Figure 2 - uploaded by Gen Yamada
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HE histology of eyes of a WT embryo and a mutant embryo with gof-β-catenin in epithelial tissues. Hematoxylin and eosin (HE) histology shows a clear difference in the cellular architecture of the eyelid anlage and the cornea between the wild-type (WT) embryo and the mutant embryo with gain-of-function (gof )-βcatenin . No obvious difference in the structure of the eye and eyelids between the WT (A) and mutant embryos (B) was observed at E13.5. At E15.5, the eyelid (arrows) development is impaired (D) compared with the WT tissue (C). At E17.5 and E18.5, the cornea is completely covered with fused eyelids (arrows) in the WT embryo (E, G), while no eyelids, but just anlage (arrow) of an eyelid, are observed in the mutant (F, H). The surface of the cornea is thicker with an irregular surface in the mutant eye while smooth curvature is seen in the WT embryo (compare H to G). Bar, 100 μm.
Source publication
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 (Ctnnb1(GOFOSE) , gof of β-catenin in the epidermis and the ocular surface epithelium) were generated by time-mating keratin 5-promoter-Cre recombinase (Kr...
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Citations
... Overactivation of Wnt/β-catenin signaling is also known to disrupt eyelid development. For example, genetic deletion of the secreted Wnt inhibitor DKK2 or constitutive activation of epithelial β-catenin both cause EOB phenotypes [18,33]. Therefore, it is crucial to precisely control activity of epithelial Wnt ligands during eyelid development. ...
Purpose:
To investigate the role of Wnt/β-catenin signaling in mouse eyelid development.
Methods:
Wnt/β-catenin signaling was disrupted by deleting supraorbital mesenchymal β-catenin or epithelial Wls. p63 was removed to determine whether the expression of Wnts is affected. The eyelid morphology was examined at different stages. Proliferation, apoptosis, and expression of Wnt ligands and their target genes were analyzed via immunofluorescence staining, TUNEL assay, and in situ hybridization.
Results:
Deletion of β-catenin in supraorbital mesenchyme abolishes eyelid growth by causing decreased proliferation in supraorbital epithelium and underlying mesenchyme. Inhibition of Wnt secretion by deleting Wls in supraorbital epithelium results in failure of eyelid development, similar to the effects of deleting mesenchymal β-catenin. Knockout of p63 results in formation of hypoplastic eyelids and reduced expression of several Wnt ligands in eyelid epithelium.
Conclusions:
Epithelial Wnt ligands activate mesenchymal Wnt/β-catenin signaling to control eyelid growth and their expression is partially regulated by p63.
... In addition to this, aberrant expression of β-catenin in presumptive corneal and conjunctival ectoderm disrupts its proper morphogenesis and results in ocular surface squamous metaplasia. [98,99] Receptors and ligands of Wnt/β-catenin signaling are consistently expressed throughout the OSE [100,101]. However, the active Wnt signaling is detected in early conjunctival epithelium and underlying mesenchyme of cornea, limbus and conjunctiva, not in presumptive corneal and limbal ectoderm [100,102,103]. ...
... It is further supported by the wide expression of Wnt inhibitors DKKs and Sfrp1 in the cornea during embryonic and postnatal development [100,101,104]. The effect of aberrant Wnt signaling depends on the stage and region of activation; CE could undergo fate switch to the epidermis [99,[104][105][106][107] dedifferentiate to more progenitor-like cells [99,105] proliferate in an unregulated fashion [98,99] or could inhibit stratification of CE cells [103,108]. ...
