The long term objective of this application is to provide a better understanding of the role of keratocyte-specific keratan sulfate proteoglycan (KSPG), keratocan, in corneal function and the role of keratocan during development and in the maintenance of corneal homeostatis. Failure to have normal developmental process of the cornea will result in diseases such as corneal dystrophy. Keratocan (Ktcn), lumican (Lum), and mimecan belong to the small leucine-rich proteoglycan (SLRP) gene family. They are major components of extracellular KSPG in vertebrate corneal stroma. It has been suggested that corneal KSPGs modulate collagen fibrillogenesis and thus contribute to the corneal transparency.
The specific aim 1 is to test the hypothesis by generating Ktcn-null mice via gene-targeting and examining the phenotypic changes in Ktcn-/- cornea. The mouse Ktcn is specifically expressed in keratocytes. In embryos, the Ktcn expression tracks the neural crest cells migrating during morphogenesis of tissue such as corneal stroma, limb and diaphragm. We hypothesize that keratocyte lineage plays a pivotal role in corneal morphogenesis and in the maintenance of corneal function.
The specific aim 2 is to elucidate the molecular basis of keratocyte-specific gene expression and to characterize keratocyte lineage. To achieve this goal, the investigator has shown that the 3.2 kb Ktcn can direct a foreign gene (beta-geo) expression specifically to keratocytes in adult transgenic mice (Tg).
Aim 2. 1 is to further define the keratocyte-specific cis-regulatory element within the 3.2 kb promoter by a series of Ktcn promoter deletion mutants using transgenic mice.
Aim 2. 2 is to confirm the cis-element with DNase I footprinting and electrophoretic mobility shift assay. The Ktcnpr3.2-betageobpA Tg allow us to trace keratocytes via X-gal staining.
The specific aim 3 is to use this Tg as a model to study cellular responses of corneal keratocytes, conjunctival keratocytes, and scleral fibroblasts during wound healing.
The specific aim 4 is to test the role of keratocytes during development and in the epithelium-mysenchyme interactions by genetic ablation of keratocytes in a tetracycline inducible Ktcnpr3.1-rtTA/tetO-DT-A Tg model.
Aim 4. 1 is to ablate the periocular neural crest cells during corneal morphogenesis and to examine the consequences on corneal and lens morphogenesis.
Aim 4. 2 is to ablate the keratocytes in adult animals, thus epithelium and endothelium response to the keratocyte cell death can be elucidated.
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