Conditional Mutations of β-Catenin and APC Reveal Roles for Canonical Wnt Signaling in Lens Differentiation

Gemma Martinez; Mary Wijesinghe; Kirsty Turner; Helen E. Abud; Makoto M. Taketo; Tetsuo Noda; Michael L. Robinson; Robb U. de Iongh

doi:10.1167/iovs.09-3567

Abstract

purpose. Previous studies indicate that the Wnt/β-catenin–signaling pathway is active and functional during murine lens development. In this study, the consequences of constitutively activating the pathway in lens during development were investigated.

methods. To activate Wnt/β-catenin signaling, β-catenin (Catnb) and adenomatous polyposis coli (Apc) genes were conditionally mutated in two Cre lines that are active in whole lens (MLR10) or only in differentiated fibers (MLR39), from E13.5. Lens phenotype in mutant lenses was investigated by histology, immunohistochemistry, BrdU labeling, quantitative RT-PCR arrays, and TUNEL.

results. Only intercrosses with MLR10 resulted in ocular phenotypes, indicating Wnt/β-catenin signaling functions in lens epithelium and during early fiber differentiation. Mutant lenses were characterized by increased progression of epithelial cells through the cell cycle, as shown by BrdU labeling, and phosphohistone 3 and cyclin D1 labeling, and maintenance of epithelial phenotype (E-cadherin and Pax6 expression) in the fiber compartment. Fiber cell differentiation was delayed as shown by reduced expression of c-maf and β-crystallin and delay in expression of the CDKI, p57^kip2. From E13.5, there were numerous cells undergoing apoptosis, and by E15.5, there was evidence of epithelial–mesenchymal transition with numerous cells expressing α-smooth muscle actin. Quantitative PCR analyses revealed large changes in expression of Wnt target genes (Lef1, Tcf7, T (Brachyury), and Ccnd1), Wnt inhibitors (Wif1, Dkk1, Nkd1, and Frzb) and also several Wnts (Wnt6, Wnt10a, Wnt8b, and Wnt11).

conclusions. These data indicate that the Wnt/β-catenin pathway plays key roles in regulating proliferation of lens stem/progenitor cells during early stages of fiber cell differentiation.

Morphogenesis of the vertebrate lens is initiated when the outgrowing optic vesicle induces overlying ectoderm to form a lens placode, which in turn invaginates to form a hollow ball of epithelial cells, the lens vesicle. Posterior lens vesicle cells elongate and differentiate into primary lens fiber cells, whereas anterior vesicle cells adopt a cuboid epithelial phenotype. During embryonic development, the anterior epithelium is highly proliferative. However, during fetal and postnatal development, proliferation becomes restricted to a small population of epithelial cells in the germinative zone, located anterior to the lens equator. The progeny from these cell divisions may contribute to the anterior epithelium or move posteriorly into the transitional zone where they differentiate into secondary lens fiber cells, a process that continues throughout life.¹How cell fate is assigned in the differentiating lens has been a question that has been the focus of studies for the past 50 years, since Coulombre and Coulombre²showed that signals emanating from the ocular media regulate lens polarity.

Several growth factor families have been shown to be involved in regulating lens induction and differentiation.³ ⁴These include the fibroblast growth factor (FGF), transforming growth factor-β (TGFβ), and bone morphogenetic protein (BMP) families.⁴ ⁵ ⁶ ⁷ ⁸ ⁹ ¹⁰ ¹¹TGFβ has also been shown to initiate the epithelial-mesenchymal transition (EMT) of lens epithelial cells, a process that underlies anterior subcapsular cataract and posterior capsule opacification.⁴ ¹²More recently, the canonical Wnt signaling pathway has also been implicated in various stages of lens development.⁴ ¹³ ¹⁴ ¹⁵

In the absence of Wnt ligand binding, cytoplasmic β-catenin is recruited to a degradation complex (axin/GSK3β/CK1 and APC) where it is phosphorylated, resulting in its ubiquitylation and subsequent proteasomal degradation. The canonical Wnt signaling pathway is activated upon binding of Wnt ligands to Frizzled (Fzd)/LDL-related protein (LRP) coreceptor complexes, leading to activation of Fzd and the phosphorylation of disheveled (Dsh) proteins. This process in turn leads to the recruitment of the β-catenin destruction complex to the membrane. Inhibition of the destruction complex components axin and GSK3β is mediated by active Dsh and leads to increased cytoplasmic levels of hypophosphorylated β-catenin, which translocates to the nucleus where it interacts with the LEF/TCF transcription factors to activate the transcription of target genes including c-myc, cyclin D1, and the ephrins. In this context, β-catenin acts as the central transcriptional regulator of the canonical Wnt signaling pathway. β-Catenin also functions as a structural protein in adherens junctions where, together with α-catenin, it links membrane cadherins to the actin cytoskeleton.

APC has been shown to be an essential component of the destruction complex that negatively regulates Wnt signaling via different functions of some of its domains (reviewed in Refs. ¹⁶ ¹⁷ ¹⁸ ). The function of the destruction complex relies on the ability of APC to promote phosphorylation of β-catenin’s centrally located amino acid repeat motifs. Failure to phosphorylate β-catenin results in its accumulation in the cytoplasm as seen in colorectal cancer exhibiting APC truncations, which do not allow the binding of APC to axin. APC has also been shown to have additional roles unrelated to regulation of β-catenin. It has been shown to bind directly and indirectly to the plus end of microtubules via its C-terminal-located basic domain, thus playing a major role in regulating the cytoskeleton as well as affecting cell migration and formation of mitotic spindles.¹⁹ ²⁰ ²¹ ²² ²³It has also been associated with the actin cytoskeleton.²¹ ²⁴ ²⁵ ²⁶

