An integrated experimental and theoretical approach to understanding corneal epithelial maintenance

Maintenance of the cornea, the transparent front surface of the eye that acts as our window on the world, is necessary for normal vision. There are many ways in which it can get permanently damaged, leading to opacity and potential loss of sight. In spite of intense study by many laboratories, major questions relating to the maintenance of the corneal surface remain. For example, although a large body of evidence exists which implies that stem cells have a major role, the actual location of these stem cells, and to what extent and under what circumstances they are active, remains highly controversial. Similarly, while it is known that cell migration occurs throughout life in the cornea, its importance and even its direction have been disputed. Because maintenance of the corneal surface is fundamental to our eyesight it is essential that we understand the basic biology about how the cornea functions normally and reacts, in the short and long term, to injury.

This project will address the significant issues about stem cell localisation and activity, cell production, migration and loss, within the corneal surface epithelium. We will build upon a computer model that we have already developed that potentially explains some of the patterns of cell migration and loss that we have previously observed. We will refine that model using new biological data, and then use it to make testable predictions about how the cornea is maintained normally, how it is affected by ageing and genetic defects and how it responds to injury. We have an array of 'reporter' mice which, because they express fluorescent or other chemical markers in subsets of their corneal epithelial cells, can be used to trace patterns of cell production and migration in the cornea throughout life, starting during gestation and continuing into the ageing adult. Using these mice, we will be able to model genetic abnormalities and corneal injuries that test the predictions of the computer model. The mice will be used to show where the stem cells are localised in the cornea - knowledge that is essential to our understanding of how the epithelium is maintained. They will show how normal patterns of cell production and migration in the corneal surface are changed by injury and genetic defects, and also reveal whether these changes are long or short term. Changes in stem cell activity and migration patterns with ageing will also be determined using these mice - it is known for example that ageing human corneas heal less quickly than those from younger people. The refined model will have lasting benefits, as we will be able to use it to predict how different types of abnormalities occur in different corneal diseases. It will also contribute to reducing future animal experiments.

Overall the project will generate key fundamental understanding of the processes that underlie corneal maintenance during normal life. We will be able to determine where the stem cells are, what they do and how replacement cells get to sites of injury. This knowledge will also help us understand why the corneal surface sometimes suffers degeneration and opacification that is difficult to treat. If new insights from our animal studies are applicable to humans this may also improve the management of corneal diseases or complications arising from operations such as laser eye surgery.

This multidisciplinary systems project studies maintenance and regeneration of the ocular surface. We have developed an in silico model of corneal epithelial maintenance incorporating cell proliferation, loss and centripetal migration, which stably recapitulates observed patterns of clonal cellular arrangement in adult life. In this project we will refine the model by fitting to experimental data, obtain new empirical data on epithelial cell loss, and hence make predictions about the response of the corneal to damage or ageing.

The in silico model will be validated in vivo using mosaic reporter transgenic mice (LacZ and GFP mosaics) and mutant mice to track the long-term patterns of cell migration and orientation in normal, wounded and ageing situations. Corneas of mouse genetic mosaics have radial striped patterns (meeting at a central spiral), consistent with centrifugal movement of cells from the peripheral limbus (putative stem cell niche). Predictions about how ocular surface wounding or ageing causes disruption to epithelial migration patterns will be tested using these reporters. Genetic disruption will be effected by crossing the mosaic reporter mice onto Pax6 and Gli3 mutant backgrounds. The location of stem cells in the ocular surface will be determined using lineage tracing with tamoxifen-inducible CAGG-CreER;loxP-reporter mice (R26R-LacZ, R26R-YFP). By applying low doses of tamoxifen we will label individual cells and, through longitudinal studies, determine whether active stem cells, producing long-term clones during normal homeostasis (without wounding), are ever found in the central cornea rather than limbus.

The cornea is an excellent model of how stem cell activity, cell movement and loss are balanced in vivo. This project is important as there are major gaps in knowledge of the basic science of corneal maintenance that are essential to resolve, both as a model for epithelial homeostasis and in relevance to corneal degenerative problems.

A. List of potential beneficiaries of this research
1. Academics.
2. Government Regulators - Home Office statistics on animal use
3. NHS and pharmaceutical industry
4. Eye charities
5. Patient support groups
6. General public

B. Ways in which potential beneficiaries will benefit from this research:
1. Academics.
As described in the academic beneficiaries section of the proposal this work will have a significant impact on academic scientists working on corneal biology. Benefits include the additional biological knowledge gained, including resolving the current dispute about the location of stem cells, and the production of a predictive model. Other theoretical scientists working in related fields will benefit from the new tools and techniques that are developed.

We expect this project to establish a core multidisciplinary group that can expand to investigate other problems. For added value, we plan to provide collaborators with samples to establish parallel research on other tissues.

2. Government Regulators - Home Office statistics on animal use
Generation of a mathematical model that enables predictions about how abnormal corneal phenotypes arise in genetic mutants will allow future experiments to be more focused and so contribute to making research more efficient and reducing the numbers of animal experiments.

3. NHS and pharmaceutical industry
In the long-term, an improved understanding of the biological basis of corneal maintenance could contribute to improvements in treatments for corneal disease using a more evidence-based approach to design clinical treatments. This would benefit the NHS and may prompt new uses of existing pharmaceuticals or even development of new products. However, the timescale for any such economic benefits is beyond the duration of this project.

4. Eye charities
A key part of the project is to refine a computer model of cornea maintenance using new biological data. We will either produce a web-based cartoon animation of what the model does or create a simplified ('toy') interactive educational computer model explaining how the cornea is maintained. This might be useful for eye charities to use on their websites to educate the public in general terms about the importance of the cornea to their eyesight, illustrate how it is maintained in a very dynamic way and explain what changes may occur with ageing.

5. Patient Support Groups
Our research would allow patient support groups to provide informed advice to those suffering from ocular surface problems. For example, a major source of uncertainty and worry for people with aniridia (resulting from PAX6 haploinsufficiency) is how to manage or decelerate corneal degeneration, and to understand how corneal surgery or chronic abrasion e.g. from contact lenses will affect them. Currently there is little basic research pertinent to these questions.

6. General public
The simplified interactive educational computer model (described in 4) could form the central component of scientific demonstrations to the public (e.g. at science festivals) to inform them about the role of stem cells in maintaining the cornea. Research on eyes, and conditions leading to blindness, is of interest to the general public and is routinely disseminated via the popular media.