Single-Cell RNA Sequencing Analysis of the Early Postnatal Mouse Lens Epithelium.

ABSTRACT

Purpose: The lens epithelium maintains the overall health of the organ. We used single-cell RNA sequencing (scRNA-seq) technology to assess transcriptional heterogeneity between cells in the postnatal day 2 (P2) epithelium and identify distinct epithelial cell subtypes. Analysis of these data was used to better understand lens growth, differentiation, and homeostasis on P2. Methods: scRNA-seq on P2 mouse lenses was performed using the 10x Genomics Chromium Single Cell 3' Kit (v3.1) and short-read Illumina sequencing. Sequence alignment and preprocessing of data were conducted using 10x Genomics Cell Ranger software. Seurat was employed for preprocessing, quality control, dimensionality reduction, and cell clustering, and Monocle was utilized for trajectory analysis to understand the developmental progression of the lens cells. CellChat and GO analyses were used to explore cell-cell communication networks and signaling interactions. Results: Lens epithelial cells (LECs) were divided into seven subclusters, classified by specific gene markers. The expression of crystallin, cell-cycle, and metabolic genes was not uniform, indicating distinct functional roles of LECs. Trajectory analysis predicted a bifurcation of differentiating and cycling cells from an Igfbp5+ progenitor pool. We also identified heterogeneity in signaling molecules and pathways, suggesting that cycling and progenitor subclusters have prominent roles in coordinating crosstalk. Conclusions: scRNA-seq corroborated many known markers of epithelial differentiation and proliferation while providing further insight into the pathways and genes directing these processes. Interestingly, we demonstrated that the developing epithelium can be divided into distinct subpopulations. These clusters reflect the transcriptionally diverse roles of the epithelium in proliferation, signaling, and maintenance.