Fig 2: 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 mice, normalized to Eogt+/+ mice. The average number of branch points for the Eogt+/+ used to compare mice with an N1- or Rbpj- allele was 237 ± 5, and for the Eogt+/+ compared to mice with an N12f or N1lbd allele was 380 ± 36 (500 X 500 µm field (n = 3–8 fields per retina), N = 2–6 mice). (C) Scatter plots showing the number of filopodia in P5 compound mutant mice as compared to Eogt-/- mice, normalized to Eogt+/+ mice. Each symbol represents the average number of filopodia per mouse (250 × 250 µm field (n = 4–12 fields per mouse), N = 2–4 mice. The average for Eogt+/+ compared to mice with a N1- or Rbpj- allele was 30 ± 1 per mm and for mice with a N112f or N1lbd allele was 36 ± 4 per mm, taken as 1.0 for normalization. (D) Images of branch points in P15 retinas comparing Eogt-/- to compound mutant mice as indicated. (E) Scatter plots shows branch points in compound mutants compared to Eogt-/- P15 retinas, normalized to Eogt+/+ mice. The average of the Eogt+/+ used to compare mice with a with a N1- or Rbpj- allele was 73 ± 3, and for mice with a N112f or N1lbd allele it was 69 ± 12 (500 x 500 µm field (n = 3–8 fields per retina), N = 3–6 mice. Data represent mean ± standard error except for P5 Eogt+/+Notch1+/- filopodia and P5 Eogt+/+Notch1+/lbd branch points and filopodia that are represented as mean ± range; p values were calculated by unpaired two-tailed Student’s t-test. *p=0.05; **p=0.01; ***p=0.001.DOI: http://dx.doi.org/10.7554/eLife.24419.017

Fig 3: 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 the deleted allele (Eogt-). Eogt exons (closed boxes); Neo and diphtheria toxin (DT3) genes served as positive and negative selection markers, respectively (open boxes); loxP sites (gray triangles); FRT sites (open triangles); KpnI (K), HindIII (H), and SalI (S) restriction sites. Homologous recombination between the WT allele and the targeting vector generated the EogtflNeo allele. The EogtF and Eogt- alleles were obtained by Flp-mediated and Ayu1-Cre-mediated recombination, respectively. Red lines indicate positions of probes used for Southern blotting. Positions of primers used for genotyping are indicated by closed triangles. (B) HindIII- or KpnI/SalI-digested genomic DNA isolated from WT (+/+) or heterozygous floxed neo (+/flNeo) ES cells was analyzed by Southern blotting using the short arm or long arm probe, respectively. In addition to signal from the WT allele (arrow), the flNeo mouse shows an additional band corresponding to the recombinant allele (arrowhead). (C) Genomic DNA isolated from WT, Eogt+/-, and Eogt-/- mice was subjected to genotyping using 3rdloxFw, 3rdloxRv, and 25307Rv primers. (D) Semi-quantitative RT-PCR analysis of total RNA from WT or Eogt-/- brain ECs using primers targeting exons 9 and 11. Minor transcripts lacking exon 10 were detected in Eogt-/- mice (arrow). Gapdh was amplified as an internal control. (E) Quantification of Eogt transcripts in Eogt-/- and WT mouse. qRT-PCR analysis of brain ECs showed a marked decrease in Eogt transcripts. Data are mean ± S.D. from three independent experiments performed in triplicate. Each experiment analyzed pooled total RNA obtained from 10 mice. (F) Lack of EOGT protein expression in Eogt-/- mouse. Lung lysates prepared from adult WT or Eogt-/- mice were analyzed in parallel with cell lysates from HEK293T cells overexpressing Eogt. Immunoblotting was performed using anti-EOGT or ß-tubulin antibodies. (G) Whole-mount in situ hybridization for Eogt in the P5 or P15 retina. Vascular staining of Eogt is evident in Eogt+/- retina. Eogt expression in vascular sprouts is indicated by arrows. (H) P5 control Tek-Cre and Tek-Cre EogtF/F retinas were subjected to in situ hybridization. Counter staining with Dylight 594-conjugated IB4 is shown below.DOI: http://dx.doi.org/10.7554/eLife.24419.01110.7554/eLife.24419.012Figure 4—source data 1.Raw data for Figure 4E.DOI: http://dx.doi.org/10.7554/eLife.24419.012

