The Wnt/β-catenin signaling pathway controls many processes during development, including cell proliferation, cell differentiation and tissue homeostasis, and its aberrant regulation has been linked to various pathologies. In this study we investigated the effect of ectopic activation of Wnt/β-catenin signaling during lens fiber cell differentiation. To activate Wnt/β-catenin signaling in lens fiber cells, the transgenic mouse referred to as αA-CLEF was generated, in which the transactivation domain of β-catenin was fused to the DNA-binding protein LEF1, and expression of the transgene was controlled by αA-crystallin promoter. Constitutive activation of Wnt/β-catenin signaling in lens fiber cells of αA-CLEF mice resulted in abnormal and delayed fiber cell differentiation. Moreover, adult αA-CLEF mice developed cataract, microphthalmia and manifested downregulated levels of γ-crystallins in lenses. We provide evidence of aberrant expression of cell cycle regulators in embryonic lenses of αA-CLEF transgenic mice resulting in the delay in cell cycle exit and in the shift of fiber cell differentiation to the central fiber cell compartment. Our results indicate that precise regulation of the Wnt/β-catenin signaling activity during later stages of lens development is essential for proper lens fiber cell differentiation and lens transparency.

Institute of Molecular Genetics Academy of Sciences of the Czech Republic Prague Czech Republic ; Department of Genetics and Microbiology Faculty of Science Charles University Prague Prague Czech Republic

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McAvoy JW, Chamberlain CG, de Iongh RU, Hales AM, Lovicu FJ (1999) Lens development. Eye (Lond) 13 (Pt 3b): 425–437. PubMed

McAvoy JW (1978) Cell division, cell elongation and the co-ordination of crystallin gene expression during lens morphogenesis in the rat. J Embryol Exp Morphol 45: 271–281. PubMed

Grindley JC, Davidson DR, Hill RE (1995) The role of Pax-6 in eye and nasal development. Development 121: 1433–1442. PubMed

Wigle JT, Chowdhury K, Gruss P, Oliver G (1999) Prox1 function is crucial for mouse lens-fibre elongation. Nat Genet 21: 318–322. PubMed

Nishiguchi S, Wood H, Kondoh H, Lovell-Badge R, Episkopou V (1998) Sox1 directly regulates the gamma-crystallin genes and is essential for lens development in mice. Genes Dev 12: 776–781. PubMed PMC

Ashery-Padan R, Marquardt T, Zhou X, Gruss P (2000) Pax6 activity in the lens primordium is required for lens formation and for correct placement of a single retina in the eye. Genes Dev 14: 2701–2711. PubMed PMC

Kim JI, Li T, Ho IC, Grusby MJ, Glimcher LH (1999) Requirement for the c-Maf transcription factor in crystallin gene regulation and lens development. Proc Natl Acad Sci U S A 96: 3781–3785. PubMed PMC

Ring BZ, Cordes SP, Overbeek PA, Barsh GS (2000) Regulation of mouse lens fiber cell development and differentiation by the Maf gene. Development 127: 307–317. PubMed

Xie Q, Cvekl A (2011) The orchestration of mammalian tissue morphogenesis through a series of coherent feed-forward loops. J Biol Chem 286: 43259–43271. PubMed PMC

Shaham O, Smith AN, Robinson ML, Taketo MM, Lang RA, et al. (2009) Pax6 is essential for lens fiber cell differentiation. Development 136: 2567–2578. PubMed PMC

Zhang P, Wong C, DePinho RA, Harper JW, Elledge SJ (1998) Cooperation between the Cdk inhibitors p27(KIP1) and p57(KIP2) in the control of tissue growth and development. Genes Dev 12: 3162–3167. PubMed PMC

Bassnett S, Beebe DC (1992) Coincident loss of mitochondria and nuclei during lens fiber cell differentiation. Dev Dyn 194: 85–93. PubMed

Lovicu FJ, McAvoy JW (2005) Growth factor regulation of lens development. Dev Biol 280: 1–14. PubMed

Zhao H, Yang T, Madakashira BP, Thiels CA, Bechtle CA, et al. (2008) Fibroblast growth factor receptor signaling is essential for lens fiber cell differentiation. Dev Biol 318: 276–288. PubMed PMC

Faber SC, Dimanlig P, Makarenkova HP, Shirke S, Ko K, et al. (2001) Fgf receptor signaling plays a role in lens induction. Development 128: 4425–4438. PubMed

Boswell BA, Overbeek PA, Musil LS (2008) Essential role of BMPs in FGF-induced secondary lens fiber differentiation. Dev Biol 324: 202–212. PubMed PMC

