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1From the Department of Biomedical Sciences, Institute of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen, Scotland, United Kingdom; the 2Division of Reproductive and Developmental Sciences, Genes and Development Group, University of Edinburgh, Edinburgh, Scotland, United Kingdom; and the 3Comparative and Developmental Genetics Section, Medical Research Council [MRC] Human Genetics Unit, Edinburgh, Scotland, United Kingdom.
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Abstract |
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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 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 ß-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.
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Materials and Methods |
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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 (
· 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 aggregation30 has been described elsewhere.23 Eight-cell embryos were obtained from Pax6Sey-Neu/+ females, homozygous for the glucose phosphate isomerase 1-b Gpi1b allele (Pax6Sey-Neu/+; Gpi1b/b), that had been mated to Pax6Sey/+ males, homozygous for the constitutive LacZ transgene TgR(ROSA26)26Sor31 (Pax6Sey/+; LacZ+/+; Gpi1b/b). The embryos were aggregated with eight-cell embryos from a Gpi1a/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 Gpi1a/a, and the eight-cell embryo derived from the Pax6+/Sey-Neu x Pax6Sey/+ mating was Gpi1b/b. Those chimeras with higher %GPI1-A are primarily composed of cells derived from the wild-type mating, and vice versa.
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Results |
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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 cells21 (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 ).
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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.
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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.
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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.
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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 |
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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 ß-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.
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Acknowledgements |
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Footnotes |
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Submitted for publication October 9, 2003; revised January 5, 2004; accepted January 6, 2004.
Disclosure: J.M. Collinson, None; S.A. Chanas, None; R.E. Hill, None; J.D. West, None
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked "advertisement" in accordance with 18 U.S.C. 
1734 solely to indicate this fact.
Corresponding author: J. Martin Collinson, Department of Biomedical Sciences, Institute of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen AB25 2ZD, Scotland, UK; m.collinson{at}abdn.ac.uk.
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References |
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diploid foetal mouse chimaeras: possible relevance to human confined mosaicism. Genet Res. 1994;63:87–99.[Web of Science][Medline][Order article via Infotrieve]
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