Mitf functions as an in ovo regulator for cell differentiation and proliferation during development of the chick RPE

Mitf has been reported to play a crucial role in regulating the differentiation of pigment cells in homeothermal animals, i.e. the melanocytes and the retinal pigment epithelium (RPE). However, less is known about the functions of Mitf in the developing RPE. To elucidate such functions, we introduced wild-type and dominant-negative Mitf expression vectors into chick optic vesicles by electroporation. Over-expression of wild-type Mitf altered neural retina cells to become RPE-like and repressed the expression of neural retina markers in vivo. In contrast, dominant-negative Mitf inhibited pigmentation in the RPE. The percentage of BrdU-positive cells decreased during normal RPE development, which was followed by Mitf protein expression. The percentage of BrdU-positive cells decreased in the wild-type Mitf-transfected neural retina, but increased in the dominant-negative Mitf-transfected RPE. p27^kip1, one of the cyclin-dependent kinase inhibitors, begins to be expressed in the proximal region of the RPE at stage 16. Transfection of wild-type Mitf induced expression of p27^kip1, while transfection of dominant-negative Mitf inhibited p27^kip1 expression. We found that Mitf was associated with the endogenous p27^kip1 5′ flanking region. These results demonstrate for the first time “in vivo” that Mitf uniquely regulates both differentiation and cell proliferation in the developing RPE.     

Extraocular dorsal signal affects the developmental fate of the optic vesicle and patterns the optic neuroepithelium

Dorsal-ventral (DV) specification in the early optic vesicle plays a crucial role in the proper development of the eye. To address the questions of how DV specification is determined and how it affects fate determination of the optic vesicle, isolated optic vesicles were cultured either in vitro or in ovo. The dorsal and ventral halves of the optic vesicle were fated to develop into retinal pigment epithelium (RPE) and neural retina, respectively, when they were separated from each other and cultured. In optic vesicles treated with collagenase to remove the surrounding tissues, the neuroepithelium gave rise to cRax expression but not Mitf, suggesting that surrounding tissues are necessary for RPE specification. This was also confirmed in in ovo explant cultures. Combination cultures of collagenase-treated optic vesicles with either the dorsal or ventral part of the head indicated that head-derived factors have an important role in the fate determination of the optic vesicle: in the optic vesicles co-cultured with the dorsal part of the head Mitf expression was induced in the neuroepithelium, while the ventral head portion did not have this effect. The dorsal head also suppressed Pax2 expression in the optic vesicle. These observations indicate that factors from the dorsal head portion have important roles in the establishment of DV polarity within the optic vesicle, which in turn induces the patterning and differentiation of the neural retina and pigment epithelium.     

The myc oncogene and transdifferentiation of the retinal pigment epithelium]

The retinal pigment epithelium (RPE) develops from the same sheet of neuroepithelium as the neuroretina. When infected with MC29, a v-myc expressing virus, the RPE cells can be induced to transdifferentiate and to take a neuroretinal epithelium fate. After a PCR-based differential screening from these cells we have identified three genes of interest. Qath5, a quail basic helix-loop-helix (bHLH) gene that is closely related to the Drosophila atonal, and whose expression is found in the developing neuroretina. A Chx10-related homeobox gene also expressed in the developing neuroretina and HuD, a RNA-binding protein not expressed in the RPE but expressed during neurogenesis. Beside these genes whose function is involved in regulating neuronal differentiation myc also induced a transient Mitf expression. Mitf is expressed in the entire optic cup, later restricted to the pigmented retina. Mitf is involved in the regulation of the pigmented differentiation. We conclude that v-myc can reverse the RPE to the bipotential retinal primordia.     

MEK mediates in vitro neural transdifferentiation of the adult newt retinal pigment epithelium cells: Is FGF2 an induction factor?

Adult newts can regenerate their entire retinas through transdifferentiation of the retinal pigment epithelium (RPE) cells. As yet, however, underlying molecular mechanisms remain virtually unknown. On the other hand, in embryonic/larval vertebrates, an MEK [mitogen‐activated protein kinase (MAPK)/extracellular signal‐regulated kinase (ERK) kinase] pathway activated by fibroblast growth factor‐2 (FGF2) is suggested to be involved in the induction of transdifferentiation of the RPE into a neural retina. Therefore, we examined using culture systems whether the FGF2/MEK pathway is also involved in the adult newt RPE transdifferentiation. Here we show that the adult newt RPE cells can switch to neural cells expressing pan‐retinal‐neuron (PRN) markers such as acetylated tubulin, and that an MEK pathway is essential for the induction of this process, whereas FGF2 seems an unlikely primary induction factor. In addition, we show by immunohistochemistry that the PRN markers are not expressed until the 1–3 cells thick regenerating retina, which contains retinal progenitor cells, appears. Our current results suggest that the activation of an MEK pathway in RPE cells might be involved in the induction process of retinal regeneration in the adult newt, however if this is the case, we must assume complementary mechanisms that repress the MEK‐mediated misexpression of PRN markers in the initial process of transdifferentiation.