Limits...
A critical function of Mad2l2 in primordial germ cell development of mice.

Pirouz M, Pilarski S, Kessel M - PLoS Genet. (2013)

Bottom Line: By the analysis of transfected fibroblasts we found that the interaction of Mad2l2 with the histone methyltransferases G9a and GLP lead to a downregulation of H3K9me2.Together, these results demonstrate the potential of Mad2l2 in the regulation of both cell cycle and the epigenetic status.The function of Mad2l2 is essential in PGCs, and thus of high relevance for fertility.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Cell Biology, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany. mehdi.pirouz@mpibpc.mpg.de

ABSTRACT
The development of primordial germ cells (PGCs) involves several waves of epigenetic reprogramming. A major step is following specification and involves the transition from the stably suppressive histone modification H3K9me2 to the more flexible, still repressive H3K27me3, while PGCs are arrested in G2 phase of their cycle. The significance and underlying molecular mechanism of this transition were so far unknown. Here, we generated mutant mice for the Mad2l2 (Mad2B, Rev7) gene product, and found that they are infertile in both males and females. We demonstrated that Mad2l2 is essential for PGC, but not somatic development. PGCs were specified normally in Mad2l2(-/-) embryos, but became eliminated by apoptosis during the subsequent phase of epigenetic reprogramming. A majority of knockout PGCs failed to arrest in the G2 phase, and did not switch from a H3K9me2 to a H3K27me3 configuration. By the analysis of transfected fibroblasts we found that the interaction of Mad2l2 with the histone methyltransferases G9a and GLP lead to a downregulation of H3K9me2. The inhibitory binding of Mad2l2 to Cyclin dependent kinase 1 (Cdk1) could arrest the cell cycle in the G2 phase, and also allowed another histone methyltransferase, Ezh2, to upregulate H3K27me3. Together, these results demonstrate the potential of Mad2l2 in the regulation of both cell cycle and the epigenetic status. The function of Mad2l2 is essential in PGCs, and thus of high relevance for fertility.

Show MeSH

Related in: MedlinePlus

Loss and apoptosis of PGCs early after specification.(A) AP-positive Mad2l2+/+ or Mad2l2−/− PGCs were detected in EHF and LHF stages. From E8.5 to E9.5, PGCs were detected by Oct4-immunostaining (arrowheads). At E13.5, Mad2l2−/− ovaries were devoid of germ cells detected by AP staining. Al: allantois; ne: neuroepithelium; he: hindgut epithelium; hg: hindgut; da: dorsal aorta; dm: dorsal mesentery; mn: mesonephros; ov: ovary. Scale bars, 100 µm. (B) Quantification of PGCs detected by AP-staining in different developmental stages. (C) Apoptosis (TUNEL assay) in E9.0 embryo sections of hindgut endoderm. SSEA1-expressing PGCs and apoptotic cells are marked by arrowheads and arrows, respectively. Note the apoptotic PGC in knockout section. Scale bar, 20 µm.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3757036&req=5

pgen-1003712-g002: Loss and apoptosis of PGCs early after specification.(A) AP-positive Mad2l2+/+ or Mad2l2−/− PGCs were detected in EHF and LHF stages. From E8.5 to E9.5, PGCs were detected by Oct4-immunostaining (arrowheads). At E13.5, Mad2l2−/− ovaries were devoid of germ cells detected by AP staining. Al: allantois; ne: neuroepithelium; he: hindgut epithelium; hg: hindgut; da: dorsal aorta; dm: dorsal mesentery; mn: mesonephros; ov: ovary. Scale bars, 100 µm. (B) Quantification of PGCs detected by AP-staining in different developmental stages. (C) Apoptosis (TUNEL assay) in E9.0 embryo sections of hindgut endoderm. SSEA1-expressing PGCs and apoptotic cells are marked by arrowheads and arrows, respectively. Note the apoptotic PGC in knockout section. Scale bar, 20 µm.

