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. 2021 Feb 8;40(6):e105123. doi: 10.15252/embj.2020105123

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

Figure 1

  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.