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Xia Wang, Cheryl Y. Gregory-Evans; Identification Of The Downstream Targets Of Pax6 In The Developing Iris Relevant To Aniridia. Invest. Ophthalmol. Vis. Sci. 2012;53(14):4932.
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© ARVO (1962-2015); The Authors (2016-present)
Aniridia is a bilateral pan-ocular eye disease characterized by partial or complete absence of the iris caused by haploinsufficiency of PAX6. The gene regulatory networks driving iris development are unknown. The aim of this study was to identify the downstream target genes of the Pax6 transcription factor by taking advantage of the Pax6Sey-Neu mouse aniridia model.
Laser capture microdissection was used to isolate the iris cells from wildtype and heterozygous Pax6Sey-Neu eyes. Differential gene expression was analyzed by TaqMan real-time RT-PCR. Spatiotemporal gene expression level was determined by in situ hybridization. Candidate downstream targets of Pax6 were tested by in vivo ChIP-PCR. Cell death and proliferation were analysed by TUNEL staining and phospho-histone H3 immunohistochemistry, respectively.
Differential gene expression levels were observed in 6 genes associated with iris development, including increased expression of Foxc1, Igf2, Tgfb2, and decreased expression of Zic2 and Bmp4 in the developing iris from E12.5 to E16.5 in heterozygous Pax6Sey-Neu mice. Changes in Pitx2 expression varied with developmental stage. These gene targets were shown to co-localize with Pax6 gene expression during iris development. Furthermore, ChIP-PCR demonstrated that the Pax6 proteinbound to the promoter sequences of Bmp4, Pitx2, Foxc1 and Tgfb2 in vivo. In the analysis of ocular phenotype of the Pax6Sey-Neu mice, increased proliferating cells were found in the developing iris between E12.5 and E16.5 as compared to wild-type, whereas there was no significant difference in cell death.
Collectively, these experiments demonstrate that Pax6 directly regulates the expression of Bmp4, Pitx2, Foxc1 and Tgfb2 in the developing iris. As Foxc1 is therefore in the same signaling pathway as Pax6, this explains the observation that mutations in FOXC1 also cause aniridia. Furthermore, since 20% of aniridia cases do not have either PAX6 or FOXC1 mutations, it suggests that PITX2, BMP4 and TGFB2 should be considered as candidates genes to be screened in these cases of aniridia. The role of these 4 downstream targets in causing the proliferation defect in the developing iris of the Pax6Sey-Neu mutant warrants further investigation.
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