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 magnification and are from the mid-periphery of the retina. (C) Flatmounts of P14 control, NdpKO, and VEcadCreER;Tgfbr1CKO/-retinas showing CLDN5 and PLVAP immunostaining and Sulfo-NHS-biotin accumulation (detected with fluorescent streptavidin). (D) Quantification of Sulfo-NHS-biotin in the retinal parenchyma. Pixel intensities for territories between vascular segments were quantified from four flatmount retinas for each of the three genotypes (13 territories per retina). For the two cohorts of control and mutant retinas, all pixel intensity values were scaled so that the mean values from the control retinas equals 1.0. Scale bar in (A), 1 mm. Scale bar in (B), 100 um. Scale bar in (C), 100 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 red 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/CKO retinas 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.

Localized CNV with EC-specific loss of TGF-beta signaling.

(A) Phenotypically WT control retina. (B) VEcadCreER;Tgfbr1CKO/- retina. Eyes from mice at ∼P90 were fresh frozen, sectioned, and immunostained. RPE65 marks the retinal pigment epithelium (RPE). In the VEcadCreER;Tgfbr1CKO/- retina in (B), PECAM1+ ECs and associated RPE cells can be seen protruding into the subretinal space in a ∼200 um wide region (white arrows). (C) Quantification of CNV tufts in frozen sections of adult VEcadCreER;Tgfbr1CKO/-retinas, VEcadCreER;Tgfbr2CKO/- retinas, NdpKOretinas, and age- matched control retinas. Each data point represents a single whole eye section. CNV tufts were seen only in VEcadCreER;Tgfbr1CKO/-retinas. (D) Retina flatmounts from VEcadCreER;Tgfbr1CKO/- ∼P90 mice show scattered CD45+ cells (upper panel) and variably-sized zones of CNV with vessel-associated CD45+ cells (lower panel). The stacked z-planes of the images are at the level of photoreceptors and RPE. Retinas were mounted photoreceptor side up. All other retina flatmount images were mounted ganglion cell side up. The confocal microscope collects images from above. Abbreviations: Ch., choroid; ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. Scale bars, 100 um.

EC-specific loss of TGF-beta signaling leads to defects in subretinal structure, with alternating zones of normal structure and CNV.

0.5 um epon sections from adult VEcadCreER;Tgfbr1CKO/- retinas show (A) essentially normal retinal structure, (B) small regions of CNV (red arrows in (B)), or (C,D) larger regions of CNV. Immune cells (red arrows in (C) and (D)) are present in the subretinal space. Ch., choroid; ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. Scale bars, 50 um.

EC-specific loss of TGF-beta signaling produces little or no change in pericyte NG2 immunostaining.

Left panels, phenotypically WT control retina. Right panels, VEcadCreER;Tgfbr2CKO/-retinas. Eyes from mice at ∼P90 were fresh frozen, sectioned, and immunostained. PLVAP marks the choroidal vasculature, CLDN5 marks the retinal vasculature, and NG2 marks pericytes. Ch., choroid; ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. Scale bar, 100 um.

Retinal hypoxia in VEcadCreER;Tgfbr1CKO/-retinas at P18.

Retina flatmounts stained for HIF1-alpha to visualize regions with hypoxia. Territories with accumulation of HIF1-alpha, localized to nuclei, are seen in VEcadCreER;Tgfbr1CKO/- retinas. Control retinas have lower levels of HIF1-alpha. Scale bar, 100 um.

No change in the density of CD45+ immune cells in VEcadCreER;Tgfbr1CKO/- heart, kidney, liver, and lung at P14.

Collagen-4 (COL4) marks ECM, including perivascular ECM. ICAM1 is unchanged. P14 correponds to the peak of CD45+ cell counts in the VEcadCreER;Tgfbr1CKO/- retina. Scale bar, 50 um.

EC specificity of VEcadCreER assessed by recombination of two loxP- stop-loxP (LSL) reporters.

