Fig 2: Endothelial Gja1 regulates β‐cell expansion Representative tile scan 3D images with α‐SMA, Isolectin, FABP4 and Emcn in the pancreas from 17‐week‐old Gja1 iΔEC mutant and control mice. Insets show higher magnifications of regions.Box whiskers, and scatter plot (left) shows the quantification of CD31+ Emcnhi vessel density in Gja1 iΔEC mutant and control pancreatic islets. (n = 7), P‐value and two‐tailed unpaired t‐test. Combo plot (right) shows the quantification of P‐Histone H3+ EC numbers per islet (f.u) counted on thick murine pancreatic sections. (n = 5), P‐value and two‐tailed unpaired t‐test.3D images show CD31 and Emcn immunostaining in the Gja1 iΔEC mutant and control pancreas. The scale bars are 50 µm.Combined violin, scatter and sample distribution plot shows the quantification of β‐cell mass (%) in pancreatic islets from Gja1 iΔEC and control mice. Data represent mean ± s.d. (n = 140 islets from seven pancreas), P‐value and two‐tailed unpaired t‐test.Representative 3D images with immunostaining of α‐SMA, insulin and VE‐Cadherin in the pancreatic islets from Gja1 iΔEC and control mice. The scale bars are 50 µm.Combo plot (left) shows insulin measurement in lysate of pancreas by ELISA. Combo plot (right) shows the percentage of insulin+ β cells per islet in Gja1 iΔEC mutant and control mice. (n = 5), P‐values and two‐tailed unpaired t‐tests.Representative 3D images of ESM‐1 and Caveolin‐1 in pancreatic islets from Gja1 iΔEC and control mice. Insets (* and #) show higher magnifications with surface rendering for ESM‐1 and Caveolin‐1 immunostaining. Combo plot with the quantification of ESM‐1+ cell numbers per islet (f.u). (n = 5), P‐value and two‐tailed unpaired t‐test. The scale bars for the 3D images are 50 µm, and 5 µm for the higher magnifications with single‐cell resolution images.3D images with CXCR4 and Emcn immunostaining in Gja1 iΔEC and control pancreas. The scale bars are 50 µm.Representative 3D images show CXCR4, Ki67 and VE‐Cadherin immunostaining in Gja1 iΔEC and control pancreas. The scale bars are 50 µm.Combo plots show the percentage of CXCR4+ β cells per islet and Ki67+ β cells normalized to the total number of β cells per islet in Gja1 iΔEC mutant versus the littermate control mice. (n = 5), P‐values and two‐tailed unpaired t‐tests.Box whiskers, and scatter plot (top) shows the quantification of CD31+ Emcnhi vessel density in aged Gja1 iΔEC mutant and control pancreatic islets. Data represent mean ± s.d. (n = 4), P‐value and two‐tailed unpaired t‐test. Combo plot (below) shows the quantification of β cells on thick murine pancreatic sections. Data represent mean ± s.d. (n = 4), P‐value and two‐tailed unpaired t‐test. Data information: The dashed lines in panel A represent the outlines of pancreas. The dashed lines in panels (C, E, G, H and I) represent the outlines of pancreatic islets. Two‐tailed Student's t‐test was performed for the statistical analysis. **P < 0.01; ****P < 0.0001. Nuclei: DAPI or TO‐PRO‐3 as indicated. Is: pancreatic islet. f.u: fluorescence unit. The line on the right side of the combo plots represents the sample distribution. Scale bars are 200 µm for tile scan images of longitudinal thick sections across the entire glands, and 50 µm for the higher magnification insets images.

Fig 3: Properties of islet endothelium and age-associated modulations Representative 3D images with insulin and Caveolin-1 immunostaining in pancreatic islets from young mice. Combined box and whiskers, and scatter plots shows the vessel density analysis in the pancreatic islets and non-islet areas from young and aged mice.Representative 3D images with adult pancreatic islets immunolabelled with Ki67 and VE-Cadherin. Inset (*) shows single-cell resolution and surface rendering for all the channels.Representative tile scan 3D image with insulin, ESM-1 and Emcn immunostaining in 19-week-old mouse pancreas. Higher magnification at the top of the right panel shows the ESM-1+ pancreatic islets. Inset (*) shows single-cell resolution and surface rendering for ESM-1 and Emcn. The white and dashed line in the tile scan image represents the outline of one lobe of the pancreas, and the dashed lines in the inset represent the pancreatic islet.Representative 3D images show CD31 and Emcn expressions in young pancreas. Insets (* and #) show higher magnifications of capillaries in the pancreatic islets and non-islet areas.Representative flow cytometry dot plots showing CD31+ Emcnhi and CD31+ Emcnlo EC populations in young and aged pancreas. Combo plot shows the population of CD31+ Emcnhi ECs in young and aged pancreas.Representative 3D images (left) with insulin, Ki67 and VE-Cadherin immunostaining on young and aged pancreas. 3D images (middle) show CXCR4, Ki67 and VE-Cadherin in young and aged pancreatic islet. Insets (yellow asterisks) show single-cell resolution and surface rendering for Ki67 and CXCR4. Arrowheads represent Ki67+ ß-cell.Combo plot shows the quantification of Ki67+ ß cells normalized to the total number of ß cells per islet in juvenile (3 weeks old), adult (12 weeks old) and aged (55 weeks old) pancreatic islets.Representative 3D images with insulin, CD31 and Emcn immunostaining in young and aged pancreas. Combo plot shows the CD31+ Emcnhi vessel density in juvenile, adult and aged pancreatic islets.Representative 3D images of adult (10 weeks old) and aged pancreas (55 weeks old) with insulin and VEGFA immunostaining. Combo plot (middle) shows the VEGFA as determined by ELISA in isolated islets from 10-week-old adult and 55-week-old aged mice. Combo plot (right) shows the percentage of VEGFA per islet (f.u). Data information: The white and dashed lines in panels (A, B, D, F, H and I) represent the outlines of pancreatic islets. (n = 5), P-values and two-tailed unpaired t-tests in panels (A, E and I) and one-way ANOVA tests with Tukey's multiple comparisons tests in panels (G and H). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Nuclei: TO-PRO-3. Is: pancreatic islet. f.u: fluorescence unit. For the combo plots, the boxes represent mean ± s.d., line in the box is the median, and the lower and upper lines show the minimum and the maximum of the values. The line on the right side of these combo plots represents the sample distribution. Scale bars are 200 µm for tile scan 3D images in panel (C), 50 µm for 3D images and 5 µm for the higher magnification single-cell resolution images in panel (B–D and F).

