Pathology And Part Of Mechanism Of Corneal Disease In Epithelial Specific Mycn Knockout Mice

Corneal disease is one of the most important cause of blindness.Mycn is a member of the Myc proto-oncogene family and is involved in many cellular processes such as cell proliferation,apoptosis and differentiation.Previous studies on Mycn mainly focused on tumorigenesis.In our previous study,a corneal opacity mouse with Mycn mutation was obtained with an N-ethyl-N-nitrosourea(ENU)mutagenesis strategy.To further confirmed that the Mycn is a corneal disease related gene,Mycn knockout mice were obtained in a cross with Mycnflox/+and EIIa-Cre from the Jackson Laboratory in the United States.In addition,it was found that some of these mice with corneal opacity displayed eyelids open at birth.These mice were divided into two types:one with eyelids closed at birth and corneal opacity;another with eyelids open at birth and corneal opacity.In this study,we obtained epithelial specific Mycn knockout mice by using the K14-Cre driver to delete Mycn.The phenotype of epithelial specific Mycn knockout mice is similar to Mycn mutation and EIIa-Cre knockout mice.We found that Mycn is necessary for proper lens vesicle separation from the surface ectoderm.In the K14-Cre Mycn knockout mice,the lens vesicle fails to separate from the ectoderm,an important reason of corneal pathology in epithelial specific Mycn knockout with eyelids closed at birth.1.A mouse with corneal disease obtained by epithelial specific Mycn knockoutBased on the principle of Cre-loxP system,epithelial specific Mycn knockout mice(Mycn-/+)were obtained in a cross with Mycnflox/+and K14-Cre from the Jackson Laboratory in the United States.Some of Mycn-/+ mice appeared corneal opacity after birth.In addition,it was found that some of these mice with corneal opacity displayed eyelids open at birth.These mice were divided into two types:one with eyelids closed at birth(ECB)and corneal opacity;another with eyelids open at birth(EOB)and corneal opacity.2.Corneal pathology in Mycn-/+ miceAfter HE staining of eyeballs of mice in three weeks old(3W),it was found that:(1)Examination of eye sections from the mice with eyelids closed at birth revealed vacuoles in the surface of the corneal epithelial cells.The endothelial cells were absent in some area of endothelium and lens pathology beneath this area;(2)Compared with the wild-type mice,the partial corneal epithelium of the mice with eyelids open at birth was thickened,and corneal neovascularization was detected in the corneal stroma.We performed staining for the corneal epithelial cells differentiation marker keratin 12(K12),the epithelial cell differentiation marker keratin 14(K14),and a specific epidermal differentiation marker,keratin 10(K10),by immunohistochemistry in eyelids open at birth,eyelids closed at birth and control littermate mice.Immunohistochemistry showed that:(1)In the control corneas,K12 was present in the corneal epithelium,K10 expression was not detected and K14 was present but weak in the basal layer of the corneal epithelium.(2)In the corneal opacity mice with eyelids closed at birth,K12 expression in part of vacuole regions of corneal epithelium was decreased.K14 was expressed most prominently in all the corneal epithelial layer.K10 was not detected.(3)In the corneal opacity mice with eyelids open at birth,K12 staining was absent in the thickened corneal regions;K14 was expressed most prominently in the corneal epithelium but weak in the thickened corneal regions;K10 expression was detected in the thickened corneal region.The immunohistochemical staining was used to detect integrin beta4 in mice corneal epithelial cells.The results showed that:The positive cells of integrin beta4 in the corneal epithelium of wild-type mice were confined mainly to the basal layer;the expressions of integrin beta 4 of corneal epithelial cells were strongly in the mice with eyelids closed and the expression of corneal epithelial thickening areas was weak in the mice with eyelids open at birth.3.Defect in lens vesicle separation in Mycn-/+miceHE staining of eyeballs of corneal opacity mice with eyelids closed at birth suggested that corneal pathology of this kind of Mycn-/+ mice may be caused by the defect in lens vesicle separation.To test this hypothesis,histological analyses of the Mycn-/+ mice were carried out at E11-13.In Mycn-/+,a persistent connection between the lens and the corneal epithelium was found,which results from failure of the lens vesicle to close and separate from the ectoderm.Immunohistochemical staining showed higher E-cadherin in Mycn-/+mice affected eye region,which results from a lens vesicle separation defect.We found that epithelium specific Mycn knockout could also lead to corneal disease.The occurrence of corneal disease in the mice with eyelids closed at birth is related to the failure of the lens vesicle separate from the ectoderm and its mechanism is preliminarily studied.This study enriched the cell biological function of Mycn and also lead to the richer understanding of the genetic basis and pathology mechanism of some corneal disease in human beings.