School of Optometry, Indiana University, Bloomington, IN, 47405, USA.
Department of Ophthalmology, Wakayama Medical University, Wakayama, Japan.
Sci Rep. 2019 Feb 13;9(1):1919. doi: 10.1038/s41598-018-36392-2.
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) in corneal keratocytes via a doxycycline (Dox)-inducible compound transgenic mouse strain. Histological analysis showed that conditional overexpression of Ctnnb1 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 Ctnnb1 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.
我们之前报道过，在小鼠角膜成纤维细胞中基因敲除β-catenin 会导致角膜上皮过早分层。在这项研究中，为了加强角膜成纤维细胞源性 Wnt/β-catenin 信号在小鼠发育过程中调节角膜上皮分层的观点，我们通过一种强力霉素（Dox）诱导的复合转基因小鼠品系，检测了角膜成纤维细胞中稳定的β-catenin 突变体（Ctnnb1）的条件过表达的结果。组织学分析表明，角膜成纤维细胞中 Ctnnb1 的条件过表达抑制了出生后发育过程中的角膜上皮分层。与同窝仔鼠对照的角膜上皮不同，后者在出生后第 21 天（P21）有 5-6 个细胞层，突变的角膜上皮在从胚胎第 0 天（E0）到 P21 以及从 E9 到 P21 分别用 Dox 诱导后，只含有 1-2 或 2-3 个细胞层。X-gal 染色显示，与同窝仔鼠对照相比，Dox 诱导的突变小鼠的角膜成纤维细胞中 Wnt/β-catenin 信号活性显著升高。此外，RT-qPCR 和免疫染色数据表明，突变角膜中 Bmp4 和 ΔNp63 的表达下调，这与突变上皮中角膜上皮增殖减少有关，如免疫荧光染色所示。然而，在用角膜成纤维细胞中 Ctnnb1 突变蛋白过表达后，突变角膜中 Krt12、Krt14 和 Pax6 的表达并未改变。总的来说，角膜成纤维细胞中突变β-catenin 的积累通过下调角膜上皮中的 Bmp4 和 ΔNp63 抑制了角膜上皮分层。
