EC-specific loss of TGF-beta signaling leads to attenuated retinal vascular development, CNV, and anastomoses between retinal and choroidal vasculatures.

(A) VEcadCreER;Tgfbr1CKO/- retinas showing CNV (white arrows in central panels), and vascular invasion of the outer nuclear layer (white arrows in lower panels), both with associated CD45+ immune cells. (B) VEcadCreER;Tgfbr2CKO/- retinas showing CNV (white arrows in central panels), and an anastomosis between retinal and choroidal vasculatures (white arrows in lower panels), with intra-retinal EC marker CLDN5, choroidal EC marker PLVAP, and pan-vascular marker COL4 (collagen4). (C) Upper right panels, in the VEcadCreER;Tgfbr1CKO/- retina, CNV (white arrows) is derived from choroidal vasculature, marked by PLVAP. Lower right panels, CNV (white arrows) is present in the subretinal space, i.e. on the retinal side of the RPE, which is marked by RPE65. Abbreviations: Ch., choroid; ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer; RPE, retinal pigment epithelium. The ages of the mice are indicated in postnatal days (P) for this and all other figures. Scale bars, 100 um.

Vascular architecture in retinas with EC-specific loss of TGF-beta signaling or global loss of Norrin/Fzd4 signaling.

(A) COL4 immunostaining of control and VEcadCreER;Tgfbr1CKO/- flatmount retinas showing arteries, veins, and capillaries in the control retina and a high density of vascular tufts in the VEcadCreER;Tgfbr1CKO/-retina. (B) False color images from the indicated genotypes and ages showing a stacked Z-series of flatmount retinas color-coded by the depth of the vasculature. For control retinas the blue-green-red color scheme corresponds to the inner two-thirds of the retina: blue, vitreal surface; green, ganglion cell layer and inner plexiform layer; and red, inner nuclear layer and outer plexiform layer. For mutant retinas, the blue-green-red color scheme corresponds to a shallower depth, as the most deeply penetrating vascular tufts go only as far as the inner edge of the inner nuclear layer: blue, vitreal surface; green, ganglion cell layer; and red, inner plexiform layer. Left column of three panels: P14 control retina (upper image; white arrows point to tip cells in the IPL) and two regions from a P14 VEcadCreER;Tgfbr1CKO/-retina (lower). Center column of three panels: P26 control retina (upper) and two regions from a P26 VEcadCreER;Tgfbr1CKO/- retina (lower). Right column of three panels, ∼P30 NdpKO retina (upper) and two regions from a ∼P30 Fzd4-/- retina (lower). All images are at the same magnfication. (C) Flatmount of a P37 VEcadCreER;Tgfbr1CKO/-retina showing PLVAP expression and Sulfo-NHS-biotin accumulation in vascular tufts. The vascular tufts are variably present with loss of TGFBR1 or TGFBR2. Scale bar in (A), 1 mm. Scale bar in (B), 100 um. Scale bar in (C), 200 um.

Immune cells in retinas with EC-specific loss of TGF-beta signaling or global loss of Norrin/Fzd4 signaling.

(A) Control and VEcadCreER;Tgfbr1CKO/-retina flatmounts (left pair of panels) with enlarged insets (right pair of panels). (B) Control and VEcadCreER;Tgfbr1CKO/-retina flatmounts (left panel) and insets (right three panels). (C) Control and VEcadCreER;Tgfbr1CKO/-;Tgfbr2CKO/-retina flatmounts, and control and Fzd4-/- retina flatmounts. (D-G) Quantification of numbers of cells positive for the indicated markers in 450 um x 450 um zones in the midperiphery of retina flatmounts from mice of the indicated genotypes. In this and other panels with statistical comparisons, the bars represent mean +/- standard deviation, and p-values, calculated using the Wilcoxon rank sum test, are shown as * <0.05, ** <0.01, *** < 0.001, and **** <0.0001. Scale bars in whole retina panels, 1 mm. Scale bars in all other panels, 100 um.

Single nucleus RNA sequencing from control and VEcadCreER;Tgfbr1CKO/- retinas at P14.

(A) Identification of cell clusters in a Uniform Manifold Approximation and Projection (UMAP) plot, based on established markers of retinal gene expression. The pooled control and VEcadCreER;Tgfbr1CKO/-data are shown. The horizontal arrow points to immune cells, almost entirely derived from the VEcadCreER;Tgfbr1CKO/-samples, as shown in (B). (B) The contributions of control and VEcadCreER;Tgfbr1CKO/- retinas to the immune cell cluster. (C) UMAP plot identifying immune cell types within the immune cell cluster, based on established markers as shown in (D) and (E). (D) Abundances of select transcripts in different immune cell types, as defined in (B). (E) UMAP plots for select markers from (D) showing immune cell type-specific expression.

Vascular anatomy and absence of immune cell infiltration in Chx10-Cre;VegfaCKO/CKO retinas.

(A) Sections of control and Chx10-Cre;VegfaCKO/CKOretinas immunostained with anti-PECAM1 to visualize the vasculature. (B) False color images of control and Chx10-Cre;VegfaCKO/CKOflatmount retinas showing a stacked Z-series color-coded by the depth of PECAM1 immunstained vasculature. Blue, vitreal surface; green, inner plexiform layer; red, outer plexiform layer. (C) Control and Chx10-Cre;VegfaCKO/CKOflatmount retinas immunostained with anti-PECAM1. (D) Control and Chx10-Cre;VegfaCKO/CKO flatmount retinas immunostained for ASC and CD45. (E) Quantification of CD45+ cells. Scale bars: (A), (B) and (D), 100 um; (C) 500 um.