Fig 4: Reduced vessel integrity in the Eogt-/- retina.(A) Immunostaining with fibrinogen (green) and a-SMA (magenta) antibodies in P15 wild-type, Eogt-/-, Tek-Cre, Tek-Cre:EogtF/F, Notch1+/-, and Rbpj+/- retinas. Arrows indicate fibrinogen staining outside vessels stained by IB4 (white). Three-dimensional images were constructed from confocal images by maximum intensity projection. (B) Higher magnification three-dimensional images of Eogt-/- retina constructed from confocal images using the Alpha-blend method. Below, single channel images showing fibrinogen (green) and IB4 (white) staining. (C) Sulfo-NHS-LC-biotin was perfused into P15 wild-type and Eogt-/- mice and extravasation determined immediately after perfusion by staining with CF488A-conjugated streptavidin (green) and Dylight594-conjugated IB4 (white). Three-dimensional reconstructions were created by maximum intensity projection. Enlarged images of boxed area are shown (right). (D) Sulfo-NHS-LC-biotin was perfused into P15 wild-type, Eogt-/-, Notch1+/-, Eogt-/-Notch1+/- mice as in (C). Quantification of the number of extravasation sites in 210 × 210 µm squares (n = 6 per retina per mouse) is shown. Note that sulfo-NHS-LC-biotin extravasation in Eogt-/- retina is augmented in compound mutant mice. Data represent mean ± standard error; p values determined by Welch's t test. ***p=0.001. (E) Whole-mount images of wild-type or Eogt-/- P15 retinas stained with IB4 (cyan) and anti-aSMA (magenta) antibody. (F) Whole-mount staining of wild-type and Eogt-/- P15 retinas using IB4 (white) together with anti-fibrinogen (green) and anti-NG2 (magenta) antibodies.DOI: http://dx.doi.org/10.7554/eLife.24419.01810.7554/eLife.24419.019Figure 7—source data 1.Raw data for Figure 7D.DOI: http://dx.doi.org/10.7554/eLife.24419.019

Fig 5: 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 Notch signaling pathway. Gene transcript levels were normalized to Gapdh. Data are mean ± S.D. from three independent experiments performed in triplicate. Each experiment analyzed pooled total RNA obtained from 10 mice. *p<0.05; **p<0.01 (Welch's t test). (B) qRT-PCR analysis of Hes1 and Hey1 in wild type or Eogt-/- lung EC cells following stimulation with immobilized DLL4-Fc or JAG1-Fc. Gene transcripts were normalized to Gapdh. Data are mean ± S.D. from three independent experiments performed in triplicate. Each experiment analyzed total RNA obtained from a single mouse. Significance determined by Welch's t-test. *p<0.05; **p<0.01; #p<0.05; ##p<0.01 compared with the left-most Hes1 (*) or Hey1 (#) histogram. (C) Cells were transfected to express Dll4 or Jag1 and subjected to flow cytometry using DLL4 or JAG1 antibody, respectively. Comparison with mock-transfected cells indicated the amount of exogenously expressed DLL4 and JAG1 on the cell surface in wild-type or EOGT-null HEK293T cells. Mock-transfected HEK293T cells were labeled without primary antibody (Cont.). (D) Dll4/Jag1-transfected cells or Mock transfectants (signal-sending cells) were co-cultured with wild type or Eogt-/- lung EC cells (signal-receiving cells). qRT-PCR analysis of mouse Hey1 expression suggested that EOGT is required for the ability to receive DLL4-mediated Notch signaling, and that EOGT is dispensable for DLL4 and JAG1 as inducers of Notch signaling. Gene transcript levels were normalized to PECAM1 (CD31). Data are mean ± S.D. from three independent experiments performed in triplicate. Significance determined by Welch's t-test. ***p<0.001.DOI: http://dx.doi.org/10.7554/eLife.24419.02110.7554/eLife.24419.022Figure 8—source data 1.Raw data for Figure 8A,B,D.DOI: http://dx.doi.org/10.7554/eLife.24419.022