Wnt/β-catenin signaling plays an essential role in the retinal pigment epithelium (RPE) determination. Since activity of Pax6 (together with Pax2) is also required for the RPE determination, we investigated a possible genetic interaction between Pax6 and Wnt/β-catenin signaling pathway by analyzing Pax6, β-catenin, and Pax6/β-catenin conditional knockout mice. Although Pax6 inactivation alone had no impact on initial specification determined by the expression of Mitf and Otx2, melanin pigmentation was reduced in the RPE. This suggests that along with Mitf and Otx2, Pax6 is required for the full differentiation of RPE. Reporter gene assays in vitro suggest that hypopigmentation is at least in part due to the direct regulation of genes encoding enzymes involved in melanin synthesis by Pax6, Mitf, and β-catenin. The RPE of a β-catenin/Pax6 double mutant was differentiated into the neural retina; however, the tissue was thinner than that of the conditional β-catenin mutant due to reduced proliferation. Together, our data demonstrate that Pax6 is required for the RPE differentiation by regulating pigmentation and accountable for hyperproliferation in the transdifferentiated RPE. In this context, Pax6 appears to function as a pleiotropic regulator, directing development of ocular tissues in concert with the signaling pathway and, at the same time, regulating expression of structural component of the eye, such as shielding pigment.
We are grateful to A. Zitova, V. Noskova, and J. Lachova for technical assistance, Drs. H. Arnheiter and F. Vaccarino for Mitf and Otx2 antibodies, Dr. Ruth Ashery-Padan for plasmids, Dr. R. Kemler for Catnblox(ex2–6) mice, Dr. P. Chambon for Trp1-Cre, and Sarka Takacova for critical reading of the manuscript. This study was supported by the Grant Agency of the Czech Republic (GAP305/11/2198 and GAP305/10/2141), BIOCEV – Biotechnology and Biomedicine Centre of the Academy of Sciences and Charles University (CZ.1.05/1.1.00/02.0109), and Czech Ministry of Education Grant LK11214.
Conditional deletion of β-catenin and Pax6 in the developing RPE. Frontal sections of wild type (a, c), Trp1-Cre;Catnblox(ex2–6)/lox(ex2–6) (b, e) and Trp1-Cre;Catnblox(ex2–6)/lox(ex2–6); Pax6fl/fl(c, f) at E11.5 showing expression of Pax6 (a-c) and β-catenin (d-f). The transdifferentiated RPE is indicated by a dashed line or an arrowhead. (GIF 124 kb)
Expression of Pax6 during eye development. Frontal cryosections of wild type embryos showing expression of Pax6 at E10.5 (a), E11.5 (b), E13.5 (c), and E15.5 (d). Pax6 is detected in the entire RPE; however, its expression is restricted to the peripheral part of the RPE at later stages. (GIF 216 kb)
Baumer N, Marquardt T, Stoykova A, Spieler D, Treichel D, Ashery-Padan R, Gruss P (2003) Retinal pigmented epithelium determination requires the redundant activities of Pax2 and Pax6. Development 130:2903–2915CrossRefPubMedGoogle Scholar
Brault V et al (2001) Inactivation of the beta-catenin gene by Wnt1-Cre-mediated deletion results in dramatic brain malformation and failure of craniofacial development. Development 128:1253–1264PubMedGoogle Scholar
Burmeister M et al (1996) Ocular retardation mouse caused by Chx10 homeobox null allele: impaired retinal progenitor proliferation and bipolar cell differentiation. Nat Genet 12:376–384CrossRefPubMedGoogle Scholar
Schwarz M, Cecconi F, Bernier G, Andrejewski N, Kammandel B, Wagner M, Gruss P (2000) Spatial specification of mammalian eye territories by reciprocal transcriptional repression of Pax2 and Pax6. Development 127:4325–4334PubMedGoogle Scholar
Vopalensky P, Kozmik Z (2009) Eye evolution: common use and independent recruitment of genetic components Philosophical transactions of the Royal Society of London Series B. Biol Sci 364:2819–2832. doi:10.1098/rstb.2009.0079CrossRefGoogle Scholar