O-GlcNAc on NOTCH1 EGF repeats regulates ligand-induced Notch signaling and vascular development in mammals
- Nagoya University Graduate School of Medicine, Japan
- Albert Einstein College of Medicine, United States
- Nagoya City University, Japan
- Institute for Molecular Science and Okazaki Institute for Integrative Bioscience, National Institutes of Natural Sciences, Japan
Figures
Figure 1 with 1 supplement
EOGT promotes NOTCH1 binding to Delta ligands.
(A) Diagram of predicted O-glycans on mouse NOTCH1: red triangle, O-fucose glycans; blue circle, O-glucose glycans; blue square, O-GlcNAc glycans. Individual sugar residues that may extend O-fucose, …
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Figure 1—source data 1
Raw data for Figure 1C,D,E,G.
- https://doi.org/10.7554/eLife.24419.004
Figure 1—figure supplement 1
Generation and characterization of EOGT- and NOTCH1-null HEK293T cells.
(A) Schematic diagram of the CRISPR/Cas9 genome editing strategy to generate EOGT-null HEK293T cells. CRISPR/Cas9-mediated DNA cleavage caused frameshift mutations in all three EOGT alleles in …
Figure 2 with 1 supplement
O-GlcNAc on NOTCH1 EGF repeats promotes DLL4-NOTCH1 interactions.
(A) Ala substitution of Thr/Ser in the O-GlcNAc consensus site C5XXG(Y/F)(T/S)GXXC6 in EGF2, 10, 17, and 20 in the NOTCH1Δ4xO-GlcNAc mutant. (B) Cell surface expression of NOTCH1Δ4xO-GlcNAc is …
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Figure 2—source data 1
Raw data for Figure 2E.
- https://doi.org/10.7554/eLife.24419.007
Figure 2—figure supplement 1
NOTCH1 O-GlcNAc site mutants.
(A) Schematic of the EGF repeats of mammalian NOTCH1 identifying the potential O-GlcNAc consensus site, C5XXG(Y/F)(T/S)GXXC6, on EGF2, 10, 11, 14, 15, 17, 20, 23, 26, 27 and 29. These consensus …
Figure 3
Notch signaling is reduced in EOGT-deficient cells.
(A) Knockdown of EOGT inhibits NOTCH1 activation cleavage. HeLa cells stably expressing shRNAs targeting GAPDH or EOGT (TA195, TA196, TA197, TA198) were co-cultured with L cells or D1/L (D1) cells …
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Figure 3—source data 1
Raw data for Figure 3E.
- https://doi.org/10.7554/eLife.24419.010
Figure 4 with 2 supplements
Generation of Eogt-targeted mice.
(A) Schematic drawing of the wild-type mouse Eogt allele (WT), the targeting vector, the floxed allele with the neomycin (Neo)-resistance gene (EogtflNeo), the floxed allele without Neo (EogtF), and …
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Figure 4—source data 1
Raw data for Figure 4E.
- https://doi.org/10.7554/eLife.24419.012
Figure 4—figure supplement 1
Deletion of exon 10 in the Eogt gene causes exon nine to be spliced to exon 11 leading to a frame shift encoding six novel amino acids before a stop codon.
Truncated EOGT protein was not detected in lung indicating nonsense-mediated decay of mutant transcripts.
Figure 4—figure supplement 2
Whole-mount in situ hybridization for Eogt.
(A) In situ hybridization was performed in Eogt+/− or Eogt−/− retinas using anti-sense or sense probes. (B) P5 control Tek-Cre and Tek-Cre EogtF/F retinas were subjected to in situ hybridization …
Figure 5 with 1 supplement
EOGT regulates retinal angiogenesis.
(A) Whole-mount images of Eogt+/+ or Eogt−/− P5 retinas stained with IB4 and anti-αSMA antibody. Bars represent 1000 μm. Scatter plots at right show vascular progression per quadrant length, per …
Figure 5—figure supplement 1
Images of control Tek-Cre and Tek-Cre EogtF/F retinas stained with isolectin B4 (IB4) and quantification of branch points in P5 (N = 6 mice) and P15 (N = 3 mice) retinas and numbers of filopodia per mm vascular front in P5 retinas (N = 6 mice).
Data were normalized from mean ± standard error; p values were calculated by unpaired two-tailed Student’s t-test. *p≤0.05; **p≤0.01.
Figure 6
Reduced Notch signaling augments the loss of Eogt in retinal angiogenesis.
(A) Images of vessel density in P5 retinas from Eogt−/− compared to compound mutant mice. (B) Scatter plots represent branch point numbers in P5 retinas from Eogt−/− compared with compound mutant …
Figure 7 with 1 supplement
Reduced vessel integrity in the Eogt−/− retina.
(A) Immunostaining with fibrinogen (green) and α-SMA (magenta) antibodies in P15 wild-type, Eogt−/−, Tek-Cre, Tek-Cre:EogtF/F, Notch1+/−, and Rbpj+/− retinas. Arrows indicate fibrinogen staining …
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Figure 7—source data 1
Raw data for Figure 7D.
- https://doi.org/10.7554/eLife.24419.019
Figure 7—figure supplement 1
Sulfo-NHS-LC-biotin was perfused into P15 wild-type, Eogt−/−, Tek-Cre, Tek-Cre;EogtF/F, Notch1+/−, Rbpj+/−, Eogt−/−Notch1+/−, Eogt−/−Rbpj+/− mice and stained with CF488A-conjugated streptavidin (green) and Dylight594-conjugated IB4 (white) immediately after perfusion.
