Knockout Of TULP1 Homologues Induced Early-Onset Retinitis Pigmentosa In Zebrafish

Retinitis pigmentosa is the main hereditary cause of blindness in humans.Mutations in TULP1（tubby-like protein 1）lead to early-onset retinitis pigmentosa,displaying night blindness,severe vision loss in infancy.The amplitude of electroretinogram decreased significantly or disappeared in affected individuals.Typical fundus features of retinitis pigmentosa,including bone spicule pigmentation,optic nerve pallor,and vascular atrophy,was present in the patients.The function of TULP1 in retinal and the exact etiology of how TULP1 mutations cause photoreceptors degeneration have not been clearly established.Currently,there is no effective treatment for the retinal diseases caused by TULP1 mutations.Exploring the function of TULP1 and revealing the mechanism of photoreceptor degeneration is the key to clinical treatment and prevention.The development and structure of the zebrafish retina is very similar to that of humans.As a model animal,zebrafish has great advantages in the research of retinal diseases.There are two homologous genes of TULP1 in zebrafish,namely tulp1a and tulp1b.Sequence alignment and in situ hybridization results showed that TULP1 was highly conserved in human and zebrafish.In this study,a zebrafish model was used to study the function of TULP1 and the pathogenesis.CRISPR/Cas9 technology was used to generate single knockout zebrafish line of tulp1a and tulp1b:tulp1a^-/-and tulp1b^-/-.tulp1a and tulp1b double knockout zebrafish line（tulp1-dKO）was generated by crossing the two mutant lines.The phenotypes of retina in tulp1a^-/-,tulp1b^-/-,and tulp1-dKO zebrafish were detected.UV cone degeneration was observed in tulp1a^-/-zebrafish compared with wild-type zebrafish by hematoxylin and eosin staining and immunofluorescence staining.The tulp1b^-/-zebrafish showed disorder and shorter outer segments of photoreceptor cells and thinner outer nuclear layer,and with the degree of degradation increasing with months.The shorter outer segments of rods and cones,decreased number of photoreceptor cells,and the thinner outer nuclear layer was observed at the early stage in tulp1-dKO zebrafish.In addition,the degree of degeneration of photoreceptor cells in tulp1-dKO zebrafish was detected at the molecular level.Western blot results showed that the expression levels of phototransduction proteins in rods and cones in tulp1-dKO zebrafish were significantly decreased in the early stage.In terms of the types of the affected photoreceptor cells and the severity of degeneration,compared with the single knockout zebrafish lines,the phenotype of retina in tulp1-dKO zebrafish is consistent with the phenotype of TULP1 mutant patients and is more suitable as a disease model to study the retinitis pigmentosa induced by TULP1 mutations.To investigate the role of TULP1 in opsin localization,it was found that protein mislocalization phenotype was present in all types of photoreceptor cells in tulp1-dKO zebrafish.Mislocalization of opsins was prior to the thinner of outer nuclear layer,which suggests that opsin mislocalization may be responsible for the photoreceptor cell death.Cilia in photoreceptors was analyzed.The results showed that shorter cilia were observed in tulp1-dKO zebrafish and was prior to the mislocalization of opsins.Transcriptomics analysis,real-time fluorescence quantitative PCR,in situ hybridization,dual luciferase reporting system was performed to research the differentially expressed genes in the retinal of wild-type and tulp1-dKO zebrafish and the potential target genes of Tulp1a and Tulp1b.It was found that the expression level of tektin 2,a gene related to ciliary length,was significantly down-regulated in tulp1-dKO zebrafish.Overexpression of Tulp1a and Tulp1b in Danio rerio fibroblast-like cells could significantly enhance the transcriptional activity of tektin 2 promoter,indicating that Tulp1a and Tulp1b may affect the development of cilia by regulating the expression of tektin 2,and leading to abnormal protein localization.The analysis performed on the transcriptome data revealed that the ferroptosis pathway may be the cytological basis of photoreceptor death.Real-time quantitative PCR showed that the expression of genes involved in the lipid peroxidation and iron homeostasis was significantly increased in the tulp1-dKO zebrafish.Accumulation of lipid,detected using BODIPY and Nile red staining,was observed in the tulp1-dKO retina.We also detected iron accumulation by Perls/DAB staining in the tulp1-dKO retina.In addition,Transmission electron microscopy results showed mitochondria exhibited changes characteristic of ferroptosis in the inner segments of tulp1-dKO zebrafish compared with wild-type zebrafish.These results suggested that ferroptosis might be the cytological basis of photoreceptor death in the tulp1-dKO zebrafish.In conclusion,there mutant zebrafish models,tulp1a^-/-,tulp1b^-/-and tulp1-dKO,were established for the first time in our study.tulp1-dKO zebrafish model well mimicked the phenotype of early-onset retinitis pigmentosa in TULP1 patients.We provide a good model for effective drug screening;mutation of Tulp1a and Tulp1b causes the downregulation of tektin 2,defects in cilia structure and opsin trafficking;our study also suggested that ferroptosis may be involved in photoreceptor degeneration.These findings offer insight into the pathogenesis and clinical treatment of retinitis pigmentosa.