Vascular endothelial-specific loss of TGF-beta signaling as a model for choroidal neovascularization and central nervous system vascular inflammation
- Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, United States
- Howard Hughes Medical Institute, Johns Hopkins University School of Medicine, United States
- Department of Ophthalmology, Johns Hopkins University School of Medicine, United States
- Department of Neuroscience, Johns Hopkins University School of Medicine, United States
Figures
Figure 1 with 8 supplements
Endothelial cell (EC)-specific loss of TGF-beta signaling leads to attenuated retinal vascular development, choroidal neovascularization (CNV), and anastomoses between retinal and choroidal vasculatures.
(A) Cdh5CreER;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) Cdh5CreER;Tgfbr2CKO/- retinas showing CNV (white arrows in central panels) and an anastomosis between retinal and choroidal vasculatures (white arrows in lower panels), with intraretinal EC marker CLDN5, choroidal EC marker PLVAP, and pan-vascular marker COL4 (collagen4). (C) Upper right panels, in the Cdh5CreER;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 µm.
Figure 1—figure supplement 1
Localized choroidal neovascularization (CNV) with endothelial cell (EC)-specific loss of TGF-beta signaling.
(A) Phenotypically wild-type (WT) control retina. (B) Cdh5CreER;Tgfbr1CKO/- retina. Eyes from mice at ~P90 were fresh frozen, sectioned, and immunostained. RPE65 marks the retinal pigment epithelium (RPE). In the Cdh5CreER;Tgfbr1CKO/- retina in (B), PECAM1+ ECs and associated RPE cells can be seen protruding into the subretinal space in an ~200 µm wide region (white arrows). (C) Quantification of CNV tufts in frozen sections of adult Cdh5CreER;Tgfbr1CKO/- retinas, Cdh5CreER;Tgfbr2CKO/- retinas, NdpKO retinas, and age-matched control retinas. Each data point represents a single whole eye section. CNV tufts were seen only in Cdh5CreER;Tgfbr1CKO/- retinas. (D) Retina flatmounts from Cdh5CreER;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; P, postnatal day. Scale bars, 100 µm.
Figure 1—figure supplement 2
Endothelial cell (EC)-specific loss of TGF-beta signaling leads to defects in subretinal structure, with alternating zones of normal structure and choroidal neovascularization (CNV).
0.5 µm epon sections from adult Cdh5CreER;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 µm.
Figure 1—figure supplement 3
Endothelial cell (EC)-specific loss of TGF-beta signaling produces little or no change in pericyte NG2 immunostaining.
Left panels, phenotypically wild-type (WT) control retina. Right panels, Cdh5CreER;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; P, postnatal day. Scale bar, 100 µm.
Figure 1—figure supplement 4
Retinal hypoxia in Cdh5CreER;Tgfbr1CKO/- retinas at postnatal day (P)18.
Retina flatmounts were stained for HIF1-alpha to visualize regions with hypoxia. Territories with accumulation of HIF1-alpha, localized to nuclei, are seen in Cdh5CreER;Tgfbr1CKO/- retinas. Control retinas have lower levels of HIF1-alpha. Scale bar, 100 µm.
Figure 1—figure supplement 5
No change in the density of CD45+ immune cells in Cdh5CreER;Tgfbr1CKO/- heart, kidney, liver, and lung at postnatal day (P)14.
Collagen-4 (COL4) marks ECM, including perivascular ECM. ICAM1 is unchanged. P14 corresponds to the peak of CD45+ cell counts in the Cdh5CreER;Tgfbr1CKO/- retina. Scale bar, 50 µm.
Figure 1—figure supplement 6
Endothelial cell (EC) specificity of Cdh5CreER assessed by recombination of two loxP-stop-loxP (LSL) reporters.
(A) Cdh5CreER;Rosa26-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) Cdh5CreER;Rosa26-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 µm.
Figure 1—figure supplement 7
Endothelial cell (EC) specificity of Cdh5CreER compared to CD45+ immune cells, assessed by recombination of the Rosa26-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 µm.
