The Mechanism Of Crb2a Domain In The Assembly Of Apical Adherens Junctions In Zebrafish Retinal Neuroepithelial Cells And Lens Epithelial Cells

Research background and purpose:The Crumbs homologue 1(CRB1)mutation is one of the causes of inherited retinal disease retinitis pigmentosa(RP).The CRB1 protein encoded by CRB1 gene contains a variable extracellular domain and a conserved intracellular domain,which is the key molecule to regulate the apical-basal polarity of epithelial cells.Vertebrate genomes encode multiple crb homologues,of which zebrafish crb2a is highly homologous with human CRB1.Adherens junctions(AJs)located at the apical region of both the zebrafish retinal neuroepithelial cells and the lens epithelial cells,which assemble into zonula adherens(ZAs).Ablation of crb2a leads to the disruption of epithelial polarity,disordering of the AJs,and induction of defects in retinal morphogenesis in zebrafish However,the specific roles of the extracellular region and the cytoplasmic tail of Crb2a in regulating AJs assembly remain unclearThe aims of the present study were to explore the mechanism of the extracellular region and the cytoplasmic tail of Crb2a on the formation and assembly of apical AJs in retinal neuroepithelial cells and lens epithelial cells,and further revealed the molecular biological mechanism of CRB1 related inherited retinal diseasesMethods:In this study,we synthesized Crb2aΔEX by using polymerase chain reaction,which absent the extracellular domain of Crb2a.AB wildtype(WT)strain and the transgenic zebrafish crb2am289,OE120;WT,Crb2aΔEX;crb2am289 were used as models to explore the integrity of outer limiting membrane(OLM),the localization of polarity proteins,the structure and the morphology of retinal lamination,as well as the adhesion and assembly of epithelial cells by using immunohistochemistry of frozen sections and high-resolution transmission electron microscopy.The phenotype and genotype relationships of zebrafish OE120;WT and CRB1 related RP patients were analyzed.The number of adhesion plaques and the assembly form of AJs in zebrafish retina and lens epithelial cells were quantitatively analyzed by using statistical method.Results:The results showed that the expression of Crb2aΔEX could not only partially rescue the AJs assembly,but also rescue the morphogenesis defects of retina caused by Crb2a loss.Specifically,in Crb2aΔEX;crb2am289 zebrafish,Crb2aΔEX substantially stabilized the membrane association of Nok in retinal photoreceptors,and effectively inhibited the abnormal proliferation caused by the loss of Crb2a.Meanwhile,Crb2aΔEX promoted the apical distribution of AJs in zebrafish retinal neuroepithelial cells and lens epithelial cells,but caused assembly into unstable punctum adherens-like adhesion plaques.Furthermore,Crb2aΔEX significantly promoted the apically localized ZO1,Nok,and pH3-positive cells,the apically-positioned adhesion plaques in retinal neuroepithelia(by 7.5 times compared with crb2am289 zebrafish),as well as the retinal patterning in Crb2aΔEX;crb2am289 zebrafish.However,Crb2aΔEX has no rescue effects on ZAs assembly in epithelia and OLM formation in photoreceptors.By contrast,Crb2aΔEX expression in WT disrupted the integrity of ZAs and the OLM,as well as the proper localization of endogenous polarity proteins in photoreceptors,which finally leads to degeneration of photoreceptors and RP-like phenotypes.Retinal degeneration started from teenagers in CRB1 related RP patients,especially in OLM and outer nuclear layer of photoreceptors.Conclusions:In this study,we revealed the different functions of the extracellular region and the intracellular tail of Crb2a in the distribution and assembly of AJs in retinal neuroepithelial cells and lens epithelial cells.This study demonstrated that both the extracellular region and the intracellular tail of Crb play an essential role in guiding the formation of apical zonula adherens.Specifically,the intracellular domain of Crb2a could promote the apical distribution of AJs;the extracellular region of Crb2a guided the transformation of AJs from the punctum adherens into stable ZAs;overexpression of the intracellular domain of Crb2a could cause RP-like retinal denegation,which mimicked the disease process and the clinical manifestations of human CRB1 related RP.