Mosaic analysis of stem cell function and wound healing in the mouse corneal epithelium
Richard L Mort1,2, Thaya Ramaesh1, Dirk A Kleinjan2, Steven D Morley3 and John D West1
of Reproductive and Developmental Sciences, Genes and Development
Group, The University of Edinburgh, Hugh Robson Building, George
Square, Edinburgh EH8 9XD, UK
2MRC Human Genetics Unit, Crewe Road, Edinburgh, EH4 2XU, UK
Biochemistry Section, Division of Reproductive & Developmental
Sciences, University of Edinburgh, Centre for Reproductive Biology,
Queen’s Medical Research Institute, 47 Little France Crescent,
Edinburgh EH16 4TJ, UK
BMC Developmental Biology 2009. This is an Open Access article distributed under the terms of the Creative Commons Attribution License.
The 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.
The 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.
Striping 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.