The genetic control of epithelial cell migration and wound healing physiology

Cell migration, the ability of a cell to get from point A to point B, is fundamental to development, growth and maintenance of the body. The most obvious example is during healing of cuts and scratches, when the epithelial cells, the top layer of the skin, have to move over to cover the wound. How do they do it? We intend to find out by studying the migration of cells across the surface of the cornea of the eye (one of the most impressive examples of regular long-distance cell migration in adults). There are only a few ways of directing a cell to move. They can follow chemical trails, or feel their way along grooves in the surface they are crawling over. They can be physically pushed by cells from behind multiplying and shoving them over to make room, or they can sense and move in the direction of endogenous electrical currents flowing through body tissues. We want to find out which factors are most important, and how problems with cell migration can lead to disease. We intend to use a mutant strain of mouse which, although it is basically healthy, exhibits eye problems associated with a failure of epithelial cells to migrate normally over the corneal surface. We have shown that there are also abnormalities with corneal wound healing in these mice. One of the potential drivers of cell migration, endogenous electric fields, are severely abnormal in our mutant mice. We will determine whether corneal cells from our mutant mice can 'see' electric fields and, if so, whether the problems with electric fields in the mutant cause the problems with cell migration. Using drugs and chemicals, we can improve cell migration in our mutant mice, and we will show whether this is mediated by improvement in electric fields. We will show whether the strength and direction of the field correlates with the strength and direction of cell migration. We will also determine whether corneal epithelial cells are steered by sensing contact with physical cues (very small grooves or chemical signposts) in the tissues they are crawling across, and whether these by themselves can push cells in the right direction. We will compare normal and mutant cells moving on grooved quartz surfaces (where they get only physical guidance) and on real corneal tissues, where they may get both chemical and physical guidance. We will investigate the molecules within the cell that are responsible for directing normal cell orientation and movement. We will show whether cell division at the outside of the cornea physically pushes cells from the edge of the cornea to the middle, and whether this goes wrong in our mutant mice Most immediately, the work will be relevant to people who suffer from corneal surface abnormalities associated with wounding, including patients who suffer the same genetic defects as our mice and patieints with long-term corneal ulceration associated with, for example, radiotherapy. Previous work has lead to new ways to try to improve healing after injuries to the spinal cord in the back or the neck, and this project will start to bring new understandings to ways of accelerating healing in the skin. Of general significance, the data will be of wide relevance to wound healing and epithelial migration studies in scientific and medical settings. For the first time, we will provide a genetic test where we give cells the opportunity to ignore electric fields, and see whether they will do so. As such, the project gets at fundamental questions about how our bodies work and how it might be possible to accelerate wound healing after injury or disease.

Factors that control epithelial cell migration are known to include chemical cues, physical (contact mediated) signals, and endogenous electric fields. The concept that electric fields drive cell migration is well supported but the most relevant 'clinching' experiment has not been performed - to genetically manipulate cells such that they receive the 'wrong' electric signals, and see whether migration is affected. In this project, we intend to perform this experiment, as part of a wider investigation into cues directing epithelial cell migration. The Pax6+/- mouse demonstrates corneal epithelial cell migration defects, and epithelial wound-healing abnormalities. Preliminary data has shown that wound-induced electric fields are reversed in about 50% of the mutants. We will assay the consequences of this for wound healing in vitro and in vivo. We will further apply electric fields to wild-type and mutant corneal epithelial cells and determine whether the mutants can respond. The roles of the EGF and Shh signalling pathways in modulation of cell migration will be assayed, and the secondary messenger signals will be determined, with particular regard to whether these are independent of the endogenous electric response. The roles of contact-mediated and physical guidance cues on wild-type and mutant cells will be investigated. Corneal epithelial cells will be allowed to migrate on grooved quartz slides that mimic the collagenous matrix of the corneal stroma, with or without chemical and electrical cues. Wild-type and mutant cells will be grown on wild-type and mutant corneal stromas and migration assayed. The role of differential and directional proliferation in maintaining a centripetal flow of cells within the corneal epithelium will be investigated. The consequences of defective cell migration for disease will be investigated in transgenic mice that overexpress Pax6 - these mice have severe wound-healing defects, but their corneas appear healthy.