Pluripotency is the remarkable capacity of a single cell to engender all the specialized cell types of an adult organism. This property can be captured indefinitely through derivation of self-renewing embryonic stem cells (ESCs), which represent an invaluable platform to investigate cell fate decisions and disease. Recent advances have revealed that manipulation of distinct signaling cues can render ESCs in a uniform "ground state" of pluripotency, which more closely recapitulates the pluripotent naive epiblast. Here we discuss the extrinsic and intrinsic regulatory principles that underpin the nature of pluripotency and consider the emerging spectrum of pluripotent states.
Cell populations can be strikingly heterogeneous, composed of multiple cellular states, each exhibiting stochastic noise in its gene expression. A major challenge is to disentangle these two types of variability and to understand the dynamic processes and mechanisms that control them. Embryonic stem cells (ESCs) provide an ideal model system to address this issue because they exhibit heterogeneous and dynamic expression of functionally important regulatory factors. We analyzed gene expression in individual ESCs using single-molecule RNA-FISH and quantitative time-lapse movies. These data discriminated stochastic switching between two coherent (correlated) gene expression states and burst-like transcriptional noise. We further showed that the "2i" signaling pathway inhibitors modulate both types of variation. Finally, we found that DNA methylation plays a key role in maintaining these metastable states. Together, these results show how ESC gene expression states and dynamics arise from a combination of intrinsic noise, coherent cellular states, and epigenetic regulation.
Germ cells are unique cell types that generate a totipotent zygote upon fertilization, giving rise to the next generation in mammals and many other multicellular organisms. How germ cells acquire this ability has been of considerable interest. In mammals, primordial germ cells (PGCs), the precursors of sperm and oocytes, are specified around the time of gastrulation. PGCs are induced by signals from the surrounding extra-embryonic tissues to the equipotent epiblast cells that give rise to all cell types. Currently, the mechanism of PGC specification in mammals is best understood from studies in mice. Following implantation, the epiblast cells develop as an egg cylinder while the extra-embryonic ectoderm cells which are the source of important signals for PGC specification are located over the egg cylinder. However, in most cases, including humans, the epiblast cells develop as a planar disc, which alters the organization and the source of the signaling for cell fates. This, in turn, might have an effect on the precise mechanism of PGC specification in vivo as well as in vitro using pluripotent embryonic stem cells. Here, we discuss how the key early embryonic differences between rodents and other mammals may affect the establishment of the pluripotency network in vivo and in vitro, and consequently the basis for PGC specification, particularly from pluripotent embryonic stem cells in vitro.
Primordial germ cells (PGCs) are the precursors of sperm and eggs, which generate a new organism that is capable of creating endless new generations through germ cells. PGCs are specified during early mammalian postimplantation development, and are uniquely programmed for transmission of genetic and epigenetic information to subsequent generations. In this Primer, we summarise the establishment of the fundamental principles of PGC specification during early development and discuss how it is now possible to make mouse PGCs from pluripotent embryonic stem cells, and indeed somatic cells if they are first rendered pluripotent in culture.
Pluripotent stem cells (PSCs) occupy a spectrum of reversible molecular states ranging from a naive ground-state in 2i, to metastable embryonic stem cells (ESCs) in serum, to lineage-primed epiblast stem cells (EpiSCs). To investigate the role of DNA methylation (5mC) across distinct pluripotent states, we mapped genome-wide 5mC and 5-hydroxymethycytosine (5hmC) in multiple PSCs. Ground-state ESCs exhibit an altered distribution of 5mC and 5hmC at regulatory elements and dramatically lower absolute levels relative to ESCs in serum. By contrast, EpiSCs exhibit increased promoter 5mC coupled with reduced 5hmC, which contributes to their developmental restriction. Switch to 2i triggers rapid onset of both the ground-state gene expression program and global DNA demethylation. Mechanistically, repression of de novo methylases by PRDM14 drives DNA demethylation at slow kinetics, whereas TET1/TET2-mediated 5hmC conversion enhances both the rate and extent of hypomethylation. These processes thus act synergistically during transition to ground-state pluripotency to promote a robust hypomethylated state.
Mouse postimplantation epiblast cultured in activin and basic fibroblast growth factor gives rise to continuously growing epiblast stem cells (EpiSCs) that share key properties with postimplantation epiblast, such as DNA methylation and an inactive X-chromosome. EpiSCs also show a distinct gene expression profile compared to embryonic stem cells (ESCs) derived from preimplantation blastocysts, and do not contribute efficiently to chimeras. EpiSCs can, however, revert to pluripotent ESC-like cells upon exposure to leukemia inhibitory factor-Stat3 signalling on feeder cells. Here we describe a protocol for the establishment of EpiSCs and their reversion to ESCs.
Germ cell development is a step-wise process that ensures the progression of the life cycle due to their unique ability to transmit their genome from one generation to the next. In the mouse, the precursors of germ cells, the Primordial Germ Cells (PGCs), arise at the onset of gastrulation. Here we discuss how PGCs acquire their fate in the epiblast and outline their development until their arrival into the gonads. Male germ cell tumors (GCTs) have a similar gene expression pattern to that of fetal germ cells and to pluripotent cells, suggesting that GCT originate from an alteration of gonocyte normal development. We evaluate coincidences and differences in germ cell development in mouse and humans and on this basis, we speculate future research perspectives.
