The transparent cornea is made up of an anterior stratified epithelium, a collagenous stromal matrix containing fibroblasts called keratocytes, Descemets membrane and a single-layered endothelium. While the extracellular stroma has been studied intensively with respect to corneal transparency, relatively little attention has been given to the molecular basis for transparency of the corneal cells. Experiments have shown that corneal epithelial cells, like lens cells, contain unexpectedly high concentrations of a few metabolic enzymes. In the mouse, aldehyde dehydrogenase 3 (ALDH3) and transketolase (TKT) comprises approximately 30% of the water-soluble protein of the corneal epithelium, an amount reminiscent of individual lens crystallins. In the last few years we have cloned the mouse ALDH3 cDNA and the 5 flanking region of the gene. Last fiscal year we used transgenic mouse experiments to identify a 4 kbp sequence composed of 1 kbp of 5 flanking region, exon 1 and intron 1 of the ALDH3 gene that directs the chloramphenicol acetyltransferase (CAT) gene selectively to the corneal epithelium (with minor expression in the liver). This was the first corneal specific promoter fragment that can function with tissue specificity in transgenic mice and opened the door to molecular analyses of corneal development and disease. The TKT gene is also expressed at least 50 times more highly in the cornea than in other tissues, although, unlike ALDH3, it is ubiquitously expressed throughout the body. In contrast to the ALDH3 promoter fragment, various TKT promoter fragments were unable to direct corneal gene expression in transgenic mice. The TKT and ALDH3 genes have numerous inducible cis-control consensus sequences in their 5flanking regions consistent with the possibility that induction by the environment (i.e. uv light) contributes significantly to their high expression in the cornea. This fits with our earlier observations that ALDH3 and TKT expression are minimal in the mouse corneal epithelial cells until after eye opening. Moreover, corneal TKT was induced several fold by exposing mice raised in the dark for 25 days after birth to light and in explanted newbborn mouse eyes exposed to light. The use of inductive mechanisms involving the environment differs from the strictly developmental control processes used by the lens for the high, preferential expression of crystallin genes. One of the major objectives this fiscal year has been to create ALDH3 and TKT null mice by homologous recombination. At present ES cells have been obtained that replace one of their ALDH3 or TKT alleles with a neomycin resistant gene. Chimeric mice were made and are being bred. One 4-week-old F1 ALDH3 hemizygote male mouse appears to have clear corneas. No F1 TKT hemizygote mice have been obtained yet. The ultimate purpose of these knockout experiments is to test whether the physical abundance of these enzymes is required for corneal transparency as crystallins are presumably necessary for transparency of the lens. This year the mouse ALDH3 promoter fragment that we identified last year has been used to direct TGFbeta and cdk5 to the cornea in transgenic mice. Mice have been obtained and are in the process of being analyzed. Last year we showed that at least 50% of the water-soluble protein in the zebrafish cornea appeared to begelsolin, a protein known to bind and cleave actin filaments. This was exciting since it suggested that gelsolin is a new corneal crystallin due to its abundance and since a gelsolin point mutation in humans hasbeen associated with lattice type II corneal dystrophy. We have now established by northern and in situ hybridization tests that this protein is intracellular gelsolin by cDNA cloning and sequencing, and that it accumulates in the epithelial cells of the zebrafish cornea. We have also shown by phalloidin staining that the abundant supply of actin in zebrafish corneal epithelial cells is not polymerized as F-actin as it is inthe mouse cornea, indicating that the corneal gelsolin is active in cleaving actin filaments. These results suggest that the abundant gelsolin and gelsolin: actin complexes accumulate as soluble protein in the zebrafish cornea, consistent with their being analagous to lens crystallins Preliminary southern blot experiments suggest that there is a single gelsolin gene in the zebrafish, and this is in the process of being cloned. Last fiscal year we startedto develop mouse corneal pithelial cell lines to use for transfection experiments for the analysis ofcorneal-specific gene expression. The approach was to use the immortomouse whose cells contain a gamma-interferon- inducible transgene encoding a temperature sensitive simian virus 40 T-antigen. We have now cloned corneal epithelial cells from these transgenic mice that grow considerably more rapidly at 33 degrees in the presence of gamma-interferon than at 37 degrees without gamma- interferon. We have concentrated on one cell line, called A6(1), and have shown that ALDH3 mRNA and enzyme activity are significantly higher at 37 than at 33 degrees, consistent with the idea that these cells are differentiating into corneal epithelial cells at the higher temperature. However, TKT mRNA and enzyme activity as well as keratin 12 mRNA content, both markers of corneal epithelial cell differentiation, are comparable at the two temperatures. By contrast, alpha-enolase, considered a marker for epithelial stem cells, has less mRNA and enzyme activity levels at 37 than at 33 degrees. Differential display experiments have also been performed this fiscal year and these have identified some genes whose expression is increased and others whose expression is lowered in the cells when the temperature is elevated from 33 to 37 degrees. Preliminary experiments indicate that the 4 kbp ALDH3 promoter will function in the A6(1) cells that have been transfected by electroporation. We anticipate that this cell line will be useful for examining the molecular basis for corneal-specific gene expression. Previous experiments have identified a 208 base pair promoter fragment from the mouse alphaB-crystallin gene that functions specifically in the lens of transgenic mice. Last fiscal year we showed that this lens-specific promoter region also functions in the corneal epithelial cells of the transgenic mice. This fiscal year we have obtained additional information supporting the low expression of this promoter fragment specifically in the corneal epithelium as well as the lens. This work is being prepared for publication. Finally, in collaborative experiments with Dr. James Jester (University of Texas Southwestern Medical Center) we showed last fiscal year that ALDH1 and TKT comprise over 30% of the water-soluble protein of the transparent rabbit corneal keratocytes. The keratocytes became reflective and specifically lost more than half of their ALDH1 and TKT after freeze-injury, a situation that persisted for several weeks and was not associated with dying cells. This year we have shown that the fibroblasts from the opaque sclera do not accumulate these two enzymes as do the transparent the keratocytes. The abundance of ALDH1 and TKT in the keratocytes exceeds their expected role for catalysis per se and extends the concept of enzyme-crystallins to these fibrocytes. This work was published this fiscal year.
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