Ocular Anterior Segment Dysgenesis upon Ablation of p120 Catenin in Neural Crest Cells

Abstract

Purpose.: Development of the ocular anterior segment depends largely on periocular mesenchyme cells, which are derived predominantly from neural crest cells (NCC). Specific and differential cell adhesion is expected to be instrumental in induction, migration, and differentiation of NCC. As p120 catenin (ctn) is an important component of cadherin–catenin cell adhesion complexes, we assessed its role in development of the anterior segment structure.

Methods.: We generated conditional p120ctn^fl/fl;Wnt1Cre knockout mice and studied the effect of this gene ablation on eye development in vivo. In addition, p120ctn was knocked down in vitro.

Results.: Wnt1Cre-mediated deletion of floxed p120ctn alleles in NCC resulted in serious ocular anterior segment dysgenesis (ASD), including iridocorneal angle closure, complete anterior chamber obliteration, iris and ciliary body hypoplasia, corneal malformation and opacity, and glaucoma-like defects. A completely penetrant phenotype was visible approximately three weeks after birth, but histologic defects were obvious at embryonal day 18.5 (E18.5). Neither migration of NCC nor expression of key transcription factors appeared to be affected. In contrast, the N-cadherin expression pattern was changed significantly in iridocorneal angle cells and corneal endothelium. A human trabecular meshwork cell line in which p120ctn was knocked down also showed decreased expression levels of N-cadherin and β-catenin at the plasma membrane, but no defect in cell migration.

Conclusions.: p120ctn has a critical role in ocular mesenchyme development. Loss of p120ctn and the associated N-cadherin downregulation in NCC leads to ASD without affecting cell migration. p120ctn abnormalities might have a role in the pathophysiology of mammalian eye development.

Introduction

Cadherins constitute a large family of cell–cell adhesion molecules involved in various morphogenetic processes, including cell sorting, motility, and signaling. ^1,2 Cadherins form cis and trans homodimers on the cell surface (via their extracellular domains), while a conserved intracellular domain of 30 amino acids interacts with armadillo proteins, either β-catenin or plakoglobin. In the classic cadherin–catenin model, β-catenin or plakoglobin creates a link with the actin cytoskeleton through α-catenin. A second strongly conserved intracellular domain, the juxtamembrane domain, provides binding sites for several p120 catenin (ctn) members, including p120ctn, ARVCF, δ-catenin/NPRAP, and p0071. ^3–5 siRNA-mediated p120ctn knockdown (KD) results in dose-dependent elimination of several classic cadherins and loss of cell–cell adhesion, suggesting that p120ctn has an important role in regulating the adhesive strength of cadherins by inhibiting their degradation and controlling their levels in cell junctions. ^6,7 In vitro studies indicate that cadherin stability is regulated posttranslationally and requires direct interaction of p120ctn with the cadherin cytoplasmic tail. ^8,9 Moreover, p120ctn is involved in modulating the activities of the Rho family guanosine triphosphatases, ^10–13 and it can regulate target gene expression by interacting with the transcriptional repressor Kaiso. ^14–16  

Neural crest cells (NCC) are pluripotent migratory cells arising from the dorsal neural tube via an epithelial-to-mesenchymal transition. ^17,18 Several cadherins have important roles, and display dynamic spatial and temporal expression patterns during NCC induction, migration, and differentiation. ^19,20 Before NCC migration, N-cadherin and cadherin-6B are expressed in the progenitor cells in the dorsal neural tube. These cadherins are down-regulated during EMT, while cadherin-7 and cadherin-11 are upregulated in migrating NCC. The latter are down-regulated again during final homing and differentiation. Disruption of normal cadherin function results in aberrant NCC delamination from the neural fold, migration abnormalities, or defective reaggregation after homing. ^21–23 NCC migrate all over the embryo, and activities of small GTPases, cytoskeleton remodeling, cell adhesion, and membrane trafficking all are necessary for their efficient directional migration. 

The highly differentiated ocular anterior segment has a critical function in the vertebrate eye, and is composed of the cornea, iris, ciliary body, lens, and trabecular meshwork. The cornea and lens are responsible for transparency and refraction. The iris controls the amount of light entering through the pupil, and the ciliary body is responsible for production of the aqueous humor, which provides most of the nutrients for the lens and cornea. The trabecular meshwork is located in the iridocorneal angle (IA) between the roots of the iris and the cornea, and is involved in draining the aqueous humor from the eye via the anterior chamber. In this way, the correct intraocular pressure is maintained and the shape of the eye is stabilized. The development of all these tissues of the anterior eye segment is a complicated process involving coordinated interactions between cells originating from surface ectoderm, neural ectoderm, and the surrounding periocular mesenchyme. ²⁴ Ocular anterior segment dysgenesis (ASD) is a developmental abnormality of the tissues of the ocular anterior segment leading to various eye disorders, including Peters's anomaly, Rieger's anomaly, Axenfeld's anomaly, aniridia, iris hypolasia, and glaucoma. ^25–28 Periocular mesenchyme is derived predominantly from the neural crest. Tissues of the anterior eye segment, including corneal stroma and endothelium, iris stroma and melanocytes, ciliary body stroma and melanocytes, and trabecular meshwork, all are of NCC origin. ²⁹ Due to the fundamental contribution of NCC to eye development, disruption of mesenchymal NCC migration or differentiation could lead to eye disorders. 

