Origin and segregation of the human germline.

Human germline-soma segregation occurs during weeks 2-3 in gastrulating embryos. Although direct studies are hindered, here, we investigate the dynamics of human primordial germ cell (PGCs) specification using in vitro models with temporally resolved single-cell transcriptomics and in-depth characterisation using in vivo datasets from human and nonhuman primates, including a 3D marmoset reference atlas. We elucidate the molecular signature for the transient gain of competence for germ cell fate during peri-implantation epiblast development. Furthermore, we show that both the PGCs and amnion arise from transcriptionally similar TFAP2A-positive progenitors at the posterior end of the embryo. Notably, genetic loss of function experiments shows that TFAP2A is crucial for initiating the PGC fate without detectably affecting the amnion and is subsequently replaced by TFAP2C as an essential component of the genetic network for PGC fate. Accordingly, amniotic cells continue to emerge from the progenitors in the posterior epiblast, but importantly, this is also a source of nascent PGCs.

Epigenetic resetting in the human germ line entails histone modification remodeling.

Epigenetic resetting in the mammalian germ line entails acute DNA demethylation, which lays the foundation for gametogenesis, totipotency, and embryonic development. We characterize the epigenome of hypomethylated human primordial germ cells (hPGCs) to reveal mechanisms preventing the widespread derepression of genes and transposable elements (TEs). Along with the loss of DNA methylation, we show that hPGCs exhibit a profound reduction of repressive histone modifications resulting in diminished heterochromatic signatures at most genes and TEs and the acquisition of a neutral or paused epigenetic state without transcriptional activation. Efficient maintenance of a heterochromatic state is limited to a subset of genomic loci, such as evolutionarily young TEs and some developmental genes, which require H3K9me3 and H3K27me3, respectively, for efficient transcriptional repression. Accordingly, transcriptional repression in hPGCs presents an exemplary balanced system relying on local maintenance of heterochromatic features and a lack of inductive cues.

Specification of human germ cell fate with enhanced progression capability supported by hindgut organoids.

Human primordial germ cells (hPGCs), the precursors of sperm and eggs, are specified during weeks 2-3 after fertilization. Few studies on ex vivo and in vitro cultured human embryos reported plausible hPGCs on embryonic day (E) 12-13 and in an E16-17 gastrulating embryo. In vitro, hPGC-like cells (hPGCLCs) can be specified from the intermediary pluripotent stage or peri-gastrulation precursors. Here, we explore the broad spectrum of hPGCLC precursors and how different precursors impact hPGCLC development. We show that resetting precursors can give rise to hPGCLCs (rhPGCLCs) in response to BMP. Strikingly, rhPGCLCs co-cultured with human hindgut organoids progress at a pace reminiscent of in vivo hPGC development, unlike those derived from peri-gastrulation precursors. Moreover, rhPGCLC specification depends on both EOMES and TBXT, not just on EOMES as for peri-gastrulation hPGCLCs. Importantly, our study provides the foundation for developing efficient in vitro models of human gametogenesis.

Organoid modeling of human fetal lung alveolar development reveals mechanisms of cell fate patterning and neonatal respiratory disease.

Variation in lung alveolar development is strongly linked to disease susceptibility. However, underlying cellular and molecular mechanisms are difficult to study in humans. We have identified an alveolar-fated epithelial progenitor in human fetal lungs, which we grow as self-organizing organoids that model key aspects of cell lineage commitment. Using this system, we have functionally validated cell-cell interactions in the developing human alveolar niche, showing that Wnt signaling from differentiating fibroblasts promotes alveolar-type-2 cell identity, whereas myofibroblasts secrete the Wnt inhibitor, NOTUM, providing spatial patterning. We identify a Wnt-NKX2.1 axis controlling alveolar differentiation. Moreover, we show that differential binding of NKX2.1 coordinates alveolar maturation, allowing us to model the effects of human genetic variation in NKX2.1 on alveolar differentiation. Our organoid system recapitulates key aspects of human fetal lung stem cell biology allowing mechanistic experiments to determine the cellular and molecular regulation of human development and disease.

SOX9 maintains human foetal lung tip progenitor state by enhancing WNT and RTK signalling.

