Connexin Molecular Biology in the Lens and Ciliary Epithelium

描述（由申请人提供）：项目摘要：目标#1。将在三种小鼠模型中研究Cx43在睫状上皮中的功能。在nestin-cre /Cx43 flox/flox系中，Cx43从睫状体上皮的色素（PE）层中选择性缺失，导致房水分泌减少和血浆蛋白回流到水室中。在GjaUrt细胞系中，睫状上皮中的Cx43免疫染色整体减少，并且还显示血浆蛋白回流。pax 6 α-cre/Cx43 flox/flox线显示非色素上皮（NPE）细胞的Cx43损失和眼内压降低。将使用光镜和电子显微镜、免疫组织化学、染料转移方法和测量小鼠眼内压来平行研究这三种模型。目标2。在出生后第一周，CxSOnull晶状体中的上皮细胞表现出有丝分裂指数的显著降低。由于Cx46取代Cx 50不能恢复正常生长速率，因此Cx 50必须提供与上皮细胞增殖相关的独特功能。我们将测试有关该函数的两个假设。首先，Cx 50通道表现出独特的选择性，允许细胞之间的调节信号的交换。第二个假设是，Cx 50，而不是Cx46，调节有丝分裂进程的C-末端结构域与细胞质蛋白的相互作用。为了区分通道特性和涉及连接蛋白胞质结构域的信号传导，我们将采用“通道死亡”Cx 50突变体，其正常地运输至质膜并组装成间隙连接斑块。具有这些特性的一种突变体已经被鉴定，其他的将被鉴定。将使用新型慢病毒方法将突变体作为转基因引入CxSOnull小鼠中。这种策略确保了快速测试多个突变基因的能力。如果这些突变体拯救了透镜生长，这将强烈暗示Cx 50的胞质结构域对于有丝分裂速率的调节至关重要。如果没有，细胞间通道的特性是关键，将通过引入无尾Cx 50突变体来测试有丝分裂速率的拯救。相关性：这些研究将探讨缝隙连接在眼内房水形成和透镜生长中的功能作用。他们将允许更好地了解青光眼和白内障的病理。

DESCRIPTION (provided by applicant): Project Summary: 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.