April 2004
Volume 45, Issue 4
Free
Cornea  |   April 2004
Corneal Development, Limbal Stem Cell Function, and Corneal Epithelial Cell Migration in the Pax6 +/− Mouse
Author Affiliations
  • J. Martin Collinson
    From the Department of Biomedical Sciences, Institute of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen, Scotland, United Kingdom; the
    Division of Reproductive and Developmental Sciences, Genes and Development Group, University of Edinburgh, Edinburgh, Scotland, United Kingdom; and the
  • Simon A. Chanas
    Division of Reproductive and Developmental Sciences, Genes and Development Group, University of Edinburgh, Edinburgh, Scotland, United Kingdom; and the
  • Robert E. Hill
    Comparative and Developmental Genetics Section, Medical Research Council [MRC] Human Genetics Unit, Edinburgh, Scotland, United Kingdom.
  • John D. West
    Division of Reproductive and Developmental Sciences, Genes and Development Group, University of Edinburgh, Edinburgh, Scotland, United Kingdom; and the
Investigative Ophthalmology & Visual Science April 2004, Vol.45, 1101-1108. doi:https://doi.org/10.1167/iovs.03-1118
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      J. Martin Collinson, Simon A. Chanas, Robert E. Hill, John D. West; Corneal Development, Limbal Stem Cell Function, and Corneal Epithelial Cell Migration in the Pax6 +/− Mouse. Invest. Ophthalmol. Vis. Sci. 2004;45(4):1101-1108. https://doi.org/10.1167/iovs.03-1118.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

purpose. To investigate the etiology of corneal dysfunction in the Pax6 +/− mouse model of aniridia-related keratopathy.

methods. Mosaic patterns of X-gal staining were compared in the corneal and limbal epithelia of female Pax6 +/− and Pax6 +/+ littermates, age 3 to 28 weeks, hemizygous for an X-linked LacZ transgene, and Pax6 +/+, LacZ Pax6 +/+, LacZ + and Pax6 +/+, LacZ Pax6 +/−, LacZ + chimeras. Histologic examination of chimeric corneas was performed.

results. Disrupted patterns of X-gal staining showed that heterozygosity for Pax6 perturbed clonal patterns of growth and development in the corneal and limbal epithelium. Centripetal migration of Pax6 +/− corneal epithelial cells was diverted. Normal patterns of centripetal Pax6 +/− cell migration and epithelial morphology were restored in Pax6 +/+Pax6 +/− chimeras. Fewer, larger clones of limbal stem cells were present in Pax6 +/− eyes, compared with wild-type. In the chimeras, Pax6 +/− limbal stem cells were cell-autonomously depleted or less efficient than wild-type cells at producing progeny to populate the corneal epithelium.

conclusions. The correct Pax6 dosage is necessary for normal clonal growth during corneal development, normal limbal stem cell activity, and correct corneal epithelial cell migration. Disruption of normal cell movement in heterozygotes may be the consequence of failure of nonautonomous guidance cues. Degeneration of the corneal surface in aniridia-related keratopathy relates to both a deficiency within the limbal stem cell niche and nonautonomous diversion of corneal epithelial cell migration.

