Cell atlas of aqueous humor outflow pathways in eyes of humans and four model species provides insight into glaucoma pathogenesis

Abstract

Increased intraocular pressure (IOP) represents a major risk factor for glaucoma, a prevalent eye disease characterized by death of retinal ganglion cells; lowering IOP is the only proven treatment strategy to delay disease progression. The main determinant of IOP is the equilibrium between production and drainage of aqueous humor, with compromised drainage generally viewed as the primary contributor to dangerous IOP elevations. Drainage occurs through two pathways in the anterior segment of the eye called conventional and uveoscleral. To gain insights into the cell types that comprise these pathways, we used high-throughput single-cell RNA sequencing (scRNAseq). From ∼24,000 single-cell transcriptomes, we identified 19 cell types with molecular markers for each and used histological methods to localize each type. We then performed similar analyses on four organisms used for experimental studies of IOP dynamics and glaucoma: cynomolgus macaque (Macaca fascicularis), rhesus macaque (Macaca mulatta), pig (Sus scrofa), and mouse (Mus musculus). Many human cell types had counterparts in these models, but differences in cell types and gene expression were evident. Finally, we identified the cell types that express genes implicated in glaucoma in all five species. Together, our results provide foundations for investigating the pathogenesis of glaucoma and for using model systems to assess mechanisms and potential interventions.

Keywords: ciliary muscle; macaque; mouse; pig; trabecular meshwork.

Conflict of interest statement

J.R.S. is a consultant at Biogen. All other authors declare no competing interest.

Figures

Fig. 1.

Human AH outflow pathways. (A) Diagram of the anterior segment of the human eye, which includes the cornea, iris, ciliary body (CB), and lens. AH is secreted by the CB and circulates (blue arrows) within the anterior chamber prior to draining from the eye through one of two pathways located within the iridocorneal angle delineated by boxed area. (B) Enlarged view of the iridocorneal angle (boxed area in A) highlighting two outflow pathways for AH. In the conventional pathway, AH traverses the TM, first through the uveal meshwork (orange highlight), then the corneoscleral meshwork (light green highlight), and finally, the JCT (dark green highlight) prior to entering SC. AH exits the SC via CCs that empty into aqueous veins (AVs) that themselves merge with episcleral veins (EVs). Nonfiltering TM is located at the insert region (yellow highlight), referred to as Schwalbe line (SL), which abuts the corneal endothelium. In the uveoscleral pathway, AH exits via the interstices of the CM. SS, scleral spur.

Fig. 2.

Cell types and gene expression in the human outflow pathways. (A) Clustering of 24,023 single-cell expression profiles from human TM and associated structures visualized by t-distributed stochastic neighbor embedding (tSNE). Arbitrary colors are used to distinguish clusters deemed to be distinct by unsupervised analysis. Clusters were numbered according to relative size, with 1 being the largest. (B) tSNE plot shown in A but with cells colored by sample of origin. Note that corneal epithelial cells (C7) and fibroblasts (C9) were derived primarily from the rim sample (H9). (C) Frequency of each cell type; numbering is the same as in A and D. (D) Violin plots showing expression of genes selectively expressed by cells of each type. The dendrogram in Left shows transcriptional relationships among cell types. Fibro, fibroblast; K-Epi, corneal epithelium; Melano, melanocyte; MØ, macrophage; SchMy, myelinating Schwann cell; SchNmy, nonmyelinating Schwann cell; Schwal, Schwalbe line; VEndo, vascular endothelium.

Fig. 3.

