Single-cell characterization of anterior segment development in the mouse reveals the cell types, pathways, and signals driving formation of the trabecular meshwork and Schlemm’s canal
- Department of Ophthalmology, Columbia University Irving Medical Center, United States
- Department of Molecular Biology and Genetics, Johns Hopkins University, United States
- Department of Ophthalmology and Visual Sciences, College of Medicine, The Ohio State University Wexner Medical Center, United States
- Department of Ophthalmology, Johns Hopkins School of Medicine, United States
- Mortimer B. Zuckerman Mind Brain Behavior Institute, Columbia University, United States
Figures
Figure 1 with 2 supplements
Overview of the single-cell RNA sequencing experiments.
(a) Diagram of developmental milestones of Schlemm’s canal (SC) and trabecular meshwork (TM) cells in mice. Red arrows indicate timepoints of sample collection. (b) Diagram of single-cell RNA sequencing experimental design. Dotted lines indicate cutting planes when dissecting the anterior segment and limbal regions. (c) Uniform Manifold Approximation and Projection (UMAP) representation of the single-cell transcriptomes from all timepoints colored by annotated cell types. (d) Barplot showing the proportion of each cell type identified across the sampled developmental stages. Some caution is needed in relating to exact in vivo proportions due to isolation and processing procedures that can alter cell-type proportions.
Figure 1—figure supplement 1
Cell-type annotation.
(a) Heatmap showing the expression of signature genes (rows) across single cells (columns), arranged by seven major cell types. (b) Uniform Manifold Approximation and Projection (UMAP) representation of all cells colored by sample collection timepoints. (c) UMAP representation of all cells colored by cluster identity at each timepoint.
Figure 1—figure supplement 2
Identification of periocular mesenchyme (POM)/neural crest (NC)-derived cell types.
(a) Visualization of selected marker genes – Tfap2b and Pitx2 – expression plotted on Uniform Manifold Approximation and Projection (UMAP) embedding of all cells. (b) Heatmap of signature gene expression across single cells arranged by POM-derived cell types.
Figure 2 with 1 supplement
Periocular mesenchyme (POM)/neural crest (NC)-derived cell-type analysis.
(a) Uniform Manifold Approximation and Projection (UMAP) representation of POM-derived cells colored by high-resolution cell-type annotations. (b) UMAP representation of POM-derived cells colored by sample timepoint. (c) Dotplot showing the expression of key POM marker genes (columns) in each cell type, where the size of dots indicates the fraction of genes expressed and color indicates the average level of expression. (d) Barplot showing the fraction of each POM/NC-derived cell type across timepoints until postnatal day 21 (P21). Data were integrated across ages, P60 omitted as integration with developing clusters obscured mature cell types at this age. Unidentified technical variation resulted in fewer TM3 being profiled at P21. (e) UMAP representation of POM/NC-derived cells at each timepoint, colored by their cell-type annotations (same as in a).
Figure 2—figure supplement 1
Developmental clusters and trabecular meshwork (TM) cell subtypes.
(a) Hierarchical clustering of annotated cell types across postnatal day 2.5 (P2.5)–P10 developmental timepoints for all periocular mesenchyme (POM)/neural crest (NC)-derived cell types. (b) Barplots demonstrating the proportion of cell cycle phases across sample collection timepoints for the five POM-derived developmental cell types.
Figure 3 with 7 supplements
Analysis of trabecular meshwork (TM) cell development.
(a) 3D view of a segment of the TM across ages represented by the Surface mode in Imaris. The morphology of the Schlemm’s canal (SC) facing side of the TM is shown. It becomes progressively more ordered along the longitudinal Y axis of the structure as development proceeds, with obvious beam-like striations at postnatal day 21 (P21). Scale bar = 50 μm. (b) Visualization of a 1-µm-thick optically sectioned portion of the TM from a 3D TM reconstruction in the orthogonal XZ plane. Nuclei (4′,6-diamidino-2-phenylindole [DAPI], blue), cell borders (GFP, yellow), and open spaces (magenta, pseudo-coloring) of the TM are shown across different developmental stages. At P6.5, the TM is dense with closely packed cells and nuclei (both rounded and elongated). Minimal spaces are evident between the cell borders. As differentiation proceeds (by P10) and inter-trabecular beams and spaces form (P14), the nuclei become elongated and more organized, with increasing volume of space between cells. Clear organization of elongated nuclei and robust inter-trabecular spaces are evident by P21 as major morphogenesis of the TM is complete. Scale bar = 50 μm, red dotted line indicates TM border. (c) Quantification of TM open space and volume with age. (d) Violin plots showing expression distributions of genes enriched in TM cell subtypes across sample timepoints. (e) Marker expression across developmental timepoints (immunofluorescence) MYOC – magenta, CRYM – red, α-SMA – green, TFAP2B – green (appears cyan because nuclear co-localization with DAPI). CD31 and yellow dotted lines mark the presumptive SC border. White lines mark TM cells. a: anterior, p: posterior. (f) Left: Uniform Manifold Approximation and Projection (UMAP) embedding of periocular mesenchyme (POM)/neural crest (NC)-derived cells between P2.5 and P10, colored by pseudo-time (Monocle) where the root node was chosen manually as a random cell in Dev5 cluster and labeled in the plot (see main text). Right: UMAP representation with monocle pseudo-time trajectory colored by cell types. (g) Sankey diagram summarizing the development of three TM cell subtypes from clusters Dev1 to Dev5.
