Computerised Image Analysis of Striping Patterns in the Corneal Epithelia of X–Inactivation Mosaic Mice, Comparing Effects of Age, Pax6 and Gli3 Genotype

Abstract

Purpose: : The dosage of the transcription factor Pax6 is crucial for normal eye development. The Pax6^Sey–Neu mutation produces a truncated form of the Pax6 protein. Animals heterozygous for Pax6^Sey–Neu are viable but have small eyes, aniridia and a range of other abnormalities. Hemizygous PAX77 transgenic mice over–express human PAX6 (Schedl et al 1996, Cell 86, 71–82) and also have small eyes. Mice heterozygous for the Gli3^XtJ mutation have a mild ocular phenotype. This study assessed the effects of age, Pax6 and Gli3 genotype on striping patterns formed by the migration of cells in the corneal epithelia of mosaic mice.

Methods: : Female X–inactivation mice, hemizygous for X–linked nLacZ, were used. Average numbers of stripes per eye, corrected with the factor 1/(1–p), were compared between ages and genotypes. In these X–inactivation mosaics, random clumps of LacZ–positive cells are seen in the corneas of young animals. In wildtype animals this pattern resolves at 8–10 weeks forming radial stripes that represent chords of clonally related, inwardly migrating cells. An automated method was developed using image analysis software to analyse these striping patterns. This method produced results that did not differ significantly from a laborious manual approach.

Results: : A reduction in corrected stripe number from ∼100 at 15 weeks to ∼50 at 39 weeks was observed with no further reduction up to 52 weeks in wildtype animals. Pax6^Sey–Neu heterozygotes did not exhibit a decline with age between 15 and 30 weeks. PAX77 positive mice had fewer corrected stripes than wildtype littermates at 15 weeks and showed no reduction up to 30 weeks. The corrected stripe number in Gli3^XtJ/+;Pax6^Sey–Neu/+ compound heterozygotes was greater than that of their Pax6^Sey–Neu littermates.

Conclusions: : Around 50 stem cell clones are sufficient to maintain the corneal epithelium. In wildtype animals it takes ∼39 weeks to reach this level. Aberrant Pax6 function (over– or under–expression) either reduces the number of functional limbal stem cell clones to this number by 15 weeks or changes the distribution of clones during development so that fewer are initially specified. Pax6^Sey–Neu and Gli3^XtJ mutations may cooperate, perhaps through Shh, to partially restore clone number.

Keywords: cornea: epithelium • mutations • imaging/image analysis: non-clinical