Mentions: For the determination of PGC numbers, embryos were collected at different time points during their early development, were staged as outlined under experimental procedures, and PGCs were identified by the presence of alkaline phosphatase (AP) or Oct4 (Figure 2A) [39]. At the early head fold (EHF) stage, the numbers of PGCs at the base of the allantois were similar in wild type, heterozygous and homozygous embryos. However, while the number of normal PGCs increased at the late head fold (LHF) stage, the number of Mad2l2−/− PGCs fell behind (Figure 2B). It decreased drastically from E8.5 onward, and at E9.0 only few instead of normally ca. 120 PGCs were found in the hindgut endoderm. At E9.5 and E10.5 Oct4-positive PGCs were no longer detected (Figure 2B). At E8.25, both wild type and remaining mutant PGCs co-expressed Oct4 together with Prdm1, Tcfap2c, and Dppa3, indicating a normal specification of mutant PGCs (Figure S2A,B,D). Oct4 and Sox2 were co-expressed in all wild type PGCs with no exception. In contrast, above 40% of Oct4-positive Mad2l2−/− PGCs did not express Sox2 at E9.0, and thus had either failed to reactivate, or at least to maintain its expression (Figure S2C). Emigration to the dorsal mesentery did not occur, and as a result, gonad primordia at E13.5 were devoid of germ cells (Figure 2A). All E9.0 Mad2l2−/− PGCs had accumulated active, acetylated p53 protein, reflecting an activated stress response and impending apoptosis (Figure S3A) [40]. As judged by the TUNEL assay (See Text S1), some SSEA1-positive PGCs undergoing cell death were detected in E9.0 hindgut endoderm (Figure 2C). In addition, the same territory contained accumulations of SSEA1-negative, apoptotic cells. Based on their size we suspected them to be germ cells having lost already expression of their typical marker, although we could not exclude that they represented mutant somatic cells. In summary, Mad2l2−/− PGCs were specified normally, but their numbers decreased progressively, and no PGCs could be detected in Mad2l2−/− embryos beyond E9.5. This time window correlates with an epigenetic transition of PGCs and cell cycle arrest between E7.5-E9.5 [3], [11].


A critical function of Mad2l2 in primordial germ cell development of mice.

Pirouz M, Pilarski S, Kessel M - PLoS Genet. (2013)

Loss and apoptosis of PGCs early after specification.(A) AP-positive Mad2l2+/+ or Mad2l2−/− PGCs were detected in EHF and LHF stages. From E8.5 to E9.5, PGCs were detected by Oct4-immunostaining (arrowheads). At E13.5, Mad2l2−/− ovaries were devoid of germ cells detected by AP staining. Al: allantois; ne: neuroepithelium; he: hindgut epithelium; hg: hindgut; da: dorsal aorta; dm: dorsal mesentery; mn: mesonephros; ov: ovary. Scale bars, 100 µm. (B) Quantification of PGCs detected by AP-staining in different developmental stages. (C) Apoptosis (TUNEL assay) in E9.0 embryo sections of hindgut endoderm. SSEA1-expressing PGCs and apoptotic cells are marked by arrowheads and arrows, respectively. Note the apoptotic PGC in knockout section. Scale bar, 20 µm.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3757036&req=5

