Connexin Molecular Biology in the Lens and Ciliary Epithelium

项目概述：目标#1。我们将在三种小鼠模型中研究Cx43在睫状上皮中的功能。在巢蛋白-cre /Cx43flox/flox系中，Cx43被选择性地从睫状上皮的色素层（PE）中删除，导致房水分泌减少，血浆蛋白回流到房水室。在GjaUrt系中，睫状体上皮的Cx43免疫染色整体降低，也显示血浆蛋白回流。pax6alpha-cre/Cx43flox/flox细胞系显示非色素上皮（NPE）细胞中Cx43缺失，眼压降低。这三种模型将同时使用光学和电子显微镜、免疫组织化学、染料转移方法和小鼠眼压测量进行研究。目标# 2。CxSOnull晶状体上皮细胞在出生后第一周有丝分裂指数显著降低。由于用Cx46替代Cx50不能恢复正常的生长速率，因此Cx50必须提供与上皮细胞增殖有关的独特功能。我们将测试关于该函数的两个假设。首先，Cx50通道表现出独特的选择性，允许细胞之间交换调节信号。第二种假设是Cx50，而不是Cx46，通过与细胞质蛋白的c端结构域相互作用调节有丝分裂进程。为了区分通道特性和涉及连接蛋白细胞质结构域的信号传导，我们将使用“通道死亡”的Cx50突变体，这些突变体通常运输到质膜并组装成间隙连接斑块。一个具有这些特性的突变体已经被鉴定出来，其他的将被鉴定出来。这些突变体将使用一种新的慢病毒方法作为转基因引入CxSOnull小鼠。这种策略确保了快速测试多个突变基因的能力。如果这些突变体挽救晶状体生长，这将强烈暗示Cx50的细胞质结构域对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，有丝分裂率的拯救将通过引入无尾Cx50突变体进行测试。相关性：这些研究将探讨眼内间隙连接在房水形成和晶状体生长中的功能作用。它们将有助于更好地了解青光眼和白内障的病理情况。

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.