Mapping the origin and fate of myeloid cells in distinct compartments of the eye by single-cell profiling

Abstract

Similar to the brain, the eye is considered an immune-privileged organ where tissue-resident macrophages provide the major immune cell constituents. However, little is known about spatially restricted macrophage subsets within different eye compartments with regard to their origin, function, and fate during health and disease. Here, we combined single-cell analysis, fate mapping, parabiosis, and computational modeling to comprehensively examine myeloid subsets in distinct parts of the eye during homeostasis. This approach allowed us to identify myeloid subsets displaying diverse transcriptional states. During choroidal neovascularization, a typical hallmark of neovascular age-related macular degeneration (AMD), we recognized disease-specific macrophage subpopulations with distinct molecular signatures. Our results highlight the heterogeneity of myeloid subsets and their dynamics in the eye that provide new insights into the innate immune system in this organ which may offer new therapeutic targets for ophthalmological diseases.

Keywords: cornea; macrophages; microglia; retina; single-cell RNA-seq.

Figure 1. Molecular census of myeloid cells in different compartments of the healthy mouse eye

1. Scheme of the murine eye.

2. Schematic diagram depicting the workflow for the isolation of single CD45⁺CD3⁻CD19⁻Ly6G⁻ cells from different eye compartments (retina, ciliary body, cornea) under healthy conditions for unbiased single‐cell RNA‐seq (scRNA‐seq). Morphological heterogeneity of sorted myeloid cells revealed by May‐Grünwald‐Giemsa stained cytospins. Scale bars represents 10 µm.

3. t‐SNE representation of individual hematopoietic cells from all eye compartments measured by scRNA‐seq. Each dot represents an individual cell. Color code indicates the respective cell types.

4. Unbiased cluster analysis of subpopulations of cells found in the steady‐state adult retina (cluster 13, 14, 15), ciliary body (cluster 1, 5, 10, 11, 12), cornea (cluster 2, 3, 6, 9, 16) and of mixed composition (4, 7, 8, 17) could be identified as microglia (cluster 13, 14, 15), macrophages (cluster 1, 2, 3, 6, 7, 8, 9, 10, 16), or peripheral blood leukocytes (cluster 4, 5, 11, 12, 17).

5. t‐SNE representation of single cells based on the tissue of origin.

6. Stacked bar plot (Marimekko chart) representation of macrophage subsets to a given cluster demonstrating pure microglia populations in clusters 13, 14, and 15. Hypergeometric testing revealed significantly enriched macrophage clusters in the cornea (2, 3, 6, 9, 16) and the ciliary body (1, 5, 10, 11, 12) as indicated by asterisks (*P < 0.05, **P < 0.01, ***P < 0.001).

7. Heatmap of the most regulated genes per cluster (adjusted P‐value < 0.05 based on the negative binomial distribution). Clusters are arranged from the left to the right and represent microglia (light green), macrophages (purple), and monocytes/granulocytes/lymphocytes (gray). The scale bar represents color‐coded z‐scores. Genes that were subsequently confirmed on protein level in (H) and (I) are highlighted by asterisks.

8. Above: t‐SNE representation of P2ry12 and Tmem119 expression. Below: immunofluorescence images for P2RY12 (red) and TMEM119 (red) in CX₃CR1⁺ (green) retinal microglia (outer plexiform layer) but not in CX₃CR1⁺ cells in the ciliary body and the peripheral stroma and epithelium of the cornea. Representative images out of three animals are shown. Scale bars represent 50 µm.

9. Above: t‐SNE plots for Cd74 and H2‐Aa expression. Below: Typical immunofluorescence pictures for CD74 (red) and MHCII (H2‐Aa) (red) in CX₃CR1⁺ (green) cells in the ciliary body and the peripheral stroma and epithelium of the cornea. Two animals were examined. Scale bars represent 50 µm.

Figure EV1. Single‐cell RNA‐seq from all cells in the healthy murine eye

1. t‐SNE representation of individual cells from all eye compartments measured by scRNA‐seq. Each dot represents an individual cell. Color code indicates the respective cell types.

2. Unbiased cluster analysis of cell subpopulations found in the retina, ciliary body, and cornea.

3. Stacked bar plot (Marimekko chart) representation of macrophage subsets to a given cluster.

4. Heatmap of the 30 most regulated genes per cluster (adjusted P‐value < 0.05 based on the negative binomial distribution). The scale bar represents the color‐coded z‐scores. Genes presented in t‐SNE plots in (E) are marked by asterisks.

