Analysis Tools
mortality/aging
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• although normal Mendelian ratios are observed at E10.5, E12.5, and E14.5, no homozygous mutant pups are detected at birth
• all homozygotes are completely resorbed by E16.5
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growth/size/body
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• at E12.5 and E14.5, mutant embryos show a marked reduction in size and cellularity relative to wild-type controls
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embryo
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• mutant placentae are hypovascular as shown by a markedly reduced H&E staining
• at E12.5, staining for smooth muscle actin revealed thin-walled and enlarged blood vessels in placental tissues
• by E14.5, placental blood vessels have collapsed and are practically absent
• large amounts of maternal blood hemorrhaging are observed around the periphery of the labyrinth, suggesting that the blood vessels are leaky
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• at E12.5 and E14.5, proper perfusion of fetal blood is observed throughout the tissue in wild-type placentae but is absent in mutant placentae
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• propidium iodide staining of embryo fractions revealed a 3-fold increase in the number of apoptotic cells at E12.5
• immunohistochemical analysis confirmed a marked increase in the levels of active caspase 3 in E12.5 but not in E10.5 mutant embryos
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• active caspase 3 staining showed a marked increase in the number of apoptotic cells within the labyrinth and junctional zones at E12.5 and E14.5
• however, no apparent decrease in placenta cellularity is observed, as shown by Ki-67 staining
• no differences in apoptosis or proliferation are observed within the maternal tissue at either E12.5 or E14.5
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• 40% of E12.5 and all of E14.5 homozygotes exhibit severe developmental defects; very rare defects are noted at E10.5
• at E14.5, marked developmental delay and abnormal structural organization is noted in the brain, eyes, muscles, viscerae, and limbs
• H&E staining of mutant embryos revealed a 33% reduction in total cellularity at E12.5
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• at E12.5 and E14.5, mutant embryos show a marked reduction in size and cellularity relative to wild-type controls
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• a large proportion of acellular lesions is seen within the junctional zone at E14.5
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• at E12.5, mutant labyrinth cells are less differentiated and do not exhibit the typical syncytiotrophoblast morphology seen in wild-type controls
• a larger proportion of acellular lesions is seen within the labyrinth at E14.5
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nervous system
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• at E12.5, several of the smaller homozygotes exhibit a larger brain size
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• betaIII-tubulin (Tuj-1) immunohistochemistry revealed a marked decrease in the size of the neuronal compartment within the posterior neural tube at E10.5
• although smaller Tuj.1+ compartments are seen at E12.5, Tuj-1 staining revealed significant degeneration and much smaller Tuj-1+ compartments at E14.5
• spatially disorganized Tuj-1+ cell clusters are noted along the vertebral axis at both E12.5 and E14.5
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• very little expansion of the DRG compartment is observed at E12.5 and E14.5
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• by E12.5 and E14.5, Tuj-1+ DRG are spatially disorganized and aligned in a dorsoventral pattern with the vertebrae, unlike in wild-type embryos
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muscle
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• at E12.5, anti-myosin heavy chain (MF20) staining revealed a less structured and smaller myogenic compartment with MF20+ myofibers scattered throughout the rib cage area and the back
• at E14.5, MF20+ myofibers are very sparse and entirely unstructured with a poorly developed back musculature
• however, no obvious defects are observed in the early premuscle masses at E10.5
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limbs/digits/tail
cardiovascular system
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• although vasculature appears normal at E12.5, mutant embryos are devoid of PECAM-1+ blood vessels by E14.5, suggesting vascular collapse and death in older embryos
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• mutant placentae are hypovascular as shown by a markedly reduced H&E staining
• at E12.5, staining for smooth muscle actin revealed thin-walled and enlarged blood vessels in placental tissues
• by E14.5, placental blood vessels have collapsed and are practically absent
• large amounts of maternal blood hemorrhaging are observed around the periphery of the labyrinth, suggesting that the blood vessels are leaky
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• at E12.5 and E14.5, proper perfusion of fetal blood is observed throughout the tissue in wild-type placentae but is absent in mutant placentae
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hemorrhage
(
J:211339
)
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• homozygotes display hemorrhaging of fetal blood around the outside of the placenta
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cellular
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• propidium iodide staining of embryo fractions revealed a 3-fold increase in the number of apoptotic cells at E12.5
• immunohistochemical analysis confirmed a marked increase in the levels of active caspase 3 in E12.5 but not in E10.5 mutant embryos
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• active caspase 3 staining showed a marked increase in the number of apoptotic cells within the labyrinth and junctional zones at E12.5 and E14.5
• however, no apparent decrease in placenta cellularity is observed, as shown by Ki-67 staining
• no differences in apoptosis or proliferation are observed within the maternal tissue at either E12.5 or E14.5
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• in culture, primary MEFs isolated from E12.5 mutant embryos show a reduced proliferation rate relative to wild-type MEFs
• however, the slower growth rate is not accompanied by a significant decrease in cell viability
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vision/eye
homeostasis/metabolism
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• SLK kinase activity toward histone H1 is reduced by ~80%, suggesting that the SLK-LacZ fusion cannot efficiently phosphorylate exogenous substrates
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