Molecular Vision 2015; 21:793-803
Received 13 October 2015 | Accepted 29 July 2015 | Published 31 July 2015
1Department of Ophthalmology, Wakayama Medical University School of Medicine, Wakayama, Japan; 2Department of Developmental Genetics, Institute of Advanced Medicine, Wakayama Medical University, Wakayama, Japan; 3Department of Ophthalmology, University of Cincinnati School of Medicine, Cincinnati, Ohio; 4Laboratory Animal Center, Wakayama Medical University, Wakayama, Japan
Correspondence to: Shin Mizoguchi, Department of Ophthalmology, Wakayama Medical University, 811-1 Kimiidera, Wakayama, 641-0012, Japan, Phone: 81-73-447-2300, FAX: 81-73-448-1991; email: email@example.com
Purpose: To examine the developmental pathobiology of the eyelid and the cornea caused by epithelial β-catenin gain-of-function (gof) during mouse embryogenesis.
Methods: Compound mutant mice (Ctnnb1GOFOSE, gof of β-catenin in the epidermis and the ocular surface epithelium) were generated by time-mating keratin 5-promoter-Cre recombinase (Krt5-Cre) and Ctnnb1fE3/WT (floxed exon 3 of Ctnnb1) mice. Eyes obtained from wild-type (WT) and mutant embryos at various gestation stages until E18.5 were examined with histology and immunohistochemistry. The ultrastructure of the ocular tissues of the E18.5 embryos was also examined.
Results: Expression of the gof-β-catenin mutant protein in the epidermis severely impaired eyelid morphogenesis at E15.5, E17.5, and E18.5. The mutant stroma exhibited impaired keratocyte differentiation with accelerated cell proliferation and reduction in the accumulation of collagen type I. The mutant embryos also showed hyperproliferative nodules in the ocular surface epithelia with anomaly of cornea-type epithelial differentiation and the absence of the epithelial basement membrane.
Conclusions: Expression of the gof-β-catenin mutant protein in basal epithelial cells disrupts eyelid and cornea morphogenesis during mouse embryonic development due to the perturbation of cell proliferation and differentiation of the epithelium and the neural crest-derived mesenchyme.
Embryonic eyelid morphogenesis is regulated by a complex interaction of epidermis and neural crest-derived mesenchyme, which is precisely orchestrated by growth factors [1,2]. A report showing that fibroblast growth factor 10 (FGF10) is essential to eyelid morphogenesis was followed by a publication that reported the bone morphogenetic protein (BMP) signal is located downstream of the FGF10 signal for eyelid morphogenesis [1,3,4]. We previously reported that transforming growth factor α (TGF-α) is involved in the migration of the neural crest–derived mesenchyme during eyelid morphogenesis, while TGF-β is also reportedly involved in eyelid morphogenesis [5,6]. At the signaling level, the loss of mitogen-activated protein kinase kinase kinase 1 (MAP3K1) reportedly impairs eyelid closure during embryonic development in mice [7,8]. Gene ablation of sphingosine 1-phosphate receptors also leads to the failure of eyelid closure during mouse embryonic development, via inhibition of MAP3K1 signaling .
Signaling-activated by β-catenin plays pivotal roles in regulating cell lineage differentiation or cell fate in tissue morphogenesis during embryonic development [10,11]. Canonical Wnt/β-catenin pathways activated by Wnt binding to Frizzled family receptors, leading to dissociation of the Dvl, GSK-3β, and Axin complex, result in the inhibition of serine/threonine phosphorylation encoded within exon 3 of the Ctnnb1 allele, which prevents β-catenin degradation via ubiquitination and leads to nuclear translocation of β-catenin . Specific target proteins of the Wnt/β-catenin signal regulate self-renewal and differentiation of various tissue-specific stem cells in adult somatic tissues, suggesting involvement in maintenance of tissue homeostasis [10-12]. Abnormal and/or excessive activation of β-catenin signaling is, in turn, reportedly involved in the development and metastasis of certain malignant tumors . We previously reported that keratin 12 promoter-driven conditional gain-of-function (gof) of β-catenin in the cornea epithelium (Krt12rtTA/Wt/tetO-Cre/Ctnnb1floxE3/Wt triple transgenic mouse) exhibited tumor-like hyperproliferation of the corneal epithelium and increased the proliferation of stromal cells in mouse embryos . In this mouse line, abnormalities were not observed in the conjunctiva and the eyelid.
