This study is a preliminary and experimental one to analyze Japan’s energy transitions to mitigate climate change from anticipatory governance aspects. Japan’s energy policy principles have been ...energy security, environmental considerations, economic efficiency, and safety (3E + S). According to the energy agency, the long-term energy outlook is also drawn up by “ambitious multiple track scenarios” and “multilayered and diversified flexible energy supply-demand structure.” This approach resonates with the aspects of anticipatory governance. It promotes the idea of preparing for multiple future scenarios, including the unthinkable worst case future scenario such as a nuclear accident (foresight), the interactions between the policymakers and the public (engagement), and the reflexive processes of policy innovations with a normative decision for the selection of energy mix (integration). However, this study finds that Japan’s energy policy lacks the aspects of anticipatory governance. It sticks to fixed energy policy institutionalized in the 1970s to promote nuclear energy and coal as oil alternatives. It rarely has interactions between the policymakers and the public and thus lacks a societal (normative) decision about a future energy path to energy transitions to mitigate climate change. Instead, Japan’s energy policy has not necessarily met its declared policy objective of 3E + S since the unprecedented Fukushima nuclear accidents occurred and cannot uphold an ambitious target for CO2 emissions reduction.
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-β3 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.
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► Generation of primordial germ cell-like cells (PGCLCs) from ESCs and iPSCs ► PGCLC induction occurs via an epiblast-like state, mimicking PGC generation in vivo ► PGCLCs contribute to spermatogenesis and produce fertile offspring ► Integrin-β3 and SSEA1 mark PGCLCs and allow purification from teratogenic cells
Reconstitution of female germ cell development in vitro is a key challenge in reproductive biology and medicine. We show here that female (XX) embryonic stem cells and induced pluripotent stem cells ...in mice are induced into primordial germ cell-like cells (PGCLCs), which, when aggregated with female gonadal somatic cells as reconstituted ovaries, undergo X-reactivation, imprint erasure, and cyst formation, and exhibit meiotic potential. Upon transplantation under mouse ovarian bursa, PGCLCs in the reconstituted ovaries mature into germinal vesicle-stage oocytes, which then contribute to fertile offspring after in vitro maturation and fertilization. Our culture system serves as a robust foundation for the investigation of key properties of female germ cells, including the acquisition of totipotency, and for the reconstitution of whole female germ cell development 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.
Germ cell specification is accompanied by epigenetic remodeling, the scale and specificity of which are unclear. Here, we quantitatively delineate chromatin dynamics during induction of mouse ...embryonic stem cells (ESCs) to epiblast-like cells (EpiLCs) and from there into primordial germ cell-like cells (PGCLCs), revealing large-scale reorganization of chromatin signatures including H3K27me3 and H3K9me2 patterns. EpiLCs contain abundant bivalent gene promoters characterized by low H3K27me3, indicating a state primed for differentiation. PGCLCs initially lose H3K4me3 from many bivalent genes but subsequently regain this mark with concomitant upregulation of H3K27me3, particularly at developmental regulatory genes. PGCLCs progressively lose H3K9me2, including at lamina-associated perinuclear heterochromatin, resulting in changes in nuclear architecture. T recruits H3K27ac to activate BLIMP1 and early mesodermal programs during PGCLC specification, which is followed by BLIMP1-mediated repression of a broad range of targets, possibly through recruitment and spreading of H3K27me3. These findings provide a foundation for reconstructing regulatory networks of the germline epigenome.
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•Chromatin dynamics were quantitatively assessed during PCG specification•EpiLCs represent a primed state with abundant bivalency with low H3K27me3•PGCLCs deplete H3K9me2 throughout the genome including from lamina-associated domains•BLIMP1 serves as a potential nucleator for H3K27me3 accumulation and spread
Kurimoto et al. perform careful analyses of chromatin remodeling during mouse germ cell specification from embryonic stem cells. Widespread epigenetic reprogramming events included re-organization of H3K27me3 and bivalent signatures as well as progressive deletion of H3K9me2 throughout the genome, creating a unique foundation for the epigenome of the next generation.
Mammalian male germ-cell development consists of three distinct phases: primordial germ cell (PGC) development, male germ-cell specification for spermatogonium development, and ensuing ...spermatogenesis. Here, we show an in vitro reconstitution of whole male germ-cell development by pluripotent stem cells (PSCs). Mouse embryonic stem cells (mESCs) are induced into PGC-like cells (mPGCLCs), which are expanded for epigenetic reprogramming. In reconstituted testes under an optimized condition, such mPGCLCs differentiate into spermatogonium-like cells with proper developmental transitions, gene expression, and cell-cycle dynamics and are expanded robustly as germline stem cell-like cells (GSCLCs) with an appropriate androgenetic epigenome. Importantly, GSCLCs show vigorous spermatogenesis, not only upon transplantation into testes in vivo but also under an in vitro culture of testis transplants, and the resultant spermatids contribute to fertile offspring. By uniting faithful recapitulations of the three phases of male germ-cell development, our study creates a paradigm for the in vitro male gametogenesis by PSCs.
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•An optimized strategy for generating spermatogonium-like cells from mouse PSCs•A faithful reconstitution for male germ-cell specification for spermatogonial development•PSC-derived germline stem cells for robust spermatogenesis in vivo and in vitro•A paradigm for the in vitro reconstitution of whole male germ-cell development
Male germ-cell development is a complex process that leads to the generation of haploid male gametes, the spermatozoa. Using the mouse as a model, Ishikura and colleagues establish a strategy to create functional spermatozoa from pluripotent stem cells in vitro by reconstituting entire male germ-cell development in a stepwise manner.
