Regulation of Lens Fiber Cell Organization

描述(申请人提供)：晶状体透明是由晶状体纤维细胞高度有序的组织、其独特的折射率以及纤维细胞中缺乏细胞器的组合而成的。晶状体纤维细胞的高度有序排列对正常的光传输至关重要，细胞-细胞相互作用的改变破坏了这种结构，很可能导致白内障。然而，调节晶状体纤维细胞相互作用的信号在很大程度上仍然未知。我们的初步研究发现了一类新的分子，Eph酪氨酸激酶受体家族，它调节晶状体细胞的组织。Eph受体的配体EPhin-A5失活会导致N-钙粘蛋白定位中断、晶状体纤维细胞形态改变、晶状体细胞结构紊乱，进而导致白内障的发生。我们推测，肾上腺素A5与其受体(S)相互作用，调节N-钙粘附素介导的纤维细胞黏附，以维持适当的晶状体细胞结构。为了验证这一假设，我们将：(1)检查ewitin-A5缺失晶状体的空间和时间特征，确定在发育过程中第一次出现晶状体缺陷的时间和地点，以及ewitin-A5的丢失是否会导致晶状体纤维细胞分化中断。将使用光学和电子显微镜技术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)通过实时定量聚合酶链式反应、原位杂交和免疫组织化学的方法，检测Eph受体在晶状体发育过程中的表达情况，阐明Ephin-A5受体在晶状体发育中的作用机制。由于Eph受体和配体之间的相互作用导致双向信号转导，我们计划通过选择性失活不同的受体结构域来分析晶状体发育是否需要受体介导的信号、配体介导的反向信号或两者兼而有之。(3)研究eaffin-A5基因缺失小鼠白内障发生的分子机制。初步研究表明，N-钙粘附素在晶状体纤维细胞中的分布受到干扰。我们将确定eaffin-A5受体是否与黏附连接分子发生物理作用，并分析该配体对N-钙粘素功能的影响。为了严格评估N-钙粘素在调节ePhin-A5功能和晶状体细胞黏附中的作用，我们还计划在表型救援实验中检测N-钙粘素-2-连环蛋白融合蛋白的表达。这项拟议的研究将确定一个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-钙粘附素功能的新调节。这些研究将加深我们对晶状体细胞相互作用如何调节以确保晶状体透明度的理解，并为白内障的发生机制提供见解。与公共卫生相关：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。这项拟议的研究将阐明ewitin-A5信号缺陷导致白内障的分子机制，并为未来人类白内障的预防和治疗提供见解。

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.