To explore the role of p120ctn in the NCC lineage, we used the Wnt1Cre system to generate mice with NCC-confined conditional knockout of p120ctn. Here, we will address specifically the function of p120ctn during ocular development. p120ctn deletion in NCC resulted in N-cadherin downregulation in the eye, implying that abnormal cell sorting following N-cadherin dysregulation could be the basis of ASD. Thus, p120ctn has an essential role in normal mammalian eye development. 

Materials and Methods

Generation of p120ctn Mutant Mice

All animal experiments were performed in accordance with the Association for Research in Vision and Ophthalmology (ARVO) statement for the Use of Animals in Ophthalmic and Vision Research, and were approved by the Experimental Animal Ethics Committee of Ghent University. Mice with floxed p120ctn alleles (p120^fl/fl) and Wnt1Cre transgenic mice have been described. ^30,31 To follow the migration of NCC in wild type (WT) and mutant mice, breeding to Rosa26 reporter (Rosa26R) mice was used. ³² X-gal staining was performed as described. ³³  

Histology

Specimens were collected from mutant and WT mice at various embryonic and adult stages. Tissues were fixed overnight in 4% paraformaldehyde in PBS at room temperature, dehydrated, embedded in paraffin, and sectioned at 5-μm thickness. The sections were stained with hematoxylin and eosin (HE) or used for immunohistochemistry experiments. Nuclei in cross-sections of optic nerves were counted at three different positions starting from where the optic nerve exits the posterior globe and separated approximately 1 mm from each other. At each position, several consecutive sections were collected, stained with HE and analyzed using Fiji software (fiji.sc; ImageJ, National Institutes of Health, Bethesda, MD).Fiji software was also used to analyze HE-stained transverse sections of adult eyes. In midsagittal sections, identified by the presence of the optic nerve, the number of nuclei of retinal ganglion cells per unit length was determined. Eyes used to prepare semithin sections were fixed for 1 hour under mild vacuum in 0.1 M cacodylate buffer containing 2% formaldehyde and 2.5% glutaraldehyde, and then rotated for 3 to 4 hours at room temperature. Fixative was changed and incubation was continued overnight at 4°C. Eyes then were washed 3 times for 30 minutes with 0.1 M cacodylate buffer and incubated overnight in 1% reduced osmium tetroxide. After washing, the eyes were dehydrated serially in ethanol (30%, 50%, 70%, 95%, and 100%), serially impregnated with Spurr's resin (1/3 Spurr in ethanol, 2/3 Spurr in ethanol, and then 100% Spurr's resin), and finally embedded in fresh Spurr's resin. Semithin sections of 1 μm were stained with toluidine blue. 

Immunostaining

For immunohistochemistry, rehydrated paraffin sections were pretreated with citrate buffer to unmask the antigen either in a Retriever apparatus (PickCell Laboratories, Amsterdam, The Netherlands) or in a microwave oven. The following mouse monoclonal antibodies were used: anti-p120ctn (1:200; BD Transduction Laboratories, San Jose, CA), anti-N-cadherin (1:500; Zymed, South San Francisco, CA), anti-E-cadherin (1:500; BD Transduction Laboratories), anti-β-catenin (1:1000; BD Transduction Laboratories), and anti-smooth muscle actin (1:400; Sigma-Aldrich, St. Louis, MO). The other antibodies are listed in the Supplementary Material. For myocilin (MYOC) immunostaining, a rabbit polyclonal antibody (used at 1:200) was kindly provided by S. Tomarev (National Eye Institute, National Institutes of Health, Bethesda, MD). The sections were incubated with the primary antibodies overnight at 4°C and then with biotinylated secondary antibody (Dako, Glostrup, Denmark), avidin-peroxidase (Dako), and 3,3′-diamino-benzidine (DAB; Biogenex, San Ramon, CA). 

For indirect immunofluorescence, the secondary antibodies were goat-anti-mouse or goat-anti-rabbit antibodies conjugated with Alexa Fluor 488 (Molecular Probes, Eugene, OR). Slides were mounted with Vectashield containing DAPI (Vector Laboratories, Burlingame, CA) and examined with an Olympus fluorescence microscope or a Leica SP5 confocal scanning microscope. 

For immunocytochemistry, cells were fixed with methanol and the following primary antibodies were used: mouse anti-p120ctn (1:200; BD Transduction Laboratories), rabbit anti-Pan-cadherin (1:300; Takara, Japan), rabbit anti-β-catenin (1:1000; Sigma-Aldrich). The secondary antibodies were goat-anti-mouse or goat-anti-rabbit antibodies conjugated with horseradish peroxidase (Molecular Probes). 

Cell Culture, Retroviral Transduction, and KD Experiments

HTM-5 cells are human trabecular meshwork cells derived from a normal non-glaucoma donor and immortalized by the SV40 large-T antigen. ³⁴ They were kindly provided by I.-H. Pang (Alcon Laboratories, Fort Worth, TX) and grown in basic Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (FCS). For KD of p120ctn by short hairpin (sh) RNA, plasmid pRS-human-sh-p120 or pRS-mouse-sh-p120, ⁶ or the empty vector pSuper-retro-neo (Oligo-Engine, Seattle, WA) was introduced into Phoenix packaging cells by calcium phosphate-mediated transfection. The virus-containing medium was harvested and incubated with DOTAP transfection reagent (Roche Diagnostics, Indianapolis, IN). HTM-5 cells were transduced by this mixture and cultured further under puromycin selection (0.6 μg/mL). Derivative clonal p120ctn KD HTM-5 cell lines were obtained by limiting dilution. KD of p120ctn expression was assessed by immunofluorescence and Western blotting. 

