• <tr id="yyy80"></tr>
  • <sup id="yyy80"></sup>
  • <tfoot id="yyy80"><noscript id="yyy80"></noscript></tfoot>
  • 99热精品在线国产_美女午夜性视频免费_国产精品国产高清国产av_av欧美777_自拍偷自拍亚洲精品老妇_亚洲熟女精品中文字幕_www日本黄色视频网_国产精品野战在线观看 ?

    Characterization of two novel knock-in mouse models of syndromic retinal ciliopathy carrying hypomorphic Sdccag8 mutations

    2022-06-07 10:50:12ZhiLinRenHouBinZhangLinLiZhengLinYangLiJiang
    Zoological Research 2022年3期

    Zhi-Lin Ren, Hou-Bin Zhang,3, Lin Li,3, Zheng-Lin Yang,3,*, Li Jiang,3,*

    1 Department of Laboratory Medicine, Sichuan Provincial People's Hospital, School of Medicine, University of Electronic Science and

    Technology of China, Chengdu, Sichuan 610072, China

    2 Sichuan Provincial Key Laboratory for Human Disease Gene Study, Sichuan Provincial People's Hospital, University of Electronic Science and Technology of China, Chengdu, Sichuan 610072, China

    3 Research Unit for Blindness Prevention of Chinese Academy of Medical Sciences (2019RU026), Sichuan Academy of Medical Sciences,Chengdu, Sichuan 610072, China

    ABSTRACT Mutations in serologically defined colon cancer autoantigen protein 8 (SDCCAG8) were first identified in retinal ciliopathy families a decade ago with unknown function.To investigate the pathogenesis of SDCCAG8-associated retinal ciliopathies in vivo, we employed CRISPR/Cas9-mediated homology-directed recombination (HDR) to generate two knock-in mouse models,Sdccag8Y236X/Y236X and Sdccag8E451GfsX467/E451GfsX467,which carry truncating mutations of the mouse Sdccag8, corresponding to mutations that cause Bardet-Biedl syndrome (BBS) and Senior-L?ken syndrome (SLS) (c.696T>G p.Y232X and c.1339-1340insG p.E447GfsX463) in humans,respectively.The two mutant Sdccag8 knock-in mice faithfully recapitulated human SDCCAG8-associated BBS phenotypes such as rod-cone dystrophy, cystic renal disorder, polydactyly, infertility, and growth retardation, with varied age of onset and severity depending on the hypomorphic strength of the Sdccag8 mutations.To the best of our knowledge,these knock-in mouse lines are the first BBS mouse models to present with the polydactyly phenotype.Major phototransduction protein mislocalization was also observed outside the outer segment after initiation of photoreceptor degeneration.Impaired cilia were observed in the mutant photoreceptors,renal epithelial cells, and mouse embryonic fibroblasts derived from the knock-in mouse embryos, suggesting that SDCCAG8 plays an essential role in ciliogenesis, and cilium defects are a primary driving force of SDCCAG8-associated retinal ciliopathies.

    Keywords: SDCCAG8; Primary cilia; Retinal ciliopathy; Bardet-Biedl syndrome (BBS); Senior-L?ken syndrome (SLS); Nephronophthisis (NPHP);Polydactyly

    lNTRODUCTlON

    Retinal ciliopathies are a group of inherited retinal degenerative diseases caused by mutations in genesencoding ciliary proteins essential for photoreceptor morphology and function (Adams et al., 2007; Bachmann-Gagescu & Neuhauss, 2019; Bujakowska et al., 2017; Chen et al., 2021).Retinal ciliopathies can present as non-syndromic retinal disorders such as Leber congenital amaurosis (LCA) or retinitis pigmentosa (RP) (Hartong et al., 2006; Koenekoop et al., 2007; Kumaran et al., 2017; Verbakel et al., 2018), as well as syndromic retinal dystrophies, such as Bardet-Biedl syndrome (BBS), Senior-L?ken syndrome (SLS), Joubert syndrome (JBTS), Meckel-Gruber syndrome (MKS), Jeune syndrome, and Alstr?m syndrome (AS) with the involvement of multiple systems and organs, including the central nervous system, kidney, skeleton, liver, and adipose tissue (Braun &Hildebrandt, 2017; Hurd & Hildebrandt, 2011; Zaghloul &Katsanis, 2009).Over 100 genes encoding retinal ciliopathy proteins are associated with retinal ciliopathies, accounting for almost 25% of all retinal dystrophies (Chen et al., 2019)(https://sph.uth.edu/retnet/).

