Fig 1.
Differential expression of Sox7, Sox17, and Sox18 in the retinal vasculature.
(A-C) Sox7 (A), Sox17 (B), and Sox18 (C) are expressed in ECs as determined by anti-Sox7, anti-Sox17 and anti-Sox18 immunostaining of P7 retinas. All retina images in this study are flat mounts. GS-lectin staining marks all blood vessels and anti-smooth muscle actin (SMA) marks arteries. The left panels were imaged in the mid-peripheral retina and the right panels were imaged near the front of the growing vascular plexus; the front is at the top of each image. Scale bar, 200 μm. (D) Mid-periphery of a P7 retina stained with anti-Sox7 (green) and anti-Sox17 (red). Sox17 is abundant in arteries and arterioles. Sox7 is abundant in arterioles but not arteries. A, artery; AO, arteriole; V, vein. Scale bar, 200 μm. (E,F) Mid-periphery of a P7 retina (E) and center of a P11 retina (F) stained with anti-Sox7 (green) and anti-Sox17 (red). Sox17 is abundant in radial arteries but not veins. Sox7 is abundant in veins but not arteries. A, artery; AO, arteriole; V, vein. Scale bar, 200 μm. (G) Summary of immunostaining intensities for SoxF proteins at P7, and extent of reduction in staining intensity in adult retinas (modest reduction, one downward arrow; large reduction, two downward arrows). 1, weak staining; 2, moderate staining; 3, strong staining. Ratings were determined by visual inspection of >100 retinas.
Fig 2.
Deletion of Sox7, Sox17, or Sox18 has little or no affect on vascular development in the retina.
(A-C) P7 retinal vasculature in WT (A-C, left panels), Sox7CKO/-;Pdgfb-CreER (A, right panels), Sox17CKO/-;Pdgfb-CreER (B, right panels), and Sox18-/- (C, right panels) shown by GS-lectin staining, with vSMC coverage of radial arteries visualized with anti-SMA. 50 μg 4HT was given at P2. Scale bar, 200 μm. (D-F) P20 retinal vasculature in WT (D-F, upper panels), Sox7CKO/-;Pdgfb-CreER (D, bottom panels), Sox17CKO/-;Pdgfb-CreER (E, bottom panels), and Sox18-/- (F, bottom panels) shown by GS-lectin staining. All three vascular layers are present in the knockout retinas. Anti-Sox7, anti-Sox17, and anti-Sox18 immunostaining show antibody specificity (the immunostaining signal is present in WT and absent in knockout retinas stained in parallel), and conditional knockout efficiency by CreER. 50–100 μg 4HT was given at P2-4. IPL, inner plexiform layer. OPL, outer plexiform layer. Scale bar, 200 μm.
Fig 3.
Sox7 expression is up-regulated in Sox17-/- ECs in mosaic Sox17CKO/-;Pdgfb-CreER retinal vasculature.
(A-E) Flat mount retinas stained with anti-Sox7 and anti-Sox17 from Sox7CKO/-;Pdgfb-CreER mice at P7 (A,B), and from Sox17CKO/-;Pdgfb-CreER mice at P7 (C,D) and at P11 (E), following 50 μg 4HT at P2. (B,D) Scatterplots of Sox7 pixel intensity vs. Sox17 pixel intensity; (B) and (D) correspond to (A) and (C), respectively. EC nuclei in Sox17CKO/-;Pdgfb-CreER retinal vasculature express either Sox17 (i.e. unrecombined Sox17+ cells; lower white arrow in E) or Sox7 (recombined Sox17- cells; upper white arrow in E). Scale bar, 50 μm.
Fig 4.
Combined loss of Sox7, Sox17, and Sox18 in ECs alters retinal vasculature.
(A) Summary of the triple SoxF cross and the resulting progeny with abbreviations for their genotypes. (B, C) Analysis of retinal vasculature in SoxF mutants at P5 (B) and P8 (C). Top panels, retina flat mounts immunostained with anti-endomucin, anti-SMA and anti-HA. Bottom panels, retina flat mounts stained with GS-lectin and anti-Sox17. Anti-HA and anti-Sox17 staining assesses CreER-mediated recombination efficiency. The retinal vasculature in Sox18+/- is indistinguishable from WT. The mutants that lack Sox17 and either one or both copies of Sox7 and Sox18 show greatly reduced vSMC coverage of radial arteries with increased capillary density. 50–100 μg 4HT was given at P0 and P2. Scale bar, 200 μm. (D) Cerebellum sections from Sox7CKO/-;Sox17CKO/CKO;Sox18-/-;Pdgfb-CreER and Sox18+/- control at P5, following 50–100 μg 4HT at P0 and P2. Vascular disorganization (arrows) is seen in Sox7CKO/-;Sox17CKO/CKO;Sox18-/-;Pdgfb-CreER. Scale bar, 200 μm.
Fig 5.
Excess tip cell production in the Sox7, Sox17, and Sox18 EC-specific triple KO (‘6-allele loss’) at P6.
(A-J) Retina flat mounts from control Sox18+/- (A, C, E, G, I) and 6-allele loss Sox7CKO/-;Sox17CKO/CKO;Sox18-/-;Pdgfb-CreER (B, D, F, H, J) mice at P6. In (I) and (J), retinas were labeled with Alexa488 GS-lectin (green) via intracardiac injection and Alexa595 GS-lectin (red) after postfixation and permeabilization. The dashed lines delineate the boundary between hypoxia/normoxia in (A and B), and perfused/non-perfused vessels in (J). In control retinas, most tip cells (arrows) express higher levels of Dll4 (C) and VEGFR3 (E) than adjacent capillaries. The larger zone of hypoxia in (B) compared to (A) reflects the retarded growth of the vascular plexus. 50–100 μg 4HT was given at P0 and P2. A, artery; V, vein. Scale bar, 200 μm.
Fig 6.
Sox7, Sox17, and Sox18 EC-specific triple KO in adulthood leads to widespread peripheral edema.
(A) A representative Sox7CKO/-;Sox17CKO/CKO;Sox18-/-;Pdgfb-CreER mouse (right) that had received 2–3 mg tamoxifen at P21, P25 and P29 developed an edematous phenotype at P40. The control Sox18+/- littermate (left) that had received the same tamoxifen treatment was unaffected. (B) Haematoxylin and eosin-stained sections of the abdominal wall from a control (left panel) mouse and a Sox7CKO/-;Sox17CKO/CKO;Sox18-/-;Pdgfb-CreER mouse (right panel) at P33, following 2–3 mg tamoxifen at P21, P23 and P25. Dermal edema is present in the 6-allele loss skin. Scale bar, 200 μm.
Fig 7.
The expression of Sox7, Sox17, and Sox18 is regulated by Norrin signaling but not by VEGF/VEGFR2/NRP1 signaling.
(A-F) Anti-Sox7, anti-Sox17, and anti-Sox18 immunostaining in flat-mount retinas from WT (A-F, left panels), NdpKO (A-C, right panels), and Nrp1CKO/-;Pdgfb-CreER (D-F, right panels) mice at P7. (Panels D and F show the same pair of retinas, double stained with anti-Sox7 and anti-Sox17, with both immunolabeling signals visualized in the green channel for ease of comparison. Some non-specific non-EC immunostaining is seen in the Sox7 immunostained retinas in panel D.) Sox17 levels are dramatically decreased in veins and capillaries in NdpKO (B), while Sox7 and Sox18 levels are modestly decreased in NdpKO (A and C). Loss of Nrp1 has little or no effect on SoxF protein levels. Arteries and veins are marked for the Sox17 mutant retinas. Scale bar, 200 μm.