device to analyse the expression of 27 transcription factors in >500 individual cells of the early mouse embryo found that cell fate transitions are progressively defined from the 16‑cell stage25. This work also identified inhibitor of DNA binding 2 (Id2) and sex determining region Y-box 2 (Sox2) as the earliest transcription factors to mark lineage allocation, with both genes showing heterogeneous and inverse expression patterns in the cells of the 16‑cell stage embryo (FIG. 1). Cells with high expression of Sox2 and low expression of Id2 occupy inner positions in the morula and are therefore precursors of ICM cells25. A similar analysis was carried out to investigate whether cell fate and lineage specification can be correlated with combinations of specific chromatin modifiers in single cells. Quantitative probing of the expression of 39 chromatin modifiers across pre-implantation development revealed a high degree of biological variability in — for example — the expression of the Polycomb gene Ring1a and strong intra-embryonic variation in the expression levels of Prdm14 at the 4‑cell stage. The quantitative nature of the single-cell data generated enabled the establishment of a mathematical model of an epigenetic landscape of cellular states in pre-implantation embryos based on the expression of a limited number of chromatin modifiers83. According to this model, the expression patterns of transcription factors delineate the lineages of the late blastocyst but not the earliest developmental stages25. By contrast, the expression of chromatin modifiers better defines early developmental transitions83. This led us to suggest that chromatin regulation might precede the resolution of expression patterns of transcription factors, which in turn enforce cell fate decisions83. A major conclusion from single-cell profiling of mechanically isolated and cultured blastomeres using microfluidics is that blastomeres retain plasticity at least until the blastocyst stage and will respond to changes in their immediate environment101. However, it is still not certain whether, in the absence of any perturbation, 4‑cell and 8‑cell stage blastomeres retain totipotency and give rise to cell lineages with equal probability, as had been previously proposed based on pioneering experimental embryology approaches107. DNA methyl ation was also investigated using a microfluidic platform combined with methylation-sensitive restriction enzyme digestion108. This study examined a handful of loci in individual 4‑cell blastomeres lacking the transcriptional co-regulator tripartite motif-containing protein 28 (TRIM28; also known as TIF1β). The results suggest that TRIM28-dependent maintenance of imprinted loci is mosaic, and therefore not all Trim28-knockout blastomeres lose DNA methylation108.

Taken together, the above studies indicate that there are intra-embryonic differences at the molecular level, even when cells are morphologically indistinguishable. However, it remains to be ascertained whether these differences reflect a stochastic process or a continued accumulation of cues towards cell fate allocation. Quantitative single-cell imaging in live embryos. Revealing the dynamics of chromatin-associated proteins can provide information about chromatin function. Timelapse analysis using live-cell fluorescence can reveal the kinetics of the process in question over broad timescales ranging from subseconds to days109. Quantitative values can be extracted to define binding parameters, diffusion, transport and protein stability, which can also help to distinguish between stochastic and regulated processes and therefore shed light on how chromatin regulates gene expression in single cells. Global chromatin dynamics in individual blastomeres have been assessed using fluorescence recovery after photobleaching (FRAP). The core histones H2A, H3.1 and H3.2 were found to be unusually mobile at the 2‑cell stage. Higher histone mobility was found in cells destined to become the ICM (that is, in cells expressing Carm1) than in those destined to become trophectoderm before lineage allocation110. It was also found that histone mobility decreases as development proceeds. These data have provided evidence that the chromatin adopts an open configuration after fertilization, but this configuration is progressively lost during development. Importantly, the data also suggest that the global state of the chromatin in single cells of the early embryo correlates with, and may affect, lineage allocation. A working model of transcription factor binding to target genes in single cells was recently proposed for OCT4. This pioneering work used fluorescence decay after photoactivation (FDAP) to determine the binding kinetics of an OCT4–GFP fusion protein in embryonic nuclei at the 8‑cell stage111. It identified two distinct populations of OCT4: one with slower diffusion kinetics, presumably corresponding to a chromatin-bound fraction, and the other with much faster kinetics, most probably reflecting the free pool of OCT4 in the nucleoplasm (FIG. 4c). Remarkably, there was a correlation between lineage allocation to the ICM and 8‑cell blastomeres that had a higher proportion of OCT4 with slower kinetics. These findings argue that transcription factor accessibility to the chromatin has a regulatory role in cell fate allocation in 8‑cell embryos. The existence of two pools of OCT4 in 8‑cell stage nuclei was confirmed by paFCS (combined photoactivation and fluorescence correlation spectroscopy (FCS)), which further showed that the diffusion dynamics of OCT4 differ in the two lineages of the early blastocyst. Notably, both OCT4 and SOX2 show slower dynamics in the ICM than in the trophectoderm, whereas CDX2 displays the opposite pattern112. Culturing embryos with trichostatin A, which is a histone deacetylase inhibitor that causes global chromatin hyperacetylation, altered the diffusion properties of OCT4 fused to photoactivated GFP, suggesting that global chromatin architecture may influence the activity of specific transcription factors on their targets.



