Analysis Tools
mortality/aging
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• mutant embryos are morphologically normal at E3.5-E4.5 and implant in the uterus but are not recovered shortly after implantation
• only cellular debris or a few trophoblast giant cells are detected in the decidua at E5.5 and E6.5
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embryo
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• at E4.5, a greater proportion of mutant embryos exhibit cleaved caspase 9-positive fragmented nuclei relative to controls, indicating increased embryo apoptosis
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• ICM derivatives fail to develop beyond E4.5 and show increased apoptosis
• however, ICM cell proliferation and early differentiation of the ICM into the epiblast and the primitive endoderm layers are normal at E4.5
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• following induction of diapause at E2.5, the number of NANOG-positive pluripotent epiblast cells is significantly reduced at 4 days and virtually absent by 7 days after induction, unlike in control embryos
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• failure of in vitro ICM growth precludes establishment of mutant embryonic stem cells
• following induction of diapause at E2.5 (to delay implantation), the number of NANOG-positive pluripotent cells marking the epiblast is significantly reduced at 4 days and virtually absent by 7 days after induction, unlike in control embryos where NANOG-positive epiblast cells persist throughout diapause
• in contrast, GATA4-positive cells marking the primitive endoderm are significantly reduced only at 7 days of diapause
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• in vitro, E3.5 mutant embryos are able to attach to the tissue culture substrate and show normal outgrowth of trophoblast giant cells but fail to form multicellular or cystic structures, unlike control embryos
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• in vitro, ICM outgrowth is normal after 2 days but is severely compromised or absent by 4 days of culture, unlike in wild-type controls
• notably, in vitro trophectoderm outgrowth is normal at 4 days after culture
• in vitro survival of mutant ICM cells is rescued by exogenous putrescine treatment, with the ICM surface area being greater than that of untreated mutant embryos and not significantly different from treated control embryos after 4 days of culture
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• only a few trophoblast giant cells are detected in the decidua at E5.5 and E6.5
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cellular
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• at E4.5, a greater proportion of mutant embryos exhibit cleaved caspase 9-positive fragmented nuclei relative to controls, indicating increased embryo apoptosis
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• ICM derivatives fail to develop beyond E4.5 and show increased apoptosis
• however, ICM cell proliferation and early differentiation of the ICM into the epiblast and the primitive endoderm layers are normal at E4.5
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homeostasis/metabolism
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• at E4.5, mutant emrbyos show decreased levels of ornithine decarboxylase 1 (Odc1) expression in the epiblast, suggesting inability to produce putrescine and lack of the end products of the polyamine synthesis pathway
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reproductive system
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• at E5.5, deciduae are empty and no homozygous mutant embryos are recovered
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