Abstract
Purpose :
This study aims to investigate the mechanisms underlying the microphthalmia and retarded lens growth in connexin 50 (Cx50, encoded by Gja8) knockout (Cx50KO), and the macrophthalmia in the mitogen-activated protein kinase kinase 1 (MEK1(E)) transgenic mice. The downstream targets of Cx50 and/or MEK1 mediated signaling pathways in lens epithelium that regulate the lens homeostasis and the lens size will be studied.
Methods :
Gene expression profiles of lens epithelial cells from wild-type (WT), Cx50KO, MEK1(E), and double mutant Cx50KO/MEK1(E) mice were determined by single-cell RNA-sequencing (scRNA-seq) analysis. Epithelial cell clusters were examined by tSNE and UMAP. Selective up- and down-regulated gene candidates in Cx50KO, MEK1(E) and Cx50KO/MEK1(E) samples were evaluated in lens epithelial cells in vivo or in vitro by using immunostaining and western blot analysis.
Results :
The tSNE plots of the scRNA-seq data of postnatal day 30 lenses reveal ten distinct cell clusters in WT, twelve clusters in Cx50KO and MEK1(E), and eleven clusters in Cx50KO/MEK1(E) lens epithelial cells. UMAP comparisons show obvious changes of specific cell clusters between WT and different mutant lens epithelial cells. Compared to WT cells, Cx50KO had drastically reduced cell numbers of the clusters 2, 3 and 4 that highly express crystallin genes, but increased cell numbers of clusters 1, 6 and 7 that highly express various non-crystallin genes; MEK1(E) showed changes of different cell clusters compared to the WT; and specific alterations of cell clusters were also detected between Cx50KO/MEK1(E) and the Cx50KO samples. Moreover, the expression profiles of distinct gene markers were identified in different cell clusters of different lens samples.
Conclusions :
Specific gene markers of distinctive cell clusters of wild-type lens epithelium reflect their cellular states and organization from the anterior pole to the equator for undergoing proliferation and differentiation to control lens homeostasis and growth. The changes of epithelial cell clusters in different mutant lenses indicate altered trajectory of these cell clusters. Identified up- or down-regulated DEG profiles from the scRNA-seq data reveal specific downstream targets of Cx50 and/or MEK1 mediated signal transduction pathways that regulate the proliferation and/or differentiation of lens epithelial cells, which ultimately affects the lens size and lens homeostasis.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.