Analysis Tools
mortality/aging
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• most homozygous mutant embryos die between E11.5 and E12.5
• only 2.9% (versus expected 25%) are obtained at E12.5, and no homozygous mutant embryos are found at P14
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growth/size/body
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• although normal at E9.5, embryos become slightly smaller in size at E10.5
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nervous system
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• at E11.5, embryos exhibit hemorrhage on the head near the neural tube opening
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• at E11.5, ~45% of live embryos exhibit reopening of a closed neural tube in the midbrain region
• by E12.5, all embryos (surviving and dead) show neural tube openings
• however, embryos appear morphologically normal at E9.5-E10.5
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• at E11.5, embryos show severe brain compression
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• at E11.5, embryos show collapse of the telencephalic vesicles
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• at E11.5, both midbrain and forebrain ventricles are smaller than normal
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• at E11.5, embryos show severe collapse of the brain ventricles
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exencephaly
(
J:237614
)
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• ~45% of live embryos exhibit exencephaly at E11.5
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• at E11.5, embryos with exencephaly show attenuated cell proliferation in the neural epithelium, as shown by Ki67 staining of cephalic neural folds in the prospective forebrain and midbrain region
• however, no difference in the number of TUNEL+ cells is observed
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embryo
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• although normal at E9.5, embryos become slightly smaller in size at E10.5
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• at E11.5, ~45% of live embryos exhibit reopening of a closed neural tube in the midbrain region
• by E12.5, all embryos (surviving and dead) show neural tube openings
• however, embryos appear morphologically normal at E9.5-E10.5
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• at E11.5, many embryonic amnions do not fully encase the embryo
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• upon dissection, most E11.5 yolk sacs appear broken, unlike wild-type yolk sacs which remain intact
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• intracellular collagen accumulation in the endoplasmic reticulum (ER) of extraembryonic membranes induces ER stress and apoptosis mainly by activating the PERK pathway of the unfolded protein response (UPR)
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• TUNEL staining showed a markedly increased number of apoptotic cells in the amnion at E11.5
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homeostasis/metabolism
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• at E11.5, embryos show secretion defects of multiple collagen types in different connective tissues
• type I collagen (Col I) and type III collagen (Col III) staining is abnormally found in the intracellular space co-localizing with KDEL (an endoplasmic reticulum marker), unlike in wild-type embryos where Col I and Col III staining is mainly observed in the extracellular space of skin fibroblasts in the embryonic head
• both Col I and Col III accumulate intracellularly in mesothelial cells of the yolk sac, with Col I co-staining with ZO1 (a tight junction protein)
• intracellular accumulation of Col I and Col III is observed in multiple other tissues, including amnion, liver, heart, blood vessel walls, and mesenchyme
• collagen-producing cells in E11.5 head skin fibroblasts and yolk sac contain grossly distended ER with no detectable collagen fibers in extracellular spaces
• impaired collagen I and III secretion is associated with reduced mRNA levels of Col1a1 and Col3a1 in the yolk sac and amnion
• type II collagen (Col II) accumulates in procartilaginous cells in the sclerotome of E11.5 embryos, unlike the extracellular staining of Col II seen in wild-type embryos
• type IV collagen (Col IV) accumulates in collagen-producing cells in the pial basement membrane in the brain and in blood vessel basement membranes, unlike the extracellular staining of Col IV seen in wild-type embryos
• intracellular accumulation of unsecreted proteins leads to strong induction of the unfolded protein response (UPR) in collagen-producing cells
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cellular
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• TUNEL staining showed a markedly increased number of apoptotic cells in the amnion at E11.5
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• RT-PCR revealed that expression of certain UPR genes associated with ER stress-induced apoptosis is progressively increased in the yolk sac from E9.5 to E11.5
• similarly, UPR genes associated with apoptosis are drastically overexpressed in the amnion at E11.5
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skeleton
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• micromass cultures of mesenchymal cells prepared from E11.5 embryos show a significant reduction in total cartilage nodule numbers relative to wild-type cells
• however, whole-mount Alcian blue staining of embryos with no neural tube phenotype revealed no obvious alterations in nascent embryonic cartilage at E11.5
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cardiovascular system
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• at E11.5, embryos exhibit hemorrhage on the head near the neural tube opening
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