Mammalian corneal development is a multistep process, including formation of corneal epithelium (CE), endothelium and stroma during embryogenesis followed by postnatal stratification of the epithelial layers, and continuous renewal of the epithelium to replace the most outer corneal cells. Herein we employed Cre- loxP system to conditionally deplete Pax6 proteins in two domains of ocular cells, including the ocular surface epithelium (cornea, limbus and conjunctiva) or postnatal CE, via K14-cre or Aldh3-cre , respectively. Earlier and broader inactivation of Pax6 in the OSE resulted in thickened OSE with CE and limbal cells adopting the conjunctival keratin expression pattern. More restricted depletion of Pax6 in postnatal CE resulted in the abnormal cornea marked by reduced epithelial thickness despite of increased epithelial cell proliferation. Immunofluorescence studies showed loss of Keratin 12, an intermediate filament and diffused expression of adherens junction components, together with reduced tight junction protein, Zonula occludens-1 (ZO-1). Furthermore, expression of Keratin 14, basal cell marker in apical layers, indicates impaired differentiation of corneal epithelial cells. Collectively, our data demonstrate that Pax6 is essential for maintaining proper differentiation and strong intercellular adhesion in postnatal corneal epithelial cells, whereas limbal Pax6 is required for preventing the outgrowth of conjunctival cells to the cornea.
... Interactions between the early neural tube and the adjacent ectoderm give rise to the neural crest cells, which are a highly migratory and multipotent cell population able to migrate between the lens and presumptive corneal epithelium to form the corneal endothelium, other epithelia of the ocular surface and the stromal keratocytes in a highly dependent process. Innervation of the corneal stroma and epithelium originates from the neural crest-and ectodermal placode-derived trigeminal ganglion (Tripathi and Tripathi, 1990;Barishak, 2001;Swamynathan, 2013;Dhouailly et al., 2014;Lwigale, 2015;Mizoguchi et al., 2015;Forrester et al., 2016). ...
Importance:
Non-invasive techniques for retrieving ocular surface cells from babies infected by zika virus (ZIKV) during the gestational period remain to be determined.
Objectives:
The aim of this study was to describe an optimized impression cytology method for the isolation of viable cells from Zika infected babies with and without Congenital Zika Syndrome (CZS) in satisfactory amount and quality to enable easy adoption in the field and application in the context of genomic and molecular approaches.
Design settings and participants:
Ocular surface samples were obtained with a hydrophilic nitrocellulose membrane (through optimized impression cytology method) from twelve babies referred to the Pediatric Service of the Antonio Pedro Hospital, Universidade Federal Fluminense (UFF), Niteroi, Rio de Janeiro, Brazil. After an authorized written informed consent from the parents, samples were collected from both eyes of 12 babies (4 babies with maternal ZIKV exposure during gestation and presence of clinical signs which included ocular abnormalities and microcephaly; 4 babies with maternal ZIKV exposure during gestation but no clinical signs; and 4 unaffected control babies with negative PCR for Zika virus and without clinical signs). Cells were used for microscopy analyses and evaluated for their suitability for downstream molecular applications in transcriptomic and proteomic experiments.
Results:
Our optimized impression cytology protocol enabled the capture of a considerable number of viable cells. The microscopic features of the conjunctival epithelial cells were described by both direct analysis of the membrane-attached cells and analysis of cytospinned captured cells using several staining procedures. Epithelial basal, polyhedral and goblet cells were clearly identified in all groups. All cases of ZIKV infected babies showed potential morphological alterations (cell keratinization, pyknosis, karyolysis, anucleation, and vacuolization). Molecular approaches were also performed in parallel. Genomic DNA and RNA were successfully isolated from all samples to enable the establishment of transcriptomic and proteomic studies.
Conclusions and relevance:
Our method proved to be a suitable, fast, and non-invasive tool to obtain ocular cell preparations from babies with and without Zika infection. The method yielded sufficient cells for detailed morphological and molecular analyses of samples. We discuss perspectives for the application of impression cytology in the context of ZIKV studies in basic and clinical research.
... Canonical Wnt signaling plays vital roles in ocular morphogenesis, homeostasis, wound healing, and pathogenesis of many ocular diseases 10,13-15,37-39 . However, to the best of our knowledge, most of the genetic studies examining the role of canonical Wnt signaling in mouse corneas were performed with corneal epithelium 21,22,[40][41][42] . For example, it was documented that repression of canonical Wnt signaling activity in corneal epithelium is an essential prerequisite for the differentiation of corneal epithelial cells from their progenitors, as reported in the Dkk2 knockout mice 21 and in Pitx2-deficient mice 20 . ...