REVIEWS Table 1 | Approaches to visualizing specific loci in single cells by time-lapse microscopy Name and schematic

Advantages and uses

Potential drawbacks

Refs

Lac operator

• Very powerful signal suitable for time-lapse microscopy • Versatile: can be easily applied to multiple biological questions (for example, to study the dynamics of DNA repair)

• Recombination of the locus is laborious owing to its repetitive nature • Difficultly in targeting specific loci

131– 134

• Visualization and tracking of lamina-associated domains (LADs) • Can be used in time-lapse microscopy to analyse the association of genomic regions with nuclear lamina

• Number of binding events required to visualize LADs is unknown, and single events or short LADs might not be detected

120

GFP Lac repressor LacR binding site Lac operator (256x repeats ~10 kb)

Putative artiﬁcial transcription start site

m6A-tracer technology (combined with Dam‑ID) LAD DNA Nucleus

m6A

methylated DNA

Lamin B1 DAM

m6A-tracer

GFP

Nuclear membrane

TGV (TALE-mediated genome visualization) Telomere

Major satellites TALE

Minor satellites

Chromocentre

GFP Nucleus

Major satellite repeat

ParB–INT DNA labelling system Oligomeric spreading ParB–GFP INT ParB2 binding site

Locus of interest (endogenous)

CRISPR–Cas9 Telomere

Major satellites

Minor satellites

dCas9 sgRNA GFP Major satellite repeat

Chromocentre

Nucleus

• Visualization of endogenous genomic loci • Adapted only for without the need to genetically modify them repetitive sequences • Very good signal-to‑noise ratio • Not yet amenable to • Highly specific binding, able to detect one single copy genes single-nucleotide polymorphism within a 15 nucleotide-long target sequence • Does not affect the cell cycle

• Ease of genome targeting given the small size of the binding site • Signal-to-noise ratio good enough to track chromatin dynamics in live cells • Can be combined with multiple INT binding sites and partitioning proteins (ParB) so that several loci can be traced simultaneously • Can be used for endogenous loci as well as for transfected plasmids

113, 135– 138

• Not yet applied in mammalian cells • Requires homologous recombination to target sequences

138

• Visualization of endogenous genomic loci • Not yet tested in without the need to genetically modify them time-lapse microscopy • Ease of design and use of multiple sgRNA for single-copy genes simultaneously for improved visualization • Cannot be used for simultaneously visualizing different loci in individual cells

139

Cas, CRISPR-associated protein; CRISPR, clustered regularly interspaced short palindromic repeats; Dam-ID, DNA adenine methyltransferase identification; INT, ~1 kb parS DNA segment from Burkholderia cenocepacia; m6A, N6-methyladenosine; sgRNA, single-guide RNA;TALE, transcription activator-like effector.

Transcription activator-like effector (TALE). A protein with hypervariable domains that recognize specific DNA bases, allowing sequence-specific targeting.

Chromosome conformation capture (3C). A technique for studying genomic organization, in which nuclei are fixed, the DNA is digested and chromosomal regions in physical proximity are ligated and identified by PCR.

