Reconstitution of the mouse germ cell specification pathway in culture by pluripotent stem cells.

The generation of properly functioning gametes in vitro requires reconstitution of the multistepped pathway of germ cell development. We demonstrate here the generation of primordial germ cell-like cells (PGCLCs) in mice with robust capacity for spermatogenesis. PGCLCs were generated from embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) through epiblast-like cells (EpiLCs), a cellular state highly similar to pregastrulating epiblasts but distinct from epiblast stem cells (EpiSCs). Reflecting epiblast development, EpiLC induction from ESCs/iPSCs is a progressive process, and EpiLCs highly competent for the PGC fate are a transient entity. The global transcription profiles, epigenetic reprogramming, and cellular dynamics during PGCLC induction from EpiLCs meticulously capture those associated with PGC specification from the epiblasts. Furthermore, we identify Integrin-ß3 and SSEA1 as markers that allow the isolation of PGCLCs with spermatogenic capacity from tumorigenic undifferentiated cells. Our findings provide a paradigm for the first step of in vitro gametogenesis.

Cohesin controls X chromosome structure remodeling and X-reactivation during mouse iPSC-reprogramming.

Reactivation of the inactive X chromosome is a hallmark epigenetic event during reprogramming of mouse female somatic cells to induced pluripotent stem cells (iPSCs). This involves global structural remodeling from a condensed, heterochromatic into an open, euchromatic state, thereby changing a transcriptionally inactive into an active chromosome. Despite recent advances, very little is currently known about the molecular players mediating this process and how this relates to iPSC-reprogramming in general. To gain more insight, here we perform a RNAi-based knockdown screen during iPSC-reprogramming of mouse fibroblasts. We discover factors important for X chromosome reactivation (XCR) and iPSC-reprogramming. Among those, we identify the cohesin complex member SMC1a as a key molecule with a specific function in XCR, as its knockdown greatly affects XCR without interfering with iPSC-reprogramming. Using super-resolution microscopy, we find SMC1a to be preferentially enriched on the active compared with the inactive X chromosome and that SMC1a is critical for the decompacted state of the active X. Specifically, depletion of SMC1a leads to contraction of the active X both in differentiated and in pluripotent cells, where it normally is in its most open state. In summary, we reveal cohesin as a key factor for remodeling of the X chromosome from an inactive to an active structure and that this is a critical step for XCR during iPSC-reprogramming.

DPPA3 facilitates genome-wide DNA demethylation in mouse primordial germ cells.

BACKGROUND: Genome-wide DNA demethylation occurs in mammalian primordial germ cells (PGCs) as part of the epigenetic reprogramming important for gametogenesis and resetting the epigenetic information for totipotency. Dppa3 (also known as Stella or Pgc7) is highly expressed in mouse PGCs and oocytes and encodes a factor essential for female fertility. It prevents excessive DNA methylation in oocytes and ensures proper gene expression in preimplantation embryos: however, its role in PGCs is largely unexplored. In the present study, we investigated whether or not DPPA3 has an impact on CG methylation/demethylation in mouse PGCs. RESULTS: We show that DPPA3 plays a role in genome-wide demethylation in PGCs even before sex differentiation. Dppa3 knockout female PGCs show aberrant hypermethylation, most predominantly at H3K9me3-marked retrotransposons, which persists up to the fully-grown oocyte stage. DPPA3 works downstream of PRDM14, a master regulator of epigenetic reprogramming in embryonic stem cells and PGCs, and independently of TET1, an enzyme that hydroxylates 5-methylcytosine. CONCLUSIONS: The results suggest that DPPA3 facilitates DNA demethylation through a replication-coupled passive mechanism in PGCs. Our study identifies DPPA3 as a novel epigenetic reprogramming factor in mouse PGCs.

Germline development in rat revealed by visualization and deletion of Prdm14.

