Cell atlas of the human ocular anterior segment: Tissue-specific and shared cell types

Abstract

The anterior segment of the eye consists of the cornea, iris, ciliary body, crystalline lens, and aqueous humor outflow pathways. Together, these tissues are essential for the proper functioning of the eye. Disorders of vision have been ascribed to defects in all of them; some disorders, including glaucoma and cataract, are among the most prevalent causes of blindness in the world. To characterize the cell types that compose these tissues, we generated an anterior segment cell atlas of the human eye using high-throughput single-nucleus RNA sequencing (snRNAseq). We profiled 195,248 nuclei from nondiseased anterior segment tissues of six human donors, identifying >60 cell types. Many of these cell types were discrete, whereas others, especially in the lens and cornea, formed continua corresponding to known developmental transitions that persist in adulthood. Having profiled each tissue separately, we performed an integrated analysis of the entire anterior segment, revealing that some cell types are unique to a single structure, whereas others are shared across tissues. The integrated cell atlas was then used to investigate cell type-specific expression patterns of more than 900 human ocular disease genes identified through either Mendelian inheritance patterns or genome-wide association studies.

Keywords: ciliary body; cornea; iris; lens; trabecular meshwork.

Fig. 1.

Human anterior segment and cells of the central cornea. (A) The human eye depicted in sagittal section. (B) The anterior segment, which includes the cornea, iris, CB, and lens. The limbus, representing the transition between peripheral cornea and sclera, houses the aqueous outflow structures including the TM and Schlemm canal. (C) Central cornea comprises three primary cellular layers: epithelium, stroma, and endothelium. The stratified epithelium is composed of basal, wing, and superficial cells delineated in the boxed area. (D) Clustering of 37,485 single-nucleus expression profiles from human central cornea visualized by uniform manifold approximation and projection (UMAP). Here and in subsequent UMAPs, arbitrary colors are used to distinguish clusters deemed to be distinct by unsupervised analysis. (E) Feature plots demonstrating DE genes corresponding to the epithelial subtypes. (F) Dot plot showing genes selectively expressed in cells of the central cornea, with gradient expression patterns noted in the epithelial subtypes. In this and subsequent figures, the size of each circle is proportional to the percentage of nuclei within a cluster expressing the gene and the color intensity depicts the average normalized transcript count in expressing cells. (G) Corneal superficial epithelium immunostained for KRT78 (green). (H) Fluorescent RNA ISH for BCAS1 (red) highlights superficial epithelium, and NECTIN4 (green) highlights both wing cells and superficial cells. (I) ISH for BCAS1 (red) highlights superficial epithelium and LAMA3 (green) highlights basal cells. (J) Transit amplifying cells are identified via ISH for TOP2A (green). (K) Corneal stromal fibroblasts are highlighted by ISH for ANGPTL7 (green); the superficial epithelium is highlighted by ISH for BCAS1 (red). (L) Corneal endothelium immunostained for CA3 (red). AF, autofluorescence; K_Epi, corneal epithelium; TA, transit amplifying; K_Fibro, corneal fibroblasts; K_Endo, corneal endothelium. Yellow bars: 20 µm.

Fig. 2.

Cell types of the internal and external limbus derived from dissection of CSW. (A) Diagram of the limbus, representing the transitional tissue between peripheral corneal and sclera. (B) Clustering of 52,309 single-nucleus expression profiles derived from CSW tissue visualized by UMAP. (C) Feature plots showing a selection of genes enriched in ocular surface epithelium subtypes. (D) ISH for BCAS1 (red) highlights the superficial epithelium in both the cornea and conjunctiva, whereas KRT12 (green) highlights basal and wing cells of the cornea. A transitional area is noted in the limbus where KRT12 expression tapers off and is absent in the conjunctival epithelium. Here, and in F, G, and I, the images represent a cropped version of a larger montage obtained from a continuous meridional section spanning cornea, limbus, and sclera, providing anatomical landmarks to support labeling. For an example of the full image, see SI Appendix, Fig. S3F. (E) ISH for KRT12 (green) highlights basal and wing cells of the cornea, and RARRES1 (green) highlights superficial and some wing cells of the conjunctiva. A transition area within the limbus is noted. (F) ISH for NTRK2 (red) highlights the basal epithelium of the cornea, and PDGFC (green) highlights mostly the basal epithelium of the conjunctiva. (G) Immunostaining against PECAM1 (red) highlights endothelial cells lining vessels in the subepithelial stromal tissues of the external limbus. Immunostaining against PDPN (green) highlights lymphatic endothelium lining a subset of these vessels. (H) Transit amplifying basal cells within the limbus are highlighted with ISH for TOP2A (green); basal and wing cells in the limbal area demonstrate KRT12 (red) expression as visualized by ISH. (I) A goblet cell in the conjunctiva visualized with immunostaining against MUC5AC (green). (J) Conjunctival melanocytes visualized by ISH for PAX3 (green) and KIT (red). (K) Uveal melanocytes visualized by ISH for PAX3 (green) and MET (red). Mø, macrophage; Conj, conjunctival; CC_VenEndo, Collector Channel/Venous Endothelium; Vasc, vascular; Endo, endothelium, K, cornea; Fibro, fibroblast. (Scale bars: 100 µm.)

Fig. 3.