Figure 1—figure supplement 8
Endothelial cell (EC) specificity of Cdh5CreER in the Cdh5CreER;Tgfbr1CKO/- genetic background, based on recombination with the Rosa26-LSL-SUN1-sfGFP-6xmyc reporter.
A retina flatmount from a P17 Cdh5CreER;Tgfbr1CKO/-;Rosa26-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 µm.
Figure 2
Vascular architecture in retinas with endothelial cell (EC)-specific loss of TGF-beta signaling or global loss of Norrin/Fzd4 signaling.
(A) COL4 immunostaining of control and Cdh5CreER;Tgfbr1CKO/- flatmount retinas showing arteries, veins, and capillaries in the control retina and a high density of vascular tufts in the Cdh5CreER;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: postnatal day (P)14 control retina (upper image; white arrows point to tip cells in the inner plexiform layer [IPL]) and two regions from a P14 Cdh5CreER;Tgfbr1CKO/- retina (lower). Center column of three panels: P26 control retina (upper) and two regions from a P26 Cdh5CreER;Tgfbr1CKO/- retina (lower). Right column of three panels, ~P30 NdpKO retina (upper) and two regions from an ~P30 Fzd4-/- retina (lower). All images are at the same magnification and are from the midperiphery of the retina. (C) Flatmounts of P14 control, NdpKO, and Cdh5CreER;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 equal 1.0. Scale bar in (A), 1 mm. Scale bar in (B), 100 µm. Scale bar in (C), 100 µm.
Figure 3 with 3 supplements
Immune cells in retinas with endothelial cell (EC)-specific loss of TGF-beta signaling or global loss of Norrin/Fzd4 signaling.
(A) Control and Cdh5CreER;Tgfbr1CKO/- retina flatmounts (left pair of panels) with enlarged insets (right pair of panels). (B) Control and Cdh5CreER;Tgfbr1CKO/- retina flatmounts (left panel) and insets (right three panels). (C) Control and Cdh5CreER;Tgfbr1CKO/-;Tgfbr2CKO/- retina flatmounts, and control and Fzd4-/- retina flatmounts. (D–G) Quantification of numbers of cells positive for the indicated markers in 450 µm × 450 µm 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 µm.
Figure 3—figure supplement 1
Density of microglia in control (wild-type) flatmount retinas.
(A) Microglia and retinal endothelial cells (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 immunostaining 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 µm, right, 50 µm.
Figure 3—figure supplement 2
Increased macrophage density in the choroid with endothelial cell (EC)-specific loss of TGF-beta signaling.
(A) After removing the retina, choroid flatmounts (sclera, choroidal vasculature, and retinal pigment epithelium [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 µm.
Figure 3—figure supplement 3
Large number of apoptotic immune cells in retinas with endothelial cell (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 Cdh5CreER;Tgfbr1CKO/- and Cdh5CreER;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 Cdh5CreER;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 µm.
Figure 4
Single-nucleus RNA sequencing from control and Cdh5CreER;Tgfbr1CKO/- retinas at postnatal day (P)14.
(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 Cdh5CreER;Tgfbr1CKO/- data are shown. The red arrow points to immune cells, almost entirely derived from the Cdh5CreER;Tgfbr1CKO/- samples, as shown in (B). (B) The contributions of control and Cdh5CreER;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. (E) UMAP plots for select markers from (D) showing immune cell-type-specific expression.
Figure 5
Vascular anatomy and absence of immune cell infiltration in Vsx2-Cre;VegfaCKO/CKO retinas.
(A) Sections of control and Vsx2-Cre;VegfaCKO/CKO retinas immunostained with anti-PECAM1 to visualize the vasculature. (B) False color images of control and Vsx2-Cre;VegfaCKO/CKO flatmount retinas showing a stacked Z-series color-coded by the depth of PECAM1 immunostained vasculature. Blue, vitreal surface; green, inner plexiform layer; red, outer plexiform layer. (C) Control and Vsx2-Cre;VegfaCKO/CKO flatmount retinas immunostained with anti-PECAM1. (D) Control and Vsx2-Cre;VegfaCKO/CKO flatmount retinas immunostained for ASC and CD45. (E) Quantification of CD45+/ASC+ cells. Scale bars, (A), (B), and (D), 100 µm; (C) 500 µm.
