Keratin 12-Deficient Mice Have Fragile Corneal Epithelia Winston W.—Y. Kao,* Chia-YangLiu,* Richard L. Converse,* Atsushi Shiraishi* Candace W.-C. Kao,* Masamichi Ishizaki* Thomas Doetschman,^ and John Duffy-f Purpose. Expression of the K3-K12 keratin pair characterizes the corneal epithelial differentia- tion. To elucidate the role of keratin 12 in the maintenance of corneal epithelium integrity, the authors bred mice deficient in keratin 12 by gene-targeting techniques. Methods. One allele of murine Krtl.12 gene was ablated in the embryonic stem cell line, E14.1, by homologous recombination with a DNA construct in which the DNA element between intron 2 and exon 8 of the keratin 12 gene was replaced by a neo-gene. The homologous recombinant embryonic stem cells were injected to mouse blastocysts, and germ lines of chimeras were obtained. The corneas of heterozygous and homozygous mice were characterized by clinical observations using stereomicroscopy, histology with light and electron microscopy, Western immunoblot analysis, immunohistochemistry, in situ hybridization, and Northern hybridization. Results. The heterozygous mice (+/—) one allele of the Krtl.12 gene appear normal and do not develop any clinical manifestations (e.g., corneal epithelial defects). Homozygous mice (—/—) develop normally and suffer mild corneal epithelial erosion. Their corneal epithelia are fragile and can be removed by gentle rubbing of the eyes or brushing with a Microsponge. The corneal epithelium of the homozygote (—/—) does not express keratin 12 as judged by immunohistochemis- try, Western immunoblot analysis with epitope-specific anti-keratin 12 antibodies, Nordiern hybrid- ization with 32P-labeled keratin 12 cDNA, and in situ hybridization with an anti-sense keratin 12 riboprobe. Light and electron microscopy revealed subtle abnormalities in the corneal epithelia of —/— mice (i.e., a decrease in number of cell layers) and cytolysis of superficial cells, but the number of hemidesmosomes and desmosomes are normal in basal and suprabasal cells. The number of keratin intermediate filaments in basal and suprabasal corneal epithelial cells in — /— mice de- creases, and they appear as dense bundles. This morphology is similar to that of keratin intermediate filaments in epidermal epithelial cells but differs from that of normal corneal epithelial cells in which the keratins form fine filamentous networks. The superficial epithelial cells are devoid of keratin intermediate filaments and often detach from the corneal surface of — /— mice. Conclusions. The presence of cornea-specific K3-K12 keratin pail's is essential for the maintenance of corneal epithelium integrity. Invest Ophthalmol Vis Sci. 1996;37:2572-2584. IVeratins are a group of water-insoluble proteins that tive charge, immunoreactivity, and sequence homolo- form 10 nm intermediate filaments in epithelial gies to types I and II wool keratins, respectively.5'6 In cells.1"'1 Approximately 30 different keratin molecules vivo, a basic keratin usually is coexpressed and have been identified.1 They can be divided into acidic "paired" with a particular acidic keratin to form a and basic-neutral subfamilies according to their rela- heterodimer.6'7 The expression of various keratin pairs is tissue specific, differentiation dependent, and devel- opmentally regulated.2'58'9 From the Departments of*Ophthalmology and ^Molecular Genetics, University of The presence of specific keratin pairs is essential Cincinnati, Ohio. Supported by National Institutes of Health grants EY10556 and HL41496 and by for the maintenance of epithelium integrity. For ex- Ohio Lions Eye Research Foundation. ample, mutations in human K14-K510"12 and K10- Submitted for publication April 16, 1996; revised July 9, 1996; accepted July 25, Kl1314 genes have been linked to the human skin dis- 1996. Proprietary interest category: N. eases, epidermolysis bullosa simplex and epidermoly- Reprint requests: Winston W.-Y. Kao, Department of Ophthalmology, University of sis hyperkeratosis, respectively. Similar clinical mani- Cincinnati, Eden and Bethesda Avenues, P.O. Box 670527, Cincinnati, OH 45267-0527. festation of these diseases have been reproduced in Investigative Ophthalmology & Visual Science, December 1996, Vol. 37, No. 13 2572 Copyright © Association for Research in Vision and Ophthalmology Downloaded from iovs.arvojournals.org on 09/29/2021 Keratin 12-Deficient Mice With Fragile Epithelia 2573 transgenic mice carrying dominant negative muta- Histologic Examination and tions of these keratin genes.15'16 In transgenic mice, Immunohistochemical Staining the dominant expression of mutated epidermal kera- The excised corneas were fixed in 4% paraformalde- tin 14 results in clinical features similar to those of hyde