Mammalian primordial germ cells (PGCs) are unipotent progenitors of the gametes. Nonetheless, they can give rise directly to pluripotent stem cells in vitro or during teratocarcinogenesis. This conversion is inconsistent, however, and has been difficult to study. Here, we delineate requirements for efficient resetting of pluripotency in culture. We demonstrate that in defined conditions, routinely 20% of PGCs become EG cells. Conversion can occur from the earliest specified PGCs. The entire process can be tracked from single cells. It is driven by leukemia inhibitory factor (LIF) and the downstream transcription factor STAT3. In contrast, LIF signaling is not required during germ cell ontogeny. We surmise that ectopic LIF/STAT3 stimulation reconstructs latent pluripotency and self-renewal. Notably, STAT3 targets are significantly upregulated in germ cell tumors, suggesting that dysregulation of this pathway may underlie teratocarcinogenesis. These findings demonstrate that EG cell formation is a robust experimental system for exploring mechanisms involved in reprogramming and cancer.
Epigenetic reprogramming of parental genomes following fertilization is important to ensure compatibility for totipotency and development thereafter. New studies by Jiang et al. and Potok et al. now demonstrate how the parental DNA methylomes are reset in zebrafish and reveal striking differences from events in mammals.
DNA methylation is dynamically remodelled during the mammalian life cycle through distinct phases of reprogramming and de novo methylation. These events enable the acquisition of cellular potential followed by the maintenance of lineage-restricted cell identity, respectively, a process that defines the life cycle through successive generations. DNA methylation contributes to the epigenetic regulation of many key developmental processes including genomic imprinting, X-inactivation, genome stability and gene regulation. Emerging sequencing technologies have led to recent insights into the dynamic distribution of DNA methylation during development and the role of this epigenetic mark within distinct genomic contexts, such as at promoters, exons or imprinted control regions. Additionally, there is a better understanding of the mechanistic basis of DNA demethylation during epigenetic reprogramming in primordial germ cells and during pre-implantation development. Here, we discuss our current understanding of the developmental roles and dynamics of this key epigenetic system.
Primordial germ cells (PGCs) and somatic cells originate from postimplantation epiblast cells in mice. As pluripotency is lost upon differentiation of somatic lineages, a naive epigenome and the pluripotency network are re-established during PGC development. Here we demonstrate that Prdm14 contributes not only to PGC specification, but also to naive pluripotency in embryonic stem (ES) cells by repressing the DNA methylation machinery and fibroblast growth factor (FGF) signalling. This indicates a critical role for Prdm14 in programming PGCs and promoting pluripotency in ES cells.
Primordial germ cells (PGCs) are the embryonic precursors of the gametes and represent the founder cells of the germline. Specification of PGCs is a critical divergent point during embryogenesis. Whereas the somatic lineages will ultimately perish, cells of the germline have the potential to form a new individual and hence progress to the next generation. It is therefore critical that the genome emerges intact and carrying the appropriate epigenetic information during its passage through the germline. To ensure this fidelity of transmission, PGC development encompasses extensive epigenetic reprogramming. The low cell numbers and relative inaccessibility of PGCs present a challenge to those seeking mechanistic understanding of the crucial developmental and epigenetic processes in this most fascinating of lineages. Here, we present an overview of PGC development in the mouse and compare this with the limited information available for other mammalian species. We believe that a comparative approach will be increasingly important to uncover the extent to which mechanisms are conserved and reveal the critical steps during PGC development in humans.
Transitions in cell states are controlled by combinatorial actions of transcription factors. BLIMP1, the key regulator of primordial germ cell (PGC) specification, apparently acts together with PRDM14 and AP2?. To investigate their individual and combinatorial functions, we first sought an in vitro system for transcriptional readouts and chromatin immunoprecipitation sequencing analysis. We then integrated this data with information from single-cell transcriptome analysis of normal and mutant PGCs. Here we show that BLIMP1 binds directly to repress somatic and cell proliferation genes. It also directly induces AP2?, which together with PRDM14 initiates the PGC-specific fate. We determined the occupancy of critical genes by AP2?-which, when computed altogether with those of BLIMP1 and PRDM14 (both individually and cooperatively), reveals a tripartite mutually interdependent transcriptional network for PGCs. We also demonstrate that, in principle, BLIMP1, AP2? and PRDM14 are sufficient for PGC specification, and the unprecedented resetting of the epigenome towards a basal state.
Interferon-induced transmembrane protein 3 (IFITM3) ?plays a crucial role in the antiviral responses of Type I interferons (IFNs). The role of IFITM3 in the central nervous system (CNS) is, however, largely unknown, despite the fact that its expression is increased in the brains of patients with neurologic and neuropsychiatric diseases. Here, we show the role of IFITM3 in long-lasting neuronal impairments in mice following polyriboinosinic-polyribocytidylic acid (polyI:C, a synthetic double-stranded RNA)-induced immune challenge during the early stages of development. We found that the induction of IFITM3 expression in the brain of mice treated with polyI:C was observed only in astrocytes. Cultured astrocytes were activated by polyI:C treatment, leading to an increase in the mRNA levels of inflammatory cytokines as well as Ifitm3. When cultured neurons were treated with the conditioned medium of polyI:C-treated astrocytes (polyI:C-ACM), neurite development was impaired. These polyI:C-ACM-induced neurodevelopmental abnormalities were alleviated by ifitm3(-/-) astrocyte-conditioned medium. Furthermore, decreases of MAP2 expression, spine density, and dendrite complexity in the frontal cortex as well as memory impairment were evident in polyI:C-treated wild-type mice, but such neuronal impairments were not observed in ifitm3(-) (/) (-) mice. We also found that IFITM3 proteins were localized to the early endosomes of astrocytes following polyI:C treatment and reduced endocytic activity. These findings suggest that the induction of IFITM3 expression in astrocytes by the activation of the innate immune system during the early stages of development has non-cell autonomous effects that affect subsequent neurodevelopment, leading to neuropathological impairments and brain dysfunction, by impairing endocytosis in astrocytes.
