Extended Data Figure 3: Inducible overexpression of a constitutively active FOXO1 mutant in ECs of mice.

a, A cassette containing the CAG promoter, a floxed STOP sequence, a cDNA encoding for Foxo1^CA, and IRES-GFP was inserted into the Rosa26 locus. A schematic representation of the wild-type Rosa26 locus, the floxed allele, and the recombined allele after cre expression is shown. b, Immunofluorescence staining for FOXO1, GFP and PECAM in P5 Foxo1^iEC-CA and control mice. c, Confocal images of mTmG⁺ control and Foxo1^iEC-CA mice that were injected with 4-OHT from P1 to P4 and analysed for GFP, ERG and IB4 expression. The right half of both images shows the GFP signal alone. d, High-magnification images of IB4-stained retinal vessels at the angiogenic front in control and Foxo1^iEC-CA pups. e, BrdU and IB4 labelling of whole-mount P5 retinas reveals reduced endothelial proliferation in Foxo1^iEC-CA animals. f, Confocal images showing MYC and PECAM immunostaining in P5 retinas of control and Foxo1^iEC-CA mice. The lower half of both images shows the MYC signal alone. g, Quantification of FOXO1 nuclear staining intensity in ECs (n = 3), radial migration (n = 10), endothelial coverage (n = 10), branch points (n = 10), and endothelial BrdU incorporation (n ≥ 6) in P5 retinas of control and Foxo1^iEC-CA mutant mice. Data represent mean ± s.d. Two-tailed unpaired t-test. **P < 0.01; ***P < 0.001; ****P < 0.0001.