Extended Data Figure 2: Endothelial FOXO1 deficiency leads to abnormal vessel size and shape.

a, Immunostaining for VECAD and ERG in Foxo1^iEC-KO and control retinas. The bottom panels show the isolated VECAD and ERG signals of the inset. b, Confocal images showing maximum intensity projections and X–Y, X–Z, and Y–Z planes of a thick stack of IB4 and collagen IV (COL) stained P5 retinas. Foxo1^iEC-KO mice develop enlarged vessels with abnormal lumen organization. White arrowheads point to areas with multiple vessel layers and intraluminal collagen strands. c, Images of IB4- (cyan) and TER119- (red) stained P5 retinas of control and Foxo1^iEC-KO mice. Note that aggregates of TER119⁺ red blood cells form in Foxo1^iEC-KO but not in control mice. d, Phospho-histone H3 (pHH3) and IB4 immunostaining of P5 Foxo1^iEC-KO and control mice. e, Images of IB4-stained retinas at P21 showing an increased vessel density in Foxo1^iEC-KO mice (same samples as in Fig. 1h). f, Higher magnification images of ERG-, ICAM2- and IB4-stained retinas at P21 showing increased numbers of ECs in the perivenous plexus of Foxo1^iEC-KO mice. g, Bar graphs showing the mean endothelial area (n ≥ 8), mean diameter of central vein (n ≥ 8), and number of ERG/IB4⁺ cells (n ≥ 4) in P21 retinas of Foxo1^iEC-KO and control mice. Data represent mean ± s.d. Two-tailed unpaired t-test. ****P < 0.0001.