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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.

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Intrinsic failure of Mad2l2 deficient PGCs.Apoptosis (TUNEL assay) in E8.75 embryo sections of hindgut endoderm after the conditional knockout of Mad2l2 by Prdm1-Cre. SSEA1-expressing PGCs are marked by arrowheads. Note the apoptotic and non-apoptotic PGC in knockout section. Arrow points to an SSEA1-negative apoptotic cell in the conditional knockout section. Scale bar, 20 µm.
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pgen-1003712-g003: Intrinsic failure of Mad2l2 deficient PGCs.Apoptosis (TUNEL assay) in E8.75 embryo sections of hindgut endoderm after the conditional knockout of Mad2l2 by Prdm1-Cre. SSEA1-expressing PGCs are marked by arrowheads. Note the apoptotic and non-apoptotic PGC in knockout section. Arrow points to an SSEA1-negative apoptotic cell in the conditional knockout section. Scale bar, 20 µm.

Mentions: Proper development of PGCs relies on their endogenous program as well as on exogenous signals emanating from surrounding somatic cells that support their induction, migration or survival in various organisms [41]–[44]. To address the cause of early PGC loss in Mad2l2 deficient embryos, we employed a Prdm1-Cre mouse line, which would be expected to delete the Mad2l2 gene specifically in nascent PGCs [4]. The TUNEL assay demonstrated apoptosis in SSEA1-positive PGCs of Prdm1-Cre+, Mad2l2fl/fl embryos at E8.75 (Figure 3). In addition, TUNEL-positive, SSEA1-negative cells with a high nuclear to cytoplasmic ratio were observed in the hindgut. Also some TUNEL-negative, SSEA1-positive PGCs were found, which is explainable by the incomplete efficiency of Prdm1-Cre mediated deletion, although the actual recombination could not be confirmed here for the few available cells [4]. In contrast, no apoptosis was observed in Prdm1-Cre+, Mad2l2fl/+ PGCs of the same age, excluding toxic effect of Cre recombinase on PGCs [45]. Together, these findings demonstrate that Mad2l2 deficient PGCs did not survive even in a wild type somatic environment. Since Mad2l2 is the subunit of a repair DNA polymerase, we asked if Mad2l2 deficient PGCs are affected by DNA damage. We applied an antibody detecting phosphorylated ATM/ATR substrates (pATM/ATR-S) including Chk1, Chk2, and MDM2, as well as specific antibodies against pChk1 and pChk2, respectively. No double-positive PGCs were detected in either wild type or knockout embryos in such staining (Figure S3B–D). Together, these observations indicate that Mad2l2 deficient PGCs are not lost due to DNA damage.


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

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

Intrinsic failure of Mad2l2 deficient PGCs.Apoptosis (TUNEL assay) in E8.75 embryo sections of hindgut endoderm after the conditional knockout of Mad2l2 by Prdm1-Cre. SSEA1-expressing PGCs are marked by arrowheads. Note the apoptotic and non-apoptotic PGC in knockout section. Arrow points to an SSEA1-negative apoptotic cell in the conditional knockout section. Scale bar, 20 µm.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1003712-g003: Intrinsic failure of Mad2l2 deficient PGCs.Apoptosis (TUNEL assay) in E8.75 embryo sections of hindgut endoderm after the conditional knockout of Mad2l2 by Prdm1-Cre. SSEA1-expressing PGCs are marked by arrowheads. Note the apoptotic and non-apoptotic PGC in knockout section. Arrow points to an SSEA1-negative apoptotic cell in the conditional knockout section. Scale bar, 20 µm.
Mentions: Proper development of PGCs relies on their endogenous program as well as on exogenous signals emanating from surrounding somatic cells that support their induction, migration or survival in various organisms [41]–[44]. To address the cause of early PGC loss in Mad2l2 deficient embryos, we employed a Prdm1-Cre mouse line, which would be expected to delete the Mad2l2 gene specifically in nascent PGCs [4]. The TUNEL assay demonstrated apoptosis in SSEA1-positive PGCs of Prdm1-Cre+, Mad2l2fl/fl embryos at E8.75 (Figure 3). In addition, TUNEL-positive, SSEA1-negative cells with a high nuclear to cytoplasmic ratio were observed in the hindgut. Also some TUNEL-negative, SSEA1-positive PGCs were found, which is explainable by the incomplete efficiency of Prdm1-Cre mediated deletion, although the actual recombination could not be confirmed here for the few available cells [4]. In contrast, no apoptosis was observed in Prdm1-Cre+, Mad2l2fl/+ PGCs of the same age, excluding toxic effect of Cre recombinase on PGCs [45]. Together, these findings demonstrate that Mad2l2 deficient PGCs did not survive even in a wild type somatic environment. Since Mad2l2 is the subunit of a repair DNA polymerase, we asked if Mad2l2 deficient PGCs are affected by DNA damage. We applied an antibody detecting phosphorylated ATM/ATR substrates (pATM/ATR-S) including Chk1, Chk2, and MDM2, as well as specific antibodies against pChk1 and pChk2, respectively. No double-positive PGCs were detected in either wild type or knockout embryos in such staining (Figure S3B–D). Together, these observations indicate that Mad2l2 deficient PGCs are not lost due to DNA damage.

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