Role of primary cilia in corneal development and repair

描述(申请人提供)：角膜的透明度、结构完整性和屏障功能取决于其复杂、分层的组织结构和填充它的细胞的功能。这种组织是通过在开发过程中发生的许多严格控制的形态发生事件来实现的。由于创伤、疾病或衰老导致的角膜层损伤和异常修复可能会损害角膜透明度，并导致失明。我们以前已经证明，初级纤毛在角膜内皮(CE)的组织中起着形态发生的作用。我们的初步数据表明，初级纤毛通过组织角膜内皮细胞(CEC)的微管细胞骨架和细胞内极性参与CE的修复，CEC参与了损伤后Descemet膜覆盖的重建。我们现在已经将这些观察扩展到其他类型的角膜细胞。我们发现睫状体的组装/解体与角膜发育的特定形态发生步骤之间存在相关性。我们已经检测到三种角膜细胞类型中初级纤毛的超微结构差异，这表明它们具有不同的功能。我们的角膜上皮修复实验表明，与完整角膜相比，损伤区域新迁移的角膜上皮中有更多的纤毛细胞。我们还检测到在角膜上皮损伤时，角膜周边的角膜上皮细胞簇中纤毛介导的通路Hedgehog(HH)激活。这一建议解决了主要假设，即角膜初级纤毛是在发育和修复过程中正常形成角膜图案所需的形态发生细胞器。在目标1中，我们将阐明纤毛蛋白在角膜发育和睫状体病变相关的ASD中的作用。首先，我们将明确纤毛组装、大小和定位如何与小鼠角膜发育过程中特定的形态发生过程相关，我们将从遗传学上剖析初级纤毛在胚胎不同的角膜细胞群中的作用，最后我们将测试纤毛是否通过Gprcr48控制ASD基因Pitx2的表达。在目标3中，我们将通过诱导体内损伤来阐明CE纤毛在正常和IFT条件突变小鼠修复过程中的作用。我们将通过慢病毒介导的基因过度表达或RNAi敲除来评估特定纤毛介导的途径在体内愈合过程中对纤毛组装/拆卸的影响。在目标3中，我们将通过评估条件睫毛基因敲除小鼠与对照动物相比上皮伤口闭合的速度来确定初级纤毛在角膜上皮修复中的作用。我们将研究角膜缘附近的角膜上皮细胞中损伤激活的HH信号的性质，并将使用假定的角膜缘上皮干细胞“标记物”来确定表达纤毛的细胞是否保持干细胞特性。这项研究将阐明所需的信号通路 对于正确的角膜细胞分化和模式，将导致识别新的治疗靶点，以对抗衰老和创伤性角膜细胞丢失，但也可能对理解其他组织中的复杂形态发生事件具有更广泛的意义。

DESCRIPTION (provided by applicant): Transparency, structural integrity and barrier function of the cornea depends on its complex, layered organization and the function of the cells that populate it. This organization is achieved through a number of tightly controlled morphogenetic events occurring during development. Damage and abnormal repair of the corneal layers due to trauma, diseases or aging can compromise corneal transparency and lead to blindness. We have previously shown that the primary cilium plays a morphogenetic role in the organization of the corneal endothelium (CE). We have preliminary data showing that the primary cilium is involved in CE repair by organizing the microtubular cytoskeleton and intracellular polarity of corneal endothelial cells (CEC) involved in re-establishment of Descemet's membrane coverage after injury. We have now extended these observations to the other corneal cell types. We have found a correlation between ciliary assembly/disassembly and specific morphogenetic steps of the corneal developmental. We have detected ultrastructural differences of the primary cilia in the three corneal cell types that suggest distinct functions. Our experiments in corneal epithelium repair show a higher number of ciliated cells in newly migrated corneal epithelium on the injured area compared to that observed in intact corneas. We have also detected an activation of the cilia-mediated pathway hedgehog (Hh) in clusters of corneal epithelial cells at the periphery of the cornea upon epithelial injury. This proposal addresses the main hypothesis that the corneal primary cilia are morphogenetic organelles required for normal patterning of the cornea during development and repair. In aim 1 we will elucidate the role of ciliary proteins in corneal development and ciliopathy-related ASD. First, we will pinpoint how ciliary assembly, size and localization correlate to specific morphogenetic processes during development of the mouse cornea, we will genetically dissect the role of primary cilia in embryologically distinct groups of corneal cells and finally we will test whether cilia control the expression of the ASD gene Pitx2, through Gprcr48. In aim 3 we will elucidate the role of the CE cilium during repair in normal and IFT conditional mutant mice by inducing in vivo injuries. We will assess the effect of specific cilia-mediated pathways on cilia assembly/disassembly during healing in-vivo via lentiviral-mediated gene over expression or RNAi knockdown. In aim 3 we will determine the role of primary cilia in corneal epithelium repair by assessing the speed of epithelial wound closure in conditional ciliary knockout mice compared to that of control animals. We will examine the nature of injury-activated Hh signaling in corneal epithelial cells in proximity of the limbus and we will use putative limbal epithelial stem cell "markers" to determine whether the cells expressing cilia maintain stem cell identity. This study will elucidate signaling pathways required for proper corneal cell differentiation and patterning and will lead to the identification of new therapeutic targets to counteract aging and traumatic corneal cell loss, but may also have wider implications for understanding complex morphogenetic events in other tissues.