Assessing the role of the transcription factor FOXC1 in the expression and regulation of the Adherens junction protein N-Cadherin during corneal endothelium development.

Abstract

The proper organization and differentiation of the anterior segment is pivotal for normal eye development. Neural crest-derived POM cells are key contributors to correct anterior segment formation, differentiating to form the monolayered corneal endothelium. Mice with homozygous null mutations in the forkhead transcription factor gene, Foxc1, fail to develop a proper corneal endothelium stabilized by adherens junctions, with the endothelium adhering to the lens, preventing anterior chamber separation. The aim of this study was to evaluate the interaction between Foxc1 and the adherens junction protein, N-cadherin, as well as an associated gene, Msx1, during key stages in corneal endothelium development. Foxc1 was over-expressed in E12.5 and E13.5 POM cells and qPCR was carried out to determine the effect of Foxc1 on N-cadherin and Msx1 gene expression. Data showed over-expression of Foxc1 in wildtype E12.5 and E13.5 POM cells to cause significant fluctuations in N-cadherin and Msx1 expression (p < 0.05). POM cells were then transfected with a Foxc1 knock-down plasmid or the Foxc1 overexpression plasmid to evaluate the effect of Foxc1 on N-cadherin protein expression by Western blot analysis, however, these results were inconsistent with the gene expression analyses with no significant differences in N-cadherin expression detected. N-cadherin protein expression and localization was then further assessed by means of immunocytochemistry (ICC) and confocal microscopy in monolayer and hanging-drop POM cell cultures. Both qPCR and confocal microscopy data showed consistency, indicating increased amounts of N-cadherin in E12.5 cells relative to E13.5 cells, with membrane-bound N-cadherin showing a clear lattice-work pattern in hanging drop culture. Foxc1 over-expression/knock-down studies on E12.5 and E13.5 POM cells together suggest that N-cadherin is transcriptionally regulated by Foxc1 and that Foxc1 has a threshold level at which it is able to exert control over N-cadherin in POM cells. Foxc1 expression is therefore essential in establishing N-cadherin adhesion junctions in the corneal endothelium. Preliminary data also suggests that Msx1 may directly interact with Foxc1 in POM cells, however, further studies must be undertaken to verify and establish the effects of Foxc1/N-cadherin/ Msx1 interaction in the development of a cohesive, integrated corneal endothelium and functional anterior segment.