    Serologically defined colon cancer autoantigen protein 8(SDCCAG8) was identified as a causative gene of retinal ciliopathy following detection of its mutation in retinal-renal ciliopathy families a decade ago (Otto et al., 2010).Genotypephenotype correlation studies have suggested that SDCCAG8-associated syndromic ciliopathies manifest predominantly as retinal-renal degeneration, accompanied by obesity, hypogonadism, recurrent pulmonary infections,cognitive defects, and mild intellectual disability in some cases, but not with polydactyly (Halbritter et al., 2013b; Kang et al., 2016; Otto et al., 2010; Schaefer et al., 2011) (Table 1).Thus, theSDCCAG8gene is alternatively referred to asNPHP10,SLS7, andBBS16.Recent genome-wide association studies have also demonstrated that genetic polymorphisms of SDCCAG8 are associated with bipolar disorder and schizophrenia (Gonzalez et al., 2016; Hamshere et al., 2013).

    The humanSDCCAG8gene encodes a 713 residue fulllength protein that contains a N-terminal globular domain(1-270 amino acids (aa)), short nuclear localization signal,and large C-terminal coiled-coil domain (CCD) (Kenedy et al.,2003; Otto et al., 2010).To date, 19 retinal ciliopathy-causingSDCCAG8mutations have been identified, including deletion,insertion, nonsense, and splicing mutations across exon5 to exon16 of the gene, resulting in a reading frame shift that produces C-terminal CCD-truncated proteins of different sizes(Table 1).Previous genotype-phenotype correlation studies have revealed thatSDCCAG8truncating mutations near the N-terminal are predominantly associated with BBS, while ones near the C-terminal are primarily associated to SLS (Table 1).However, it has been difficult to correlate specific truncating mutations to phenotypic variants in SDCCAG8-associated syndromic retinal ciliopathies, and the underlying pathogenesis remains largely unknown.

    Table 1 SDCCAG8 mutations identified in patients with retinal ciliopathies

    Vertebrate photoreceptors possess a highly specialized primary sensory cilium, composed of a basal body (BB),connecting cilium (CC)/transition zone (TZ), and outer segment (OS), where retinal ciliopathy proteins localize and function (May-Simera et al., 2017).SDCCAG8 localizes at the BB/centrosome and CC/TZ in photoreceptors, and in other ciliated cell types in other systems, and directly interacts with other ciliopathy-associated proteins, including OFD1, NPHP5,RP1, and RPGRIP1 (Di Gioia et al., 2012; Insolera et al.,2014; Otto et al., 2010; Patil et al., 2012).Suppression of SDCCAG8 expression in zebrafish causes developmental defects in the kidney, brain, and body axis (Otto et al., 2010).Sdccag8mutant mouse models carrying distinct gene-trap alleles, i.e.,Sdccag8gt,Sdccag8tm1e(EUCOMM)Wtsi, andSdccag8SBT, show loss of full-length SDCCAG8 protein expression (Airik et al., 2014; Insolera et al., 2014; Weihbrecht et al., 2018).Sdccag8gt/gtmice present with early-onset retinal degeneration, late-onset nephronophthisis (NPHP), as well as developmental and structural abnormalities of the skeleton and limbs, mimicking disease phenotypes in humans (Airik et al., 2016, 2014 ).In addition, theSdccag8tm1e(EUCOMM)Wtsi/tm1e(EUCOMM)WtsiandSdccag8SBT/SBTmouse lines exhibit neonatal lethality with developmental defects in the central nervous system, limbs, and lungs, but without retinal-renal involvement, a major feature of SDCCAG8-assciated ciliopathies (Insolera et al., 2014;Weihbrecht et al., 2018).Thus, these threeSdccag8mutant mouse models show significant phenotype variation and genotype-phenotype discrepancies.

    To investigate how differentSDCCAG8truncating mutations may cause syndromic retinal ciliopathies with different severity and system involvement, we employed CRISPR/Cas9-mediated homology-directed recombination (HDR) technology to generate knock-in mouse modelsSdccag8Y236X/Y236XandSdccag8E451GfsX467/E451GfsX467carrying truncating mutations of the mouseSdccag8gene, which correspond to BBS- and SLS-causing mutations in humans, respectively.Results showed that the two mutantSdccag8knock-in mice closely phenocopied retinal and renal degeneration of SDCCAG8-associated retinal ciliopathies, with varying disease onset and severity.In addition, the mice displayed preaxial polydactyly,which is absent in retinal ciliopathies caused bySDCCAG8mutations.They also showed major phototransduction protein mislocalization outside the OS after initiation of photoreceptor death.Retinal photoreceptors, renal epithelial cells, and mouse embryonic fibroblasts (MEFs) from the knock-in mice exhibited impaired biogenesis and structural defects in cilia,suggesting that SDCCAG8 plays an essential role in ciliogenesis, and its dysfunction is an underlying mechanism driving retinal and renal degeneration inSdccag8knock-in mice.

