Abstract

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.

Highlights

  • Bidirectional mesenchymal-epithelial interactions play essential roles in the development of organs with an epithelial parenchyma

  • We reported that deletion of β-catenin, in keratocytes of the triple transgenic mice (KR29; TC30; Ctnnb1flox/flox 31), resulted in precocious corneal epithelial stratification during morphogenesis and postnatal development[9]

  • We discovered that a stabilized β-catenin mutant, Ctnnb1ΔE3, aberrantly expressed in corneal keratocytes by means of a Dox-inducible compound transgenic mouse model (KeraRT; TC; Ctnnb1fE3) caused the inhibition of corneal epithelial stratification during ocular surface postnatal morphogenesis, which may be attributed to decreased proliferation of the basal corneal epithelial cells, caused by down-regulation of the expression of Bmp[4] and ΔNp63 in the mutant mice

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Summary

Introduction

Bidirectional mesenchymal-epithelial interactions play essential roles in the development of organs with an epithelial parenchyma. To establish a functional cornea, complex developmental processes must be precisely coordinated by intrinsic regulators and reciprocal signal communication between the epithelium and stroma through signaling transduction, such as Wnt/β-catenin and BMP signaling pathways[9,10,11,12]. Both of these two signaling pathways play critical roles in ocular morphogenesis[13,14,15]. Our data indicated that corneal keratocyte-derived Wnt/β-catenin signaling plays indispensable roles in corneal epithelial maturation during mouse ocular surface development

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