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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.

Key Points Regulation of pluripotency and early post-implantation embryonic development have diverged between humans and mice, which might affect the mechanism of primordial germ cell (PGC) specification. Specification of human and mouse PGCs occurs in response to extrinsic signals, including bone morphogenetic protein 2 (BMP2) and BMP4. Models of human PGC specification from pluripotent stem cells suggest that human PGCs originate from mesodermal precursors at the posterior epiblast during the onset of gastrulation, whereas mouse PGCs originate from the pre-gastrulation epiblast. The gene regulatory network for PGC specification and maintenance in humans and mice has diverged. Notably, SRY-box 17 (SOX17), a key endoderm specifier, is critical for PGC specification in humans but not in mice. PGCs undergo genome-wide DNA demethylation, which erases parental epigenetic memories and facilitates germ cell differentiation in humans and mice. Repressive histone modifications might safeguard PGC genome stability during global DNA demethylation. In early germline development, extra-embryonic signals trigger a regulatory network that induces the specification and subsequent epigenetic reprogramming of primordial germ cells, the precursors of sperm and eggs. Here, the authors review germline specification and reprogramming in humans, and discuss the crucial mechanistic differences between these processes in humans and mice. Primordial germ cells (PGCs), the precursors of sperm and eggs, are established in perigastrulation-stage embryos in mammals. Signals from extra-embryonic tissues induce a unique gene regulatory network in germline-competent cells for PGC specification. This network also initiates comprehensive epigenome resetting, including global DNA demethylation and chromatin reorganization. Mouse germline development has been studied extensively, but the extent to which such knowledge applies to humans was unclear. Here, we review the latest advances in human PGC specification and epigenetic reprogramming. The overall developmental dynamics of human and mouse germline cells appear to be similar, but there are crucial mechanistic differences in PGC specification, reflecting divergence in the regulation of pluripotency and early development.

Germline–soma segregation is a fundamental event during mammalian embryonic development. Here we establish the epigenetic principles of human primordial germ cell (hPGC) development using in vivo hPGCs and stem cell models recapitulating gastrulation. We show that morphogen-induced remodelling of mesendoderm enhancers transiently confers the competence for hPGC fate, but further activation favours mesoderm and endoderm fates. Consistently, reducing the expression of the mesendodermal transcription factor OTX2 promotes the PGC fate. In hPGCs, SOX17 and TFAP2C initiate activation of enhancers to establish a core germline programme, including the transcriptional repressor PRDM1 and pluripotency factors POU5F1 and NANOG. We demonstrate that SOX17 enhancers are the critical components in the regulatory circuitry of germline competence. Furthermore, activation of upstream cis -regulatory elements by an optimized CRISPR activation system is sufficient for hPGC specification. We reveal an enhancer-linked germline transcription factor network that provides the basis for the evolutionary divergence of mammalian germlines. Tang et al. report that sequential epigenetic remodelling of mesendoderm enhancers is required for the segregation of human primordial germ cell and mesendodermal cell fates.

Epiblasts regain pluripotency The pluripotent state, first established in the primitive ectoderm cells of the blastocyst-stage embryo, is lost during subsequent development. Bao et al . now show that advanced post-implantation epiblast cells from mouse embryos aged up to 7.5 days can be reprogrammed back to pluripotency by exposure to LIF/STAT3 signalling. They show accompanying changes in the transcriptome (the total mRNA in the cell) results in a loss of the phenotypic and epigenetic memory found in epiblast cells. Cells reprogrammed in this way can contribute to somatic tissues and germ cells in chimaeras, unlike self-renewing epiblast stem cells. This work provides a model for studying how signalling and epigenetic reprogramming may promote reacquisition of pluripotency. The pluripotent state is first established in the primitive ectoderm cells of blastocysts but is progressively and irreversibly lost during development. For example, the transition from primitive ectoderm cell to epiblast cell, post implantation, involves significant transcriptional and epigenetic changes. A technique for reprogramming advanced epiblast cells from embryonic day 5.5–7.5 mouse embryos to embryonic-stem-cell-like cells — representing a return to pluripotency — is now demonstrated. The pluripotent state, which is first established in the primitive ectoderm cells of blastocysts, is lost progressively and irreversibly during subsequent development 1 . For example, development of post-implantation epiblast cells from primitive ectoderm involves significant transcriptional and epigenetic changes, including DNA methylation and X chromosome inactivation 2 , 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 3 , 4 and that differ from embryonic stem (ES) cells derived from primitive ectoderm 5 . 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.

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.

