Yukihiro Yabuta

Critical function of Prdm14 for the establishment of the germ cell lineage in mice

Specification of germ cell fate is fundamental in development and heredity. Recent evidence indicates that in mice, specification of primordial germ cells (PGCs), the common source of both oocytes and spermatozoa, occurs through the integration of three key events: repression of the somatic program, reacquisition of potential pluripotency and ensuing genome-wide epigenetic reprogramming. Here we provide genetic evidence that Prdm14, a PR domain–containing transcriptional regulator with exclusive expression in the germ cell lineage and pluripotent cell lines, is critical in two of these events, the reacquisition of potential pluripotency and successful epigenetic reprogramming. In Prdm14 mutants, the failure of these two events manifests even in the presence of Prdm1 (also known as Blimp1), a key transcriptional regulator for PGC specification. Our combined evidence demonstrates that Prdm14 defines a previously unknown genetic pathway, initiating independently from Prdm1, for ensuring the launching of the mammalian germ cell lineage.

Cellular dynamics associated with the genome-wide epigenetic reprogramming in migrating primordial germ cells in mice

We previously reported that primordial germ cells (PGCs) in mice erase genome-wide DNA methylation and histone H3 lysine9 dimethylation (H3K9me2), and instead acquire high levels of tri-methylation of H3K27 (H3K27me3) during their migration, a process that might be crucial for the re-establishment of potential totipotency in the germline. We here explored a cellular dynamics associated with this epigenetic reprogramming. We found that PGCs undergo erasure of H3K9me2 and upregulation of H3K27me3 in a progressive, cell-by-cell manner, presumably depending on their developmental maturation. Before or concomitant with the onset of H3K9 demethylation, PGCs entered the G2 arrest of the cell cycle, which apparently persisted until they acquired high H3K27me3 levels. Interestingly, PGCs exhibited repression of RNA polymerase II-dependent transcription, which began after the onset of H3K9me2 reduction in the G2 phase and tapered off after the acquisition of high-level H3K27me3. The epigenetic reprogramming and transcriptional quiescence were independent from the function of Nanos3. We found that before H3K9 demethylation, PGCs exclusively repress an essential histone methyltransferase, GLP, without specifically upregulating histone demethylases. We suggest the possibility that active repression of an essential enzyme and subsequent unique cellular dynamics ensures successful implementation of genome-wide epigenetic reprogramming in migrating PGCs.

Complex genome-wide transcription dynamics orchestrated by Blimp1 for the specification of the germ cell lineage in mice

Specification of germ cell fate is fundamental in development. With a highly representative single-cell microarray and rigorous quantitative PCR analysis, we defined the genome-wide transcription dynamics that create primordial germ cells (PGCs) from the epiblast, a process that exclusively segregates them from their somatic neighbors. We also analyzed the effect of the loss of Blimp1, a key transcriptional regulator, on these dynamics. Our analysis revealed that PGC specification involves complex, yet highly ordered regulation of a large number of genes, proceeding under the strong influence of mesoderm induction but specifically avoiding developmental programs such as the epithelial-mesenchymal transition, Hox cluster activation, cell cycle progression, and DNA methyltransferase machinery. Remarkably, Blimp1 is essential for repressing nearly all the genes normally down-regulated in PGCs relative to their somatic neighbors. In contrast, it is dispensable for the activation of approximately half of the genes up-regulated in PGCs, uncovering the Blimp1-independent events for PGC specification. Notably, however, highly PGC-specific genes exhibited distinct correlations to Blimp1 in wild-type embryos, and these correlations faithfully predicted their expression impairments in Blimp1 mutants. Moreover, their expression overlaps within single cells were severely damaged without Blimp1, demonstrating that Blimp1 exerts positive influence on their concerted activation. Thus, Blimp1 is not a single initiator but a dominant coordinator of the transcriptional program for the establishment of the germ cell fate in mice.

Gene Expression Dynamics During Germline Specification in Mice Identified by Quantitative Single-Cell Gene Expression Profiling