Western Blotting

Cells were grown to confluence and lysed in 1× Laemmli buffer. Protein concentrations were measured by a detergent-compatible colorimetric assay kit (Bio-Rad Laboratories, Hercules, CA) according to the manufacturer's instructions. A total of 20 μg proteins was separated by SDS-PAGE in 8% polyacrylamide gels and transferred onto polyvinylidene difluoride membranes (PVDF; Millipore), which were blocked for 1 hour in 5% nonfat dry milk and then incubated overnight at 4°C with primary antibodies. The following antibodies were used: mouse anti-p120ctn (1:500; BD Transduction Laboratories), rabbit anti-pan-cadherin (1:500; Sigma), rabbit anti-β-catenin (1:4000; Sigma), and mouse anti-β-tubulin (1:10,000; Sigma). After washing, the membranes were incubated for 1 hour with peroxidase-coupled secondary antibody. Luminol enhancer solution (Thermo Scientific, Waltham, MA) was applied to visualize the proteins after exposure to an autoradiography film. 

Cell Migration Measurements

A cell migration assay was performed in chambered coverglass 8-well plates (Nunc; VWR International, Radnor, PA) in which were placed biocompatible Culture-inserts (ibidi; Proxylab). This generates two culture areas of 0.22 cm² separated by a cell-free gap 500 μm wide. HTM-5 derivative cells were seeded at 3.5 × 10⁴ cells per well insert. Cells were incubated for 24 hours at 37°C under 5% CO₂. Before imaging, the inserts were removed and the cells were incubated for 30 minutes with Hoechst 33342 stain at 0.5 μg/ml. 

A time lapse experiment was performed by using a Leica SP5 AOBS confocal microscope (Leica, Mannheim, Germany). Images were taken every 15 minutes during 16 hours. A contrast based autofocus was performed at each time point to maintain the right focal plane during the whole period. Hoechst and bright field images were taken using an HCX PL APO lambda blue 20.0 × 0.70 IMM UV objective. The Hoechst stain was excited with a 405 UV diode laser and detected with a spectral bandwidth of 415 to 520 nm in front of the photomultiplier tube. The velocity of the cells was determined using the “track objects” algorithm in Volocity 5.5.1 software (Perkin Elmer). The objects are first defined using an intensity threshold on the Hoechst signal. Afterwards, the program links the same objects found at different time points. In the protocol settings we specified that static objects are to be ignored. Each dot in the resulting graph corresponded to the velocity (in μm/sec) of one cell or object. 

Results

Conditional Loss of p120ctn during Eye Development by Wnt1Cre-Mediated Recombination

In vivo fate mapping experiments revealed that NCC contribute substantially to the periocular mesenchyme. ²⁹ To study the function of p120ctn in eye development, we bred mice inheriting a floxed p120ctn allele ³⁰ to Wnt1Cre transgenic mice. ³¹ This generated p120^fl/fl;Wnt1Cre mice with p120ctn alleles inactivated specifically in NC-derived tissues, including ocular mesenchyme. The phenotype of these mice included complex malformations of the ocular anterior segment structures with complete penetrance, neural tube closure defects with incomplete penetrance, and a limited melanocyte cell development defect. This report focuses on the ocular defects. 

Expression analysis of p120ctn by immunohistochemistry at embryonic day 13.5 (E13.5) showed that its expression in developing ocular mesenchyme cells was normal in WT embryos (Fig. 1A) but reduced substantially in p120^fl/fl;Wnt1Cre embryos (Fig. 1B), indicating successful NCC-specific ablation of the p120ctn protein. In WT (Fig. 1C) and p120^fl/fl;Wnt1Cre (Fig. 1D) embryos, E-cadherin was expressed in the epithelial cells of cornea, retina, and lens, where p120ctn was still expressed normally in the mutant embryos (Fig. 1B). This indicates that the Wnt1-driven Cre functions properly in NCC derived cells, but not in other cell types. Similarly, compared to expression of p120ctn protein in WT eyes at postnatal day 1 (P1) stage (Figs. 2A, 2C), its expression in p120^fl/fl;Wnt1Cre mice was virtually absent within corneal stroma and endothelium cells, IA cells, and stromal cells of ciliary body and iris (Figs. 2B, 2D). 

Figure 1. 

 

Depletion of p120ctn in neural crest (NC)-derived ocular cells. Immunohistochemical analysis of the expression of p120ctn (A, B) and E-cadherin (C, D) in the developing mouse eye at E13.5. (A) p120ctn was expressed homogeneously in the periocular mesenchyme (Me; arrow) in WT eyes. (B) In p120^fl/fl;Wnt1Cre mutant eyes, p120ctn was deleted from the Me (arrows), but retained in lens (L) and retina (R). (C, D) E-cadherin was expressed in the surface epithelial cells (SE; which will become the corneal epithelium at E14.5), anterior lens epithelium (ALE), and retinal pigment epithelium (RPE) in WT and p120^fl/fl;Wnt1Cre mutant eyes. In these tissues also p120ctn was retained indicating lack of Wnt1-Cre activity (see B).

Figure 2. 

