Aim #1. The function of Cx43 in the ciliary epithelium will be studied in three mouse models. In the nestin-cre /Cx43flox/flox line, Cx43 is selectively deleted from the pigmented (PE) layer of the ciliary epithelium resulting in a decrease in aqueous humor secretion and backflow of plasma proteins into the aqueous compartment. In the GjaUrt line, Cx43 immunostaining in the ciliary epithelium is globally reduced and also shows backflow of plasma proteins. The pax6alpha-cre/Cx43flox/flox line shows a loss of Cx43 from the non-pigmented epithelial (NPE) cells and a decrease in intraocular pressure. The three models will be studied in parallel using light and electron microscopy, immunohistochemistry, dye transfer methods and measurement of mouse intraocular pressure.
Aim #2. Epithelial cells in CxSOnull lenses exhibit a striking reduction in mitotic index during the first postnatal week. Since replacement of Cx50 with Cx46 does not restore the normal growth rate, Cx50 must provide a unique functionality relating to epithelial cell proliferation. We will test two hypotheses regarding that function. The first is that Cx50 channels exhibit unique properties of selectivity, permitting the exchange of regulatory signals between cells. The second hypothesis is that Cx50, but not Cx46, regulates mitotic progression by C-terminal domain interactions with cytoplasmic proteins. To distinguish between channel properties and signaling involving connexin cytoplasmic domains, we will employ """"""""channel-dead"""""""" Cx50 mutants that traffic normally to the plasma membrane and assemble into gap junctional plaques. One mutant with these properties is already characterized and others will be identified. The mutants will be introduced into CxSOnull mice as transgenes using a novel lentivirus approach. This strategy insures an ability to test multiple mutant genes very quickly. If these mutants rescue lens growth, this will strongly implicate the cytoplasmic domains of Cx50 as critical for the regulation of mitotic rate. If not, the properties of the intercellular channel are key and a rescue of mitotic rate will be tested by the introduction of tailless Cx50 mutants. Relevance: These studies will investigate the functional roles of gap junctions in the formation of aqueous humor and lens growth in the eye. They will permit a better understanding of the pathologies involved in glaucoma and cataract. ? ? ?
|Chai, Zhifang; Goodenough, Daniel A; Paul, David L (2011) Cx50 requires an intact PDZ-binding motif and ZO-1 for the formation of functional intercellular channels. Mol Biol Cell 22:4503-12|
|Magnotti, Laura M; Goodenough, Daniel A; Paul, David L (2011) Deletion of oligodendrocyte Cx32 and astrocyte Cx43 causes white matter vacuolation, astrocyte loss and early mortality. Glia 59:1064-74|
|Magnotti, Laura M; Goodenough, Daniel A; Paul, David L (2011) Functional heterotypic interactions between astrocyte and oligodendrocyte connexins. Glia 59:26-34|
|Hou, Jianghui; Goodenough, Daniel A (2010) Claudin-16 and claudin-19 function in the thick ascending limb. Curr Opin Nephrol Hypertens 19:483-8|
|Hou, Jianghui; Renigunta, Aparna; Gomes, Antonio S et al. (2009) Claudin-16 and claudin-19 interaction is required for their assembly into tight junctions and for renal reabsorption of magnesium. Proc Natl Acad Sci U S A 106:15350-5|
|Goodenough, Daniel A; Paul, David L (2009) Gap junctions. Cold Spring Harb Perspect Biol 1:a002576|
|Calera, Mónica R; Wang, Zhao; Sanchez-Olea, Roberto et al. (2009) Depression of intraocular pressure following inactivation of connexin43 in the nonpigmented epithelium of the ciliary body. Invest Ophthalmol Vis Sci 50:2185-93|
|Himmerkus, Nina; Shan, Qixian; Goerke, Boeren et al. (2008) Salt and acid-base metabolism in claudin-16 knockdown mice: impact for the pathophysiology of FHHNC patients. Am J Physiol Renal Physiol 295:F1641-7|
|Hou, Jianghui; Renigunta, Aparna; Konrad, Martin et al. (2008) Claudin-16 and claudin-19 interact and form a cation-selective tight junction complex. J Clin Invest 118:619-28|
|Calera, Monica R; Topley, Heather L; Liao, Yongbo et al. (2006) Connexin43 is required for production of the aqueous humor in the murine eye. J Cell Sci 119:4510-9|
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