There is considerable evidence that inhibition of canonical Wnt signaling is essential for proper eye field specification in various species (reviewed in Ref. ¹⁴ ). Consistent with this, conditional loss of β-catenin, in murine periocular ectoderm, before eye formation, results in the formation of ectopic lenses by the loss of Wnt signals and in abnormal ocular morphogenesis by the disruption of adherens junctions.¹⁵ ²⁷By contrast, conditional activation of β-catenin by deletion of exon3, which harbors the phosphorylation/ubiquitination sites, results in suppression of lens cell fate in periocular ectoderm.¹⁵ ²⁸Other studies have also indicated that Wnt signaling plays roles during later stages of lens differentiation. Components of the Wnt pathway, including genes for Wnts, Fzd receptors, and LRPs, as well as modulators of Wnt activity, such as the secreted frizzled-related proteins (Sfrps) and Dikkopf (Dkks), have been found to be expressed primarily in the lens epithelium from the early lens vesicle stages through to the postnatal stages (reviewed in Ref. ¹⁴ ). Moreover, analysis of the Lef/TCF reporter lines has shown activity of Wnt signaling in embryonic lens epithelium.²⁹ ³⁰The requirement for Wnt signaling during lens differentiation was first demonstrated in Lrp6 null mice, which have lenses with a deficient anterior epithelium.³¹More recent studies of mice with a conditional null mutation of β-catenin¹³show that Wnt signaling affects lens epithelial stem/progenitor cells; these mice also have a deficient epithelial layer with loss of E-cadherin expression, epithelial cell cycle arrest at the G₁-S transition and premature cell cycle exit. Fiber cell differentiation in these β-catenin-null lenses was also compromised, with disrupted cell elongation, decreased β-crystallin expression, and abnormal extracellular matrix (ECM) expression.

In this study, we report that activation of the Wnt pathway in the lens by truncation of APC or by an activating mutation of β-catenin, results in the expansion of lens progenitor cells, inhibition of fiber cell differentiation and partial commitment to a mesenchymal fate. These effects are only observed if the pathway is activated in lens epithelium and fiber cells but not in differentiated fiber cells alone. The results indicate that the Wnt pathway plays an essential role in the regulation of lens stem/progenitor cells and early phases of fiber cell differentiation.

Materials and Methods

All experimental procedures on animals conformed to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and were approved by the Animal Ethics Committees of the University of Melbourne.

Generation of Conditional Mutants

Transgenic mice expressing Cre recombinase under the control of the αA-crystallin promoter (fiber cells only; MLR39) or with the inclusion of a Pax6 enhancer element (epithelial and fiber cells; MLR10) have been described previously.³²The floxed APC mouse (Apc ^lox ⁽ ^580S ⁾) in which LoxP sites have been inserted into the introns flanking exon 14 of the Apc gene have also been described previously.³³Cre-mediated recombination at this locus results in deletion of exon 14 and a downstream frameshift mutation at codon 580. The resultant mutant APC protein is unable to participate in β-catenin phosphorylation, ubiquitylation and degradation. Mice harboring the floxed exon 3 of β-catenin (Catnb ^lox ⁽ ^Ex3 ⁾) have also been described previously.³⁴Cre-mediated recombination at this locus removes the phosphorylation and ubiquitination sites in exon 3 and results in a stable form of β-catenin that constitutively activates Wnt/β-catenin signaling.

MLR10 or MLR39 mice were mated with Apc ^lox ⁽ ^580S ⁾ and Catnb ^lox ⁽ ^Ex3 ⁾ mice and the resultant progeny were screened by PCR for the desired genotype. Heterozygous progeny (Apc ^lox ⁽ ^580S ⁾ ^/wt/Cre⁺ and Catnb ^lox ⁽ ^Ex3/wt/Cre⁺) were crossed until mice homozygous for the Cre transgene and the floxed alleles were generated. Resultant mutant mice were maintained by inbreeding to obtain mice with desired genotypes: APC10 (Apc ^lox ⁽ ^580S ⁾ ^/lox ⁽ ^580S ⁾/MLR10Cre⁺), APC39 (Apc ^lox ⁽ ^580S ⁾ ^/lox ⁽ ^580S ⁾/MLR39Cre⁺), βCAT^Ex310 (Catnb ^lox ⁽ ^Ex3 ⁾ ^/lox ⁽ ^Ex3 ⁾/MLR10Cre⁺ or Catnb ^Ex3/lox ⁽ ^Ex3 ⁾/MLR10Cre⁺), or βCAT^Ex339 (Catnb ^Ex3/lox ⁽ ^Ex3 ⁾/MLR39Cre⁺).

Histology and Immunofluorescence

Eyes (postnatal day [P]1–P21) and embryos (embryonic day [E]13.5–E17.5) were obtained from superovulated female mutant and Wt mice. Superovulations were performed as previously described.⁶Eyes or embryo heads were fixed in either 10% neutral buffered formalin (NBF) and embedded in paraffin or in fresh 4% paraformaldehyde (PFA) in PBS before cryoprotection with 30% sucrose and embedding in OCT compound (Tissue-Tek; Sakura Fintek, Tokyo, Japan).

Paraffin sections (4–7 μm) were dewaxed and rehydrated through decreasing graded ethanol series to water before staining with hematoxylin and eosin, periodic acid Schiff or immunofluorescence. Paraffin-embedded sections used for immunostaining were subjected to heat-mediated antigen retrieval with 0.01 M citrate buffer (pH 6.0) containing 0.05% Tween-20, as described previously.¹³Frozen and paraffin-embedded sections were blocked with 3% to 5% normal goat serum (NGS) in 0.1% BSA/PBS for 20 minutes before they were incubated with primary antibodies diluted in blocking solution. The primary antibodies used were mouse anti-E-cadherin (1:200; cat. no. 610182; BD Transduction, Lexington, KY), mouse anti-β-crystallin (Clone 3H9; hybridoma supernatant, the kind gift of Robert C. Augusteyn, Vision CRC, Sydney, Australia), mouse anti-active β-catenin (1:100; 05-665; Millipore, Billerica MA), rabbit anti-APC (kind gift of Inke Nathke, University of Dundee),³⁵rabbit anti-phospho-histone H3 (1:200, 07-424; Millipore), mouse anti-α-smooth muscle actin (1:200, A2547; Sigma-Aldrich, Castle Hill, NSW, Australia), rabbit anti-p57^kip2 (1:200; 4058-500; AbCam, Cambridge, UK) and rabbit monoclonal anti-cyclin D1 (cat no. 21699 (SP4); Abcam) at 4°C overnight. APC immunohistochemistry required sections to be permeabilized with CSK buffer (50 mM NaCl, 300 mM sucrose, 10 mM PIPES [pH 6.3], 3 mM MgCl₂ with 0.5% Triton X-100) at room temperature for 10 minutes and blocked with 20% NGS and 1% BSA in PBS before incubation with primary antibody. Secondary antibodies used were HRP-conjugated goat-anti mouse (1:500, 65-6120; Invitrogen, Mt. Waverley, VIC, Australia), HRP-conjugated goat-anti rabbit (1:200, 65-6520; Invitrogen) or AlexaFluor488-conjugated anti-mouse or anti-rabbit IgG (1:500; A-11008, A-11001; Invitrogen) diluted in blocking solution for 2 hours at room temperature. Sections labeled with HRP-conjugated secondary antibodies were developed with diaminobenzidine (DAB) and were counterstained with hematoxylin, dehydrated, and mounted in DPX. For fluorescent labeling of nuclei, the sections were stained with Hoechst dye (Sigma-Aldrich) for 10 minutes before they were washed with PBS and mounted with fluorescent mounting medium (Dako Cytomation, Inc., Carpinteria, CA).