Naive pluripotent embryonic stem cells (ESCs) and embryonic germ cells (EGCs) are derived from the preimplantation epiblast and primordial germ cells (PGCs), respectively. We investigated whether differences exist between ESCs and EGCs, in view of their distinct developmental origins. PGCs are programmed to undergo global DNA demethylation; however, we find that EGCs and ESCs exhibit equivalent global DNA methylation levels. Inhibition of MEK and Gsk3b by 2i conditions leads to pronounced reduction in DNA methylation in both cell types. This is driven by Prdm14 and is associated with downregulation of Dnmt3a and Dnmt3b. However, genomic imprints are maintained in 2i, and we report derivation of EGCs with intact genomic imprints. Collectively, our findings establish that culture in 2i instills a naive pluripotent state with a distinctive epigenetic configuration that parallels molecular features observed in both the preimplantation epiblast and nascent PGCs.
Steel factor, the protein product of the Steel locus in the mouse, is a multifunctional signal for the primordial germ cell population. We have shown previously that its expression accompanies the germ cells during migration to the gonads, forming a "travelling niche" that controls their survival, motility, and proliferation. Here we show that these functions are distributed between the alternatively spliced membrane-bound and soluble forms of Steel factor. The germ cells normally migrate as individuals from E7.5 to E11.5, when they aggregate together in the embryonic gonads. Movie analysis of Steel-dickie mutant embryos, which make only the soluble form, at E7.5, showed that the germ cells fail to migrate normally, and undergo "premature aggregation" in the base of the allantois. Survival and directionality of movement is not affected. Addition of excess soluble Steel factor to Steel-dickie embryos rescued germ cell motility, and addition of Steel factor to germ cells in vitro showed that a fourfold higher dose was required to increase motility, compared to survival. These data show that soluble Steel factor is sufficient for germ cell survival, and suggest that the membrane-bound form provides a higher local concentration of Steel factor that controls the balance between germ cell motility and aggregation. This hypothesis was tested by addition of excess soluble Steel factor to slice cultures of E11.5 embryos, when migration usually ceases, and the germ cells aggregate. This reversed the aggregation process, and caused increased motility of the germ cells. We conclude that the two forms of Steel factor control different aspects of germ cell behavior, and that membrane-bound Steel factor controls germ cell motility within a "motility niche" that moves through the embryo with the germ cells. Escape from this niche causes cessation of motility and death by apoptosis of the ectopic germ cells.
Despite recent critical insights into the pluripotent state of embryonic stem cells (ESCs), there is little agreement over the inaugural and subsequent steps leading to its generation [1-4]. Here we show that inner cell mass (ICM)-generated cells expressing Blimp1, a key transcriptional repressor of the somatic program during germ cell specification [5, 6], emerge on day 2 of blastocyst culture. Single-cell gene expression profiling indicated that many of these Blimp1-positive cells coexpress other genes typically associated with early germ cell specification. When genetically traced in vitro, these cells acquired properties normally associated with primordial germ cells. Importantly, fate-mapping experiments revealed that ESCs commonly arise from Blimp1-positive precursors; indeed, prospective sorting of such cells from ICM outgrowths increased the rate of ESC derivation more than 9-fold. Finally, using genetic ablation or distinct small molecules [7, 8], we show that epiblast cells can become ESCs without first acquiring Blimp1 positivity. Our findings suggest that the germ cell-like state is facultative for the stabilization of pluripotency in vitro. Thus, the association of Blimp1 expression with ESC development furthers understanding of how the pluripotent state of these cells is established in vitro and suggests a means to enhance the generation of new stem cell lines from blastocysts.
Pluripotency and self-renewal are the hallmarks of embryonic stem cells. This state is maintained by a network of transcription factors and is influenced by specific signalling pathways. Current evidence indicates that multiple pluripotent states can exist in vitro. Here we review the recent advances in studying the transcriptional regulatory networks that define pluripotency, and elaborate on how manipulation of signalling pathways can modulate pluripotent states to varying degrees.
Dissecting the relationship between genotype and phenotype is one of the central goals in developmental biology and medicine. Transcriptome analysis is a powerful strategy to connect genotype to phenotype of a cell. Here we review the history, progress, potential applications and future developments of single-cell transcriptome analysis. In combination with live cell imaging and lineage tracing, it will be possible to decipher the full gene expression network underlying physiological functions of individual cells in embryos and adults, and to study diseases.
Stochastic and deterministic allele specific gene expression (ASE) might influence single cell phenotype, but the extent and nature of the phenomenon at the onset of early mouse development is unknown. Here we performed single cell RNA-Seq analysis of single blastomeres of mouse embryos, which revealed significant changes in the transcriptome. Importantly, over half of the transcripts with detectable genetic polymorphisms exhibit ASE, most notably, individual blastomeres from the same two-cell embryo show similar pattern of ASE. However, about 6% of them exhibit stochastic expression, indicated by altered expression ratio between the two alleles. Thus, we demonstrate that ASE is both deterministic and stochastic in early blastomeres. Furthermore, we also found that 1,718 genes express two isoforms with different lengths of 3UTRs, with the shorter one on average 5-6 times more abundant in early blastomeres compared to the transcripts in epiblast cells, suggesting that microRNA mediated regulation of gene expression acquires increasing importance as development progresses.
