Figure 1.
Electron microscopy of whole eyes and corneal epithelium.
A–C. SEM micrographs of (A) WT, (B) Pax6+/− and (C) PAX77Tg/− eyes showing differences in size and gross morphology. Scale bars = 500 µm. (Diameters of WT, Pax6+/− and PAX77Tg/− eyes used for EM were approximately 2.9, 2.5 and 2.2 mm respectively and corneal diameters (dome base diameters) were approximately 2.5, 2.3 and 1.6 mm respectively.) D–F. SEM micrographs of (D) WT, (E) Pax6+/− and (F) PAX77Tg/− surfaces of corneal epithelial cells showing polygonal cell shapes and cell junctions. The surface of the cells appears more irregular in the PAX77Tg/− corneas than WT. Scale bars = 10 µm. G–I. Higher power SEM micrographs of (G) WT, (H) Pax6+/− and (I) PAX77Tg/− surfaces showing microvilli on cell surfaces. Microvilli are larger in the PAX77Tg/− corneas than WT. Scale bars = 5 µm.
Table 1.
Main new abnormalities in Pax6+/− and PAX77Tg/− corneas identified by electron microscopy.
Figure 2.
Electron microscopy of corneal endothelium.
A–C. SEM micrographs of (A) WT, (B) Pax6+/− and (C) PAX77Tg/− corneal endothelial cells. WT endothelial cells have a regular hexagonal shape, Pax6+/− cells have an irregular vacuolated appearance and are larger than normal and PAX77Tg/− endothelial cells have an irregular vacuolated appearance, are irregular in shape and the cell borders are difficult to resolve. Scale bars = 10 µm. D–F. Higher power SEM micrographs of (D) WT, (E) Pax6+/− and (F) PAX77Tg/− corneal endothelial cells showing that although both Pax6+/− and PAX77Tg/− corneal endothelial cells are vacuolated and irregular in shape the cell borders are more distinct in Pax6+/− cells. Scale bars = 10 µm. G–I. TEM micrographs of (G) WT, (H) Pax6+/− and (I) PAX77Tg/− corneal endothelial cells (shown above Descemet's membrane and stroma) at the periphery of the cornea. Pax6+/− and PAX77Tg/− endothelial cells contain large vacuoles. Scale bars = 1 µm.
Figure 3.
Electron microscopy of corneal stroma.
TEM micrographs of (A) WT corneal stroma showing normal keratocyte morphology with no vacuoles; (B,C) Pax6+/− corneal stroma showing keratocytes with very large vacuoles; (D) PAX77Tg/− corneal stroma showing keratocyte with small vacuoles; (E) Pax6+/− corneal stroma showing nerve cell; (F) PAX77Tg/− corneal stroma showing nerve cell. Abbreviations: *v, vacuole; *n, nerve cell. Scale bars = 500 nm in A–D and 1 µm in E and F.
Figure 4.
Mosaic patterns in the corneal epithelium.
Representative images of β-gal staining in the corneal epithelia of X-inactivation mosaic eyes showing variation in mosaic patterns among the four different genotypes: (A,B) WT, XLacZTg/−; (C,D) Pax6+/−, XLacZTg/−; (E,F) PAX77Tg/−, XLacZTg/− and (G,H) Pax6Leca4/+, XLacZTg/−. Scale bar = 1 mm.
Table 2.
Comparison of maintenance of corneal epithelium in adult Pax6+/− and PAX77Tg/− mice with low and high doses of Pax6 respectively.
Figure 5.
Sections of β-gal-stained mosaic corneal epithelia.
Representative images of β-gal-stained corneal sections showing vertical alignment of β-gal-positive epithelial cells across the full thickness of the epithelium in eyes expressing various levels of Pax6. (A,B) WT, XLacZTg/−; (C,D) Pax6+/−, XLacZTg/− and (E,F) PAX77Tg/−, XLacZTg/−. The higher frequency of β-gal-positive cells in XLacZTg/−, Pax6+/− corneal stromas (C,D) probably reflects the greater permeability of the thin Pax6+/− corneal epithelium to X-gal stain during whole-mount staining. Scale bar = 100 µm.
Table 3.
Preliminary comparison of corrected stripe number in corneal epithelia of wild-type, XLacZTg/− and PAX77Tg/−, XLacZTg/− X-inactivation mosaics at 15 weeks.