    MATERlALS AND METHODS

    Mutant Sdccag8 knock-in mice

    All procedures for animal experiments were approved by the Animal Care and Use Committee of Sichuan Provincial People’s Hospital and conformed to the recommendations of the Association for Research in Vision and Ophthalmology(Approval No.2014NSF(09)).Mice were maintained under 12 h cyclic dark/light conditions.

    We generated two knock-in mouse models carrying either a point mutationSdccag8-Y236X (c.708C>G p.Y236X) in exon7 or a 1 bp insertionSdccag8-E451GfsX467 (c.1 351-1352insG p.E451GfsX467) in exon11 with CRISPR/Cas9-mediated HDR technology (Viewsolid Biotech, China).The correspondingSDCCAG8mutations in humans, i.e.,c.696T>G p.Y232X and c.1339-1340insG p.E447GfsX463,are known to cause BBS and SLS, respectively (Otto et al.,2010).We first designed two mutation-specific guide RNA(gRNA) targets, i.e.,Sdccag8-Y236X-g (5'-GGCTGAA ACTCACATACGAGG-3') andSdccag8-E451GfsX467-g (5'-ACGTTGCGTCTCAGGAAATGG-3'), to guide sequencespecific cutting near each mutation, as well as two corresponding donor DNA oligos, i.e.,Sdccag8-Y236X-d (5'-TCCTGCCTTGTTCTGCAGGAGAGGCTGAAACTCACATAG GATCCGGCGAAGACTGACCTTCTGGAATCTCAGCTGATG CTT-3') andSdccag8-E451GfsX467- d (5'-GTCACTTAGAGGAAATTCAGAACCCGTTGCGTCTCAAGG ATCCAATGGACGTCACAAAGGTCCGAGAAAGTTTTGCTTT AA-3') to introduce the mutations into mouseSdccag8genomic DNA through HDR.In addition, we designed a restriction endonuclease (BamHI) site (GGATCC) next to each mutation to facilitate mouse genotyping, as well as a synonymous mutation 1350G>A inSdccag8-E451GfsX467-d to destroy the protospacer adjacent motif sequence to prevent recurrence of gRNA-mediated cutting following recombination.Each gRNA and donor DNA oligo pair was microinjected into the fertilized eggs of C57BL/6 mice together with CRISPR plasmid encoding Cas9 nuclease to generateSdccag8knockin chimeras through CRISPR/Cas9-mediated HDR.Founders with successful recombination were selected by Sanger sequencing with mouse tail DNA, and subsequently used to produce heterozygous and homozygous knock-in miceSdccag8Y236X/Y236XandSdccag8E451GfsX467/E451GfsX467for our study.

    Mouse genotyping

    Knock-in allelesSdccag8-Y236X andSdccag8-E451GfsX467 were identified by polymerase chain reaction (PCR)amplification with subsequent BamHI restriction enzyme digestion due to the introduction of a BamHI site next to each mutation.Two pairs of primers were designed for allelespecific PCR: i.e.,Sdccag8-Y236X-F:ACAGCAGAGTGGAGTGAGCTAGT andSdccag8-Y236X-R:TTGAGCACAGGAGACACCTAAC;Sdccag8-E451GfsX467-F:GCTGAGAAGGTAGAGAAGTG andSdccag8-E451GfsX467-R: CACACCACACCACATACAT.PCR amplification was conducted in a total reaction volume of 20 μL.Subsequently,8.7 μL of PCR raw products were directly digested using the BamHI restriction enzyme (New England Biolabs (NEB), USA)in a 10 μL reaction system at 37 °C for 1 h, and then analyzed by 2% agarose gel electrophoresis.

