X chromosome activity in mouse XX primordial germ cells.

In the early epiblast of female mice, one of the two X chromosomes is randomly inactivated by a Xist-dependent mechanism, involving the recruitment of Ezh2-Eed and the subsequent trimethylation of histone 3 on lysine 27 (H3K27me3). We demonstrate that this random inactivation process applies also to the primordial germ cell (PGC) precursors, located in the proximal region of the epiblast. PGC specification occurs at about embryonic day (E)7.5, in the extraembryonic mesoderm, after which the germ cells enter the endoderm of the invaginating hindgut. As they migrate towards the site of the future gonads, the XX PGCs gradually lose the H3K27me3 accumulation on the silent X chromosome. However, using a GFP transgene inserted into the X chromosome, we observed that the XX gonadal environment (independently of the gender) is important for the substantial reactivation of the inactive X chromosome between E11.5 and E13.5, but is not required for X-chromosome reactivation during the derivation of pluripotent embryonic germ cells. We describe in detail one of the key events during female PGC development, the epigenetic reprogramming of the X chromosome, and demonstrate the role of the XX somatic genital ridge in this process.

Generation of primordial germ cells from pluripotent stem cells.

Embryonic stem (ES) cells, derived from pre-implantation embryo, embryonic germ (EG) cells, derived from embryonic precursors of gametes, primordial germ cells (PGCs), can differentiate into any cell type in the body. Moreover, ES cells have the capacity to differentiate into PGCs in vitro. In the present study we have shown the differentiation capacity of six EG cell lines to form PGCs in vitro, in comparison to ES cells. Cell lines were differentiated via embryoid body (EB) formation using the co-expression of mouse vasa homolog (Mvh) and Oct-4 to identify newly formed PGCs in vitro. We found an increase of PGC numbers in almost all analysed cell lines in 5-day-old EBs, thus suggesting that EG and ES cells have similar efficiency to generate PGCs. The addition of retinoic acid confirmed that the cultures had attained a PGC-like identity and continued to proliferate. Furthermore we have shown that the expression pattern of Prmt5 and H3K27me3 in newly formed PGCs is similar to that observed in embryonic day E11.5 PGCs in vivo. By co-culturing EBs with Chinese hamster ovary (CHO) cells some of the PGCs entered into meiosis, as judged by Scp3 expression. The derivation of germ cells from pluripotent stem cells in vitro could provide an invaluable model system to study both the genetic and epigenetic programming of germ cell development in vivo.

Heterogeneity in imprinted methylation patterns of pluripotent embryonic germ cells derived from pre-migratory mouse germ cells.

Pluripotent stem cells, termed embryonic germ (EG) cells, have been generated from both human and mouse primordial germ cells (PGCs). Like embryonic stem (ES) cells, EG cells have the potential to differentiate into all germ layer derivatives and may also be important for any future clinical applications. The development of PGCs in vivo is accompanied by major epigenetic changes including DNA demethylation and imprint erasure. We have investigated the DNA methylation pattern of several imprinted genes and repetitive elements in mouse EG cell lines before and after differentiation. Analysed cell lines were derived soon after PGC specification, "early", in comparison with EG cells derived after PGC colonisation of the genital ridge, "late" and embryonic stem (ES) cell lines, derived from the inner cell mass (ICM). Early EG cell lines showed strikingly heterogeneous DNA methylation patterns, in contrast to the uniformity of methylation pattern seen in somatic cells (control), late EG cell and ES cell lines. We also observed that all analysed XX cell lines exhibited less methylation than XY. We suggest that this heterogeneity may reflect the changes in DNA methylation taking place in the germ cell lineage soon after specification.

DNA methyltransferase expression in the mouse germ line during periods of de novo methylation.

DNA methyltransferase (DNMT) 3A and DNMT3B are both active de novo DNA methyltransferases required for development, whereas DNMT3L, which has no demonstrable methyltransferase activity, is required for methylation of imprinted genes in the oocyte. We show here that different mechanisms are used to restrict access by these proteins to their targets during germ cell development. Transcriptional control of the Dnmt3l promoter guarantees that message is low or absent except during periods of de novo activity. Use of an alternative promoter at the Dnmt3a locus produces the shorter Dnmt3a2 transcript in the germ line and postimplantation embryo only, whereas alternative splicing of the Dnmt3b transcript ensures that Dnmt3b1 is absent in the male prospermatogonia. Control of subcellular protein localization is a common theme for DNMT3A and DNMT3B, as proteins were seen in the nucleus only when methylation was occurring. These mechanisms converge to ensure that the only time that functional products from each locus are present in the germ cell nuclei is around embryonic day 17.5 in males and after birth in the growing oocytes in females.