PMC

Induction of epithelial genes in embryo fibroblasts derived from wild-type, Zeb1 heterozygous and Zeb1 null litter mates. Real time PCR analysis of mRNA levels is shown.

Induction of COL4A3 and E-cadherin mRNAs in the cornea in Zeb1 null mice. mRNA levels in corneas isolated from liter mates at E18.5 were quantified by real time PCR, and compared to β-actin and GAPDH mRNAs as a control. Corneas from three embryos were pooled for each genotype.

Iridocorneal and corneolenticular adhesions in adult Zeb1 heterozygous mice. H&E stained sections eyes from adult wild type (panels A–A′) and Zeb1 heterozygous (panel B–B′) mice. Arrows in Panels B and B′ show corneolenticular and iridocorneal adhesions. EP, corneal epithelium; EN, corneal endothelium, LE, lens epithelium; I, iris; R, retina; I, iris; CB. ciliary body; R, retina. Bars are 100 μm.

Iridocorneal adhesion leads to loss of the iridocorneal angle in adult Zeb1 heterozygous mice. H&E section through the eye of an adult Zeb1 heterozygous mouse showing iridocorneal adhesion and loss of the iridocorneal angle. The inset shows the iridocorneal angle in a wild-type litter mate. EP, corneal epithelium; EN, corneal endothelium; LE, lens epithelium; I, iris; CB, ciliary body; R, retina. Arrows indicate iridocorneal adhesions. Bars are 200 μm.

E-cadherin becomes ectopically expressed in corneal endothelium, and cytokeratin and COL4A3 become ectopically expressed in corneal endothelium and keratocytes in Zeb1 null mice. (A–A′). Immunostaining for Zeb1 is evident in corneal endothelium and keratocytes in wild-type mice. EP, corneal epithelium; EN, corneal endothelium; K, keratocytes; LE, lens epithelium. A Nomarski image is shown on the right in each panel. Arrows indicate the same location. (B–B′). Immunostaining for COL4A3 in the corneal epithelium in wild-type mice. (C–C′). Immunostaining for E-cadherin in the corneal epithelium in wild-type mice. (D–D′). Immunostaining for pan cytokeratin in the corneal epithelium in wild-type mice. (E–E′). Immunostaining for COL4A3 expands to corneal keratocytes and endothelium in Zeb1 null mice. EL, eye lid. (F–F′). Immunostaining for E-cadherin expands to the corneal endothelium in Zeb1 null mice. (G–G′). Cytokeratin immunostaining expands to corneal keratocytes and endothelium in Zeb1 null mice. Sections of mice at E16.5 are shown. The bar is 100 μm.

Iridocorneal, corneolenticular, and iridolenticular adhesions in Zeb1 null mice. H&E stained sections of eyes from wild type (panels A–B) and Zeb1 null (panel C–F) mice. C, cornea; EL, eye lid; I, iris; LE, lens epithelium, R, retina; I, iris; EN, corneal endothelium; EP, corneal epithelium. The “*” in panels A and C indicates the region of the cornea analyzed for BrdU incorporation () and corneal thickness (). Arrows in panel D indicate iridocorneal adhesion. Arrows in panels E and F show corneolenticular and iridolenticular adhesions, respectively. Bars are 100 μm. Sections at E17.5 are shown.

Corneal thickening occurs in Zeb1 null mice at E17.5 and in adult heterozygotes (but not in Zeb1 null mice at E13.5), and increased keratocyte numbers is seen in heterozygous adults. (A). Corneal thickness was assessed in the regions indicated in below. Corneal thickness was measured at E13.5, E17.5 and in 4 month old adults. (B). The number of keratocyte nuclei in a 200 μM length of cornea (in areas used to measure corneal thickness in panel A) was counted. Error bars are standard deviations. Litter mates were examined for these measurements, and at least two different mice were examined for each genotype, and 5 adjacent 5 micron sections were analyzed for each mouse.

Abnormal corneal proliferation occurs in Zeb1 null mice at E15.5 but not at E13.5. Mice at E13.5 or E15.5 were injected with BrdU and embryos were harvested 2 hours later and sectioned for immunostaining with anti-BrdU antibody. (A). Immunofluorescent staining for BrdU and quantification of BrdU incorporation in corneal epithelium (EP), endothelium (EN), keratocytes (K), and lens epithelium (LE) is shown at E15.5. Arrows indicate the same location in the images. Nomarski images are shown at the left in each panel. (B). Immunoproxidase staining for BrdU and quantification of BrdU incorporation into the cornea at E13.5. Bars are 100 μm. The corneal areas counted in panels A and B are denoted below by the “*” in . Error bars are standard deviations. Litter mates were examined, and at least two different mice were examined for each genotype, and 5 adjacent 5 micron sections were counted for each mouse.