    DNA constructs and transfection

    We obtained pEGFP-Sdccag8plasmids expressing the mouse wild-type SDCCAG8 protein with the N-terminal fluorescent protein marker enhanced green fluorescent protein (EGFP)(OriGene, USA).For the generation of pEGFP-Sdccag8(Y236X) and pEGFP-Sdccag8(E451GfsX467) mutant constructs, we designed two pairs of mutagenesis primers,pSdccag8(Y236X)-F: TCCGGCGAAGACTGACCTTCT and pSdccag8(Y236X)-R: TCCTATGTGAGTTTCAGCCTCTC;pSdccag8(E451GfsX467)-F: GATCCAATGGACGTCACAAA GGTG and pSdccag8(E451GfsX467)-R:CTTGGGACGCAACGTGGTTCTGA.We then conducted QuikChange XL site-directed mutagenesis with mutagenesisprimers following the manufacturer’s protocols (Stratagene,USA).The wild-type and mutant plasmids were transfected into HEK293T cells (cultured with 10% fetal bovine serum(FBS)/Dulbecco’s Modified Eagle Medium (DMEM) containing 1% penicillin-streptomycin solution, 37 °C, 5% CO2) with a Lipofectamine 3 000 Kit (Invitrogen, USA).Expression of SDCCAG8 wild-type and mutant proteins in the transfected cells was analyzed by western blotting.

    Preparation of MEFs

    The MEFs were derived from wild-type andSdccag8knock-in E13.5 embryos, as described previously (Dong et al., 2015),then cultured with 20% FBS/DMEM containing 2% penicillinstreptomycin at 37 °C in humidified 5% CO2.To analyze cilium biogenesis, the MEFs were seeded in 24-well plates and serum-starved for 24 h before immunofluorescence staining.

    Histology

    The eyeballs and kidneys were dissected from euthanized(CO2inhalation) mice and fixed in fixative solution of 1.22%glutaraldehyde and 0.8% paraformaldehyde in 0.08 mol/L phosphate buffer at 4 °C overnight.Subsequently, the fixed tissues were dehydrated through an ethanol series and embedded in paraffin.Sections were taken at 5 μm for both retinas and kidneys.Hematoxylin and eosin staining was performed for retinal and kidney sections, and Masson trichrome staining was conducted for kidney sections following standard protocols.Images of the stained sections were acquired with a Zeiss Axiovert 200 microscope (Carl Zeiss,USA) under 63× objective.Images across the entire retinal sections were acquired with a 20× objective and imported into ImageJ v1.8.0 software with 3.8 pixels/mm scaling.The outer nuclear layer (ONL) thicknesses were measured at 500 μm intervals from the optic nerve head.

    Transmission electron microscopy (TEM)

    Photoreceptor ultrastructure inSdccag8knock-in mice was investigated using standard protocols, as described previously(Jiang et al., 2011).Dissected mouse eyecups were first fixed in 2.5% glutaraldehyde and 1% paraformaldehyde in 0.1 mol/L cacodylate buffer overnight at 4 °C, then postfixed with 1%osmium tetroxide in 0.1 mol/L cacodylate for 1 h.The fixed eyecups were staineden blocwith uranyl acetate after bufferwashing and dehydrated with methanol solutions.The eyecups were subsequently embedded in Epon812 resin (Ted Pella, USA) and cut into 60 nm sections with an ultramicrotome (Leica EM UC7, Germany) near the optic nerve.The sections were placed onto carbon-coated copper grids and stained with both uranyl acetate and lead citrate for contrast enhancement.TEM was performed at 75 kV using a H-7 650 electron microscope (Hitachi-Science & Technology,Japan) to observe the morphology of the photoreceptor ciliary compartments.

    Western blotting

    Mouse retinas and transfected cells were lysed by sonication in standard RIPA buffer (150 mmol/L NaCl, 1% Triton X-100,0.5% sodium deoxycholate, 0.1% SDS, 50 mmol/L Tris-HCl pH 7.4) supplemented with complete protease inhibitor cocktail tablets (Roche, USA).The supernatant of protein lysates (~20 μg) was resolved using 10% sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE), and then transferred to a nitrocellulose membrane (0.45 μmol/L,Millipore, Germany).Subsequently, the membrane was blocked with 8% non-fat dry milk in Tris-buffered saline with 0.1% Triton X-100 (TBST) for 2 h at room temperature and then probed with primary antibodies overnight at 4 °C,followed by horseradish peroxidase (HRP)-conjugated second antibodies at room temperature for 1 h.

    lmmunochemistry

    Mouse eyeballs were dissected from euthanized mice and immediately fixed with 4% paraformaldehyde (PFA) in 0.1 mol/L phosphate buffer (pH 7.4) for 2 h on ice, then dehydrated sequentially with 15% and 30% sucrose in 0.1 mol/L phosphate buffer (pH 7.4) for 2 h each.The eyecups were then embedded in optimal cutting temperature compound (OCT, Sakura Finetek, USA) after lens removal and cut into 12 μm retinal sections.Mouse kidneys were first dissected and fixed with 4% PFA.After dehydration with 30%sucrose for 24 h, the kidneys were embedded in OCT and cut into 5 μm sections.MEFs cultured on coverslips were fixed in 4% PFA for 10 min and permeabilized with 1% Triton X-100 for immunostaining.

