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P. D. Westenskow, J. B. McKean, C. Zou, S. Fuhrmann; Regulation of RPE development by β-catenin and Otx2. Invest. Ophthalmol. Vis. Sci. 2009;50(13):4013.
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The transcription factors Microphthalmia associated transcription factor (Mitf) and orthodenticle homolog 2 (Otx2) are key transcription factors for terminal differentiation of the retinal pigment epithelium (RPE). The manner in which these factors are regulated, however, remains elusive. We have shown previously that conditional deletion of β-catenin induces many defects including RPE-to-retina transdifferentiation and downregulation of Mitf expression. Furthermore, we observed that β-catenin associates with and activates the RPE-specific Mitf-D isoform through the Wnt/β-catenin pathway transcription factors TCF/LEF. However, RPE differentiation does not occur when dominant-stable β-catenin is ectopically expressed in the retina implying that another factor is needed. Here, we identify OTX2 as a candidate for the missing factor.
The mouse Mitf-D promoter was cloned and inserted into pGL3-B, which contains a luciferase gene. This construct, along with those encoding dominant-stable β-catenin and/or Otx2, was tested in HEK293T cells. Additionally, dominant-stable β-catenin and Otx2 were misexpressed in stage HH10 chicken optic vesicles using in ovo electroporation. Two days later the embryos were harvested and stained to identify transfected cells ectopically expressing Mitf.
β-catenin and Otx2 transfection in HEK293T cells both activate the Mitf-D promoter when introduced alone, and in an additive manner when co-expressed. In the electroporated chicken retinas, a modest increase in the number of cells that express Mitf is observed in those transfected only with β-catenin, and virtually no cells transfected only with Otx2. In cells in which both β-catenin and Otx2 are misexpressed, we observed a dramatic increase in the number of Mitf expressing cells.
These data suggest that β-catenin and Otx2 can activate the Mitf-D promoter in vitro and that they cooperate to induce Mitf expression in vivo. These results provide insight into the genetic network responsible for Mitf regulation during RPE development.
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