Regulation of Lens Fiber Cell Organization

描述（由申请人提供）：透镜的透明性是由透镜纤维细胞的高度有序组织、其独特的折射率和纤维细胞中细胞器的缺乏的组合而实现的。透镜纤维细胞的高度有序排列对于适当的光传输是至关重要的，并且通过细胞-细胞相互作用的改变而破坏这种结构可能导致白内障。然而，调节透镜纤维细胞相互作用的信号在很大程度上仍然未知。我们的初步研究已经确定了一类新的分子，Eph酪氨酸激酶受体家族，调节透镜细胞的组织。肝配蛋白-A5（Eph受体的配体）的失活导致N-钙粘蛋白定位的破坏、透镜纤维细胞形状的改变、透镜细胞的解体和白内障的发展。我们假设肝配蛋白A5与其受体相互作用，调节N-钙粘蛋白介导的纤维细胞粘附，以维持适当的透镜细胞组织。为了检验这一假设，我们将：（1）检查肝配蛋白-A5-空透镜的空间和时间特征，确定在发育过程中何时何地首次出现透镜缺陷，以及肝配蛋白-A5的缺失是否导致透镜纤维细胞分化的中断。将使用光学和电子显微镜技术分析不同发育阶段的透镜形态。针对透镜细胞分化标志物的抗体将用于免疫组织化学实验以研究分化标志物的表达。(2)通过使用实时PCR、原位杂交和免疫组织化学检查哪种特异性Eph受体在发育中的透镜中表达以及它们在何处表达，阐明ephrin-A5在透镜发育中的受体机制。由于Eph受体和配体之间的相互作用导致双向信号传导，我们计划使用不同受体结构域的选择性失活来分析透镜发育是否需要受体介导的信号传导、配体介导的反向信号传导或两者。(3)研究导致ephrin-A5缺失小鼠白内障的分子改变。初步研究表明，在透镜纤维细胞中的N-钙粘蛋白分布的破坏。我们将确定肝配蛋白A5受体是否与粘附连接分子发生物理相互作用，并分析配体对N-钙粘蛋白功能的影响。为了严格评估N-钙粘蛋白在介导肝配蛋白-A5功能和透镜细胞粘附中的作用，我们还计划在表型拯救实验中检测N-钙粘蛋白-2-连环蛋白融合蛋白的表达。拟议的研究将建立一个以前未被怀疑的分子家族在透镜发育中的作用，并揭示N-钙粘蛋白功能的新规定。这些研究将加深我们对透镜细胞相互作用如何调节以确保透镜透明度的理解，并为白内障发生机制提供见解。

DESCRIPTION (provided by applicant): Lens transparency is made possible by a combination of the highly ordered organization of the lens fiber cells, their unique refractive index, and the lack of organelles in the fiber cells. The highly ordered arrangement of the lens fiber cells is critical for proper light transmission, and disruption of this structure by alterations of cell-cell interactions is likely to lead to cataracts. However, signals that regulate lens fiber cell interaction remain largely unknown. Our preliminary studies have identified a new class of molecules, the Eph tyrosine kinase receptor family that regulates lens cell organization. Inactivation of ephrin-A5, a ligand of the Eph receptors, leads to the disruption of N-cadherin localization, change in lens fiber cell shape, disorganization of lens cells, and the development of cataracts. We hypothesize that ephrin-A5, interacting with its receptor(s), regulates N-cadherin-mediated fiber cell adhesion to maintain proper lens cell organization. To test this hypothesis, we will: (1) Examine the spatial and temporal characteristics of the ephrin-A5-null lens, determine when and where during development the lens defects first occur, and whether the loss of ephrin-A5 results in disruptions of lens fiber cell differentiation. The morphology of the lens at different developmental stages will be analyzed using both light and electron microscope techniques. Antibodies against markers of lens cell differentiation will be used in immunohistochemical experiments to study the expression of differentiation markers. (2) Elucidate receptor mechanisms of ephrin-A5 in lens development by examining which specific Eph receptors are expressed in the developing lens and where they are expressed, using Real-Time PCR, in situ hybridization, and immunohistochemistry. Since the interaction between Eph receptors and ligands leads to bidirectional signaling, we plan to analyze whether receptor- mediated signaling, the ligand-mediated reverse signaling or both are required for lens development using selective inactivation of different receptor domains. (3) Study the molecular alterations that lead to cataracts in ephrin-A5-null mice. Preliminary studies have revealed a disruption of N-cadherin distribution in the lens fiber cells. We will determine whether ephrin-A5 receptors interact physically with adherens junction molecules, and analyze effects of the ligand on N-cadherin functions. To critically evaluate roles of N-cadherin in mediating ephrin-A5 function and lens cell adhesion, we plan also to examine expression of a N-cadherin-2-catenin fusion protein in a phenotypic rescue experiment. The proposed studies will establish roles of a previously unsuspected family of molecules in lens development and reveal novel regulations of N-cadherin functions. These studies will enhance our understanding of how lens cell interaction is regulated to ensure lens transparency and provide insights into the mechanisms of cataractogenesis.