The role of lens-derived signals in the development of the corneal endothelium.
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Date
2013
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Abstract
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.
Description
Thesis (M.Sc.)-University of KwaZulu-Natal, Westville, 2013.
Keywords
Cornea., Anterior segment (Eye), Theses--Microbiology.