Close association between immune cells and retinal vasculature with EC-specific loss of TGF-beta signaling.

(A) Upper six panels show a control retina flatmount. Lower six panels show a VEcadCreER;Tgfbr1CKO/+;Tgfbr2CKO/-retina flatmount. ASC and CD45 label immune cells, including microglia. The Z-plane is indicated by the numbers at the bottom of each image. The nerve fiber layer (Z-plane 3) and the inner plexiform layer (Z-planes 10-11) are shown schematically in (B) and (C). (B) and (C), retinal schematics showing the relationship of the vasculature and the three retinal layers. Confocal Z-planes are numbered at right. (D) Immune cells and venous ECs in control and VEcadCreER;Tgfbr1CKO/-retinas. In the lower image, three of the “impressions” of CD45+ immune in the distribution of PECAM1 on the EC surface are highlighted with white arrows. A, artery; V, vein. NFL, nerve fiber layer; GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer; IS/OS, inner segment/outer segment. Scale bars in (A), 100 um. Scale bar in (D), 50 um.

ICAM1 in ECs and SMA in pericytes in retinas with EC-specific loss of TGF-beta signaling.

(A) ICAM1 in the retinal vasculature with EC-specific loss of TGF-beta signaling. Immunostaining conditions and image capture settings were identical across genotypes. (B) Quantification of the relative intensities of ICAM1 immunostaining in control vs. VEcadCreER;Tgfbr1CKO/- , control vs Fzd4-/- retinas, and control vs NdpKO retinas. (C) Left, control retina flatmount showing strong SMA immunstaining in arteries, weak/patchy SMA immunstaining in veins, and undetectable SMA immunostaining in capillaries. Right, VEcadCreER;Tgfbr1CKO/- retina flatmount showing SMA immunostaining in all vessels, including vascular tufts. NG2 immunostaining (a pericyte marker) is shown in the images below. (D) Quantification of the relative intensities of PECAM1, NG2, and SMA immunostaining in flatmount control and VEcadCreER;Tgfbr1CKO/- retinas. Bars represent mean +/- standard deviation, and p-values, calculated using the Wilcoxon rank sum test, are shown as * <0.05, ** <0.01, *** < 0.001, and **** <0.0001. In (A) and (C), the retinal periphery is at the right. Scale bars in (A) and (C), 500 um.

Transient IgG extravasation, SMA accumulation in pericytes, and immune cell infiltration in the brains of mice with EC-specific loss of TGF-beta signaling.

(A) Endogenous IgG in control and VEcadCreER;Tgfbr1CKO/-brains at P14 and P24. IgG accumulation is minimal in control brains at P14 and P24, but is readily detectable in VEcadCreER;Tgfbr1CKO/- brains at P14 but not at P24. (B) ECs (visualized with PECAM1) and pericytes (visualized with NG2) in control and VEcadCreER;Tgfbr1CKO/-brains at P14. SMA immunostaining visualizes arterioles (continuous staining) and veins (patchy staining) in control and VEcadCreER;Tgfbr1CKO/-brains, and a subset of capillary-associated pericytes in VEcadCreER;Tgfbr1CKO/- brains. (C) Immune cells (CD45+ and F4-80+) in control and VEcadCreER;Tgfbr1CKO/- brains at at P35. Control brains have minimal numbers of immune cells other than resident microglia. VEcadCreER;Tgfbr1CKO/-brains show localized regions with concentrated accumulations of immune cells. White squares marked with letters in the sagittal brain images (upper) are enlarged below. Scale bars, 1 mm for whole brain images and 200 um for enlarged images.

snRNAseq of control and VEcadCreER;Tgfbr1CKO/- vascular fragments enriched from the brain at P14.

(A) UMAP plots showing cell clusters encampassing the major cell types in the mouse brain, enriched for ECs, pericytes, and vascular smooth muscle cells (vSMCs). The locations of the EC clusters in the VEcadCreER;Tgfbr1CKO/-and control UMAP plots (vertical red arrows) are shifted, indicating substantial changes in their transcriptomes. Other cell cluster locations are largely unchanged. (B) Volcano plot showing transcripts from the EC cluster in control vs. VEcadCreER;Tgfbr1CKO/- snRNAseq. The labeled transcripts have adjusted -log10 p-values greater than 50. (C) UMAP plots as in (A) highlighting individual transcripts, with the EC cluster indicated by a vertical red arrow. Left column, UMAP plots for transcripts that are up-regulated in VEcadCreER;Tgfbr1CKO/-ECs. Central column, UMAP plots for transcripts that are down-regulated in VEcadCreER;Tgfbr1CKO/- ECs. Right column, Icam1, Icam2, and Tgfbr3. (D) Gene set enrichment analysis (GSEA) for the EC, glial, and astrocyte clusters in (A) showing the degree of enrichment in VEcadCreER;Tgfbr1CKO/-brains. The data used to generate the volcano plot in (B) and the GSEA in (D) are in Supplemental Table 1. NES, normalized enrichment score. *, adjusted p-value <0.05.