Because MAP3K1 positively modulates Wnt-dependent gene expression via interaction with axin1, it is likely that Wnt/β-catenin signaling is involved in the impairment of eyelid morphogenesis in MAP3K1 null mice [7,8,15]. Inhibition of Tcf3-β-catenin by the homozygous Tcf3 knock-in mutant (Tcf3ΔN/ΔN) also results in a defect in embryonic eyelid closure in mice, which supports the previous notion . A mouse line introduced with a null mutation of Dkk-2 also shows impaired eyelid morphogenesis during embryonic development . These reports suggest that the involvement of epithelial β-catenin signaling in mouse eyelid tissue morphogenesis should be investigated further. To understand the effects of gof-β-catenin signaling in the epithelium on mouse eyelid morphogenesis, we created a conditional mutant mouse line with the gof allele of β-catenin by deleting exon 3 (floxed E3 β-catenin) with keratin 5 promoter-driven Cre recombinase in basal epithelial cells and examined morphogenesis of the eyelid and the ocular surface, that is, the cornea and the conjunctiva during embryonic development. Our data indicate that the keratin 5 promoter mutants exhibit a corneal phenotype similar to that of keratin 12-deriven gof-β-catenin, but they manifest marked impairment in eyelid morphogenesis.
The experiments were approved by the DNA Recombination Experiment Committee and the Animal Care and Use Committee of Wakayama Medical University. The experiments performed in accordance with the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research.
Genetically modified mouse lines of Krt5-Cre and Ctnnb1fE3/WT (Ctnnb1-floxed Exon3) were previously described [18,19]. Cre excises exon 3 of the Ctnnb1fE3 allele, leading to the accumulation of active β-catenin in the cells. The genotypes were identified with PCR using oligonucleotide primers specific for each transgene as previously reported . Primers were designed based on the mouse β-catenin sequence (DDBJ/EMBL/GenBank accession No. M90364): BCAT-S1 (5’-ATG GCT ACT CAA GCT GAC CTG ATG G-3’) and BCAT-AS1 (5’-TTA CAG GTC AGT ATC AAA CCA GGC C-3’). PCR was for 30 cycles of 94 °C for 30 s, 60 °C for 90 s and 72 °C for 2 min. In all experiments, the detection time of the vaginal plug was defined as E0.5 of embryonic development. The E13.5, E15.5, E17.5, and E18.5 embryos are described below. Embryos at E13.5 and E15.5 were labeled with maternal i.p., administration of bromo-deoxyuridine (BrdU) as previously reported .
The mutant (Krt5-Cre/ Ctnnb1(ex3)fl/WT) mouse dies at birth due to epidermis defects since Krt5 is ubiquitously expressed by all stratified epithelia. The eyes and eyelids of the mutant embryos and wild-type (WT) littermates at E13.5, 15.5, and 17.5 were processed for paraffin embedding, and E18.5 embryos were used for the electron microscopic examination. The number of embryos used were E13.5 (n=2), E15.5 (n=2), E17.5 (n=5), and E18.5 (n=4) in the WT and E13.5 (n=2), E15.5 (n=2), E17.5 (n=5), and E18.5 (n=4) in mutants. Deparaffinized sections (5 μm) were processed for hematoxylin and eosin (HE) staining. Immunohistochemistry was also performed with antibodies against β-catenin (1:1,000 in PBS [1X; 137 mM NaCl, 2.70 mM KCl, 7.75 mM Na2HPO4, 1.47 mM KH2PO4, pH 7.5], BD Biosciences, San Jose, CA), keratin 12 , keratin 14 (1:200 in PBS, Sigma-Aldrich, St. Louis, MO), collagen type I (1:200 in PBS, Southern Biotech, Birmingham, AL), laminin (1:25 in PBS, Sigma-Aldrich), and keratocan . Secondary antibodies were conjugated with either peroxidase or Alexa-Fluor 555. Nuclei were counterstained with either methylgreen or 4,6-diamidino-2-phenylindole (DAPI), respectively. Immunodetection of BrdU in cell nuclei was performed according to the manufacturer’s protocol (Roche Diagnostics, Mannheim, Germany) as previously reported . The eyes and eyelids of E18.5 embryos of both genotypes (n=2) were observed under routine transmission electron microscopy as previously reported . Briefly, tissues were fixed in 2% glutaraldehyde in 0.1 M phosphate-buffer (pH 7.4) for 48 h and then post-fixed in 1% osmium tetroxide. Then the samples were embedded in Epon mixture. Ultrathin sections were cut, electron-stained, and observed under transmission electron microscopy.