Faber SC, Robinson ML, Makarenkova HP, Lang RA (2002) Bmp signaling is required for development of primary lens fiber cells. Development 129: 3727–3737. PubMed

de Iongh RU, Lovicu FJ, Overbeek PA, Schneider MD, Joya J, et al. (2001) Requirement for TGFbeta receptor signaling during terminal lens fiber differentiation. Development 128: 3995–4010. PubMed

Belecky-Adams TL, Adler R, Beebe DC (2002) Bone morphogenetic protein signaling and the initiation of lens fiber cell differentiation. Development 129: 3795–3802. PubMed

Klaus A, Birchmeier W (2008) Wnt signalling and its impact on development and cancer. Nat Rev Cancer 8: 387–398. PubMed

Cain S, Martinez G, Kokkinos MI, Turner K, Richardson RJ, et al. (2008) Differential requirement for beta-catenin in epithelial and fiber cells during lens development. Dev Biol 321: 420–433. PubMed

Kreslova J, Machon O, Ruzickova J, Lachova J, Wawrousek EF, et al. (2007) Abnormal lens morphogenesis and ectopic lens formation in the absence of beta-catenin function. Genesis 45: 157–168. PubMed

Machon O, Kreslova J, Ruzickova J, Vacik T, Klimova L, et al. (2010) Lens morphogenesis is dependent on Pax6-mediated inhibition of the canonical Wnt/beta-catenin signaling in the lens surface ectoderm. Genesis 48: 86–95. PubMed

Miller LA, Smith AN, Taketo MM, Lang RA (2006) Optic cup and facial patterning defects in ocular ectoderm beta-catenin gain-of-function mice. BMC Dev Biol 6: 14. PubMed PMC

Smith AN, Miller LA, Song N, Taketo MM, Lang RA (2005) The duality of beta-catenin function: a requirement in lens morphogenesis and signaling suppression of lens fate in periocular ectoderm. Dev Biol 285: 477–489. PubMed

Stump RJ, Ang S, Chen Y, von Bahr T, Lovicu FJ, et al. (2003) A role for Wnt/beta-catenin signaling in lens epithelial differentiation. Dev Biol 259: 48–61. PubMed

Martinez G, Wijesinghe M, Turner K, Abud HE, Taketo MM, et al. (2009) Conditional mutations of beta-catenin and APC reveal roles for canonical Wnt signaling in lens differentiation. Invest Ophthalmol Vis Sci 50: 4794–4806. PubMed

Clevers H (2006) Wnt/beta-catenin signaling in development and disease. Cell 127: 469–480. PubMed

Galceran J, Hsu SC, Grosschedl R (2001) Rescue of a Wnt mutation by an activated form of LEF-1: regulation of maintenance but not initiation of Brachyury expression. Proc Natl Acad Sci U S A 98: 8668–8673. PubMed PMC

Chen Q, Liang D, Fromm LD, Overbeek PA (2004) Inhibition of lens fiber cell morphogenesis by expression of a mutant SV40 large T antigen that binds CREB-binding protein/p300 but not pRb. J Biol Chem 279: 17667–17673. PubMed

Gerido DA, Sellitto C, Li L, White TW (2003) Genetic background influences cataractogenesis, but not lens growth deficiency, in Cx50-knockout mice. Invest Ophthalmol Vis Sci 44: 2669–2674. PubMed

Gat U, DasGupta R, Degenstein L, Fuchs E (1998) De Novo hair follicle morphogenesis and hair tumors in mice expressing a truncated beta-catenin in skin. Cell 95: 605–614. PubMed

Machon O, Backman M, Machonova O, Kozmik Z, Vacik T, et al. (2007) A dynamic gradient of Wnt signaling controls initiation of neurogenesis in the mammalian cortex and cellular specification in the hippocampus. Dev Biol 311: 223–237. PubMed

Overbeek PA, Chepelinsky AB, Khillan JS, Piatigorsky J, Westphal H (1985) Lens-specific expression and developmental regulation of the bacterial chloramphenicol acetyltransferase gene driven by the murine alpha A-crystallin promoter in transgenic mice. Proc Natl Acad Sci U S A 82: 7815–7819. PubMed PMC

Graw J (2009) Genetics of crystallins: cataract and beyond. Exp Eye Res 88: 173–189. PubMed

Nielsen PA, Baruch A, Shestopalov VI, Giepmans BN, Dunia I, et al. (2003) Lens connexins alpha3Cx46 and alpha8Cx50 interact with zonula occludens protein-1 (ZO-1). Mol Biol Cell 14: 2470–2481. PubMed PMC