Bromodeoxyuridine (BrdU) Labeling

BrdU labeling was performed to quantify cell proliferation in lenses. Pregnant mice were injected with 0.1 mg 5-bromo-2′-deoxyuridine (BrdU) in PBS/g body weight and 0.1 mg 5-fluoro-2′deoxyuridine (FldU) in PBS per 10g body weight 1 to 1.5 hours before collection and fixation of embryos. Rehydrated paraffin-embedded sections were treated with 2 M HCl at room temperature for 30 minutes to denature DNA followed by neutralization with 0.1 M sodium borate at room temperature for 10 minutes. The sections were washed with PBS and incubated with 0.12% trypsin in PBS at 37°C for 25 minutes and then processed for anti-BrdU immunofluorescence. They were blocked with 3% NGS in 0.1% BSA in PBS at room temperature for 20 minutes and incubated with monoclonal anti-BrdU antibody (1:50; Bioclone, Sydney, NSW, Australia) at 4°C overnight. The secondary antibody was HRP-conjugated goat anti-mouse (1:300) incubated at room temperature for 2 hours. Color development was performed with DAB, and the sections were counterstained with hematoxylin, dehydrated, and mounted in DPX. BrdU⁺ and total cell counts were quantified in the epithelial and fiber compartments in sections from three to six E13.5 embryos. Data are expressed as the mean (± SEM) percentage of BrdU⁺ cells in each compartment, analyzed by ANOVA and Student’s t-test.

TUNEL Assay

To detect cells undergoing apoptosis, a TUNEL system (DeadEnd Fluorometric; Promega, Sydney, NSW, Australia) was used on rehydrated paraffin-embedded sections, according to the manufacturer’s instructions.

PCR Array Profiling

Mouse Wnt PCR arrays (Signaling Pathway RT² Profiler PCR Arrays; SuperArray Bioscience Corp., Frederick, MD) were used to interrogate the expression of 84 genes involved in Wnt-mediated signal transduction. The arrays include primers coding for members of the Frizzled and Wnt families, Wnt signaling pathway regulators and competitive antagonists, intracellular signaling mediators, pathway target genes, and genes involved in protein modification downstream of Wnt signaling, including genes involved in kinase and phosphatase activity and ubiquitination. The arrays also contain primers coding for a panel of five housekeeping genes to normalize PCR array data, genomic DNA control primers, reverse transcription controls and positive PCR controls.

Embryonic lenses were dissected from at least two litters of wild-type, APC10 or βCAT^Ex310 embryos at E14.5, taking care to remove surrounding optic cup tissue. As the embryonic lens is invested with the tunica vasculosa lentis, inclusion of endothelial cells and blood cells could not be excluded. Total RNA was isolated from pooled lens vesicles (RNeasy Mini Kit; Qiagen Pty Ltd., Doncaster, VIC, Australia) with on-column DNase-digestion. RNA integrity and concentration were quantified with a spectrophotometer (NanoDrop; ThermoFisher Scientific, Wilmington, DE). For each array, 1 μg RNA from each sample was reverse transcribed (RT² First Strand kit; SuperArray Bioscience) and the resultant cDNA was amplified by real-time PCR (RT² Real-Time SYBR Green PCR master mix; SuperArray Bioscience) as follows: melting for 10 minutes at 95°C, 40 cycles of two-step PCR including melting for 15 seconds at 95°C, and annealing for 1 minute at 60°C. The cycling program was immediately followed by a melting curve analysis to confirm generation of a single amplicon. All reverse transcriptions and real-time PCR analyses were performed in triplicate and average C _t values for each gene were analyzed by the ΔΔC _t method using commercial analysis template (Excel; SuperArray Bioscience). Relative expression values were determined between wild-type and APC10 or βCAT^Ex310 samples by normalizing differences in ΔC _t values (ΔΔC _t) to the average of five housekeeping genes. The change (x-fold) of gene expression in samples was calculated as 2^{−ΔΔC _t}.

Results

Ocular Phenotype of Mutant Mice

Mutant mice derived from MLR10 crosses (βCAT^Ex310, APC10,) had microphthalmia that was evident from eye opening (Figs. 1B 1D) . By contrast, mice derived from MLR39 crosses (APC39, Fig. 1C ; βCAT^Ex339, not shown) had apparently normal ocular phenotype similar to that of wild-type mice (Fig 1A) . Histologic examination of neonatal (P2) eyes revealed gross disturbances of lens differentiation in APC10 and βCAT^Ex310, but not APC39 or βCAT^Ex339 lenses. βCAT^Ex310 lenses were characterized by an irregular multilayered anterior epithelium and an abnormal fiber compartment that contained numerous epithelioid cells and poorly elongated fiber cells (Fig. 1F) . The hyaloid vasculature appeared hypertrophic. The APC10 lenses at P2 were characterized by the lack of a well defined epithelium and highly vacuolated fiber mass that often ruptured through the posterior lens capsule into the vitreous chamber (Fig. 1F) .

Epithelial Hypertrophy and Disrupted Fiber Differentiation in Embryonic Lenses

Previous studies have shown that Cre-mediated recombination in MLR10- and MLR39-derived lines is complete at E13.5.¹³Histologic analysis of wild-type and mutant lenses at E13.5 and at E15.5 showed that MLR39-derived mice had the characteristic epithelial and fiber cell arrangement similar to wild-type lenses (Figs. 2A 2B 2E 2F) . In these lenses, the epithelium covered the anterior surface of the lens and cells at the equator began to elongate into lens fiber cells. However, in E13.5 βCAT^Ex310 and APC10 lenses, the cells at the equator failed to elongate, maintained an epithelial phenotype, and continued to migrate along the posterior capsule (Figs. 2C 2D) . Fiber cell elongation appeared to be inhibited in both lines and there was no evidence of the characteristic bow zone. The structure of the optic cup neuroepithelium in both lines appeared normal and similar to wild-type.