Figure 6 with 3 supplements
Close association between immune cells and retinal vasculature with endothelial cell (EC)-specific loss of TGF-beta signaling.
(A) Upper six panels show a control retina flatmount. Lower six panels show a Cdh5CreER;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 Cdh5CreER;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 µm. Scale bar in (D), 50 µm.
Figure 6—figure supplement 1
Close association between immune cells and a blood vessel forming a retinal-choroidal anastomosis in a retina with endothelial cell (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 Cdh5CreER;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 µm.
Figure 6—figure supplement 2
Low-magnification view of the close association between immune cells and a blood vessel forming a retinal-choroidal anastomosis in a retina with endothelial cell (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 12 panels show a series of Z-planes through the full thickness of a Cdh5CreER;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 µm.
Figure 6—figure supplement 3
High-magnification view of the close association between immune cells and a blood vessel forming a retinal-choroidal anastomosis in a retina with endothelial cell (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 µm.
Figure 7 with 1 supplement
Intercellular adhesion molecule 1 (ICAM1) in endothelial cells (ECs) and smooth muscle actin (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. Cdh5CreER;Tgfbr1CKO/-, control vs. Fzd4-/- retinas, and control vs. NdpKO retinas. (C) Left, control retina flatmount showing strong SMA immunostaining in arteries, weak/patchy SMA immunostaining in veins, and undetectable SMA immunostaining in capillaries. Right, Cdh5CreER;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 Cdh5CreER;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 µm.
Figure 7—figure supplement 1
Examples of intercellular adhesion molecule 1 (ICAM1) immunostaining in individual experiments with control vs. Cdh5CreER;Tgfbr1CKO/- retinas, control vs. Fzd4-/- retinas, and control vs. NdpKO retinas used for the quantification shown in Figure 7.
One quadrant of a retina flatmount from four representative experiments is shown, each with a phenotypically wild-type (WT) control processed in parallel (top panel in each set). Different experiments are separated by a white space. Each image is from a different 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 µm.
Figure 8 with 1 supplement
Transient IgG extravasation, smooth muscle actin (SMA) accumulation in pericytes, and immune cell infiltration in the brains of mice with endothelial cell (EC)-specific loss of TGF-beta signaling.
(A) Endogenous IgG in control and Cdh5CreER;Tgfbr1CKO/- brains at postnatal day (P)14 and P24. IgG accumulation is minimal in control brains at P14 and P24 but is readily detectable in Cdh5CreER;Tgfbr1CKO/- brains at P14 but not at P24. (B) ECs (visualized with PECAM1) and pericytes (visualized with NG2) in control and Cdh5CreER;Tgfbr1CKO/- brains at P14. SMA immunostaining visualizes arterioles (continuous staining) and veins (patchy staining) in control and Cdh5CreER;Tgfbr1CKO/- brains, and a subset of capillary-associated pericytes in Cdh5CreER;Tgfbr1CKO/- brains. (C) Immune cells (CD45+ and F4-80+) in control and Cdh5CreER;Tgfbr1CKO/- brains at P35. Control brains have minimal numbers of immune cells other than resident microglia. Cdh5CreER;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 µm for enlarged images.
Figure 8—figure supplement 1
Intercellular adhesion molecule 1 (ICAM1) immunostaining in Cdh5CreER;Tgfbr1CKO/- brains.
Sagittal sections of one control brain (top row) and two Cdh5CreER;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 Cdh5CreER;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 µm.
Figure 9 with 2 supplements
Single-nucleus RNAseq (snRNAseq) of control and Cdh5CreER;Tgfbr1CKO/- vascular fragments enriched from the brain at postnatal day (P)14.