Expression of gof-β-catenin driven by the Krt5 promoter in basal epidermal cells perturbed eyelid morphogenesis under the examination of the gross appearance of the eyelids and the external ocular structure (Figure 1). At E13.5, no obvious abnormalities were observed in eyelid elongation (not shown). As early as E15.5, elongation of eyelid tissues was somewhat impaired (Figure 1B) compared with a WT littermate (Figure 1A). At E17.5 (Figure 1C) and E18.5 (Figure 1D), the eyelid fused in the WT littermate, while the mutant of gof-β-catenin in basal epidermal cells exhibited an eye-open phenotype at these embryonic day points (Figure 1E, F).
HE histology of the eyelid anlage seemed to be similar between the WT and mutant embryos at E13.5 (Figure 2A,B). However, the mesenchymal cells in the periocular tissue of the mutant embryo (Figure 2B) seemed to be more densely packed compared with the WT littermate at this embryonic stage (Figure 2A). Morphogenesis of the upper and lower eyelids was severely impaired in the mutant embryo of gof-β-catenin at E15.5 (Figure 2D) compared with the WT embryo (Figure 2C). At E17.5 and E18.5, eyelid fusion can be readily seen in the wild-type embryo (Figure 2E,G), while in the mutant the cornea is not covered by fused eyelids (Figure 2F,H) with an uneven corneal epithelium (described in detail below). Higher magnification observation showed that the mesenchymal cells in the periocular tissue of a mutant embryo (Figure 2H) still seemed to be more densely packed compared with the WT littermate (Figure 2G) at E18.5 similar to embryos at earlier embryonic day points.
Characterization of tissue phenotype was then performed with ultrastructural observation and immunohistochemistry. Transmission electron microscopy showed abnormal epidermal cell differentiation (loss of stratification or cornification in the superficial epithelial layer) and reduction in the collagenous matrix in the dermis of an E18.5 mutant embryo (Figure 3D) compared with that of a WT littermate (Figure 3C).
Immunohistochemistry detected nuclear accumulation of β-catenin in the focal nodular structures of the proliferating epidermal keratinocytes of the eyelid skin of the mutant embryo at E18.5 (Figure 3H), while faint staining for β-catenin was seen in the epidermis of the WT embryos (Figure 3G). The eyelid epidermis in the E18.5 WT and knockout (KO) embryos (Figure 3I, J) was not labeled for vimentin, and vimentin-labeled cells (presumably fibroblasts) were distributed in the dermis. At E15.5, the cell proliferation rate of the eyelid epidermis determined with BrdU labeling was comparable between the WT (Figure 3 K) and mutant embryos (Figure 3L).
We then examined abnormalities of the ocular surface epithelium in detail. HE staining histology showed that the thickness of the corneal epithelium was similar between the WT (Figure 4A) and mutant (Figure 4B) embryos at E13.5. The cell density of the stromal cells was higher in the mutant embryo compared with the stroma of the WT littermate throughout the embryonic stages (Figure 4A-H, discussed later). At E15.5, the WT corneal epithelium remained thin (Figure 4C). At this time point, the epithelium of a mutant mouse embryo seemed thicker with multilayerization than that in the WT control, and localized multilayerization was observed (Figure 4D). Gross examination of the cornea of the E17.5 embryos showed opaque tumor-like nodules on the open corneal surface without overlaying eyelids (Figure 1E); in contrast, the eyelids were well developed in the WT littermate (Figure 1C). Focal nodular epithelial hyperproliferation was observed in the corneal epithelium in the mutant embryo (Figure 4F,H) but not in the WT embryo (Figure 4E,G).