Prmt5, an arginine methyltransferase, has multiple roles in germ cells, and possibly in pluripotency. Here we show that loss of Prmt5 function is early embryonic-lethal due to the abrogation of pluripotent cells in blastocysts. Prmt5 is also up-regulated in the cytoplasm during the derivation of embryonic stem (ES) cells together with Stat3, where they persist to maintain pluripotency. Prmt5 in association with Mep50 methylates cytosolic histone H2A (H2AR3me2s) to repress differentiation genes in ES cells. Loss of Prmt5 or Mep50 results in derepression of differentiation genes, indicating the significance of the Prmt5/Mep50 complex for pluripotency, which may occur in conjunction with the leukemia inhibitory factor (LIF)/Stat3 pathway.
Genome-wide active DNA demethylation in primordial germ cells (PGCs), which reprograms the epigenome for totipotency, is linked to changes in nuclear architecture, loss of histone modifications, and widespread histone replacement. Here, we show that DNA demethylation in the mouse PGCs is mechanistically linked to the appearance of single-stranded DNA (ssDNA) breaks and the activation of the base excision repair (BER) pathway, as is the case in the zygote where the paternal pronucleus undergoes active DNA demethylation shortly after fertilization. Whereas BER might be triggered by deamination of a methylcytosine (5mC), cumulative evidence indicates other mechanisms in germ cells. We demonstrate that DNA repair through BER represents a core component of genome-wide DNA demethylation in vivo and provides a mechanistic link to the extensive chromatin remodeling in developing PGCs.
Mouse and rat embryonic stem cells can be sustained in defined medium by dual inhibition (2i) of the mitogen-activated protein kinase (Erk1/2) cascade and of glycogen synthase kinase 3. The inhibitors suppress differentiation and enable self-renewal of pluripotent cells that are ex vivo counterparts of naïve epiblast cells in the mature blastocyst. Pluripotent stem cell lines can also be derived from unipotent primordial germ cells via a poorly understood process of epigenetic reprogramming. These are termed embryonic germ (EG) cells to denote their distinct origin. Here we investigate whether EG cell self-renewal and derivation are supported by 2i. We report that mouse EG cells can be established with high efficiency using 2i in combination with the cytokine leukaemia inhibitory factor (LIF). Furthermore, addition of fibroblast growth factor or stem cell factor is unnecessary using 2i-LIF. The derived EG cells contribute extensively to healthy chimaeric mice, including to the germline. Using the same conditions, we describe the first derivations of EG cells from the rat. Rat EG cells express a similar marker profile to rat and mouse ES cells. They have a diploid karyotype, can be clonally expanded and genetically manipulated, and are competent for multilineage colonisation of chimaeras. These findings lend support to the postulate of a conserved molecular ground state in pluripotent rodent cells. Future research will determine the extent to which this is maintained in other mammals and whether, in some species, primordial germ cells might be a more tractable source than epiblast for the capture of naïve pluripotent stem cells.
Mouse Embryonic Stem (ES) cells express a unique set of microRNAs (miRNAs), the miR-290-295 cluster. To elucidate the role of these miRNAs and how they integrate into the ES cell regulatory network requires identification of their direct regulatory targets. The difficulty, however, arises from the limited complementarity of metazoan miRNAs to their targets, with the interaction requiring as few as six nucleotides of the miRNA seed sequence. To identify miR-294 targets, we used Dicer1-null ES cells, which lack all endogenous mature miRNAs, and introduced just miR-294 into these ES cells. We then employed two approaches to discover miR-294 targets in mouse ES cells: transcriptome profiling using microarrays and a biochemical approach to isolate mRNA targets associated with the Argonaute2 (Ago2) protein of the RISC (RNA Induced Silencing Complex) effector, followed by RNA-sequencing. In the absence of Dicer1, the RISC complexes are largely devoid of mature miRNAs and should therefore contain only transfected miR-294 and its base-paired targets. Our data suggest that miR-294 may promote pluripotency by regulating a subset of c-Myc target genes and upregulating pluripotency-associated genes such as Lin28.
We describe here a protocol for digital transcriptome analysis in a single mouse oocyte and blastomere using a deep-sequencing approach. In this method, individual cells are isolated and transferred into lysate buffer by mouth pipette, followed by reverse transcription carried out directly on the whole cell lysate. Free primers are removed by exonuclease I and a poly(A) tail is added to the 3 end of the first-strand cDNAs by terminal deoxynucleotidyl transferase. Single-cell cDNAs are then amplified by 20 + 9 cycles of PCR. The resulting 100-200 ng of amplified cDNAs are used to construct a sequencing library, which can be used for deep sequencing using the SOLiD system. Compared with cDNA microarray techniques, our assay can capture up to 75% more genes expressed in early embryos. This protocol can generate deep-sequencing libraries for 16 single-cell samples within 6 d.
