The Histological Characteristics And Molecular Mechanism Of An ENU-induced Eye Open-at-birth Mouse Model

During normal development in mammals,the eyelids grow across the eye,fuse and subsequently reopen.The development of the eyelid requires coordinated cellular processes of proliferation,cell shape changes,migration and cell death.A number of genes encoding growth factors,growth factor receptors,cytoplasmic effectors and transcription factors have been shown to regulate eyelid growth and closure.Failure of the eyelids to grow across the eye and fuse during the fetal stage in mice leads to a birth defect of open-eyelids at birth,result in a variety of eye defects including corneal opacity in humans and mice.Transient lid closure and reopening is a common morphogenetic event that also takes place in humans.Unlike in mice,however,eyelid closure and reopening in humans is accomplished in utero.This has made the detection of lid closure defects a major challenge,and as a result.little is truly understood about the incidences of eyelid closure defects and the associated diseases in humans.A mouse mutant line with open-eyelids at birth(EOB)phenotype was induced with an N-ethyl-N-nitrosourea(ENU)mutagenesis strategy.Upon closer examination of the adulthood mice,we observed that EOB mice were uniformly characterized by corneal opacity phenotype.Here,we report the histological characteristics and molecular mechanism of the open-at-birth mouse model.1.Histological examination of EOB miceH.E analysis of the eyeballs showed that the epithelium of corneal of wild mouse is non-keratinized and have no neovascularization.Comparing with wild mouse,the epithelium of corneal of mutant mouse have stratum granulosum and keratinized layer.The neovascularization is also obvious.Further immunohistochemistry analysis showed that cytokeratinl2,14 and 10 were all abnormal in mutant mice.2.Fine mapping and identification of the mutant gene that cause EOBIn initial mapping,the mutant gene was mapped to chromosome 13 and near D13Mit76.In order to further refine the map position,we crossed heterozygotes on the B6 genetic background to D2 to obtain F1 mice and then backcross B6 mice to obtain N2.Genomic DNA from 143 N2 mutants was analyzed using microsatellite and SNP markers.The mutation was determined to be between markers D13Mit291 and rs3697199,which were located at 62.07cM and 112598216bp,respectively.The region contains the Map3klgene and the phenotypes of other Map3k1 mutants resemble that of our mutant.Therefore,the Map3k1 gene was considered a good candidate for the EOB.3.Mechamism analysis of EOB caused by Map3kl mutation in miceImmunohistochemistry analysis of E14.5 mice showed significant decrease expression of c-Jun and P-c-Jun in EOB mice.Conclusion:These studies may help to decipher the "genetic codes" underlying eyelid developmental defects in humans,leading to unravel the origins of congenital developmental disorders in children.The EOB mice are also valuable resources of animal model of human eye disease.