 

Immunostaining of p120ctn in P1 mouse eyes. (A) p120ctn is expressed in corneal epithelium (Epi), stroma (Str) and endothelium (End), in stroma of the iris (I), in the ciliary body (CB, arrowhead), and in the IA (arrow), L and R. (B) In p120^fl/fl;Wnt1Cre mice, p120ctn expression is abolished in Str and End (upward arrows), stroma of the I, and the CB (arrowhead), and the IA region (IAR, arrow in C), all of which are derived from NCC. (C, D) Magnifications of the IAR (white rectangles in A and B, respectively). Bars, 50 μm.

Inactivation of p120ctn in NC-Derived Periocular Mesenchyme Induces Multiple Ocular Anomalies

p120^fl/fl;Wnt1Cre mice experienced corneal opacification (Fig. 3A), a completely penetrant phenotype that was visible macroscopically three weeks after birth. It was evident histologically as early as E18.5, when in WT animals the ciliary body, iris, and trabecular meshwork are well defined, which results in a clearly defined anterior chamber (Fig. 3B). The eyes of mutant mice were of various sizes and often different in the same animal (Fig. 3C). No eye defects were found in any of the heterozygous p120^fl/+;Wnt1Cre mice analyzed. 

Figure 3. 

 

p120ctn ablation in mouse NC-derived cells results in opacification of the eyes at the age of 2 months. (A) Eye of p120^fl/fl;Wnt1Cre mouse with opaque cornea (frontal view). (B) Eye of WT mouse showing a transparent cornea. (C) Left and right eyes from the same p120^fl/fl;Wnt1Cre mouse are of different size, but both are opaque.

Histologic analysis revealed multiple ocular ASD in postnatal p120^fl/fl;Wnt1Cre mice. The iris was fused with the posterior side of the cornea (arrow in Fig. 4B; compare to WT in Fig. 4A), resulting in complete loss of the anterior chamber. A defective separation between iris and cornea, already apparent in E18.5 embryos, resulted in a closed IA (Figs. 4C, 4D). At P5, in WT mice a loosely arranged network of mesenchymal cells formed in the IA (Fig. 5A), which develops over a few weeks into the trabecular meshwork (Fig. 5C). ²⁴ In contrast, in p120^fl/fl;Wnt1Cre mice a group of cells aggregated closely between the root of the iris and the ciliary body (Fig. 5B). Also, the root of the developing iris in p120^fl/fl;Wnt1Cre mice was fused with the inner side of the cornea instead of being detached as in WT mice (Figs. 5A, 5B). The ciliary body in these mutant mice was hypoplastic and much less folded than in WT litter mates (Figs. 5C, 5D). In the eyes of adult p120^fl/fl;Wnt1Cre mice, the trabecular meshwork was not recognizable and Schlemm's canal was small or missing (Fig. 5D, compared to Fig. 5C). This phenotype may explain partly the corneal opacification at this age (Fig. 3). 

Figure 4. 

 

Compound ocular defects in p120^fl/fl;Wnt1Cre mice. (A, B) HE staining of midsagittal sections of eyes of mice at the age of 2 months demonstrates histologic differences between WT and p120^fl/fl;Wnt1Cre eyes. (A) In the WT eye, the anterior chamber (AC) develops normally, with a well developed I in front of the L and separated from the cornea (C) by an open IA (arrows). R, retina. (B) In the p120^fl/fl;Wnt1Cre eye, the AC disappears completely because of adhesion of the iris to the posterior side of the cornea (arrows). The iris is hypoplastic. (C) HE staining of eyes of WT embryos at E18.5 reveals that the IA (arrow) is well defined at that stage. (D) In p120^fl/fl;Wnt1Cre mice at E18.5, the iris is fused to the cornea and the IA is lost (arrow). Bars, 50 μm.

Figure 5. 

 

IA anomalies in p120^fl/fl;Wnt1Cre mice. (A, B) IA mesenchyme was analyzed on postnatal day 5 by staining thin sections with toluidine blue. (A) In WT eyes, the IA is occupied by a group of loose mesenchymal cells (arrow). The AC is formed between the I and C. (B) In p120^fl/fl;Wnt1Cre eyes, a group of aggregated cells (Agg, arrow) is seen in the presumptive iridocorneal site between the outside of I/CB and the inner side of cornea/sclera. The root of the iris attaches to the posterior of the cornea with loss of the AC (arrowhead). (C, D) IA morphology (HE staining) in WT and p120^fl/fl;Wnt1Cre mice of 2 months. (C) In WT eyes, both trabecular meshwork (TM, open arrow) and Schlemm's canal (SC, solid arrow) are well differentiated. The AC develops normally between I (arrowhead) and C. The CB develops fully with several foldings. Black signal is due to natural pigment. (D) Loss of differentiated trabecular meshwork and Schlemm's canal in p120^fl/fl;Wnt1Cre eyes. The anterior chamber did not develop because of adhesion of the I (arrowhead) to the C. The CB (arrow) is hypoplastic with only one notable fold. (E) Immunostaining revealed that MYOC is expressed strongly in the trabecular meshwork of WT eyes (white rectangle). (F) In contrast, in p120^fl/fl;Wnt1Cre eyes MYOC was undetectable in the region where trabecular meshwork cells might be expected (white rectangle). Bars, 50 μm.

To explore whether the lack of structural differentiation is associated with a lack of typical trabecular meshwork gene expression, we analyzed expression of MYOC by immunostaining. In WT eyes at the P24 stage, MYOC was expressed in the epithelium of the ciliary body and more strongly in trabecular meshwork, in line with a previous report ³⁵ (Fig. 5E). In contrast, MYOC could not be detected in the region of the closed IA of p120^fl/fl;Wnt1Cre mice (Fig. 5F). 

