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Z. Ding, T. Truong, S. Deng; Differential Expression Pattern of Wnt Signaling Molecules in the Human Limbus. Invest. Ophthalmol. Vis. Sci. 2009;50(13):4544.
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© ARVO (1962-2015); The Authors (2016-present)
To investigate the expression pattern of genes involved in the Wnt signaling pathway in the limbus compared to that in the cornea in human and the presence of Wnt signaling in the limbus stem cell culture.
The epithelium along with 1/3 of the underlying stroma from the limbus and cornea were dissected from human sclerocorneal tissues. RNA was isolated using the Qiagen RNeasy kit. RNA quality was assessed by gel electrophoresis. Total RNA was used for reverse transcription. Gene specific primers were designed. Quantitative RT-PCR was performed to detect the relative abundance of transcripts. The average value of the triplicates from each transcript was used for comparison. Selected gene expression was further confirmed by immunohistochemistry (IHC) in human cornea tissues. Primary limbus epithelium cells was cultured in KSFM medium with NIH 3T3 feeder layer. Expression of some Wnt signaling molecules including Wnt3, Wnt2, Fzd3, Wnt5a and β-Catenin in cultured limbus stem cell colony were examined by IHC.
The QRT-RCR results showed that 4 members of the Wnt protein family and 4 members of the Frizzled protein family had 1.5 fold higher expression levels in the limbus compared to those in the cornea. While there were 4 Wnt genes and 2 Frizzled genes expressed 1.5 fold higher levels in the cornea than in the limbus. Other important genes belong to Wnt signalling pathway, including Wnt inhibitory factor 1, β-Catenin, Lef1, Lrp5, Lrp6, C-Myc, and Dickkopf-1 also showed preferred expression in the limbus. The expression level and cellular localization of β -Catenin, c-Myc and Wnt2 was further confirmed by IHC. Among 5 Wnt signaling molecules investigated, 3 of them as Wnt2, Fzd3 and β-Catenin were strongly expressed in the cultured limbus stem cell colony.
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