Development Genes and Evolution

, Volume 225, Issue 2, pp 121–128 | Cite as

Genetic interaction between Pax6 and β-catenin in the developing retinal pigment epithelium

  • Naoko Fujimura
  • Lucie Klimova
  • Barbora Antosova
  • Jana Smolikova
  • Ondrej Machon
  • Zbynek KozmikEmail author
Short Communication


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.


Pax6 β-Catenin Retina Pigmentation Transdifferentiation 



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 Catnb lox(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.

Supplementary material

427_2015_493_Fig7_ESM.gif (124 kb)
Fig. S2

Conditional deletion of β-catenin and Pax6 in the developing RPE. Frontal sections of wild type (a, c), Trp1-Cre;Catnb lox(ex2–6)/lox(ex2–6) (b, e) and Trp1-Cre;Catnb lox(ex2–6)/lox(ex2–6) ; Pax6 fl/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)

427_2015_493_MOESM1_ESM.tif (1.8 mb)
High resolution image (TIFF 1815 kb)
427_2015_493_Fig8_ESM.gif (216 kb)
Fig. S2

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)

427_2015_493_MOESM2_ESM.tif (3.1 mb)
High resolution image (TIFF 3173 kb)


  1. 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
  2. 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
  3. 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
  4. Das G, Choi Y, Sicinski P, Levine EM (2009) Cyclin D1 fine-tunes the neurogenic output of embryonic retinal progenitor cells. Neural Dev 4:15. doi: 10.1186/1749-8104-4-15 CrossRefPubMedCentralPubMedGoogle Scholar
  5. Fuhrmann S, Zou C, Levine EM (2014) Retinal pigment epithelium development, plasticity, and tissue homeostasis. Exp Eye Res 123:141–150. doi: 10.1016/j.exer.2013.09.003 CrossRefPubMedGoogle Scholar
  6. Fujimura N, Taketo MM, Mori M, Korinek V, Kozmik Z (2009) Spatial and temporal regulation of Wnt/beta-catenin signaling is essential for development of the retinal pigment epithelium. Dev Biol 334:31–45. doi: 10.1016/j.ydbio.2009.07.002 CrossRefPubMedGoogle Scholar
  7. Klimova L, Kozmik Z (2014) Stage-dependent requirement of neuroretinal Pax6 for lens and retina development. Development 141:1292–1302. doi: 10.1242/dev.098822 CrossRefPubMedGoogle Scholar
  8. Martinez-Morales JR, Signore M, Acampora D, Simeone A, Bovolenta P (2001) Otx genes are required for tissue specification in the developing eye. Development 128:2019–2030PubMedGoogle Scholar
  9. Mori M, Metzger D, Garnier JM, Chambon P, Mark M (2002) Site-specific somatic mutagenesis in the retinal pigment epithelium. Invest Ophthalmol Vis Sci 43:1384–1388PubMedGoogle Scholar
  10. Nguyen M, Arnheiter H (2000) Signaling and transcriptional regulation in early mammalian eye development: a link between FGF and MITF. Development 127:3581–3591PubMedGoogle Scholar
  11. Raviv S et al (2014) PAX6 regulates melanogenesis in the retinal pigmented epithelium through feed-forward regulatory interactions with MITF. PLoS Genet 10:e1004360. doi: 10.1371/journal.pgen.1004360 CrossRefPubMedCentralPubMedGoogle Scholar
  12. 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
  13. Shaham O, Menuchin Y, Farhy C, Ashery-Padan R (2012) Pax6: a multi-level regulator of ocular development. Prog Retin Eye Res 31:351–376. doi: 10.1016/j.preteyeres.2012.04.002 CrossRefPubMedGoogle Scholar
  14. 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.2009.0079 CrossRefGoogle Scholar
  15. Westenskow P, Piccolo S, Fuhrmann S (2009) Beta-catenin controls differentiation of the retinal pigment epithelium in the mouse optic cup by regulating Mitf and Otx2 expression. Development 136:2505–2510. doi: 10.1242/dev.032136 CrossRefPubMedCentralPubMedGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2015

Authors and Affiliations

  • Naoko Fujimura
    • 1
  • Lucie Klimova
    • 1
  • Barbora Antosova
    • 1
  • Jana Smolikova
    • 1
  • Ondrej Machon
    • 1
  • Zbynek Kozmik
    • 1
    Email author
  1. 1.Institute of Molecular GeneticsAcademy of Sciences of the Czech RepublicPrague 4Czech Republic

Personalised recommendations