The gene encoding the transcription factor Pax6 is expressed at high levels in all cells of the developing and adult conjunctival, limbal, and corneal epithelia. 1 2 3 4 Corneal opacities in PAX6 +/− patients 5 6 that correlate with vascularization and epithelial fragility, have been termed aniridic keratopathy 7 or aniridia-related keratopathy (ARK). 8 The Pax6 +/− mouse is an excellent model of ARK, recapitulating all the morphologic defects exhibited by humans. 4 8 9 Pax6 +/− mice have thin, irregular, vacuolated, fragile corneal epithelia, within which the presence of goblet cells may indicate encroachment of conjunctival cells into the corneal field. 8 9  
The adult corneal epithelium is maintained by a population of limbal stem cells (LSCs). 10 11 LSCs produce undifferentiated progeny with limited proliferative potential that migrate centripetally from the periphery of the corneal epithelium to replace cells desquamated during normal life. 12 13 14 15 16 17 18 Epithelial thinning and the possible encroachment of conjunctival epithelium could infer a deficiency of LSC activity in ARK or a defective wound-healing response, such that cells lost from the corneal surface are not adequately replaced. 19 20 LSC activation and function and patterns of corneal epithelial migration, have not been directly studied in Pax6 +/− mice or humans. 
We created an assay for LSC function and corneal epithelial migration, using patterns of β-galactosidase activity in LacZ +LacZ chimeras and in female mice carrying an X-linked LacZ transgene. 21 The assay revealed that, in wild-type mice, development of the corneal epithelium, with activation of LSCs and centripetal streaming of their progeny into the cornea, is not completed before the 10th postnatal week. 
In this study, new series of chimeras and X-inactivation mosaics were produced to define defects in the Pax6 +/− corneal epithelium that are relevant to the etiology of ARK. We found development of the corneal epithelium was disrupted, and Pax6 +/− LSCs were autonomously defective or depleted. Patterns of Pax6 +/− corneal epithelial cell migration were disrupted in Pax6 +/− mice but could be corrected in Pax6 +/+Pax6 +/− chimeras, suggesting that the corneal epithelial defects are not mediated in a cell-autonomous manner. 
Materials and Methods
Mosaic Analysis of LSC Function and Corneal Epithelial Cell Movement
All animal procedures were performed in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Pax6 Sey/+ and Pax6 Sey-Neu/+ stocks and the H253 transgenic line, carrying X-linked LacZ (hereafter XlacZ) expressed from a housekeeping promoter have already been described. 22 23 24 25  
H253 males (XlacZ +/Y) or homozygous females (XlacZ +/+) were bred with Pax6 +/Sey-Neu (Pax6 +/−) mates. The eyes of their progeny were fixed and stained with X-gal at 3 to 28 weeks of age. 21 Corneal diameters were measured. Mice were genotyped by PCR. 23 26 Mosaic patterns of X-gal staining in hemizygous XlacZ +/− females were compared for Pax6 +/+ and Pax6 +/− littermates. 
In the wild-type, each LacZ-expressing blue patch of LSCs produces a blue radial stripe of corneal epithelial cells migrating centripetally in the mature eye. 21 Nonexpressing cells appear as white patches or stripes. The pattern of radial stripes in the cornea is a direct assay of the arrangement and size of blue and white patches of LSCs in the limbus. Each blue or white patch derives from one or more independently specified LacZ + or LacZ clones of LSCs. As the percentage contribution of LacZ + cells to the limbus increases, the probability that any single blue stripe is derived from more than one adjacent clone of LacZ + LSCs increases. The number of coherent clones of LSCs was estimated as described fully in Collinson et al. 21 by counting the number of radial stripes and correcting for the total percentage contribution of blue cells to the peripheral cornea using the 1/(1 − p) correction factor described previously, 21 27 28 based on Roach. 29 The mean width of LSC clones at the corneal boundary was calculated as (π · corneal diameter)/number of clones. 
A near-identical protocol was used to estimate clone size in the randomly oriented blue and white patches in corneal epithelia of young mice. A circle corresponding to a diameter of 1 mm was overlaid on scaled photographs of each stained eye. The number and width of blue and white patches cut by this circle were measured, and clone width was calculated. Statistical analyses were calculated on computer (Prism, ver. 3.0; GraphPad, San Diego, CA).  
Chimeras
Production of chimeras by aggregation 30 has been described elsewhere. 23 Eight-cell embryos were obtained from Pax6 Sey-Neu/+ females, homozygous for the glucose phosphate isomerase 1-b Gpi1 b allele (Pax6 Sey-Neu/+; Gpi1 b/b ), that had been mated to Pax6 Sey/+ males, homozygous for the constitutive LacZ transgene TgR(ROSA26)26Sor 31 (Pax6 Sey/+; LacZ +/+; Gpi1 b/b ). The embryos were aggregated with eight-cell embryos from a Gpi1 a/a , Pax6 +/+ wild-type cross (BALB/c x A/J)F2). Chimeras were mixtures of Pax6 +/+ LacZ −/− cells, and LacZ +/− cells of various Pax6 genotypes: control chimeras (Pax6 +/+, LacZ −/−Pax6 +/+, LacZ +/−), heterozygous chimeras (Pax6 +/+, LacZ −/−Pax6 +/−, LacZ +/−), and homozygous mutant chimeras (Pax6 +/+, LacZ −/−Pax6 −/−, LacZ +/−), which could be distinguished by PCR. 23 26  
Eyes were dissected at 15 weeks and stained with X-gal. The percentage contribution of LacZ + cells to the peripheral cornea, Bouter was measured as for the mosaic mice. 21 The percentage contribution of blue cells to the central cornea, Binner, measured around a centered circle, two fifths of the corneal radius, was also calculated. Tissues from the forebrain, heart, lungs, spleen, liver, and kidney of each chimera were taken for quantitative GPI1 analysis. 32 33 For all aggregations, the eight-cell embryo derived from the wild-type mating was Gpi1 a/a , and the eight-cell embryo derived from the Pax6 +/Sey-Neu x Pax6 Sey/+ mating was Gpi1 b/b . Those chimeras with higher %GPI1-A are primarily composed of cells derived from the wild-type mating, and vice versa. 
Results
Clonal Analysis of the Pax6 +/− Corneal Epithelium
Pax6 +/+ H253 male mice, hemizygous for an X-linked LacZ reporter transgene (XlacZ +/Y), 25 were mated to nontransgenic Pax6 Sey-Neu/+ mice on a CBA/Ca genetic background. Their female progeny were XlacZ +/− and either Pax6 Sey-Neu/+ (Pax6 +/−) or Pax6 +/+, with mosaic XlacZ expression due to random epigenetic X-inactivation in early development. Patterns of X-gal staining in the Pax6 +/+ and Pax6 +/− female littermates aged 3 to 28 weeks were compared (Fig. 1) to determine whether the development or maintenance of the Pax6 +/− corneal epithelium were abnormal. XlacZ −/Y males were used as negative control subjects (these showed no blue staining), and XlacZ +/Y males or XlacZ +/+ females as positive control animals (corneal epithelia were entirely blue). 
Abnormal Patterns of Growth during Postnatal Development of the Pax6 +/− Cornea.
Three-week old Pax6 +/+ mice showed fine-scale patterns of randomly oriented blue and white patches in the corneal epithelium, identical with those seen previously 21 (12/12 corneas; Fig. 1A ). In contrast, Pax6 +/− littermates were extremely variable (eight corneas; Figs. 1B 1C 1D ), both between individuals and between different eyes in the same individual. Pax6 +/− patch sizes were larger (Figs. 1B 1C 1D) . For each eye, blue and white patch sizes were corrected on the basis of the percentage of LacZ + cells, to estimate the mean coherent clone width at 0.5 mm from the center of the cornea (see the Methods section). Coherent clones of corneal epithelial cells were larger and more variable for Pax6 +/− (mean, 60.9 ± 7.6 μm; n = 6) than for Pax6 +/+ corneas (mean, 28.2 ± 2.1 μm; n = 7; t-test: P = 0.006) even though the Pax6 +/− corneas were of smaller diameter (2.26 ± 0.06 mm) than wild-type (2.39 ± 0.02 mm). 