Xu L, Overbeek PA, Reneker LW (2002) Systematic analysis of E-, N- and P-cadherin expression in mouse eye development. Exp Eye Res 74: 753–760. PubMed

Kawauchi S, Takahashi S, Nakajima O, Ogino H, Morita M, et al. (1999) Regulation of lens fiber cell differentiation by transcription factor c-Maf. J Biol Chem 274: 19254–19260. PubMed

Duncan MK, Xie L, David LL, Robinson ML, Taube JR, et al. (2004) Ectopic Pax6 expression disturbs lens fiber cell differentiation. Invest Ophthalmol Vis Sci 45: 3589–3598. PubMed

Blixt A, Mahlapuu M, Aitola M, Pelto-Huikko M, Enerback S, et al. (2000) A forkhead gene, FoxE3, is essential for lens epithelial proliferation and closure of the lens vesicle. Genes Dev 14: 245–254. PubMed PMC

Brownell I, Dirksen M, Jamrich M (2000) Forkhead Foxe3 maps to the dysgenetic lens locus and is critical in lens development and differentiation. Genesis 27: 81–93. PubMed

Niehrs C, Acebron SP (2012) Mitotic and mitogenic Wnt signalling. EMBO J 31: 2705–2713. PubMed PMC

Griep AE (2006) Cell cycle regulation in the developing lens. Semin Cell Dev Biol 17: 686–697. PubMed PMC

Harada N, Tamai Y, Ishikawa T, Sauer B, Takaku K, et al. (1999) Intestinal polyposis in mice with a dominant stable mutation of the beta-catenin gene. EMBO J 18: 5931–5942. PubMed PMC

Lovicu FJ, Overbeek PA (1998) Overlapping effects of different members of the FGF family on lens fiber differentiation in transgenic mice. Development 125: 3365–3377. PubMed

Chen Q, Hung FC, Fromm L, Overbeek PA (2000) Induction of cell cycle entry and cell death in postmitotic lens fiber cells by overexpression of E2F1 or E2F2. Invest Ophthalmol Vis Sci 41: 4223–4231. PubMed

Duncan MK, Kozmik Z, Cveklova K, Piatigorsky J, Cvekl A (2000) Overexpression of PAX6(5a) in lens fiber cells results in cataract and upregulation of (alpha)5(beta)1 integrin expression. J Cell Sci 113 (Pt 18): 3173–3185. PubMed

Yang Y, Cvekl A (2005) Tissue-specific regulation of the mouse alphaA-crystallin gene in lens via recruitment of Pax6 and c-Maf to its promoter. J Mol Biol 351: 453–469. PubMed PMC

Piatigorsky J (1980) Intracellular ions, protein metabolism, and cataract formation. Curr Top Eye Res 3: 1–39. PubMed

Graw J (2004) Congenital hereditary cataracts. Int J Dev Biol 48: 1031–1044. PubMed

Cvekl A, Duncan MK (2007) Genetic and epigenetic mechanisms of gene regulation during lens development. Prog Retin Eye Res 26: 555–597. PubMed PMC

Kralova J, Czerny T, Spanielova H, Ratajova V, Kozmik Z (2002) Complex regulatory element within the gammaE- and gammaF-crystallin enhancers mediates Pax6 regulation and is required for induction by retinoic acid. Gene 286: 271–282. PubMed

Yang Y, Chauhan BK, Cveklova K, Cvekl A (2004) Transcriptional regulation of mouse alphaB- and gammaF-crystallin genes in lens: opposite promoter-specific interactions between Pax6 and large Maf transcription factors. J Mol Biol 344: 351–368. PubMed

Musgrove EA, Caldon CE, Barraclough J, Stone A, Sutherland RL (2011) Cyclin D as a therapeutic target in cancer. Nat Rev Cancer 11: 558–572. PubMed

Shtutman M, Zhurinsky J, Simcha I, Albanese C, D′Amico M, et al. (1999) The cyclin D1 gene is a target of the beta-catenin/LEF-1 pathway. Proc Natl Acad Sci U S A 96: 5522–5527. PubMed PMC

Tetsu O, McCormick F (1999) Beta-catenin regulates expression of cyclin D1 in colon carcinoma cells. Nature 398: 422–426. PubMed

Yamamoto N, Majima K, Marunouchi T (2008) A study of the proliferating activity in lens epithelium and the identification of tissue-type stem cells. Med Mol Morphol 41: 83–91. PubMed

Abe T, Sakaue-Sawano A, Kiyonari H, Shioi G, Inoue K, et al. (2012) Visualization of cell cycle in mouse embryos with Fucci2 reporter directed by Rosa26 promoter. Development 140: 237–246. PubMed