Complete lack of NF-kappaB activity in IKK1 and IKK2 double-deficient mice: additional defect in neurulation - PubMed (original) (raw)
. 2000 Jul 15;14(14):1729-33.
Affiliations
- PMID: 10898787
- PMCID: PMC316792
Complete lack of NF-kappaB activity in IKK1 and IKK2 double-deficient mice: additional defect in neurulation
Q Li et al. Genes Dev. 2000.
Abstract
NF-kappaB activity is induced by cytokines, stress, and pathogens. IKK1 and IKK2 are critical IkappaB kinases in NF-kappaB activation. In this study mice lacking IKK1 and IKK2 died at E12. Additional defect in neurulation associated with enhanced apoptosis in the neuroepithelium was also observed. MEF cells from IKK1(-/-)/IKK2(-/-) embryos did not respond to NF-kappaB inducers. Upon crossing with kappaB-lacZ transgenic mice, double-deficient embryos also lost lacZ transgene expression in vascular endothelial cells during development. Our data suggest that IKK1 and IKK2 are essential for NF-kappaB activation in vivo and have an important role in protecting neurons against excessive apoptosis during development.
Figures
Figure 1
Phenotypes of _IKK1_−/−/_IKK2_−/− embryos. Side (A) and views back (B) of wild type (left) and _IKK1_−/−/_IKK2_−/− (right) embryos at E11.5 (A) and E9.5 (B). The neural tube in the hindbrain region failed to close (arrow). H&E-stained transverse sections of E9.5 wild type (C) and _IKK1_−/−/_IKK2_−/− mutant (D) at the hindbrain level showing that the roof of the hindbrain (arrow) is present in the wild type but absent in the mutant. TUNEL assay on transverse section of E9.5 wild type (E) and double mutant (F), revealing increased apoptosis (green) in the neuroepithelium of the hindbrain (arrow). Enhanced apoptosis (red arrowhead) was observed in an H&E-stained liver section from _IKK1_−/−/_IKK2_−/− at E11.5 (H) in comparison with that of a wild type littermate (G). (I—L) Whole-mount pictures of wild-type, _IKK2_−/−, _IKK1_−/−, and _IKK1_−/−/_IKK2_−/− embryos in _TNFR1_−/− genetic background at E14.5. No morphologic differences were detected between _TNFR1_−/− (I) and _TNFR1_−/−/_IKK2_−/− (J) embryos. Phenotypes of dumpy limb buds and curled tail were observed in both _TNFR1_−/−/_IKK1_−/− (K) and _TNFR1_−/−/_IKK1_−/−/_IKK2_−/− (L) embryos. In addition, _TNFR1_−/−/_IKK1_−/−/_IKK2_−/− embryos also had NTD (arrow in L). Scale bar, 1 mm in A, B, and I–L; 100 μm in C–F; and 24 μm in G and H.
Figure 2
NF-κB activation is blocked in _IKK1_−/−/_IKK2_−/− MEFs. (A) No NF-κB DNA binding activity was detectable in _IKK1_−/−/_IKK2_−/− MEFs upon induction. Nuclear extracts (5 μg) were used for electrophoretic mobility shift analysis with 32P-end-labeled HIV-κB oligonucleotide. (B) Induced IκB degradation was blocked in the absence of IKK1 and IKK2. Cytoplasmic extract (40 μg) was used for immunoblotting with IκBα, IκBβ, IKK1, and IKK2 antisera (Santa Cruz Biotechnology). (C) TNFα-induced kinase activity of the IKK complex for IκB and p65 was abolished in the absence of IKK1 and IKK2. Cells from three 15-cm plates, untreated or treated with TNFα for 7 min, were lysed and immunoprecipitated with anti-NEMO serum. IP complexes were eluted from the antibody by the synthetic peptide. Equal amounts of eluates were used for kinase assays using the substrate indicated. (D,E) IκBα induction by TNFα was abolished in IKK1 and IKK2 deficient MEFs. RNA (10 μg) from wild-type, _IKK1_−/−, _IKK2_−/−, and _IKK1_−/−/_IKK2_−/− MEFs, untreated or treated with 10 ng/ml hTNFα for 1 hr was subjected to Northern blot analysis with probe specific for IκBα and GAPDH. Quantification and original data are shown in D and E, respectively.
Figure 3
Endothelial cell-specific expression of lacZ in κB–lacZ trangenic mice during early development. Whole-mount X-gal staining of κB–lacZ transgenic embryos at E9.5 (A) and E10.5 (B). (C) Sagittal sections of an X-gal-stained embryo in B counterstained with nuclear fast red. lacZ expression was readily detected everywhere, especially in vascular vessels such the DA and ISV. (End) Endocardium; (HB) hindbrain; (NE) neuroepithelium. (D–G) β-Gal (green) and PECAM-1 (red) double staining on the sagittal sections of an E11.5 transgenic embryo. The transgene, NLS-containing β-gal, is expressed in the nuclei of PECAM-1-positive cells in blood vessels (D, E) and primary capillary plexus at the neuroepithelium (F) but not in PECAM-1-positive endocardium (G). Scale bar, 1 mm in A–C and 24 μm in D–G.
Figure 4
IKK1 and IKK2 are required for NF-κB activity in vascular endothelial cells. Whole-mount X-gal staining showing different expression levels of transgenes in different IKK mutants at E11.5 (A) and E10.5 (C). (B) Sagittal section of embryos in A. X-gal expression in _IKK1_−/−/_IKK2_−/− embryos is lost almost completely; its expression in _IKK1_−/−and _IKK2_−/− embryos is also attenuated in comparison with wild-type controls. (See text and legend to Fig. 3 for abbreviations.) Whole-mount PECAM-1 staining of an _IKK1_−/−/_IKK2_−/− mutant (E) and a control littermate (D) at E10.5 revealed normal vasculogenesis in the absence of IKK1 and IKK2 kinases. Scale bar, 1 mm in A–E.
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