Primordial germ cells (PGCs), the founder cells of the germline, are specified in pre-gastrulating embryos in mammals, and subsequently migrate towards gonads to mature into functional gametes. Here, we investigated PGC development in rats, by genetically modifying Prdm14, a unique marker and an essential PGC transcriptional regulator. We trace PGC development in rats, for the first time, from specification until the sex determination stage in fetal gonads using Prdm14 H2BVenus knock-in rats. We uncover that the crucial role of Prdm14 in PGC specification is conserved between rat and mice, by analyzing Prdm14-deficient rat embryos. Notably, loss of Prdm14 completely abrogates the PGC program, as demonstrated by failure of the maintenance and/or activation of germ cell markers and pluripotency genes. Finally, we profile the transcriptome of the post-implantation epiblast and all PGC stages in rat to reveal enrichment of distinct gene sets at each transition point, thereby providing an accurate transcriptional timeline for rat PGC development. Thus, the novel genetically modified rats and data sets obtained in this study will advance our knowledge on conserved versus species-specific features for germline development in mammals.

In vitro expansion of mouse primordial germ cell-like cells recapitulates an epigenetic blank slate.

The expansion of primordial germ cells (PGCs), the precursors for the oocytes and spermatozoa, is a key challenge in reproductive biology/medicine. Using a chemical screening exploiting PGC-like cells (PGCLCs) induced from mouse embryonic stem cells (ESCs), we here identify key signaling pathways critical for PGCLC proliferation. We show that the combinatorial application of Forskolin and Rolipram, which stimulate cAMP signaling via different mechanisms, expands PGCLCs up to ~50-fold in culture. The expanded PGCLCs maintain robust capacity for spermatogenesis, rescuing the fertility of infertile mice. Strikingly, during expansion, PGCLCs comprehensively erase their DNA methylome, including parental imprints, in a manner that precisely recapitulates genome-wide DNA demethylation in gonadal germ cells, while essentially maintaining their identity as sexually uncommitted PGCs, apparently through appropriate histone modifications. By establishing a paradigm for PGCLC expansion, our system reconstitutes the epigenetic "blank slate" of the germ line, an immediate precursory state for sexually dimorphic differentiation.

Cyclosporin A and FGF signaling support the proliferation/survival of mouse primordial germ cell-like cells in vitro†.

Primordial germ cells (PGCs) are the founding population of the germ cell lineage that undergo a multistep process to generate spermatozoa or oocytes. Establishing an appropriate culture system for PGCs is a key challenge in reproductive biology. By a chemical screening using mouse PGC-like cells (mPGCLCs), which were induced from mouse embryonic stem cells, we reported previously that forskolin and rolipram synergistically enhanced the proliferation/survival of mPGCLCs with an average expansion rate of ~20-fold. In the present study, we evaluated other chemicals or cytokines to see whether they would improve the current mPGCLC culture system. Among the chemicals and cytokines examined, in the presence of forskolin and rolipram, cyclosporin A (CsA) and fibroblast growth factors (FGFs: FGF2 and FGF10) effectively enhanced the expansion of mPGCLCs in vitro (~50-fold on average). During the expansion by CsA or FGFs, mPGCLCs comprehensively erased their DNA methylation to acquire a profile equivalent to that of gonadal germ cells in vivo, while maintaining their highly motile phenotype as well as their transcriptional properties as sexually uncommitted PGCs. Importantly, these mPGCLCs robustly contributed to spermatogenesis and produced fertile offspring. Furthermore, mouse PGCs (mPGCs) cultured with CsA ex vivo showed transcriptomes and DNA methylomes similar to those of cultured mPGCLCs. The improved culture system for mPGCLCs/mPGCs would be instructive for addressing key questions in PGC biology, including the mechanisms for germ cell migration, epigenetic reprogramming, and sex determination of the germline.

Klf5 maintains the balance of primitive endoderm versus epiblast specification during mouse embryonic development by suppression of Fgf4.

