Regulation of Lens Fiber Cell Organization

描述（由申请人提供）：透镜透明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确的光传输至关重要，细胞间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。我们的初步研究已经确定了一类新的分子，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）失活可导致n-钙粘蛋白定位的破坏，晶状体纤维细胞形状的改变，晶状体细胞的紊乱和白内障的发生。我们假设ephrin-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晶状体细胞的正常组织。为了验证这一假设，我们将：(1)检查ephrin-A5缺失晶状体的时空特征，确定晶状体缺陷在发育过程中首次发生的时间和地点，以及ephrin-A5的缺失是否会导致晶状体纤维细胞分化的中断。晶状体在不同发育阶段的形态将使用光学和电子显微镜技术进行分析。针对晶状体细胞分化标记物的抗体将用于免疫组织化学实验，研究分化标记物的表达。(2)利用Real-Time PCR、原位杂交和免疫组织化学技术，通过检测发育中的晶状体中表达的特异性Eph受体及其表达位置，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缺失小鼠白内障发生的分子改变。初步研究揭示了晶状体纤维细胞中n -钙粘蛋白分布的破坏。我们将确定ephrin-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-cadherin在介导ephrin-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测N-cadherin-2-catenin融合蛋白的表达。提出的研究将建立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 -钙粘蛋白功能的新调控。这些研究将增强我们对晶状体细胞相互作用如何被调节以确保晶状体透明度的理解，并为白内障发生的机制提供见解。公共卫生相关性：白内障是致盲的主要原因。白内障形成的分子机制尚不完全清楚。这些研究将阐明ephrin-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.