Immunohistochemistry detected obvious β-catenin staining in the corneal epithelium of the mutant embryo (Figure 4J), but not in the WT embryo, at E13.5 (Figure 4I). At E15.5, β-catenin was detected in the WT corneal epithelium, which showed a monolayer appearance (Figure 4K). β-catenin staining was markedly observed in the corneal epithelium, which was thicker than the normal one. Immunoreactivity for β-catenin was observed in cell–cell borders among each epithelial cell and was prominently accumulated in the cytoplasm and/or nuclei of the focal proliferative nodules in the corneal epithelium of the mutant embryos at E15.5 (Figure 4L). At E17.5, β-catenin was detected in the cell–cell border of the basal cells of the WT corneal epithelium (Figure 4M). At this embryonic day point, accumulation of β-catenin was still observed in the cytoplasm and/or nuclei of the epithelial cell nodule but not observed in the thick epithelium among each nodules (Figure 4N).
Ultrastructural observation showed a regular arrangement of stratified epithelial cells on the surface of the WT cornea at E18.5 (Figure 5C). In the E18.5 mutant embryos, the superficial epithelial cells of the corneal epithelium failed to assume the flattened characteristic shape of the stratified superficial epithelium seen in the WT cornea (compare Figure 5D to Figure 5C). The E18.5 mutant epithelium lacked a subepithelial basement membrane at the interface with the underlying stroma (Figure 5F), while the WT epithelium had an obvious basement membrane structure (arrows, Figure 5E).
Because the β-catenin signal is closely related to cell differentiation and cell phenotype control, the epithelial–mesenchymal transition (EMT), we examined whether gof-β-catenin affected differentiation of the corneal epithelium by using immunohistochemical analysis of intermediate filament components. We first examined the characteristics of epithelial differentiation by examining the expression pattern of keratin 12, the marker of cornea-type epithelium differentiation, and keratin 14. In WT embryos, the keratin 12 protein was negative in the cornea at E13.5 (Appendix 1, panel A), and expression in the corneal epithelium was detected at E15.5 (Appendix 1, panel B); marked expression was detected at E17.5 (Figure 6A). However, the keratin 12 protein was not detected in the corneal epithelium throughout E13.5–17.5 (Figure 6B). Keratin 14 was observed in the basal cells of the WT corneal epithelium (Appendix 1, panel C). Keratin 14 was markedly expressed in epithelial nodules (Appendix 1, panel D). Focal hyperproliferation of the conjunctival epithelium was also observed in the mutant embryos (Appendix 1, panel E).
Impairment of intraepithelial differentiation from the basal layer toward the superficial layer might be associated with changes in cell proliferation, which can be examined with BrdU labeling. At E13.5, the incidence of BrdU-labeling in the embryonic corneal epithelium was similar between the WT and mutant embryos (Appendix 1, panel F,G). At E15.5, many epithelial cells were labeled with BrdU immunohistochemistry in the mutant corneal epithelium (Figure 7B,D), while such BrdU-labeled cells were less frequently observed in the WT corneal epithelium (Figure 7A,C).
