Primordial germ cell specification: a context-dependent cellular differentiation event [corrected].

During embryonic development, the foundation of the germline is laid by the specification of primordial germ cells (PGCs) from the postimplantation epiblast via bone morphogenetic protein (BMP) and WNT signalling. While the majority of epiblast cells undergo differentiation towards somatic cell lineages, PGCs initiate a unique cellular programme driven by the cooperation of the transcription factors BLIMP1, PRDM14 and AP2γ. These factors synergistically suppress the ongoing somatic differentiation and drive the re-expression of pluripotency and germ cell-specific genes accompanied by global epigenetic changes. However, an unresolved question is how postimplantation epiblast cells acquire the developmental competence for the PGC fate downstream of BMP/WNT signalling. One emerging concept is that transcriptional enhancers might play a central role in the establishment of developmental competence and the execution of cell fate determination. Here, we discuss recent advances on the specification and reprogramming of PGCs thereby highlighting the concept of enhancer function.U.G. is supported by a Marie Curie Intra-European fellowship. E.M. is supported by the Icelandic Research Fund. M.A.S. is supported by the Wellcome Trust (WT096738).This is the final version. It was first published by Royal Society Publishing at http://rstb.royalsocietypublishing.org/content/369/1657/2013054

PRMT5 protects genomic integrity during global DNA demethylation in primordial germ cells and preimplantation embryos.

Primordial germ cells (PGCs) and preimplantation embryos undergo epigenetic reprogramming, which includes comprehensive DNA demethylation. We found that PRMT5, an arginine methyltransferase, translocates from the cytoplasm to the nucleus during this process. Here we show that conditional loss of PRMT5 in early PGCs causes complete male and female sterility, preceded by the upregulation of LINE1 and IAP transposons as well as activation of a DNA damage response. Similarly, loss of maternal-zygotic PRMT5 also leads to IAP upregulation. PRMT5 is necessary for the repressive H2A/H4R3me2s chromatin modification on LINE1 and IAP transposons in PGCs, directly implicating this modification in transposon silencing during DNA hypomethylation. PRMT5 translocates back to the cytoplasm subsequently, to participate in the previously described PIWI-interacting RNA (piRNA) pathway that promotes transposon silencing via de novo DNA remethylation. Thus, PRMT5 is directly involved in genome defense during preimplantation development and in PGCs at the time of global DNA demethylation.U.G. was supported by a Marie Sk1odowska Curie Intra-European Fellowship. J.J.Z. was a recipient of a Wellcome Trust PhD Studentship (RG44593). This research was supported by grants from the Wellcome Trust to M.A.S. (WT096738).This is the final published version. It first appeared at http://www.cell.com/molecular-cell/abstract/S1097-2765%2814%2900787-4

NANOG alone induces germ cells in primed epiblast in vitro by activation of enhancers.

Nanog, a core pluripotency factor in the inner cell mass of blastocysts, is also expressed in unipotent primordial germ cells (PGCs) in mice, where its precise role is yet unclear. 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. 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. 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, 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.This is the author accepted manuscript. The final version is available from Nature Publishing Group via http://dx.doi.org/10.1038/nature1648

A genetic system to analyze the function of histones and histone variants in Drosophila melanogaster

