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The mouse corneal epithelium is a continuously renewing 5–6 cell thick protective layer …

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- Mosaic analysis of stem cell function and wound healing in the mouse corneal epithelium

Female mice, hemizygous for the H253 X-linked nLacZ transgene (here termed XLacZ+/-), are X-inactivation mosaics and show variegated patterns of β-galactosidase (β-gal) reporter expression in all of their tissues [1]. These mosaics have been widely used to study lineage relationships during development [2,3] but they can also be used to analyse maintenance of adult tissues by stem cells [4,5].

Maintenance of the corneal epithelium by stem cells

The corneal epithelium is an excellent model system for the study of tissue maintenance and repair because it is a discrete 5–6 cell thick epithelium replenished by a regionalised stem cell population, which is confined to the basal layer of the limbus at the periphery of the cornea [6,7]. These limbal stem cells (LSCs) produce transient (or transit) amplifying cells (TACs), which proliferate rapidly and migrate centripetally in the basal epithelial layer until their final division when both daughter cells move into the superficial layers, differentiate and are eventually lost from the epithelial surface by desquamation [8-11]. Previous studies with XLacZ+/- mosaics from our group have shown that LSCs become active after birth [4] and identified possible genetic influences on LSC function [5].

Our previous mosaic analysis, suggesting that LSCs become active after birth, was based on a transition from a pattern of patches to one of radial stripes. The sequence of events shown in Fig. 1A–D proposes that some of the basal limbal epithelial cells are specified as LSCs after the limbal epithelium has been determined. Subsequently LSCs become activated and the cornea is maintained by centripetal migration of TACs. Thus, it is predicted that the initial mosaic pattern of patches is established during fetal and early postnatal development and the emergence of stripes indicates when stem cell function begins. Stem cells are required to maintain tissues throughout life and the idea that stem cell function may decline with age and so contribute to age-related changes in tissue homeostasis is currently of great interest [12-14]. However, this possibility has not yet been investigated systematically for LSCs maintaining the corneal epithelium.

Wound healing in the corneal epithelium

LSCs are involved in wound healing, as well as normal tissue homeostasis, and they are up-regulated to replace the lost cells [15]. Corneal epithelial wound healing proceeds through three stages: (i) an initial migratory stage to cover the wound with a cell monolayer, (ii) a proliferative stage to restore the epithelial thickness and (iii) a period of differentiation to restore the complex epithelial structure [16]. Several possible mechanisms could drive cell movement during the initial migratory stage, including population pressure of streams of cells moving centripetally from the limbus, population pressure from the wound-margin or other forces, such as chemotaxis or electric fields [17]. Cell proliferation is stimulated in the peripheral limbal epithelium and to a lesser extent in the corneal epithelium, within 12 hours of wounding, to replace lost cells [6]. However, proliferation at the wound margin may be suppressed to maintain tissue integrity [18]. Thus, many new cells will arise in the periphery and migrate centripetally, as in normal tissue homeostasis, to restore cell numbers and tissue morphology.

The source of the cells and the extent of cell mixing during the early phase of wound healing can be investigated experimentally by a combination of mosaic analysis and organ culture. Wound healing during 24-hour organ cultures reproduces the initial rapid movement of surrounding corneal epithelial cells to cover the exposed stroma but after 24-hours the experimental wound is only covered by a single layer of cells and the epithelium does not stratify during this initial ex vivo healing response [19-21].

The alternative experimental approach of wounding cultured corneal epithelial cell monolayers is thought to cause a loss of spatial constraints and induce motility of sheets of epithelial cells rather than individual cells [22]. However, this may not reflect the situation in vivo or in organ culture where wounding of a multi-layered stratified epithelium allows more scope for cell mixing during wound healing. This is because cells from the upper epithelial layers could contribute to the cell monolayer that forms during the initial movement phase. It has also been suggested that cells at the wound margin become less adhesive and may detach from the epithelial sheet [16], so promoting cell mixing. Distributions of retrovirus-labelled skin epithelial cells during ex-vivo wound healing [23] have been interpreted to suggest cell mixing is quite extensive [24] but it has yet to be determined to what extent cell mixing also occurs during corneal epithelial wound healing.

Quantitative mosaic analysis of limbal stem cell function

Quantitative analysis of distributions of the two cell populations in mosaic tissues (e.g. analysis of the relative numbers, size, shape and distribution of patches) can provide more information than qualitative mosaic analysis [25-27] but this has not yet been widely exploited. The striping patterns in the adult cornea are produced by LSC function and cell movement in the epithelium. LSC function can be compared in different experimental groups by quantitative analysis of stripe numbers.

In an adult corneal epithelium, showing mosaic expression of a LacZ transgene, the stripes of β-gal-positive cells are elongated patches formed from one or more β-gal-positive coherent clones whose descendents have migrated centripetally. (The terms 'patch' and 'coherent clone' are defined in the Methods section.) A stripe spans the corneal radius, so its length is not affected by the number of LSCs and is not relevant to the analysis. The stripe width, however, is variable and depends in part on the number of adjacent corneal epithelial coherent clones belonging to the same cell population (either β-gal-positive or β-gal-negative). Clearly, an individual stripe is more likely to be made up of multiple adjacent β-gal-positive corneal epithelial coherent clones when the proportion of β-gal-positive cells in the corneal epithelium is higher. This source of variation in stripe width can be factored out by dividing the observed mean width of β-gal-positive stripes by the function 1/(1-p), where p is the proportion of β-gal-positive cells around the circumference [25,28]. The resultant 'corrected mean stripe width' can be used to derive a 'corrected stripe number' (see Methods section). This is proportional to the number of corneal epithelial coherent clones and can, therefore, be used to compare LSC function in different groups. A coherent clone of β-gal-positive limbal stem cells will produce a coherent clone of cells in the basal layer of the corneal epithelium that extends to the centre as cells move centripetally and extends to the suprabasal and outer epithelial layers as cells leave the basal layer. Each corneal epithelial coherent clone is assumed to be formed from a single active coherent clone of LSCs. Thus, the number of active LSC coherent clones can be compared in different groups of mosaic eyes by comparing the corrected stripe numbers.

Although, the corrected stripe number is related to the number of active LSC coherent clones it does not provide a direct estimate of LSC numbers. This is partly because the proportion of LSCs that are active may vary and also because the number of LSCs per LSC coherent clone may vary. For example, variation in the number of LSCs per LSC coherent clone may occur because of differences in the extent of cell mixing during development of the surface ectoderm, from which the corneal and limbal epithelia develop (compare Fig. 1A–D, showing the consequences of extensive cell mixing during development, with Fig. 1E–H, showing the consequences of less cell mixing).


The aims of this study were to better characterise LSC function and the streaming and mixing behaviour of the cells they produce during maintenance, repair and ageing of the mouse corneal epithelium. Analysis of mosaic patterns in intact and wounded corneas demonstrated that (i) LSC function declines with age, (ii) little cell mixing occurs either during normal maintenance of the corneal epithelium or during wound healing, (iii) the main driving force during wound closure is not population pressure from centripetally streaming cells produced by LSCs and (iv) quantitative and temporal mosaic analyses provide new possibilities for studying stem cell function in tissue maintenance and repair.

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