Regulation of Lens Fiber Cell Organization

描述（由申请人提供）：晶状体透明度是通过晶状体纤维细胞的高度有序组织、其独特的折射率以及纤维细胞中缺乏细胞器的组合而成为可能的。晶状体纤维细胞的高度有序排列对于正确的光传输至关重要，并且通过改变细胞与细胞相互作用来破坏这种结构可能会导致白内障。然而，调节晶状体纤维细胞相互作用的信号仍然很大程度上未知。我们的初步研究发现了一类新的分子，即调节晶状体细胞组织的 Eph 酪氨酸激酶受体家族。 Ephrin-A5（Eph 受体的配体）失活会导致 N-钙粘蛋白定位破坏、晶状体纤维细胞形状改变、晶状体细胞解体以及白内障的发生。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晶状体细胞组织。为了检验这一假设，我们将：（1）检查无肝配蛋白 A5 的晶状体的空间和时间特征，确定发育过程中晶状体缺陷首次发生的时间和地点，以及肝配蛋白 A5 的缺失是否会导致晶状体纤维细胞分化的破坏。将使用光学和电子显微镜技术来分析不同发育阶段的晶状体的形态。针对晶状体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发育中的晶状体中表达哪些特定 Eph 受体以及它们的表达位置，阐明 ephrin-A5 在晶状体发育中的受体机制。由于Eph受体和配体之间的相互作用导致双向信号传导，我们计划通过选择性失活不同受体结构域来分析晶状体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都需要。 (3) 研究ephrin-A5缺失小鼠中导致白内障的分子改变。初步研究表明，晶状体纤维细胞中 N-钙粘蛋白的分布受到破坏。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 功能和晶状体细胞粘附中的作用，我们还计划在表型拯救实验中检查 N-钙粘蛋白-2-连环蛋白融合蛋白的表达。拟议的研究将确定以前未被怀疑的分子家族在晶状体发育中的作用，并揭示 N-钙粘蛋白功能的新调控。这些研究将增强我们对如何调节晶状体细胞相互作用以确保晶状体透明度的理解，并提供对白内障发生机制的见解。公共卫生相关性：白内障是导致失明的主要原因。白内障发生的分子机制尚不完全清楚。拟议的研究将阐明肝配蛋白-A5信号传导缺陷导致白内障的分子机制，并为未来预防和治疗人类白内障提供见解。

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. PUBLIC HEALTH RELEVANCE: Cataracts are a leading cause of blindness. The molecular mechanisms underlie cataractogenesis are incompletely understood. The proposed studies will elucidate molecular mechanisms by which defects in ephrin-A5 signaling lead to cataracts and provide insights into future prevention and treatment of human cataracts.