5. t‐SNE plot of typical genes for microglia subsets (P2ry12, Tmem119, Hexb), macrophage cluster (Cd74, H2‐Aa, Apoe), Rods/Cone cluster (Rho, Prph2, Pdc), and epithelial cells cluster (Emp1, Gsto1, Krt5).

Figure EV2. Gene ontology enrichment (GO) analysis across all cell clusters of different eye compartments

GO pathways were selected and depicted for all cell cluster identified in Fig 1 for the eye compartments retina, ciliary body, and cornea.

Figure EV3. Comparison of brain and eye macrophages by single‐cell RNA‐seq

1. t‐SNE representation of single cells based on the tissue of origin.

2. Unbiased cluster analysis of subpopulations of cells found in the steady‐state adult brain and eye.

3. Stacked bar plot (Marimekko chart) representation of the proportional contribution of macrophage subsets from different tissues to a given cluster demonstrating a pure microglia from both brain and retina in clusters 0. Hypergeometric testing revealed significantly enriched microglia (cluster 0), and macrophage clusters in the ciliary body (3, 4, 5) are indicated by asterisks (*P < 0.05, **P < 0.01, ***P < 0.001).

4. Heatmap of the most regulated genes per cluster (adjusted P‐value < 0.05 based on the negative binomial distribution). Clusters are arranged from the left to the right. The scale bar represents the color‐coded z‐scores.

5. t‐SNE expression and line plot of homeostatic gene signatures of microglia (Tmem119, Hexb, Slc2a5, P2ry12, Siglech, Trem2), macrophages (Mrc1, Cd163, Lyve1, Siglec1, Stab1, Pf4, Ms4a7, Cbr2, Apoe), boarder‐associated macrophages (Irf7, Crip1, Ccl6, Ccl9, Clec4b1, Ccr2, Vim, Lsp1, Lgals3), monocytes (Ly6c2, Ccr2, Anxa8, Plac8, Nr4a1), dendritic cells (Flt3, Zbtb46, Batf3, Clec9a, Itgae), and antigen‐presenting cells (APCs) (Cd74, H2−Aa, H2−Eb1, H2−Ab1, Cd80, Cd86, Cd40).

Figure EV4. Flow cytometric gating strategy for eye macrophages and monocytes

1. Gating strategy of eye macrophages. CD11b⁺ or CD45⁺ cells underwent doublet exclusion by FSC‐W, ‐A, and ‐H gating and subsequent dead cell exclusion (fixable viability dye, FVD). Retinal microglia were specified as CD45^loCD11b⁺, macrophages in the ciliary body and cornea as CD45^loCD11b⁺CD64⁺F4/80⁺.

2. Gating strategy of blood monocytes. Leukocytes underwent doublet exclusion by FSC‐W, ‐A, and ‐H gating and subsequent dead cell exclusion (fixable viability dye, FVD). CD45⁺CD11b⁺ myeloid blood cells were further subdivided in CD45⁺CD11b⁺CD115⁺Ly6C^hi and CD45⁺CD11b⁺CD115⁺Ly6C^lo monocytes.

Figure EV5. Molecular survey of all retinal cells during neovascularization

1. t‐SNE representation of cells from control, CNV d3, and CNV d7 conditions.

2. t‐SNE plot showing identity of individual cells. Color code indicates different cell types.

3. Unbiased cluster analysis of cell populations found in the retina upon CNV induction.

4. Heatmap of the 30 most regulated genes per cluster (adjusted P‐value < 0.05 based on the negative binomial distribution). Scale bar represents the color‐coded z‐scores. Selected genes presented in t‐SNE plots shown in (E) are marked by asterisks.

5. t‐SNE presentations of genes characteristic for microglia subsets (P2ry12, Tmem119), dendritic cell cluster (Cd74, H2‐Aa), Rods/Cone cluster (Rho, Pdc), and epithelial cells cluster (Krt12, Gsto1).

Figure EV6. Gene ontology enrichment (GO) analysis across all myeloid cell clusters during neovascularization

GO pathways were selected and depicted for all cell cluster identified in Fig 7.

