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Shivalingappa K. Swamynathan, Doreswamy Kenchegowda, Stephen A. Harvey, Kira Lathrop, Sudha Swamynathan; Dynamic changes in Klf5 functions during corneal maturation and maintenance. Invest. Ophthalmol. Vis. Sci. 2012;53(14):4190.
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Profound changes in the Klf5-conditional null (Klf5CN) ocular surface demonstrated the critical role of Klf5 in the mouse ocular surface following eyelid opening (Kenchegowda D. et al., Dev Biol. 356: 5-18 (2011)). Here, we identify the changes in Klf5CN corneal gene expression at postnatal day-11 (PN11) and PN56, and compare them with the changes (i) in Klf4-conditional null (Klf4CN) corneas and (ii) during wild type (WT) corneal maturation between PN11 and PN56.
PN11 and PN56 WT and Klf5CN corneal transcriptomes were compared by Affymetrix microarrays, validated by QPCR and analyzed by "Microsoft Excel", "BRB ArrayTools" as well as "Ingenuity Pathway Analysis". Whole mount immunofluorescent staining examined corneal lymphangiogenesis and CD45+ cells influx.
Microarrays identified 788 and 767 genes with increased expression and 321 and 223 genes with decreased expression in PN11 and PN56 Klf5CN corneas, respectively. Modulated genes common to both stages comprised 357 concordant increases, 68 concordant decreases and 3 discordant changes, revealing differences between PN11 and PN56 corneal Klf5-target genes. In the WT corneas, expression of 1586 genes was increased and that of 1926 genes decreased between PN11 and PN56, consistent with striking changes in gene expression during corneal maturation. Contingency analysis of all possible expression profiles (Χ2 test: p = 5 x 10-184) revealed that a significant portion of the genes modulated between PN11 and PN56 was also affected in the Klf5CN corneas, consistent with a major role for Klf5 in corneal maturation. Canonical pathway analysis identified 35 and 34 pathways significantly (p<0.001) enriched at PN11 and PN56, respectively, with 24 pathways represented in both stages. Angiogenesis- and inflammation-related genes were affected in the Klf5CN corneas consistent with spontaneous lymphangiogenesis and CD45+ cell influx, respectively. Comparison of the 990 Klf5-target genes with 867 Klf4-target genes selected from the previously published data (Swamynathan SK, et al., IOVS 49, 3360-70 (2008)) identified only 264 common target genes, consistent with the non-redundant functions of Klf4 and Klf5 in the cornea.
We identified the corneal Klf5-target genes at PN11 and PN56, revealing the molecular basis for post-eyelid opening defects in Klf5CN corneas. Significant differences in Klf5-target genes at PN11 and PN56 suggest dynamic changes in Klf5 functions during corneal maturation and maintenance. Striking differences in the corneal Klf4- and Klf5-target genes are consistent with the non-redundant functions of Klf4 and Klf5, two structurally related transcription factors highly expressed in the mouse cornea.
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