The balance between self-renewal and differentiation in human foetal lung epithelial progenitors controls the size and function of the adult organ. Moreover, progenitor cell gene regulation networks are employed by both regenerating and malignant lung cells, where modulators of their effects could potentially be of therapeutic value. Details of the molecular networks controlling human lung progenitor self-renewal remain unknown. We performed the first CRISPRi screen in primary human lung organoids to identify transcription factors controlling progenitor self-renewal. We show that SOX9 promotes proliferation of lung progenitors and inhibits precocious airway differentiation. Moreover, by identifying direct transcriptional targets using Targeted DamID, we place SOX9 at the centre of a transcriptional network, which amplifies WNT and RTK signalling to stabilise the progenitor cell state. In addition, the proof-of-principle CRISPRi screen and Targeted DamID tools establish a new workflow for using primary human organoids to elucidate detailed functional mechanisms underlying normal development and disease.

Sequential enhancer state remodelling defines human germline competence and specification.

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.

Tracing the emergence of primordial germ cells from bilaminar disc rabbit embryos and pluripotent stem cells.

Rabbit embryos develop as bilaminar discs at gastrulation as in humans and most other mammals, whereas rodents develop as egg cylinders. Primordial germ cells (PGCs) appear to originate during gastrulation according to many systematic studies on mammalian embryos. Here, we show that rabbit PGC (rbPGC) specification occurs at the posterior epiblast at the onset of gastrulation. Using newly derived rabbit pluripotent stem cells, we show robust and rapid induction of rbPGC-like cells in vitro with WNT and BMP morphogens, which reveals SOX17 as the critical regulator of rbPGC fate as in several non-rodent mammals. We posit that development as a bilaminar disc is a crucial determinant of the PGC regulators, regardless of the highly diverse development of extraembryonic tissues, including the amnion. We propose that investigations on rabbits with short gestation, large litters, and where gastrulation precedes implantation can contribute significantly to advances in early mammalian development.

Specification and epigenomic resetting of the pig germline exhibit conservation with the human lineage.

Investigations of the human germline and programming are challenging because of limited access to embryonic material. However, the pig as a model may provide insights into transcriptional network and epigenetic reprogramming applicable to both species. Here we show that, during the pre- and early migratory stages, pig primordial germ cells (PGCs) initiate large-scale epigenomic reprogramming, including DNA demethylation involving TET-mediated hydroxylation and, potentially, base excision repair (BER). There is also macroH2A1 depletion and increased H3K27me3 as well as X chromosome reactivation (XCR) in females. Concomitantly, there is dampening of glycolytic metabolism genes and re-expression of some pluripotency genes like those in preimplantation embryos. We identified evolutionarily young transposable elements and gene coding regions resistant to DNA demethylation in acutely hypomethylated gonadal PGCs, with potential for transgenerational epigenetic inheritance. Detailed insights into the pig germline will likely contribute significantly to advances in human germline biology, including in vitro gametogenesis.

A critical role of PRDM14 in human primordial germ cell fate revealed by inducible degrons.

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.

Pluripotency and X chromosome dynamics revealed in pig pre-gastrulating embryos by single cell analysis.

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.

Author Correction: Segregation of mitochondrial DNA heteroplasmy through a developmental genetic bottleneck in human embryos.

In the version of this Letter originally published, an author error led to the affiliations for Brendan Payne, Jonathan Coxhead and Gavin Hudson being incorrect. The correct affiliations are: Brendan Payne: 3Wellcome Trust Centre for Mitochondrial Research, Institute of Genetic Medicine, Newcastle University, Newcastle upon Tyne, UK. 6Institute of Neuroscience, Newcastle University, Newcastle upon Tyne, UK; this is a new affiliation 6 and subsequent existing affiliations have been renumbered. Jonathan Coxhead: 11Genomic Core Facility, Institute of Genetic Medicine, Newcastle University, Newcastle upon Tyne, UK; this is a new affiliation 11 and subsequent existing affiliations have been renumbered. Gavin Hudson: 3Wellcome Trust Centre for Mitochondrial Research, Institute of Genetic Medicine, Newcastle University, Newcastle upon Tyne, UK. In addition, in Fig. 2d, the numbers on the x-axis of the left plot were incorrectly labelled as negative; they should have been positive. These errors have now been corrected in all online versions of the Letter.

Esrrb Complementation Rescues Development of Nanog-Null Germ Cells.