The phenotypes of the βCAT^Ex310 and APC10 lenses began to diverge at E15.5. In E15.5 βCAT^Ex310 lenses, there was a distinct anterior epithelium but the fiber compartment was populated by numerous epithelioid cells surrounding a large space containing degenerate cells (Fig 2G) . By contrast, the E15.5 APC10 lenses comprised a multilayered anterior epithelium that was indistinguishable from the overlying corneal endothelium and overlaid a mass of enlarged swollen cells. The fiber mass was largely degenerated and vacuolated with numerous clumps of cells populating the posterior capsule (Fig. 2H) . In many cases the lens fiber mass also contained numerous red blood cells indicating penetration of the hyaloid vessels into the lens.

Activation of the Wnt Pathway in Mutant Lenses

In Apc ^lox ⁽ ^580S ⁾ mice, the Apc gene has LoxP sites flanking exon 14. Cre-mediated excision of exon 14 in APC10 conditional mutants results in a frameshift truncation mutation of the APC protein that can no longer bind β-catenin in lens epithelial and fiber cells. As a result β-catenin is not ubiquitinated or degraded and accumulates in the cytoplasm and nuclei. To confirm effective recombination of the Apc gene in APC10 lenses, immunohistochemistry was performed with an APC antibody raised to amino acid residues 1034 to 2130 of the human APC protein that recognizes part of the protein downstream of the frameshift mutation.³⁵ ³⁶APC immunostaining of E13.5 wild-type lenses showed abundant expression of wild-type APC in epithelial and fiber cells (Fig. 3A) . By contrast, APC10 lenses had a marked loss of APC reactivity in both epithelial and fiber cells but not in the overlying cornea (Fig. 3B) , indicating efficient truncation of APC in the lens. Localization patterns of APC in E15.5 wild-type and APC10 lenses were similar to that observed at E13.5 (data not shown). To confirm that there was activation of Wnt signaling we used an antibody that recognizes active β-catenin. In wild-type lenses, this antibody recognizes membrane-associated β-catenin (Fig. 3E) , with very little nuclear reactivity, similar to previous studies³¹ ; however, in APC10 lenses, there was increased cytoplasmic staining, particularly in the epithelioid cells populating the fiber compartment (Fig. 3F) . Distinct nuclear β-catenin reactivity was evident in many lens cells but not in cells outside the lens (Figs. 3G 3H) .

In Catnb ^lox ⁽ ^Ex3 ⁾ mice, exon3 of the β-catenin gene was flanked by LoxP sites, and Cre-mediated recombination resulted in a form of β-catenin that cannot be phosphorylated and targeted for proteasomal destruction. In wild-type lenses, β-catenin was detectable in all cells but was particularly strong along epithelial cell membranes (Fig. 3C) . However, in βCAT^Ex310 lenses, there was dramatically increased localization of β-catenin throughout the lens, particularly in the epithelioid cells in the fiber compartment (Fig. 3D) . The reactivity was localized along membranes, in the cytoplasm and also in nuclei. The localization patterns strongly indicate that in these lenses there is activation of the Wnt pathway, consistent with previous studies using Apc ^lox ⁽ ^580S ⁾ and Catnb ^lox ⁽ ^Ex3 ⁾ mice.³³ ³⁴ ³⁷ ³⁸ ³⁹ ⁴⁰

To further confirm activation of the Wnt pathway, RNA was isolated from E14.5 APC10 and βCAT^Ex310 lens vesicles and assayed using quantitative PCR arrays. When this approach was used, 38 of 84 Wnt pathway-associated genes in the array showed at least a twofold significant (P < 0.05) difference in expression in either APC10 or βCAT^Ex310 compared with wild-type (Table 1and Supplementary Data). The data showed remarkably concordant results for both APC10 and βCAT^Ex310 mice, with 20 genes upregulated and 14 genes downregulated significantly (P < 0.05) in both samples. Of genes showing a greater than 1.5-fold significant change, only five genes (Wnt1, Wnt4, Fshb, Fzd2, and Fzd5) showed discordant patterns, being significantly upregulated in one and significantly downregulated in the other (Supplementary Data). Consistent with the activation of canonical Wnt signaling in both lines, there was a similar significant upregulation of many Wnt target genes such as Lef1 (59- and 31-fold, respectively), Brachyury (T; 16- and 11-fold), Tcf7 (7- and 5-fold) and Ccnd1 (2.8, 1.5-fold). There was also dramatic upregulation of Wnt inhibitors such as Wif1 (392, 287-fold), Dkk1 (70, 21-fold), Frzb (14-fold) and the naked cuticle homologue, Nkd1 (25- and 13-fold). Of interest, some inhibitors such as Sfrp1, Sfrp2, the Groucho-related gene (Aes), and the ubiquitin ligase complex gene (B trc) were significantly downregulated. Many of the Wnt ligands were also highly upregulated, including Wnts associated with canonical (Wnt10a, Wnt8b) and noncanonical (Wnt4, Wnt5a, and Wnt11) signaling and Wnt6, which has been implicated in EMT.⁴¹ ⁴²Three Wnt genes, Wnt7a, Wnt7b, and Wnt8a were significantly downregulated; both Wnt7b and Wnt8a are known to have canonical effects, whereas Wnt7a appears to initiate both canonical and noncanonical signals, depending on the Frizzled receptor (Fzd5 or Fzd10) it binds.⁴³Of the Wnt receptors, Fzd8 and Lrp5 were upregulated but Fzd5 and Fzd3 were moderately downregulated.

Maintenance of Epithelial Phenotype by Constitutive Activation of Wnt Signaling

A predominant feature of the lens phenotypes in APC10 and βCAT^Ex310 lenses was the persistence of epithelial-like cells in the posterior fiber compartment. To determine whether these cells retained epithelial cell markers, we examined the expression of E-cadherin and Pax6 by immunofluorescence at E13.5. In wild-type and βCAT^Ex339 lenses, E-cadherin expression was detected uniquely in the epithelium, with little or no staining below the equator in the transitional zone or fiber cell compartment (Figs. 4A 4B) . βCAT^Ex310 and APC10 lenses also expressed E-cadherin in the epithelium, but in both mutants, persistent E-cadherin reactivity was detected in transitional zone cells below the equator and in the fiber compartment (Figs. 4C 4D) . Intriguingly, E-cadherin was downregulated in many cells at the posterior pole, suggesting that other factors may downregulate E-cadherin. In older βCAT^Ex310 lenses at E15.5, similar aggregations of E-cadherin-positive, epithelial-like cells were observed in the fiber compartment (data not shown).

