Effects of age and Pax6 deficiency on mouse limbal stem cell function
The conventional view for corneal epithelial maintenance suggests that a stem cell population found in the limbus (at the rim of the cornea) produces daughter cells, called transient amplifying cells, which migrate centripetally. This limbal stem cell (LSC) hypothesis was recently questioned and the alternative model suggests that stem cells are present throughout the corneal epithelium. The main aims of this thesis were to investigate whether age and Pax6 genotype affect LSC function. Previous work with X-inactivation mosaics revealed radial stripes of β-galactosidase-expressing cells in the corneal epithelium (from about 5 weeks of age), which decreased with age and were reduced in Pax6+/- mice (a model for aniridia, a human eye disease). The reduction in Pax6+/- mice could be due to either reduced LSCs function or a more coarse-grained mosaicism caused by reduced cell mixing during development. Comparison of patch sizes in Pax6+/- and wild-type X-inactivation mosaics showed that patches were smaller in Pax6+/- cornea epithelia before the initiation of stripes (3 weeks of age). This implies that stripe-number reduction is not caused by reduced cell mixing, so an effect on LSC function remained a possibility. Thus, the numbers of label-retaining cells (putative stem cells) in Pax6+/- were compared to controls at 15 and 30 weeks old but they were not reduced at 30 weeks or in Pax6+/- mice, as had been predicted. The failure to demonstrate the predicted result suggests either that the hypothesis was incorrect or the experimental approach was inappropriate. Furthermore, it was discovered that mice expressing β-galactosidase under the keratin 5 promoter produced rare stripes in the corneal epithelium, which are likely to represent clonal lineages derived from individual stem cells. Older mice demonstrated a significantly lower frequency of stripes, a result compatible with the predicted reduction of active LSC with age. Pax6+/- corneas were highly abnormal and stripes were not formed properly, so direct comparison was not possible. Finally, pilot experiments with conditional expression of a reporter gene revealed the successful formation of a stripe, and hence provide a plausible alternative approach to compare stripe numbers reflecting active LSCs but the method has yet to be optimised. Overall, the results suggest that LSCs are reduced with age and support the limbal location of stem cells maintaining the corneal epithelium.