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.

Corneal nerve fibers serving sensory, reflex and neurotrophic functions sustains the corneal homeostasis and transparency to warrant the normal visual function. It remains unknown whether corneal epithelium is also reciprocally important for the corneal innervation. Herein, we generated a compound transgenic mouse strain, K14rtTA;tetO-Cre (TC);Shp2 flox/flox , in which Shp2 was conditionally knocked out from the K14-positive cells including corneal epithelium (Shp2 K14ce-cko ) upon doxycycline (dox) administration. Our data revealed that Shp2 K14ce-cko resulted in corneal denervation. More specifically, the corneal epithelium thickness and corneal sensitivity reduced dramatically in Shp2 K14ce-cko mice. In addition, corneal epithelial debridement wound healing was delayed substantially in the mutant mice. These defects manifested in Shp2 K14ce-cko mice resemble the symptoms of human neurotrophic keratopathy. Our In vitro study unveiled that neurite outgrowth of the mouse primary trigeminal ganglion cells (TGCs) was inhibited as co-cultured with mouse corneal epithelial cells (TKE2) transfected by Shp2-, Mek1/2-, or ∆Np63-targeted siRNA but not by Akt1/2-targeted siRNA. Furthermore, ∆Np63 RNA interference down-regulated Ngf expression in TKE2 cells. More interesting, co-transfection experiments revealed that Shp2 tightly monitored ΔNp63 protein level in HEK293 and TKE2 cells. Taken together, our data suggested that Shp2-mediated MAPK pathway regulated ΔNp63, which in turn positively regulated Ngf in epithelium to warrant corneal innervation and epithelial