PRDM14 is a crucial regulator of mouse primordial germ cells (mPGCs), epigenetic reprogramming and pluripotency, but its role in the evolutionarily divergent regulatory network of human PGCs (hPGCs) remains unclear. Besides, a previous knockdown study indicated that PRDM14 might be dispensable for human germ cell fate. Here, we decided to use inducible degrons for a more rapid and comprehensive PRDM14 depletion. We show that PRDM14 loss results in significantly reduced specification efficiency and an aberrant transcriptome of hPGC-like cells (hPGCLCs) obtained in vitro from human embryonic stem cells (hESCs). Chromatin immunoprecipitation and transcriptomic analyses suggest that PRDM14 cooperates with TFAP2C and BLIMP1 to upregulate germ cell and pluripotency genes, while repressing WNT signalling and somatic markers. Notably, PRDM14 targets are not conserved between mouse and human, emphasising the divergent molecular mechanisms of PGC specification. The effectiveness of degrons for acute protein depletion is widely applicable in various developmental contexts. PRDM14 is a critical transcription factor for mouse primordial germ cell specification, but its role in human remains unclear. Here, PRDM14 protein depletion using auxin-inducible degron uncovers a critical role for human germ cell specification, but regulation of a different set of target genes than in mouse.

High-resolution molecular programmes delineating the cellular foundations of mammalian embryogenesis have emerged recently. Similar analysis of human embryos is limited to pre-implantation stages, since early post-implantation embryos are largely inaccessible. Notwithstanding, we previously suggested conserved principles of pig and human early development. For further insight on pluripotent states and lineage delineation, we analysed pig embryos at single cell resolution. Here we show progressive segregation of inner cell mass and trophectoderm in early blastocysts, and of epiblast and hypoblast in late blastocysts. We show that following an emergent short naive pluripotent signature in early embryos, there is a protracted appearance of a primed signature in advanced embryonic stages. Dosage compensation with respect to the X-chromosome in females is attained via X-inactivation in late epiblasts. Detailed human-pig comparison is a basis towards comprehending early human development and a foundation for further studies of human pluripotent stem cell differentiation in pig interspecies chimeras. Lineage segregation from conception to gastrulation has been mapped at the single cell level in mouse, human and monkey. Here, the authors provide a comprehensive analysis of porcine preimplantation development using single cell RNA-seq; mapping metabolic changes, X chromosome inactivation and signalling pathways.

Primordial germ cells (PGCs) are the precursors of sperm and eggs. They undergo genome-wide epigenetic reprogramming to erase epigenetic memory and reset the genomic potential for totipotency. Global DNA methylation erasure is a crucial part of epigenetic resetting when DNA methylation levels decrease across the genome to <5%. However, certain localized regions exhibit slower demethylation or resistance to reprogramming. Since DNA methylation plays a crucial role in transcriptional regulation, this depletion in PGCs requires mechanisms independent of DNA methylation to regulate transcriptional control during PGC reprogramming. Histone modifications are predicted to compensate for the loss of DNA methylation in gene regulation. Different histone modifications exhibit distinct patterns in PGCs undergoing epigenetic programming at the genomic level during PGC development in conjunction with changes in DNA methylation. Together, they contribute to PGC-specific genomic regulation. Recent findings related to these processes provide a comprehensive overview of germline epigenetic reprogramming and its importance in mouse and human PGC development. Additionally, we evaluated the extent to which in vitro culture techniques have replicated the development processes of human PGCs. A Unique Journey of Germ Cells with Dramatic Epigenetic Changes Primordial germ cells (PGCs), which eventually become eggs or sperm, undergo unique and important changes early in their development that are crucial for forming a complete organism after fertilization. Recent studies have advanced our understanding of these changes. This review explains how certain processes, such as adding chemical markers to DNA (DNA methylation) and modifying proteins around DNA (histone modifications), control the development of PGCs in humans and mice. It also explores the replication of these processes in the lab using human stem cells. This review provides important insights into the impact of these changes on reproduction and offers potential new avenues for treating infertility. This summary was initially drafted using artificial intelligence, then revised and fact-checked by the author.

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.

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 AP2γ. 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 AP2γ, which together with PRDM14 initiates the PGC-specific fate. We determined the occupancy of critical genes by AP2γ—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, AP2γ and PRDM14 are sufficient for PGC specification, and the unprecedented resetting of the epigenome towards a basal state. Surani and colleagues use single-cell transcriptomics analysis in a model of mouse primordial germ cell specification to analyse the collaboration between three transcription factors, BLIMP1, PRDM14 and AP2γ, in determining germ cell fate. They find that BLIMP1 binds directly to repress somatic and cell proliferation genes, and at the same time induces AP2γ, which acts together with PRDM14. The three factors are sufficient for specification and form a tripartite interdependent transcriptional network.