at 4°C overnight and embedded in paraffin as human epidermolysis bullosa simplex.1516 Recently, a previously described.20'22 Five-micrometer-thick sec- null mutation of K8 keratin by gene targeting has been tions were mounted on Super Frost slides (Fisher Sci- reported.17 Many of the homozygous mutant mice are entific, Pittsburgh, PA). Histologic examination was embryonic lethal at 10 days of gestation, when the K8- performed after Harris hematoxylin and eosin stain- K18 pair normally would be expressed during em- ing. Immunohistochemical staining was performed us- bryogenesis.17 These results demonstrate that keratin ing epitope-specific anti-keratin 1222 and anti-keratin intermediate filaments are vital for the integrity of 14 antibodies23 (a generous gift of Dr. D. R. Roop, epithelium. Baylor College of Medicine, Houston, TX) as pre- Expression of the K3-K12 keratin pair has been 20 22 viously described. ' found in human, bovine, guinea pig, rabbit, mouse, and chicken corneas and is regarded as a marker for In Situ Hybridization corneal-type epithelial differentiation. 1~3'5'718'19 We previously cloned the murine Krtl.12 gene and dem- The plasmid DNA containing a full-length keratin 12 onstrated that its expression is corneal epithelial cell cDNA insert was linearized with EcoR I and Kpn I. T- specific, differentiation dependent, and developmen- 7 and T-3 RNA polymerase were used to synthesize tally regulated.20'21 The cornea-specific nature of kera- antisense and sense riboprobes with digoxigenin using procedures recommended by the manufacturer (Boe- tin 12 gene expression signifies that keratin 12 plays hringer-Mannheim, Indianapolis, IN). Tissue sec- a unique role in maintaining normal corneal epithe- tions (5 fim) were incubated with riboprobes (1 //g/ lium functions. No hereditary human corneal epithe- ml using procedures recommended by Boehringer— lial disorder has been linked direcdy to a mutation of Mannheim). The nonspecific binding of riboprobes the keratin 12 gene, although such a human genetic was removed by RNase (20 //g/ml) digestion and a defect may exist. Nevertheless, the exact function of stringent wash in 0.2 X SSC at 65°C.20'22 Tissue sections keratin 12 remains unknown. To elucidate the func- were incubated with anti-digoxigenin antibody alka- tion of keratin 12, we have created knockout mice line phosphatase conjugate at 4°C overnight. The hy- lacking the Krtl.12 gene by gene targeting techniques. bridization signal was visualized with 5-nitroblue tetra- The homozygous mutant mice (Krtl.12, —/ — ) are zolium chloride as recommended by Boehringer— characterized by fragile corneal epithelium. Mannheim. MATERIALS AND METHODS Western Blot Analysis Animal Experiments To isolate keratins, frozen tissues were first homoge- Animal experiments were performed in compliance nized in an extraction solution containing 0.1% Tri- with the ARVO Statement for the Use of Animals in ton X-100 and a mixture of protease inhibitors in Tris- Ophthalmic and Vision Research. Adult mice were saline (0.15 M NaCl, 20 mM Tris-HCl, pH 7.5) with anesthetized by intraperitoneal injections of 70 mg/ a Tissuemizer (Tekmar, Cincinnati, OH) as previously 22 24 kg sodium pentobarbital. Under a stereomicroscope, described. ' The homogenate was centrifuged at a partial epithelial defect was created in both eyes by 12,000 rpm for 15 minutes. The supernatant was dis- scraping the corneal surface with a number 69 Beaver carded, and the pellet was rehomogenized in extrac- blade (Becton-Dickinson, Franklin Lakes, NJ) or tion buffer containing 9 M urea in Tris-saline to ex- brushing with a wet Microsponge (Alcon, Dallas, TX). tract the keratin. After centrifugation, equal volumes Partial defects occupied approximately 60% to 70% of 2 X sodium dodecyl sulfate-polyacrylamide gel of the total corneal epithelium. Neomycin ointment electrophoresis sample buffer was added to the super- was applied on the eyes immediately after surgery. natant. The samples were boiled for 10 minutes and Eyes were examined using a stereomicroscope (Olym- were then subjected to Western immunoblot analysis pus, Melville, NY) every other day beginning on the in 7% or 8% acrylamide using epitope-specific anti- first day after wounding to evaluate reepithelialization keratin 12 antibodies, anti-K12n, and anti-K12c as pre- and to detect any signs of infection.22 The animals viously described.22'24 were killed in a CO2 chamber, and the corneas were removed. Corneas either were embedded in paraffin Northern Blot Analysis for histology, immunohistochemistry, or in situ hy- Total RNAs were isolated from mouse corneas with bridization, or they were prepared for Western blot TRI reagent (MRC, Cincinnati, OH), using proce- analysis as described below. dures recommended