Extended Data Fig. 1: GLUL knockout impairs vessel sprouting.

a, GLUL mRNA levels in HUVECs (n = 9 donors), lung ECs (n = 5), colon ECs (n = 4), liver ECs (n = 3), human umbilical artery ECs (HUAECs) (n = 2) and human blood outgrowth ECs (BOECs) (n = 2); (mean ± s.e.m.; *P < 0.05 versus HUVEC, Student’s t-test) and in HEPG2 cells (mean ± s.e.m.; n = 3; *P < 0.05 versus HUVEC, Student’s t-test). b, c, Western blot of GS protein levels in HUVECs and HEPG2 cells in medium containing 0.6 mM glutamine (+) or 0.025 mM glutamine (−) (b), and in isolated mouse liver ECs (mLiECs) and mouse astrocytes (c) (representative immunoblots of two independent experiments are shown). d, e, Genomic organization of the loxP-flanked Glul allele before and after Cre-mediated excision (d) and correct recombination of the lox allele (L) in Glul^vECKO and Glul^pECKO mice upon tamoxifen (Tam) treatment, as assessed by genomic DNA PCR (e; the PCR to amplify the loxP-flanked Glul allele (lox) or to amplify the cre-recombined allele (∆) were run in separate reactions but loaded in the same lane; the gel picture shown is representative of all control, Glul^vECKO and Glul^pECKO mice used in this study). f, Quantification of branchpoints at the rear of the plexus in Glul^vECKO mice (mean ± s.e.m.; n = 10 mice for Glul^vECKO and 11 for WT controls from 3 litters; *P < 0.05 versus WT littermates, mixed-models R statistics). g, Pericyte coverage of retinal microvessels in WT and Glul^vECKO littermates determined by NG2 staining and shown as the NG2⁺ area as a percentage of the vessel area (mean ± s.e.m.; n = 4 mice for WT and 3 for Glul^vECKO from 1 litter; NS, P > 0.05 versus WT, Student’s t-test). h, Reduced complexity of the retinal vascular front in P5 Glul^vECKO compared with WT mice, determined by the number of branches on distal sprouts (mean ± s.e.m.; n = 13 mice for WT and 21 for Glul^vECKO from 5 litters; *P < 0.05 versus WT, Student’s t-test). i, Quantification of EdU⁺ ECs at the rear of the plexus (mean ± s.e.m.; n = 12 mice for WT and 22 for Glul^vECKO from 4 litters; NS, P > 0.05 versus WT littermates, Student’s t-test). j–m, IB4 staining of P5 retinal vascular plexuses from WT (j) and Glul^pECKO (k) mice (representative pictures with magnification shown in the inset; A, artery; V, vein) and quantification of branch points at the front (l) and the rear (m) of the plexus (mean ± s.e.m.; n = 10 mice for WT and 18 for Glul^pECKO from 4 litters; *P ≤ 0.05 versus WT littermates, Student’s t-test). n–u, IB4 staining of the retinal microvasculature of three-week-old (P21) (n, o) and six-week-old (P42) (r, s) WT and Glul^vECKO littermates (A, artery; V, vein). The lower left insets display a higher magnification of the IB4-stained superficial plexus, whereas the lower right insets display a higher magnification of the deep plexus. The corresponding quantification of the vascular area (p, t) and the branch point density (q, u) in the superficial and the deep layer is also shown (mean ± s.e.m.; n = 8 mice for WT and 8 for Glul^vECKO at P21, from two litters; n = 10 mice for WT and 14 for Glul^vECKO at P42, from four litters; NS, P > 0.05 versus WT, Student’s t-test). v–ag, Representative micrographs of heart (v, z), liver (w, aa) and kidney (x, ab) sections from WT and Glul^vECKO littermates immunostained for the EC marker endoglin and of lung (y, ac) sections immunostained for the EC marker CD34 and corresponding quantifications of endoglin⁺ (ad, heart; ae, liver; af, kidney) or CD34⁺ (ag, lung) vascular area (mean ± s.e.m.; n = 5 mice (4 for heart) for WT and 7 (6 for heart) for Glul^vECKO, from two litters, NS, P > 0.05 versus WT, Student’s t-test). ah–ai, Images of flat-mounted retinas from control (ah) and MSO-treated (ai) ROP mice (vaso-obliterated area in white). Images shown are representative of 7 (ah) and 6 (ai) mice. Exact P values: HUVEC versus lung ECs: 0.0278; HUVEC versus colon ECs: 0.1086; HUVEC versus liver ECs: 0.3334; HUVEC versus HEPG2: <0.0001 (a); <0.0001 (f); 0.3491 (g); <0.0001 (h); 0.8247 (i); 0.0012 (l); 0.050 (m); superficial: 0.1218; deep: 0.1720 (p); superficial: 0.9995; deep: 0.4289 (q); superficial: 0.9792; deep: 0.6602 (t); superficial: 0.7979; deep: 0.1275 (u); 0.9021 (ad); 0.2279 (ae); 0.7647 (af); 0.3614 (ag). Scale bars: 200 μm (j, k, n, o, r, s), 20 μm (v–ac), 1 mm (ah–ai). For gel source images, see Supplementary Fig. 1.

Source data