In wild-type and βCAT^Ex339 lenses, Pax6 was localized in the nuclei and cytoplasm of epithelial and transitional zone cells, with a sharp reduction in expression in differentiating fiber cells (Figs. 4E 4F) . In βCAT^Ex310 and APC10 mutant lenses, nuclear Pax6 reactivity was detected in all cells, including the epithelioid cells in the fiber cell compartment (Figs. 4G 4H) .

Delayed Fiber Cell Differentiation in βCAT^Ex310 and APC10 Lenses

The epithelial marker expression indicated that cells in the fiber compartment retained aspects of an epithelial phenotype. To examine whether these cells had undergone fiber cell differentiation, we examined the expression of β-crystallin, c-Maf, and Prox1. β-Crystallin is a characteristic marker for initiation of fiber differentiation. At E13.5, β-crystallin is normally expressed in the transitional zone cells undergoing extensive elongation and in terminally differentiated fibers in the center of the lens (Fig. 5A) . A similar expression was detected in βCAT^Ex339 lenses (Fig. 5B) . However, in βCAT^Ex310 and APC10 mutant lenses, β-crystallin expression was significantly delayed, with most epithelioid cells not showing any reactivity (Figs. 5C 5D) . Only cells in the center of the lens showed distinct β-crystallin expression.

The transcription factors, c-Maf (Figs. 5E 5F)and Prox1 (not shown) are predominantly expressed in fiber cell nuclei of wild-type lenses.⁴⁴ ⁴⁵In mutant lenses, expression of c-Maf appeared to be markedly reduced and delayed (Figs. 5G 5H) . By contrast, Prox1 expression did not seem to be greatly affected, being clearly detectable in nuclei of fiber compartment cells of βCAT^Ex310 mutant lenses (not shown).

Abnormal ECM Protein Expression in βCAT^Ex310 and APC10 Lenses

As the phenotype at postnatal ages suggested insufficiency of the lens capsule we examined the expression of two key lens capsule proteins: laminin and collagen IV. In wild-type lenses, both laminin and collagen IV were predominantly detected in epithelial cells and in the posterior capsule (Figs. 6A 6D) . However, in both βCAT^Ex310 and APC10 mutant lenses (Figs. 6B 6C 6E 6F) , cellular expression of these proteins was increased and extended into the fiber compartment. Paradoxically, the distinct expression in the basement membrane of the posterior lens capsule was decreased and difficult to detect. This result suggests that, although there is increased cellular production of ECM proteins, there appears to be altered deposition or organization of the basement membrane.

Dysregulated Cell Cycle in βCAT^Ex310 and APC10 Lenses

Wnt signals are known to regulate the cell cycle and, as the phenotype of mutant lenses suggested altered regulation of lens epithelial progenitor cells, we examined cell cycle markers. BrdU incorporation assays showed that wild-type and βCAT^Ex339 lenses had normal patterns of epithelial cell proliferation, similar to that documented previously (Figs. 7A 7B) . Only an occasional BrdU⁺ cell was detected, just below the lens equator. However, in mutant βCAT^Ex310 and APC10 lenses, BrdU incorporation was detected, not only in the anterior epithelium but also in cells of the posterior fiber compartment (Figs. 7C 7D) . Quantification of BrdU incorporation in wild-type lenses showed that, compared with wild-type lenses, there was a significant (P < 0.05) increase in cell proliferation in the anterior epithelial layer of βCAT^Ex310 (39.1% ± 3.4% vs. 24.2% ± 1.8%), but not APC10 (18.6% ± 1.6% vs. 14.8% ± 1.7%) lenses. The most dramatic changes occurred in the fiber compartment (Figs. 7E 7F) , where both mutants showed significant increases in BrdU⁺ cells compared to wild-type (βCAT^Ex310, 20.2% ± 2.3% vs. 0.2% ± 0.2%; APC10, 14.0% ± 1.2% vs. 1.9% ± 0.5%).

To assess changes in the cell cycle, we also examined markers for M-phase (phosphohistone 3; PH3), G₁-S phase (cyclin D1), and cell cycle exit (p57^kip2). PH3 expression is associated with condensed chromosomes at the G₂-M transition. In wild-type (Fig. 8A)and βCAT^Ex339 (Fig. 8B)lenses, PH3 expression was detected in a few anterior epithelial cells. By contrast, βCAT^Ex310 (Fig. 8C)and APC10 (Fig. 8D)lenses contained PH3-positive cells in both the epithelial layer and in the fiber compartment, suggesting persistence of cell cycle activity in this normally postmitotic lens compartment.

In E13.5 wild-type and βCAT^Ex339 lenses, cyclin D1 was strongly expressed in cells of the germinative and early transitional zones (Figs. 8E 8F)with expression decreasing as cells undergo terminal differentiation. Lenses of βCAT^Ex310 and APC10 mice, however, displayed abnormal and persistent cyclin D1 expression in cells of the fiber compartment (Figs. 8G 8H) , suggesting persistence of the cell cycle. Consistent with these observations of BrdU, PH3, and cyclin D1, analysis of the cyclin-dependent kinase inhibitor p57^kip2 showed that there was delayed cell cycle exit in these cells. In wild-type and βCAT^Ex339 lenses, p57^kip2 expression was detected in cells just below the lens equator (Figs. 8I 8J) . However, in βCAT^Ex310 and APC10 mutants, many of the epithelioid cells below the equator were negative for p57^kip2 expression (Figs. 8K 8L)reflecting a delay in cell cycle exit and concomitant fiber differentiation.

Increased Apoptosis in βCAT^Ex310 and APC10 Lenses

Disruptions of cell cycle often lead to apoptosis. TUNEL assays showed that in wild-type lenses at E13.5 and at E15.5, apoptosis was virtually absent (Figs. 9A 9D) . However, in both mutant lenses at E13.5 and E15.5 there were abundant TUNEL⁺ cells in both the epithelial and fiber cell compartments (Figs. 9B 9C 9E 9F) .