Cells of the human conventional outflow pathway. (A) Dot plot showing genes selectively expressed in cells of the conventional outflow pathway. In this and subsequent figures, the size of each circle is proportional to the percentage of cells expressing the gene, and the shade intensity depicts the average normalized transcript count in expressing cells. Fibro, fibroblast; K-Epi, corneal epithelium; Melano, melanocyte; MØ, macrophage; SchMy, myelinating Schwann cell; SchNmy, nonmyelinating Schwann cell; Schwal, Schwalbe line; VEndo, vascular endothelium. (B) Labeled meridional section of a human corneoscleral rim visualized using autofluorescence (AF) demonstrates outflow anatomy at the iridocorneal angle visualized after removal of iris and ciliary body. The dashed line indicates scleral spur and arrowhead indicates region of CCs. (C) TM (beam and JCT) cells immunostained for PDPN (green) and RARRES1 (red). (D) Fluorescent RNA in situ hybridization against TMEFF2 (red) and PPPR1B1 (green) highlights beam cells. (E) Fluorescent RNA in situ hybridization against ANGPTL7 (green) and CHI3L1 (red) highlights cells in the JCT. (F) Immunostaining for PECAM1/CD31 (green) highlights SC, CCs (arrowheads), and CM capillaries, while ACKR1/DARC (red) costains only the CCs. The same field as in B. (G) Corneoscleral rim section demonstrates the tissue left behind after TM dissection. Very few cells in this region are positive for PDPN (green) or PECAM1/CD31 (red), indicating successful removal of relevant structures (TM and SC) during dissection protocol. (H) Scleral fibroblasts identified in the corneoscleral rim collection immunostained for ADH1B. (I) Schwalbe line cells at the junction of TM and corneal endothelium costain with PDPN (green) and AQP1 (red). (J) Fluorescent RNA in situ hybridization against POSTN (red) and TFF3 (green) highlights SC endothelium. DAPI, 4′,6-diamidino-2-phenylindole (nuclear stain). (Scale bars: 50 µm.)

Fig. 4.

Cells of the human uveoscleral pathway. (A) Dot plot showing genes selectively expressed in cells of the uveoscleral outflow pathway. Fibro, fibroblast; K-Epi, corneal epithelium; Melano, melanocyte; MØ, macrophage; SchMy, myelinating Schwann cell; SchNmy, nonmyelinating Schwann cell; Schwal, Schwalbe line; VEndo, vascular endothelium. (B) Smooth muscle cells immunostained for DES (red) and melanocytes immunostained for MLANA (green) in CM. (C) Capillaries in the CM immunostained for PECAM1 (green) and ALPL (red). Occasional PECAM1+ALPL+ staining was also noted in SC, suggesting that this structure contains more than one cell type. (D) Immunostaining for CALB2 (red) highlights intrinsic neurons of the CM. (E) Immunostaining for LYVE1 (green) and CD27 (red) identifies macrophages in the TM. (F) Schwann cells in the CM immunostained for CDH19 (red) amid CM cells stained for DES (green). (G) Higher magnification of the area bracketed in B demonstrates an MLANA+ melanocyte (green). AF, autofluorescence; DAPI, 4′,6-diamidino-2-phenylindole (nuclear stain). (Scale bars: B and C, 50 µm; D–G, 25 µm.)

Fig. 5.

Human disease genes. Cell type-specific expression of several genes implicated in glaucoma as illustrated by t-distributed stochastic neighbor embedding (A; arranged as in Fig. 2A) and a dot plot (B). B also includes data from a retinal cell atlas, showing expression in RGCs and three types of retinal glia. Green shading highlights genes that genetics studies suggest may confer additional IOP-independent glaucoma risk; many of these genes are also associated with high IOP. K-Epi, corneal epithelium; MØ, macrophage; ScEndo, Schlemm canal endothelium; Schwann-my, myelinating Schwann cell; Schwann-nmy, nonmyelinating Schwann cell; VascularEndo, vascular endothelium.

Fig. 6.

