Abstract
BackgroundThe mouse corneal epithelium is a continuously renewing 5–6 cell thick protective layer covering the corneal surface, which regenerates rapidly when injured. It is maintained by peripherally located limbal stem cells (LSCs) that produce transient amplifying cells (TACs) which proliferate, migrate centripetally, differentiate and are eventually shed from the epithelial surface. LSC activity is required both for normal tissue maintenance and wound healing. Mosaic analysis can provide insights into LSC function, cell movement and cell mixing during tissue maintenance and repair. The present study investigates cell streaming during corneal maintenance and repair and changes in LSC function with age.ResultsThe initial pattern of corneal epithelial patches in XLacZ+/- X-inactivation mosaics was replaced after birth by radial stripes, indicating activation of LSCs. Stripe patterns (clockwise, anticlockwise or midline) were independent between paired eyes. Wound healing in organ culture was analysed by mosaic analysis of XLacZ+/- eyes or time-lapse imaging of GFP mosaics. Both central and peripheral wounds healed clonally, with cells moving in from all around the wound circumference without significant cell mixing, to reconstitute striping patterns. Mosaic analysis revealed that wounds can heal asymmetrically. Healing of peripheral wounds produced stripe patterns that mimicked some aberrant striping patterns observed in unwounded corneas. Quantitative analysis provided no evidence for an uneven distribution of LSC clones but showed that corrected corneal epithelial stripe numbers declined with age (implying declining LSC function) but stabilised after 39 weeks.ConclusionStriping patterns, produced by centripetal movement, are defined independently and stochastically in individual eyes. Little cell mixing occurs during the initial phase of wound healing and the direction of cell movement is determined by the position of the wound and not by population pressure from the limbus. LSC function declines with age and this may reflect reduced LSCs numbers, more quiescent LSCs or a reduced ability of older stem cells to maintain tissue homeostasis. The later plateau of LSC function might indicate the minimum LSC function that is sufficient for corneal epithelial maintenance. Quantitative and temporal mosaic analyses provide new possibilities for studying stem cell function, tissue maintenance and repair.
Highlights
The mouse corneal epithelium is a continuously renewing 5–6 cell thick protective layer covering the corneal surface, which regenerates rapidly when injured
Analysis of mosaic patterns in intact and wounded corneas demonstrated that (i) limbal stem cell (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
Qualitative characterisation of X-inactivation mosaic patterns in the corneal epithelium Examination of mosaic corneal epithelia of 3–52 week old female XLacZ+/- mosaic mice showed that the initial pattern of randomly orientated patches seen at 3 weeks was replaced by radial stripes by postnatal week 10 (Fig. 2A– D)
Summary
The mouse corneal epithelium is a continuously renewing 5–6 cell thick protective layer covering the corneal surface, which regenerates rapidly when injured. 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 [8,9,10,11]. This possibility has not yet been investigated systematically for LSCs maintaining the corneal epithelium
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