EMT in Mutant Lenses

The morphology of cells in the mutant lenses, particularly the APC10 lenses at E15.5, was clearly abnormal (Fig 2H) . As activation of the Wnt/β-catenin pathway has been associated with EMT, we examined the expression of the mesenchymal marker, α-smooth muscle actin (α-sma). Normally, distinct fibrillar reactivity for α-sma was not detectable in wild-type lenses (Fig. 10A) . However, immunostaining for α-sma of βCAT^Ex310 and APC10 lenses showed discrete expression in numerous cells (Figs. 10B 10D) , which had a highly elongated morphology and resembled myofibroblasts. In βCAT^Ex310 mutants, α-sma-positive cells were detected in both the anterior epithelial and posterior fiber compartments. By contrast in APC10 lenses, α-sma-positive cells were detected only in the anterior part of the lens.

Discussion

In several studies, β-catenin and the canonical Wnt signaling have been found to participate in lens development.¹³ ¹⁵ ²⁷ ³¹ ⁴⁶During early stages of lens induction, Wnt signals in the presumptive lens ectoderm must be suppressed for lens formation to occur.¹⁵ ²⁷However, during lens differentiation, inhibition of Wnt signals by loss of Lrp6 ³¹or by loss of β-catenin¹³results in abnormal lens differentiation, characterized by deficits in the epithelium due to diminished cell proliferation and defective fiber cell differentiation with reduced levels of β-crystallin. These studies suggest that there are distinct stages of Wnt activity during lens induction and differentiation. In this study, we used conditional mutations of Catnb and Apc alleles to examine the effects of overactivating the Wnt/β-catenin signaling pathway during early lens differentiation.

Effect of Wnt Pathway Activation on Epithelial and Early Differentiating Fiber Cells

Cre-mediated recombination at the APC^580S allele³³results in a mutant APC protein that inactivates the β-catenin degradation complex and thus constitutively activates the Wnt/β-catenin pathway. Similarly, deletion of exon-3 of β-catenin results in a protein that is resistant to ubiquitination and degradation and also constitutively activates this pathway. In this study, we took advantage of the specificity of two lens-specific transgene promoters¹³ ³²that drive Cre expression in the whole lens (MLR10) or in the lens fibers only (MLR39) to determine whether there were any lens compartment-specific effects of activating the Wnt pathway. The lack of phenotype in mice derived from the MLR39 line indicates that activation of the Wnt pathway in differentiating fiber cells of the lens cortex does not alter their fate or the differentiation process. Similarly, our previous study has shown that loss of β-catenin in these cells, which are already committed to differentiate, also does not affect lens fiber differentiation.¹³Lyu and Joo⁴⁷showed that in vitro, Wnt signals can act synergistically with FGFs to mediate expression of βB2-crystallin in lens epithelial cells undergoing fiber differentiation. In the present study we did not investigate whether there were any alterations in the ratio of crystallins expressed in these lenses. However, the normal total β-crystallin reactivity and the lack of any morphologic effects on fiber differentiation in APC39 and βCAT^Ex339 mice suggest that any synergistic effects of Wnt signals with FGFs are likely to occur before the stage of fiber differentiation at which Cre becomes active in these mice.

By contrast, both lines generated with the MLR10 line, APC10, and βCAT^Ex310, showed severe microphthalmia, indicating that overactivation of the Wnt pathway has profound effects on lens epithelial cells and early differentiating fibers in the transitional zone. The end-stage phenotypes observed in weanling and newborn mice suggest that truncation of APC and loss of β-catenin exon-3 result in different pathologies. The APC mutation resulted in more severely degenerate lenses, whereas the β-catenin exon-3 mutation resulted in lenses with recognizable structure and numerous epithelial-like cells in the fiber compartment. However, examination of the phenotypes at E13.5, just after gene recombination occurs, revealed that initially the phenotypes were highly similar and appeared to be due to Wnt-pathway–mediated effects. Subsequently, from E15.5 onward, the phenotypes in these mice started to diverge. Cells in E15.5 APC10 lenses ceased to proliferate, whereas cells in the E15.5 βCAT^Ex310 lens continued to proliferate. The patterns of cell differentiation and apoptosis also varied. The APC mutation resulted in the loss of a large part of the carboxyl domains of the APC protein, with functions not only in Wnt signaling but also in microtubule association (via Kap3/KIF and EB1 domains), actin binding (via ASEF and PDZ domains) and nuclear export and localization (via nuclear export sequences and nuclear localization sequences).¹⁸ ⁴⁸ ⁴⁹Therefore, APC10 lenses may have Wnt-pathway–independent defects in processes regulated by these domains, such as cell migration, proliferation, and death, thus accounting for the divergence in phenotype between APC10 and βCAT^Ex310 lenses. Consistent with these putative roles of APC in cytoskeletal dynamics and migration, there are subtle differences in phenotype already apparent at E15.5 (Figs. 2 10) , suggestive of migration defects. In APC10 lenses, the epithelial cells accumulate at the anterior margin as opposed to the posterior compartment in βCAT^Ex310 lenses. Similarly, the α-sma-positive cells in the APC10 lenses are restricted to the anterior compartment but are distributed in both compartments of βCAT^Ex310 lenses. Studies are in progress to investigate in more detail the phenotypes of these mice at stages after E15.5 to determine the contribution of these other functions of APC to the mutant phenotypes.

Consistent with previous studies, Cre-mediated mutation of Apc and Catnb ^Ex3 alleles resulted in overactivation of the Wnt signaling pathway in βCAT^Ex310 and APC10 lenses. In both models, there was increased expression and nuclear localization of β-catenin at E13.5, which is when the recombination is complete. Further analyses by real-time PCR at E14.5 were also consistent with activation of Wnt signaling, showing dramatic upregulation of Wnt target genes (Lef1, Brachyury, Tcf7, and cyclin D1), canonical Wnt inhibitor genes (Wif1, Dkk1, Frzb, kremen1, and Nkd1) and putative noncanonical Wnt pathway genes (Rhou, Wnt4, Wnt5a, Wnt11, and Fz8). The upregulation of noncanonical Wnt signaling components is intriguing, as these pathways have been reported to be antagonistic to canonical Wnt signaling.⁵⁰It has been suggested that the Wnt/planar cell polarity pathway functions in regulating cytoskeletal polarity and cell adhesion during fiber cell differentiation,⁵¹ ⁵²thus increased expression of components in this pathway could compound the phenotypes observed in βCAT^Ex310 and APC10 mice. It is unclear whether the observed upregulation of several Wnt inhibitors (Wif1, Dkk1, Nkd1, Frzb, and Sfrp4) in these lenses is due to direct activation by β-catenin at Lef/Tcf sites or whether it reflects a more general feedback response to the overactivation of β-catenin signaling. An intriguing finding is that, Sfrp2, a Wnt inhibitor that was not upregulated in βCAT^Ex310 and APC10 lenses, has been shown in transgenic overexpression experiments to bind Wnt7a and to affect cytoskeletal organization of differentiating lens fiber cells,⁵²suggesting that distinct Wnt-Frz and antagonist combinations regulate canonical and noncanonical signals in the lens.

