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In humans, germline competency and the specification of primordial germ cells (PGCs) are thought to occur in a restricted developmental window during early embryogenesis. Despite the importance of specifying the appropriate number of PGCs for human reproduction, the molecular mechanisms governing PGC formation remain largely unexplored. Here, we compared PGC-like cell (PGCLC) differentiation from 18 independently derived human embryonic stem cell (hESC) lines, and discovered that the expression of primitive streak genes were positively associated with hESC germline competency. Furthermore, we show that chemical inhibition of TGFβ and WNT signaling, which are required for primitive streak formation and CRISPR/Cas9 deletion of Eomesodermin (EOMES), significantly impacts PGCLC differentiation from hESCs. Taken together, our results suggest that human PGC formation involves signaling and transcriptional programs associated with somatic germ layer induction and expression of EOMES.

DNA methyltransferases and Ten‐Eleven Translocation (TET) proteins regulate the DNA methylation and demethylation cycles during mouse embryonic development. Although DNMT1 mainly plays a role in the maintenance of DNA methylation after DNA replication, it is also reported to possess de novo methyltransferase capacity. However, its physiological significance remains unclear. Here, we demonstrate that full‐length DNMT1 (FL) and a mutant lacking the N‐terminus necessary for its maintenance activity (602) confer the differentiation potential of mouse Dnmt1, Dnmt3a, and Dnmt3b (Dnmts‐TKO) embryonic stem cells (ESCs). Both FL and 602 inhibit the spontaneous differentiation of Dnmts‐TKO ESCs in the undifferentiated state. Dnmts‐TKO ESCs showed loss of DNA methylation and de‐repression of primitive endoderm‐related genes, but these defects were partially restored in Dnmts‐TKO + FL and Dnmts‐TKO + 602 ESCs. Upon differentiation, Dnmts‐TKO + FL ESCs show increased 5mC and 5hmC levels across chromosomes, including pericentromeric regions. In contrast, Dnmts‐TKO + 602 ESCs didn't accumulate 5mC, and sister chromatids showed 5hmC asynchronously. Furthermore, in comparison with DNMT1_602, DNMT1_FL effectively promoted commitment to the epiblast‐like cells and beyond, driving cell‐autonomous mesendodermal and germline differentiation through embryoid body‐based methods. With precise target selectivity achieved by its N‐terminal region, DNMT1 may play a role in gene regulation leading to germline development. [ABSTRACT FROM AUTHOR]

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 [...]

Genome-wide active DNA demethylation in primordial germ celts (PGCs), which reprograms the epigenome for totipotency, is linked to changes in nuclear architecture, loss of histone modifications, and widespread histone replacement. Here, we show that DNA demethylation in the mouse PGCs is mechanistica|ly linked to the appearance of single-stranded DNA (ssDNA) breaks and the activation of the base excision repair (BER) pathway, as is the case in the zygote where the paternal pronucleus undergoes active DNA demethylation shortly after fertilization. Whereas BER might be triggered by deamination of a methylcytosine (5mC), cumulative evidence indicates other mechanisms in germ cells. We demonstrate that DNA repair through BER represents a core component of genome-wide DNA demethylation in vivo and provides a mechanistic link to the extensive chromatin remodeling in developing PGCs. 10.1126/science.1187945

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 [...]