Transcriptomic profiling of Schlemm's canal cells reveals a lymphatic-biased identity and three major cell states
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. Overall design: The anterior segment of the eye including the limbus was dissected from each of ten 3-month-old Prox1-GFP mice to make 1 biological replicate of each cell type. The iris, cornea, and ciliary body were gently and carefully removed using forceps to prevent damage to the delicate aqueous humor drainage structures. A strip of limbal tissue 1-2 mm wide consisting of the aqueous humor outflow tissues SC and TM, limbal blood vessels, and lymphatics was dissected from each anterior segment. Limbal strips were dissected in ice-cold PBS. Strips from 20 eyes were pooled and dissociated paying strict attention to osmolarity (290-300 mOSm) and mechanical force, using 1mg/ml Collagenase Type 4 (Worthington Biochemical Cat. no. LS004188) to preserve membrane proteins for the staining cell surface marker for flow cytometry.  Single-cell suspension was stained with Lyve1 conjugated to eFluor 660 (ALY7 eBioscience Cat. no. 14-0443-82), Endomucin conjugated to phycoerythrin (eBio V.7.C7 Cat. no.14-5851-82), and propidium iodide to mark dead cells. Forward and side scatter gates were first used to eliminate events with low scatter which include derbies, cell fragments and pyknotic cells. Then propidium iodide positive dead cells were gated out. Further gating on the viable cells was applied such that distinct population of cells were isolated a) SECs -GFP+Lvye1-collected as, b) lymphatic endothelial cells (LECs)-GFP+ Lyve1+, c) GFP- blood endothelial cells (BECs)-Endomucin+. A total of 1500-1700 cells for SEC, 200-300 cells for LEC and 2000-2500 cells for BEC were collected. Three biological replicates were processed for each of SECs, BECs, and LECs.