Analysis Tools
mortality/aging
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• very few homozygous mutant pups are born alive, and these die within a few hours of birth
• no homozygotes are obtained from heterozygous intercrosses at P7
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• very few homozygous mutant pups are born alive
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craniofacial
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• P0 neonates exhibit craniofacial malformation
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pigmentation
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• loss of eye pigmentation at E14.5
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nervous system
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• 52% increase in the number of Pax6+ radial glia/apical progenitors in the brain at E14.5
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• at E14.5, fewer BrdU+ Ki67- cells have exited from the cell cycle in the cortex relative to wild-type controls
• number of Tuj1+ early differentiated neurons is significantly reduced at E14.5, suggesting impaired early neuronal differentiation of neural progenitor cells (NPCs)
• Notch signaling is upregulated in neural progenitors, inhibiting cortical neurogenesis
• in culture, % of Tuj1+ (differentiating) cells is significantly decreased in neurospheres derived from E14.5 NPCs relative to those in wild-type neurospheres; however, % of Tuj1+ cells is restored to wild-type levels by treatment with DAPT (a gamma-secretase inhibitor)
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• increased BrdU labelling index (% of Ki67+ cells that have incorporated BrdU) in cortical progenitors following a 2-hr BrdU pulse at E14.5, suggesting increased proliferation of NPCs
• markedly increased density of phosphohistone-3 positive (PH3+) mitotic cells in the cortical ventricular zone at E13.5 and E14.5, suggesting that NPCs are arrested in a proliferative state, inhibiting normal neurogenesis
• in culture, average diameter of neurospheres derived from E14.5 NPCs and % of nestin-positive (proliferating) cells are significantly increased relative to those in wild-type neurospheres; however, average diameter of neurospheres and % of nestin-positive cells are restored to wild-type levels by treatment with DAPT (a gamma-secretase inhibitor)
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• markedly thinner cortical intermediate zone at E14.5
• however, ventricular zone thickness is relatively normal
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• markedly thinner cortical plate at E14.5
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• smaller brain size at E14.5
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• enlarged lateral ventricles at E14.5
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• severe defects in early neurogenesis and morphogenesis in the developing cerebral cortex
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• at E12.5 and E14.5, Ctip2+ early-born layer V and VI neurons and Tbr1+ layer VI neurons are all significantly reduced in number
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• at E12.5 and E14.5, Ctip2+ early-born layer V and VI neurons and Tbr1+ layer VI neurons are all significantly reduced in number
• number of Tuj1+ early differentiated neurons is significantly reduced at E14.5
• however, no significant change is noted in the number of apoptotic cells in the neocortex at E14.5, as shown by TUNEL analysis
• thickness of the Tuj1+ neuronal layer is significantly reduced at E14.5
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• thinner cerebral cortex (neocortex) at E14.5
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hematopoietic system
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• c-Kit+CD34+ HSPCs show increased proliferation in the aorta-gonad-mesonephros region, as shown by Ki67+ 7AAD+ (7-amino-actinomycin D) staining
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• impaired hematopoiesis at E12.5
• Notch signaling is enhanced in the aorta-gonad-mesonephros region
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• drastically decreased CFU-GM in aorta-gonad-mesonephros tissues
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• a colony-forming unit-cell (CFU-C) assay revealed reduced colony formation ability (CFU-Mix) with relatively unchanged CFU-E and drastically decreased CFU-GM in aorta-gonad-mesonephros tissues, suggesting that differentiation of HSPCs is greatly inhibited
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• expression of Runx1, an hematopoietic stem and progenitor cell (HSPC) marker, is increased in the aorta-gonad-mesonephros region (AGM) region and even expanded into the dorsal region of the dorsal aorta
• at E11, the % of c-Kit+CD34+ HSPCs located in the AGM region is significantly higher than in wild-type embryos; however, treatment with DBZ (a gamma-secretase inhibitor) inhibits the expansion of c-Kit+CD34+ HSPCs from E11 AGM
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cellular
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• mouse embryonic fibroblasts (MEFs) derived from E14.5 embryos show a significant increase in the number of endolysosomes relative to wild-type MEFs
• however, the numbers of multivesicular bodies, late endosomes, and lysosomes are not significantly altered
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• 52% increase in the number of Pax6+ radial glia/apical progenitors in the brain at E14.5
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• at E14.5, fewer BrdU+ Ki67- cells have exited from the cell cycle in the cortex relative to wild-type controls
• number of Tuj1+ early differentiated neurons is significantly reduced at E14.5, suggesting impaired early neuronal differentiation of neural progenitor cells (NPCs)
• Notch signaling is upregulated in neural progenitors, inhibiting cortical neurogenesis
• in culture, % of Tuj1+ (differentiating) cells is significantly decreased in neurospheres derived from E14.5 NPCs relative to those in wild-type neurospheres; however, % of Tuj1+ cells is restored to wild-type levels by treatment with DAPT (a gamma-secretase inhibitor)
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• c-Kit+CD34+ HSPCs show increased proliferation in the aorta-gonad-mesonephros region, as shown by Ki67+ 7AAD+ (7-amino-actinomycin D) staining
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• increased BrdU labelling index (% of Ki67+ cells that have incorporated BrdU) in cortical progenitors following a 2-hr BrdU pulse at E14.5, suggesting increased proliferation of NPCs
• markedly increased density of phosphohistone-3 positive (PH3+) mitotic cells in the cortical ventricular zone at E13.5 and E14.5, suggesting that NPCs are arrested in a proliferative state, inhibiting normal neurogenesis
• in culture, average diameter of neurospheres derived from E14.5 NPCs and % of nestin-positive (proliferating) cells are significantly increased relative to those in wild-type neurospheres; however, average diameter of neurospheres and % of nestin-positive cells are restored to wild-type levels by treatment with DAPT (a gamma-secretase inhibitor)
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• MEFs exhibit impaired lysosomal degradation of Notch1; treatment with leupeptin (an inhibitor of lysosomal proteolysis) has no effect on intracellular Notch1 (NTM) levels, unlike in wild-type MEFs
• however, NTM degradation is normally suppressed by treatment with MG132 (a proteasome inhibitor) as in wild-type MEFs
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• intracellular Notch1 (NTM) distribution shows a marked increase in the EEA1 (early endosomes) and CD63-labeled (multivesicular bodies and late endosomes) fractions in E14.5 brain tissues relative to wild-type controls, indicating accumulation of Notch1 (NTM) in endosomes
• MEFs show altered endolysosomal trafficking of Notch1, with increased endogenous Notch1 localization in EEA1-positive (early endosome) and LBPA-positive (late endosome) vesicles, and decreased Notch1 localization in LAMP1-labeled (lysosome) vesicles relative to wild-type MEFs
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vision/eye
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• loss of eye pigmentation at E14.5
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