Analysis Tools
mortality/aging
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• homozygous mutant embryos can implant into the uterine wall but die between 4.5 and 5.5 dpc, shortly after implantation
• no mutant embryos are observed after 5.5 dpc; most embryo resorptions occur at a stage prior to 5.5 dpc
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embryo
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• an obvious reduction in the growth rate of the ICM region is first noted at 96 hrs of culture and becomes more severe between day 4 and 8 of culture
• a dramatic reduction in the rate of ICM proliferation is confirmed at days 6 and 8 of culture by phosphorylated histone 3 staining
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• between day 4 and 8 of culture, ICM cells from mutant embryos fail to proliferate while wild-type ICM cells continue to expand
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• embryoid bodies (EBs) derived from homozygous mutant ES cells lack cells of the embryonic ectoderm origin; instead, mutant EBs retain cells of the endoderm lineage
• re-introduction of the Serpinb5 gene by adenovirus infection partially rescues the defect in mutant EBs; importantly, the embryonic ectoderm appears complete with a layer of visceral endodrem cells surrounding the ectoderm in the rescued EBs
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• EBs derived from homozygous mutant ES cells exhibit a disorganized, endodermal cell mass and lack a basement membrane layer
• however, early endoderm differentiation (from primitive endoderm to visceral endoderm (VE) or to parietal endoderm (PE) is not blocked
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• homozygous mutant embryos fail to develop into gastrulated embryos
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• the epiblast (embryonic ectoderm), which gives rise to all cell types of the embryo, is gradually lost after 4-8 days in culture
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• EBs derived from homozygous mutant ES cells display defective lumen formation (embryoid body cavitation)
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• in vitro embryo outgrowth analysis indicates that mutant blastocysts show inner cell mass failure during outgrowth and differentiation
• unlike most of the wild-type ICM outgrowths which are typically spherical, mutant ICM outgrowths appear irregular in shape, contain less GATA4-positive endoderm cells at day 4 of culture, and lack an obvious monolayer of endodermal cells during days 4-8 of culture
• in contrast, mutant embryos develop a trophoblast region equal to the size of wild-type embryos, suggesting that initial trophoblast differentiation and morphogenesis is normal
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• EBs derived from homozygous mutant ES cells are smaller in size and display a disorganized layer of visceral endoderm relative to wild-type EBs
• the visceral endoderm (VE) cells in both the EBs and blastocyst outgrowths derived from mutant mice display decreased proliferation relative to wild-type VE cells
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cellular
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• EBs derived from homozygous mutant ES cells display a significantly reduced rate of cell proliferation relative to wild-type EBs, as determined by phosphorylated histone 3 staining
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• an obvious reduction in the growth rate of the ICM region is first noted at 96 hrs of culture and becomes more severe between day 4 and 8 of culture
• a dramatic reduction in the rate of ICM proliferation is confirmed at days 6 and 8 of culture by phosphorylated histone 3 staining
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• between day 4 and 8 of culture, ICM cells from mutant embryos fail to proliferate while wild-type ICM cells continue to expand
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