During the transition from the inner cell mass (ICM) cells of blastocysts to pluripotent embryonic stem cells (ESCs) in vitro, a normal developmental program is replaced in cells that acquire a capacity for infinite self-renewal and pluripotency. We explored the underlying mechanism of this switch by using RNA-Seq transcriptome analysis at the resolution of single cells. We detected significant molecular transitions and major changes in transcript variants, which include genes for general metabolism. Furthermore, the expression of repressive epigenetic regulators increased with a concomitant decrease in gene activators that might be necessary to sustain the inherent plasticity of ESCs. Furthermore, we detected changes in microRNAs (miRNAs), with one set that targets early differentiation genes while another set targets pluripotency genes to maintain the unique ESC epigenotype. Such genetic and epigenetic events may contribute to a switch from a normal developmental program in adult cells during the formation of diseased tissues, including cancers.
Given the explosion of research on induced pluripotent stem (iPS) cells, it is timely to consider the various ethical, legal, and social issues engaged by this fast-moving field. Here, we review issues associated with the procurement, basic research, and clinical translation of iPS cells.
We have developed a sequencing-based gene expression profiling assay at single-cell resolution by combining a modified single-cell whole transcriptome amplification method with the next generation sequencing technique, the SOLiD system. Using this assay, we have shown that blastomeres in a four-cell stage embryo have similar gene expression, which is compatible with the fact that they have similar developmental potential. We proved that compared with cDNA microarray technique, our single-cell cDNA SOLiD sequencing assay can detect expression of thousands of more genes. Moreover, for the genes detected by microarray and SOLiD sequencing, our assay detected new transcript variants for a large proportion of them, which confirms unambiguously at single-cell resolution that the transcriptome complexity is higher than expected traditionally. Finally, by using our assay to Dicer knockout (KO) and Ago2 KO oocytes, we showed that a significant amount of transposons were up-regulated abnormally in Dicer/Ago2 KO mature oocytes compared with wild-type controls.
Pluripotent epiblast stem cells (EpiSCs) derived from postimplantation embryos exhibit properties that are characteristically different when compared with pluripotent embryonic stem cells (ESCs) derived from mouse blastocysts. However, EpiSCs are relatively less well characterised compared with ESCs. In particular, the relationship between EpiSCs and primordial germ cells (PGCs) is unknown, and is worthy of investigation because PGCs originate from postimplantation epiblast cells in vivo. We show that EpiSCs have an infinite capacity for generating PGCs, under conditions that sustain their pluripotency and self-renewal. These PGCs generated in vitro show appropriate transcriptional and epigenetic reprogramming events and are able to develop further into late germ cells. Notably, the PGCs can, in turn, be induced to undergo dedifferentiation into pluripotent embryonic germ cells (EGCs), which resemble ESCs and not the EpiSC from which they are derived. Our observations demonstrate intrinsic reprogramming during specification of PGCs that results in the erasure of epigenetic memory of EpiSCs following reactivation of the X-chromosome, DNA demethylation and re-expression of key pluripotency genes. This study provides novel insights into the nature and properties of EpiSCs, and introduces an in vitro model system that will be useful for investigations on PGC specification and on mechanisms regulating epigenetic reprogramming in germ cells.
The pluripotent state, which is first established in the primitive ectoderm cells of blastocysts, is lost progressively and irreversibly during subsequent development. For example, development of post-implantation epiblast cells from primitive ectoderm involves significant transcriptional and epigenetic changes, including DNA methylation and X chromosome inactivation, which create a robust epigenetic barrier and prevent their reversion to a primitive-ectoderm-like state. Epiblast cells are refractory to leukaemia inhibitory factor (LIF)-STAT3 signalling, but they respond to activin/basic fibroblast growth factor to form self-renewing epiblast stem cells (EpiSCs), which exhibit essential properties of epiblast cells and that differ from embryonic stem (ES) cells derived from primitive ectoderm. Here we show reprogramming of advanced epiblast cells from embryonic day 5.5-7.5 mouse embryos with uniform expression of N-cadherin and inactive X chromosome to ES-cell-like cells (rESCs) in response to LIF-STAT3 signalling. Cultured epiblast cells overcome the epigenetic barrier progressively as they proceed with the erasure of key properties of epiblast cells, resulting in DNA demethylation, X reactivation and expression of E-cadherin. The accompanying changes in the transcriptome result in a loss of phenotypic and epigenetic memory of epiblast cells. Using this approach, we report reversion of established EpiSCs to rESCs. Moreover, unlike epiblast and EpiSCs, rESCs contribute to somatic tissues and germ cells in chimaeras. Further studies may reveal how signalling-induced epigenetic reprogramming may promote reacquisition of pluripotency.
Pluripotency, the capacity for indefinite self-renewal and differentiation into diverse cell types is a unique state exhibited by embryonic stem (ES) cells. Transcriptional regulators, such as Oct4, are critical for pluripotency, but the role of epigenetic modifiers remains to be fully elucidated.
The imprinted H19 gene produces a non-coding RNA of unknown function. Mice lacking H19 show an overgrowth phenotype, due to a cis effect of the H19 locus on the adjacent Igf2 gene. To explore the function of the RNA itself, we produced transgenic mice overexpressing H19. We observed postnatal growth reduction in two independent transgenic lines and detected a decrease of Igf2 expression in embryos. An extensive analysis of several other genes from the newly described imprinted gene network (IGN) was performed in both loss- and gain-of-function animals. We found that H19 deletion leads to the upregulation of several genes of the IGN. This overexpression is restored to the wild-type level by transgenic expression of H19. We therefore propose that the H19 gene participates as a trans regulator in the fine-tuning of this IGN in the mouse embryo. This is the first in vivo evidence of a functional role for the H19 RNA. Our results also bring further experimental evidence for the existence of the IGN and open new perspectives in the comprehension of the role of genomic imprinting in embryonic growth and in human imprinting pathologies.