In addition, the corneal epithelium was much thinner in p120^fl/fl;Wnt1Cre mice: 2 to 3 cell layers instead of 5 to 7 cell layers in WT mice (Figs. 6A, 6B). On the other hand, the corneal stroma in mutant mice was thicker and uneven with disorganized collagen layers, which also could contribute to corneal opacification (Figs. 6A, 6B). Moreover, the mutant corneal endothelium was disorganized or partly absent, especially in young postnatal mice (Figs. 6C, 6D). 

Figure 6. 

 

Defects in the cornea of p120^fl/fl;Wnt1Cre mice as revealed by HE staining. (A, B) Comparison of corneal morphology between WT and p120^fl/fl;Wnt1Cre eyes at the age of 2 months. (A) Cornea of WT mouse eye. The corneal epithelium (Epi) is 5 to 7 cell layers thick, with flattened squamous apical cells and several layers of cuboidal-basal cells. Corneal stroma (Str) consists of regularly arranged collagen fibers with sparsely distributed keratocytes. Corneal endothelium (Endo) is a well arranged cell monolayer. (B) In a p120^fl/fl;Wnt1Cre mouse eye, the corneal epithelium is reduced to only 2 to 3 cell layers. The stromal collagen fibers are less well arranged and keratocyte morphology is aberrant. The corneal endothelial cells in mutant eyes are less regularly arranged. (C, D) Comparison of corneas on P1. (C) Organized corneal endothelium in WT eye with clear separation from the lens (L). (D) Less organized corneal endothelium in p120^fl/fl;Wnt1Cre eye with dispersed cells between cornea and lens. Bars, 50 μm.

Abnormalities in the ocular anterior segment can lead to glaucoma. ²⁸ Trabecular meshwork and Schlemm's canal are essential for draining aqueous humor into the episcleral vessels outside the eye to maintain normal intraocular pressure. As we could not measure this pressure reproducibly, we analyzed the potential onset of glaucoma by looking for morphologic changes in retinas and optic nerves of p120^fl/fl;Wnt1Cre mice. Compared to WT mice, mutant mice at the age of four months displayed, besides apparent disorganization of the inner nuclear cell layer of the retina, a significant decrease in the number of retinal ganglion cells (Figs. 7A–7C), which points to development of moderately advanced glaucoma. ³⁶ This finding was substantiated by analysis of cross-sections of the optic nerves (Figs. 7D–7F). Optic nerves of mutant mice at the age of 3 months contained significantly more nuclei, indicative of replacement of nerve fibers by glial cells. This finding was strengthened by further analysis of representative optic nerve sections: there was a clear loss of myelin in the mutant optic nerves and the observed nuclei were located in GFAP-positive cytoplasm (Supplementary Fig. S1). There also was evidence for optic nerve head cupping in the mutant eyes (not shown). Thus, p120ctn loss might have an important role in inducing glaucoma. 

Figure 7. 

 

Depletion of p120ctn induces defects in retina and optic nerve, pointing to glaucoma onset at the age of 3 to 4 months. (A, B) Compared to retina from the WT eye, the retinal ganglion cells (RGC) seen in a midsagittal section of a p120^fl/fl;Wnt1Cre eye are affected and cells in the inner nuclear layer (INL) are disorganized. ONL, outer nuclear layer. Bars, 50 μm. (C) The number of RGC per unit width was decreased significantly (***P = 0.0003, 99% confidence interval [CI]) in retinas of p120ctn-depleted four-month-old mice. Each dot represents a measurement at a location on average 1.1 mm from the optic nerve, which was present in each midsagittal section analyzed. Two WT and two mutant eyes were analyzed. (D, E) Representative cross-sections through the optic nerves of WT and mutant mice. Sections were at 1 mm from where the nerve exits the eye globe. (F) The number of nuclei in p120^fl/fl;Wnt1Cre optic nerves is significantly higher (***P < 0.0001, 99% CI) than in WT. Optic nerves of three WT and two mutant mice were analyzed at two or three defined locations per nerve. Each dot represents the total number of nuclei seen in the optic nerve section. These nuclei reside in GFAP-positive cytoplasm (Supplementary Fig. S1). Bars, 100 μm.

p120ctn Ablation Does Not Result in Extensive NCC Migration Defects

In vivo fate mapping revealed that NCC contribute to the anterior ocular segment during development. ^29,37 To determine if the observed ocular ASD is due to impaired NCC migration or to a differentiation defect, we introduced the Rosa26R allele ³² into mice with p120ctn floxed alleles. Then, by crossing with Wnt1Cre lines, the fate of eye cells with p120ctn ablation could be monitored in the progeny by histochemical staining for β-galactosidase. Heterozygous p120^fl/+;Wnt1Cre;Rosa26R mice were used as control because they displayed no eye defects. 

The X-gal staining in the eyes of p120^fl/fl;Wnt1Cre;Rosa26R mice at different developmental stages was similar to staining of p120^fl/+;Wnt1Cre;Rosa26R eyes; this observation is in agreement with published data ^29,37 (Fig. 8). This was observed at E10.5, when NC-derived mesenchymal cells began to migrate to the cornea, and also at E11.5, when these mesenchymal cells occupied the whole corneal region (Figs. 8A–8D). Also at E14.5, when NC-derived mesenchymal cells began to migrate into the stroma of iris and ciliary body, and when the corneal endothelium started to form, no migration defect was observed in p120^fl/fl;Wnt1Cre;Rosa26R mice (Figs. 8E, 8F). No such defect was seen also at the P1 stage, when the IA was occupied in the mutant eye by a dense mass of NC-derived mesenchymal cells (Fig. 8H, compared to Fig. 8G). Therefore, genesis of the NC cells and their migration to the corneal stroma, corneal endothelium, stromal cells of iris and ciliary body, and anterior chamber angle all were unaffected in p120^fl/fl;Wnt1Cre;Rosa26R mice, indicating that the ocular malformations observed might have arisen from NCC differentiation defects rather than from a migration defect. 