Figure 2. Comparative bulk RNA‐seq analysis of microglia from brain and retina, cornea macrophages, and bone marrow‐derived monocytes

1. Heatmap of differentially expressed genes between rMG, bMG, cMΦ, or bone marrow‐derived monocytes (BM‐Mo). The mean centered and s.d. scaled expression values for genes that were significantly and at least twofold more or less abundantly expressed are shown. See Fig EV4 for gating strategy. Data are derived from four independent experiments with 5–10 pooled mice per sample and shown as mean ± s.e.m.

2. Principal component analysis of myeloid cell transcripts analyzed by RNA‐seq. Data are derived from four independent experiments with 5–10 pooled mice per sample and shown as mean ± s.e.m.

3. Comparison of functional gene clusters between BM‐Mo, cMΦ, and rMG in comparison with bMG (reference population). Data are derived from four independent experiments with 5–10 pooled mice per sample and shown as mean ± s.e.m.

4. Left, spider plots showing commonly expressed genes across macrophages in comparison with bMG (reference population, center). Bold genes were plotted as bar graph on the right. Four samples were analyzed per cell type. Data are derived from four independent experiments with 5–10 pooled mice per sample and shown as mean ± s.e.m.

5. Left, spider plots showing genes highly enriched in microglia in comparison with bMG (reference population, center). Bold genes are plotted as bar graphs on the right. Data are derived from four independent experiments with 5–10 pooled mice per sample and shown as mean ± s.e.m.

6. Left, spider plots showing genes commonly expressed by monocytes and/or cMΦ in comparison with bMG (reference population, center). Bold genes are plotted as bar graphs (right). Data are derived from four independent experiments with 5–10 pooled mice per sample and shown as mean ± s.e.m.

7. Left, bar graphs showing expression of the genes Mrc1, Itgax, and Hexb in rMG, bMG, cMΦ, or bone marrow‐derived monocytes (BM‐Mo). Right, validation of Mrc1 (CD206) in Cx3cr1‐GFP mice, Itgax in Itgax‐GFP mice, and Hexb in Hexb‐Tomato mice in the cornea and the retina. Arrows point to CX₃CR1⁺CD206⁺ cMΦ (top row) or Iba1⁺CD11c⁺ cMΦ or rMG (middle row) or Iba1⁺Hexb⁺ cMΦ or rMG (bottom row). Asterisks point CX₃CR1⁺CD206⁻ cMΦ (top row) or Iba1⁺CD11c⁻ cMΦ or rMG (middle row) or Iba1⁺Hexb⁻ cMΦ or rMG (bottom row). Data are derived from four independent experiments with 5–10 pooled mice per sample and shown as mean ± s.e.m. Scales bar represents 50 µm.

Figure 3. Prenatal source of eye macrophages

1. Left: Creation of Acta1 ^GFP/+ :Acta1 ^+/+ bone marrow chimeras. Right: Donor‐derived GFP⁺CD45^loCD11b⁺ were detectable in the recipient retina by flow cytometry 20 weeks after bone marrow reconstitution. FACS Plots are representative for five animals from one experiment.

2. Quantification of GFP⁺ cells among CD45⁺CD11b⁺ retinal microglia by flow cytometry. Data are presented as mean ± s.e.m. One symbol represents one mouse.

3. Typical retinal flat mount from Acta1 ^GFP/+ :Acta1 ^+/+ bone marrow chimeras 20 weeks after reconstitution. Donor‐derived GFP⁺Iba1⁺ cells (arrow) and GFP⁻Iba1⁺ resident microglia (asterisks) are shown. Pictures are representative for three animals from one experiment. Scale bars represent 50 µm.

4. Microscopy‐based quantification of GFP⁺Iba1⁺ retinal microglia in the inner (IPL) and outer plexiform layer (OPL). One symbol represents one mouse. Data are presented as mean ± s.e.m.

5. Scheme of a fate mapping experiment using Cx3cr1^CreERT2:Rosa26‐YFP female mice. Tamoxifen (TAM) and progesterone injection were performed at embryonic day 9.0 (E9.0). Mice were subsequently evaluated at postnatal day 0 (P0). Administration of TAM leads to intra‐embryonic excision of a stop sequence flanked by loxP sites (gray triangles) in Cx3cr1 expressing cells which causes stable and steady YFP expression under the control of the Rosa26 promotor.

6. Direct fluorescence microscopic visualization for YFP (green), the macrophage marker Iba1 (red) and DAPI for the nuclei (blue) at P0. YFP⁺Iba1⁺ double‐positive cells are marked by arrows. YFP⁻Iba1⁺ single‐positive cells are labeled by asterisks. Representative images out of five examined animals are shown. Scale bars represent 25 µm.

