Analysis Tools
mortality/aging
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• all homozygotes die between E10.5 and E11.5
• spontaneous heart beating is noted until E10.5, consistent with a normal primitive heart morphology
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growth/size/body
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• starting at E8.5, homozygous mutant embryos appear retarded, lagging at least 0.5 days behind the development of wild-type and heterozygous controls
• severe growth defects are observed by E9.5 and E10.5
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• a significant reduction in embryo size is noted by E9.5 and E10.5
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embryo
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• at E9.0, apoptotic or necrotic cells are detected in the notochord, neural tube, hindgut, and their surrounding mesenchyme
• at E9.5, significantly increased caspase-3 staining is detected in the notochord, neural tube and somites relative to wild-type controls
• in contrast, no changes in proliferation are observed, as shown by Ki-67 staining
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• at E9.5, significantly increased caspase-3 staining is detected in the allantois relative to wild-type controls
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• at E9.5, almost all homozygotes show absence of embryo turning
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• starting at E8.5, homozygous mutant embryos appear retarded, lagging at least 0.5 days behind the development of wild-type and heterozygous controls
• severe growth defects are observed by E9.5 and E10.5
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• a significant reduction in embryo size is noted by E9.5 and E10.5
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• at E9.0, H&E staining revealed notochord hypertrophy; the border of the notochord appears disorganized
• at E9.5, caspase-3+ apoptotic cells are scattered beyond the normal bordered layer of the notochord, whereas only a few apoptotic cells are detected in wild-type embryos
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• at E9.0, the mutant extraembryonic endoderm and mesoderm layers are more widely separated than wild-type layers with no apoptotic cells
• however, the endothelial cell layers appear normal in the extraembryonic endoderm and mesoderm
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• at E9.0, the mutant labyrinth layer lacks immature embryonic nucleated erythrocytes and embryonic blood vessels derived from mesoderm, possibly due to the absence of chorionic fusion
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• at E9.0, mutant yolk sac vessels are dramatically enlarged
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• at E9.0, the mutant allantois appears as an abnormally large, swollen sac not fused with the chorion
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• by E9.5 and E10.5, homozygotes show defects in the yolk sac extraembryonic region
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• despite the presence of an allantois at E9.5 and E10.5, chorioallantoic fusion fails to occur
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• at E9.5, mutant yolk sacs display abnormal patterning of vascularization, lacking the numerous large, branching vessels seen in wild-type yolk sacs
• although an initial primitive vascular plexus is formed at E8.5, PECAM-1 immunostaining confirmed the absence of any mature or remodeled vitelline blood vessels at ~E9.5
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cardiovascular system
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• at E9.0, the mutant labyrinth layer lacks immature embryonic nucleated erythrocytes and embryonic blood vessels derived from mesoderm, possibly due to the absence of chorionic fusion
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• although mutant yolk sacs show normal localization of PECAM-1 and VE-cadherin in cell-cell adhesion sites, JAM-A (a component of tight junctions as well as adherens junctions) is not found in cell-cell adhesion sites, suggesting abnormal angiogenesis, possibly due to defects in cell-cell adhesion-related tissue remodeling
• however, no defects in the junctional complex function are observed, as shown by a paracellular barrier assay of mutant yolk sacs at E9.5
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• at E9.0, mutant yolk sac vessels are dramatically enlarged
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cellular
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• in contrast, no changes in proliferation are observed, as shown by Ki-67 staining
• at E9.0, apoptotic or necrotic cells are detected in the notochord, neural tube, hindgut, and their surrounding mesenchyme
• at E9.5, significantly increased caspase-3 staining is detected in the notochord, neural tube and somites relative to wild-type controls
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• at E9.5, significantly increased caspase-3 staining is detected in the allantois relative to wild-type controls
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• at E9.5, significantly increased caspase-3 staining is detected in the neural tube, leading to disorganization of the ventricular side of the neural tube
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nervous system
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• at E9.5, significantly increased caspase-3 staining is detected in the neural tube, leading to disorganization of the ventricular side of the neural tube
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