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Nanog, a core pluripotency factor in the inner cell mass of blastocysts, is also expressed in unipotent primordial germ cells (PGCs) in mice1, where its precise role is yet unclear (2-4). 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 (5). 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 (6-8). 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 (9,10), 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., Transcription factors and epigenetic changes confer competence for somatic and PGC fates when naive pluripotent inner cell mass (ICM) from embryonic day (E)3.5-4.5 blastocysts develop to primed epiblast at ~E6.0 [...]

The rarity and inaccessibility of the earliest primordial germ cells (PGCs) in the mouse embryo thwart efforts to investigate molecular mechanisms of germ-cell specification. stella (also called Dppa3) marks the rare founder population of the germ lineage (1,2). Here we differentiate mouse embryonic stem cells carrying a stella-transgenic reporter into putative PGCs in vitro. The [Stella.sup.+] cells possess a transcriptional profile similar to embryo-derived PGCs, and like their counterparts in vivo, lose imprints in a time-dependent manner. Using inhibitory RNAs to screen candidate genes for effects on the development of [Stella.sup.+] cells in vitro, we discovered that Lin28, a negative regulator of let-7 microRNA processing (3-6), is essential for proper PGC development. Furthermore, we show that Blimp1 (also called Prdm1), a let-7 target and a master regulator of PGC specification (7-9), can rescue the effect of Lin28 deficiency during PGC development, thereby establishing a mechanism of action for Lin28 during PGC specification. Overexpression of Lin28 promotes formation of [Stella.sup.+] cells in vitro and PGCs in chimaeric embryos, and is associated with human germ-cell tumours. The differentiation of putative PGCs from embryonic stem cells in vitro recapitulates the early stages of gamete development in vivo, and provides an accessible system for discovering novel genes involved in germ-cell development and malignancy., The transcriptional program responsible for specifying PGCs has been investigated through molecular analysis of cDNA created from single cells (10,11), but because transgenic or knockout methods required to verify roles [...]

RNA interference (RNAi) is a mechanism by which double-stranded RNAs (dsRNAs) suppress specific transcripts in a sequence-dependent manner. dsRNAs are processed by Dicer to 21-24-nucleotide small interfering RNAs (siRNAs) and [...]

Author(s): Toshihiro Kobayashi [1, 2]; Haixin Zhang [3]; Walfred W. C. Tang [1, 2]; Naoko Irie [1, 2]; Sarah Withey [3]; Doris Klisch [3]; Anastasiya Sybirna [1, 2, 4]; Sabine [...]

Author(s): Yasuhide Ohinata [1, 7]; Bernhard Payer [2, 7]; Dónal O'Carroll [3, 7]; Katia Ancelin [2]; Yukiko Ono [1]; Mitsue Sano [1]; Sheila C. Barton [2]; Tetyana Obukhanych [4]; Michel [...]

Author(s): Mitinori Saitou; Sheila C. Barton; M. Azim Surani (corresponding author) August Weismann proposed that embryonic cells that inherit preformed germ plasm acquire germ cell fate while the rest become [...]

Author(s): M. Azim Surani (corresponding author) Development is a remarkably orderly process -- it begins with a totipotent zygote and ends with an array of specific, differentiated cell types in [...]

Author(s): Andreas P. Russ (corresponding author) [1, 2, 3]; Sigrid Wattler [4]; William H. Colledge [2]; Samuel A. J. R. Aparicio [1, 3, 6]; Mark B. L. Carlton [1, 2, [...]

Parental imprinting in the expression of the H19 gene in mouse embryos was examined. In normal embryos, the H190 gene expresses a developmentally regulated product of unknown function. The gene lies on chromosome 7 and is susceptible to trancriptional regulation by parental imprinting, causing the expression of the maternally inherited gene and suppression of the paternally inherited gene. The study protocol comparison of CpG methylation and nuclease sensitivity of chromatin in MatDi7 embryos with those of normal littermates. The results that the H19 promoter gene region is hypermethylated only in the paternal allele.

Pluripotent cells have the potential to differentiate into any cell type in the body. Their isolation and propagation from mouse embryos was pivotal for advances in understanding human development and [...]

An analysis of methylation effects on active parental gene selection explains the theory that strategic DNA methylation, a molecular marker, is necessary for mutant mammal survival. The test genes, Igf-2r, Igf-2 and H19, lose the molecular imprints that allow parent recognition when subjected to impaired DNA methylation. The imprints' presence implies that the gene containing the imprint is an active, paternal or maternal copy.

The pluripotent state, which is first established in the primitive ectoderm cells of blastocysts, is lost progressively and irreversibly during subsequent development. For example, development of post-implantation epiblast cells from primitive ectoderm involves significant transcriptional and epigenetic changes, including DNA methylation and X chromosome inactivation, 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 and that differ from embryonic stem (ES) cells derived from primitive ectoderm. 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. [ABSTRACT FROM AUTHOR]