The mechanism for sex determination in mammalian germ cells remains unclear. Here, we reconstitute the female sex determination in mouse germ cells in vitro under a defined condition without the use ...of gonadal somatic cells. We show that retinoic acid (RA) and its key effector, STRA8, are not sufficient to induce the female germ‐cell fate. In contrast, bone morphogenetic protein (BMP) and RA synergistically induce primordial germ cells (PGCs)/PGC‐like cells (PGCLCs) derived from embryonic stem cells (ESCs) into fetal primary oocytes. The induction is characterized by entry into the meiotic prophase, occurs synchronously and recapitulates cytological and transcriptome progression in vivo faithfully. Importantly, the female germ‐cell induction necessitates a proper cellular competence—most typically, DNA demethylation of relevant genes—which is observed in appropriately propagated PGCs/PGCLCs, but not in PGCs/PGCLCs immediately after induction. This provides an explanation for the differential function of BMP signaling between PGC specification and female germ‐cell induction. Our findings represent a framework for a comprehensive delineation of the sex‐determination pathway in mammalian germ cells, including humans.
Synopsis
In vitro reconstitution of female sex determination using ESC‐derived germ cells demonstrates requirement of integrated signaling inputs and epigenetic background for fetal oocyte induction.
Female mouse germ‐cell sex specification is reconstituted under a defined set of conditions.
Retinoic acid (RA) and its effector STRA8 are not sufficient to induce the fetal oocyte phenotype from ESC‐derived primordial germ cells.
Bone morphogenetic protein (BMP) and RA act synergistically to instruct female germ‐cell fate.
Cellular competence for acquiring female germ‐cell fate includes DNA demethylation of key genes.
In vitro reconstitution of female sex determination using ESC‐derived germ cells demonstrates requirement of integrated signaling and epigenetic background for fetal oocyte induction.
Specification of the germ cell lineage is vital to development and heredity. In mice, the germ cell fate is induced in pluripotent epiblast cells by signaling molecules, yet the underlying mechanism ...remains unknown. Here we demonstrate that germ cell fate in the epiblast is a direct consequence of Bmp4 signaling from the extraembryonic ectoderm (ExE), which is antagonized by the anterior visceral endoderm (AVE). Strikingly, Bmp8b from the ExE restricts AVE development, thereby contributing to Bmp4 signaling. Furthermore, Wnt3 in the epiblast ensures its responsiveness to Bmp4. Serum-free, defined cultures revealed that, in response to Bmp4, competent epiblast cells uniformly expressed key transcriptional regulators Blimp1 and Prdm14 and acquired germ-cell properties, including genome-wide epigenetic reprogramming, in an orderly fashion. Notably, the induced cells contributed to both spermatogenesis and fertility of offspring. By identifying a signaling principle in germ cell specification, our study establishes a robust strategy for reconstituting the mammalian germ cell lineage in vitro.
In serum, mouse embryonic stem cells (mESCs) fluctuate between a naive inner cell mass (ICM)-like state and a primed epiblast-like state, but when cultured with inhibitors of the mitogen-activated ...protein kinase (MAPK) and glycogen synthase kinase 3 pathways (2i), they are harnessed exclusively in a distinct naive pluropotent state, the ground state, that more faithfully recapitulates the ICM. Understanding the mechanism underlying this naive pluripotent state will be critical for realizing the full potential of ESCs. We show here that PRDM14, a PR-domain-containing transcriptional regulator, ensures naive pluripotency through a dual mechanism: antagonizing activation of the fibroblast growth factor receptor (FGFR) signaling by the core pluripotency transcriptional circuitry, and repressing expression of de novo DNA methyltransferases that modify the epigenome to a primed epiblast-like state. PRDM14 exerts these effects by recruiting polycomb repressive complex 2 (PRC2) specifically to key targets and repressing their expression.
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► Prdm14 expression is important for maintenance of naive pluripotency ► PRDM14 antagonizes FGFR signaling and activates Akt-mTORC1 signaling ► PRDM14 represses de novo DNA methyltransferase expression ► This regulatory input is mediated through recruitment of PRC2
PRDM14 targets FGF signaling, Akt-mTORC1 signaling, and DNA methyltransferases through recruitment of PRC2 to hold ESCs in a naive pluripotent state.
Background
Two‐dimensional shear wave elastography (2D‐SWE) provides information on hepatic elastic modulus as shear wave velocity (SWV).
Hypothesis/Objectives
To assess SWV using 2D‐SWE in dogs with ...induced volume overload, investigate the relationship between this information and right atrial pressure (RAP) measured by invasive right heart catheterization, and also evaluate the difference in SWV before and after diuretic administration.
Animals
Six healthy beagles.
Methods
Prospective experimental study. Right heart catheterization and 2D‐SWE were performed in 6 anesthetized beagles at baseline and after the induction of volume overload. Volume overload was induced by IV hydroxyethyl starch 70/0.5 infusion (100 mL/kg/h). Furosemide (4‐6 mg/kg, IV) was administered, and the SWVs were measured.
Results
Shear wave velocity showed a significant gradual increase during acute volume overload compared to baseline. SWV was significantly positively correlated with RAP (P < .0001, ρ = 0.9729). The area under the curve of SWV to predict RAP at >10, >15, and >20 mm Hg was 0.9896 (95% confidence interval 95% CI, 0.9690‐1.000), 0.9907 (95% CI, 0.9701‐1.000), and 0.9722 (95% CI, 0.9280‐1.000), respectively. The SWV after diuretic use decreased significantly.
Conclusions and Clinical Importance
Two‐dimensional shear wave elastography might be useful for noninvasive and reliable estimation of RAP in dogs with acute volume overload and has potential as a quantitative biomarker for evaluating therapeutic response in dogs with right sided congestive heart failure.
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