Abstract
Purpose :
To investigate the mechanism for how connexin 50 (Cx50, encoded by Gja8), a protein subunit of intercellular gap junctions, regulates the proliferation, differentiation, and homeostasis of lens epithelium; to study how Cx50 mediated-intrinsic signals and downstream molecules modulate lens epithelial cell heterogeneity and lens growth.
Methods :
Gene expression profiles of lens epithelial cells from both wild-type (WT) and Cx50 knockout (Cx50KO) mice were determined by single-cell RNA-sequencing (scRNA-seq) analysis. Both up- and down-regulated gene candidates in Cx50KO samples were evaluated in lens epithelial cells in vivo or in vitro by using immunostaining and western blot analysis. The lens growth in live knockout mice was longitudinally studied in vivo by using the Leica Envisu R4310 spectral-domain OCT (SD-OCT) system.
Results :
The scRNA-seq data reveal differentially expressed genes between Cx50KO and WT epithelial cells. Altered epithelial cell clusters are associated with changes of Cx50-mediated downstream genes in the Cx50KO lens epithelium. 164 upregulated and 29 downregulated genes have been identified as significant candidates in Cx50KO epithelium, including upregulation of Aqp1 and several anti-proliferative transcription factors and downregulation of transcription factor Maf and 11 crystallin genes. The longitudinal study of lens growth shows significantly reduced lens anterior chamber depth (ACD) and lens thickness (LT) in Cx50KO compared to WT at different ages during postnatal development.
Conclusions :
The scRNA-seq data indicate distinctive cell clusters of wild-type monolayer epithelium, which are likely organized to regulate the lens growth for mediating signals of surrounding ocular tissues. Cx50-mediated gap junction communication maintains water homeostasis and lens epithelial cell proliferation by regulating aquaporin 1 and several anti-proliferation factors. The upregulation of anti-proliferation factors may directly control lens epithelial cells proliferation leading to small lens phenotype in CX50KO. The OCT measurement reveals new ocular pathological changes in Cx50KO mice.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.