LSCs become active at 5 to 6 weeks in wild-type mice. 21 Six-week old Pax6 +/− XlacZ + female mice had thin corneal epithelia with large, randomly oriented patches of LacZ + cells (Figs. 1F 1G) that contrasted with the patterns of dense, small, randomly oriented clones with radial stripes emerging from the limbus shown by their wild-type littermates (Fig. 1E) . Clone size was significantly larger in Pax6 +/− eyes (131.47 ± 9.35 μm, n = 6) than Pax6 +/+ (83.88 ± 6.63 μm, n = 8; P = 0.002). 
LSC Clones and Patterns of Migration in the Pax6 +/− Cornea.
From 9 to 28 weeks, Pax6 +/+ XlacZ +/− corneal epithelia developed patterns of radial stripes due to centripetal immigration of LSC-derived cells 21 (Figs. 1I 1J 1K 1L 1M 1N 1O 1P 1Q 1R 1S 1T) . Tissue sections confirmed that staining was epithelial and that apical cells were clonally related to underlying basal cells (at any point on the cornea, the entire thickness of the epithelium was either blue or white). Nearly all Pax6 +/− XlacZ +/− eyes showed evidence of radial patterns, sometimes almost normal (Figs. 1P 1T) . In most Pax6 +/− eyes, although clear radial stripes emerged from the limbus, they became disorganized, giving patchwork or deviating patterns toward the center of the cornea that reflected diversions from normal centripetal migration (Figs. 1O 1S)
Measurement of the width and number of the blue and white stripes near the corneal periphery allowed an estimate of the number and width of coherent clones of LSCs responsible for producing the striped patterns in eyes of both genotypes (see the Methods section). At every age, significantly fewer, larger LSC clones were active in the Pax6 +/− corneas than in their Pax6 +/+ littermates (Table 1 ; Fig. 2 ). 
The experiments were repeated with the Pax6 Sey allele, 22 on C57BL x CBA and inbred 129/Sv backgrounds. On both backgrounds, at 15 weeks, the Pax6 Sey/+ mice had fewer coherent LSC clones than their Pax6 +/+ littermates, similar to those for Pax6 Sey-Neu/+ 15-week mice (Table 1)
There was therefore a strong developmental component to the Pax6 +/− corneal abnormalities. Fewer LSC clones were activated in Pax6 +/− mice. Migration patterns of Pax6 +/− corneal epithelial cells were disrupted. The defects were robust across age, genetic background, and the specific null allele of Pax6
Correction of Corneal Epithelial Thickness and Cell Movement in Chimeras
The disruption of centripetal migration of corneal epithelial cells may be either cell autonomous (a requirement for correct Pax6 dosage in those cells) or nonautonomous (a secondary consequence of a requirement for a normal Pax6 dosage in other cells or tissues). A transcription factor, such as Pax6, may have both cell-autonomous functions (through regulation of genes effecting the differentiation of cells in which it is expressed) and nonautonomous gene functions (e.g., through regulation of expression of secreted molecules that control differentiation of surrounding cells). Chimeras are the classic tool for determining cell autonomy and nonautonomy of gene action. If a gene acts cell autonomously in a particular tissue, in chimeras comprising a mixture of wild-type and mutant cells, only the mutant cells show the mutant phenotypic effect (e.g., they may be absent, depleted in number, or abnormally distributed in that tissue). If the gene does not act cell autonomously, the wild-type cells may rescue the mutant cells so that neither show an abnormal phenotype. 34 Comparison of the behavior of Pax6 +/− cells in X-inactivation XlacZ mosaics (where all cells are Pax6 +/−) and Pax6 +/−Pax6 +/+ chimeras (where only one population of cells is Pax6 +/−) provides a test to distinguish between cell autonomous and nonautonomous gene action. 
A series of chimeras was made, using a constitutive autosomal LacZ transgene as a marker by which cells from the aggregated embryos could be differentiated. Eight-cell embryos from a Pax6 +/+, LacZ −/− line were aggregated to eight-cell embryos from a Pax6 +/− x Pax6 +/−, LacZ +/+ mating. Twenty-three chimeras were analyzed at postnatal week 15. Nine were Pax6 +/+Pax6 +/+, LacZ +/−; 12 were Pax6 +/+Pax6 +/−, LacZ +/−; and 2 were Pax6 +/+Pax6 −/−, LacZ +/−. Cells derived from the Pax6 +/− x Pax6 +/−, LacZ +/+ aggregated embryo, irrespective of their Pax6 genotype, were identifiable by X-gal staining. The ratio of GPI1-A to GPI1-B allozymes in nonocular tissues from each chimera was determined (see the Methods section): mean %GPI1-B was taken to be an estimate of the percentage of cells in the chimera that were derived from the Pax6 +/− x Pax6 +/− eight-cell embryo. 
Eyes were dissected and stained with X-gal (Fig. 3) . Thirteen of 16 Pax6 +/+Pax6 +/+, LacZ +/− chimeric corneas showed normal radial blue and white stripes. Three corneas had no contribution of LacZ + cells. 
Blue radial stripes of Pax6 +/−, LacZ +/− cells were observed in 19 of 24 Pax6 +/+Pax6 +/− corneal epithelia. Five corneas had no LacZ +/− cells. The stripes were identical in character with those found in the Pax6 +/+Pax6 +/+, LacZ +/− control corneas, with none of the irregularity or deviation that was characteristic of the Pax6 +/−, XlacZ +/− mosaic mice described earlier. The data suggest that normal patterns of corneal epithelial replenishment and cell migration were restored in chimeras. Quantitative analyses were performed to test this, as described later. 
Cell-Autonomous Defect in Pax6 +/− LSC Function
The contribution of blue and white cells in the peripheral corneal epithelium of each chimeric eye (Bouter) was measured to estimate the proportion of the limbus that was populated by active LacZ + LSCs. These data are summarized in Table 2 . For each eye, Bouter was compared with the global composition of the chimera, as estimated by mean %GPI1-B. It was reasoned that if the distribution of LacZ + cells to chimeric corneal epithelia was unbiased and not affected by Pax6 genotype, then Bouter minus %GPI1-B would approximate 0. This was true for Pax6 +/+Pax6 +/+ chimeras in which mean Bouter minus %GPI1-B was 6.81 ± 4.35 (n = 16). The contribution of LacZ + cells to Pax6 +/+Pax6 +/− corneas, however, was significantly less than expected: mean Bouter minus the %GPI1-B was −25.46 ± 3.11; n = 24 (t-test comparing control and experimental chimeras: P < 0.0001). A similar underrepresentation was demonstrated by comparing the mean Bouter/%GPI1-B ratios (Table 2) . This underrepresentation of Pax6 +/− cells is evidence for an autonomous requirement for correct Pax6 dosage in LSCs during normal replenishment of the corneal epithelium. 
The underrepresentation of Pax6 +/− cells in the corneal epithelium of chimeras suggests that Pax6 +/− LSCs are autonomously depleted or defective, yet the heterozygous Pax6 +/− X-inactivation mosaics had larger coherent LSC clones than wild-type Pax6 +/+ mosaics. This apparent paradox could be reconciled if Pax6 +/− LSCs are at a competitive disadvantage, compared with wild-type cells, in chimeras, but that this defect does not manifest in XlacZ, Pax6 +/− X-inactivation mosaics because all LSCs in these eyes are heterozygous. Pax6 +/− eyes may have larger LSC clones than wild-type Pax6 +/+ mosaics as a result of expansion of a smaller number of specified LSCs to fill the limbal epithelium and/or reduced cell mixing. The possibility that Pax6 +/− LSCs are at a competitive disadvantage in chimeras predicts that coherent clones of Pax6 +/− LSCs may be smaller than wild-type clones in Pax6 +/+Pax6 +/− chimeras. Our analysis of clone size and number produces average values for blue and white clones in each eye. Consequently, any differences between mutant and wild-type cells would be masked, and we cannot test directly whether the mutant clones are smaller. The Pax6 +/+Pax6 +/− experimental chimeras had slightly more (smaller) LSC clones per eye (82.92 ± 7.97; n = 19) than the Pax6 +/+Pax6 +/+ control chimeras (65.11 ± 7.65; n = 13) of equivalent diameter, but the difference was not significant (t-test: P = 0.09). Thus, although our data suggest that LSC function is controlled autonomously by Pax6 dosage within the LSCs, the data do not yet demonstrate whether Pax6 +/− LSC-coherent clones are fewer or smaller than wild-type clones in chimeras. 
Corneal Epithelial Function
Epithelial Cell Migration.