In this study we limited our analyses predominantly to E13.5, the stage at which gene recombination in the lens cells is complete and thus only investigated the immediate effects of activating the Wnt/β-catenin pathway. This method also allowed comparison with our previous analyses of mice with conditional deletion of β-catenin in epithelium and fibers to contrast with loss of Wnt/β-catenin signals.¹³

Effect of Activation of the Wnt Pathway on Epithelial Phenotype and Fiber Differentiation

In both APC10 and βCAT^Ex310 mice at E13.5, the lens phenotype was characterized by overproliferation of lens epithelial cells and their delayed differentiation. The cells maintained expression of the epithelial markers E-cadherin and Pax6, but had delayed expression of the fiber cell markers β-crystallin and c-maf. Moreover analysis of cell cycle markers indicated that these epithelioid cells in the fiber compartment failed to exit the cell cycle (lack of p57^Kip2 expression) and continued to progress through the G₁, S, and M phases of the cell cycle. Similarly, our previous studies¹³showed that loss of β-cateinin in the lens results in cell cycle arrest in G₁. These observations are consistent with other studies showing that Wnt/β-catenin signals regulate cell proliferation in the brain⁵³and other tissues.³⁷ ³⁸ ⁵⁴

The maintenance of lens epithelial phenotype by activation of Wnt signaling in this study is consistent with in vitro studies,⁵⁵which assessed the effects of LiCl on lens epithelial explants. LiCl is known to inhibit GSK3β and thus potentially activates Wnt signaling. In an intriguing finding, although addition of LiCl induced migratory lens cells to adopt a more epithelial phenotype, with stabilization of tight junctions and E-cadherin-based adhesion junctions, there was decreased cell proliferation and inhibition of α-sma expression in response to TGFβ. This result contrasts with the increased proliferation and induction of α-sma in this study in response to activation of Wnt signaling. As indicated by these workers, it is likely that LiCl has effects other than just activating the Wnt pathway and may also affect signaling via Src kinase and expression of Slug and Smad4.⁵⁵Similarly, the in vivo context of the current studies cannot account for other signaling pathways that influence lens cell responses. In addition, active Wnt signaling in vivo, detected using Lef/TCF reporters, only occurs in lens at E13–14. The lack of activity of these reporters in the lens at later postnatal stages, from which explants are usually obtained, suggests that the differences may also be stage dependent.

The results presented herein indicate that inappropriate Wnt/β-catenin signals also impact on early stages of fiber cell differentiation. Exit from the cell cycle is essential for fiber cell differentiation and is temporally linked with β-crystallin expression during normal fiber differentiation.⁵⁶Key regulators of crystallin expression are the Maf transcription factors.⁵⁷Overactivation of Wnt/β-catenin signals in APC10 and βCAT^Ex310 lenses results in delayed cell cycle exit (p57^kip2 expression), reduced c-maf and delayed β-crystallin expression. By contrast, loss of β-catenin results in precocious expression of p57^kip2 and c-maf in epithelial cells, yet normal initiation of β-crystallin expression in fiber cells, albeit at decreased levels.¹³These results indicate that Wnt signals regulate cell cycle exit and c-maf expression. Although c-maf expression and cell cycle exit are associated with β-crystallin expression in the fiber compartment, precocious expression of c-maf and p57^kip2 is insufficient to induce β-crystallin expression in the epithelial compartment, indicating that other signals (FGF and BMPs) and transcription factors are necessary to initiate β-crystallin expression. Indeed, previous studies of Maf proteins indicate that they can act as repressors or activators of crystallin expression, depending on context and expression of other transcription factors such as Pax6 and Sox1 to -3.⁵⁷ ⁵⁸Consistent with the notion that Wnt signals regulate crystallin expression, Lyu and Joo⁴⁷showed, in epithelial explants, that Wnt3a-conditioned medium synergizes with low levels of FGF2 to promote expression of βB2-crystallin. Taken together, these data suggest that Wnt signals are essential for regulating cell cycle progression and the exit from the cell cycle (via p57^kip2) necessary for differentiation. However, initiation of β-crystallin expression during fiber differentiation is independent of Wnt/β-catenin signals and is probably dependent on FGF⁴and BMP⁵signals. Once differentiation is initiated, low levels of endogenous Wnt/β-catenin signals appear to be necessary to enhance β-crystallin expression.¹³ ⁴⁷However, overactivation of Wnt/β-catenin signals inhibits β-crystallin expression. Thus, FGF and Wnt signals must be tightly regulated and coordinated during lens development, particularly at the lens equator, to ensure progression/arrest of cell cycle and optimal differentiation of lens fiber cells.

Effect of Wnt Pathway on EMT and Apoptosis

The abnormal levels of cell proliferation in APC10 and βCAT^Ex310 lenses were accompanied by elevated levels of apoptosis. Similar induction of apoptosis has been reported in various other tissues with similar conditional mutations of APC and β-catenin.³⁷ ⁵⁹The mechanism by which active Wnt signals induces apoptosis in lens cells is unclear. It may be due to dysregulation of cell cycle checkpoints and DNA damage, as active β-catenin and APC truncations have been show to cause spindle and chromosomal segregation abnormalities.²³ ⁶⁰ ⁶¹ ⁶² ⁶³Alternatively, alterations in the composition of the basement membrane may have initiated an anoikis response. Consistent with this, most epithelioid cells undergoing apoptosis are not in contact with the lens capsule (Fig. 9)and there are alterations in the expression of laminin and collagen IV (Fig. 6) , which may result in altered cell-matrix interactions. Truncated APC also targets to mitochondria where it binds Bcl2 and thus may directly affect intrinsic apoptotic pathways.⁶⁴In colon cancer cell lines, truncated APC appears to have antiapoptosis effects⁶⁴ ⁶⁵and binding of truncated APC with Bcl2 is hypothesized to provide a survival advantage. In APC10 lenses, the levels of apoptosis were less extensive than in βCAT^Ex310 lenses. However, it seems unlikely that truncated APC confers improved cell survival in these lenses, as the phenotype at postnatal stages is more severe.

