Ectopic Mitf in the embryonic chick retina by co-transfection of β-catenin and Otx2.

PURPOSE: Development of the retinal pigment epithelium (RPE) is controlled by intrinsic and extrinsic regulators including orthodenticle homeobox 2 (Otx2) and the Wnt/β-catenin pathway, respectively. Otx2 and β-catenin are necessary for the expression of the RPE key regulator microphthalmia-associated transcription factor (Mitf); however, neither factor is sufficient to promote Mitf expression in vivo. The study was conducted to determine whether Otx2 and β-catenin act in a combinatorial manner and tested whether co-expression in the presumptive chick retina induces ectopic Mitf expression.
METHODS: The sufficiency of Wnt/β-catenin activation and/or Otx2 expression to induce RPE-specific gene expression was examined in chick optic vesicle explant cultures or in the presumptive neural retina using in ovo-electroporation. Luciferase assays were used to examine the transactivation potentials of Otx2 and β-catenin on the Mitf-D enhancer and autoregulation of the Mitf-D and Otx2T0 enhancers.
RESULTS: In optic vesicles explant cultures, RPE-specific gene expression was activated by lithium chloride, a Wnt/β-catenin agonist. However, in vivo, Mitf was induced only in the presumptive retina if both β-catenin and Otx2 are co-expressed. Furthermore, both Mitf and Otx2 can autoregulate their own enhancers in vitro.
CONCLUSIONS: The present study provides evidence that β-catenin and Otx2 are sufficient, at least in part, to convert retinal progenitor cells into presumptive RPE cells expressing Mitf. Otx2 may act as a competence factor that allows RPE specification in concert with additional RPE-promoting factors such as β-catenin.