The transcription factors (TFs) Nanog and Esrrb play important roles in embryonic stem cells (ESCs) and during primordial germ-cell (PGC) development. Esrrb is a positively regulated direct target of NANOG in ESCs that can substitute qualitatively for Nanog function in ESCs. Whether this functional substitution extends to the germline is unknown. Here, we show that germline deletion of Nanog reduces PGC numbers 5-fold at midgestation. Despite this quantitative depletion, Nanog-null PGCs can complete germline development in contrast to previous findings. PGC-like cell (PGCLC) differentiation of Nanog-null ESCs is also impaired, with Nanog-null PGCLCs showing decreased proliferation and increased apoptosis. However, induced expression of Esrrb restores PGCLC numbers as efficiently as Nanog. These effects are recapitulated in vivo: knockin of Esrrb to Nanog restores PGC numbers to wild-type levels and results in fertile adult mice. These findings demonstrate that Esrrb can replace Nanog function in germ cells.

Segregation of mitochondrial DNA heteroplasmy through a developmental genetic bottleneck in human embryos.

Mitochondrial DNA (mtDNA) mutations cause inherited diseases and are implicated in the pathogenesis of common late-onset disorders, but how they arise is not clear1,2. Here we show that mtDNA mutations are present in primordial germ cells (PGCs) within healthy female human embryos. Isolated PGCs have a profound reduction in mtDNA content, with discrete mitochondria containing ~5 mtDNA molecules. Single-cell deep mtDNA sequencing of in vivo human female PGCs showed rare variants reaching higher heteroplasmy levels in late PGCs, consistent with the observed genetic bottleneck. We also saw the signature of selection against non-synonymous protein-coding, tRNA gene and D-loop variants, concomitant with a progressive upregulation of genes involving mtDNA replication and transcription, and linked to a transition from glycolytic to oxidative metabolism. The associated metabolic shift would expose deleterious mutations to selection during early germ cell development, preventing the relentless accumulation of mtDNA mutations in the human population predicted by Muller's ratchet. Mutations escaping this mechanism will show shifts in heteroplasmy levels within one human generation, explaining the extreme phenotypic variation seen in human pedigrees with inherited mtDNA disorders.

Derivation of hypermethylated pluripotent embryonic stem cells with high potency.

Naive hypomethylated embryonic pluripotent stem cells (ESCs) are developmentally closest to the preimplantation epiblast of blastocysts, with the potential to contribute to all embryonic tissues and the germline, excepting the extra-embryonic tissues in chimeric embryos. By contrast, epiblast stem cells (EpiSCs) resembling postimplantation epiblast are relatively more methylated and show a limited potential for chimerism. Here, for the first time, we reveal advanced pluripotent stem cells (ASCs), which are developmentally beyond the pluripotent cells in the inner cell mass but with higher potency than EpiSCs. Accordingly, a single ASC contributes very efficiently to the fetus, germline, yolk sac and the placental labyrinth in chimeras. Since they are developmentally more advanced, ASCs do not contribute to the trophoblast. ASCs were derived from blastocysts in two steps in a chemically defined medium supplemented with Activin A and basic fibroblast growth factor, followed by culturing in ABCL medium containing ActA, BMP4, CHIR99021 and leukemia inhibitory factor. Notably, ASCs exhibit a distinct transcriptome with the expression of both naive pluripotency genes, as well as mesodermal somatic genes; Eomes, Eras, Tdgf1, Evx1, hand1, Wnt5a and distinct repetitive elements. Conversion of established ESCs to ASCs is also achievable. Importantly, ASCs exhibit a stable hypermethylated epigenome and mostly intact imprints as compared to the hypomethylated inner cell mass of blastocysts and naive ESCs. Properties of ASCs suggest that they represent cells at an intermediate cellular state between the naive and primed states of pluripotency.

Principles of early human development and germ cell program from conserved model systems.

