Conditional Mutations of ␤-Catenin and APC Reveal Roles for Canonical Wnt Signaling in Lens Differentiation Gemma Martinez,1,2 Mary Wijesinghe,1,2 Kirsty Turner,1 Helen E. Abud,3 Makoto M. Taketo,4 Tetsuo Noda,5 Michael L. Robinson,6 and Robb U. de Iongh1 PURPOSE. Previous studies indicate that the Wnt/␤-catenin–signal- orphogenesis of the vertebrate lens is initiated when the ing pathway is active and functional during murine lens develop- Moutgrowing optic vesicle induces overlying ectoderm to ment. In this study, the consequences of constitutively activating form a lens placode, which in turn invaginates to form a hollow the pathway in lens during development were investigated. ball of epithelial cells, the lens vesicle. Posterior lens vesicle cells elongate and differentiate into primary lens fiber cells, METHODS. To activate Wnt/␤-catenin signaling, ␤-catenin (Catnb) and adenomatous polyposis coli (Apc) genes were whereas anterior vesicle cells adopt a cuboid epithelial pheno- type. During embryonic development, the anterior epithelium conditionally mutated in two Cre lines that are active in whole is highly proliferative. However, during fetal and postnatal lens (MLR10) or only in differentiated fibers (MLR39), from development, proliferation becomes restricted to a small pop- E13.5. Lens phenotype in mutant lenses was investigated by ulation of epithelial cells in the germinative zone, located histology, immunohistochemistry, BrdU labeling, quantitative anterior to the lens equator. The progeny from these cell RT-PCR arrays, and TUNEL. divisions may contribute to the anterior epithelium or move RESULTS. Only intercrosses with MLR10 resulted in ocular phe- posteriorly into the transitional zone where they differentiate notypes, indicating Wnt/␤-catenin signaling functions in lens into secondary lens fiber cells, a process that continues epithelium and during early fiber differentiation. Mutant lenses throughout life.1 How cell fate is assigned in the differentiating were characterized by increased progression of epithelial cells lens has been a question that has been the focus of studies for through the cell cycle, as shown by BrdU labeling, and phos- the past 50 years, since Coulombre and Coulombre2 showed phohistone 3 and cyclin D1 labeling, and maintenance of that signals emanating from the ocular media regulate lens epithelial phenotype (E-cadherin and Pax6 expression) in the polarity. fiber compartment. Fiber cell differentiation was delayed as Several growth factor families have been shown to be in- shown by reduced expression of c-maf and ␤-crystallin and volved in regulating lens induction and differentiation.3,4 delay in expression of the CDKI, p57kip2. From E13.5, there These include the fibroblast growth factor (FGF), transforming were numerous cells undergoing apoptosis, and by E15.5, growth factor-␤ (TGF␤), and bone morphogenetic protein 4–11 there was evidence of epithelial–mesenchymal transition with (BMP) families. TGF␤ has also been shown to initiate the numerous cells expressing ␣-smooth muscle actin. Quantita- epithelial-mesenchymal transition (EMT) of lens epithelial cells, tive PCR analyses revealed large changes in expression of Wnt a process that underlies anterior subcapsular cataract and pos- 4,12 target genes (Lef1, Tcf7, T (Brachyury), and Ccnd1), Wnt terior capsule opacification. More recently, the canonical Wnt signaling pathway has also been implicated in various inhibitors (Wif1, Dkk1, Nkd1, and Frzb) and also several Wnts 4,13–15 (Wnt6, Wnt10a, Wnt8b, and Wnt11). stages of lens development. ␤ ␤ In the absence of Wnt ligand binding, cytoplasmic -catenin CONCLUSIONS. These data indicate that the Wnt/ -catenin pathway is recruited to a degradation complex (axin/GSK3␤/CK1 and plays key roles in regulating proliferation of lens stem/progenitor APC) where it is phosphorylated, resulting in its ubiquitylation cells during early stages of fiber cell differentiation. (Invest Oph- and subsequent proteasomal degradation. The canonical Wnt thalmol Vis Sci. 2009;50:4794–4806) DOI:10.1167/iovs.09-3567 signaling pathway is activated upon binding of Wnt ligands to Frizzled (Fzd)/LDL-related protein (LRP) coreceptor com- plexes, leading to activation of Fzd and the phosphorylation of 1 disheveled (Dsh) proteins. This process in turn leads to the From the Ocular Development Laboratory, Department of Anatomy ␤ and Cell Biology, University of Melbourne, Victoria, Australia; and 3Anat- recruitment of the -catenin destruction complex to the mem- omy and Cell Biology, Monash University, Clayton, Victoria, Australia; the brane. Inhibition of the destruction complex components axin 4Department of Pharmacology Graduate School of Medicine, Kyoto Uni- and GSK3␤ is mediated by active Dsh and leads to increased