): Due to its unique structure and function, the ocular lens depends absolutely on the lens fibers remaining interconnected to the surface epithelial cells by gap junctional intercellular communication pathways. Ionic homeostasis is essential in order to avoid cataract: precipitation of the high concentration of soluble proteins in the lens fiber cytoplasm. The long-term objectives of this application are to experimentally demonstrate the function of intercellular communication via gap junctions in lens development and homeostasis. Gap junctional intercellular channels are composed of connexins, a family of integral membrane proteins. Three connexins are expressed in the lens: connexin43 (Cx43), Cx46 and Cx50. Mouse lines are now available with specific deletions of each of these connexin genes. The Cx43 knock-out animals die at birth due to heart defects; the other two knock-out lines are viable and fertile, although unique and specific lens pathology results from each connexin deletion. Experiments are proposed to develop mouse lines in which Cx43 is specifically ablated in the lens by using the Cre/loxP system to remove the Cx43 gene. These animals will be bred with the Cx46 and Cx50 knock-out lines to produce double and triple knock-out animals, permitting an in-depth analysis of connexin function in lens development and homeostasis. As the Cx50 knock-out results in a small lens, the mechanisms underlying this decrease in the cell cycle will be experimentally investigated. First, the coding region of Cx46 will be """"""""knocked in"""""""" to the Cx50 locus. These animals will ostensibly produce the normal numbers of gap junctional channels, but the channels will be composed of only Cx46, thus lacking wild-type connexin diversity. If these animals show normal lenses, then an absolute number of channels is required to insure normal cell cycle timing. If not, then unique Cx50 channel properties are required. Cell cycle rates in wild-type and knock-out lenses will be measured with [3]H-thymidine and BrdU labeling, and the dividing cells correlated with connexin expression patterns. Primary cultures will be used to attempt to reconstruct altered cell cycle timing in vitro. Finally, a novel mutant of Cx50 has been identified which reveals dominant-negative activity when co-expressed with wild-type Cx50 in Xenopus oocyte pairs. A transgenic mouse line expressing this mutant driven by the alphaA-crystallin promoter will be developed and studied.
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