Cell types and gene expression in the outflow pathways of two macaque species (M. mulatta, MM; and M. fascicularis, MF). (A) A t-distributed stochastic neighbor embedding (tSNE) plot showing 15 cell types derived from TM and associated structures of M. mulatta. (B) Transcriptional correspondence between human and M. mulatta cell types summarized as a ‘‘confusion matrix.’’ In this and subsequent figures, the size of the circle and its intensity indicate the percentage of cells of a given cluster from the model species (column) assigned to a corresponding human cluster (row) by a classification algorithm trained on the human cells. Fibro, fibroblast; K-Epi, corneal epithelium; Melano, melanocyte; MØ, macrophage; ScEndo, Schlemm canal endothelium; SchMy, myelinating Schwann cell; SchNmy, nonmyelinating Schwann cell; Schwal, Schwalbe line; VEndo, vascular endothelium. (C) A tSNE plot showing 20 cell types derived from TM and associated structures of M. fascicularis. (D) Transcriptional correspondence between human and M. fascicularis shown as in B. (E) Violin plot showing examples of genes selectively expressed by each cell type in M. mulatta.

Fig. 7.

Cell types and gene expression in the outflow pathways of the pig. (A) A t-distributed stochastic neighbor embedding (tSNE) plot showing 18 cell types derived from TM and associated structures of pig. (B) Transcriptional correspondence between human and pig cell types shown as in Fig. 6B. Fibro, fibroblast; K-Epi, corneal epithelium; Melano, melanocyte; MØ, macrophage; SchMy, myelinating Schwann cell; SchNmy, nonmyelinating Schwann cell; Schwal, Schwalbe line; VEndo, vascular endothelium. (C) Violin plot showing examples of genes selectively expressed by each cell type in pig. KEndo, corneal endothelium; K Epi, corneal epithelium; ScEndo, Schlemm canal endothelium; Sch-my, myelinating Schwann cell; Sch-nmy, nonmyelinating Schwann cell.

Fig. 8.

Cell types and gene expression in the outflow pathways of the mouse. (A) A t-distributed stochastic neighbor embedding (tSNE) plot showing 20 cell types derived from TM and associated structure of mouse. (B) Transcriptional correspondence between human and mouse cell types shown as in Fig. 6B. Fibro, fibroblast; K-Epi, corneal epithelium; Melano, melanocyte; MØ, macrophage; SchMy, myelinating Schwann cell; SchNmy, nonmyelinating Schwann cell; Schwal, Schwalbe line; VEndo, vascular endothelium. (C) Violin plot showing examples of genes selectively expressed by each cell type in mouse. K-Endo, corneal endothelium; NPCE, nonpigmented ciliary epithelium; PE, pigmented epithelium; Schwn, Schwann cell. (D and E) Pdpn (red) is present in multiple cell types, including pigmented and nonpigmented epithelium of the iris and ciliary body (CB) as well as a subset of TM cells, K-Endo, and K-Epi, whereas Chil1 (green; ortholog to CHI3L1 in humans) stains a different subset of TM cells and to a lesser extent, cells within the CB. (E) Higher magnification of the boxed area in D. (F and G) Immunostaining against Postn (green), a secreted protein, highlights SC and JCT cells. Pecam1 (red) highlights SC as well as vascular endothelial clusters. AF, autofluorescence. DAPI, 4′,6-diamidino-2-phenylindole (nuclear stain). (Scale bars: 50 µm.)

Fig. 9.

Comparison of gene expression across species. (A and B) Key genes are shown in dot plots for cell types comprising the TM (A) and uveoscleral outflow pathway (B). (C) Heat map showing expression of genes implicated in human POAG in aqueous outflow cells of humans (replotted from SI Appendix, Fig. S3) and model species. In some cases (i.e. TM, cEndo), multiple cell types from each species have been merged into their broader class to facilitate comparison. cEndo, canal endothelium (includes both Schlemm canal and collector channel clusters where available); Melano, melanocyte; Sch-my, myelinating Schwann cell; Sch-nmy, nonmyelinating Schwann cell; TM, trabecular meshwork (includes beam cells and JCT where available), vEndo, vascular endothelium.