KERATIN 12-DEFICIENT MICE HAVE FRAGILE CORNEAL EPITHELIA

Purpose. Expression of the K3-K12 keratin pair characterizes the corne al epithelial differentiation. 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 al lele of murine Krt1.12 gene was ablated in the embryonic stem cell lin e, E14.1, by homologous recombination with a DNA construct in which th e DNA element between intron 2 and exon 8 of the keratin 12 gene was r eplaced by a neogene. The homologous recombinant embryonic stem cells were injected to mouse blastocysts, and germ lines of chimeras were ob tained, The corneas of heterozygous and homozygous mice were character ized by clinical observations using stereomicroscopy, histology with l ight and electron microscopy. Western immunoblot analysis, immunohisto chemistry, in situ hybridization, and Northern hybridization. Results. The heterozygous mice (+/-) one allele of the Krt1.12 gene appear nor mal and do not develop any clinical manifestations (e.g., corneal epit helial defects). Homozygous mice (-/-) develop normally and suffer mil d corneal epithelial erosion, Their corneal epithelia are fragile and can be removed by gentle rubbing of the eyes or brushing with a Micros ponge. The corneal epithelium of the homozygote (-/-) does not express keratin 12 as judged by immunohistochemistry, Western immunoblot anal ysis with epitope-specific anti-keratin 12 antibodies, Northern hybrid ization with P-32-labeled keratin 12 cDNA, and in situ hybridization w ith 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 b asal and suprabasal cells. The number of keratin intermediate filament s in basal and suprabasal corneal epithelial cells in -/- mice decreas es, and they appear as dense bundles. This morphology is similar to th at of keratin intermediate filaments in epidermal epithelial cells but differs from that of normal corneal epithelial cells in which the ker atins form fine filamentous networks. The superficial epithelial cells are devoid of keratin intermediate filaments and often detach from th e corneal surface of -/- mice. Conclusions. The presence of cornea-spe cific K3-K12 keratin pairs is essential for the maintenance of corneal epithelium integrity.