Some blue radial stripes failed to reach the center of the cornea (e.g., Fig. 3C ). If there were any autonomous migration defects of Pax6 +/− cells, this may manifest as an increased tendency for Pax6 +/− LacZ + blue stripes to become depleted in heterozygous chimeras before reaching the center of the cornea. This possibility was tested. For each chimeric corneal epithelium that contained LacZ + cells, the proportion of blue cells cut by a centered circle of radius two fifths that of the cornea, %Binner, was calculated as for Bouter. Values of Binner − Bouter and Binner/Bouter were calculated for each eye. Means were calculated for control and heterozygous chimeras. The functions Binner − Bouter and Binner/Bouter did not differ significantly between heterozygous and control chimeras (Table 2) . No quantitative or qualitative autonomous defect in the migratory potential of Pax6 +/− corneal epithelial cells was therefore detected. The restoration of normal migration of Pax6 +/− cells in Pax6 +/+Pax6 +/− chimeras is evidence that the migration defect noted in (nonchimeric) heterozygotes is mediated in a nonautonomous manner. This implies that the migration pattern of any individual corneal epithelial cell is controlled, not by Pax6 dosage in that cell, but by Pax6 dosage in surrounding cells or tissues. 
Epithelial Thickness.
Chimeric corneas and those of nonchimeric Pax6 +/+ and Pax6 +/− mice, were sectioned. Nonchimeric Pax6 +/− corneal epithelia were thinner, with fewer cell layers (n = 2–4) than wild-type (n = 5–8; Figs. 4A 4B ). 4 8 9 The thicknesses (apical-basal depth) of 20 blue stripes from five Pax6 +/+Pax6 +/+, LacZ +/− chimeras and 38 blue stripes from 8 Pax6 +/+Pax6 +/−, LacZ +/− chimeric corneas were measured and compared with the immediately adjacent white stripes. In control chimeras, for which both the blue and white stripes were Pax6 +/+, blue stripes were slightly thicker than white (mean blue-to-white depth ratio = 1.239 ± 0.025, n = 20; Fig. 4C ). A similar artifactual apical-basal thickening of blue stripes was found for the X-inactivation mosaics (mean blue-to-white depth ratio = 1.27 ± 0.022 [n = 29] from three Pax6 +/+ and one Pax6 +/− H253 mosaic eye), and implies differential shrinkage of stained LacZ + and LacZ epithelia during wax processing. 
In the Pax6 +/+Pax6 +/−, LacZ +/− chimeric corneas, mean blue-to-white depth ratio was 1.247 ± 0.022 (n = 38), not significantly different from control chimeras (t-test: P = 0.83; Figs. 4D 4E ). The sectors of Pax6 +/− cells in the chimeric corneal epithelia were five to seven cells deep, equivalent to wild-type. Thus, both absolute thickness and normal cellular stratification were restored in Pax6 +/− sectors of Pax6 +/+Pax6 +/− chimeras. The control of corneal epithelial stratification is poorly understood, but the chimeric data suggest that, as for epithelial migration, the mutant phenotype in Pax6 +/− mice is mediated through nonautonomous functions of Pax6 in other cells or tissues. 
Discussion
Our results demonstrated that (1) the pattern of clonal growth is disrupted in the developing Pax6 +/− corneal epithelium (before activation of LSCs); (2) fewer, larger clones of LSCs are active in Pax6 +/− eyes, compared with Pax6 +/+; (3) centripetal cell movement in the Pax6 +/− corneal epithelium is frequently disrupted; (4) defects in Pax6 +/− corneal epithelial thickness and cell movement are nonautonomous and can be corrected in chimeras; (5) Pax6 +/− cells are underrepresented in the corneal epithelium of chimeras, and so Pax6 +/− LSCs are autonomously depleted or less efficient than wild-type at populating the limbal epithelium. 
Development of the Pax6 +/− Corneal Epithelium
Pax6 controls the expression of a number of adhesion-related molecules in the cornea and elsewhere and has been identified as a central component of proliferation-differentiation pathways in the developing central nervous system. 9 35 36 The mechanistic basis of the disturbance of normal clonal arrangement and growth in Pax6 +/− mice has not yet been identified, but the larger clone sizes may reflect defective proliferation-differentiation decisions, or induction of a smaller corneal field in the Pax6 +/− mice. One possibility, consistent with known adhesive roles of Pax6, is that cell mixing is reduced in Pax6 +/− corneal epithelia compared with Pax6 +/+
LSCs in Pax6 +/− Mice
The data presented herein for XlacZ-mosaic mice show that fewer coherent clones of LSCs are present in Pax6 +/− animals than in wild-types. If we postulate that a relatively small number of LSCs are specified during development and that clones of these founders expand to fill the limbal niche, 21 it is possible therefore that fewer LSCs are specified in the heterozygotes, but Pax6 +/− LSCs remain capable of long-term survival and production of functional progeny. Analysis of the contribution of Pax6 +/− cells to the corneal epithelia of Pax6 +/+Pax6 +/− chimeras showed that Pax6 +/− LSCs are nevertheless less efficient than wild-type at populating the limbal epithelium and producing active migrating progeny, demonstrating the power of the chimeras for revealing subtle phenotypic effects. Previous evidence of Pax6 +/− LSC deficiency has been circumstantial or correlative—the work presented herein is the first direct demonstration of autonomous LSC deficiency. This will be central to further experiments to elucidate the molecular defects underlying the Pax6 +/− LSC phenotype. 
Rescue of Pax6 +/− Corneal Epithelial Cells in Chimeras
Epithelial cell migration in the Pax6 +/− cornea was abnormal. Results from the mosaic mice did not distinguish between a model wherein Pax6 +/− cells are autonomously incapable of responding appropriately to signals that guide their migration, or a model in which the cells are capable of responding to signals, but that these signals are disrupted in Pax6 +/− eyes. The complete normalization of epithelial thickness and cell migration in the chimeric mice suggests that the latter model—failure of normal guidance cues in Pax6 +/− eyes—is likely. 
The source of the rescue signals for Pax6 +/− cells in the chimeric corneal epithelium is yet to be determined. Pax6 +/− cells are eliminated from the embryonic lens of chimeras, and gross corneal opacities, iris hypoplasia, and lens defects do not subsequently occur. 33 We suggest that Pax6 +/− cells in chimeric corneas are rescued in a nonautonomous manner by (unknown) signals from the wild-type lens. The possibility that they are supported by the surrounding streams of Pax6 +/+ epithelial cells or by Pax6 +/+ keratocytes cannot be discounted. 
It is not known how centripetal migration in the uninjured corneal epithelium is controlled, although several mechanisms of differential proliferation, desquamation, chemotropic guidance, and electrical cues are suggested. 17 37 38 39 40 Further work will determine how putative guidance cues may be affected in Pax6 +/− eyes and whether they are corrected in the chimeras. 
Aniridia-Related Keratopathy
Nishida et al. 19 reported that symptoms normally associated with LSC deficiency (absence of palisades of Vogt and incursion of goblet cells into the peripheral cornea) occurred in 16 of 16 patients with aniridia. It was suggested that the early postnatal corneal epithelium (produced before LSC activation) was possibly normal, but that depletion of LSCs resulted in a failure of corneal maintenance later in life. 19  
Our work has implications for the etiology of ARK and does not suggest that ARK can be explained solely by failure of LSCs. We showed that the developing corneal epithelium is disrupted before LSC activation, and described defects in the migration of corneal epithelial cells. Defects in cell migration may relate to downregulation of adhesion-related molecules such as integrin subunits or β-catenin in the Pax6 +/− corneal epithelium. 9 ARK is associated with fragility of the corneal epithelium, perhaps due to downregulation of the Pax6 target, cytokeratin-12. 8 9 Because epithelial cells move toward and fill in sites of corneal abrasion, 40 it is possible that the disruption of normal cell movement in the Pax6 +/− cornea is exacerbated by chronic damage to the epithelium and persistent small-scale diversion of radial migration. 
 