Germ cells undergo comprehensive epigenetic reprogramming toward acquiring fitness for pluripotency and totipotency. Notably, the full extent of the epigenetic reprogramming experienced by germ cells remains unmatched by attempts to experimentally restore pluripotency in somatic cells. We propose that the defects present in experimentally generated cells are corrected upon differentiation into the germ cell lineage, as has been observed in cases of germline transmission. Unraveling the mechanisms responsible for germ cell-specific epigenetic reprogramming will likely have important implications for both basic and clinical stem cell research.
Argonaute2 protein (Ago2) is a key component of RNA-induced gene silencing complex, which is crucial for microRNA-mediated repression of target genes. The function of Ago2 in the mouse oocyte and early embryonic development is less well characterized but it is likely to have an important role in regulating maternally inherited mRNA. We have examined the role of Ago2 by conditional deletion of the gene in developing oocytes.
The rarity and inaccessibility of the earliest primordial germ cells (PGCs) in the mouse embryo thwart efforts to investigate molecular mechanisms of germ-cell specification. stella (also called Dppa3) marks the rare founder population of the germ lineage. Here we differentiate mouse embryonic stem cells carrying a stella transgenic reporter into putative PGCs in vitro. The Stella(+) cells possess a transcriptional profile similar to embryo-derived PGCs, and like their counterparts in vivo, lose imprints in a time-dependent manner. Using inhibitory RNAs to screen candidate genes for effects on the development of Stella(+) cells in vitro, we discovered that Lin28, a negative regulator of let-7 microRNA processing, is essential for proper PGC development. Furthermore, we show that Blimp1 (also called Prdm1), a let-7 target and a master regulator of PGC specification, can rescue the effect of Lin28 deficiency during PGC development, thereby establishing a mechanism of action for Lin28 during PGC specification. Overexpression of Lin28 promotes formation of Stella(+) cells in vitro and PGCs in chimaeric embryos, and is associated with human germ-cell tumours. The differentiation of putative PGCs from embryonic stem cells in vitro recapitulates the early stages of gamete development in vivo, and provides an accessible system for discovering novel genes involved in germ-cell development and malignancy.
Steel factor is an essential survival and proliferation factor for primordial germ cells (PGCs) during their migration in the early mouse embryo. PGCs arise during gastrulation, and migrate into the posterior endoderm that becomes the hindgut. Previous reports have suggested that PGCs become dependent on Steel factor when they colonize the hindgut. However, in the absence of a good marker for living PGCs, their behavior before hindgut colonization has not been previously studied. We report here the normal behavior of PGCs in live embryos before hindgut colonization, and the roles of Steel factor, using a reporter line in which GFP is driven by the promoter of the Stella gene, whose activation accompanies the initial specification of PGCs. We show first that PGCs are surrounded by Steel factor-expressing cells from their first appearance in the allantois to the time they enter the genital ridges. Second, fewer PGCs are found in the allantois in Steel-null embryos, but this is not due to a failure of PGC specification. Third, the analysis of cultured Steel-null early embryos shows that Steel factor is required for normal PGC motility, both in the allantois and in the hindgut. Germ cells migrate actively in the allantois, and move directionally from the allantois into the proximal epiblast. In the absence of Steel factor, caused by either null mutation or antibody blockade, PGC motility is dramatically decreased, but directionality is maintained, demonstrating a primary role for Steel factor in PGC motility. This was found both before and after colonization of the hindgut. These data, together with previously published data, show that PGCs are Steel factor dependent from their initial specification until they colonize the genital ridges, and suggest the existence of a ;spatio-temporal niche that travels with this important pluripotential cell population in the embryo.
Next-generation sequencing technology is a powerful tool for transcriptome analysis. However, under certain conditions, only a small amount of material is available, which requires more sensitive techniques that can preferably be used at the single-cell level. Here we describe a single-cell digital gene expression profiling assay. Using our mRNA-Seq assay with only a single mouse blastomere, we detected the expression of 75% (5,270) more genes than microarray techniques and identified 1,753 previously unknown splice junctions called by at least 5 reads. Moreover, 8-19% of the genes with multiple known transcript isoforms expressed at least two isoforms in the same blastomere or oocyte, which unambiguously demonstrated the complexity of the transcript variants at whole-genome scale in individual cells. Finally, for Dicer1(-/-) and Ago2(-/-) (Eif2c2(-/-)) oocytes, we found that 1,696 and 1,553 genes, respectively, were abnormally upregulated compared to wild-type controls, with 619 genes in common.
Embryonic stem (ES) cells, derived from pre-implantation embryo, embryonic germ (EG) cells, derived from embryonic precursors of gametes, primordial germ cells (PGCs), can differentiate into any cell type in the body. Moreover, ES cells have the capacity to differentiate into PGCs in vitro. In the present study we have shown the differentiation capacity of six EG cell lines to form PGCs in vitro, in comparison to ES cells. Cell lines were differentiated via embryoid body (EB) formation using the co-expression of mouse vasa homolog (Mvh) and Oct-4 to identify newly formed PGCs in vitro. We found an increase of PGC numbers in almost all analysed cell lines in 5-day-old EBs, thus suggesting that EG and ES cells have similar efficiency to generate PGCs. The addition of retinoic acid confirmed that the cultures had attained a PGC-like identity and continued to proliferate. Furthermore we have shown that the expression pattern of Prmt5 and H3K27me3 in newly formed PGCs is similar to that observed in embryonic day E11.5 PGCs in vivo. By co-culturing EBs with Chinese hamster ovary (CHO) cells some of the PGCs entered into meiosis, as judged by Scp3 expression. The derivation of germ cells from pluripotent stem cells in vitro could provide an invaluable model system to study both the genetic and epigenetic programming of germ cell development in vivo.
