Fig 2: ERG regulates endothelial Notch signalling.(a) Western blot (WB) analysis of Notch intracellular domain (NICD) expression in control (siCtrl) and ERG-deficient (siERG) HUVEC (n=4). (b) RBP-J TP-1 Notch reporter activity in control and ERG-deficient HUVEC (n=4). (c) qPCR of Notch target gene expression in siCtrl and siERG-treated HUVEC: Hes1, Hey1, Nrarp and p21 (n=4). (d) Microarray and PCR screen analysis of differential gene expression in HUVEC was performed at 24 and 48 h after ERG inhibition29, with fold change of selected genes represented as high (red) and low (blue) expression compared to the median (grey). (e) qPCR analysis of Hes1 and Hey1 Notch target gene expression in siCtrl and siERG-transfected HUVEC stimulated with Dll4 or control BSA (n=4). (f) RBP-J TP-1 Notch reporter activity in control and ERG-deficient HUVEC plated on Dll4 or BSA (n=4). (g) WB analysis and quantification of NICD expression in siCtrl and siERG-transfected HUVEC stimulated with Dll4 or BSA (n=4). (h) qPCR analysis of Notch1 and Notch4 mRNA expression in siCtrl and siERG-transfected HUVEC stimulated with Dll4 or BSA (n=4). All graphical data are mean±s.e.m., *P<0.05, **P<0.01, ***P<0.001, Student’s t-test.

Fig 3: Individual ECs with loss of Rbpj/Notch signalling often express p21 and occupy the leading edge. a–d The induction of Rbpj genetic deletion from P1 to P3 results in a strong increase of p21 and P-ERK levels at the AF in P6 retinas. Charts show quantification of several large microscopic fields (n = 3 mice per group). Scale bars, 200 µm. e Confocal micrographs of P6 retinas from animals with inducible mosaic deletion of Rbpj in ECs (RbpjiECMosaic-KO) expressing MbTomato-2A-Cre. Images to the right are high-magnification views of boxed areas, showing the indicated signals in more detail. Scale bar, 115 µm. f, g Quantification of mutant (Tomato+) and control (Tomato-) ECs (ERG+) from RbpjiECMosaic-KO retinas (n = 6). Individual cells without Rbpj/Notch signalling (MbTomato-2A-Cre+) are more frequently p21+, especially if they are at the leading edge of retina vessels. In the mature vascular area, most ECs with Rbpj deletion do not upregulate p21. Note that a fraction of Tomato- cells may have Rbpj deletion (compare white bars in f with g). h Cells without Rbpj/Notch signalling (MbTomato-2A-Cre+), are more frequently found at the leading edge of vessels than at the angiogenic front. Error bars indicate std. dev; ***p < 0.0005. Two-tailed unpaired t test. Source data are provided as a Source Data file

Fig 4: Angiogenic ECs with high VEGFR2 and ERK signalling exit the cell cycle. a Confocal micrographs of the retinal angiogenic front in iChr-Cerulean/GFP x Tie2-Cre reporter mice (see also Supplementary Fig. 4a), showing labelling of EC nuclei (Cerulean/GFP+). Activated ERK levels (P-ERK, red) are high in the nuclei and cytoplasm of endothelial tip cells (left image). Dll4/Notch inhibition increases P-ERK levels, particularly in stalk cells. b Quantification of P-ERK signals in tip and stalk cells from animals 24 h after injection with control IgG or anti-Dll4. Comparison of indicated parameters in large microscopic fields of IgG control (n = 3) or anti-Dll4 (n = 4) treated mice. c iMb-Vegfr2 Mosaic construct with genetic distances in kilobases (kb). d ECs expressing VEGFR2Ac (MbTomato + , yellow arrows) have high P-ERK levels, like some endothelial tip cells (white arrowheads). See also Supplementary Fig. 5b. e–g ECs expressing VEGFR2Ac (MbTomato+) or VEGFR2TKMut (MbYFP+) exit the cell cycle. Only a small fraction of MbTomato+ or MbYFP+ ECs are in S-phase (Erg+/EdU+-white nuclei), indicated with white arrowheads. Chart shows quantification of several microscopic fields from retinas of different mice (n = 5). h, i Most single ECs with very high (MbTomato+) or low (MbYFP+) VEGFR2 signalling do not divide over a 3 day period. The iChr-Control-Mosaic allele was used to increase single-cell pulse–chase clonal resolution. Chart shows quantification of several clones in large microscopic fields (n = 4 retinas per group). j, k The increase in VEGFR2 activity in iMb-Vegfr2-Mosaic Cdh5-CreERT2 retinal vessels from P2 to P6 arrests physiological angiogenesis. MbTomato labelling shows the mosaic induction of the transgene in the endothelium (isolectinB4+), resulting in a significant decrease in vascular development. Chart shows quantification of large microscopic fields from several retinas of control (n = 3) and mutant (n = 2) mice. Scale bars in all panels, 100 μm. Error bars indicate std. dev.; NS nonsignificant; ***p < 0.0005; **p < 0.005. One-way ANOVA with Tukey’s post hoc test (b, g) or two-tailed unpaired t test (k). Source data are provided as a Source Data file

Fig 5: Inducible iMb-Vegfr2-Mosaic ES Cells and Mice(A) iMb-Vegfr2-Mosaic DNA construct. Below the genetic distance (kb) between different LoxP sites and the relative recombination ratios obtained after Cre transfection.(B) Representative picture of iMb-Vegfr2-Mosaic ES cells expressing the different fluorescent proteins.(C) Confocal micrographs showing the MbYFP- and MbTomato-expressing cells in a vasculature of a P6 mouse retina, 3 days after tamoxifen injection. IsolectinB4 labels the surface and ERG the nuclei of all ECs. Each dot in the chart indicates the identified ratio of EC surface area occupied by MbTomato- and MbYFP-expressing cells in each microscopic field.(D) Immunostaining of retinal vessels for phospho-ERK. Charts show that P-ERK signals are higher in most tip cells (arrowhead) and in MbTomato+ cells (arrows).(E) Immunostaining of retina ECs (nuclei, ERG+) for the indicated markers 3 days after the tamoxifen pulse. Most YFP+ cells have no EdU labeling (arrows) and few are EdU+ (pink nuclei, arrowhead inset).(F) Embryoid bodies derived from ES cells were plated on a OP9 monolayer to induce EC differentiation and sprouting. Colored bars indicate occurrence of each cell population among all recombined cells at the ES cell stage and after differentiation to ECs.In (D) and (E), error bars SD, ∗p < 0.05.