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Yujin Zhang, Lung-Kun Yeh, Winston W-Y Kao, Chia-yang Liu; Conditional Ablation Of beta-catenin In Keratocytes Triggers Precocious Corneal Morphogenesis During Development. Invest. Ophthalmol. Vis. Sci. 2012;53(14):4740.
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Mesenchyme-epithelial interaction plays a pivotal role on corneal morphogenesis during embryonic development. In this study, attempts were made to examine the hypothesis that perturbation of β-catenin-BMP-4 signaling axis resulting from the loss of β-catenin gene (Ctnnb1) in corneal stromal keratocytes (Ctnnb1cskΔ/cskΔ) leads to anomaly of the mTOR signaling pathway in corneal epithelium.
Kera-rtTA/ tet-O-Cre/Ctnnb1f/f (KR/TC/ Ctnnb1f/f) triple transgenic mice were induced with doxycycline from embryonic 12.5 (E12.5) to various gestation stages e.g., E16.5, E18.5 and postnatal day 0 (P0) or from P0 to P10. Embryos and neonates were collected. The specimens were subjected to histology and immunohistochemistry examination. RT-PCR and western blotting were performed to exam BMP4 expression in stroma and p70S6K and pAKT, components involving in mTOR pathway, in cultured epithelial cells and epithelium of Ctnnb1cskΔ/cskΔ mice, respectively.
The Ctnnb1cskΔ/cskΔ mutants were precocious in corneal epithelium stratification (4-5 cell layers) as compared to that of wild-type mice (1-2 cell layers) at P0 concomitant with increased number of proliferative epithelial cells as demonstrated by PCNA and Cyclin D1 immunostaining. The size of basal cells is significantly larger in the mutant corneas as compared to those of wild type littermates at P0. Immunofluorescence staining showed that p70S6K and pAKT were up-regulated, suggesting mTOR signaling pathway was ectopically activated in corneal epithelium. Moreover, scanning electron microscopy showed that the collagen fibrils and stromal cells are better organized in mutant corneal stroma, resembling the collagenous matrix seen in adult mice. Biochemical analyses showed that expression of BMP-4, known to be able to stimulate mTOR signaling, was up-regulated in cultured fibroblasts from Ctnnb1cskΔ/cskΔ mice in vitro. Furthermore, RT-PCR analysis revealed that CDK inhibitor of P21 was up-regulated and the extracellular matrix (ECM) components such as TGF-β, collagen I alpha 2 and keratocan were increased when Ctnnb1 was ablated in primary cultured corneal stromal cells from Ctnnb1cskΔ/cskΔ mice.
The precocious corneal epithelium stratification in Ctnnb1cskΔ/cskΔ mutants may result from the enhanced expression of BMP-4 in developing corneal stroma, which subsequently caused enhanced mTOR signaling in corneal epithelial cells, leading to precocious epithelium stratification. This supports the notion that cross-talk between β-catenin/BMP-4 axis (in stroma) and mTOR signaling (in epithelium) may play a pivotal role in corneal epithelium morphogenesis during embryonic development.
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