Activation of the Wnt/β-catenin signaling pathway has been associated previously with EMT during embryonic development and cancer metastasis.⁶⁶ ⁶⁷In both APC10 and βCAT^Ex310 lenses, evidence for EMT was present at E15.5, with numerous cells showing α-sma expression. However, as this occurred 2 days after genetic recombination and initiation of Wnt signaling at E13.5, it is not clear whether the EMT in these lenses is a direct effect of Wnt/β-catenin signaling or secondary to potential activation of TGFβ signaling in these abnormal lenses. Although TGFβ signaling is the major inducer of EMT in the lens,⁴ ¹²there is increasing evidence that EMT is mediated by a network of various signaling pathways.⁶⁷ ⁶⁸TGFβ signaling can result in the activation of Wnt-responsive genes by translocation of β-catenin to the nucleus after E-cadherin downregulation. Similarly, TGFβ has also been shown to upregulate Wnts and Frizzleds in lens and cause nuclear translocation of β-catenin in lens epithelial cells.⁶⁹The results presented herein suggest that Wnt/β-catenin signaling is an initiator of EMT in the lens, which is a characteristic of subcapsular cataract and posterior capsule opacification.

In conclusion, this study showed that Wnt/β-catenin signaling plays important roles in regulating lens stem/progenitor cell populations and in maintaining lens epithelial cell phenotype. Wnt/β-catenin signaling also plays important roles during early stages of fiber differentiation, regulating c-maf and β-crystallin expression. In this context, it remains to be determined how Wnt and FGF signals combine to regulate the precise temporal and spatial sequence of events during lens fiber cell differentiation. Dysregulation of Wnt signaling also affects the EMT of lens cells and thus may afford targets for pharmaceutical intervention for cataract and posterior capsule opacification.

² Contributed equally to the work and therefore should be considered equivalent authors.

Supported by Project Grant 400174 from the National Health and Medical Research Council (NHMRC) (RUdI, HEA) and the Rebecca L. Cooper Research Foundation for equipment.

Submitted for publication February 13, 2009; revised April 9, 2009; accepted July 2, 2009.

Disclosure: G. Martinez, None; M. Wijesinghe, None; K. Turner, None; H.E. Abud, None; M.M. Taketo, None; T. Noda, None; M.L. Robinson, None; R.U. de Iongh, None

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Corresponding author: Robb U. de Iongh, Anatomy and Cell Biology, The University of Melbourne, Parkville VIC 3010, Australia; [email protected].

Figure 1.

Gene	Change (x-Fold) APC10/Wt	t-Test P	Change (x-Fold) βCAT^Ex310/Wt	t-Test P	Average ΔCt (C _t[GOI] − C _t[HKG])
Gene	Change (x-Fold) APC10/Wt	t-Test P	Change (x-Fold) βCAT^Ex310/Wt	t-Test P				Wt	APC10	βCAT^Ex310
Wnt target genes
Lef1	58.8	8.5E-07	30.8	2.7E-06	9.69	3.81	4.74
T	16.5	4.4E-06	11.1	1.0E-06	10.78	6.73	7.31
Tcf7	7.5	4.2E-07	5.0	1.1E-05	4.72	1.82	2.39
Ccnd1	2.8	9.1E-05	1.5	2.3E-03	3.33	1.83	2.71
Jun	2.4	5.8E-05	2.0	1.4E-04	4.49	3.21	3.50
Pitx2	1.2	1.5E-01	−2.6	4.2E-03	8.40	8.11	9.80
Foxn1	−3.2	7.4E-04	−2.6	4.3E-04	7.52	9.17	8.88
Wnt pathway components
Rhou	2.2	2.2E-03	1.6	2.0E-02	7.91	6.80	7.26
Kremen1	2.1	2.7E-04	1.7	7.6E-03	4.91	3.82	4.18
Ep300	−1.0	7.2E-01	−2.0	2.5E-03	4.84	4.90	5.84
Ctnnb1	1.2	3.5E-01	−2.4	9.7E-04	1.36	1.12	2.63
Wnt Inhibitors
Wif1	392.5	1.2E-06	287.4	1.E-06	12.04	3.43	3.88
Dkk1	69.7	1.9E-06	21.3	7.1E-06	11.82	5.70	7.41
Nkd1	25.3	2.8E-06	13.3	4.8E-06	5.29	0.63	1.55
Frzb	13.8	8.6E-05	13.6	1.1E-04	11.11	7.32	7.34
Tle1	1.9	3.0E-04	2.1	5.7E-05	6.26	5.33	5.16
Sfrp4	7.8	1.0E-04	1.8	5.6E-03	13.28	10.32	12.40
Sfrp2	−2.1	9.8E-04	−1.1	4.2E-01	1.23	2.27	1.34
Aes	−2.4	1.0E-03	−1.1	4.9E-01	2.72	4.04	2.87
Btrc	−1.9	5.5E-06	−2.6	1.2E-06	5.75	6.68	7.12
Sfrp1	−3.0	3.1E-05	−3.1	8.4E-06	0.01	1.61	1.64
Wnts
Wnt6	508.2	8.7E-08	353.9	6.0E-07	12.49	3.50	4.02
Wnt10a	100.3	1.3E-06	62.9	2.4E-05	15.41	8.77	9.44
Wnt8b	18.6	2.2E-05	2.7	2.0E-02	15.41	11.19	13.98
Wnt11	4.2	3.1E-02	3.4	9.6E-02	13.60	11.55	11.82
Wnt4	4.1	7.8E-04	−2.1	2.4E-02	11.72	9.69	12.75
Wnt5a	2.5	7.4E-03	1.7	3.4E-02	9.94	8.62	9.16
Wnt7a	−2.3	1.1E-03	−6.5	1.3E-04	4.30	5.51	7.00
Wnt7b	−2.4	1.1E-03	−4.1	2.3E-04	2.99	4.27	5.01
Wnt8a	−13.5	2.7E-01	−2.8	6.5E-01	12.52	16.28	13.99
Wnt Receptors
Lrp5	2.4	2.E-04	2.4	9.8E-04	7.84	6.56	6.57
Fzd8	2.1	7.4E-02	2.0	6.7E-03	13.18	12.10	12.17
Fzd5	−1.5	3.7E-03	5.7	2.2E-05	6.76	7.37	4.26
Fzd3	−1.6	7.1E-03	−2.0	1.3E-03	3.26	3.92	4.23

Jump To...

This feature is available to authenticated users only.

Related Articles

From Other Journals

Related Topics

To View More...

You must be signed into an individual account to use this feature.