Figure 3—figure supplement 1
3D analysis of trabecular meshwork (TM) development.
This figure describes the anatomic landmarks used to define the TM for Figure 3 and Figure 3—figure supplement 2. (a) En face view of the entire circumference of the aqueous humor drainage structures and surrounding tissues. Fluorescent labeling was achieved by breeding together the Wnt1-Cre2 and mTmG alleles. This gene combination results in the Schlemm’s canal (SC) and surrounding vasculature expressing tdTomato (red), while the TM, corneal endothelium, ciliary muscle, and lymphatics express GFP (green; see Materials and methods). The ocular tissue was prepared as a whole mount and imaged through the Z-depth using confocal microscopy. Scale bar = 500 µm. (b) En face view of a cropped region of the aqueous humor (AH) drainage structures, as indicated by gray lines in (a). The orientation icon in the upper-right corner corresponds to that used in subsequent figures. (c–e) YZ views spanning the full depth of the tissue, from the outer to inner surface of the eye. The limbal vascular plexus (LVP), containing both blood and lymphatic vessels, is located closer to the outer surface of the eye compared to the SC and TM. The LVP was optically removed to view the SC and TM. Collector channels (asterisks) are cropped to within 10 µm of their connection to the outer SC surface. (f–g) En face views of TM and SC along the tissue plane between the dotted lines in (d) and (e), with other regions cropped out. The SC is visible in (f), while the TM and adjacent structures are shown in (g). The TM exhibits the highest GFP signal intensity. At the posterior boundary of the TM, ciliary muscle fibers (arrows) run orthogonally to the TM. At the anterior boundary of the TM, larger, circular cell borders of the corneal endothelium are visible (hashtags). Dotted lines mark the anterior and posterior boundaries of the TM. (h) For analysis, the structures surrounding the TM were further cropped out at the dotted lines in (g). All scale bars (b–h) = 100 µm.
Figure 3—figure supplement 2
Trabecular meshwork (TM) cell subtype classification.
(a) View of the aqueous humor (AH)-facing side of the TM segments shown in Figure 3 (Imaris Surface mode). As development progresses, the structure becomes increasingly organized along its longitudinal axis. (b) Separated panels of TM XZ optical sections from Figure 3. Empty spaces that lacked both nuclei (4′,6-diamidino-2-phenylindole [DAPI], blue) and cells (GFP, yellow Materials and methods) are marked (magenta, pseudo-color), the red dotted line indicates TM border. (c) Violin plot showing Crym expression across developmental clusters and ages. Crym is expressed in the Dev1 cluster from postnatal day 2.5 (P2.5) to P10, which may explain the detection of CRYM protein in the developing TM prior to P6.5 (before TM2 cells are robustly identified). (d) Immunofluorescence visualization of TM cell markers shown in Figure 3 at additional developmental ages. MYOC (TM1Hi, magenta) is absent in the TM at early stages but becomes detectable by P10. CRYM (TM2Hi, red) is present at all examined ages, though early expression may reflect Dev1 identity (see above). α-SMA (TM3Hi, green) is consistently expressed across all stages. CD31 and yellow dotted lines demarcate the presumptive SC boundary. White lines outline the TM cell group. a: anterior, p: posterior.
Figure 3—figure supplement 3
Gene ontology (GO) enrichment analysis of trabecular meshwork (TM) developmental marker genes.
(a, b) GO biological processes of TM3 sub-cluster marker genes (compared to other periocular mesenchyme [POM]/neural crest [NC]-derived cell types) at postnatal day 2.5 (P2.5) and P4.5. (c) GO molecular function of TM2 sub-cluster marker genes at P6.5. (d) GO biological processes of TM2 sub-cluster marker genes at P10. (e) GO molecular function of TM1 sub-cluster marker genes at P14. (f) GO biological processes of TM1 sub-cluster marker genes at P21.
