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X chromosome activity in mouse XX primordial germ cells.

Chuva de Sousa Lopes SM, Hayashi K, Shovlin TC, Mifsud W, Surani MA, McLaren A - PLoS Genet. (2008)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: The Wellcome Trust/Cancer Research UK Gurdon Institute, University of Cambridge, Cambridge, United Kingdom. smc80@cam.ac.uk

ABSTRACT
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.

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Xi Reactivates in XX PGCs between E11.5 and E13.5(A,B) FACS-analysis of XX E13.5 WT (A) and ΔPEOct4:gfp (B) PGCs show that anti-PECAM1 is a suitable antibody to separate PGCs from the surrounding somatic tissue. Anti-SSEA1 was used to separate XX E11.5 and E12.5 PGCs from the surrounding somatic tissue.(C) FACS-analysis of XX E13.5 XGFP homozygous genital ridges containing 100% GFP-positive cells, used as positive control.(D–F) Representative dot-plots showing FACS-analysis of XX E11.5 (D), E12.5 (E), and E13.5 (F) genital ridges from individual Xp-XGFP embryos.(G,H) The percentage of FACS-analysed GFP-expressing PGCs (G) and surrounding somatic tissue (H) in the genital ridges of individual XX E11.5, E12.5, and E13.5 Xp-XGFP embryos. PGCs and somatic cells were respectively positive and negative for SSEA1 (E11.5, 12.5) or PECAM1 (E13.5). Red bars depict the median, n is the total number of embryos analysed.(I) Transcriptional levels of X-coded genes and autosomal genes at E9.5, E11.5, and E13.5. Shown are the relative transcription levels of each gene compared to the expression of that same gene observed at E9.5. The localization of the X-coded genes analysed and Xist are shown on the cartoon of the X chromosome (from Ensembl).
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pgen-0040030-g005: Xi Reactivates in XX PGCs between E11.5 and E13.5(A,B) FACS-analysis of XX E13.5 WT (A) and ΔPEOct4:gfp (B) PGCs show that anti-PECAM1 is a suitable antibody to separate PGCs from the surrounding somatic tissue. Anti-SSEA1 was used to separate XX E11.5 and E12.5 PGCs from the surrounding somatic tissue.(C) FACS-analysis of XX E13.5 XGFP homozygous genital ridges containing 100% GFP-positive cells, used as positive control.(D–F) Representative dot-plots showing FACS-analysis of XX E11.5 (D), E12.5 (E), and E13.5 (F) genital ridges from individual Xp-XGFP embryos.(G,H) The percentage of FACS-analysed GFP-expressing PGCs (G) and surrounding somatic tissue (H) in the genital ridges of individual XX E11.5, E12.5, and E13.5 Xp-XGFP embryos. PGCs and somatic cells were respectively positive and negative for SSEA1 (E11.5, 12.5) or PECAM1 (E13.5). Red bars depict the median, n is the total number of embryos analysed.(I) Transcriptional levels of X-coded genes and autosomal genes at E9.5, E11.5, and E13.5. Shown are the relative transcription levels of each gene compared to the expression of that same gene observed at E9.5. The localization of the X-coded genes analysed and Xist are shown on the cartoon of the X chromosome (from Ensembl).

Mentions: As the XX PGCs migrated into the endoderm of the invaginating hindgut and subsequently to the genital ridges, the proportion showing a clear H3K27me3 accumulation on the X chromosome decreased although the overall levels of nuclear H3K27me3 were increasing (Figure 3). At E9.5, the percentage of PGCs showing a nuclear H3K27me3 accumulation decreased to about 30%. This suggested that either H3K27me3 is not involved in the maintenance of X-chromosome inactivation or alternatively that the Xi is being reactivated specifically in the PGCs during their migratory journey to the genital ridges. To distinguish between these two possibilities, we analysed the number of E9.5 PGCs expressing the X-linked GFP transmitted either by the father or the mother and observed that the PGCs still showed random X-chromosome inactivation (Figure 4). Once in the genital ridges, the number of PGCs expressing the X-borne GFP transgene increased from just over 50% at E11.5 (random X-chromosome inactivation) to nearly 95% at E13.5, while the somatic cell population over the same period remained at about 50% expression (Figure 5A–5H).