Figure 1.
 
Clonal patterns of cell growth and movement in Pax6 +/− eyes. Patterns of X-gal staining in corneal epithelia of (A, E, I, M, Q) Pax6 +/+ and (BD, FH, JL, NP, RT) Pax6 +/− female littermates from crosses of Pax6 +/− mice with H253 transgenic mice carrying an X-linked LacZ transgene. Representative eyes are presented at (AD) 3, (EH) 6, (IL) 9, (MP) 15, and (QT) 28 weeks. Scale bar, 500 μm.
Figure 1.
 
Clonal patterns of cell growth and movement in Pax6 +/− eyes. Patterns of X-gal staining in corneal epithelia of (A, E, I, M, Q) Pax6 +/+ and (BD, FH, JL, NP, RT) Pax6 +/− female littermates from crosses of Pax6 +/− mice with H253 transgenic mice carrying an X-linked LacZ transgene. Representative eyes are presented at (AD) 3, (EH) 6, (IL) 9, (MP) 15, and (QT) 28 weeks. Scale bar, 500 μm.
Table 1.
 
LSC Clone Number and Width in Pax6 +/+ and Pax6 +/− Mice
Table 1.
 
LSC Clone Number and Width in Pax6 +/+ and Pax6 +/− Mice
Age (wk) LSC Clone Number LSC Clone Width (μm)
Pax6 +/+ Pax6 +/− P (t-test) Pax6 +/+ Pax6 +/− P (t-test)
Pax6 Sey-Neu Allele, (CBA × H253 Background)
  9 95.27 ± 5.88 (10) 49.03 ± 5.30 (7) <0.00001 91.30 ± 5.88 (10) 228.76 ± 47.5 (7) 0.0273
 15 85.04 ± 4.25 (15) 46.43 ± 3.70 (12) <0.00001 118.86 ± 5.97 (15) 202.83 ± 17.96 (11) <0.0001
 28 73.66 ± 4.32 (27) 37.83 ± 3.97 (25) <0.00001 149.71 ± 11.21 (27) 270.16 ± 22.01 (25) <0.0001
Pax6 Sey Allele, [(C57BL × CBA) × H253 Background]
 15 87.12 ± 4.62 (12) 59.14 ± 4.54 (12) 0.00028
Pax6 Sey Allele, (129/Sv × H253 Background)
 15 83.21 ± 5.95 (12) 53.09 ± 3.54 (14) 0.00037
Figure 2.
 
Size and number of LSC clones in Pax6 +/− and wild-type eyes. Mean number ± SEM of coherent clones of LSCs per eye and circumferential width of coherent clones of LSCs for Pax6 +/+ and Pax6 +/− corneas at 9 to 28 weeks. In some cases, the SE bars are smaller than the symbols.
Figure 2.
 