Imaging chromatin dynamics in real time at specific loci. The few studies described above that used quantitative imaging of chromatin components and transcription factors have provided several insights into cell fate allocation, but the specific genes involved remain unknown. Imaging specific loci is crucial to understanding how nuclear positioning can effectively regulate specific gene expression. Tools for addressing the spatial and temporal dynamics of specific genes in the nucleus have only recently been developed (TABLE 1). Transcription activator-like effector (TALE)-mediated genome visualization (TGV; TABLE 1) has been successfully used to image major satellites in pre-implantation embryos113. These analyses have revealed that these heterochromatic regions are highly mobile in the zygote and 2‑cell embryos. Furthermore, they have provided insights into how the condensation of the pericentromeric chromatin

in mitotic chromosomes from the paternal and maternal genomes occurs. Important questions remain to be addressed, such as whether gene activity drives changes in nuclear positioning during development or whether global chromatin dynamics affect the nuclear positioning of important lineage-specific transcription factors in some cells but not in others. Single-cell analysis of nuclear architecture. Nuclear organization can be addressed using chromosome conformation capture (3C) approaches in fixed samples to reveal favoured interactions between genomic regions in the three-dimensional (3D) space of the interphase nucleus114. Interactions of specific genomic regions with the nuclear lamina have also been mapped using DNA adenine methyltransferase identification (Dam‑ID). The proximity to the nuclear lamina is thought to



REVIEWS DNA adenine methyltransferase identification (Dam‑ID). A technique for mapping protein binding to DNA by fusing proteins to a bacterial adenine methylase, which is not endogenous to eukaryotes. Binding can be mapped based on the position of methylated adenines.

Lamina-associated domains (LADs). Regions of the genome that have been demonstrated to interact with the nuclear lamina.

Hi-C A variation of chromosome conformation capture in which all interacting regions of the genome can be mapped by high-throughput sequencing of the ligated products.

regulate gene expression115, and in somatic cells, laminaassociated domains (LADs) are enriched with methylated H3K9 and are associated with gene silencing116–118. By contrast, recent evidence suggests that proximity to the nuclear periphery might not necessarily impose gene silencing in the mouse embryo after fertilization, which implies that early embryos have different nuclear organization from somatic cells65. The systematic interrogation of lamina and chromosome interactions will thus be important for determining whether nuclear compartmentalization changes functionally during development, and whether the interaction of regulatory regions in the 3D space can affect lineage allocation in vivo. Two different, complementary approaches for mapping chromosomal interactions and visualizing LADs in single cells are single-cell Hi‑C and a modified Dam‑ID‑based tracer approach for tracking LAD dynamics119,120 (TABLE 1). For example, when combined with detailed live-cell microscopy to track specific loci or with electron microscopy, these approaches could provide a comprehensive map of the nuclear architecture of individual cells in the embryo during development and lineage allocation.

Concluding remarks The period of development immediately after fertilization is characterized by a unique organization of the DNA in the nuclear space. The composition of the embryonic chromatin is markedly atypical, characterized by the incorporation of several histone variants and a lack of most of the histone post-translational modifications that are normally found associated with constitutive heterochromatin. Moreover, there is now evidence of the global open configuration of the chromatin in individual cells of the early embryo, particularly in cells that are destined to become pluripotent ICM cells.

1.

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Single-cell experimental approaches have provided important contributions towards understanding the considerable plasticity of embryonic cells. Significant variability in the expression patterns of transcription factors and chromatin components has been observed before lineage allocation. Intriguingly, in this context, changes in chromatin dynamics and structure seem to occur before transcription factors become lineagerestricted. We postulate that individual cells in the early embryo develop a chromatin configuration that is permissive for cell fate allocation. Single-cell approaches have also underscored the extent of cell heterogeneity in the early embryo and the temporal dynamics of these differences between cells. Combining the different technologies discussed above with mathematical modelling should provide novel insights into the pathways that regulate cell fate allocation during early development. Functional approaches should now be applied at the single-cell level to identify the importance (or otherwise) of any identified intra-embryonic differences in chromatin composition at discrete developmental stages. Finally, the basis of these differences at the epigenetic level should be addressed to identify important players in cell differentiation. Many features of epigenetic reprogramming are conserved in plants and animals121 and across Mammalia. By contrast, mechanisms that drive lineage fate decisions do not seem to be well conserved, with an apparent increase in flexibility occurring in mammals, which may be related to the process of implantation. It will be interesting to identify which features of epigenetic reprogramming and cell fate allocation are more or less conserved, as this will provide novel insights into the functionality of various factors during these important developmental processes.

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Acknowledgements

M.E.T.-P. acknowledges funding from EpiGeneSys NoE, ERCStg ‘NuclearPotency’, the FP7 Marie-Curie Actions ITN Nucleosome4D, the EMBO YIP and the Fondation Schlumberger pour l’Education et la Recherche. A.B. was a recipient of a fellowship from the Fondation pour la Recherche Médicale.

Competing interests statement

The authors declare no competing interests.


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