Figure 6.
Quantitative comparisons of PAX77Tg/− XLacZTg/− and PAX77−/− XLacZTg/− eyes at 15 and 30-weeks.
(A) WT Eye mass increased significantly between 15 and 30 weeks (2-way ANOVA P<0.0001, results of relevant post-hoc tests are shown in the figure). (B) Corneal circumference differed significantly between WT and PAX77Tg/− at 15 and 30 weeks but the increase in circumference between 15 and 30 weeks was only significant for WT (2-way ANOVA P<0.0001, results of relevant post-hoc tests are shown in the figure). (C) The mean corrected stripe number was significantly higher in the 15-week WT corneas than the 30-week WT or 15-week PAX77Tg/− groups, there was a significant decline in stripe number between 15 and 30 weeks in the WT but not the PAX77Tg/− group (2-way ANOVA P<0.0001, results of relevant post-hoc tests are shown in the figure). (D) The mean corrected stripe number was significantly higher in the 15-week WT corneas than the 30-week WT or 15-week PAX77Tg/− groups, there was a significant decline in stripe number between 15 and 30 weeks in the WT but not the PAX77Tg/− group (2-way ANOVA P<0.0001, results of relevant post-hoc tests are shown in the figure). For each comparison there were 14–36 eyes per group: 22 15-week WT, 36 30-week WT, 14 15-week PAX77Tg/− and 20 30-week PAX77Tg/−. Significant P-values for Tukey's HSD post-hoc tests are shown: ns = not significant; *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001. For all post-hoc tests see Tables S1, S2, S3, S4.
Figure 7.
Representative images of β-gal staining in the corneal epithelia of X-inactivation mosaic eyes expressing different levels of Pax6.
(A) WT (PAX77−/−, Pax6+/+, XLacZTg/−) eyes exhibit ordered radial stripes of clonally related epithelial cells. (B) PAX77−/−, Pax6+/−, XLacZTg/− eyes are smaller and striping patterns are disrupted, normal radial stripes are only rarely observed. (C) In eyes over-expressing PAX6 (PAX77Tg/−, Pax6+/+, XLacZTg/−) the corneal epithelial diameter is smaller in comparison to the overall eye size (microcornea) but normal radial stripe patterns are visible. (D) PAX77Tg/−, Pax6+/−, XLacZTg/− corneas appear normal both in size and striping phenotype. Scale bar = 1 mm.
Figure 8.
Quantitative comparison of stripe patterns in corneal epithelia of X-inactivation mosaics of four genotypes.
Corneal circumferences and corrected stripe numbers were compared in WT (PAX77−/−, Pax6+/+, XLacZTg/−); Pax6+/− (PAX77−/−, Pax6+/−, XLacZTg/−); PAX77Tg/− (PAX77Tg/−, Pax6+/+, XLacZTg/−); and combined PAX77Tg/−, Pax6+/− (PAX77Tg/−, Pax6+/−, XLacZTg/−) mosaic eyes at both 15 and 30-weeks. (A) The corneal circumference was significantly smaller in both 15-week and 30-week old PAX77Tg/− (Pax6+/+, PAX77Tg/−, XLacZTg/−) mice than in the three other genotypes at both ages (2-way ANOVA P<0.0001, results of relevant post-hoc tests are shown in the figure). (B) The Pax6+/−, PAX77Tg/− corrected stripe number did not differ from WT at 15 weeks. The WT corrected stripe number declined significantly between 15 and 30 weeks. This was not the case for any other group. (2-way ANOVA P<0.0001, results of relevant post-hoc tests are shown in the figure). (C) On this genetic background correcting the mean corrected stripe number for circumference abrogated the significant results observed in B (2-way ANOVA P<0.01, but results of relevant post-hoc tests were all non-significant). For each comparison there were 7–25 eyes per group: 25 15-week WT, 12 30-week WT, 20 15-week Pax6+/−, 12 30-week Pax6+/−, 7 15-week PAX77Tg/−, 16 30-week PAX77Tg/−, 22 15-week Pax6+/+, PAX77Tg/− and 22 30-week Pax6+/+, PAX77Tg/− eyes. Significant P-values for Tukey's HSD post-hoc tests are shown: ns = not significant; *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001. † P<0.0001 for differences with all other genotypes at both ages. For all post-hoc tests see Tables S5, S6, S7.