Human primordial germ cells (hPGCs), the precursors of sperm and eggs, originate during weeks 2-3 of early post-implantation development. Using in vitro models of hPGC induction, recent studies have suggested that there are marked mechanistic differences in the specification of human and mouse PGCs. This may be due in part to the divergence in their pluripotency networks and early post-implantation development. As early human embryos are not accessible for direct study, we considered alternatives including porcine embryos that, as in humans, develop as bilaminar embryonic discs. Here we show that porcine PGCs originate from the posterior pre-primitive-streak competent epiblast by sequential upregulation of SOX17 and BLIMP1 in response to WNT and BMP signalling. We use this model together with human and monkey in vitro models simulating peri-gastrulation development to show the conserved principles of epiblast development for competency for primordial germ cell fate. This process is followed by initiation of the epigenetic program and regulated by a balanced SOX17-BLIMP1 gene dosage. Our combinatorial approach using human, porcine and monkey in vivo and in vitro models provides synthetic insights into early human development.

Specification and epigenetic programming of the human germ line.

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.

NANOG alone induces germ cells in primed epiblast in vitro by activation of enhancers.

Nanog, a core pluripotency factor in the inner cell mass of blastocysts, is also expressed in unipotent primordial germ cells (PGCs) in mice, where its precise role is yet unclear. We investigated this in an in vitro model, in which naive pluripotent embryonic stem (ES) cells cultured in basic fibroblast growth factor (bFGF) and activin A develop as epiblast-like cells (EpiLCs) and gain competence for a PGC-like fate. Consequently, bone morphogenetic protein 4 (BMP4), or ectopic expression of key germline transcription factors Prdm1, Prdm14 and Tfap2c, directly induce PGC-like cells (PGCLCs) in EpiLCs, but not in ES cells. Here we report an unexpected discovery that Nanog alone can induce PGCLCs in EpiLCs, independently of BMP4. We propose that after the dissolution of the naive ES-cell pluripotency network during establishment of EpiLCs, the epigenome is reset for cell fate determination. Indeed, we found genome-wide changes in NANOG-binding patterns between ES cells and EpiLCs, indicating epigenetic resetting of regulatory elements. Accordingly, we show that NANOG can bind and activate enhancers of Prdm1 and Prdm14 in EpiLCs in vitro; BLIMP1 (encoded by Prdm1) then directly induces Tfap2c. Furthermore, while SOX2 and NANOG promote the pluripotent state in ES cells, they show contrasting roles in EpiLCs, as Sox2 specifically represses PGCLC induction by Nanog. This study demonstrates a broadly applicable mechanistic principle for how cells acquire competence for cell fate determination, resulting in the context-dependent roles of key transcription factors during development.

A Unique Gene Regulatory Network Resets the Human Germline Epigenome for Development.

Resetting of the epigenome in human primordial germ cells (hPGCs) is critical for development. We show that the transcriptional program of hPGCs is distinct from that in mice, with co-expression of somatic specifiers and naive pluripotency genes TFCP2L1 and KLF4. This unique gene regulatory network, established by SOX17 and BLIMP1, drives comprehensive germline DNA demethylation by repressing DNA methylation pathways and activating TET-mediated hydroxymethylation. Base-resolution methylome analysis reveals progressive DNA demethylation to basal levels in week 5-7 in vivo hPGCs. Concurrently, hPGCs undergo chromatin reorganization, X reactivation, and imprint erasure. Despite global hypomethylation, evolutionarily young and potentially hazardous retroelements, like SVA, remain methylated. Remarkably, some loci associated with metabolic and neurological disorders are also resistant to DNA demethylation, revealing potential for transgenerational epigenetic inheritance that may have phenotypic consequences. We provide comprehensive insight on early human germline transcriptional network and epigenetic reprogramming that subsequently impacts human development and disease.

SOX17 is a critical specifier of human primordial germ cell fate.

Specification of primordial germ cells (PGCs) marks the beginning of the totipotent state. However, without a tractable experimental model, the mechanism of human PGC (hPGC) specification remains unclear. Here, we demonstrate specification of hPGC-like cells (hPGCLCs) from germline competent pluripotent stem cells. The characteristics of hPGCLCs are consistent with the embryonic hPGCs and a germline seminoma that share a CD38 cell-surface marker, which collectively defines likely progression of the early human germline. Remarkably, SOX17 is the key regulator of hPGC-like fate, whereas BLIMP1 represses endodermal and other somatic genes during specification of hPGCLCs. Notable mechanistic differences between mouse and human PGC specification could be attributed to their divergent embryonic development and pluripotent states, which might affect other early cell-fate decisions. We have established a foundation for future studies on resetting of the epigenome in hPGCLCs and hPGCs for totipotency and the transmission of genetic and epigenetic information.