Abstract Germ cell fate in mice is induced in proximal epiblast cells at Embryonic Day (E) 6.5 by signaling molecules. Prdm1(also known as Blimp1)-positive lineage-restricted precursors of primordial germ cells (PGCs) initiate the formation of a cluster that differentiates into Dppa3 (also known as stella)-positive PGCs from around E7.0 onwards in the extra-embryonic mesoderm. Around E7.5, these PGCs begin migrating towards the definitive endoderm, with concomitant extensive epigenetic reprogramming. To gain a more precise insight into the mechanism of PGC specification and its subsequent development, we exploited quantitative, single-cell, gene expression profiling to explore gene expression dynamics during the 36 h of PGC differentiation from E6.75 to E8.25, in comparison with the corresponding profiles of somatic neighbors. This analysis revealed that the transitions from Prdm1-positive PGC precursors to Dppa3-positive PGCs and to more advanced migrating PGCs involve a highly dynamic, stage-dependent transcriptional orchestration that begins with the regaining of the pluripotency-associated gene network, followed by stepwise activation of PGC-specific genes, differential repression of the somatic mesodermal program, as well as potential modulations of signal transduction capacities and unique control of epigenetic regulators. The information presented here regarding the cascade of events involved in PGC development should serve as a basis for detailed functional analyses of the gene products associated with this process, as well as for appropriate reconstitution of PGCs and their descendant cells in culture.

PRDM14 Ensures Naive Pluripotency through Dual Regulation of Signaling and Epigenetic Pathways in Mouse Embryonic Stem Cells

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.

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.

Global single-cell cDNA amplification to provide a template for representative high-density oligonucleotide microarray analysis

We describe here a protocol for the representative amplification of global mRNAs from typical single mammalian cells to provide a template for high-density oligonucleotide microarray analysis. A single cell is lysed in a tube without purification and first-strand cDNAs are synthesized using a poly(dT)-tailed primer. Unreacted primer is specifically eliminated by exonuclease treatment and second strands are generated with a second poly(dT)-tailed primer after poly(dA) tailing of the first-strand cDNAs. The cDNAs are split into four tubes, which are independently directionally amplified by PCR, and then recombined. The amplified products (∼100 ng) show superior representation and reproducibility of original gene expression, especially for genes expressed in more than 20 copies per cell, compared with those obtained by a conventional PCR protocol, and can effectively be used for quantitative PCR and EST analyses. The cDNAs are then subjected to another PCR amplification with primers bearing the T7 promoter sequence. The resultant cDNA products are gel purified, amplified by one final cycle and used for isothermal linear amplification by T7 RNA polymerase to synthesize cRNAs for microarray hybridization. This protocol yields cDNA templates sufficient for more than 80 microarray hybridizations from a single cell, and can be completed in 5–6 days.

Robust In Vitro Induction of Human Germ Cell Fate from Pluripotent Stem Cells.

Mechanisms underlying human germ cell development are unclear, partly due to difficulties in studying human embryos and lack of suitable experimental systems. Here, we show that human induced pluripotent stem cells (hiPSCs) differentiate into incipient mesoderm-like cells (iMeLCs), which robustly generate human primordial germ cell-like cells (hPGCLCs) that can be purified using the surface markers EpCAM and INTEGRINα6. The transcriptomes of hPGCLCs and primordial germ cells (PGCs) isolated from non-human primates are similar, and although specification of hPGCLCs and mouse PGCs rely on similar signaling pathways, hPGCLC specification transcriptionally activates germline fate without transiently inducing eminent somatic programs. This includes genes important for naive pluripotency and repression of key epigenetic modifiers, concomitant with epigenetic reprogramming. Accordingly, BLIMP1, which represses somatic programs in mice, activates and stabilizes a germline transcriptional circuit and represses a default neuronal differentiation program. Together, these findings provide a foundation for understanding and reconstituting human germ cell development in vitro.

A Mesodermal Factor, T, Specifies Mouse Germ Cell Fate by Directly Activating Germline Determinants

Germ cells ensure reproduction and heredity. In mice, primordial germ cells (PGCs), the precursors for spermatozoa and oocytes, are induced in pluripotent epiblast by BMP4 and WNT3, yet the underlying mechanism remains unclear. Here, using an in vitro PGC specification system, we show that WNT3 induces many transcription factors associated with mesoderm in epiblast-like cells through β-CATENIN. Among these, T (BRACHYURY), a classical and conserved mesodermal factor, was essential for robust activation of Blimp1 and Prdm14, two of the germline determinants. T, but not SMAD1 or TCF1, binds distinct regulatory elements of both Blimp1 and Prdm14 and directly upregulates these genes, delineating the downstream PGC program. Without BMP4, a program induced by WNT3 prevents T from activating Blimp1 and Prdm14, demonstrating a permissive role of BMP4 in PGC specification. These findings establish the key signaling mechanism for, and a fundamental role of a mesodermal factor in, mammalian PGC specification.

TDRD5 is required for retrotransposon silencing, chromatoid body assembly, and spermiogenesis in mice

Tdrd5-deficient mice develop a functional haploid genome despite spermiogenesis arrest at the round spermatid stage.