In our previous study, we did not fully examine corneal stroma abnormalities in the keratin 12 promoter-driven conditional expression of gof-β-catenin (Krt12rtTA/Wt/tet-O-Cre/Ctnnb1floxE3/Wt triple transgenic) mouse embryos . In the present study, the mutant embryo with Krt5-Cre-driven gof-β-catenin overexpression induced hypercellularity with increased thickness in the stroma as early as E13.5 (Figure 4A,B) along with epithelial β-catenin immunostaining (Figure 4I-P). An ultrastructural examination was performed with the E18.5 embryos to examine the potential anomaly of keratocytes and the extracellular matrix. The WT corneal stroma exhibited a well-organized lamellar structure of keratocytes and collagen fibers at E18.5 (Figure 8A), while the keratocytes of the mutant stroma showed a disorganized distribution with less extracellular matrix in between the cells (Figure 8B). Such abnormality was more prominent in the anterior stroma than in the posterior stroma (Figure 5D). Neovascularization was also observed in the mutant stroma (Figure 8B).
To characterize the level of differentiation or maturation of keratocytes and the nature of the extracellular matrix of the corneal stroma, we then performed immunohistochemistry for the extracellular matrix components. Expression of keratocan is a hallmark of keratocyte-type differentiation. At E13.5, the corneal stroma of the WT and mutant littermates was not labeled with anti-keratocan antibodies (Appendix 1, panel H,I). At E15.5 (Appendix , panel J) and E17.5, immunoreactivity for keratocan was detected in the stroma of the wild-type mouse (Figure 9A) but completely absent in the mutant stroma (Figure 9B). We then evaluated the maturation of the stromal extracellular matrix by examining the protein expression of collagen type I (the major collagen in the corneal stroma).
Collagen type I was not detected at E13.5 but was readily observed at E15.5 (Appendix 1, panel E) and E17.5 in the WT embryos (Figure 9C). Epithelial gof-β-catenin completely abolished accumulation of collagen I in the corneal stroma at E17.5 (Figure 9D). These findings indicate that gof-β-catenin disrupted the keratocyte-type differentiation of corneal stromal cells.
Our primary interest was to understand how augmented β-catenin signaling in the basal epithelial cells of the stratified epithelium, for example, the epidermis and the ocular surface epithelia, affects the embryonic development of whole ocular tissues. We used Krt5-Cre mice to drive Ctnnb1fE3/WT for this purpose because our previous study with Krt12-Cre mice showed an increase in the β-catenin signal in the corneal epithelium, but not in the conjunctiva and the eyelid epidermis. Our major purpose included understanding the mechanisms of pathogenic consequences induced by stabilized epidermal β-catenin-mediated signals in mouse eyelid morphogenesis. Our results showed the constitutive excess β-catenin-mediated signal in the epithelial tissues disrupted morphogenesis of the eyelid in addition to ocular surface epithelia and the underlying mesenchyme.
Embryonic morphogenesis of the eyelid is a complex, yet orchestrated, process governed by the interaction between epithelium derived from the surface ectoderm and mesenchymal cells of neural crest origin. Several investigations including literature mentioned in the Introduction suggest β-catenin-mediated signals in the epidermis are involved in eyelid morphogenesis. Null mutation of Dkk-2 impairs eyelid morphogenesis, which also prompted us to hypothesize that expression of β-catenin in periocular epidermal keratinocytes might have adverse consequences on eyelid formation . Eyelid formation is essential to the morphogenesis of ocular adnexal tissues, for example, the meibomian gland or the extraocular muscle.
The present study showed that constitutive activation of β-catenin signaling in the basal epidermal epithelial cells interrupted eyelid morphogenesis. In the WT mouse embryo, the eyelid folds begin to grow at E15.5 and fuse by E17.5 as observed with the gross appearance. Eyelid morphogenesis was morphologically impaired in the mutant with epidermal gof-β-catenin at E15.5, although gof-β-catenin was activated at E13.5. The exact mechanism(s) by which the excess β-catenin signal in the epidermis impairs the eyelid formation must be investigated. Ultrastructural observation showed abnormal epidermal cell differentiation (loss of organized stratification in the upper layers and ectopic vimentin expression). The finding suggests epidermal keratinocytes with excess β-catenin signal might induce the epithelial–mesenchymal transition and might affect organized keratinocyte movement for eyelid morphogenesis. Accumulation of dermal collagen bundles also seemed to be impaired in the eyelid mesenchyme of the mutant embryo compared with the WT embryo, suggesting epidermal–mesenchymal (neural crest–derived cell) crosstalk; growth factors/cytokines expressed by mutant epithelial cells might affect the behavior of mesenchymal cells. Further studies are needed to investigate whether this abnormal structure of the epidermis is the direct consequence of impaired keratinocyte migration during eyelid formation.
