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In order to study the determination of corneal epithelial cells, rabbit corneal epithelium of 12- to 24-day embryos, newborn and 12-day-old offspring were recombined with mouse embryo upper-lip or dorsal dermis. Epithelial differentiation was analyzed using immunohistology with corneal monospecific monoclonal antibody AK12 (anti-keratin K12). Recombinants involving 12-day embryo undifferentiated corneal ectoderm formed a typical epidermis with hair follicles after 10 days of culture on the chick chorioallantoic membrane. Central corneal epithelium from 23- to 24-day embryos and newborn, which express suprabasally both K3 and K12 keratins and basally the K12 keratin alone, when grown in association with trichogenic dermis first failed to produce K12 in its new forming basal layer and then stratified after 11 days of culture above a differentiating epidermis with hair buds. When the culture period was increased up to 21 days by grafting under the kidney capsule from athymic mice, even the central corneal epithelium of 12-day-old off-spring gave rise to a complete epidermis with emerging hairs. The vibrissal- or pelage-type of hairs was in conformity with the regional origin of the mouse dermis. The species origin of the epithelial cells of the recombinants was discriminated incontestably using the Hoechst staining of interphase nuclei. Thus, the rabbit corneal epithelial cells can transdifferentiate into epidermal keratinocytes and trichocytes at least until 12 days after birth, despite the fact that from the fifth postnatal day the cells of its basal layer express both the K3 and the K12 keratins, a keratin pair marker of corneal cell-type terminal differentiation.

Corneal epithelial cells synthesize an acidic (55 kDa) K12 and a basic (64 kDa) K3 keratin as their major differentiation products during an advanced stage of differentiation. In this paper, we describe the cDNA cloning of rabbit K12 keratin. We used a 36 base pairs (bp) oligonucleotide corresponding to a consensus sequence of many known acidic keratins as a probe to screen a cDNA library of normal rabbit corneal epithelium. Several partial cDNA clones were isolated. Hybrid-selection showed that the 3'keratin chain-specific portion of the cDNA hybridizes with K12 mRNA. A rabbit antiserum raised against the C-terminus of the cDNA-deduced amino acid sequence recognizes, in immunoblotting, the K12 keratin. In situ hybridization showed that K12 mRNA is present in all cell layers of central corneal epithelium, but in only the suprabasal cells of limbal epithelium indicating a parallel expression pattern between K12 and K3. Cultured rabbit corneal epithelial cells initially synthesize K14/K5 keratins, but later when the cells become heavily stratified they synthesize large quantities of K12 and K3 mRNAs, as detected by Northern blotting. Cultured esophageal epithelial cells do not make K12 mRNA confirming the tissue-specificity of K12 expression. Although it has been suggested that conjunctival epithelial cells can trans-differentiate into a bona fide corneal epithelium, we showed here that cultured conjunctival cells do not synthesize significant amounts of K12/K3 mRNAs. These results strongly suggest that conjunctival epithelial cells, whose differentiation can be modulated significantly by the extracellular matrix, form a lineage intrinsically distinct from the corneal/limbal epithelial lineage.

Keratins form the intermediate filaments in almost all epithelial cells in vertebrates. The keratins of the rabbit corneal epithelium have previously been shown to be composed of a major keratin pair, formed by an acidic keratin K12 and a basic keratin K3, together with a minor keratin pair (acidic K14 and basic K5). Using electrophoretic and immunological analyses with two monoclonal antibodies, AK12 and AE5, that are monospecific for K12 and K3 respectively, we showed that the acidic keratin K12 is highly conserved throughout all the species studied except the trout, whereas the basic keratin K3 shows interspecies variations and was unequivocally identified only in mammals. Furthermore, in dog and human cornea K3 is present in small amounts compared with K12, while in mouse it is undetectable, suggesting that, in those species, the acidic keratin K12 may interact with the basic keratin K5 to form the corneal intermediate filaments.

Sequential expression of the K3/K12 keratin pair was studied during epithelial corneal differentiation, using monoclonal antibodies coupled with electrophoretic analysis. In the chick embryo, K12 appears on day 12, while K3 is present at least from day 11. By contrast, in the rabbit embryo, the expression of K12 (at 17 days) precedes that of K3 (at 21 days). In the adult rabbit, K3 is expressed without K12 in part of the limbus. Thus, within the corneal-type keratin pair, either the acidic or the basic partner appears first, according to the developmental stage or the species.