(A) VEcadCreER;R26-LSL-mtdT-2A-nlsGFP shows membrane-localized tdTomato exclusively in EC cell bodies and nuclear-localized GFP exclusively in EC nuclei in flatmounts of retina (left) and vibratome sections of cerebellum (right). (B) VEcadCreER;R26-LSL-SUN1-sfGFP-6xmyc shows co-localization of the nuclear membrane fusion protein SUN1-super-folder (sf)GFP-6xmyc with nuclear-localized ERG, an EC-specific transcription factor, in flatmounts of retina and in vibratome sections of cerebellum, heart, kidney, and lung. PECAM1 marks ECs, and the SUN1-GFP reporter is seen to localize exclusively to ECs. In the kidney and lung images, a few nuclei are ERG-positive but GFP- negative (white arrows); these presumably represent EC nuclei in which Cre-mediated recombination did not occur. Scale bars, 100 um.

EC specificity of VEcadCreER compared to CD45+ immune cells, assessed by recombination of the R26-LSL-SUN1-sfGFP-6xmyc reporter.

(A,B) Co-localization of the nuclear- localized GFP reporter with the EC transcription factor ERG and non-co-localization with the macrophage marker CD45 in (A) a flatmount of choroid and in (B) a vibratome section of small intestine. In the rightmost panel, GFP and CD45 are seen to have distinct localizations in both tissues. (C) Retina flatmounts showing non-overlapping patterns of GFP (elongated EC nuclei) and CD45 (microglia). Scale bars, 100 um.

EC specificity of VEcadCreER in the VEcadCreER;Tgfbr1CKO/- genetic background, based on recombination with the R26-LSL-SUN1-sfGFP-6xmyc reporter.

A retina flatmount from a P17 VEcadCreER;Tgfbr1CKO/-;R26-LSL-SUN1-sfGFP-6xmyc mouse shows the phenotype of CD45+ immune cell accumulation following loss of TGF-beta signaling. GFP expressed from the LSL reporter co- localizes with ERG and shows no co-localization with CD45. Scale bar, 100 um.

Density of microglia in control (wild type) flatmount retinas.

(A) Microglia and retinal ECs were visualized by immunostaining for CD45 and PECAM1 (upper two panels). By visual inspection, the distributions of the two cell types appear to be uncorrelated. For quantification, microglia were visualized by immuostaining for CD45 (plasma membrane) and ASC1 (nucleus) (lower two panels). The regions in the white squares in the left two panels are shown enlarged in the right two panels. (B) Quantification of microglial density in each of the three layers of the inner retina, the regions where microglia are found. Bars represent mean +/- standard deviation. RGC, retinal ganglion cell layer; IPL, inner plexiform layer; OPL, outer plexiform layer. Scale bars in (A): left, 100 um, right, 50 um.

Increased macrophage density in the choroid with EC-specific loss of TGF-beta signaling.

(A) After removing the retina, choroid flatmounts (sclera, choroidal vasculature, and RPE) were imaged from the RPE side. (B) The number of CD45+ cells in choroid flatmounts. 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 bar, 100 um.

Large number of apoptotic immune cells in retinas with EC-specific loss of TGF-beta signaling.

(A) Retina flatmounts from mice with the indicated genotypes were immunostained for cleaved Caspase 3, CD45, and PECAM1. In VEcadCreER;Tgfbr1CKO/- and VEcadCreER;Tgfbr1CKO/-;Tgfbr2CKO/-retinas, more than 20% of CD45+ cells are also positive for cleaved caspase 3. (B) Quantifying the number of cleaved caspase 3+ cells in control, Fzd4-/-, 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. n.s., not significant. Scale bar, 100 um.

Close association between immune cells and a blood vessel forming a retinal-choroidal anastomosis in a retina with EC-specific loss of TGF-beta signaling.

(A) Upper three panels show a control retina flatmount with sparse CD45+ microglia and PECAM1+ vasculature, imaged at three Z-planes (indicated by the numbers in the lower left corner of each image) corresponding to the NFL, IPL, and OPL. The lower nine panels show a series of Z-planes through the full thickness of a VEcadCreER;Tgfbr1CKO/- retina flatmount with many CD45+ immune cells in contact with the vasculature. (B) and (C), retinal schematics showing the relationship of the vasculature and the three retinal layers. Confocal Z-planes are numbered at right. 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.

Low magnification view of the close association between immune cells and a blood vessel forming a retinal-choroidal anastomosis in a retina with EC-specific loss of TGF-beta signaling.