We previously reported the expression of gof-β-catenin in the limited area of the corneal epithelium in mice by using tet-On (Krt12-rtTA/tet-O-Cre/Ctnnb1fE3/WT) mice that exhibited intraepithelial neoplasm-like lesions in the corneal epithelium similar to that seen in the present study using Krt5-Cre/ Ctnnb1fE3/WT mice . In addition, we observed lesions in the epidermis of the eyelid and the conjunctival epithelium in the present study. The mutant gof-β-catenin (Krt5-Cre/Ctnnb1fE3/WT) embryos also manifested abnormalities in conjunctival differentiation; focal hyperproliferative nodules formed in the mutant conjunctival epithelium such as the corneal epithelium (Appendix 1, panel E).
An in vitro study conducted by other investigators showed that Wnt/β-catenin signaling has an inhibitory effect on the differentiation of corneal epithelium–stem cells, and promotes epithelial cell proliferation and default epithelium differentiation . Results of the current in vivo experiments do not contradict these cell culture findings. Namely, gof-β-catenin impaired differentiation of the corneal epithelium in association with the presence of hyperproliferative spots and increased epithelial intracellular layerization in the corneal epithelium as evaluated with immunohistochemistry and ultrastructural observation . The phenotype seen in the corneal epithelium of the current mutant mouse embryos was similar to that observed in gof-β-catenin in the corneal epithelium. Krt12rtTA/WT/TC/Ctnnb1fE3/WT triple transgenic gof-β-catenin mice did not completely abolish the expression of keratin 12, the cornea-type epithelium differentiation marker . However, expression of keratin 12 was not detected in the current mutant embryos at E17.5 while it was readily seen in WT embryos at E15.5, suggesting that gof-β-catenin in limbal basal cells shuts down cornea-type epithelial differentiation. Ultrastructural observation showed the subepithelial basement membrane disappeared, which coincides with a previous finding that β-catenin signaling leads to upregulated expression of matrix metalloproteinase-7, which may account for the EMT of the corneal epithelium . Targeting β-catenin as a strategy for treating ocular surface neoplasms should be examined.
The current finding of the increased proliferation of corneal stromal cells with epithelial gof-β-catenin is consistent with our previous result of hyperproliferation in stromal cells in Krt12rtTA/Wt/tet-O-Cre/Ctnnb1floxE3/Wt triple transgenic mouse embryos . However, we failed to fully examine the nature of the corneal stromal matrix in the previous mutants. The present study showed an abnormally increased cell population in the mutant corneal stroma at E13.5. This abnormality is somewhat different from those observed in Krt12-rtTA-driven mutants, which can in part be explained by the fact that keratin 5 is expressed at E12.5 several days ahead of keratin 12 around E13.5 to 14.5. Thereafter, perturbation of the peridermal epithelium by gof-β-catenin may potentially affect the migration and differentiation of periocular mesenchymal cells of neural crest origin, which contribute to the formation of ocular surface stroma, as well as the eyelid, during embryonic development. This possibility is substantiated by observations with transmission electron microscopy and immunohistochemistry: an irregular arrangement of embryonic keratocytes without expression of keratocan and collagen type I in the mutant embryo stroma . The corneal endothelium seemed ultrastructurally well developed even in the mutant embryos at E17.5.
In Summary, further study is needed to uncover the roles of β-catenin signaling in ocular Development. But we conclude that excess β-catenin signaling in the epidermis and corneal epithelium impairs eyelid morphogenesis in addition to affecting morphogenesis of the cornea and the conjunctiva in mice.
This study was supported by a grant of Wakayama Medical Award for Young Researchers (to SM), Grants from the Ministry of Education, Science, Sports and Culture of Japan (C24592638 to YO and C25462729 to SS), and NEI Eye Research Foundation.