Figure 3—figure supplement 4
Identification of epithelial cell types.
(a) Visualization of selected marker gene expression plotted on Uniform Manifold Approximation and Projection (UMAP) embedding of all cells. (b) UMAP representation of epithelial cells colored by subtypes. (c) UMAP representation of epithelial cells colored by collection timepoint. (d) Heatmap of signature gene expression across single cells arranged by epithelial cell types.
Figure 3—figure supplement 5
Identification of ciliary body/iris/retinal pigment epithelium (RPE) cell types.
(a) Visualization of selected marker gene expression plotted on Uniform Manifold Approximation and Projection (UMAP) embedding of all cells. (b) UMAP representation of ciliary body/iris/RPE cells colored by subtypes. (c) UMAP representation of ciliary body/iris/RPE cells colored by collection timepoint. (d) Heatmap of signature gene expression across single cells arranged by ciliary body/iris/RPE cell types.
Figure 3—figure supplement 6
Identification of immune cell types.
(a) Visualization of selected marker gene expression plotted on Uniform Manifold Approximation and Projection (UMAP) embedding of all cells. (b) UMAP representation of immune cells colored by subtypes. (c) UMAP representation of immune cells colored by collection timepoint. (d) Heatmap of signature gene expression across single cells arranged by immune cell types.
Figure 3—figure supplement 7
Identification of progenitor cell types.
(a) Visualization of selected marker gene expression plotted on Uniform Manifold Approximation and Projection (UMAP) embedding of all cells. (b) UMAP representation of progenitor cells colored by subtypes. (c) UMAP representation of progenitor cells colored by collection timepoint. (d) Heatmap of signature gene expression across single cells arranged by progenitor cell types.
Figure 4 with 1 supplement
Analysis of Schlemm’s canal endothelial cell (SEC) development.
(a) Uniform Manifold Approximation and Projection (UMAP) representation of endothelial cells colored by cell subtypes. (b) Dotplot summarizing expression of marker genes across endothelial cell types where dot size indicates fraction of cells expressing each gene and color indicates average level of expression. (c) UMAP representation of endothelial cells colored by sample timepoints. (d) UMAP representation of endothelial cells colored by high-resolution cell-type annotations across individual sample timepoints. Asterisk indicates the emergence of SECs. (e) Independent analysis of endothelial cells across developmental timepoints – dotplots of expression of key marker genes across each timepoint for each of the dynamically changing endothelial cell types. (f) Dimensional reduction (UMAP visualization) and cell-type annotation as defined separately for each timepoint.
Figure 4—figure supplement 1
Identification of endothelial cell subtypes.
(a) Visualization of selected marker genes plotted on Uniform Manifold Approximation and Projection (UMAP) embedding of all cells. (b) Heatmap of signature gene expression across single cells arranged by endothelial cell types. (c) Barplots demonstrating the fraction of cell cycle phase across sample collection timepoints for the five endothelial cell types. (d) Dotplots representing the expression of selected marker genes (columns) across sample collection timepoints in each of the five endothelial cell types (rows). (e) Normalized expression of genes along pseudotime with individual dots colored by chronological time.
Figure 5
Differentiation of inner wall (IW) and outer wall (OW) of Schlemm’s canal endothelial cells (SECs).
(a) Uniform Manifold Approximation and Projection (UMAP) representation of endothelial cells colored by developmental pseudo-time inferred by Monocle. (b) Monocle trajectory superimposed on the UMAP representation of endothelial cells colored by cell-type annotation, root node being programmatically determined. (c) Visualization of marker gene expression plotted on UMAP embedding of endothelial cells. (d) Scatter plot showing the average expression level of marker genes in a combined population of vascular progenitor (VP) and SECs across sample collection timepoints. (e) Immunofluorescence labeling of key SEC marker genes across developmental and adult timepoints, top panels showing the vascular plane, bottom panels showing the Schlemm’s canal (SC) plane from the same Z stack.
Figure 6 with 3 supplements
Dynamic gene expression analysis during Schlemm’s canal endothelial cell (SEC) development.
