PMC

Changes in chromatin structure may be seen as prerequisite for the DNA demethylation process (accessibility model.) Alternatively, DNA demethylation in PGCs can occur via DNA repair pathway as described in plants ^{, ,} . The resulting DNA damage and repair could potentially induce chromatin remodelling, which would occur as a consequence of DNA demethylation (for details see the text).

A) Dissapearance of linker histone H1 in PGCs at E11.5. PGC7/Stella (in red) was used as germ cell specific marker. PGCs are depicted by arrows. B) Increase in the nuclear size and disappearance of chromocenters in PGCs at E11.5 as observed by DAPI staining of cell suspension from genital ridges. PGCs are depicted by arrows. The lower panel shows identification of PGCs by staining with germ cell specific marker (SSEA1 or Oct4). Scale bars: 10μm. C) Measurement of the nuclear size on the cryosections from E10.5-E13.5 genital ridges shows transient change in the size of germ cell nuclei. The increase is highly statistically significant using t-test. The measured PGC nuclei were identified on the cryosections using Oct4 stainings (data not shown).

A) Transient appearance of two populations of GFP expressing PGCs at E11.5. The X and Y axis represent the FSC (forward scatter) and GFP intensity values, respectively. The lower panels show the profiles of the GFP (germ cell specific) signal. Note the appearance of two distinct peaks at E11.5. B) Separation and analysis of chromatin configuration of the two populations of PGC, denoted as A and B, at E11.5. The population B is more advanced and shows loss of histone H1 and H2A.Z. Note the cytoplasmic localisation of CAF-1 in these populations and relocalisation of NAP1 from the cytoplasm to the nucleus in population B (for details see the text). Cells shown here are representative of the populations. Between 50 and 100 PGCs were examined for each population. Scale bar: 10μm. C) Analysis of DNA methylation of peg3 and lit1 (kcnqtot1) DMRs in dinstict populations of E11.5 PGCs using bisulphite genomic sequencing. Each line represents an independent clone. Open and closed circles represent non-methylated and methylated CpGs, respectively.

A) Presence of H3K27me3 in PGCs. At E10.5 nuclei of PGCs contain high levels of H3K27me3 as part of their germline chromatin signature (for details see text). This chromatin mark is lost in PGCs at E11.5 and re-gained between E12.5 and E13.5. Notably the loss of H3K27me3 occurs despite the continuous presence of Ezh2 responsible for this modification in PGCs (data not shown). B) Loss of H3K9me3 in PGCs at E11.5. Stainings were performed on single cell suspensions from genital ridges between E10.5-E13.5. The shown epigenetic changes are detectable in 70-90% of PGCs depending on the developmental stage of the embryo. Germ cells were stained in green using the germ cell specific markers SSEA1 and Oct4, respectively, and are depicted by arrows. Scale bar: 10μm. C) The immunofluorescence stainings of cryosections of embryonic gonads (as described in Methods section). Oct4 positive germ cells (depicted by arrows) are stained in green. Note high levels of H2A.Z in early postmigratory PGCs at E10.5 and the disappearance of the H2A.Z staining in later developmental stages (E11.5 and E12.5), while the signal in surrounding somatic cells remains relatively constant. Scale bar: 10μm. D) Quantification of the H2A.Z signal in germ cells. In order to normalize between different samples, the intensity of H2A.Z staining was calculated as a mean pixel intensity in every PGC divided by the mean pixel intensity in surrounding somatic cells. The Y axis values thus represent the intensity of H2A.Z staining in PGCs in comparison with surrounding somatic cells.