versity, Kyoto, Japan; the 5Department of Cell Biology, The Cancer Insti- cytoplasmic levels of hypophosphorylated ␤-catenin, which tute, Japanese Foundation for Cancer Research, Tokyo, Japan; and the translocates to the nucleus where it interacts with the LEF/TCF 6Department of Zoology, Miami University, Oxford, Ohio. 2 transcription factors to activate the transcription of target Contributed equally to the work and therefore should be consid- genes including c-myc, cyclin D1, and the ephrins. In this ered equivalent authors. context, ␤-catenin acts as the central transcriptional regulator Supported by Project Grant 400174 from the National Health and ␤ Medical Research Council (NHMRC) (RUdI, HEA) and the Rebecca L. of the canonical Wnt signaling pathway. -Catenin also func- Cooper Research Foundation for equipment. tions as a structural protein in adherens junctions where, Submitted for publication February 13, 2009; revised April 9, together with ␣-catenin, it links membrane cadherins to the 2009; accepted July 2, 2009. actin cytoskeleton. Disclosure: G. Martinez, None; M. Wijesinghe, None; K. APC has been shown to be an essential component of the Turner, None; H.E. Abud, None; M.M. Taketo, None; T. Noda, destruction complex that negatively regulates Wnt signaling None; M.L. Robinson, None; R.U. de Iongh, None via different functions of some of its domains (reviewed in The publication costs of this article were defrayed in part by page Refs. 16–18). The function of the destruction complex relies charge payment. This article must therefore be marked “advertise- ␤ ment” in accordance with 18 U.S.C. §1734 solely to indicate this fact. on the ability of APC to promote phosphorylation of -cate- Corresponding author: Robb U. de Iongh, Anatomy and Cell Biol- nin’s centrally located amino acid repeat motifs. Failure to ogy, The University of Melbourne, Parkville VIC 3010, Australia; phosphorylate ␤-catenin results in its accumulation in the cy- [email protected]. toplasm as seen in colorectal cancer exhibiting APC trunca- Investigative Ophthalmology & Visual Science, October 2009, Vol. 50, No. 10 4794 Copyright © Association for Research in Vision and Ophthalmology Downloaded from iovs.arvojournals.org on 09/25/2021 IOVS, October 2009, Vol. 50, No. 10 Role of Wnt Signaling in Lens Development 4795 tions, which do not allow the binding of APC to axin. APC has ␤-catenin (Catnblox(Ex3)) have also been described previously.34 Cre- also been shown to have additional roles unrelated to regula- mediated recombination at this locus removes the phosphorylation tion of ␤-catenin. It has been shown to bind directly and and ubiquitination sites in exon 3 and results in a stable form of indirectly to the plus end of microtubules via its C-terminal- ␤-catenin that constitutively activates Wnt/␤-catenin signaling. located basic domain, thus playing a major role in regulating MLR10 or MLR39 mice were mated with Apclox(580S) and the cytoskeleton as well as affecting cell migration and forma- Catnblox(Ex3) mice and the resultant progeny were screened by PCR tion of mitotic spindles.19–23 It has also been associated with for the desired genotype. Heterozygous progeny (Apclox(580S)/wt/ the actin cytoskeleton.21,24–26 Creϩ and Catnblox(Ex3/wt/Creϩ) were crossed until mice homo- There is considerable evidence that inhibition of canonical zygous for the Cre transgene and the floxed alleles were gener- Wnt signaling is essential for proper eye field specification in ated. Resultant mutant mice were maintained by inbreeding to various species (reviewed in Ref. 14). Consistent with this, obtain mice with desired genotypes: APC10 (Apclox(580S)/lox(580S)/ conditional loss of ␤-catenin, in murine periocular ectoderm, MLR10Creϩ), APC39 (Apclox(580S)/lox(580S)/MLR39Creϩ), ␤CATEx310 before eye formation, results in the formation of ectopic lenses (Catnblox(Ex3)/lox(Ex3)/MLR10Creϩ or CatnbEx3/lox(Ex3)/MLR10Creϩ), by the loss of Wnt signals and in abnormal ocular morphogen- or ␤CATEx339 (CatnbEx3/lox(Ex3)/MLR39Creϩ). esis by the disruption of adherens junctions.15,27 By contrast, conditional activation of ␤-catenin by deletion of exon3, which Histology and Immunofluorescence harbors the phosphorylation/ubiquitination sites, results in suppression of lens cell fate in periocular ectoderm.15,28 Other Eyes (postnatal day [P]1–P21) and embryos (embryonic day [E]13.5– E17.5) were obtained from superovulated female mutant and Wt mice. studies have also indicated that Wnt signaling plays roles dur- 6 ing later stages of lens differentiation. Components of the Wnt Superovulations were performed as previously described. Eyes or pathway, including genes for Wnts, Fzd receptors, and LRPs, as embryo heads were fixed in either 10% neutral buffered formalin (NBF)