M. Mochii et al., ROLE OF MITF IN DIFFERENTIATION AND TRANSDIFFERENTIATION OF CHICKEN PIGMENTED EPITHELIAL-CELL, Developmental biology, 193(1), 1998, pp. 47-62
Mitf encodes a basic helix-loop-helix-leucine-zipper (bHLHzip) protein
that is known to function in the development of melanocytes, pigmente
d epithelial cells (PECs), osteoclasts, and mast cells. In this paper,
we report on the isolation, expression, and overexpression of the chi
cken Mitf and discuss the role of its protein product in the different
iation and transdifferentiation of PECs. Northern blotting showed that
chicken Miff is predominantly expressed in embryonic retinal pigmente
d epithelium (PE), but is expressed at low levels in other tissues. A
5' RACE analysis revealed differences in the 5' region of Mitf mRNA in
PE and other tissues. Immunological analysis revealed that Mitf, the
protein encoded by Miff, is first detected in the nuclei of the optic
vesicle cells at embryonic stage 13 in a restricted region covered wit
h mesenchymal cells. From stage 14 to 24, the specific staining is obs
ervable in the PE and precursor of the PE, the outer layer of the opti
c cup. In embryos at stages later than stage 25, 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 PE
Cs (rPECs) was induced by phenylthiourea and testicular hyaluronidase,
bFGF, or TGF-beta. Miff expression was inhibited by these factors and
reactivated during redifferentiation of the dedifferentiated cells in
to rPECs, showing the correlation between Miff expression and rPEC dif
ferentiation. Retrovirus-mediated overexpression of Miff inhibited bFG
F-induced dedifferentiation and transdifferentiation of rPECs to both
lens and neural cells. These findings showed that downregulation of Mi
ff expression is essential for the transdifferentiation of rPEC. Miff
overexpression caused hyperpigmentation in cultured rPECs and suppress
ed the changes in gene expression induced by bFGF. Miff overexpression
promoted expression of mmp115 and tyrosinase in bFGF-treated rPECs su
ggesting a critical role for Mitf in rPEC differentiation. Miff overex
pression, 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 express
ion patterns of pax6 and Mitf are complementary both in vivo and in vi
tro. Overexpression of Miff inhibited expression of pax6, in cultured
rPECs. These observations suggest that Mitf regulates pax6 expression
negatively. (C) 1998 Academic Press.