(A) Uniform Manifold Approximation and Projection (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 Cdh5CreER;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. Cdh5CreER;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 upregulated in Cdh5CreER;Tgfbr1CKO/- ECs. Central column, UMAP plots for transcripts that are downregulated in Cdh5CreER;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 Cdh5CreER;Tgfbr1CKO/- brains. The data used to generate the volcano plot in (B) and the GSEA in (D) are in Supplementary file 1. Additional analyses of differential expression are in Supplementary file 2. NES, normalized enrichment score. *, adjusted p-value<0.05.
Figure 9—figure supplement 1
NF-kappaB, integrin, and other immune-related transcripts and proteins in retinal and brain endothelial cells (ECs) in control and Cdh5CreER;Tgfbr1CKO/- mice.
(A) Abundance of transcripts coding for NF-kappaB pathway components in brain ECs and in the major classes of central nervous system (CNS) cells based on single-nucleus RNAseq (snRNAseq) of control and Cdh5CreER;Tgfbr1CKO/- mice at postnatal day (P)14 (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). Control or Con., phenotypically WT control. EC KO, Cdh5CreER;Tgfbr1CKO/-. (B) Abundance of transcripts coding for integrin subunits and integrin binding proteins in brain ECs based on snRNAseq of control and Cdh5CreER;Tgfbr1CKO/- mice at P14. The data used to generate the dotplots in (A) and (B) are in Supplementary file 2. (C) Abundance of transcripts coding for immune regulators that show statistically significant differences in brain EC snRNAseq from control vs. Cdh5CreER;Tgfbr1CKO/- mice at P14. The gene sets examined include chemokines, cell-surface adhesion proteins, integrins, and TNF receptor superfamily members. (D) Retina flatmounts immunostained for PECAM1 and NFkB-p65 (upper panels), ITGA2 (middle panels), or ITGA4 (lower panels). Scale bar in (C), 500 µm.
Figure 9—figure supplement 2
Increased TOX levels in retinal endothelial cells (ECs) in Cdh5CreER;Tgfbr1CKO/- mice.
(A) Uniform Manifold Approximation and Projection (UMAP) plots for Tox and Tox3, two members of the Tox transcription factor (TF) family that show increases in transcript abundance in Cdh5CreER;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 Cdh5CreER;Tgfbr1CKO/- retinas compared to control retinas. For each genotype, three retinal depths are shown: nerve fiber layer (left), ganglion cell layer (center), and inner plexiform layer (right). Many non-ECs express TOX.
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Genetic reagent (Mus musculus) | Cdh5CreER | Monvoisin et al., 2006 | Cdh5CreER | |
| Genetic reagent (Mus musculus) | Tgfbr1CKO | Larsson et al., 2001 | JAX 028701 | |
| Genetic reagent (Mus musculus) | Tgfbr2CKO | Levéen et al., 2002 | JAX 012603 | |
| Genetic reagent (Mus musculus) | Fzd4KO | Wang et al., 2001 | JAX 012823 | |
| Genetic reagent (Mus musculus) | NdpKO | Ye et al., 2009 | JAX 012287 | |
| Genetic reagent (Mus musculus) | VEGFCKO | Gerber et al., 1999 | VEGFCKO | |
| Genetic reagent (Mus musculus) | Vsx2-Cre | Rowan and Cepko, 2004 | Vsx2-Cre | |
| Genetic reagent (Mus musculus) | Rosa26-LSL-SUN1-sfGFP | Mo et al., 2015 | JAX 030952 | |