Mouse primordial germ cells (PGCs) undergo sequential epigenetic changes and genome-wide DNA demethylation to reset the epigenome for totipotency. Here, we demonstrate that erasure of CpG methylation (5mC) in PGCs occurs via conversion to 5-hydroxymethylcytosine (5hmC), driven by high levels of TET1 and TET2. Global conversion to 5hmC initiates asynchronously among PGCs at embryonic day (E) 9.5 to E10.5 and accounts for the unique process of imprint erasure. Mechanistically, 5hmC enrichment is followed by its protracted decline thereafter at a rate consistent with replication-coupled dilution. The conversion to 5hmC is an important component of parallel redundant systems that drive comprehensive reprogramming in PGCs. Nonetheless, we identify rare regulatory elements that escape systematic DNA demethylation in PGCs, providing a potential mechanistic basis for transgenerational epigenetic inheritance.
The discovery that phenotypic diversity among differentiated cells results from epigenetic and not genetic differences, and can be reset to restore pluripotency, promises revolutionary advances in medicine. I discuss how this and related seminal discoveries have brought us to an exciting future.
Gene expression analysis at single-cell-resolution is a powerful tool for uncovering individual cell differences within heterogeneous cell populations and complex tissues, which can provide invaluable insights into the extent of gene expression variability. Multi-dimensional information of gene expression at the level of the individual cell can help to identify distinct and rare molecular cell states within populations and aid in unravelling genetic regulatory circuits. Gene expression analysis at the single-cell-level will also enhance our understanding of the molecular basis of aberrant cell states and disease development and holds great promise for the advancement of personalized medicine. We present approaches that provide large-scale views of gene expression at the level of the individual cell.
How cell fate becomes restricted during somatic cell differentiation is a long-lasting question in biology. Epigenetic mechanisms not present in pluripotent cells and acquired during embryonic development are expected to stabilize the differentiated state of somatic cells and thereby restrict their ability to convert to another fate. The histone variant macroH2A acts as a component of an epigenetic multilayer that heritably maintains the silent X chromosome and has been shown to restrict tumor development. Here we show that macroH2A marks the differentiated cell state during mouse embryogenesis. MacroH2A.1 was found to be present at low levels upon the establishment of pluripotency in the inner cell mass and epiblast, but it was highly enriched in the trophectoderm and differentiated somatic cells later in mouse development. Chromatin immunoprecipitation revealed that macroH2A.1 is incorporated in the chromatin of regulatory regions of pluripotency genes in somatic cells such as mouse embryonic fibroblasts and adult neural stem cells, but not in embryonic stem cells. Removal of macroH2A.1, macroH2A.2 or both increased the efficiency of induced pluripotency up to 25-fold. The obtained induced pluripotent stem cells reactivated pluripotency genes, silenced retroviral transgenes and contributed to chimeras. In addition, overexpression of macroH2A isoforms prevented efficient reprogramming of epiblast stem cells to naïve pluripotency. In summary, our study identifies for the first time a link between an epigenetic mark and cell fate restriction during somatic cell differentiation, which helps to maintain cell identity and antagonizes induction of a pluripotent stem cell state.
Mouse primordial germ cells (PGCs) erase global DNA methylation (5mC) as part of the comprehensive epigenetic reprogramming that occurs during PGC development. 5mC plays an important role in maintaining stable gene silencing and repression of transposable elements (TE) but it is not clear how the extensive loss of DNA methylation impacts on gene expression and TE repression in developing PGCs. Using a novel epigenetic disruption and recovery screen and genetic analyses, we identified a core set of germline-specific genes that are dependent exclusively on promoter DNA methylation for initiation and maintenance of developmental silencing. These gene promoters appear to possess a specialised chromatin environment that does not acquire any of the repressive H3K27me3, H3K9me2, H3K9me3 or H4K20me3 histone modifications when silenced by DNA methylation. Intriguingly, this methylation-dependent subset is highly enriched in genes with roles in suppressing TE activity in germ cells. We show that the mechanism for developmental regulation of the germline genome-defence genes involves DNMT3B-dependent de novo DNA methylation. These genes are then activated by lineage-specific promoter demethylation during distinct global epigenetic reprogramming events in migratory (~E8.5) and post-migratory (E10.5-11.5) PGCs. We propose that genes involved in genome defence are developmentally regulated primarily by promoter DNA methylation as a sensory mechanism that is coupled to the potential for TE activation during global 5mC erasure, thereby acting as a failsafe to ensure TE suppression and maintain genomic integrity in the germline.
Development of mammalian primordial germ cells (PGCs) presents a unique example of a cell fate specification event that is intimately linked with epigenetic reprogramming. Cell fate commitment is governed by transcription factors which, together with epigenetic regulators, instruct lineage choice in response to signalling cues. Similarly, the reversal of epigenetic silencing is driven by the combinatorial action of transcriptional regulators, resulting in an increase in cellular plasticity. PGCs constitute a paradox, since their development as a unipotent specialised lineage is coupled with extensive reprogramming, which eventually leads to an increase in cellular potency. In this review we discuss the role of key factors in the specification of the germ cell lineage that are also important for the comprehensive erasure of epigenetic modifications, which provides the foundation for regeneration of totipotency. We further discuss current concepts of transcriptional and epigenetic control of cell fate decisions, with a particular focus on emerging principles of enhancer activity and their potential implications for the transcriptional control of PGC specification.
