Expression of β-Catenin Gain-Of-Function Mutant Disrupts Fetal Eyelid Closure in the Mouse

Abstract

Purpose: : Eyelid closure is an important event for eye morphogenesis during embryonic development of all mammals, as the eyelid closure may provide a protective micro environment for other ocular tissues. Currently, most of mouse models have shown that the signaling pathways within eyelid epithelial cells play pivotal roles in eyelid closure, but little is known regarding stromal cells. The canonical Wnt signaling pathway has a number of critical functions in tissue morphogenesis. To test whether Wnt signaling pathway has a role of eyelid morphogenesis, we examined the consequence of constitutive activation of Wnt signaling by expressing β-catenin gain-of-function (gof) mutant in eyelid closure using a novel Kera-rtTA/tet-O-Cre/Ctnnb1^{floxed Ex3} (KR/TC/Ctnnb1^{f Ex3}) triple transgenic mice.

Methods: : A novel KR mouse strain carrying rtTA transgene whose expression is controlled by mouse keratocan promoter was generated. Triple transgenic mice KR/TC/Ctnnb1^fEx3 were achieved by cross-breeding of the KR and TC/Ctnnb1^fEx3 bitransgenic mice. The genotypes of the offspring were determined by PCR. To activate Wnt signaling by expressing β-catenin gof mutant missing exon 3 region, mouse embryos were exposed to Doxycycline (Dox) at embryonic (E) day 12.5 by feeding pregnant mother with Dox-chow and embryos and neonates were collected at E14.5, E15.5, E16.5, birth, and postnatal (P) day 21 and subjected to immunohistological examinations.

Results: : The β-catenin gof mutant embryos and neonates exhibited anomaly of bilateral concaved eyelids at birth and severe eyelid margin malformation at P21. Histological and immunofluorescence examinations showed that eyelid epithelial sheet from both β-catenin wild-type and gof mutant mice began to migrate and expressed pJun at E14.5. However, the subsequent eyelid stromal elongation and eyelid closure which normally occurred at E16.5 was disrupted in the β-catenin gof mutant. It was noteworthy that the expression of β-catenin gof mutant caused the formation of a highly proliferative cell mass accumulated in the eyelid stroma which was characterized by the malformation of tarsal plate and Meibomian gland.

Conclusions: : Our results indicated that constitutively activation of Wnt/β-catenin signaling pathway in the eyelid mesenchymal cells might perturb the coordinated movement along with the migrating epithelium during eyelid morphogenesis. The KR/TC/Ctnnb1^{f Ex3} triple mice may be a novel model to study the signaling within stroma during eyelid morphogenesis.

Keywords: eyelid • gene/expression • development