The Mechanism Investigation Of HSF4 In Lens Development And Cataract Formation In Zebrafish

Cataracts can be described as opacities of the lens which impede the passage of light.The causes of cataract including aging,inherited genetic factors,trauma,radiation exposure,intoxication and malnutrition.Congenital cataract is a major cause of childhood blindness.About 20 genes have currently been linked to congenital or early-onset cataract.These causative genes can be classified into the following groups by their functions,lens crystallins,growth or transcription factors,connexins,intermediate filament proteins,membrane proteins,protein degradation apparatus,other functional divergent gene.HSF4 belongs to heat shock transcription factor(HSF)family whose mutations have been associated with isolated cataract.It has been discovered that HSF4 can transactivate the expression of ?-crystallin,vimentin,beaded filament structural protein(BFSP)and deoxyribonuclease 2 beta(DLAD).All of these proteins play important roles in maintain lens normal function,but the the mechanisms regarding how HSF4 regulates lens development still needs to be further illustrated.Thus,we constructed an hsf4 knockout zebrafish model using transcription activator-like effector nuclease technology in this study.We wanted to discover how hsf4 is involved in regulating lens development and cataract formation by analyzing this hsf4 knockout zebrafish model.The mutant zebrafish developed an early onset cataract.Histological analysis revealed that knockout hsf4 resulted in multiple developmental defects in zebrafish lens.In hsf4 null zebrafish,the lens epithelial cells were over-proliferated which resulting in the overabundance of lens fiber cells depositing in lens.As a consequence,the arrangement of the lens fiber cells became more disordered and irregular with age.More importantly,through the observations using transmission electron microscope,we discovered that the organelles including nucleus,mitochondrial,lysosome and endosome cannot be cleaved in due time.Which indicated that hsf4 is responsible for the regulation of lens fiber cell terminal differentiation.To further uncovered the mechanisms of how hsf4 was involved in regulating organelle loss during the lens fiber cell differentiation,we focused on roles of p53 in regulating lens development.Our previous work revealed that HSF4 can stabilize and retain p53 in the nucleus.In this study,we discovered that HSF4 could stabilize and retain p53 in the nucleus to activate its target genes such as fas cell surface death receptor(Fas)and Bcl-2-associated X apoptosis regulator(Bax)in the cultured human lens epithelial cells.Consequently,the executor caspase3 was also activated by HSF4.In the hsf4 null zebrafish,both p53 and activated-caspase3 were significantly decreased.Combined with the finding that the denucleation defect could be partially rescued through microinjection of p53,fas and bax m RNA into the mutant embryos,we directly proved that HSF4 promotes lens fiber cell differentiation by activating p53 and its downstream regulators.The data we presented suggest that apoptosis-related genes are involved in the lens fiber cell differentiation.Our finding that HSF4 functions in the upstream to activate these genes highlighted the new regulatory modes of HSF4 in regulating lens fiber cell differentiation.In conclusion,by constructing hsf4 knockout zebrafish we observed the pathological progression of the cataract caused by deleting hsf4.Our work revealed that HSF4 could maintain the banlanced cell proliferatin and differentiation activity in lens,promote lens fiber cell terminal differentianion,keep regular arrangement of the lens fiber cell.On the molecular level,we discovered HSF4 can regulate both extrinsic and intrinsic apoptotic pathways to mediate its control of lens differentiation by stabilizing p53.Loss of HSF4 leads to downregulation of p53,inactivation of the p53-dependent death with clear attenuation of caspase3 activation and eventual halt of the lens differentiation.Together,our results reveal a fundamental mechanism by which HSF4 controls normal lens development and prevents cataractogenesis.