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Tharkika Nagendran, Rupalatha Maddala, Vasanth Rao; Double deficiency of Rap1A and B reveals a crucial role for Rap1 in cell adhesive interactions and cell survival, plasticity, polarity and migration events during lens morphogenesis. Invest. Ophthalmol. Vis. Sci. 2013;54(15):3193.
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© ARVO (1962-2015); The Authors (2016-present)
Lens development, differentiation and cytoarchitecture are dependent on temporally and spatially regulated cell-cell and cell-extracellular matrix based adhesive interactions. However, the molecular mechanisms regulating different cell adhesive interactions during lens development and maturation are not completely clear. Here we explored the role of Rap1 GTPase in lens development, architecture and function by targeting the expression of both Rap1A and B simultaneously in a lens-specific manner.
Since the deficiency of Rap1A or Rap1B individually did not result in defects of lens morphogenesis and function, we evaluated the impact of a lens-specific double knock down approach using the Rap1A and B double floxed mice and Le-Cre transgenic mice. The Rap1A and B double conditional knockdown mouse lenses generated were characterized from the embryonic stage E11.5 to P1 using histological, immunofluorescence and biochemical analyses.
Both Rap1A and B are expressed abundantly in mouse lens epithelium and fibers, as confirmed by cDNA microarray, q-PCR and immunofluorescence analyses. Rap1 is distributed predominantly to the lens membrane fraction and localized to the cell-cell junctions. Double deficiency of Rap1A (70% reduction) and Rap1B (45% reduction) caused a severe lens phenotype starting from E11.5. Many of the Rap1a/b deficient P1 eyes were microphthalmic with characteristic defects in the ocular lens. Adult mice exhibited completely degenerated and microphthalmic eyes. Histological analysis of Rap1a/b-cKO mouse lenses revealed delayed lens vesicle closure, loss of E-cadherin, ZO-1, increased apoptosis and expression of alpha-smooth muscle actin in the lens epithelium. Some lenses with intact lens epithelium exhibited distinct abnormalities in primary fiber cell polarity, migration with reduced levels of N-cadherin, integrin-1 and beta-catenin. However, lens fiber cell differentiation was found to be normal based on the distribution and expression profile of fiber cell differentiation markers.
Taken together, these data uncover a vital role for Rap1 GTPase in regulating lens epithelial and fiber cell adhesive interactions, survival, transdifferentiation, polarity and migration which are essential for lens morphogenesis, architecture and function.
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