Murine Wee1 plays a critical role in cell cycle regulation and pre-implantation stages of embryonic development - PubMed (original) (raw)
Murine Wee1 plays a critical role in cell cycle regulation and pre-implantation stages of embryonic development
Yohei Tominaga et al. Int J Biol Sci. 2006.
Abstract
Wee1 kinase regulates the G2/M cell cycle checkpoint by phosphorylating and inactivating the mitotic cyclin-dependent kinase 1 (Cdk1). Loss of Wee1 in many systems, including yeast and drosophila, leads to premature mitotic entry. However, the developmental role of Wee1 in mammals remains unclear. In this study, we established Wee1 knockout mice by gene targeting. We found that Wee-/- embryos were defective in the G2/M cell cycle checkpoint induced by gamma-irradiation and died of apoptosis before embryonic (E) day 3.5. To study the function of Wee1 further, we have developed MEF cells in which Wee1 is disrupted by a tamoxifen inducible Cre-LoxP approach. We found that acute deletion of Wee1 resulted in profound growth defects and cell death. Wee1 deficient cells displayed chromosome aneuploidy and DNA damage as revealed by gamma-H2AX foci formation and Chk2 activation. Further studies revealed a conserved mechanism of Wee1 in regulating mitotic entry and the G2/M checkpoint compared with other lower organisms. These data provide in vivo evidence that mammalian Wee1 plays a critical role in maintaining genome integrity and is essential for embryonic survival at the pre-implantation stage of mouse development.
Conflict of interest statement
Conflict of interest: The authors have declared that no conflict of interest exists.
Figures
Figure 1
Generation of Wee1 knockout mice. (A) Structure of Wee1 targeting vector, ploxPneoWee1 (upper) and map of a part of normal mouse Wee1 gene (lower). Be: BbeI, Ev: EcoRV, S: SalI, and Xh: XhoI. Probes used were as indicated. Probes “a”, which is 5' flanking the targeting vector, and probe “b”, which is located in the 3' arm of the targeting vector, were used in Southern blot analysis. (B) Structure of targeted allele (upper), and exons 9/10 deleted allele (lower). (C) Southern blot showed targeted allele (lanes 2 and 3) after EcoRV digestion and probed with probe b. (F) PCR based genotype of offspring obtained from crosses between _Wee1+/neo_and EIIa-Cre mice. Primer 1: 5' TGT CTA CAA GTT GTC TTG TCA TGA 3', Primer 2: 5' ACT GTG GAG AGC TCT CAA TG 3', Primer 3: 5' CTC CAG GTG TGT CAT ATA CC 3', Primer 4: 5' CCG TTC CTC AGC TGC AAC TT 3'.
Figure 2
Abnormal appearance of Wee1-/- embryos at pre-implantation stages. (A,B) Images of Wee1+/+ (A) and Wee1-/- (B) embryos. (C) Genotyping of E3.5 embryos by PCR. (D,E) Morphology (D) of blastocysts after they were cultured after different times as indicated. All embryos showing proliferation were either Wee1+/+ or Wee1+/- embryos. Genotypes (E) were determined by PCR 4 days after culture.
Figure 3
Apoptosis and impaired G2/M cell cycle checkpoint in Wee1-/- embryos. (A, B) TUNEL assay revealed increased apoptosis in Wee1-/- embryos. (C,D) Mitotic cells in E2.5 embryos. (E,F) Mitotic index of E2.5 and E3.5 embryos. Each graph bar summarizes data obtained from at least 4 embryos.
Figure 4
Generation of tamoxifen inducible Wee1 knockout MEF cells. (A) Structure of conditional allele (upper) and exons 9/10 deleted allele (lower). (B) PCR based genotyping of Wee1Co/+ and Wee1Co/-;Tm-Cre MEFs in the presence (+) or absence (-) of 4-ht. The same primers were used for genotyping as shown in Fig. 1.
Figure 5
Increased cell death and impaired cell cycle regulation of Wee1-deficient cells (A) MTT assay of Wee1Co/-;TM-cre MEF cells that were treated with ethanol or no treatment (upper curve), or 1μM of 4-HT. (B) Apoptotic fraction of Wee1Co/-;TM-cre MEF cells after 4-ht treatment revealed by annexin V and EtBr staining. (C) Defective G2/M cell cycle checkpoint of Wee1Co/-;TM-cre MEF cells. Cells were treated with 1 μM 4-HT for 24 hours and fixed at 1-4 hours after they were irradiated with 10 Gy γ-ray. (D,E) Premature entry into mitosis revealed by BrdU and histone H3 double staining. Wee1Co/-;TM-cre MEFs were incubated in the absence (D) or presence (E) of 4-HT for 24 hours prior to the staining. (F) Percentage of BrdU and histone H3 double positive cell. The percentage is calculated by the following formula [(BrdU++H3Pi+)/H3Pi+]. (G) Flow cytometry analysis of mitotic cells in ethanol or 4-ht treated Wee1Co/-;TM-cre MEFs by PI and H3Pi staining. (H) Western blotting of Wee1Co/-;TM-cre MEFs. The arrow points to the phosphorylated form of Chk2 after lighter expossure of the gel. (I,J,K) Effect of purralanol A (10 μM) on mitotic entry of Wee1Co/-;Tm-Cre cells in the absence (-) and presence (+) of 4-HT. Percentages of BrdU (I), H3Pi (J), and double (K) positive cells were shown.
Figure 6
Morphology of Wee1Co/-;Tm-Cre MEFs in the presence (A) and absence (B) of 4-HT for 48 hours.
Figure 7
DNA damage and chromosome instability of Wee1Co/-;TM-cre MEF cells. (A,B) Immunofluorescent staining of Wee1Co/-;TM-cre MEF cells using an antibody to γ-H2AX in the absence (A) or presence (B) of 1 μM 4-ht for 24 hours. (C,D) Chromosome spread of Wee1Co/-;TM-cre MEF cells after they were grown in the absence (C) or presence (D) of 1 μM 4-ht for 24 hours. (E) A summary of chromosomal spreading data from more than 60 cells in the absence (black bar) and presence (open bar) of 4-HT.
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