The inner cell mass of the mouse blastocyst gives rise to the pluripotent epiblast (EPI), which forms the embryo proper, and the primitive endoderm (PrE), which forms extra-embryonic yolk sac tissues. All inner cells coexpress lineage markers such as Nanog and Gata6 at embryonic day (E) 3.25, and the EPI and PrE precursor cells eventually segregate to exclusively express Nanog and Gata6, respectively. Fibroblast growth factor (FGF)-extracellular signal-regulated kinase (ERK) signalling is involved in segregation of the EPI and PrE lineages; however, the mechanism involved in Fgf4 regulation is poorly understood. Here, we identified Klf5 as an upstream repressor of Fgf4Fgf4 was markedly upregulated in Klf5 knockout (KO) embryos at E3.0, and was downregulated in embryos overexpressing Klf5 Furthermore, Klf5 KO and overexpressing blastocysts showed skewed lineage specification phenotypes, similar to FGF4-treated preimplantation embryos and Fgf4 KO embryos, respectively. Inhibitors of the FGF receptor (Fgfr) and ERK pathways reversed the skewed lineage specification of Klf5 KO blastocysts. These data demonstrate that Klf5 suppresses Fgf4-Fgfr-ERK signalling, thus preventing precocious activation of the PrE specification programme.

Induction of mouse germ-cell fate by transcription factors in vitro.

The germ-cell lineage ensures the continuity of life through the generation of male and female gametes, which unite to form a totipotent zygote. We have previously demonstrated that, by using cytokines, embryonic stem cells and induced pluripotent stem cells can be induced into epiblast-like cells (EpiLCs) and then into primordial germ cell (PGC)-like cells with the capacity for both spermatogenesis and oogenesis, creating an opportunity for understanding and regulating mammalian germ-cell development in both sexes in vitro. Here we show that, without cytokines, simultaneous overexpression of three transcription factors, Blimp1 (also known as Prdm1), Prdm14 and Tfap2c (also known as AP2γ), directs EpiLCs, but not embryonic stem cells, swiftly and efficiently into a PGC state. Notably, Prdm14 alone, but not Blimp1 or Tfap2c, suffices for the induction of the PGC state in EpiLCs. The transcription-factor-induced PGC state, irrespective of the transcription factors used, reconstitutes key transcriptome and epigenetic reprogramming in PGCs, but bypasses a mesodermal program that accompanies PGC or PGC-like-cell specification by cytokines including bone morphogenetic protein 4. Notably, the transcription-factor-induced PGC-like cells contribute to spermatogenesis and fertile offspring. Our findings provide a new insight into the transcriptional logic for PGC specification, and create a foundation for the transcription-factor-based reconstitution and regulation of mammalian gametogenesis.

Principles for the regulation of multiple developmental pathways by a versatile transcriptional factor, BLIMP1.

Single transcription factors (TFs) regulate multiple developmental pathways, but the underlying mechanisms remain unclear. Here, we quantitatively characterized the genome-wide occupancy profiles of BLIMP1, a key transcriptional regulator for diverse developmental processes, during the development of three germ-layer derivatives (photoreceptor precursors, embryonic intestinal epithelium and plasmablasts) and the germ cell lineage (primordial germ cells). We identified BLIMP1-binding sites shared among multiple developmental processes, and such sites were highly occupied by BLIMP1 with a stringent recognition motif and were located predominantly in promoter proximities. A subset of bindings common to all the lineages exhibited a new, strong recognition sequence, a GGGAAA repeat. Paradoxically, however, the shared/common bindings had only a slight impact on the associated gene expression. In contrast, BLIMP1 occupied more distal sites in a cell type-specific manner; despite lower occupancy and flexible sequence recognitions, such bindings contributed effectively to the repression of the associated genes. Recognition motifs of other key TFs in BLIMP1-binding sites had little impact on the expression-level changes. These findings suggest that the shared/common sites might serve as potential reservoirs of BLIMP1 that functions at the specific sites, providing the foundation for a unified understanding of the genome regulation by BLIMP1, and, possibly, TFs in general.

Replication-coupled passive DNA demethylation for the erasure of genome imprints in mice.