We previously reported that genetic deletion of β-catenin in mouse corneal keratocytes resulted in precocious corneal epithelial stratification. In this study, to strengthen the notion that corneal keratocyte-derived Wnt/β-catenin signaling regulates corneal epithelial stratification during mouse development, we examined the consequence of conditional overexpression of a stabilized β-catenin mutant (Ctnnb1ΔE3) in corneal keratocytes via a doxycycline (Dox)-inducible compound transgenic mouse strain. Histological analysis showed that conditional overexpression of Ctnnb1ΔE3 in keratocytes inhibited corneal epithelial stratification during postnatal development. Unlike the corneal epithelium of the littermate controls, which consisted of 5-6 cell layers at postnatal day 21 (P21), the mutant corneal epithelium contained 1-2 or 2-3 cell layers after Dox induction from embryonic day 0 (E0) to P21 and from E9 to P21, respectively. X-gal staining revealed that Wnt/β-catenin signaling activity was significantly elevated in the corneal keratocytes of the Dox-induced mutant mice, compared to the littermate controls. Furthermore, RT-qPCR and immunostaining data indicated that the expression of Bmp4 and ΔNp63 was downregulated in the mutant corneas, which was associated with reduced corneal epithelial proliferation in mutant epithelium, as revealed by immunofluorescent staining. However, the expression of Krt12, Krt14 and Pax6 in the mutant corneas was not altered after overexpression of Ctnnb1ΔE3 mutant protein in corneal keratocytes. Overall, mutant β-catenin accumulation in the corneal keratocytes inhibited corneal epithelial stratification probably through downregulation of Bmp4 and ΔNp63 in the corneal epithelium.
Mammalian corneal development is a multistep process, including formation of the corneal epithelium (CE), endothelium and stroma during embryogenesis, followed by postnatal stratification of the epithelial layers and continuous renewal of the epithelium to replace the outermost corneal cells. Here, we employed the Cre-loxP system to conditionally deplete Pax6 proteins in two domains of ocular cells, i.e., the ocular surface epithelium (cornea, limbus and conjunctiva) (OSE) or postnatal CE via K14-cre or Aldh3-cre, respectively. Earlier and broader inactivation of Pax6 in the OSE resulted in thickened OSE with CE and limbal cells adopting the conjunctival keratin expression pattern. More restricted depletion of Pax6 in postnatal CE resulted in an abnormal cornea marked by reduced epithelial thickness despite increased epithelial cell proliferation. Immunofluorescence studies revealed loss of intermediate filament Cytokeratin 12 and diffused expression of adherens junction components, together with reduced tight junction protein, Zonula occludens-1. Furthermore, the expression of Cytokeratin 14, a basal cell marker in apical layers, indicates impaired differentiation of CE cells. Collectively, our data demonstrate that Pax6 is essential for maintaining proper differentiation and strong intercellular adhesion in postnatal CE cells, whereas limbal Pax6 is required to prevent the outgrowth of conjunctival cells to the cornea.
Purpose:
Defects in neural crest development are a major contributing factor in corneal dysgenesis, but little is known about the genetic landscape during corneal development. The purpose of this study was to provide a detailed transcriptome profile and evaluate changes in gene expression during mouse corneal development.
Methods:
RNA sequencing was used to uncover the transcriptomic profile of periocular mesenchyme (pNC) isolated at embryonic day (E) 10.5 and corneas isolated at E14.5 and E16.5. The spatiotemporal expression of several differentially expressed genes was validated by in situ hybridization.
Results:
Analysis of the whole-transcriptome profile between pNC and embryonic corneas identified 3815 unique differentially expressed genes. Pathway analysis revealed an enrichment of differentially expressed genes involved in signal transduction (retinoic acid, transforming growth factor-β, and Wnt pathways) and transcriptional regulation.
Conclusions:
Our analyses, for the first time, identify a large number of differentially expressed genes during progressive stages of mouse corneal development. Our data provide a comprehensive transcriptomic profile of the developing cornea. Combined, these data serve as a valuable resource for the identification of novel regulatory networks crucial for the advancement of studies in congenital defects, stem cell therapy, bioengineering, and adult corneal diseases.