Size and number of LSC clones in Pax6 +/− and wild-type eyes. Mean number ± SEM of coherent clones of LSCs per eye and circumferential width of coherent clones of LSCs for Pax6 +/+ and Pax6 +/− corneas at 9 to 28 weeks. In some cases, the SE bars are smaller than the symbols.
Figure 3.
 
Robust directed centripetal migration in Pax6 +/+Pax6 +/− chimeras. Patterns of X-gal staining in the corneal epithelia of 15-week-old (AC) Pax6 +/+Pax6 +/+ chimeras (both aggregated embryos Pax6 +/+, one embryo LacZ +) and (DF) Pax6 +/+Pax6 +/− chimeras (LacZ + cells are Pax6 +/−, LacZ are Pax6 +/+). Scale bar, 1 mm.
Figure 3.
 
Robust directed centripetal migration in Pax6 +/+Pax6 +/− chimeras. Patterns of X-gal staining in the corneal epithelia of 15-week-old (AC) Pax6 +/+Pax6 +/+ chimeras (both aggregated embryos Pax6 +/+, one embryo LacZ +) and (DF) Pax6 +/+Pax6 +/− chimeras (LacZ + cells are Pax6 +/−, LacZ are Pax6 +/+). Scale bar, 1 mm.
Table 2.
 
Contribution of Blue Cells to Corneal Epithelia of Pax6 +/+Pax6 +/+, LacZ +/− Control Chimeras and Pax6 +/+Pax6 +/−, LacZ +/− Heterozygous Chimeras
Table 2.
 
Contribution of Blue Cells to Corneal Epithelia of Pax6 +/+Pax6 +/+, LacZ +/− Control Chimeras and Pax6 +/+Pax6 +/−, LacZ +/− Heterozygous Chimeras
Eye GPI Bouter Binner Bouter/GPI Bouter − GPI Binner/Bouter Binner − Bouter
Control chimeras
 MC379L 2.97 0.00 0.00 0.00 −2.97
 MC379R 2.97 0.00 0.00 0.00 −2.97
 MC382L 39.97 76.63 89.42 1.92 36.66 1.17 12.79
 MC382R 39.97 50.71 58.43 1.27 10.74 1.15 7.72
 MC383L 40.86 56.00 69.11 1.37 15.14 1.23 13.11
 MC383R 40.86 49.94 62.10 1.22 9.08 1.24 12.16
 MC388L 80.93 94.19 88.92 1.16 13.26 0.94 −5.26
 MC388R 80.93 * * * *
 MC405L 29.29 21.30 1.15 0.73 −7.99 0.05 −20.15
 MC405R 29.29 21.52 0.00 0.73 −7.77 0.00 −21.52
 MC406L 4.85 7.69 6.92 1.59 2.84 0.90 −0.77
 MC406R 4.85 0.00 0.00 0.00 −4.85
 MC430L 8.95 * * * *
 MC430R 8.95 5.45 9.68 0.61 −3.50 1.78 4.23
 MC434L 20.72 67.15 35.54 3.24 46.43 0.53 −31.61
 MC434R 20.72 47.99 34.96 2.32 27.27 0.73 −13.03
 MC438L 21.03 9.09 1.93 0.43 −11.94 0.21 −7.16
 MC438R 21.03 10.58 5.67 0.50 −10.45 0.54 −4.91
 Mean ± SEM 1.07 ± 0.22 6.81 ± 4.35 0.81 ± 0.15 −4.18 ± 3.98
Heterozygous chimeras
 MC364L 32.24 4.58 1.63 0.14 −27.66 0.36 −2.95
 MC364R 32.24 7.24 1.93 0.22 −25.00 0.27 −5.30
 MC367L 26.20 0.00 0.00 0.00 −26.20
 MC367R 26.20 1.24 4.20 0.05 −24.96 3.38 2.96
 MC368L 3.40 9.38 9.81 2.76 5.98 1.05 0.43
 MC368R 3.40 1.51 0.00 0.44 −1.89 0.00 −1.51
 MC377L 27.66 2.99 0.00 0.11 −24.67 0.00 −2.99
 MC377R 27.66 7.04 1.86 0.25 −20.63 0.26 −5.18
 MC378L 40.98 3.91 2.55 0.10 −37.08 0.65 −1.35
 MC378R 40.98 4.82 5.29 0.12 −36.16 1.10 0.47
 MC385L 31.01 11.73 1.96 0.38 −19.28 0.17 −9.77
 MC385R 31.01 20.03 17.29 0.65 −10.98 0.86 −2.74
 MC391L 40.85 22.44 24.95 0.55 −18.41 1.11 2.51
 MC391R 40.85 31.88 17.93 0.78 −8.97 0.56 −13.95
 MC397L 20.33 0.00 0.00 0.00 −20.33
 MC397R 20.33 0.00 0.00 0.00 −20.33
 MC408L 67.14 34.75 17.20 0.52 −32.39 0.49 −17.55
 MC408R 67.14 31.96 12.67 0.48 −35.18 0.40 −19.29
 MC409L 17.87 0.00 0.00 0.00 −17.87
 MC409R 17.87 0.00 0.00 0.00 −17.87
 MC431L 74.00 7.78 4.34 0.11 −66.21 0.56 −3.44
 MC431R 74.00 43.49 24.74 0.59 −30.51 0.57 −18.75
 MC436L 52.31 3.65 7.27 0.07 −48.66 1.99 3.62
 MC436R 52.31 6.49 7.56 0.12 −45.82 1.16 1.07
 Mean ± SEM 0.35 ± 0.12 −25.46 ± 3.11 0.79 ± 0.18 −4.93 ± 1.70
t-Test (control v. heterozygous chimeras) P = 0.009 <0.0001 0.93 0.86
Figure 4.
 