7. Quantitative analysis of regional YFP expression in Iba1⁺ macrophages in TAM‐induced and untreated Cx3cr1^CreERT2:Rosa26‐YFP mice. Bars represent means ± s.e.m. Quantification was done from three (untreated) or five (TAM) mice obtained from one (untreated) or two (TAM) independent experiments. Level of significance determined by Mann–Whitney test between TAM and untreated revealed *P < 0.05 and Kruskal–Wallis test between retina, ciliary body, and cornea revealed *P = 0.0204.

Figure 4. Contribution of definitive hematopoiesis to eye macrophage subsets during steady‐state

1. Scheme of a fate mapping experiment using adult Cx3cr1^CreERT2:Rosa26‐YFP mice. Tamoxifen (TAM) injection was performed at postnatal day 42 (P42). Mice were evaluated at 2, 12, and 24 weeks post‐injections. Administration of TAM leads to the excision of a stop sequence flanked by loxP sites (gray triangles) in Cx3cr1 expressing cells in the eye which causes stable YFP expression under the control of the Rosa26 promotor.

2. Flow cytometric measurement of the persistence of YFP⁺ retinal microglia (rMG), cliliary body (cb) MΦ, and corneal (c) MΦ in adult Cx3cr1^CreERT2:Rosa26‐YFP mice. Doublets and dead cells were excluded by FSC‐W and viability dye. Representative flow cytometry plots from two independent experiments with at least six mice are displayed.

3. Kinetics of YFP labeling in macrophages of the healthy eye. Symbols represent means ± s.e.m. rMG are shown as squares (2 weeks: n = 10 mice, 12 weeks: n = 9 mice, 24 weeks: n = 12 mice, Kruskal–Wallis ns P > 0.05), cbMΦ are depicted as triangles (2 weeks: n = 4 samples from eight mice, 12 weeks: n = 3 samples from six mice, 24 weeks: n = 6 samples from twelve mice, Kruskal–Wallis ns P > 0.05) and cMΦ as circles (2 weeks: n = 6 mice, 12 weeks: n = 8 mice, 24 weeks: n = 12 mice, one‐way ANOVA ***P < 0.0001). Data were obtained from two (cMΦ: 2 weeks, cbMΦ: 2 and 12 weeks), three (rMG: 2 weeks, cbMΦ: 24 weeks), or four (rMG: 12 and 24 weeks, coMΦ: 12 and 24 weeks) independent experiments.

4. Sketch of the Flt3‐dependent Cre‐mediated recombination system with excision of the loxP‐flanked stop‐sequences leading to expression of YFP under the control of the Rosa26 promotor in Flt3^Cre:Rosa26‐YFP mice.

5. Left: Representative flow cytometric characterization of rMG by CD45 and CD11b and cbMΦ and cMΦ by CD45, CD11b, CD64, and F4/80 in Flt3^Cre:Rosa26‐YFP mice. Doublets and dead cell were excluded. Right: representative flow cytometric images depicting YFP expression in eye tissue macrophages of 12‐ or 52‐week‐old Flt3^Cre:Rosa26‐YFP mice. Typical images were taken from two independent experiments with six to seven mice.

6. Quantification of the percentage of YFP⁺ eye macrophages at 12 and 52 weeks of age. rMG are shown as squares (12 weeks: n = 7 mice, 52 weeks: n = 6 mice), cbMΦ as triangles (12 weeks: n = 3 samples from six mice, 52 weeks: n = 3 samples from six mice), and cMΦ as circles (12 weeks: n = 7 mice, 52 weeks: n = 6 mice). Data are presented as means ± s.e.m. and were acquired in two independent experiments.

Figure 5. Peripheral blood‐derived origin of myeloid cells contributing to homeostatic turnover of eye macrophages

1. A

   Left, Experimental setup of surgically connected parabiotic mice. Acta1 ^GFP/+ and Acta1^+/+ mice underwent parabiosis for 2, 12, and 20 weeks before analysis. Right, quantification of GFP⁺Iba1⁺ microglia in the retina (rMG, squares, n.d. = not detectable), ciliary body (cbMΦ, triangles, Mann–Whitney ns P > 0.05), and cornea (cMΦ, circles, Kruskal–Wallis **P = 0.0024) of parabiotic mice. Blood chimerism of CD45⁺CD11b⁺Ly6C^hiGFP⁺ cells in the analyzed wild‐type mice was 37.7 ± 3.2% (2 weeks), 27.5 ± 2.7% (12 weeks), and 34.7 ± 3.7% (20 weeks). Symbols represent mean ± s.e.m. of three (2 weeks), four (12 weeks) or five (20 weeks) individual mice. Scale bars represent 50 µm.

