The role of lens-derived signals in the development of the corneal endothelium.
Corneal endothelial development is an intricate process driven by finely tuned gene expression. Its formation is necessary for the continued normal development of the anterior segment of the eye. The presence of an inductive lens able to secrete factors such as TGFβ2 as well as the expression of Foxc1 and Pitx2 is essential to corneal endothelial development, as in the absence of any of these; the corneal endothelium fails to form. Corneal endothelial development begins as peri-ocular mesenchyme (POM) cells migrate into the space between the lens and surface ectoderm at E11.5. From E12.5, these cells begin to transition from a mesenchymal to an epithelial/endothelial (MET) phenotype, differentiating into a monolayered endothelium by E15 characterised by inter-cellular junctions. To study the initial process of development, immortalised POM cell lines from E12.5 and E13.5 embryos were used. Expression of the key genes, the transcription factors, Foxc1 and Pitx2 and two genes involved in EMT/MET, Slug and Tsc22, were analysed at these stages to establish the developmental norm. The effect of the lens on these expression levels was then determined. To establish whether TGFβ2 is the lens secreted signal responsible for gene expression changes, cells were subjected to TGFβ2 treatment. In all these experiments, the role of Foxc1 in regulating gene expression was determined by Foxc1 overexpression and knockdown. The effect of the lens on cellular proliferation and on the expression and cellular arrangement of N-cadherin, a junction protein was also determined. The results showed that, at E12.5, the lens downregulates Foxc1 and Pitx2 expression, is a potent inducer of Tsc22 expression and is required for maintaining Slug levels. TGFβ2 was shown to play a role in Foxc1 and Pitx2 downregulation. Analysis suggests that Tsc22 expression is responsive to lens signals, but that TGFβ2 is not the signal responsible for its downregulation between E12.5 and E13.5. The lens has no effect on Slug expression in the presence of Foxc1, but when Foxc1 is silenced, Slug is induced. Thus, Foxc1 plays a crucial regulatory role in Slug expression. At E13.5, as differentiation is initiated, Foxc1 expression remains responsive to the lens and to TGFβ2. Pitx2 expression is still induced by the lens but, at this stage, TGFβ2 does not play a part in Pitx2 regulation suggesting involvement of other unknown lens secreted signals. Other lens secreted signal/s were also shown to downregulate Tsc22 and Slug at this stage. The lens was implicated in MET as it was shown to have an effect on N-cadherin localisation in 3-dimensional culture. E12.5 Spheroids exposed to E6 lenses formed a distinct lattice arrangement of N-cadherin compared to the uniform distribution in control cells. Although the 13.5 control cell aggregates also showed a lattice framework, it was more pronounced in the lens treated cells. The transcriptional role of Foxc1 was determined by overexpression and knockdown experiments where Foxc1 overexpression and knockdown upregulated Tsc22 and downregulated Pitx2 and Slug at E12.5. At E13.5, Pitx2 was downregulated and Slug was upregulated in response to aberrant expression of Foxc1. This was illustrative of the sensitivity these genes have to Foxc1 expression during development. It is known that the presence of a functioning lens and Foxc1 are essential for proper development of the corneal endothelium, which in turn is necessary for normal eye development. The understanding of the precise molecular mechanisms required for corneal endothelial development and the processes requisite for cell proliferation and differentiation has important consequences for providing further insight into the pathophysiology of anterior segment dysgenesis and glaucoma. Previous studies suggest that stem-cell like qualities are conferred in cells undergoing EMT. Such an investigation may lead to application in regenerative medicine such as the bioengineering of corneal tissue.