Spontaneous Transdifferentiation of Quail Pigmented Epithelial Cell Is Accompanied by a Mutation in theMitfGene

Abstract

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 hybridization 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 that the ectopic neural retina is formed via transdifferentiation of differentiated pigmented epithelial cells (PECs). An in vitro study revealed 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, while B/B PECs continued to proliferate. Some B/B cells overlaid other B/B cells and formed lentoid bodies. Immunological analysis revealed that B/B rPECs transdifferentiated to lens cells and neural cells in vitro with no addition of basic FGF (bFGF), while B/+ rPECs required bFGF to transdifferentiate. Expression of PEC-specific genes, mmp115, tyrosinase, and TRP-1, was downregulated, but that of Mitf and pax6 was upregulated in B/B PECs. Antibody against Mitf stained the nucleus of B/+ PECs but not that of B/B cells, suggesting that the normal Mitf is not present in the silver homozygote due to mutation. Sequence analysis revealed that Mitf from the silver homozygote has an amino acid substitution in the basic region and is truncated in the C-terminal region. Transient transfection analysis revealed that Mitf from the silver homozygote exhibits a lower level of activity than wild-type Mitf with respect to transactivation of the mmp115 promoter. Furthermore, overexpression of chicken Mitf induced normal pigmentation in B/B rPECs. These results strongly suggest that the silver phenotype is caused by the mutation of Mitf and that Mitf plays a critical role in rPEC differentiation and transdifferentiation.

Retinal pigmented epithelium determination requires the redundant activities of Pax2 and Pax6

“…3C,F,I, arrows). Similar defects have been described following loss of function of Mitf in eyes of the mi/mi mice (Scholtz and Chan, 1987;Mochii et al, 1998;Bumsted and Burnstable, 2000). Therefore, we examined the expression of Mitf in Pax2 -/-; Pax6 +/-embryos.…”

“…Later on Mitf and Chx10 continue to be expressed in adjacent regions of the eye, suggesting a reciprocal regulation between these two factors (Nguyen and Arnheiter, 2000). Interestingly, in Mitf-mutant eyes, portions of the outer RPE layer transdifferentiate into NR instead of RPE, reflecting the important roles of Mitf in promoting the RPE differentiation and suppressing NR characteristics (Mochii et al, 1998;Bumsted and Burnstable, 2000;Nguyen and Arnheiter, 2000). Chx10-mutant NRs are generally hypocellular and lack bipolar cells (Ferda Perkin et al, 2000;Burmeister et al, 1996).…”

The hedgehog pathway is a modulator of retina regeneration

“…Transdifferentiating retina shows visinin-positive cells in the presumptive photoreceptor layer and no Mitf expression (Fig. 3B3), consistent with downregulation of Mitf upon transdifferentiation (Mochii et al, 1998a;Mochii et al, 1998b). CB/CMZ regeneration shows strong Mitf expression in the RPE, but shows no photoreceptor differentiation at this point (Fig.…”

Characterization of Japanese Quail yellow as a Genomic Deletion Upstream of the Avian Homolog of the Mammalian ASIP (agouti) Gene

“…In contrast, several genes did not show the predicted pattern from studies in mammals. In particular, we found no evidence for downregulation of the genes encoding the melanogenic enzymes of the Tyrosinase family (TYR, TYRP1, and DCT), as has been documented in response to ASIP in mammalian melanocytes (Kobayashi et al 1995;Furumura et al 1998Furumura et al , 2001; but see Voisey et al 2003), although these are present in domestic fowl, with functions similar to those in mammals (Mochii et al 1998;Chang et al 2006;Nadeau et al 2007b). One possible explanation for this is temporal variation in ASIP expression (see discussion above).…”