Fig 4: Abnormal sprouting during retinal angiogenesis in Pacsin2-/- mice.a Immunofluorescent images of whole-mount retinas stained for Isolectin B4 (IB4, red, as marker of blood vessels) and the endothelial transcription factor ERG (cyan, marker of endothelial nuclei) from control (Pacsin2+/+) and Pacsin2-/- mouse littermates at P6. Representative images of at least ten retinas per genotype from three independent littermates. White rectangles indicate regions of interest (ROI) of which magnifications are shown below. b Representative images of the sprouting front of retinas from control and Pacsin2-/- mouse littermates at P6 stained for ERG (red), IB4 (blue), and VE-cadherin (green). Punctuated lines indicate the sprouting front boundary. The red arrows indicate nuclear clusters in abnormal sprouts. c Quantification of the number of branch points per unit of area, the number of sprouts per 100 µm of sprouting front border, and the average length of sprouts (at least n = 10 retinas per genotype, from three independent littermates). P = 0.0295 when comparing the sprout length in Pacsin2+/+ to Pacsin2-/- retinas. d Quantification of the number of nuclei per 100 µm of sprouting front border, the number of endothelial cell clusters per sprout, and the percentage of proliferating cells based on cytoplasmic (dividing cells) versus nuclear ERG (non-dividing cells) stainings per unit area in the sprouting front of control and Pacsin2-/- P6 retinas (at least ten retinas per genotype, from at least three independent littermates). P = 0.0015 when comparing the number of nuclei, and P = 0.0242 when comparing the number of endothelial clusters in Pacsin2+/+ to Pacsin2-/- retinas. Quantification of the number of tip cells (ESM1+) per total number of ECs at the sprouting front (at least four retinas per genotype, from two independent littermates). e Representative high-resolution images of the sprouting front from control and Pacsin2-/- mice stained for VE-cadherin (green), ERG (red), and IB4 (blue). The black dotted squares indicate the ROIs that are magnified in the panels below. f Quantification of the intensity of cytoplasmic VE-cadherin at the vascular sprouts of control and Pacsin2-/- retinas (at least seven retinas per genotype, from two independent littermates). P = 0.0401 when comparing Pacsin2+/+ to Pacsin2-/- retinas. All quantifications represent mean ± SEM (error bars) and the statistics were performed by the two-sided Mann–Whitney test. ns non-significant; *P < 0.05; **P < 0.01. Scale bars, 100 and 50 µm (a), 40 µm (b), 20 and 5 µm (e). Arb. units arbitrary units. Source data are provided as a Source Data file.

Fig 5: Age-associated vascular modulations in mouse endocrine glands Young and aged adrenal gland immunolabelled with HSPG2, NG2 and Podocalyxin. Higher magnification insets show cortex (Co) and medulla (Me). Combined box and whiskers, and scatter plot with quantification of vessel density in young and aged adrenal glands.Young and aged ovary immunolabelled with Sca-1, PDGFRß and Emcn. Higher magnification insets show cortex and medulla. Combo plot with quantification of vessel density in young and aged ovary.Young (10 weeks old) and aged pituitary gland immunolabelled with ESM-1, collagen IV and Emcn. Insets with higher magnifications of pars distalis and pars nervosa regions. Combo plot shows quantification of vessel density.Young and aged testis immunolabelled with Tie-2 and HSPG2. Higher magnifications of cortex and medulla. Combo plot shows quantification of vessel density.Young and aged pancreas immunolabelled with SM22a and Endoglin. Insets representative of higher magnifications of regions in young and aged pancreas. Combo plot shows quantification of vessel density.Young and aged thyroid gland immunolabelled with CXCR4, laminin and Emcn. Combo plot shows quantification of vessel density in young and aged thyroid glands. Insets: periphery and centre of the thyroid gland. Data information: The white and dashed lines in each panel represent the outlines of each organ. (n = 5), P-values and two-tailed unpaired t-tests. ns: not significant; **P < 0.01; ***P < 0.001. Nuclei: TO-PRO-3. For the combo plots, the boxes represent mean ± s.d., line in the box is the median, and the lower and upper lines show the minimum and the maximum of the values. The line on the right side of these combined plots represents the sample distribution. Co, cortex; Ct, centre; Me, medulla; Pd, pars distalis; Pe, periphery; Pn, pars nervosa. Scale bars for tile scan 3D images 200 µm, for insets 50 µm.