Figure 8. 

 

In vivo fate mapping of NC-derived ocular cells shows no defect in genesis or migration of NCC in p120^fl/fl;Wnt1Cre mutant mice. (A, B) At stage E10.5, when NC-derived mesenchymal cells begin to migrate into the presumptive cornea (arrows), no difference in migration pattern is seen between heterozygous (p120^fl/+;Wnt1Cre:Rosa26R) control mice and homozygous (p120^fl/fl;Wnt1Cre;Rosa26R) mutant mice, as revealed by β-gal positivity. (C, D) At stage E11.5, the cornea (C) in heterozygous control and homozygous mutant mice is occupied by NC-derived cells (arrows). (E, F) There also is no staining difference between heterozygous control and homozygous mutant mice at stage E14.5, when mesenchymal cells close to the lens (L) begin to differentiate into corneal endothelium cells (arrows). (G, H) At stage P1, when the IA is formed, stroma of the CB and the iris (I) of homozygous mutant eyes show β-gal positivity resembling that in heterozygous control eyes, including positivity in the presumptive IA region (PIA). Bars, 50 μm.

Reduced N-Cadherin and β-Catenin in p120ctn Mutant Mice

To determine whether the morphologic defects in the anterior part of the eyes in p120^fl/fl;Wnt1Cre mice were associated with reduced cadherin levels, we analyzed the expression patterns of E- and N-cadherin and their associated catenins. Immunofluorescence staining revealed that the level of N-cadherin in mutant eyes was lower than in controls (Figs. 9A–9D). In WT mice, N-cadherin was expressed strongly at the cell membranes of corneal endothelium, IA, and lens (Figs. 9A, 9C). However. in p120^fl/fl;Wnt1Cre mice it was almost undetectable in the IA and corneal endothelium, in which p120ctn was depleted (Figs. 9B, 9D). In contrast, N-cadherin was expressed normally in the lens of mutant eyes (Fig. 9B), confirming that N-cadherin downregulation was due to p120ctn ablation. E-cadherin was expressed at normal levels in the corneal epithelium and lens epithelium of mutant mice, in which p120ctn also was expressed normally (Fig. 1D). Expression of β-catenin was examined because it is associated directly with the cytoplasmic domains of classic cadherins, including N-cadherin. β-Catenin was observed at the cell membrane in the IA and corneal endothelium in WT mice, but it was down-regulated in these tissues in mutant mice (Figs. 9E–9H). To provide evidence at the cellular level for the analysis described above, HTM5, a cell line derived from normal human trabecular meshwork, was transduced with a retrovirus encoding human p120ctn shRNA, and stable p120ctn KD cell lines were generated successfully. Immunostaining showed that in these p120ctn KD trabecular meshwork cells, expression levels of classic cadherins and β-catenin were decreased dramatically at the plasma membrane and cell–cell contacts (Fig. 10 shows representative stainings), which is consistent with our in vivo results. Western blotting analysis also confirmed the reduction of N-cadherin and β-catenin levels in the p120ctn KD cells (Fig. 11A). Cell migration assays showed further that the migration velocity of three clonal p120ctn KD HTM5 derivatives was not different from that of control HTM5 cells, which were transduced with an empty vector (Fig. 11B). 

Figure 9. 

 

N-cadherin and β-catenin are down regulated in the eyes of p120^fl/fl;Wnt1Cre mice. (A) In WT mice at P1, immunofluorescent staining shows that N-cadherin is expressed in IA cells (white rectangle) and corneal endothelium (CE, arrow). (B) N-cadherin expression levels in p120^fl/fl;Wnt1Cre mice appear to be decreased strongly in the region where IA cells are expected (IAR, white rectangle) and in the CE (arrow). (C, D) Rectangles: magnifications of the IAR are seen in (A) and (B), respectively. (E, F) Compared to the WT situation, β-catenin expression levels also appear to be decreased strongly in the region of IA cells (white rectangle) and CE (arrow) of p120^fl/fl;Wnt1Cre eyes. (G, H) Rectangles: magnifications of the IAR seen in (E) and (F), respectively. Bars, 50 μm.

Figure 10. 

 

KD of p120ctn in the normal human trabecular meshwork cell line HTM-5 leads to decreased expression levels of classic cadherins and β-catenin. (A–C) p120ctn and classic cadherins (N-cadherin in this case) are co-expressed at the plasma membrane of control HTM-5 cells transduced with an empty vector. (D–F) After transduction with a vector expressing shRNA targeting human p120ctn, the obvious p120ctn KD is associated with loss of classic cadherins from the plasma membrane. (G–I) p120ctn and β-catenin are co-expressed at the plasma membrane of control HTM-5 cells transduced with an empty vector. (J–L) In p120ctn-KD cells, β-catenin expression levels also are down-regulated at the plasma cell membrane. Bars, 20 μm.

Figure 11. 