(a–b) Gene ontology (GO) enrichment of biological process genes selectively expressed during early development in a: vascular progenitors (VPs); b: SECs. In all GO plots, x-axis represents the fraction of genes in each pathway, which are also identified in the corresponding differentially expressed gene set. Colors indicate adjusted p-value of enrichment, and dot size indicates number of genes in the pathway. (c, d) Average expression level of genes in selected GO biological processes enriched in VPs and SECs during early development, across sample collection timepoints. (e) GO enrichment of genes expressed during mid-developmental timepoints in SECs. (f–i) Average expression level of genes in select processes enriched in VPs or SECs across sample collection timepoints. (j) GO enrichment of genes expressed in SECs at late development. (k–m) Average expression level of genes in select processes enriched in SECs across sample collection timepoints.
Figure 6—figure supplement 1
Gene ontology (GO) molecular pathway enrichment analysis of genes across Schlemm’s canal endothelial cell (SEC) development.
(a–d) Enrichment of molecular function pathway genes selectively expressed during SEC development.
Figure 6—figure supplement 2
Gene ontology (GO) biological process enrichment analysis of genes across Schlemm’s canal endothelial cell (SEC) development.
(a–d) Enrichment of biological process pathway genes selectively expressed during SEC development.
Figure 6—figure supplement 3
Expression of pericyte marker genes.
Violin plots representing the expression profile of four selected pericyte marker genes across sample collection timepoints. Dots represent individual cells.
Figure 7 with 6 supplements
Ligand-target interaction analysis between trabecular meshwork (TM) and Schlemm’s canal (SC) cells.
(a–f) Circos plots demonstrating inferred interactions between TM (sender) and SC (receiver) cells across sample collection timepoints stratified by early (postnatal day 2.5 [P2.5] and P4.5), mid (P6.5 and P10), and late (P14 and P21) timepoints.
Figure 7—figure supplement 1
Predicted ligand-target interactions between trabecular meshwork (TM) and Schlemm’s canal (SC) cells during development.
Heatmaps represent the regulatory potential between TM ligands (rows) and predicted receptors/targets in SC cells (columns) across three sample collection timepoints (a: postnatal day 2.5 [P2.5]; b: P10; c: P21). NicheNet prioritized extracellular signaling molecules (y-axis) and their predicted downstream direct and indirect targets in the receiving cell population (x-axis), which may include receptors, signaling mediators, extracellular matrix (ECM) components, and transcriptional targets. Color intensity reflects the NicheNet regulatory potential score.
Figure 7—figure supplement 2
Expression levels of trabecular meshwork (TM) ligands during Schlemm’s canal (SC) development.
(a–f) Dotplots indicating the expression pattern of ligands (ligands) which were predicted to contribute to SC development in each of the three TM cell types (columns) across sample collection timepoints. Dot size is indicative of percent of cells expressing the ligand (not number of cells – due to low numbers of TM1 and TM2 cells at postnatal day 2.5 [P2.5] and P4.5, the dots should be interpreted with caution at these ages).
Figure 7—figure supplement 3
Predicted ligand-target interaction between vascular progenitor (VP)-Schlemm’s canal (SC) and SC-SC interactions.
(a) Heatmaps representing the regulatory potential between VP ligands (rows) and predicted receptors/targets in SC endothelial cells (SECs) (columns) at postnatal day 4 (P4). (b) Heatmaps representing the regulatory potential between SC ligands (rows) and predicted receptors on SC cells (columns) at P4.
Figure 7—figure supplement 4
Ligand-target interaction analysis between macrophages/pericytes and Schlemm’s canal endothelial cells (SECs).
(a, b) Circos plot demonstrating inferred interactions between macrophages (sender) and SECs (receiver) at postnatal day 4.5 (P4.5) and P21. (b, c) Circos plot demonstrating inferred interactions between pericytes (sender) and SECs (receiver) at P4.5 and P21.
Figure 7—figure supplement 5
Ligand-target interaction analysis macrophages-trabecular meshwork (TM) and Schlemm’s canal (SC)-TM.
(a, b) Circos plot demonstrating inferred interactions between macrophages (sender) and TM (receiver) cells at postnatal day 10 (P10) and P21. (c, d) Circos plot demonstrating inferred interactions between SC endothelial cells (SECs) (sender) and TM (receiver) cells at P10 and P21.
Figure 7—figure supplement 6
Analysis of expression profile of glaucoma risk genes in Schlemm’s canal (SC) and trabecular meshwork (TM) development.
(a) Dot plot summarizing expression of glaucoma risk genes from intraocular pressure (IOP) and primary open angle glaucoma (POAG) GWAS across all anterior segment (AS) cell types. (b, c) Gene ontology (GO) molecular function and biological process gene enrichment of genes that cause developmental glaucoma. (d, e) Average expression level of glaucoma risk genes in SC endothelial cells (SECs) and TM cells across sample collection timepoints; developmental glaucoma genes are marked by a rectangular box.