X chromosome activity in mouse XX primordial germ cells.

Chuva de Sousa Lopes SM, Hayashi K, Shovlin TC, Mifsud W, Surani MA, McLaren A - PLoS Genet. (2008)

Xi Reactivates in XX PGCs between E11.5 and E13.5(A,B) FACS-analysis of XX E13.5 WT (A) and ΔPEOct4:gfp (B) PGCs show that anti-PECAM1 is a suitable antibody to separate PGCs from the surrounding somatic tissue. Anti-SSEA1 was used to separate XX E11.5 and E12.5 PGCs from the surrounding somatic tissue.(C) FACS-analysis of XX E13.5 XGFP homozygous genital ridges containing 100% GFP-positive cells, used as positive control.(D–F) Representative dot-plots showing FACS-analysis of XX E11.5 (D), E12.5 (E), and E13.5 (F) genital ridges from individual Xp-XGFP embryos.(G,H) The percentage of FACS-analysed GFP-expressing PGCs (G) and surrounding somatic tissue (H) in the genital ridges of individual XX E11.5, E12.5, and E13.5 Xp-XGFP embryos. PGCs and somatic cells were respectively positive and negative for SSEA1 (E11.5, 12.5) or PECAM1 (E13.5). Red bars depict the median, n is the total number of embryos analysed.(I) Transcriptional levels of X-coded genes and autosomal genes at E9.5, E11.5, and E13.5. Shown are the relative transcription levels of each gene compared to the expression of that same gene observed at E9.5. The localization of the X-coded genes analysed and Xist are shown on the cartoon of the X chromosome (from Ensembl).
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Related In: Results  -  Collection

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pgen-0040030-g005: Xi Reactivates in XX PGCs between E11.5 and E13.5(A,B) FACS-analysis of XX E13.5 WT (A) and ΔPEOct4:gfp (B) PGCs show that anti-PECAM1 is a suitable antibody to separate PGCs from the surrounding somatic tissue. Anti-SSEA1 was used to separate XX E11.5 and E12.5 PGCs from the surrounding somatic tissue.(C) FACS-analysis of XX E13.5 XGFP homozygous genital ridges containing 100% GFP-positive cells, used as positive control.(D–F) Representative dot-plots showing FACS-analysis of XX E11.5 (D), E12.5 (E), and E13.5 (F) genital ridges from individual Xp-XGFP embryos.(G,H) The percentage of FACS-analysed GFP-expressing PGCs (G) and surrounding somatic tissue (H) in the genital ridges of individual XX E11.5, E12.5, and E13.5 Xp-XGFP embryos. PGCs and somatic cells were respectively positive and negative for SSEA1 (E11.5, 12.5) or PECAM1 (E13.5). Red bars depict the median, n is the total number of embryos analysed.(I) Transcriptional levels of X-coded genes and autosomal genes at E9.5, E11.5, and E13.5. Shown are the relative transcription levels of each gene compared to the expression of that same gene observed at E9.5. The localization of the X-coded genes analysed and Xist are shown on the cartoon of the X chromosome (from Ensembl).
Mentions: As the XX PGCs migrated into the endoderm of the invaginating hindgut and subsequently to the genital ridges, the proportion showing a clear H3K27me3 accumulation on the X chromosome decreased although the overall levels of nuclear H3K27me3 were increasing (Figure 3). At E9.5, the percentage of PGCs showing a nuclear H3K27me3 accumulation decreased to about 30%. This suggested that either H3K27me3 is not involved in the maintenance of X-chromosome inactivation or alternatively that the Xi is being reactivated specifically in the PGCs during their migratory journey to the genital ridges. To distinguish between these two possibilities, we analysed the number of E9.5 PGCs expressing the X-linked GFP transmitted either by the father or the mother and observed that the PGCs still showed random X-chromosome inactivation (Figure 4). Once in the genital ridges, the number of PGCs expressing the X-borne GFP transgene increased from just over 50% at E11.5 (random X-chromosome inactivation) to nearly 95% at E13.5, while the somatic cell population over the same period remained at about 50% expression (Figure 5A–5H).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: The Wellcome Trust/Cancer Research UK Gurdon Institute, University of Cambridge, Cambridge, United Kingdom. smc80@cam.ac.uk

ABSTRACT
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.

Show MeSH