Histone sind das Ziel von verschiedenen posttranslationalen chemischen Modifikationen, die für die Regulation der Genexpression eine Rolle spielen. Die Funktion der Histone jedoch konnte bislang nicht direkt in höheren Organismen untersucht werden, da es keine entsprechenden Histonmutanten gab. Dies lässt sich darauf zurückführen, dass die Histongene bei höheren Eukaryoten jeweils in hoher Kopienzahl und im Genom verteilt sind. Bei Drosophila melanogaster sind alle Histongene in einer distinkten Chromosomenregion, im sogenannten Histonkomplex, lokalisiert, der aus 23 sich wiederholenden Histongeneinheiten (His-GE) besteht, wobei jede Einheit für jeweils eine Kopie der Histongene codiert. Diese Genomorganisation ermöglichte es, den gesamten Histonkomplex zu deletieren und damit die erste definierte Histonnullmutation (HisC) in einem eukaryotischen Organismus herzustellen. HisC-mutante Embryonen, die über maternal eingelagerte Histon-mRNAs und Histonproteine verfügen, führen die ersten 14 embryonalen Zellzyklen aus. Danach sind die mutanten Zellen zwar noch in der Lage, die DNA-Replikation in der S-Phase 15 vollständig durchzuführen, arretieren dann aber im Zellzyklus. Die Ergebnisse lassen den Schluss zu, dass Histonneusynthese und DNA-Replikationsrate gekoppelte Prozesse sind, und dass es einen bislang unbekannten Mechanismus gibt, der während oder nach der DNA-Replikation die Nukleosomenassemblierung überwacht und gegebenenfalls einen Zellzyklusarrest bewirkt, der von der Repression der Phosphatase String abhängig ist. Die Ergebnisse zeigen auch, dass die HisC-Deletion durch transgene Histongene dosisabhängig komplementiert wird. Dieses Histondeletion/Transgen-System ermöglicht es, die Funktion von Histon-abhängigen Prozessen und die biologische Relevanz von Histonmodifikationen in einem multizellulären Organismus zu untersuchen und damit auch erstmalig die „Histoncode“-Hypothese kritisch zu testen.Histones are target of post-translational chemical modifications that modulate chromatin structure and control genome function. It was not possible, however, to directly assess the in vivo function of histones in higher eukaryotes, since corresponding histone mutants were not available. The lack of such histone mutants, and in particular a histone null mutant, is due to the reiteration and wide genomic distribution of histone genes in most of the higher organisms. In order to establish an experimental system that allows testing designed histone mutants, I made use of the unique histone gene organization in Drosophila melanogaster, where all histone genes are clustered at a single chromosomal site in the ‘histone complex’. Here I report the generation and characterization of a deletion that uncovers the histone complex, representing a histone null mutation (termed ‘HisC’). In HisC mutant embryos, the maternally derived histones are sufficient to drive the first 14 embryonic cell cycles. In the absence of zygotic histone expression, embryos fail to complete cell cycle 15 and die. The mutant cells are able to duplicate their DNA at very low rate in S phase 15 but they fail to enter the subsequent mitosis and arrest instead. The results suggest that histone supply determines the rate of S phase progression to ensure a balance between DNA replication and nucleosome assembly. In addition, the data imply a novel surveillance mechanism that is able to stall the cell cycle by repressing the expression of the phosphatase String when proper nucleosome assembly fails. Moreover, both cellular and embryonic lethality phenotypes could be rescued in a gene dose-dependent manner by transgenes carrying histone genes. This novel histone deletion/transgene system provides a unique tool to explore histone-based processes and to test the significance of histone modifications in terms of the proposed ‘histone code’ in a multicellular organism

Developmental Competence for Primordial Germ Cell Fate.

During mammalian embryonic development, the trophectoderm and primitive endoderm give rise to extraembryonic tissues, while the epiblast differentiates into all somatic lineages and the germline. Remarkably, only a few classes of signaling pathways induce the differentiation of these progenitor cells into diverse lineages. Accordingly, the functional outcome of a particular signal depends on the developmental competence of the target cells. Thus, developmental competence can be defined as the ability of a cell to integrate intrinsic and extrinsic cues to execute a specific developmental program toward a specific cell fate. Downstream of signaling, there is the combinatorial activity of transcription factors and their cofactors, which is modulated by the chromatin state of the target cells. Here, we discuss the concept of developmental competence, and the factors that regulate this state with reference to the specification of mammalian primordial germ cells

Bällchen participates in proliferation control and prevents the differentiation of Drosophila melanogaster neuronal stem cells

Stem cells continuously generate differentiating daughter cells and are essential for tissue homeostasis and development. Their capacity to self-renew as undifferentiated and actively dividing cells is controlled by either external signals from a cellular environment, the stem cell niche, or asymmetric distribution of cell fate determinants during cell division. Here we report that the protein kinase Bällchen (BALL) is required to prevent differentiation as well as to maintain normal proliferation of neuronal stem cells of Drosophila melanogaster, called neuroblasts. Our results show that the brains of ball mutant larvae are severely reduced in size, which is caused by a reduced proliferation rate of the neuroblasts. Moreover, ball mutant neuroblasts gradually lose the expression of the neuroblast determinants Miranda and aPKC, suggesting their premature differentiation. Our results indicate that BALL represents a novel cell intrinsic factor with a dual function regulating the proliferative capacity and the differentiation status of neuronal stem cells during development