Pax6 +/− epithelial sectors of normal thickness in Pax6 +/+Pax6 +/− chimeric corneas. (A, B) Representative histologic sections of the corneal epithelia of adult (15 weeks) Pax6 +/+ (A) and Pax6 +/− (B) mice from the Pax6 Sey-Neu/+ x Pax6 Sey/+ crosses that were used to make chimeras. Heterozygotes have thinner corneal epithelia with fewer cell layers. (CE) Sections of the corneal epithelia of a 15-week Pax6 +/+Pax6 +/+, LacZ + control chimera (C), and Pax6 +/+Pax6 +/−, LacZ + littermates (D, E). Pax6 +/−, LacZ + cells produced multicellular corneal epithelia of normal thickness in chimeric situations, equivalent to Pax6 +/+, LacZ + cells in the controls. (A, B, E) Hematoxylin/eosin staining; (C, D, E) X-gal staining; (C, D) phase-contrast micrographs. Scale bar, 35 μm.
Figure 4.
 
Pax6 +/− epithelial sectors of normal thickness in Pax6 +/+Pax6 +/− chimeric corneas. (A, B) Representative histologic sections of the corneal epithelia of adult (15 weeks) Pax6 +/+ (A) and Pax6 +/− (B) mice from the Pax6 Sey-Neu/+ x Pax6 Sey/+ crosses that were used to make chimeras. Heterozygotes have thinner corneal epithelia with fewer cell layers. (CE) Sections of the corneal epithelia of a 15-week Pax6 +/+Pax6 +/+, LacZ + control chimera (C), and Pax6 +/+Pax6 +/−, LacZ + littermates (D, E). Pax6 +/−, LacZ + cells produced multicellular corneal epithelia of normal thickness in chimeric situations, equivalent to Pax6 +/+, LacZ + cells in the controls. (A, B, E) Hematoxylin/eosin staining; (C, D, E) X-gal staining; (C, D) phase-contrast micrographs. Scale bar, 35 μm.
The authors thank Jean Flockhart for providing technical expertise; Baljean Dhillon, Kanna Ramaesh, and Thaya Ramaesh for helpful discussions; Seong-Seng Tan for the H253 mice; Maureen Ross, Denis Doogan, and Jim Macdonald for specialist assistance; and Sarah McDonald for performing the preliminary analysis of the Pax6-H253 strains. 
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Figure 1.
 
Clonal patterns of cell growth and movement in Pax6 +/− eyes. Patterns of X-gal staining in corneal epithelia of (A, E, I, M, Q) Pax6 +/+ and (BD, FH, JL, NP, RT) Pax6 +/− female littermates from crosses of Pax6 +/− mice with H253 transgenic mice carrying an X-linked LacZ transgene. Representative eyes are presented at (AD) 3, (EH) 6, (IL) 9, (MP) 15, and (QT) 28 weeks. Scale bar, 500 μm.
Figure 1.
 
Clonal patterns of cell growth and movement in Pax6 +/− eyes. Patterns of X-gal staining in corneal epithelia of (A, E, I, M, Q) Pax6 +/+ and (BD, FH, JL, NP, RT) Pax6 +/− female littermates from crosses of Pax6 +/− mice with H253 transgenic mice carrying an X-linked LacZ transgene. Representative eyes are presented at (AD) 3, (EH) 6, (IL) 9, (MP) 15, and (QT) 28 weeks. Scale bar, 500 μm.
Figure 2.
 
Size and number of LSC clones in Pax6 +/− and wild-type eyes. Mean number ± SEM of coherent clones of LSCs per eye and circumferential width of coherent clones of LSCs for Pax6 +/+ and Pax6 +/− corneas at 9 to 28 weeks. In some cases, the SE bars are smaller than the symbols.
Figure 2.
 
Size and number of LSC clones in Pax6 +/− and wild-type eyes. Mean number ± SEM of coherent clones of LSCs per eye and circumferential width of coherent clones of LSCs for Pax6 +/+ and Pax6 +/− corneas at 9 to 28 weeks. In some cases, the SE bars are smaller than the symbols.
Figure 3.
 
Robust directed centripetal migration in Pax6 +/+Pax6 +/− chimeras. Patterns of X-gal staining in the corneal epithelia of 15-week-old (AC) Pax6 +/+Pax6 +/+ chimeras (both aggregated embryos Pax6 +/+, one embryo LacZ +) and (DF) Pax6 +/+Pax6 +/− chimeras (LacZ + cells are Pax6 +/−, LacZ are Pax6 +/+). Scale bar, 1 mm.
Figure 3.
 
Robust directed centripetal migration in Pax6 +/+Pax6 +/− chimeras. Patterns of X-gal staining in the corneal epithelia of 15-week-old (AC) Pax6 +/+Pax6 +/+ chimeras (both aggregated embryos Pax6 +/+, one embryo LacZ +) and (DF) Pax6 +/+Pax6 +/− chimeras (LacZ + cells are Pax6 +/−, LacZ are Pax6 +/+). Scale bar, 1 mm.
Figure 4.
 
Pax6 +/− epithelial sectors of normal thickness in Pax6 +/+Pax6 +/− chimeric corneas. (A, B) Representative histologic sections of the corneal epithelia of adult (15 weeks) Pax6 +/+ (A) and Pax6 +/− (B) mice from the Pax6 Sey-Neu/+ x Pax6 Sey/+ crosses that were used to make chimeras. Heterozygotes have thinner corneal epithelia with fewer cell layers. (CE) Sections of the corneal epithelia of a 15-week Pax6 +/+Pax6 +/+, LacZ + control chimera (C), and Pax6 +/+Pax6 +/−, LacZ + littermates (D, E). Pax6 +/−, LacZ + cells produced multicellular corneal epithelia of normal thickness in chimeric situations, equivalent to Pax6 +/+, LacZ + cells in the controls. (A, B, E) Hematoxylin/eosin staining; (C, D, E) X-gal staining; (C, D) phase-contrast micrographs. Scale bar, 35 μm.
Figure 4.
 
Pax6 +/− epithelial sectors of normal thickness in Pax6 +/+Pax6 +/− chimeric corneas. (A, B) Representative histologic sections of the corneal epithelia of adult (15 weeks) Pax6 +/+ (A) and Pax6 +/− (B) mice from the Pax6 Sey-Neu/+ x Pax6 Sey/+ crosses that were used to make chimeras. Heterozygotes have thinner corneal epithelia with fewer cell layers. (CE) Sections of the corneal epithelia of a 15-week Pax6 +/+Pax6 +/+, LacZ + control chimera (C), and Pax6 +/+Pax6 +/−, LacZ + littermates (D, E). Pax6 +/−, LacZ + cells produced multicellular corneal epithelia of normal thickness in chimeric situations, equivalent to Pax6 +/+, LacZ + cells in the controls. (A, B, E) Hematoxylin/eosin staining; (C, D, E) X-gal staining; (C, D) phase-contrast micrographs. Scale bar, 35 μm.
Table 1.
 
