Mapping the expression of key functional genes associated with murine corneal endothelial development.

Abstract

The cornea is a complex, layered, transparent structure that is responsible for transmitting and refracting light to ensure vision. Of all the layers, the corneal endothelium is physiologically the most important layer. Its principle function is to regulate deturgescence, a requirement for corneal transparency. Corneal deturgescence is regulated by key functional genes that include a network of tight junctions, water channels and ion pumps. In this study, we created an in vitro model of murine corneal endothelial development by immortalising endothelial cells at significant stages in corneal endothelial morphogenesis. Primary cultures of presumptive corneal endothelial cells were immortalised from wild-type mouse embryos at embryonic day (E) 14.5 and E15.5, and mature corneal endothelial cells were immortalised from wild-type pups at post-natal day (P) 13. These cell lines, together with already established cell lines at earlier stages in corneal endothelial development (E12.5 and E13.5), were used to map the expression patterns of three functional genes associated with corneal endothelial function, Zo1, Aqp1 and Slc4a4. Zo1 is a tight junction protein that facilitates the formation of the resilient monolayer that is characteristic of the corneal endothelium. Aqp1 is a water channel protein that mediates transcellular fluid transport while Slc4a4 is an ion pump that transports cellular bicarbonate ions across plasma membranes. Aqp1 and Slc4a4 are disease target genes which is indicative of their functional significance in corneal endothelial physiology. Quantitative real-time PCR (qPCR) and western blot analysis showed that Zo1 expression at both mRNA and protein level was progressively down-regulated from E12.5 to E15.5, after which it was up-regulated at P13 (p˂0.05 relative to E15.5 at mRNA level). Aqp1 gene expression was up-regulated at E13.5, with subsequent down-regulation at E14.5. Thereafter, Aqp1 gene expression was up-regulated at E15.5. Interestingly, Aqp1 gene expression was significantly down-regulated at P13 when the endothelium is completely matured (p˂0.05 relative to all other cell lines). Western blot analysis demonstrated Aqp1 expression throughout development in this model. Densitometric analysis showed that glycosylated Aqp1 protein expression levels decreased gradually from E12.5 to P13. Unglycosylated Aqp1 protein expression was only present at E12.5 and E13.5, and was up-regulated at E13.5. Slc4a4 was expressed at low levels during corneal endothelial development. However, it was highly expressed at P13 (p˂0.05 relative to all other cell lines). Protein expression for Slc4a4 could not be determined due to the lack of a commercially available antibody specific for murine Slc4a4. Confocal microscopy analysis demonstrated the protein localisation patterns for Zo1 and Aqp1 in monolayer and hanging drop culture, mimicking the 3D environment in which corneal endothelial development occurs. The results of this study provide valuable insight into the expression patterns of key functional genes associated with corneal endothelial development.