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C. Liu, M. Azhar, T.C. Doetschman, B.J. Aronow, S. Kong, W.W. Kao; Gene Expression Profiling of the Mouse Eye Lacking Tgfb2 . Invest. Ophthalmol. Vis. Sci. 2003;44(13):410.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose. Transforming growth factor beta 2 (TGFß2) plays a pivotal role in mouse eye development. Mice lacking Tgfb2 reveal profound malformations including thin corneal stroma, absence of corneal endothelium, fusion of cornea to lens, and accumulation of hyaline cells in vitreous during embryonic eye morphogenesis. To understand possible mechanism underlying the pathogenesis of the Tgfb2-/- eye, we compared ocular gene expression profile between Tgfb2-/- and Tgfb2+/- using oligonucleotide probe arrays. Methods. Embryonic eyes (E16.5) of Tgfb2+/- and Tgfb2-/- were pooled, respectively, and the total RNA were isolated. Total RNA samples were used to make biotinylated RNAs for gene chip hybridization. Each labeled RNA was sequentially hybridized to GeneChip probe arrays (murine genome U74Av2) representing 15099 genes from Affymetrix. Scanning of probe arrays and data analysis was performed according to standard Affymetrix protocols. Genes which were found statistically significant and Tgfb2-/-/Tgfb2+/- expression levels over 1.4X (301 genes) and under 0.5X (293 genes) were filtered and pooled to generate 594-genelist of regulated genes. Real-time RT-PCR, Northern blotting, and immunohistochemistry were used to validate the microarray data. Results. Tgfb2 expression is severely down-regulated in the Tgfb2-/- eye indicating the reliability of the microarray analysis. In the Tgfb2-/- eye, genes related to corneal ECM component i.e., collagens (types I, V, VI, XI, and XIV) and leucine-rich proteoglycans (keratocan, lumican, mimecan and fibromodulin) are down-regulated. Major intrinsic lens calpain protease genes such as calpain lp82 (lens-specific) and calpain3 are also down-regulated. Real-time RT-PCR and northern blot analyses confirmed that the overall mRNA levels of col1a1, kera, lum, mimecan, and calpain lp82 decrease in the Tgfb2-/- eye. On the other hand, genes specific to mast cells such as histidine decarboxylase, mast cell protease-5 and 6, and mast cell surface marker AA4, etc. are up-regulated. Immunohistochimal staining using polyclonal antibody to histidine decarboxylase was able to detect positive cells in the Tgfb2-/- but not in the Tgfb+/- eyes. Conclusions. TGFß2 has two roles in normal ocular morphogenesis: 1) regulating corneal ECM synthesis and lens calpain proteases gene expression; 2) modulating eye development by preventing invasion of mast cells.
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