Transcriptomic profiling of Schlemm's canal cells reveals a lymphatic-biased identity and three major cell states

Abstract

Schlemm's canal (SC) is central in intraocular pressure regulation but requires much characterization. It has distinct inner and outer walls, each composed of Schlemm's canal endothelial cells (SECs) with different morphologies and functions. Recent transcriptomic studies of the anterior segment added important knowledge, but were limited in power by SEC numbers or did not focus on SC. To gain a more comprehensive understanding of SC biology, we performed bulk RNA sequencing on C57BL/6J SC, blood vessel, and lymphatic endothelial cells from limbal tissue (~4500 SECs). We also analyzed mouse limbal tissues by single-cell and single-nucleus RNA sequencing (C57BL/6J and 129/Sj strains), successfully sequencing 903 individual SECs. Together, these datasets confirm that SC has molecular characteristics of both blood and lymphatic endothelia with a lymphatic phenotype predominating. SECs are enriched in pathways that regulate cell-cell junction formation pointing to the importance of junctions in determining SC fluid permeability. Importantly, and for the first time, our analyses characterize 3 molecular classes of SECs, molecularly distinguishing inner wall from outer wall SECs and discovering two inner wall cell states that likely result from local environmental differences. Further, and based on ligand and receptor expression patterns, we document key interactions between SECs and cells of the adjacent trabecular meshwork (TM) drainage tissue. Also, we present cell type expression for a collection of human glaucoma genes. These data provide a new molecular foundation that will enable the functional dissection of key homeostatic processes mediated by SECs as well as the development of new glaucoma therapeutics.

Figure 1:. SEC bulk transcriptome is more similar to LEC than BEC transcriptomes.

A. Pairwise comparison of bulk RNA sequencing data showing differentially expressed genes (DEGs) between cell groups, note larger number of non-DEGs in gray in plot of LEC vs. SEC compared to others. B. Hierarchical clustering of BECs, SECs, and LECs. C. Pathways enriched in SECs vs BECs (top panel), LECs vs. SECs (bottom panel) by GSEA analysis. BEC: Blood endothelial cell, LEC: Lymphatic endothelial cell, SEC: Schlemm’s canal endothelial cell.

Figure 2.. scRNA-seq data reveals robust SEC signature genes.

A. Clusters of cells from the limbal tissue represented on a UMAP space B. Expression of Egfl7 in endothelial cells. C. Endothelial cell cluster is sub-clustered into BEC, LEC, and SEC. D. Expression of Flt1 enriched in BEC, Prox1 in LEC and SEC, and Lvye1 in LEC identifying the three cell clusters. E. Violin plot of genes expressed BEC, SEC, and LEC from scRNA-seq data. F. Heatmap of signature genes from BEC, SEC, and LEC from scRNA-seq data. BEC: Blood endothelial cell, LEC: Lymphatic endothelial cell, SEC: Schlemm’s canal endothelial cell.

Figure 3:. Integrating scRNA and snRNA-seq enables detection of outer and inner wall transcriptomes.

A. snRNA-seq of C57BL/6J limbal tissue identifies similar cell types as the sc RNA-seq but captures more endothelial cells (left panel). Expression of Egfl7 and Cdh5 in snRNA-seq endothelial cells (right panel). B. Integration of sc and snRNA-seq endothelial cells followed by sub-clustering identifies BECs, LECs, IW SECs, OW SECs, and CC. Integration of sc- and sn- RNA sequencing shows distribution across clusters (bottom panel). C. Heatmap of differentially expressed genes of the identified sub-clusters. D. SECs and LEC sub-clusters identified in B, expressing Prox1. In the SEC cluster, IW cells express Npnt and OW cells Selp, CCs express Ackr1 and BECs robustly express Flt1. IW: Inner wall, OW: Outer wall, CC: Collector channels, BEC: Blood endothelial cell, LEC: Lymphatic endothelial cell, SEC: Schlemm’s canal endothelial cell.

Figure 4.. Integration of C57BL/6J and 129/Sj endothelial cells identifies IW states.

A. scRNA-seq of 129/Sj anterior segment tissue identifies various cell types similar to that in C57BL/6J single-cell RNA sequencing (left panel). Expression of Egfl7 and Cdh5 in snRNA-seq endothelial cells (right panel). B. Integration of B6 and 129/Sj endothelial cells followed by sub-clustering identifies BECs, LECs, IW1 SECs, IW2 SECs, OW SECs, and CCs (top panel). Integration of B6 and 129/Sj endothelial cells distributed across clusters (bottom panel). C. Sub-clustering identifies complementary expression patterns of Npnt and Ccl21a in IW1 and IW2 SECs, Ackr1 in CCs, and Selp in OW SECs. D. Heatmap of differentially expressed genes of the identified sub-clusters. E. Violin plot showing differences in expression levels of various genes which as a combination defines individual sub-clusters. IW: Inner wall, OW: Outer wall, CC: Collector channels, BEC: Blood endothelial cell, LEC: Lymphatic endothelial cell, SEC: Schlemm’s Canal endothelial cell.

Figure 5.. Immunofluorescence validates cell types and discovers bias for discrete localization of IW1 and IW2 cells.

A. Npnt expression in a subgroup of SEC in scRNA-seq data (i) and corresponding immunofluorescence (IF) reveals high level of expression of NPNT in anterior portion of IW of SC in a frozen section (ii) and whole mount (iii and iv). B. Ccl21a is expressed in SECs and LECs (i) and corresponding IF reveals high expression in posterior portion of IW of SC in a frozen section (ii) and whole mount (iii and iv). C. Selp is expressed in OW SECs and CCs, a subgroup of SECs in single-cell (i) and corresponding IF (ii frozen section, iii-iv whole mount). D. Ackr1 expression in a subset of CC cells (i) and corresponding IF (ii frozen section, iii whole mount). DAPI in blue labels nuclei in all panels. IW: Inner wall, OW: Outer wall, CC: Collector channels, CB: Ciliary body, LY: Lymphatic vessels, BV: Blood vessels SC: Schlemm’s canal. Ant.: Anterior SC, Post.: Posterior SC.

Figure 6.. Cell-type assignment of high IOP and glaucoma genes.

A. Expression levels of gene list obtained from GWAS and genetic analyses associated with glaucoma and elevated IOP. B. Expression levels of genes involved in developmental forms of glaucoma. C. Disease relevance score (DRS) of genes from GWAS studies associated with glaucoma and elevated IOP. D. Expression levels of specific genes in subtypes of endothelial cells.

Figure 7:. Predicted ligand-target analysis reveals signaling pairs between TM and SC.

A, B. Circos plots depicting predicted ligand-target interaction between genes expressed in SECs and those in surrounding trabecular meshwork cells. C. Gene ontology analysis of genes enriched in SC. D. Heatmap of genes in individual enriched pathways showing their expression in endothelial cell subsets.