SPONTANEOUS TRANSDIFFERENTIATION OF QUAIL PIGMENTED EPITHELIAL-CELL IS ACCOMPANIED BY A MUTATION IN THE MITF GENE

An ectopic neural retina is formed at the outer layer of the retina in the silver homozygote (B/B) of the Japanese quail. In situ hybridizat ion and immunohistochemical analysis revealed that cells in the outer layer of retina first expressed a pigment-cell-specific gene, mmp115, and then began to express a neural marker in B/B embryos, indicating t hat the ectopic neural retina is formed via transdifferentiation of di fferentiated pigmented epithelial cells (PECs). An in vitro study reve aled that cultured retinal PECs (rPECs) from B/B embryos exhibit less pigment granule and a higher growth rate than cells from heterozygotes (B/+). B/+ PECs stopped proliferating when confluency was reached, wh ile BIB PECs continued to proliferate. Some BIB cells overlaid other B IB cells and formed lentoid bodies. Immunological analysis revealed th at BIB rPEGs transdifferentiated to lens cells and neural cells in vit ro with no addition of basic FGF (bFGF), while B/+ rPEGs required bFGF to transdifferentiate. Expression of PEG-specific genes, mmp115, tyro sinase, and TRP-1, was downregulated, but that of Mitf and pax6 was up regulated in B/B PECs. Antibody against Mitf stained the nucleus of B/ + PECs but not that of Bl B cells, suggesting that the normal Mitf is not present in the silver homozygote due to mutation. Sequence analysi s revealed that Mitf from the silver homozygote has an amino acid subs titution in the basic region and is truncated in the C-terminal region . Transient transfection analysis revealed that Mitf from the silver h omozygote exhibits a lower level of activity than wild-type Mitf with respect to transactivation of the mmp115 promoter. Furthermore, overex pression of chicken Mitf induced normal pigmentation in B/B rPECs. The se results strongly suggest that the silver phenotype is caused by the mutation of Mitf and that Mitf plays a critical role in rPEC differen tiation and transdifferentiation. (C) 1998 Academic Press.