Mitf encodes a basic helix-loop-helix-leucine-zipper (bHLHzip) protein that is known to function in the development of melanocytes, pigmented epithelial cells (PECs), osteoclasts, and mast cells. In this paper, we report on the isolation, expression, and overexpression of the chicken Mitf and discuss the role of its protein product in the differentiation and transdifferentiation of PECs. Northern blotting showed that chicken Mitf is predominantly expressed in embryonic retinal pigmented epithelium (PE), but is expressed at low levels in other tissues. A 5' RACE analysis revealed differences in the 5' region Mitf nRNA in PE and other tissues. Immunological analysis revealed that Mitf, the protein encoded by Mitf, is first detected in the nuclei of the optic vesicle cells at embryonic stage 13 in a restricted region covered with mesenchymal cells. From stage 14 to 24, the specific staining is observable in the PE and precursor of the PE, the outer layer of the optic cup. In embryos at stages later than stage 29 the signals for Mitf in the future iris, ciliary body, and posterior retinal regions become faint. These results show that expression of Mitf starts at the optic vesicle stage at which no other marker genes for PECs such as mmp115 and tyrosinase are expressed. Dedifferentiation of cultured retinal PECs (rPECs) was induced by phenylthiourea and testicular hyaluronidase, bFGF, or TGF-beta. Mitf expression was inhibited by these factors and reactivated during redifferentiation of the dedifferentiated cells into rPECs, showing the correlation between Mitf expression and rPEC differentiation. Retrovirus-mediated overexpression of Mtif inhibited bFGF-induced dedifferentiation and transdifferentiation of rPECs to both lens and neural cells. These findings showed that downregulation of Mitf expression is essential for the transdifferentiation of rPEC. Mitf overexpression caused hyperpigmentation in cultured rPECs and suppressed the changes in gene expression induced by bFGF. Mitf overexpression promoted expression of mmp115 and tyrosinase in bFGF-treated rPECs suggesting a critical role for Mitf in rPEC differentiation. Mitf overexpression, however, did not promote expression of another rPEC-specific gene, pP344, in bFGF-treated rPECs. This result suggests the presence of other regulatory genes promoting rPEC differentiation. The expression patterns of pax6 and Mitf are complementary both in vivo in vitro. Overexpression of Mitf inhibited expression of pax6 in cultured rPECs. These observations suggest that Mitf regulates pax6 expression negatively.

Lens regeneration in newts occurs exclusively from the dorsal aspect of the iris pigment epithelium. Although the phenomenon has been a paradigm of experimental tissue regeneration, little is understood about how it is initiated and restricted to the dorsal iris. Here we show among various growth factors injected in an intact eye, a single injection of FGF2 specifically caused morphological changes of the iris characteristic of lens regeneration, induced expression of transcription factor genes Pax6, Sox2 and MafB, as well as endogenous Fgf2 in both dorsal and ventral halves, and provoked second lens development only from the dorsal iris. FGF2 protein accumulated in the iris tissue after the lens was removed, and injection of a soluble form of FGF receptor titrating FGF2 inhibited all reactions observed after the lens removal or after administration of FGF2. These results indicate that FGF2 and/or related molecules trigger lens regeneration from the dorsal iris in the newt. The observations also indicate that the absence of lens regeneration from the ventral iris is due to a block in a later phase of lens developmental pathway.

We have previously shown that lens regeneration from the pigmented epithelium of the dorsal iris in the adult newt eye proceeds in two steps after lens removal or intraocular FGF2 injection. The FGF2-dependent proliferation of iris pigmented epithelium and activation of early lens genes that occur over the entire circumference of the iris comprise the first step, while subsequent dorsally confined lens development marks the second step. Here, we investigated the expression of Wnt and Wnt receptor Frizzled genes in lens-regenerating iris tissues. Wnt2b and Frizzled4 were activated only in the dorsal half of the iris in synchrony with the occurrence of the second step, whereas Wnt5a and Frizzled2 were activated in both halves throughout the period of the first and second steps. Cultured explants of the iris-derived pigmented epithelium in the presence of FGF2 underwent dorsal-specific lens development fully recapitulating the in vivo lens regeneration process. Under these conditions, Wnt inhibitors Dkk1, which specifically inhibits the canonical signal pathway, and/or sFRP1 repressed the lens development, while exogenous Wnt3a, which generally activates the canonical pathway like Wnt2b, stimulated lens development from the dorsal iris epithelium and even caused lens development from the ventral iris epithelium, albeit at a reduced rate. Wnt5a did not elicit lens development from the ventral epithelium. These observations indicate that dorsal-specific activation of Wnt2b determines the dorsally limited development of lens from the iris pigmented epithelium.