Genome-wide DNA demethylation, including the erasure of genome imprints, in primordial germ cells (PGCs) is a critical first step to creating a totipotent epigenome in the germ line. We show here that, contrary to the prevailing model emphasizing active DNA demethylation, imprint erasure in mouse PGCs occurs in a manner largely consistent with replication-coupled passive DNA demethylation: PGCs erase imprints during their rapid cycling with little de novo or maintenance DNA methylation potential and no apparent major chromatin alterations. Our findings necessitate the re-evaluation of and provide novel insights into the mechanism of genome-wide DNA demethylation in PGCs.

SC3-seq: a method for highly parallel and quantitative measurement of single-cell gene expression.

Single-cell mRNA sequencing (RNA-seq) methods have undergone rapid development in recent years, and transcriptome analysis of relevant cell populations at single-cell resolution has become a key research area of biomedical sciences. We here present single-cell mRNA 3-prime end sequencing (SC3-seq), a practical methodology based on PCR amplification followed by 3-prime-end enrichment for highly quantitative, parallel and cost-effective measurement of gene expression in single cells. The SC3-seq allows excellent quantitative measurement of mRNAs ranging from the 10,000-cell to 1-cell level, and accordingly, allows an accurate estimate of the transcript levels by a regression of the read counts of spike-in RNAs with defined copy numbers. The SC3-seq has clear advantages over other typical single-cell RNA-seq methodologies for the quantitative measurement of transcript levels and at a sequence depth required for the saturation of transcript detection. The SC3-seq distinguishes four distinct cell types in the peri-implantation mouse blastocysts. Furthermore, the SC3-seq reveals the heterogeneity in human-induced pluripotent stem cells (hiPSCs) cultured under on-feeder as well as feeder-free conditions, demonstrating a more homogeneous property of the feeder-free hiPSCs. We propose that SC3-seq might be used as a powerful strategy for single-cell transcriptome analysis in a broad range of investigations in biomedical sciences.

Persistent Requirement and Alteration of the Key Targets of PRDM1 During Primordial Germ Cell Development in Mice.

Primordial germ cells (PGCs) are the foundation of totipotency and vital for reproduction and heredity. PGCs in mice arise from the epiblast around Embryonic Day (E) 7.0, migrate through the hindgut endoderm, and colonize and proliferate in the embryonic gonads until around E13.5 prior to their differentiation either into prospermatogonia or oogonia. PRDM1, a transcriptional repressor, plays an essential role in PGC specification that includes robustly repressing a somatic mesodermal program. Using an inducible conditional knockout system, we show here that PRDM1 is critically required throughout PGC development. When Prdm1 was deleted in migrating PGCs at E9.5 or E10.5, or in male gonadal PGCs at E11.5, PGCs were eliminated by apoptosis from around E10.5, E11.5, or E13.5, respectively. When Prdm1 was deleted in female gonadal PGCs at E11.5, PGCs progressed into the first meiotic prophase in an apparently normal fashion, but the oogonia exhibited an aberrant pachytene phenotype, undergoing abrupt apoptosis from around E16.5. The escape of a fraction of PGCs (∼10%) from the Prdm1 deletion was sufficient to recover fairly normal germ cell pools, both in male and female adults. The key targets of PRDM1 in migrating and/or gonadal PGCs, including genes for development, apoptosis, and prospermatogonial differentiation, showed only a modest overlap with those upon PGC specification, and were enriched with histone H3 lysine 27 trimethylation (H3K27me3). Our findings provide critical insight into the mechanism for maintaining the transcriptional integrity of PGCs.

Epigenetic reprogramming in mouse pre-implantation development and primordial germ cells.

Epigenetic modifications are crucial for the identity and stability of cells, and, when aberrant, can lead to disease. During mouse development, the genome-wide epigenetic states of pre-implantation embryos and primordial germ cells (PGCs) undergo extensive reprogramming. An improved understanding of the epigenetic reprogramming mechanisms that occur in these cells should provide important new information about the regulation of the epigenetic state of a cell and the mechanisms of induced pluripotency. Here, we discuss recent findings about the potential mechanisms of epigenetic reprogramming, particularly genome-wide DNA demethylation, in pre-implantation mouse embryos and PGCs.