pgen-1003712-g002: Loss and apoptosis of PGCs early after specification.(A) AP-positive Mad2l2+/+ or Mad2l2−/− PGCs were detected in EHF and LHF stages. From E8.5 to E9.5, PGCs were detected by Oct4-immunostaining (arrowheads). At E13.5, Mad2l2−/− ovaries were devoid of germ cells detected by AP staining. Al: allantois; ne: neuroepithelium; he: hindgut epithelium; hg: hindgut; da: dorsal aorta; dm: dorsal mesentery; mn: mesonephros; ov: ovary. Scale bars, 100 µm. (B) Quantification of PGCs detected by AP-staining in different developmental stages. (C) Apoptosis (TUNEL assay) in E9.0 embryo sections of hindgut endoderm. SSEA1-expressing PGCs and apoptotic cells are marked by arrowheads and arrows, respectively. Note the apoptotic PGC in knockout section. Scale bar, 20 µm.
Mentions: For the determination of PGC numbers, embryos were collected at different time points during their early development, were staged as outlined under experimental procedures, and PGCs were identified by the presence of alkaline phosphatase (AP) or Oct4 (Figure 2A) [39]. At the early head fold (EHF) stage, the numbers of PGCs at the base of the allantois were similar in wild type, heterozygous and homozygous embryos. However, while the number of normal PGCs increased at the late head fold (LHF) stage, the number of Mad2l2−/− PGCs fell behind (Figure 2B). It decreased drastically from E8.5 onward, and at E9.0 only few instead of normally ca. 120 PGCs were found in the hindgut endoderm. At E9.5 and E10.5 Oct4-positive PGCs were no longer detected (Figure 2B). At E8.25, both wild type and remaining mutant PGCs co-expressed Oct4 together with Prdm1, Tcfap2c, and Dppa3, indicating a normal specification of mutant PGCs (Figure S2A,B,D). Oct4 and Sox2 were co-expressed in all wild type PGCs with no exception. In contrast, above 40% of Oct4-positive Mad2l2−/− PGCs did not express Sox2 at E9.0, and thus had either failed to reactivate, or at least to maintain its expression (Figure S2C). Emigration to the dorsal mesentery did not occur, and as a result, gonad primordia at E13.5 were devoid of germ cells (Figure 2A). All E9.0 Mad2l2−/− PGCs had accumulated active, acetylated p53 protein, reflecting an activated stress response and impending apoptosis (Figure S3A) [40]. As judged by the TUNEL assay (See Text S1), some SSEA1-positive PGCs undergoing cell death were detected in E9.0 hindgut endoderm (Figure 2C). In addition, the same territory contained accumulations of SSEA1-negative, apoptotic cells. Based on their size we suspected them to be germ cells having lost already expression of their typical marker, although we could not exclude that they represented mutant somatic cells. In summary, Mad2l2−/− PGCs were specified normally, but their numbers decreased progressively, and no PGCs could be detected in Mad2l2−/− embryos beyond E9.5. This time window correlates with an epigenetic transition of PGCs and cell cycle arrest between E7.5-E9.5 [3], [11].

Bottom Line: By the analysis of transfected fibroblasts we found that the interaction of Mad2l2 with the histone methyltransferases G9a and GLP lead to a downregulation of H3K9me2.Together, these results demonstrate the potential of Mad2l2 in the regulation of both cell cycle and the epigenetic status.The function of Mad2l2 is essential in PGCs, and thus of high relevance for fertility.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Cell Biology, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany. mehdi.pirouz@mpibpc.mpg.de

ABSTRACT
The development of primordial germ cells (PGCs) involves several waves of epigenetic reprogramming. A major step is following specification and involves the transition from the stably suppressive histone modification H3K9me2 to the more flexible, still repressive H3K27me3, while PGCs are arrested in G2 phase of their cycle. The significance and underlying molecular mechanism of this transition were so far unknown. Here, we generated mutant mice for the Mad2l2 (Mad2B, Rev7) gene product, and found that they are infertile in both males and females. We demonstrated that Mad2l2 is essential for PGC, but not somatic development. PGCs were specified normally in Mad2l2(-/-) embryos, but became eliminated by apoptosis during the subsequent phase of epigenetic reprogramming. A majority of knockout PGCs failed to arrest in the G2 phase, and did not switch from a H3K9me2 to a H3K27me3 configuration. By the analysis of transfected fibroblasts we found that the interaction of Mad2l2 with the histone methyltransferases G9a and GLP lead to a downregulation of H3K9me2. The inhibitory binding of Mad2l2 to Cyclin dependent kinase 1 (Cdk1) could arrest the cell cycle in the G2 phase, and also allowed another histone methyltransferase, Ezh2, to upregulate H3K27me3. Together, these results demonstrate the potential of Mad2l2 in the regulation of both cell cycle and the epigenetic status. The function of Mad2l2 is essential in PGCs, and thus of high relevance for fertility.

Show MeSH
Related in: MedlinePlus