FoxC1 activates limbal epithelial stem cells following corneal epithelial debridement

Abstract

Limbal epithelial stem cells are not only critical for corneal epithelial homeostasis but also have the capacity to change from a relatively quiescent mitotic phenotype to a rapidly proliferating cell in response to population depletion following corneal epithelial wounding. Pax6^+/- mice display many abnormalities including corneal vascularization and these aberrations are consistent with a limbal stem cell deficiency (LSCD) phenotype. FoxC1 has an inhibitory effect on corneal avascularity and a positive role in stem cell maintenance in many tissues. However, the role of FoxC1 in limbal epithelial stem cells remains unknown. To unravel FoxC1's role(s) in limbal epithelial stem cell homeostasis, we utilized an adeno-associated virus (AAV) vector to topically deliver human FOXC1 proteins into Pax6 ^+/- mouse limbal epithelium. Under unperturbed conditions, overexpression of FOXC1 in the limbal epithelium had little significant change in differentiation (PAI-2, Krt12) and proliferation (BrdU, Ki67). Conversely, such overexpression resulted in a marked increase in the expression of putative limbal epithelial stem cell markers, N-cadherin and Lrig1. After corneal injuries in Pax6 ^+/- mice, FOXC1 overexpression enhanced the behavior of limbal epithelial stem cells from quiescence to a highly proliferative status. Overall, the treatment of AAV8-FOXC1 may be beneficial to the function of limbal epithelial stem cells in the context of a deficiency of Pax6 function.

Keywords: Corneal wounding; FoxC1; Limbal stem cell deficiency; Pax6; Proliferation.

Figure 1.

Delivery of Foxc1 in limbal epithelium of Pax6+/− mice using AAV increases the expression of stem cell markers. (A-G) 21-day-old mice were infected with AAV-Foxc1 or AAV empty vector (control). Fourteen days after infection, mice received an I.P. injection of BrdU (50 mg/kg). One hour later, mouse eyes were harvested and subjected to immunostaining for Foxc1 (A), N-cadherin (N-cad, B), Lrig1 (C), PAI-2 (D), Krt12 (E), Ki67 (F), BrdU (G). Quantification of the relative fluorescent intensity was conducted using ImageJ. n = 12; *, p < 0.05.

Figure 2.

Delivery of Foxc1 in limbal epithelium of Pax6+/− mice using AAV promotes the response of limbal epithelial stem cells to wounding. Twenty-one-day old mice were infected with AAV-Foxc1 or AAV-GFP (control). Fourteen days after infection, mice were subjected to debridement wounding in the central corneal epithelium using a diamond burr. Twenty-four hours later, mice received an I.P. injection of BrdU (50 mg/kg). One hour later, mouse eyes were harvested and subjected to immunostaining for BrdU. Quantification of the BrdU+ cells was conducted using ImageJ (L). n = 8; *, p < 0.05.