Protocol for high-quality single-cell RNA-seq from tissue sections with DRaqL.

Single-cell RNA sequencing (scRNA-seq) combined with laser capture microdissection (LCM) offers a versatile framework for comprehensive transcriptomics from tissue sections. Here, we present a detailed protocol for DRaqL (direct RNA recovery and quenching for LCM) in combination with Smart-seq2 (DRaqL-Smart-seq2), which enables high-quality RNA sequencing for single cells obtained from alcohol-fixed murine ovarian sections. Additionally, we provide an optional procedure for scRNA-seq from formalin-fixed sections (DRaqL-Protease-Smart-seq2). We outline key steps for cell lysis, cDNA amplification, and sequencing library preparation. For complete details on the use and execution of this protocol, please refer to Ikeda et al.1.

High-quality single-cell transcriptomics from ovarian histological sections during folliculogenesis.

High-quality, straightforward single-cell RNA sequencing (RNA-seq) with spatial resolution remains challenging. Here, we developed DRaqL (direct RNA recovery and quenching for laser capture microdissection), an experimental approach for efficient cell lysis of tissue sections, directly applicable to cDNA amplification. Single-cell RNA-seq combined with DRaqL allowed transcriptomic profiling from alcohol-fixed sections with efficiency comparable with that of profiling from freshly dissociated cells, together with effective exon-exon junction profiling. The combination of DRaqL with protease treatment enabled robust and efficient single-cell transcriptome analysis from formalin-fixed tissue sections. Applying this method to mouse ovarian sections, we were able to predict the transcriptome of oocytes by their size and identified an anomaly in the size-transcriptome relationship relevant to growth retardation of oocytes, in addition to detecting oocyte-specific splice isoforms. Furthermore, we identified differentially expressed genes in granulosa cells in association with their proximity to the oocytes, suggesting distinct epigenetic regulations and cell-cycle activities governing the germ-soma relationship. Thus, DRaqL is a versatile, efficient approach for high-quality single-cell RNA-seq from tissue sections, thereby revealing histological heterogeneity in folliculogenic transcriptome.

Rat post-implantation epiblast-derived pluripotent stem cells produce functional germ cells.

In mammals, pluripotent cells transit through a continuum of distinct molecular and functional states en route to initiating lineage specification. Capturing pluripotent stem cells (PSCs) mirroring in vivo pluripotent states provides accessible in vitro models to study the pluripotency program and mechanisms underlying lineage restriction. Here, we develop optimal culture conditions to derive and propagate post-implantation epiblast-derived PSCs (EpiSCs) in rats, a valuable model for biomedical research. We show that rat EpiSCs (rEpiSCs) can be reset toward the naive pluripotent state with exogenous Klf4, albeit not with the other five candidate genes (Nanog, Klf2, Esrrb, Tfcp2l1, and Tbx3) effective in mice. Finally, we demonstrate that rat EpiSCs retain competency to produce authentic primordial germ cell-like cells that undergo functional gametogenesis leading to the birth of viable offspring. Our findings in the rat model uncover principles underpinning pluripotency and germline competency across species.

Conservation and divergence of canonical and non-canonical imprinting in murids.