The extravasation sites are shown by arrows.
Figure 8 with 1 supplement
EOGT acts on Notch receptors to regulate ligand-induced Notch signaling in ECs.
(A) Relative mRNA expression in purified brain EC cells. WT (gray) and Eogt−/− (dark gray) EC cells isolated from cerebrum using anti-CD31 antibody were analyzed for gene expression related to the …
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Figure 8—source data 1
Raw data for Figure 8A,B,D.
- https://doi.org/10.7554/eLife.24419.022
Figure 8—figure supplement 1
Dll4 expression is suppressed by inhibiting Notch signaling in ECs.
Cultured lung ECs from wild-type or Eogt−/− lungs were treated with DAPT (2 μM) or DMSO for 16 hr. Dll4 expression was analyzed by qRT-PCR. Gene transcripts were normalized to Gapdh. Data are mean ± …
Author response image 1
Tables
Table 1
Primers used for cloning DNA fragments, screening ES cells, or genotyping by PCR.
| Target region Exon10 Fw 5’–ATACGAAGTTATCACCGAACCTAGCCCATATTT–3’ Exon10 Rv2 5’–ACGAAGTTATGTCGACGACTGAGCATTGCTGTT–3’ |
| Long arm region Long arm Fw1 5’–CGAATCAAGCTGTTTGGTCCATTCTCTGCTCCA–3’ Long arm Rv 5’–ACGAAGTTATGGTACGGTCAACTTGAAGAAGTA–3’ |
| Short arm region Short arm Fw 5’–TAGGAACTTCCTCGAAATTCAGTGCTTAGAAGT–3’ Short arm Rv1 5’–GCGCGCCTTTCTCGAACACTGTGTACAGTGACA–3’ |
| Long-arm probe larm16380Fw 5’–CTGCCTCAGCTTCCTGAGTG–3’ larm17196Rv 5’–CATGTCAGATCAGACAGTTC–3’ |
| Short-arm probe sarm26294Fw 5’–CTGAGCTATGTACTGGATGC–3’ AscI-sarmRv2 5’–TGAAGAGGCGCGCCCAGAGACAGAAAAAGCAC–3’ |
| ES cells first screening PGK S1 5’–CCTCCCCTACCCGGTAGAATTGACC–3’ GL1 typing RV2 5’–GAACTGTCAGATTTGGTGACACAGAAAGGC–3’ |
| ES cells second screening 3rdlox Fw 5’–CCACCCGACCCCTGCCAGAACATAATGCTCTCTTGCATC–3’ 3rdlox Rv 5’–GCTGTCGCCAGAGGAGAGAGTGGGTGCTTACTTAC–3’ |
| Eogt mice genotyping 3rdloxFw 5’–CCACCCGACCCCTGCCAGAACATAATGCTCTCTTGCATC–3’ 3rdloxRv2 5’–GCTGTCGCCAGAGGAGAGAGTGGGTGCTTACTTAC–3’ 25307Rv 5’–CCAAGGCGGTCTTGGCCCAT–3’ |
| RT-PCR for Eogt Exon 9 Fw 5’– AGGCTACACGCAGCTCAATT –3’ Exon 11 Rv 5’– AGAAGCCGTGTTTTCGTTGC –3’ |
| qRT-PCR for Eogt Exon 1 Fw 5’–AAGCTGCAGGTCCGTGAAAA–3’ Exon 2 Rv 5’–TAGGTTAGGCTACCGCGTCT–3’ |
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Underlined sequences are 15 bp homologous overlaps required for In-Fusion cloning.
Table 2
Primers used for qRT-PCR.
| Target | Forward | Reverse |
|---|---|---|
| Notch1 | AGTGTGACCCAGACCTTGTGA | AGTGGCTGGAAAGGGACTTG |
| Notch2 | CCCAAGGACTGAGAGTCAGG | GGCAGCGGCAGGAATAGTGA |
| Notch3 | ATTTGAGGGGTGCTGAAGTG | GAAGGCTGGGACAGAGAGAA |
| Notch4 | TCCGGACTTTTAAGGCCAAA | TTCCCATTGCTGTGCATACTCT |
| Dll4 | CCCACAATGGCTGTCGTCAT | AACCCTTTGGCCCACTGTTG |
| N-cadherin | AGGGTGGACGTCATTGTAGC | CTGTTGGGGTCTGTCAGGAT |
| CD31 | AGCCAACAGCCATTACGGTTA | AGCCTTCCGTTCTCTTGGTG |
| GAPDH | GGTGCTGAGTATGTCGTGGA | CCTTCCACCATGCCAAAGTT |
| Jag1 | TCTCTGACCCCTGCCATAAC | TTGAATCCATTCACCAGATCC |
| Hes1 | ACACCGGACAAACCAAAGAC | CGCCTCTTCTCCATGATAGG |
| Hey1 | CATGAAGAGAGCTCACCCAGA | CGCCGAACTCAAGTTTCC |
| Hey1 (mouse specific) | TGAATCCAGATGACCAGCTACTGT | TACTTTCAGACTCCGATCGCTTAC |
| Vegfr1 | ACATTGGTGGTGGCTGACTCTC | CCTCTCCTTCGGCTGGCATC |
| Vegfr2 | GCGGGCTCCTGACTACAC | CCAAATGCTCCACCAACTCTG |
| Vegfr3 | CCGCAAGTGCATTCACAGAG | TCGGACATAGTCGGGGTCTT |