 

Effects of p120ctn KD in clonal HTM-5 cell derivatives. (A) Western blotting confirmed that, upon KD of p120ctn in HTM-5 cells, N-cadherin and β-catenin are also down-regulated. Lanes 1–3: different HTM-5 derivative cell clones transduced with a retroviral vector expressing shRNA targeting human p120ctn. Controls include HTM-5 parental cells (lane 4), HTM-5 cells transduced with a vector expressing shRNA targeting mouse p120ctn (lane 5), and HTM-5 cells transduced with an empty vector (lane 6). Size markers are indicated on the right. (B) Cell migration assay revealing that the three different p120ctn KD cell clones (cf. lanes 1–3 of A) display a migration rate comparable to that of control cells transduced with an empty vector.

Discussion

We demonstrated that NCC-specific inactivation of p120ctn in mice perturbs the proper development of NC-derived eye structures and causes a spectrum of ASD, including closure of the IA, adhesion of the iris to the cornea, hypoplasia of iris and ciliary body, and dysgenesis of cornea, trabecular meshwork, and Schlemm's canal. All the adult mutant mice analyzed displayed corneal opacity, which can result from attachment of the iris to the cornea, and disorganization of corneal stroma and endothelial cells. Trabecular meshwork and Schlemm's canal are the ocular drainage structures, and their malformation is a likely cause of the observed glaucoma-related loss of retinal ganglion cells, disorganization of the inner nuclear cell layer in the retina, and apparent gliosis of the optic nerve. To understand the molecular mechanisms of p120ctn-induced ASD, we analyzed the N-cadherin expression patterns and found them to be down-regulated in the IA and corneal endothelial cells of p120^fl/fl;Wnt1Cre mice. p120ctn KD in the human trabecular meshwork cell line HTM5 also resulted in decreased N-cadherin. Hence, abnormal cell sorting and differentiation due to decreased N-cadherin levels might have caused the complex ocular ASD phenotype. 

Studies on mouse models have shown that NC-derived mesenchymal cells have a major role in eye development and contribute substantially to development of the anterior ocular segment. ^29,37,38 In the mouse eye, three different waves of mesenchymal cells migrate consecutively into the ocular anterior segment. ²⁴ Mesenchymal cells start to migrate into the presumptive cornea at approximately E10, and by E15 the corneal stroma and endothelium are occupied by these cells. During corneal endothelium differentiation, the second wave of mesenchymal cells begins to migrate, and eventually they differentiate into the stroma of the iris and the ciliary body. Finally, the IA is occupied by the third wave of mesenchymal cells. By following the migration of NC-derived mesenchymal cells at different developmental stages, we did not find any evidence for severe defects in either NCC genesis or NCC migration in p120^fl/fl;Wnt1Cre mice. On the other hand, we used HTM5 cells as a tool to confirm our in vivo findings. After KD of p120ctn, we found that HTM5 cells did not display any defect in a simple cell migration assay. In contrast, MYOC expression by the trabecular meshwork was much affected in the p120^fl/fl;Wnt1Cre eyes. Hence, ASD in p120^fl/fl;Wnt1Cre mice might be due to a differentiation failure of NCC despite correct migration and homing. 

In adult p120^fl/fl;Wnt1Cre eyes, the corneal stromal cells were disorganized and the corneal epithelium was thinner than in WT eyes. Since corneal stroma originates from NCC and p120ctn is ablated in the corneal stroma of p120^fl/fl;Wnt1Cre mice, the observed stromal disorganization might be related directly to p120ctn depletion. However, we have no conclusive evidence for this hypothesis and disorganization of the corneal stroma could also be secondary to incomplete formation of corneal endothelium. In any event, the thinner corneal epithelium in mutant mice reflects an indirect nonautonomous effect because these cells are not derived from NCC. Interestingly, properly formed corneal endothelium has an important role during the development of the anterior segment. ^39,40 In the chicken eye, the progressive organization of NCC-derived mesenchymal cells in the anterior chamber, including the induction of N-cadherin expression and a mesenchymal-to-epithelial transition to corneal endothelial cells, all were shown to be dependent on early signals from the anterior lens epithelial cells before they mature into lens fibers (up to E15). ³⁹ This apparent association between N-cadherin induction and correct organization of the anterior chamber is most interesting because N-cadherin downregulation is obvious in our mutant mice, as discussed further below. Ectopic expression of TGFα or epidermal growth factor (EGF) in the lens leads to defective differentiation of the corneal endothelium, and thereby initiates a cascade of malformations in the anterior segment: adhesion of the iris and lens to the cornea, loss of the anterior chamber, abnormal differentiation of corneal stroma, and reduced thickness of the corneal epithelium. ⁴⁰ Based on that report, the major reason for the wide-spectrum ASD we observed in p120^fl/fl;Wnt1Cre mice could be the partial formation of corneal endothelium owing to poor N-cadherin expression. Indeed, the study by Reneker et al. also stresses the importance of N-cadherin expression in the corneal endothelium for bringing about separation of cornea from lens and iris. ⁴⁰ N-cadherin downregulation is obvious in our mutant mice, as discussed further below. 