Cells of the iris and CB. (A) Diagram of the human iris, consisting of the anterior border layer, the stroma, the sphincter muscle, APE, and PPE, shown in greater detail within the box. (B) Clustering of transcriptomesderived from iris tissue visualized by UMAP. (C) Dot plot showing genes selectively expressed in cells of the iris. (D) Iris sphincter muscle cells immunostained for desmin (DES; red). (E) Uveal melanocytes within the anterior border layer and stroma of the iris immunostained for Melan-A (MLANA; red). (F) Iris fibroblasts and vessel endothelium within the iris stroma immunostained with PDPN (green) and PECAM1 (red), respectively. (G) Immunostaining against RELN (red) highlights the iris PPE and to a lesser extent iris APE; the latter is also positive for DES (green), highlighting its contractile role as the iris dilator. (H) Diagram of the human CB, consisting of the ciliary muscle, ciliary stroma, and ciliary processes, shown in greater detail within the box. (I) Clustering of 34,132 single-nucleus expression profiles derived from CB tissue visualized by UMAP. (J) Dot plot showing genes selectively expressed in cells of the CB. (K) Ciliary stromal fibroblasts immunostained with PDPN (green) and vessel endothelium with PECAM1 (red). (L) Uveal melanocytes within the ciliary stroma immunostained for MLANA (red). (M) CB-NPCE and CB-PCE immunostained with LRP2 (green) and RELN (red), respectively. (N) Immunostaining against CRB1 (green) highlights a subset of NPCE situated in proximity with neighboring ciliary processes. (O) RNA ISH for MECOM (green) highlights NPCE lining a ciliary process. Vasc_Endo, vascular endothelium; CM, ciliary muscle, CP, ciliary process; White scale bars: 100 µm; yellow bars: 20 µm.

Fig. 4.

Cells of the crystalline lens. (A) The human crystalline lens consists of lens epithelium and lens fiber cells. (B) Clustering of 13,900 single-nucleus expression profiles derived from lens tissue visualized by UMAP. A continuum is observed ranging from anterior epithelium to lens fiber cells. (C) Dot plot showing genes selectively expressed in cells of the lens. (D) Feature plots demonstrating DE genes corresponding to cells of the lens. (E and F) Lens epithelial cells visualized with ISH. Expression of CACNA1A (green) is evident in both anterior and equatorial epithelial cells, whereas ATP8B4 (red) is confined to the anterior epithelium. (G) ISH demonstrates the expression of GPR160 (green) by early lens fiber cells and CAV1 (red) by more mature fiber cells. (H and I) Transitional lens epithelial cells in the post equatorial region are positive for SLC1A2 (red) as visualized by ISH; early fiber cells are positive for GPR160 (green); and fiber cells are positive for UCHL1 (green). (White scale bars; 100 µm; yellow bars: 20 µm.)

Fig. 5.

Integrated analysis of cells populating the human anterior segment. (A) Clustering of expression profiles pooled for the integrated analysis and visualized by UMAP. (B) Stacked bar chart indicating proportions within each cluster contributed by separate tissue sources. Transcriptional relatedness indicated by dendrogram. (C–E) Dot plot showing common and DE genes in fibroblast types (C),vessel endothelial types (D), and uveal epithelium. (F) Violin plots showing common and selectively expressed genes in the two pericyte clusters. Here and in other violin plots, the scale on y axis represents expression level, calculated as a normalized log(UMI+1) value. (G) Feature plots demonstrating expression patterns within the macrophage cluster. (H) Dot plot showing cell-type–specific expression of PAX3, MET, KIT, and LEF1. (I) Iris fibroblasts selectively expressing ETNPPL (red), visualized with RNA ISH. (J) TM fibroblasts selectively expressing NEB (red), visualized with RNA ISH. (K) Schlemm canal (SC) endothelium expressing FN1 (red), visualized with RNA ISH. (L) SC endothelium expressing PKHD1L1 (red), visualized with RNA ISH. (M) Melanocytes within the basal layer of the conjunctiva visualized with ISH for PAX3 (red) and LEF1 (green). Consistent with expression pattern seen in H, vascular endothelium lining a vessel within the conjunctival stroma is also noted to be positive for LEF1 (green). (N) Melanocytes within the iris stroma, visualized with RNA ISH for PAX3 (red), do not express LEF1 (green, absent). The iris sphincter is instead positive for LEF1, consistent with expression noted in H. (O) Uveal melanocytes within the iris anterior border layer and stroma visualized with ISH for MET (red) and PAX3 (green). (P) Melanocytes within the iris stroma, visualized with ISH for PAX3 (red), do not express KIT (green, absent). L, Limbus; Conj Epi, conjuctival epithelium. Remaining abbreviations as in previous figures. (White scale bars: 100 µm.)

Fig. 6.

Expression of ocular disease-associated genes. (A) Feature plots demonstrating enriched expression of glaucoma-associated genes within cell types localized to the iridocorneal angle drainage structures. (B) Dot plot showing cataract- and EL-associated genes. (C) Feature plot showing expression patterns of genes implicated in corneal dystrophies. (D) Dot plot showing cell-type–specific enrichment scores of genes identified through GWAS for common ocular conditions or traits. Major retinal cell types from normal macula are also included. Cell type abbreviations are as in previous figures. H1/H2, horizontal cells; BC, bipolar cells; AC, amacrine cells; RGC, retinal ganglion cells.