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.
Damage of the cornea due to trauma, diseases or aging followed by abnormal repair of corneal tissues can compromise corneal transparency and lead to blindness. 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 pathological and traumatic corneal cell loss, but may also have wider implications for understanding complex morphogenetic events in other tissues.
|Grisanti, Laura; Revenkova, Ekaterina; Gordon, Ronald E et al. (2016) Primary cilia maintain corneal epithelial homeostasis by regulation of the Notch signaling pathway. Development 143:2160-71|
|Want, Andrew; Gillespie, Stephanie R; Wang, Zheng et al. (2016) Autophagy and Mitochondrial Dysfunction in Tenon Fibroblasts from Exfoliation Glaucoma Patients. PLoS One 11:e0157404|
|Balmer, Sophie; Dussert, Aurore; Collu, Giovanna M et al. (2015) Components of Intraflagellar Transport Complex A Function Independently of the Cilium to Regulate Canonical Wnt Signaling in Drosophila. Dev Cell 34:705-18|
|Gentile, Ronald C; Iomini, Carlo (2015) Beating Cilia and Whipping Flagella: More Than Meets the Eye. J Pediatr Ophthalmol Strabismus 52:139-40|
|Luo, Na; Conwell, Michael D; Chen, Xingjuan et al. (2014) Primary cilia signaling mediates intraocular pressure sensation. Proc Natl Acad Sci U S A 111:12871-6|
|Aguilar, Andrea; Becker, Lars; Tedeschi, Thomas et al. (2014) ?-tubulin K40 acetylation is required for contact inhibition of proliferation and cell-substrate adhesion. Mol Biol Cell 25:1854-66|