p120ctn is an important multifunctional protein that stabilizes junctions at cell–cell contacts, ^6,7 modulates Rho GTPases in the cytoplasm, ^9–13 and regulates gene activity in the nucleus by binding to the transcriptional repressor Kaiso. ^14–16 At the cell surface, p120ctn acts as a major mediator of junctional stability by preventing the recruitment of classic cadherins into endocytotic vesicles. ^41,42 Conditional p120ctn ablation in mice reduces the levels of these cadherins on the cell surface in the targeted tissues, as reported for skin, submaxillary gland, forebrain, teeth, and intestine. ^30,43–46 Thus, the observed downregulation of N-cadherin in p120^fl/fl;Wnt1Cre mouse eyes is in agreement with these studies. Classic cadherins are important mediators of specific cell–cell adhesion, and properly localized expression of cadherins is essential for cell sorting during embryogenesis. For instance, combined lens-specific N-cadherin and E-cadherin ablation results in lens epithelial cell sorting defects during development and failure of the lens vesicle to separate from the surface ectoderm. ⁴⁷ We found that IA cells expressing N-cadherin separate from the surrounding cells at E18.5, probably followed by differentiation to fates such as trabecular meshwork and Schlemm's canal. However, in p120^fl/fl;Wnt1Cre mice, the expression level of N-cadherin probably dropped below the threshold needed for this differentiation process. A similar abnormality likely happened to the corneal endothelium of p120^fl/fl;Wnt1Cre mice, in which N-cadherin downregulation was often seen to be associated with defective lens–cornea separation (Supplementary Fig. S2), which resembles the situation in the lens-specific N-cadherin–E-cadherin double mutant mice. ⁴⁷ It might be worthwhile to investigate if specific depletion of N-cadherin from NC-derived cells results in similar eye defects. However, ablation of N-cadherin by use of the Wnt1Cre system results in embryonic death by E13, which precludes the proposed investigation because NC cells do not migrate into the IA before E17–E19. ^23,24 In summary, the published data together with our findings underscore the importance of p120ctn-mediated N-cadherin stabilization in NCC-derived ocular mesenchyme. 

At adherens junctions, the intracellular domain of cadherins interacts with β-catenin and, thus, links the extracellular environment to the actin cytoskeleton. ⁴⁸ In our study, β-catenin displayed strong plasma membrane staining in the IA and corneal endothelium of WT mice, but a weaker expression pattern in the same eye structures of p120^fl/fl;Wnt1Cre mice, possibly as a direct result of N-cadherin downregulation. However, cytoplasmic β-catenin levels did not increase in NC-derived ocular cells in p120^fl/fl;Wnt1Cre mice. Therefore, any excess cytoplasmic β-catenin in mutant eyes seems to be counteracted by proteasomal degradation triggered by formation of the GSK3β/Axin/APC complex. ⁴⁹ Less β-catenin at the cell junctions due to decreased N-cadherin might lead to disorganization of the cytoskeleton, disruption of cell–cell contacts, and failure of cell sorting. Likewise, conditional deletion of β-catenin in the presumptive lens had a significant effect on lens epithelial cell adhesion and coordinated morphogenesis. ⁵⁰  

Many transcription factors are involved in eye development, and mutations in the corresponding genes are known to cause ASD. These genes include Foxc1, Foxc2, and Pax6. ^28,51,52 Since p120ctn can inhibit transcriptional repression by Kaiso, ^14–16 we investigated if p120ctn depletion affects the expression of the above-mentioned eye transcription factors and, thus, results in the ASD phenotype. Protein expression analysis by immunodetection did not reveal any distinct differences between E13.5 p120ctn KO and WT eyes, neither in expression intensity nor in subcellular localization (Supplementary Fig. S3). Although this analysis is not exhaustive, it indicates that gene transcription regulation by p120ctn is unlikely to be involved in the eye defects of p120^fl/fl;Wnt1Cre mice. 

In conclusion, we demonstrated that the cadherin-associated protein p120ctn has an important role during ocular anterior segment differentiation. N-cadherin is expressed in the IA and corneal endothelium. A threshold level of N-cadherin expression at the cell surface, achieved by p120ctn-mediated protein stabilization, promotes proper eye development. If N-cadherin expression levels go awry, eyes will display a complex ASD phenotype. Our results show that defects of p120ctn might be involved in mammalian eye disorders, in which case p120ctn could become a target in diagnosis and treatment. For instance, the human anterior segment disorder Peters' anomaly (OMIM #604229) includes corneal opacity due to abnormalities in the corneal structure, and different degrees of iris and lenticular attachments to the posterior cornea. This phenotype is similar to the ocular defects in p120^fl/fl;Wnt1Cre mice. Peters' anomaly has been linked to mutations in PAX6 on human chromosome 11p13, PITX2 on 4q25-q26, CYP1B1 on 2p22-p21, and FOXC1 on 6p25. However, so far it has not been linked to mutations in the p120ctn-encoding CTNND1 gene (on 11q12.1). Nonetheless, our findings justify the analysis of the p120ctn status in human ocular disorders. 

Supplementary Materials

pdf S1 

Acknowledgments

Stanislav Tomarev (National Eye Institute, NIH, Bethesda, MD) and I.-H. Pang (Alcon Laboratories, Fort Worth, TX) provided gifts of reagents. Riet de Rycke and Chris Guerin provided expert help with microscopy, and Amin Bredan edited the manuscript. 

Footnotes

 Supported by the Concerted Research Actions (GOA) of Ghent University, by the Queen Elisabeth Medical Foundation (G.S.K.E.), Belgium, and by the Research Foundation – Flanders (FWO-Vlaanderen).

Footnotes

 Disclosure: H. Tian, None; E. Sanders, None; A. Reynolds, None; F. van Roy, Alcon (F); J. van Hengel, None