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V.I. Shestopalov, D. Ivanov, G. Dvoriantchikova, L. Nathanson; Microarray Analysis of the Fiber Cell Maturation in the Lens . Invest. Ophthalmol. Vis. Sci. 2005;46(13):1874.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: The mammalian lens consists of an aged core of quiescent cells enveloped by a layer of younger, continuously maturing fiber cells that are transcriptionally active.. Fiber cell maturation temporally coincides with formation of the core syncytium and commences within a thin layer of fiber cells after elongation is complete. To identify molecular pathways associated with these events, we utilized LCM microdissection and RNA amplification techniques to characterize changes in gene expression by differential microarray profiling. Methods: In this study, we compared global gene expression profiles in young elongating and maturing fiber cells to those that are fully elongated. These two sub–populations of fibers possess differential patterns of GFP labeling in the TgN(GFPU5)Nagy mouse strain, reflecting incorporation of maturing fibers into the core syncytium. One lens from each animal was used for LCM and RNA extraction; the lens from the contralateral eye was used to the define position of the GFP border. Methanol–fixed cells from both sub–populations were microdissected from paraffin–embedded lens sections using laser capture microdissection (LCM). Following two rounds of aRNA amplification on the LCM samples, gene expression profiling was performed using the 22K mouse oligo microarray. Microarray data from three independent dye–swap experiments were analyzed for differential gene expression. Results: The high quality of mRNA extracted from LCM microdissected lens tissue assured successful amplification, labeling, hybridization and reproducibility of microarray profiles. At a cut–off factor of 2, a total of 73 differentially expressed genes were identified. The group of 22 upregulated genes included Stx11, Gadd45b, Dlad and Pacsin3. The group of 51 downregulated genes included Tgfb2, Id1, Id2 and Pfkfb3. Changes in expression levels were confirmed by quantitative RT–PCR of randomly selected genes. Conclusions: We tested a novel technique of RNA probe preparation that allowed us to analyze ultra–small tissue samples in order to characterize closely spaced differentiation events. The changes in the profile of gene expression reflected a shift in cell physiology, characteristic of the beginning of fiber maturation process. Genes previously implicated in the syncytium formation were not detected in this study, suggesting that post–transcriptional regulation plays a major role.
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