(A) The upper four panels show a control retina flatmount with PECAM1+ vasculature and sparse ASC+/CD45+ microglia, imaged at two Z-planes (indicated by the numbers in the lower left corner of each image) corresponding to the NFL and OPL. The lower twelve panels show a series of Z-planes through the full thickness of a VEcadCreER;Tgfbr1CKO/- retina flatmount with a retinal-choroidal anastomosis (in white squares) and many CD45+ immune cells in contact with the vasculature. (B) and (C), retinal schematics showing the relationship of the vasculature and the three retinal layers. Confocal Z-planes are numbered at right. 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), 200 um.

High magnification view of the close association between immune cells and a blood vessel forming a retinal-choroidal anastomosis in a retina with EC-specific loss of TGF-beta signaling.

The boxed regions in each of the panels in Figure 6 – figure supplement 2 are enlarged here. The figure layout is otherwise identical to that of Figure 6 – figure supplement 2. Scale bars in (A), 50 um.

Examples of ICAM1 immunostaining in individual experiments with control vs. VEcadCreER;Tgfbr1CKO/- retinas, control vs. Fzd4-/- retinas, and control vs. NdpKOretinas used for the quantification shown in Figure 7.

One quadrant of a retina flatmount from four representative experiments are shown, each with a phenotypically WT control processed in parallel (top panel in each set). Different experiments are separated by a white space. Each image is from a diferent retina. The immunostaining procedure was identical within an experiment and matched as closely as possible across experiments. Confocal microscope settings were identical across experiments. Scale bar, 500 um.

ICAM1 immunostaining in VEcadCreER;Tgfbr1CKO/-brains.

Sagittal sections of one control brain (top row) and two VEcadCreER;Tgfbr1CKO/- brains (second and third rows). The regions in the white squares are shown at higher magnification in the lower nine panels. ICAM1 immunostaining is present in a larger fraction of capillaries in the VEcadCreER;Tgfbr1CKO/-brains compared to controls. ICAM1 is also present on the clustered immune cells, as seen in the bottom row of enlarged images. Scale bar for full brain sections, 1 mm. Scale bar for magnified regions, 100 um.

NF-kappaB, integrin, and other immune-related transcripts and proteins in retinal and brain ECs in control and VEcadCreER;Tgfbr1CKO/-mice.

(A) Abundance of transcripts coding for NF-kappaB pathway components in brain ECs and in the major classes of CNS cells based on snRNAseq of control and VEcadCreER;Tgfbr1CKO/-mice at P14 (see Figure 9). Listed are transcripts for I- kappaB kinase subunits (Ikbke, Ikbkb, and Ikbkg), NFkB subunits (Nfkkb1, Nfkb2, Rel, Rela, and Relb), and NFkB inhibitors (Nfkbia, Nfkbiz, Nfkbil1, Nfkbib, and Nfkbid). (B) Abundance of transcripts coding for integrin subunits and integrin binding proteins in brain ECs based on snRNAseq of control and VEcadCreER;Tgfbr1CKO/-mice at P14. The data used to generate the dotplots in (A) and (B) are in Supplemental Table 2. (C) Retina flatmounts immunostained for PECAM1 and NFkB-p65 (upper panels), ITGA2 (middle panels), or ITGA4 (lower panels). (D) Abundance of transcripts coding for immune regulators that show statistically significant differences in brain EC snRNAseq data from control vs. VEcadCreER;Tgfbr1CKO/- mice at P14. The gene sets examined include chemokines, cell-surface adhesion proteins, integrins, and TNF receptor superfamily members. Con., phenotypically WT control. CKO, VEcadCreER;Tgfbr1CKO/-. Scale bar in (C), 500 um.

Increased TOX levels in retinal ECs in VEcadCreER;Tgfbr1CKO/-mice.

(A) UMAP plots for Tox and Tox3, two members of the Tox TF family that show increases in transcript abundance in VEcadCreER;Tgfbr1CKO/- CNS ECs. Red arrows, EC cluster. UMAP plots are similar to those in Figure 9C. (B) Retina flatmounts immunostained for TOX and PECAM1. Arrows indicate TOX+ EC nuclei. The TOX signal is several-fold more intense in the EC nuclei in VEcadCreER;Tgfbr1CKO/- Retinas compared to control retinas.