Knockout Of USH2A Gene In Zebrafish Causes Hearing Impairment And Late Onset Rod-cone Dystrophy

Usher syndrome,also known as hereditary deafness-retinitis pigmentosa syndrome,is characterized by visual loss owing to retinitis pigmentosa（RP）,congenital hearing loss and in some cases vestibular dysfunction.It is an autosomal recessive genetic disease.Typically,there are three subtypes of USH:USH1,USH2 and USH3,which are classified according to the auditory and vestibular symptoms.USH is highly genetically heterogeneous,and is associated with 13 genes.USH2 is the most prevalent form and accounts for well over one-half of all USH cases;importantly,the USH2A gene mutations are responsible for up to70%of USH2 cases.In 1994,USH2A gene was accurately located at the long arm of chromosome 1.USH2A was identified by Eudy et al.in 1998.According to the databases reported in Leiden Open Variation Database,more than 400 mutations of USH2A have been identified so far.These mutations spread all over the exons and introns of USH2A.Two main isoforms of USH2A,isoform_a and isoform_b,have been reported in humans.Isoform_a contains 21exons and encodes an extracellular protein of 170 kDa;isoform_b contains 72 exons and encodes a huge protein product called usherin,which was predicted to be the major isoform in the retina and inner ear.Isoform_a is an N-terminal 1546 amino acid protein fragment of isoform_b.Most of the usherin protein（USH2A）is located extracellularly and this extracellular section has multiple functional domains that are common in cell adhesion proteins and extracellular matrix（ECM）proteins.USH2A also has a membrane-spanning segment followed by an intracellular PDZ binding domain at the C-terminus.USH2 caused by USH2A mutation seriously affects patients’quality of life.However,the pathogenesis of USH2 is unknown and there are no effective therapies either.The studies in the USH2A knockout mouse model provided some clues for uncovering the function of the USH2A gene,but the exact function of USH2A in the retina was still obscure.In order to gain deeper insights into the disease causing gene USH2A,we constructed a USH2A knockout(USH2A^-/-)zebrafish model using TALEN technology to investigate the molecular pathology of USH2.The predicted protein of zebrafish USH2A share a 52%homology with the human USH2A long variant,and they have the same motif arrangement.Hearing tests were performed in USH2A^-/-zebrafish via startle response assay.An early-onset auditory disorder and abnormal morphology of inner ear stereocilia were identified.Consequently,the disruption of USH2A in zebrafish led to a hearing impairment,like that in mammals.Electroretinography（ERG）test indicated that deletion of USH2A caused mild visual defects at an early stage,and histological analysis revealed that the photoreceptors progressively degenerated.Photoreceptor degeneration was identified at about 10 months of age and progressed very slowly,for there was no obvious further deterioration until 20 months of age than that of 10 months.Using high resolution TEM assay,we found that the gradual thinning of photoreceptor layer in USH2A^-/-zebrafish was due to the damage of the outer segments（OSs）.Rod degeneration occurred prior to cone degeneration in USH2A^-/-zebrafish,which is consistent with the classical description of the progression of retinitis pigmentosa.Next,we investigated the function of USH2A in the retina.We have detected the expression of phototransduction cascade proteins on mRNA as well as protein levels,and confirmed that USH2A may not directly contribute to phototransduction in zebrafish photoreceptors.However,destruction of the outer segments of rods led to the down-regulation of phototransduction cascade proteins at late stage,and that might be one of the causal factors of RP in the mutants.Previous studies have demonstrated that the murine or human USH2A interacted with a basement membrane protein,fibronectin,via the LE domains in vitro.Fibronectin is a constituent of extracellular matrix（ECM）and regulates its stability.Based on these findings,we would like to know whether knockout of the USH2A gene will affect the fibronectin as well as the basement membrane in zebrafish.Through the immunostaining assay,we found that in the USH2A mutants,although the location of fibronectin signals was not altered,the intensity of the signals was clearly attenuated.At 10 months,when the photoreceptor deficiency was apparent,the fibronectin signals had almost disappeared completely.Meanwhile,the expression of fibronectin showed a down-regulation which was confirmed by western blot analysis.Integrin is an important cellular adhesion molecule,using western blot assay,we also found that the protein level of ITGB1 was reduced.The above findings suggest that ablation of USH2A probably led to improper assembly of fibronectin at the retinal basement membrane and resulted in declining cell adhesion to the basement membrane.Moreover,through immunoblotting assay,we detected an up-regulation of the expression of USH1B and USH1C proteins.USH proteins are believed to form a supramolecular complex and function cooperatively in vivo,the increased expression of USH1 proteins might reduce the dysfunction of the USH protein complex caused by USH2A ablation.However,the relevant molecular mechanism is elusive and requires further study.In summary,for the first time we generated a USH2A knockout zebrafish line with auditory disorder and retinal degeneration which mimicked the symptoms of patients,and revealed that disruption of fibronectin assembly may be one of the factors underlying RP.This model may help us to better understand the pathogenic mechanism and find treatment for USH2 in the future.