2. B–D

   Representative immunofluorescence images from the retina (20 weeks), ciliary body (12 weeks), and cornea (20 weeks) from flat mounts of Acta1 ^+/+ parabiotic mice. GFP⁺Iba1⁺ double‐positive cells are marked by arrows, GFP⁻Iba1⁺ single‐positive cells are labeled by asterisks and GFP⁺Iba1⁻ leukocytes are indicated by arrow heads. Pictures are representative of three animals.

3. E

   Flow cytometric quantification of RFP⁺ cells in Ccr2‐RFP mice among CD45⁺CD11b⁺CD115⁻Ly6C^int granulocytes (triangles, n = 4), CD45⁺CD11b⁺CD115⁺Ly6C^lo monocytes (filled squares, n = 4 mice), CD45⁺CD11b⁺CD115⁺Ly6C^hi monocytes (open squares, n = 4 mice), CD45^loCD11b⁺ rMG (squares, n = 6 mice), CD45⁺CD11b⁺CD64⁺F4/80⁺ cbMΦ (triangles, n = 6 samples from 12 mice), and CD45⁺CD11b⁺CD64⁺F4/80⁺ cMΦ (filled circles, n = 12 mice, unpaired t‐test ***P < 0.0001). Data were obtained from one (rMG) or two independent experiments (blood, cbMΦ, cMΦ). Data are presented as means ± s.e.m.

4. F–H

   Flow cytometry of eye macrophages from healthy Ccr2 ^RFP/+ mice (left) and representative histograms (right, Ccr2 ^RFP/+ solid red line, Ccr2 ^+/+ controls dotted gray line).

5. I

   Flow cytometry of myeloid blood cells from Ccr2 ^RFP/+ mice. Left: CD45⁺CD11b⁺ cells were further subdivided according to the expression of Ly6C and CD115 into CD45⁺CD11b⁺CD115⁻Ly6C^int granulocytes (gate 1), CD45⁺CD11b⁺CD115⁺Ly6C^hi inflammatory (gate 2), and CD45⁺CD11b⁺CD115⁺Ly6C^lo resident monocytes (gate 3), respectively. Right: representative histograms are shown (Ccr2 ^RFP/+ solid red line, Ccr2 ^+/+ controls dotted gray line). Four mice were investigated.

6. J

   Top left, sketch of Ccr2‐RFP construct. Top right, typical confocal picture for CCR2 (red), Iba1 (green), and collagen IV (Coll IV, white) revealing Ccr2‐RFP expression in a blood vessel (arrow, left image, scale bar represents 20 µm) and no RFP signal in retinal microglia (right images, scale bar represents 100 µm). Bottom, representative pictures of the ciliary body and cornea immunolabeled with F4/80 (green), CCR2 (red), and DAPI (blue). Arrows indicate RFP⁺F4/80⁺ cells. Asterisks point to RFP⁻F4/80⁺ cells. Representative images from two independent experiments with three mice are displayed. Scale bars represents 20 µm.

Figure 6. Spatiotemporal characteristics of myeloid subsets during experimental neovascularization

1. A

   Experimental setup. TAM was applied to 6‐week‐old Cx3cr1^CreERT2:Rosa26‐tdTomato mice leading to the excision of the stop sequence followed by robust Tomato expression in microglia. At the age of 14 weeks (8 weeks post‐TAM), three focal argon laser burns were applied to each retina to induce microglia activation and subsequent choroidal neovascularization (CNV). Analysis was performed on days (d) 3, 7, 14, 28, and 56, respectively.

2. B

   Above: Representative funduscopic pictures from living healthy Cx3cr1^CreERT2:Rosa26‐tdTomato mice on d0. Funduscopy and red fluorescence visualize the fundus and regular distribution of tomato⁺ microglia before the laser‐induced lesion formation. Below: Corresponding immunofluorescence pictures. Non‐lesioned retina show a regular pattern of Iba1⁺ (green) tomato⁺ (red) retinal microglia while macrophages are absent on the retinal pigment epithelium (RPE) under native conditions. Pictures are representative for six mice analyzed in one experiment. Scale bars represent 50 µm.

