The transparent cornea is made up of an anterior stratified epithelium, a collagenous stromal matrix containing fibroblasts called keratocytes, Descemet's membrane and a single-layered endothelium. We have put forth the refracton hypothesis attempting to relate cornea to lens. The cornerstone of this hypothesis is that the exceptionally abundant intracellular proteins in the cornea share properties with the multifunctional crystallins of the lens. Our current corneal research addresses the molecular basis for corneal-specific gene expression and the roles of the abundant, intracellular corneal proteins. For corneal-specific gene expression, we are continuing to investigate the mouse aldehyde dehydrogenase 3 (ALDH3), rabbit ALDH1 and zebrafish gelsolin promoter in both transfection and transgenic mouse experiments. In FY2002 we continued our studies showing that the corneal epithelial cells of Pax6+/- Small eye mice carrying two different mutations (SeyNeu or SeyDey) are poorly stratified and have reduced amounts of ALDH3, transketolase (TKT) and cytokeratin 12 (K12), all characteristic of mouse corneas. This indicates that high expression of these proteins in the corneal epithelium require proper amounts of Pax6 during development. The 4 kb ALDH3 promoter/CAT reporter gene (which comprises 1 kb 5? flanking region and the 3 kb intron 1 of the ALDH3 gene), shown earlier to be expressed specifically in corneal epithelial cells of transgenic mice, was stimulated 10-fold in co-transfection experiments with Pax6 in Cos 7 cells. Surprisingly, Pax5a, a Pax6 isoform, repressed promoter activation in co-transfection experiments. Thus, Pax6/5a ratios, which we show change during corneal development, may have a role in corneal ALDH3 expression in vivo. We also showed this year that corneal epithelial cell adhesion is reduced in the Small eye mice. This is associated with reductions in desmoglein, and in beta and gamma-catenins. We showed earlier in collaborative studies with Dr. James Jester (Southwestern Medical Center, Dallas, Texas) that ALDH1 is the principal protein in rabbit corneal keratocyes. It is also a major protein of the rabbit corneal epithelium. This year we showed that a 1 kb promoter fragment is active in cornea and, especially, in retina of transgenic mice. By contrast, a 3.5 kb promoter fragment is active principally in the cornea in transgenic mice. Current mutagenesis experiments suggest that XRE cis-control elements play a role in the regulation of the rabbit ALDH1 gene in the cornea. We showed earlier that gelsolin, an actin binding protein, comprises approximately half of the water-soluble protein of the zebrafish corneal epithelium, making it a putative corneal crystallin in this species. Morever, we showed by in situ hybridization that gelsolin undergoes a burst of expression at the blastula stage in zebrafish embryos, becomes expressed in the notocord, and then becomes concentrated in the eye. We established in FY2002 by microinjection of gelsolin-specific morpholino oligonucleotides to eliminate gelsolin expression that zebrafish gelsolin is required for dorsalization during embryogenesis. Ventralized embryos were rescued by coinjection of zebrafish gelsolin or chordin mRNAs, or human gelsolin protein. Moreover, injection of gelsolin mRNA or human gelsolin protein dorsalized the developing embryo, often resulting in axis duplication. These and other tests indicated that gelsolin, which is specialized for corneal expression in adults, also modulates embryonic dorsal/ventral pattern formation in zebrafish. We continued our studies reported last year on our ALDH3 null and TKT heterozygote mice in order to publish the results. In short, we established that ALDH3 null mice show no abnormal phenotype, and that there is no evidence for the appearance of a compensatory protein in the cornea. In addition, we showed that TKT null mice die in the early cleavage stages of development. However, the heterozygote TKT mice are viable and show no corneal abnormalities. Unexpectedly, the TKT mice were on average about 30% smaller than wild type mice. The liver, testis, ovary and, especially, fat tissue were proportionately smaller in the TKT+/- mice, but the size of the brain and kidney appeared normal. A dramatic loss of body fat is responsible for the reductions in size of the TKT heterozygous mice. TKT heterozygote females gave small litters when mated with wild type males. These unexpected dose-dependent TKT phenotypes have clinical relevance. From a corneal perspective, it remains baffling that such profound reductions and eliminations of soluble proteins and metabolic enzymes have no affect of corneal morphology or clarity. In FY2002 we explored global gene expression in 6-week-old and 9-day-old mouse corneas by making and analyzing SAGE libraries. 60,000 tags, each representing the expression of a cDNA, were sequenced from each stage. In short, the pattern of genes expressed is characteristic of the tissue and is in good agreement with what is known about the expression of corneal proteins. Numerous genes expressed in these libraries are not represented in published SAGE libraries from other tissues (e.g. retina, lymphocytes, etc.). Many genes were identified that either increase or decrease in expression between these two stages of corneal development. Approximately 60,000 tags were also sequenced from SAGE libraries made from the central or limbal region of the rat corneal epithelium. The genes encoding WDNM1 and marapsin are particularly interesting. Both are expressed significantly in the corneal limbus, however, the former is absent in the central corneal epithelium, while the latter is highly enriched in the limbus and poorly expressed in the central corneal epithelium or conjunctiva. These, then, are putative markers for corneal limbal stem cells. Many other interesting features of corneal gene expression presently under investigation were found in these experiments. Finally, we have continued our experiments attempting to obtain transgenic mice that express the Cre gene specifically in corneal epithelial cells in order to perform corneal-specific gene knockouts. We have used a -339/+44 aB-crystallin promoter/Pax6 enhancer to express the Cre gene in the cornea of transgenic mice. Transgenic lines have been obtained that express Cre in the corneal epithelium, and experiments are in progress to determine whether other tissues in the adult or embryo also express this transgene.
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