LSC Clone Number and Width in Pax6 +/+ and Pax6 +/− Mice
Table 1.
 
LSC Clone Number and Width in Pax6 +/+ and Pax6 +/− Mice
Age (wk) LSC Clone Number LSC Clone Width (μm)
Pax6 +/+ Pax6 +/− P (t-test) Pax6 +/+ Pax6 +/− P (t-test)
Pax6 Sey-Neu Allele, (CBA × H253 Background)
  9 95.27 ± 5.88 (10) 49.03 ± 5.30 (7) <0.00001 91.30 ± 5.88 (10) 228.76 ± 47.5 (7) 0.0273
 15 85.04 ± 4.25 (15) 46.43 ± 3.70 (12) <0.00001 118.86 ± 5.97 (15) 202.83 ± 17.96 (11) <0.0001
 28 73.66 ± 4.32 (27) 37.83 ± 3.97 (25) <0.00001 149.71 ± 11.21 (27) 270.16 ± 22.01 (25) <0.0001
Pax6 Sey Allele, [(C57BL × CBA) × H253 Background]
 15 87.12 ± 4.62 (12) 59.14 ± 4.54 (12) 0.00028
Pax6 Sey Allele, (129/Sv × H253 Background)
 15 83.21 ± 5.95 (12) 53.09 ± 3.54 (14) 0.00037
Table 2.
 
Contribution of Blue Cells to Corneal Epithelia of Pax6 +/+Pax6 +/+, LacZ +/− Control Chimeras and Pax6 +/+Pax6 +/−, LacZ +/− Heterozygous Chimeras
Table 2.
 
Contribution of Blue Cells to Corneal Epithelia of Pax6 +/+Pax6 +/+, LacZ +/− Control Chimeras and Pax6 +/+Pax6 +/−, LacZ +/− Heterozygous Chimeras
Eye GPI Bouter Binner Bouter/GPI Bouter − GPI Binner/Bouter Binner − Bouter
Control chimeras
 MC379L 2.97 0.00 0.00 0.00 −2.97
 MC379R 2.97 0.00 0.00 0.00 −2.97
 MC382L 39.97 76.63 89.42 1.92 36.66 1.17 12.79
 MC382R 39.97 50.71 58.43 1.27 10.74 1.15 7.72
 MC383L 40.86 56.00 69.11 1.37 15.14 1.23 13.11
 MC383R 40.86 49.94 62.10 1.22 9.08 1.24 12.16
 MC388L 80.93 94.19 88.92 1.16 13.26 0.94 −5.26
 MC388R 80.93 * * * *
 MC405L 29.29 21.30 1.15 0.73 −7.99 0.05 −20.15
 MC405R 29.29 21.52 0.00 0.73 −7.77 0.00 −21.52
 MC406L 4.85 7.69 6.92 1.59 2.84 0.90 −0.77
 MC406R 4.85 0.00 0.00 0.00 −4.85
 MC430L 8.95 * * * *
 MC430R 8.95 5.45 9.68 0.61 −3.50 1.78 4.23
 MC434L 20.72 67.15 35.54 3.24 46.43 0.53 −31.61
 MC434R 20.72 47.99 34.96 2.32 27.27 0.73 −13.03
 MC438L 21.03 9.09 1.93 0.43 −11.94 0.21 −7.16
 MC438R 21.03 10.58 5.67 0.50 −10.45 0.54 −4.91
 Mean ± SEM 1.07 ± 0.22 6.81 ± 4.35 0.81 ± 0.15 −4.18 ± 3.98
Heterozygous chimeras
 MC364L 32.24 4.58 1.63 0.14 −27.66 0.36 −2.95
 MC364R 32.24 7.24 1.93 0.22 −25.00 0.27 −5.30
 MC367L 26.20 0.00 0.00 0.00 −26.20
 MC367R 26.20 1.24 4.20 0.05 −24.96 3.38 2.96
 MC368L 3.40 9.38 9.81 2.76 5.98 1.05 0.43
 MC368R 3.40 1.51 0.00 0.44 −1.89 0.00 −1.51
 MC377L 27.66 2.99 0.00 0.11 −24.67 0.00 −2.99
 MC377R 27.66 7.04 1.86 0.25 −20.63 0.26 −5.18
 MC378L 40.98 3.91 2.55 0.10 −37.08 0.65 −1.35
 MC378R 40.98 4.82 5.29 0.12 −36.16 1.10 0.47
 MC385L 31.01 11.73 1.96 0.38 −19.28 0.17 −9.77
 MC385R 31.01 20.03 17.29 0.65 −10.98 0.86 −2.74
 MC391L 40.85 22.44 24.95 0.55 −18.41 1.11 2.51
 MC391R 40.85 31.88 17.93 0.78 −8.97 0.56 −13.95
 MC397L 20.33 0.00 0.00 0.00 −20.33
 MC397R 20.33 0.00 0.00 0.00 −20.33
 MC408L 67.14 34.75 17.20 0.52 −32.39 0.49 −17.55
 MC408R 67.14 31.96 12.67 0.48 −35.18 0.40 −19.29
 MC409L 17.87 0.00 0.00 0.00 −17.87
 MC409R 17.87 0.00 0.00 0.00 −17.87
 MC431L 74.00 7.78 4.34 0.11 −66.21 0.56 −3.44
 MC431R 74.00 43.49 24.74 0.59 −30.51 0.57 −18.75
 MC436L 52.31 3.65 7.27 0.07 −48.66 1.99 3.62
 MC436R 52.31 6.49 7.56 0.12 −45.82 1.16 1.07
 Mean ± SEM 0.35 ± 0.12 −25.46 ± 3.11 0.79 ± 0.18 −4.93 ± 1.70
t-Test (control v. heterozygous chimeras) P = 0.009 <0.0001 0.93 0.86
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