3. C

   Above: In vivo funduscopy on d7 post‐lesion. Funduscopic and red fluorescence image depict the lesions (encircled with dashed white lines) and accumulation of tomato⁺ microglia in Cx3cr1^CreERT2:Rosa26‐tdTomato mice. Intraperitoneal fluorescein application was performed to label retinal vessels and areas of choroidal neovascularization. Below: Representative immunofluorescence for Iba1 (green) in Cx3cr1^CreERT2:Rosa26‐tdTomato mice. Resident retinal microglia are Iba1⁺tomato⁺ (asterisks) whereas blood‐derived myeloid cells are Iba1⁺tomato⁻ (arrows) and accumulate at sites of laser‐induced CNV. Overlay is shown left. Typical pictures from six mice obtained from one independent experiment are shown. Scale bars represent 50 µm.

4. D

   Percentage (left) and absolute numbers (right) of myeloid subsets in the retina at different time points post lesion. Red columns, red lines, and red symbols represent tomato⁺Iba1⁺ microglia in Cx3cr1^CreERT2:Rosa26‐tdTomato mice whereas green columns, green lines, and green symbols represent blood‐derived tomato⁻Iba1⁺ myeloid cells. Left, Wilcoxon test at d3 (ns P = 0.0625), d28 (ns, P = 0.25), and d56 (ns P = 0.125) and paired t‐test at d7 (***P < 0.0001), and d14 (***P < 0.0001; right, absolute numbers Kruskal–Wallis test (tomato⁺Iba1⁺ *P < 0.05, tomato⁻Iba1⁺ **P < 0.01). Data represent means ± s.e.m. from at least three mice per group (two to six lesion per mouse) out of one (d0, d14, d28, d56) or two (d7, d14) independent experiments.

5. E

   Distribution (left) and absolute numbers (right) of myeloid cells in the RPE at different time points following laser‐induced lesion. Red columns, red lines, and red symbols represent tomato⁺Iba1⁺ microglia in Cx3cr1^CreERT2:Rosa26‐tdTomato mice. Green columns, green lines, and green symbols represent blood‐derived tomato⁻Iba1⁺ myeloid cells. Left, paired t‐test at d3, d7, d14 (**P < 0.01, ***P < 0.001), Wilcoxon test at d28 and d56 (ns P > 0.05); right, absolute numbers Kruskal–Wallis test (ns P > 0.05). Data represent means ± s.e.m. from at least three mice per group (two to six lesion per mouse) out of one (d0, d14, d28, d56) or two (d7, d14) independent experiments.

6. F, G

   Flow cytometric measurement of tomato expression in 14‐week‐old Cx3cr1^CreERT2:Rosa26‐tdTomato mice 8 weeks after TAM treatment (F) or with no treatment (G). Red lines represent the tomato signal and black lines the corresponding CreER‐negative control. Data are presented as mean ± s.e.m. from four mice analyzed in one experiment.

Figure 7. Detection of disease‐associated myeloid cell clusters

1. Schematic diagram depicting the experimental setup.

2. Isolation of single myeloid cells from the retina under healthy conditions (d0) and three (d3) or 7 days (d7) after laser pulse for unbiased sampling and single‐cell RNA‐seq (scRNA‐seq). Increased cellular heterogeneity in lesioned mice compared with healthy controls (d0), shown by May‐Grünwald‐Giemsa stained cytospins. Scale bars represents 10 µm.

3. t‐SNE representation of individual cells from all conditions analyzed (control represents homeostatic microglial cells), CNV d3, CNV d7, with scRNA‐seq. Each dot represents an individual cell. Color code indicates the different experimental conditions.

4. t‐SNE plot depicting different cell types.

5. Unbiased cluster analysis of hematopoietic cell subpopulations found at different conditions.

6. Marimekko chart showing the clusterwise distribution of 511 cells color‐coded for the experimental conditions (control 122 cells, CNV d3 209 cells, CNV d7 180 cells). Hypergeometric testing revealed significantly enriched clusters for the different conditions (control: cluster 4 and 5; CNV d3: cluster 1, 6 and 7; CNV d7: cluster 11, 12, and 14), indicated by asterisks (*P < 0.05, **P < 0.01, ***P < 0.001).

7. Marimekko chart presenting the proportion of cell types among all assigned cells (see Fig 5D) during different experimental conditions demonstrating almost pure microglia populations present at d0, whereas clusters of dendritic cells (DC), lymphocytes, monocytes, granulocytes, and macrophages are present at d3 and d7 of CNV.

