Limbal stem cells maintain the corneal epithelium through a process of clonal growth and
ordered migration. In X-inactivation mosaic female mice, that express LacZ from one of
their X-chromosomes, random clumps of LacZ-positive cells are seen in the cornea at 3-6
weeks of life. This pattern resolves between 6-10 weeks forming radial stripes thought to
represent chords of clonally related, inwardly migrating cells. By measuring the number and
width of stripes and correcting for the effects of different proportions of LacZ-positive cells,
an estimate of the number of coherent stem cell clones maintaining the tissue can be derived.
Analysis at 5 ages demonstrated that the estimated number of coherent stem cell clones is
reduced from ~100 at 15 weeks to ~50 at 39 weeks and is then stable at least until 52 weeks.
An automated method was developed using image analysis software to analyse these striping
patterns. This method produced results that did not differ significantly from the above. The
dosage of the transcription factor Pax6 is crucial for normal eye development. In Pax6
heterozygous animals the estimated number of coherent stem cell clones is reduced to ~50 at
15 weeks with no further reduction up to 30 weeks. Mice hemizygous for the PAX77
transgene over-express human PAX6. In PAX77 hemizygous X-inactivation mosaics,
estimated clone number was similarly reduced to ~50 with no further decline. Mice
heterozygous for both Gli3 and Pax6 have a distinct striping phenotype, highlighted by an
increase in coherent clones. When the corneal epithelium is injured the surrounding
epithelial cells migrate along the corneal stroma to cover the wound. X-gal staining of
healed, centrally wounded X-inactivation eyes reveals that striping patterns are reconstituted
during wound healing in ex-vivo culture. In GFP mosaics the healing process can be imaged
using time-lapse confocal microscopy. This technique demonstrated that clones remain
contiguous throughout their migration. Healing of peripheral wounds was observed to form
de-novo whorling patterns, revealing that basal cells in the epithelium can migrate both away
from and towards the limbal region