| Genetic reagent (Mus musculus) | Rosa26-LSL-tdTomato-2A-H2B-GFP | Wang et al., 2018 | JAX 030867 | |
| Antibody | Rat anti-mouse mAb PLVAP/MECA-32 | BD Biosciences | BD Biosciences # 553849 | (1:400) |
| Antibody | Rat anti-mouse mAb CD31 | BD Biosciences | BD Biosciences # 553370 | (1:400) |
| Antibody | Rat anti-mouse mAb ICAM-1 | Invitrogen | Invitrogen # 14-0542-82 | (1:400) |
| Antibody | Rat anti-mouse mAb F4/80 | Bio-Rad | Bio-Rad # MCA497G | (1:400) |
| Antibody | Rat anti-mouse mAb CD206 | Bio-Rad | Bio-Rad # MCA2235 | (1:400) |
| Antibody | Rat anti-mouse mAb PU.1/Spi-1 | R&D Systems | R&D Systems # MAB7124 | (1:400) |
| Antibody | Mouse mAb anti-alpha SMA, Cy3 conjugate | Sigma-Aldrich | Sigma-Aldrich # C6198 | (1:400) |
| Antibody | Mouse mAb anti-CLDN5, Alexa Fluor 488 conjugate | Thermo Fisher Scientific | Thermo Fisher Scientific # 352588 | (1:400) |
| Antibody | Mouse mAb anti-RPE65, Dylight 550 conjugate | Invitrogen | Invitrogen # MA5-16044 | (1:400) |
| Antibody | Rabbit polyclonal anti-Collagen IV | Novus Biologicals | Novus Biologicals # NB120-6586 | (1:400) |
| Antibody | Rabbit polyclonal anti-NG2 Chondroitin Sulfate Proteoglycan | Millipore | Millipore # AB5320 | (1:400) |
| Antibody | Rabbit mAb anti-ASC/TMS1 | Cell Signaling | Cell Signaling # 67824S | (1:400) |
| Antibody | Rabbit mAb anti-cleaved Caspase-3 | Cell Signaling | Cell Signaling # 9664S | (1:400) |
| Antibody | Rabbit mAb anti-HIF-1alpha | Cell Signaling | Cell Signaling # 36169S | (1:400) |
| Antibody | Rabbit mAb anti-P-SMAD1/5/9 | Cell Signaling | Cell Signaling # 13820S | (1:400) |
| Antibody | Armenian hamster mAb anti-CD3e | Invitrogen | Invitrogen # 14-0031-82 | (1:400) |
| Antibody | Goat polyclonal anti-CD45 | R&D Systems | R&D Systems # AF114 | (1:400) |
| Antibody | Goat polyclonal anti-Iba1 | Novus Biologicals | Novus Biologicals # NB100-1028 | (1:400) |
| Antibody | Chicken polyclonal anti-GFP | Aves Labs | Aves Labs # GFP-1020 | (1:400) |
| Antibody | Rabbit mAb anti-NF-κB p65. D14E12 | Cell Signaling Technology | Cell Signaling Technology # 8242S | (1:400) |
| Antibody | Rabbit mAb anti-Integrin alpha 2 (ITGA2); clone GEB | BosterBio | BosterBio # M01933 | (1:400) |
| Antibody | Rabbit mAb anti-Integrin alpha 4 (ITGA4); D2E1 | Cell Signaling Technology | Cell Signaling Technology # 8440 | (1:400) |
| Antibody | Rabbit mAb anti-TOX/TOX2; E6G5O | Cell Signaling Technology | Cell Signaling Technology # 36778S | (1:400) |
Additional files
-
Supplementary file 1
Differential gene expression data used to generate the volcano plot in Figure 9B and the gene set enrichment analysis (GSEA) plot in Figure 9D.
- https://cdn.elifesciences.org/articles/107018/elife-107018-supp1-v1.xlsx
-
Supplementary file 2
Differential gene expression data used to generate the dotplots in Figure 9—figure supplement 1A and B.
- https://cdn.elifesciences.org/articles/107018/elife-107018-supp2-v1.xlsx
-
MDAR checklist
- https://cdn.elifesciences.org/articles/107018/elife-107018-mdarchecklist1-v1.docx
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Vascular endothelial-specific loss of TGF-beta signaling as a model for choroidal neovascularization and central nervous system vascular inflammation
eLife 14:RP107018.
https://doi.org/10.7554/eLife.107018.3
