Role of Mitf in Differentiation and Transdifferentiation of Chicken Pigmented Epithelial Cell

Abstract

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.

A novel function forHedgehogsignalling in retinal pigment epithelium differentiation

“…The deduced amino acid sequence of three clones was 73% identical to human Mitf (138-247 amino acids; protein reference number I38024). Thus, these cDNAs contained sequence common to heart, neural retina and RPE Mitf clones (Mochii et al, 1998). In order to isolate a sequence unique to retinal pigment epithelial Mitf, or Mitf-A, the Xenopus Mitf sequence data was used to design primers for a 5′ RACE reaction, MixGSP-5′RACE CTT CGC CTT CTT TCA ATG AGG TTG TG and 3Mix262 ATT GTC CTT CTT TTG CCG TTC.…”

“…To investigate this further, we performed in situ hybridisation on cyclopamine-treated embryos with several other RPE markers. We first wanted to look at an early marker of RPE cell differentiation, such as Mitf (Mochii et al, 1998). We therefore cloned by RT-PCR the Xenopus homologue of Mitf (see Materials and Methods).…”

“…Cells in the distalmost part of the vesicle, immediately beneath the surface ectoderm, undergo neural differentiation, while cells in the proximal part differentiate into RPE. This patterning could involve intrinsic cues such as the transcription factors Mitf and Otx2 that are differentially expressed in the prospective neural and RPE domains of the optic vesicle (Mochii et al, 1998;MartinezMorales et al, 2001). But the patterning of the optic vesicle also depends on interactions with the overlaying surface ectoderm (Hyer et al, 1998).…”

Rx for tissue restoration: Regenerative biology and medicine

“…Overexpression of Mitf inhibits FGF-2-induced dedifferentiation and transdetermination of cultured RPE cells. Conversely, induction of dedifferentiation and transdetermination of RPE cells by FGF-2 inhibits Mitf activity, suggesting that downregulation of this gene is essential for NR (and lens) regeneration (Mochii et al, 1998). It will be important to assess the expression patterns, in the newt embryonic and regenerating retina, of other genes known to be essential to retinal development.…”

Compartmentalization of Vertebrate Optic Neuroephithelium: External Cues and Transcription Factors

“…Ectopic expression of Mitf in cultured avian NR cells results in the induction of pigmentation by initiating the expression of two markers of differentiated pigment cells: melanosomal matrix protein 115 kDa (MMP115) and tyrosinase (Mochii et al, 1998;Planque et al, 1999). By contrast, inhibition of Mitf by small interfering RNAs (siRNAs) decreases MMP115 expression and promotes dedifferentiation of the RPE (Iwakiri et al, 2005).…”