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Aberrant expression of a stabilized ?-catenin mutant in keratocytes inhibits mouse corneal epithelial stratification.

ABSTRACT: We previously reported that genetic deletion of ?-catenin in mouse corneal keratocytes resulted in precocious corneal epithelial stratification. In this study, to strengthen the notion that corneal keratocyte-derived Wnt/?-catenin signaling regulates corneal epithelial stratification during mouse development, we examined the consequence of conditional overexpression of a stabilized ?-catenin mutant (Ctnnb1^?E3) in corneal keratocytes via a doxycycline (Dox)-inducible compound transgenic mouse strain. Histological analysis showed that conditional overexpression of Ctnnb1^?E3 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/?-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 ?Np63 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 Ctnnb1^?E3 mutant protein in corneal keratocytes. Overall, mutant ?-catenin accumulation in the corneal keratocytes inhibited corneal epithelial stratification probably through downregulation of Bmp4 and ?Np63 in the corneal epithelium.

SUBMITTER: Zhang L

PROVIDER: S-EPMC6374483 | biostudies-literature | 2019 Feb

REPOSITORIES: biostudies-literature

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Aberrant expression of a stabilized β-catenin mutant in keratocytes inhibits mouse corneal epithelial stratification.

Zhang Lingling L Wang Yen-Chiao YC Okada Yuka Y Zhang Suohui S Anderson Matthew M Liu Chia-Yang CY Zhang Yujin Y

Scientific reports 20190213 1

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 (Ctnnb1<sup>ΔE3</sup>) in corneal keratocytes via a doxycycline (Dox)-inducible compound transgeni ...[more]

PMID: 30760729

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Project description:BackgroundAltered dosage of the transcription factor PAX6 causes multiple human eye pathophysiologies. PAX6⁺/⁻ heterozygotes suffer from aniridia and aniridia-related keratopathy (ARK), a corneal deterioration that probably involves a limbal epithelial stem cell (LESC) deficiency. Heterozygous Pax6(+/Sey-Neu) (Pax6⁺/⁻) mice recapitulate the human disease and are a good model of ARK. Corneal pathologies also occur in other mouse Pax6 mutants and in PAX77(Tg/-) transgenics, which over-express Pax6 and model human PAX6 duplication.Methodology/principal findingsWe used electron microscopy to investigate ocular defects in Pax6⁺/⁻ heterozygotes (low Pax6 levels) and PAX77(Tg/-) transgenics (high Pax6 levels). As well as the well-documented epithelial defects, aberrant Pax6 dosage had profound effects on the corneal stroma and endothelium in both genotypes, including cellular vacuolation, similar to that reported for human macular corneal dystrophy. We used mosaic expression of an X-linked LacZ transgene in X-inactivation mosaic female (XLacZ(Tg/-)) mice to investigate corneal epithelial maintenance by LESC clones in Pax6⁺/⁻ and PAX77(Tg/-) mosaic mice. PAX77(Tg/-) mosaics, over-expressing Pax6, produced normal corneal epithelial radial striped patterns (despite other corneal defects), suggesting that centripetal cell movement was unaffected. Moderately disrupted patterns in Pax6⁺/⁻ mosaics were corrected by introducing the PAX77 transgene (in Pax6⁺/⁻, PAX77(Tg/-) mosaics). Pax6(Leca4/+), XLacZ(Tg/-) mosaic mice (heterozygous for the Pax6(Leca4) missense mutation) showed more severely disrupted mosaic patterns. Corrected corneal epithelial stripe numbers (an indirect estimate of active LESC clone numbers) declined with age (between 15 and 30 weeks) in wild-type XLacZ(Tg/-) mosaics. In contrast, corrected stripe numbers were already low at 15 weeks in Pax6⁺/⁻ and PAX77(Tg/-) mosaic corneas, suggesting Pax6 under- and over-expression both affect LESC clones.Conclusions/significancePax6⁺/⁻ and PAX77(Tg/-) genotypes have only relatively minor effects on LESC clone numbers but cause more severe corneal endothelial and stromal defects. This should prompt further investigations of the pathophysiology underlying human aniridia and ARK.

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Aberrant expression of a stabilized ?-catenin mutant in keratocytes inhibits mouse corneal epithelial stratification.

Publications

Aberrant expression of a stabilized β-catenin mutant in keratocytes inhibits mouse corneal epithelial stratification.

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