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A murine AP-endonuclease gene-targeted deficiency with post-implantation embryonic progression and ionizing radiation
D L Ludwig1, M A MacInnes, Y Takiguchi
1Life Sciences Division, Los Alamos National Laboratory, NM 87545, USA.
Abstract:
Apurinic/apyrimidinic endonuclease (here designated APE/REF) carries out repair incision at abasic or single-strand break damages in mammals. This multifunctional protein also has putative role(s) as a cysteine 'reducing factor' (REF) in cell-stress transcriptional responses. To assess the significance of APE/REF for embryonic teratogenesis we constructed a more precisely targeted Ape/Ref-deficient genotype in mice. Ape/Ref gene replacement in ES cells eliminated the potential of APE/REF protein synthesis while retaining the Ape/Ref bi-directional promoter that avoided potential inactivation of an upstream gene. Chimeric animals crossed into Tac:N:NIHS-BC produced germline transmission. Homozygous null Ape/Ref-embryos exhibited successful implantation and nearly normal developmental progression until embryonic day 7.5 followed by morphogenetic failure and adsorption of embryos by day 9.5. We characterized the cellular events proceeding to embryonic lethality and examined ionizing radiation sensitivity of pre-implantation Ape/Ref-null embryos. After intermating of heterozygotes, Mendelian numbers of putative Ape/Ref-null progeny embryos at day 6.5 displayed a several-fold elevation of pycnotic, fragmenting cell nuclei within the embryo proper-the epiblast. Increased cell-nucleus degeneration occurred within epiblast cells while mitosis continued and before obvious morphogenetic disruption. Mitogenic response to epiblast cell death, if any, was ineffective for replacement of lost cells. Extra-embryonic yolk sac, a trophectoderm derived lineage retained normal appearance to day 9. Explanted homozygous Ape/Ref-null blastocysts displayed increased sensitivity to gamma-irradiation, most likely a manifestation of APE/REF incision defect. Our study establishes that this new Ape/Ref deficiency genotype is definitely capable of post-implantation developmental progression to the onset of gastrulation. Function(s) of APE/REF in base damage incision and also conceivably in mitogenic responses towards epiblast cell death are critical for transit through the gastrulation stage of embryonic growth and development.
Insights
Apurinic/apyrimidinic endonuclease (APE/REF) deficiency in mice causes embryonic death by day 9.5 due to cell death during gastrulation. This highlights APE/REF
Area of Science:
- Molecular Biology
- Developmental Biology
- Genetics
Background:
- Apurinic/apyrimidinic endonuclease (APE/REF) is crucial for DNA repair in mammals.
- APE/REF also functions as a reducing factor (REF) in cellular stress responses.
- The role of APE/REF in embryonic development and teratogenesis requires precise investigation.
Purpose of the Study:
- To investigate the significance of APE/REF in embryonic development and teratogenesis.
- To create and analyze a precisely targeted Ape/Ref-deficient mouse model.
- To understand the cellular events leading to embryonic lethality in Ape/Ref-null embryos.
Main Methods:
- Generated a gene-replacement strategy in ES cells to create a homozygous null Ape/Ref genotype.
- Produced chimeric animals and achieved germline transmission for homozygous null embryos.
- Analyzed embryonic development, cell death, mitosis, and radiation sensitivity of Ape/Ref-null embryos.
Main Results:
- Homozygous null Ape/Ref embryos showed normal implantation but failed morphogenetically by day 9.5.
- Significant increase in pycnotic, fragmenting cell nuclei (apoptosis) observed in the epiblast by day 6.5.
- Ape/Ref-null blastocysts exhibited increased sensitivity to gamma-irradiation, indicating an incision defect.
Conclusions:
- APE/REF is critical for embryonic development, particularly during the gastrulation stage.
- The DNA incision function of APE/REF is essential for preventing epiblast cell death.
- APE/REF may also play a role in mitigating cell death through mitogenic responses during embryogenesis.