8. Heatmap of the 30 most regulated genes per cluster (adjusted P‐value < 0.05 based on the negative binomial distribution). Asterisks highlight genes presented in t‐SNE plots shown in (I).The scale bar represents the color‐coded z‐scores.

9. Selection of t‐SNE plots representative for different cell subsets using Hexb, C1qa, and Sparc to show differential expression of these genes between microglia subpopulations; Grp34, Csf1r, and Gapdh to depict cluster 6 as the highest activated microglia cluster; Cd74, H2‐Aa, and Clec10a to present dendritic cell cluster; Ly6c2, Plac8, and Ms4a4b to illustrate blood‐derived cell cluster.

Figure 8. Disease stage‐dependent gene expression pattern in myeloid cells during experimental CNV

1. Trajectory analysis reveals alterations in gene expression pattern over time and disease state (d0, d3, d7). Above: Trajectory heatmap depicting loss of homeostatic microglial gene expression signatures (P2ry12, Tmem119, Csf1r, SiglecH) and induction of genes involved in cytoskeleton modifications (Actb, Tuba1c), cell cycle progression (Cdk1, Ptma), and antigen presentation (Cd74, H2‐Aa). Highlighted molecules (asterisks) were confirmed by immunohistochemistry shown in (B–D). Below: Trajectory plot showing the trajectory followed by myeloid cells upon CNV progress.

2. Above: P2ry12 expression is shown by trajectory analysis and represented in a t‐SNE plot. Expression is high in homeostatic microglia (see Figs 1G and H and 7H). Below: Typical immunohistochemistry for P2RY12 (green) in unlesioned and lesioned areas. Arrows indicate lost P2RY12 expression. Asterisks mark microglial cells that maintained P2RY12 expression. Iba1 immunohistochemistry (red) to show microglia. Pictures are representative of two mice. Scale bars represent 25 µm.

3. Above: Tmem119 expression visualized by trajectory analysis and represented in a t‐SNE plot. High signals in homeostatic microglia (see Fig 1G and H). Below: TMEM119 immunofluorescence (green) by Iba1⁺ (red) microglia inside lesioned and non‐lesioned areas in the retina and RPE. Arrows indicate microglia with lost TMEM119 expression whereas asterisks mark microglial cells that maintained TMEM119 expression. Picture is representative of two mice. Scale bars represent 25 µm.

4. Above: Ptma expression presented by a t‐SNE plot and its trajectory analysis over the disease course. Ptma expression correlates with proliferation of rMGs at d7 during CNV in Cx3cr1^CreERT2:Rosa26‐tdTomato mice. Below: Representative immunofluorescence of proliferating EdU⁺ (white) Iba1⁺ (red, Tomato) microglia at sites of CNV on d7 (arrow) next to an EdU⁻Tomato⁺Iba1⁻ cell (asterisks) after EdU treatment from d0 to d6. Arrowhead points toward an intravascular myeloid EdU⁻Tomato⁻Iba1⁺ cell. Typical pictures from three mice obtained from one independent experiment are shown. Scale bar represents 25 µm.

Figure 9. Correlative analysis between murine experimental and human neovascular CNV

1. H2‐Aa expression visualized by trajectory analysis and represented in a t‐SNE plot.

2. H2‐Aa immunohistochemistry (green) by Iba1⁺ (red) microglia inside lesioned and non‐lesioned areas in the retina and RPE. Arrows indicate microglia with acquired MHCII expression whereas asterisks mark microglial cells that remained MHCII negative. Picture is representative of two mice. In the RPE, arrow heads label Tom⁻MHCII⁺ cells. Scale bars represent 25 µm.

3. HLA‐DRA mRNA expression examined by RNA‐Seq via Massive analysis of cDNA ends (MACE) analysis of human formalin‐fixed and paraffin‐embedded (FFPE) membranes of choroidal neovascularization (CNV). Bars represent means ± s.e.m. of four investigated human samples of age‐related macular degeneration associated with choroidal neovascularization and four control tissues consisting of choroid and RPE. Level of significance was calculated using DESeq (P = 3.61 × 10⁻²¹).

4. Immunohistochemistry for HLA‐DR in the RPE/choroid of one control and the CNV membrane of one patient suffering from age‐related macular degeneration associated with choroidal neovascularization.