Blimp1 (Prdm1), the key determinant of primordial germ cells (PGCs), plays a combinatorial role with Prdm14 during PGC specification from postimplantation epiblast cells. They together initiate epigenetic reprogramming in early germ cells toward an underlying pluripotent state, which is equivalent to embryonic stem cells (ESCs). Whereas Prdm14 alone can promote reprogramming and is important for the propagation of the pluripotent state, it is not known whether Blimp1 is similarly involved. By using a genetic approach, we demonstrate that Blimp1 is dispensable for the derivation and maintenance of ESCs and postimplantation epiblast stem cells (epiSCs). Notably, Blimp1 is also dispensable for reprogramming epiSCs to ESCs. Thus, although Blimp1 is obligatory for PGC specification, it is not required for the reversion of epiSCs to ESCs and for their maintenance thereafter. This study suggests that reprogramming, including that of somatic cells to ESCs, may not entail an obligatory route through a Blimp1-positive PGC-like state.
Epigenetic reprogramming in early germ cells is critical toward the establishment of totipotency, but investigations of the germline events are intractable. An objective cell culture-based system could provide mechanistic insight on how the key determinants of primordial germ cells (PGCs), including Prdm14, induce reprogramming in germ cells to an epigenetic ground state. Here we show a Prdm14-Klf2 synergistic effect that can accelerate and enhance reversion of mouse epiblast stem cells (epiSCs) to a naive pluripotent state, including X reactivation and DNA demethylation. Notably, Prdm14 alone has little effect on epiSC reversion, but it enhances the competence for reprogramming and potentially PGC specification. Reprogramming of epiSCs by the combinatorial effect of Prdm14-Klf2 involves key epigenetic changes, which might have an analogous role in PGCs. Our study provides a paradigm toward a systematic analysis of how other key genes contribute to complex and dynamic events of reprogramming in the germline.
Germ cells possess the extraordinary and unique capacity to give rise to a new organism and create an enduring link between all generations. To acquire this property, primordial germ cells (PGCs) transit through an unprecedented programme of sequential epigenetic events that culminates in an epigenomic basal state that is the foundation of totipotency. This process is underpinned by genome-wide DNA demethylation, which may occur through several overlapping pathways, including conversion to 5-hydroxymethylcytosine. We propose that the epigenetic programme in PGCs operates through multiple parallel mechanisms to ensure robustness at the level of individual cells while also being flexible through functional redundancy to guarantee high fidelity of the process. Gaining a better understanding of the molecular mechanisms that direct epigenetic reprogramming in PGCs will enhance our ability to manipulate epigenetic memory, cell-fate decisions and applications in regenerative medicine.
Tissue-specific stem cells are considered to have a limited differentiation potential. Recently, this notion was challenged by reports that showed a broader differentiation potential of neural stem cells, in vitro and in vivo, although the molecular mechanisms that regulate plasticity of neural stem cells are unknown. Here, we report that neural stem cells derived from mouse embryonic cortex respond to Lif and serum in vitro and undergo epithelial to mesenchymal transition (EMT)-mediated dedifferentiation process within 48 h, together with transient upregulation of pluripotency markers and, more notably, upregulation of mesendoderm genes, Brachyury (T) and Sox17. These induced putative mesendoderm cells were injected into early gastrulating chick embryos, which revealed that they integrated more efficiently into mesoderm and endoderm lineages compared to non-induced cells. We also found that TGF? and Jak/Stat pathways are necessary but not sufficient for the induction of mesendodermal phenotype in neural stem cells. These results provide insights into the regulation of plasticity of neural stem cells through EMT. Dissecting the regulatory pathways involved in these processes may help to gain control over cell fate decisions.
Analysis of transcription at the level of single cells in prokaryotes and eukaryotes has revealed the existence of heterogeneities in the expression of individual genes within genetically homogeneous populations. This variation is an emerging hallmark of populations of Embryonic Stem (ES) cells and has been ascribed to the stochasticity associated with the biochemical events that mediate gene expression. It has been suggested that these heterogeneities play a role in the maintenance of pluripotency. However, for the most part, studies have focused on individual genes in large cell populations. Here we use an existing dataset on the expression of eight genes involved in pluripotency in eighty-three ES cells to create Gene Regulatory Networks (GRNs) at the single cell level. We observe widespread heterogeneities in the expression of the eight genes, but analysis of correlations within individual cells reveals three distinct classes centered on the expression of Nanog, a marker of pluripotency, and Fgf5, a gene associated with differentiation: high levels of Nanog and low levels of Fgf5, low levels of Nanog and high levels of Fgf5, and low levels of both. Each of these classes is associated with a collection of active sub-networks, with differing degrees of connectivity between their elements, which define a cellular state: self-renewal, primed for differentiation or transition between the two. Though every cell should be governed by the same network, the active sub-networks may emerge due to considerations such as variation in (i) the expression level of active transcription factors (e.g. through post-translational modification or ligand/co-factor availability) or (ii) access to the target gene locus (e.g. via changes in chromatin status or epigenetic modifications). We conclude that heterogeneities in gene expression should not be interpreted as representing different states of a single unique network, but as a reflection of the activity of different sub-networks in sub-populations of cells.
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