BACKGROUND: Genomic imprinting affects gene expression in a parent-of-origin manner and has a profound impact on complex traits including growth and behavior. While the rat is widely used to model human pathophysiology, few imprinted genes have been identified in this murid. To systematically identify imprinted genes and genomic imprints in the rat, we use low input methods for genome-wide analyses of gene expression and DNA methylation to profile embryonic and extraembryonic tissues at allele-specific resolution. RESULTS: We identify 14 and 26 imprinted genes in these tissues, respectively, with 10 of these genes imprinted in both tissues. Comparative analyses with mouse reveal that orthologous imprinted gene expression and associated canonical DNA methylation imprints are conserved in the embryo proper of the Muridae family. However, only 3 paternally expressed imprinted genes are conserved in the extraembryonic tissue of murids, all of which are associated with non-canonical H3K27me3 imprints. The discovery of 8 novel non-canonical imprinted genes unique to the rat is consistent with more rapid evolution of extraembryonic imprinting. Meta-analysis of novel imprinted genes reveals multiple mechanisms by which species-specific imprinted expression may be established, including H3K27me3 deposition in the oocyte, the appearance of ZFP57 binding motifs, and the insertion of endogenous retroviral promoters. CONCLUSIONS: In summary, we provide an expanded list of imprinted loci in the rat, reveal the extent of conservation of imprinted gene expression, and identify potential mechanisms responsible for the evolution of species-specific imprinting.

Conditional knockdown of Nanog induces apoptotic cell death in mouse migrating primordial germ cells.

The pluripotency factor Nanog is expressed in peri-implantation embryos and primordial germ cells (PGCs). Nanog-deficient mouse embryos die soon after implantation. To explore the function of Nanog in germ cells, Nanog RNA was conditionally knocked down in vivo by shRNA. Nanog shRNA transgenic (NRi-Tg) mice were generated through the formation of germline chimeras with NRi-Tg embryonic stem cells. In E12.5 Cre-induced ER-Cre/NRi-Tg and TNAP-Cre/NRi-Tg double-transgenic embryos, the number of alkaline phosphatase-positive and SSEA1-positive PGCs decreased significantly. In the E9.5 and E10.5 migrating Nanog-knockdown PGCs, TUNEL-positive apoptotic cell death became prominent in vivo and in vitro, despite Oct4 expression. Single-cell microarray analysis of E10.5 Nanog-knockdown PGCs revealed significant up- and downregulation of a substantial number of genes, including Tial1, Id1 and Suz12. These data suggest that Nanog plays a key role in the proliferation and survival of migrating PGCs as a safeguard of the PGC-specific molecular network.

Functional primordial germ cell-like cells from pluripotent stem cells in rats.

The in vitro generation of germ cells from pluripotent stem cells (PSCs) can have a substantial effect on future reproductive medicine and animal breeding. A decade ago, in vitro gametogenesis was established in the mouse. However, induction of primordial germ cell-like cells (PGCLCs) to produce gametes has not been achieved in any other species. Here, we demonstrate the induction of functional PGCLCs from rat PSCs. We show that epiblast-like cells in floating aggregates form rat PGCLCs. The gonadal somatic cells support maturation and epigenetic reprogramming of the PGCLCs. When rat PGCLCs are transplanted into the seminiferous tubules of germline-less rats, functional spermatids-that is, those capable of siring viable offspring-are generated. Insights from our rat model will elucidate conserved and divergent mechanisms essential for the broad applicability of in vitro gametogenesis.

Filopodium-derived vesicles produced by MIM enhance the migration of recipient cells.

Extracellular vesicles (EVs) are classified as large EVs (l-EVs, or microvesicles) and small EVs (s-EVs, or exosomes). S-EVs are thought to be generated from endosomes through a process that mainly depends on the ESCRT protein complex, including ALG-2 interacting protein X (ALIX). However, the mechanisms of l-EV generation from the plasma membrane have not been identified. Membrane curvatures are generated by the bin-amphiphysin-rvs (BAR) family proteins, among which the inverse BAR (I-BAR) proteins are involved in filopodial protrusions. Here, we show that the I-BAR proteins, including missing in metastasis (MIM), generate l-EVs by scission of filopodia. Interestingly, MIM-containing l-EV production was promoted by in vivo equivalent external forces and by the suppression of ALIX, suggesting an alternative mechanism of vesicle formation to s-EVs. The MIM-dependent l-EVs contained lysophospholipids and proteins, including IRS4 and Rac1, which stimulated the migration of recipient cells through lamellipodia formation. Thus, these filopodia-dependent l-EVs, which we named as filopodia-derived vesicles (FDVs), modify cellular behavior.