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (Mus musculus, both sexes) | C57BL/6J | Jackson Labs | IMSR_JAX: 000664 | |
| Strain, strain background (M. musculus, both sexes) | B6.Cg-E2f1Tg(Wnt1-cre)2Sor/J | Jackson Labs | Stock 022501 | |
| Strain, strain background (M. musculus, both sexes) | B6.129(Cg)-Gt(ROSA)26Sortm4(ACTB-tdTomato,-EGFP)Luo/J | Jackson Labs | Stock 007676 | |
| Antibody | α-SMA (Rabbit polyclonal) | Abcam | Cat# ab5694; RRID:AB_2223021 | WM (1:50) Sections (1:200) |
| Antibody | α-SMA (Mouse monoclonal) | Sigma-Aldrich | Cat# A5228; RRID:AB_262054 | IF sections (1:200) |
| Antibody | Goat polyclonal MYOC | R&D Systems | Cat# AF2537; RRID:AB_2266857 | WM (1:50) Sections (1:200) |
| Antibody | Rabbit polyclonal Mu-Crystallin | Proteintech | Cat# 12495–1-AP RRID:AB_2084620 | IF sections (1:100), TSA |
| Antibody | Rabbit polyclonal TFAP2B | Novus Biologicals | Cat# NBP1-89063; AB_11005304 | IF sections (1:200) |
| Antibody | FLT4 (Goat polyclonal) | R&D Systems | Cat# AF743; RRID:AB_355563 | WM (1:50) Sections (1:200) |
| Antibody | NPNT (Goat polyclonal) | R&D Systems | Cat# AF4298; RRID:AB_10645643 | WM (1:50) Sections (1:200) |
| Antibody | PECAM-1 (CD31) (Rat monoclonal) | eBioscience | Cat# 14-0311-82; RRID:AB_467201 | WM (1:50) Sections (1:200) |
| Antibody | SELP (Goat polyclonal) | R&D Systems | Cat# AF737-SP RRID:AB_2285644 | WM (1:50) Sections (1:100) |
| Commercial assay or kit | Papain Dissociation System | Worthington Biochemical | Cat# LK003153 | |
| Commercial assay or kit | Tyramide signal amplification | Akoya Biosciences | Cat # NEL744001KT | |
| Chemical compound, drug | 4% PFA | Thermo Fisher Scientific | Cat# 50-980-495 | |
| Chemical compound, drug | Propidium iodide | Thermo Fisher Scientific | Cat# P1304MP | |
| Chemical compound, drug | SYTOX green Nucleic Acid Stain | Thermo Fisher Scientific | Cat# S7020 | |
| Chemical compound, drug | Collagenase Type 4 | Worthington Biochemical | Cat# LS004188 | 1 mg/ml |
| Other | DAPI | Thermo Fisher Scientific | Cat# 62248 | (1:5000) Section ‘Immunohistochemistry’ |
| Other | 40 and 100 μm Falcon cell strainers | Thermo Fisher Scientific | 08-771-1/08-771-19 | Section ‘Single-cell RNA sequencing’ |
Appendix 1—table 1
Markers for developing and adult trabecular meshwork (TM) and Schlemm’s canal (SC) cell types.
| Markers | Target cell type |
|---|---|
| MYOC, CHIL1 | TM1 |
| CRYM, INMT, EDN3 | TM2 |
| a-SMA, TFAP2B, LYPD1, LMX1B | TM3 |
| CXCL1, CCL2, HMCN1 | Dev1 |
| COL14A1, DLK1 | Dev2 |
| CLU, COL8A1 | Dev3 |
| IGF1, ECM1, LMX1B | Dev4 |
| HES1 | Dev5 |
| APLNR, CLEC14A | Vascular progenitors |
| IGF1, MMP14 | Early/developing Schlemm’s canal cells |
| NPNT, CCL21A, ITGA9 | Schlemm’s canal endothelial cells (inner wall) |
| SELP, LCN2 | Schlemm’s canal endothelial cells (outer wall) |
-
Marker genes are not completely specific to each cell type indicated above. Combination of markers and their levels of expression must be considered. Markers listed above are most highly expressed in the indicated cell types.
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Single-cell characterization of anterior segment development in the mouse reveals the cell types, pathways, and signals driving formation of the trabecular meshwork and Schlemm’s canal
eLife 15:RP109230.
https://doi.org/10.7554/eLife.109230.3
