Overview of the single-cell RNA-sequencing experiments.

a) Diagram of developmental milestones of SC and TM cells in mice. Red arrows indicate timepoints of sample collection. b) Diagram of single-cell RNA-sequencing experimental design. 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.

POM/NC-derived cell type analysis.

a) 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 size of dots indicates fraction of genes expressed and color indicates average level of expression. d) Barplot showing the fraction of each POM/NC-derived cell type across timepoints until P21. Un-identified 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).

Analysis of 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 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 P21. Scale bar = 50µm b) Visualization of a 1 um thick optically sectioned portion of the TM from a 3D TM reconstruction in the orthogonal XZ plane. Nuclei (DAPI, blue) and cell borders (GFP, yellow pseudo-coloring) of the TM are shown across different developmental stages. At P6.5, the TM is a densely packed with nuclei (both rounded and elongated), and minimal spaces are evident between 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 cell borders. Clear organization of elongated nuclei and robust inter-trabecular spaces is evident by P21 as major morphogenesis of the TM is complete. Scale bar = 50µm c) Violin plots showing expression distributions of genes enriched in TM cell subtypes across sample timepoints. d) Marker expression across developmental timepoints (immunofluorescence) MYOC – magenta, CRYM – red, a-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. e) Top: UMAP embedding of POM/NC-derived cells between P2.5-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). Bottom: UMAP representation with monocle pseudo-time trajectory colored by cell types. f) Sankey diagram summarizing the development of three TM cell subtypes from clusters Dev1-Dev5.

Analysis of SEC development.

a) 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 Schlemm’s canal endothelial cell cluster (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.

Differentiation of IW and OW of SECs.

a) UMAP representation of endothelial cells colored by developmental pseudotime 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 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 SC plane from the same Z-stack.

Dynamic gene expression analysis during Schlemm’s canal endothelial cell development.

a-b) Gene ontology (GO) enrichment of biological process genes selectively expressed during early development in a: 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.

Ligand-target interaction analysis between TM and SC cells.

a-f) Circos plots demonstrating inferred interactions between TM (sender) and SE (receiver) cells across sample collection timepoints stratified by early (P2.5 and P4.5), mid (P6.5 and P10), and late (P14 and P21) timepoints.