    For immunofluorescence staining, retinal and kidney sections, as well as MEF slides, were blocked with 10%normal donkey serum and 0.2% Triton X-100 in phosphate buffer at room temperature for 1 h, then incubated with primary antibodies overnight at 4 °C.After washing three times with phosphate buffer, the tissue sections and MEF slides were incubated with fluorescence-conjugated secondary antibodies at room temperature for 1 h.To investigate apoptosis of mouse retinas, TUNEL staining of retinal sections was performed with anIn SituCell Death Detection Kit (Roche Diagnostics, China).Fluorescence images were acquired using a confocal microscope (LSM800,Carl Zeiss, Germany).

    Antibodies

    The primary antibodies used for western blotting and immunofluorescence analysis included: SDCCAG8 (WB 1:2 000, IHC 1:50, 13 471-1-AP; Proteintech Group, USA),GRK1 (1:400, 24 606-1-AP; Proteintech Group, USA),rhodopsin (1:400, 1D4, clone D4B9B; Cell Signaling Technology, CST, USA), S-opsin (1:300, Ab5407; Abcam,UK), GFP (1:3 000, 50 430-2-AP; Proteintech Group, USA),GAPDH (1:5 000, 10 494-1-AP; Proteintech Group, USA), βactin (1:5 000, 20 536-1-AP; Proteintech Group, USA), PDE6B(1:400, T13343; Thermo, USA), Alexa Fluor 594 conjugated peanut agglutinin (PNA) (1:200, L32459; Thermo, USA), cone arrestin (1:300, AB15282; Sigma, USA), and anti-alpha tubulin(acetyl K40) (1:1 000, ab24610; Abcam, UK).Secondary antibodies included: goat anti-rabbit Alexa Fluor 488 and 594(1:1 000; Invitrogen, USA), goat anti-mouse Alexa Fluor 594(1:1 000; Invitrogen, USA), and HRP-conjugated Affinipure goat anti-rabbit IgG (H+L) (1:5 000, SA00001-2; Proteintech Group, USA).

    Electroretinogram (ERG) analysis

    As described previously (Jiang et al., 2011), experimentalmice were dark-adapted overnight, with subsequent procedures performed under dim red light.The dark-adapted mice were first anesthetized with a combination of ketamine(16 mg/kg body weight) and xylazine (80 mg/kg body weight)by intraperitoneal injection, and their eyes were then dilated with tropicamide, phenylephrine, and tetracaine (0.5%).Body temperature was maintained at 37 °C with a heating pad.Both scotopic and photopic ERG responses were recorded from 3-5 mice with the Espion Visual Electrophysiology System(Diagnosis, AbelConn, LLC, USA).Scotopic ERG,representing rod visual function, was first conducted with light stimuli at intensities ranging from -2.52 log cd·s/m2to 1.30 log cd·s/m2, while photopic ERG, representing cone visual function, was performed with light stimuli at intensities ranging from 0.48 log cd·s/m2to 1.30 log cd·s/m2after light adaptation for 20 mins.Antibiotic ointment was applied to the eyes after the ERG procedure to prevent infection.

    Analysis of urine albumin to creatinine ratio (uACR)

    Proteinuria, which is a major feature of chronic kidney disease(CKD), was assessed based on the uACR.We obtained 24 h urine samples from mice at P180 using metabolic cages (TSE systems, Germany).The concentrations of urine microalbumin and creatinine were measured by immunoturbidimetric assay(Mindray, China) and kinetic enzymatic assay (Maccura,China), respectively, on an AU5800 automatic biochemical analyzer (Beckman Coulter, Japan).uACR was calculated by dividing the microalbumin concentration in micrograms by the creatinine concentration in milligrams, reported as μg/mg.

    Statistical analysis

    T-test was used to compare data between two groups.Multiple comparisons involving more than three groups were analyzed using analysis of variance (ANOVA).Significance was determined atP<0.05.Data were analyzed using GraphPad Prism v8 (GraphPad Software, USA) and are presented as mean±standard error of the mean (SEM).

    RESULT S

    Generation of two knock-in mouse models expressing hypomorphic alleles, Sdccag8-Y236X and Sdccag8-E451GfsX467

    Two humanSDCCAG8recessive mutations, i.e., nonsense mutation 696T>G (p.Y232X) and 1 bp insertion 1 339-1340insG (p.E447GfsX463), are known to cause syndromic retinal ciliopathies BBS and SLS, respectively (Table 1)(Otto et al., 2010).To investigate genotype-phenotype correlation and pathogenesis of SDCCAG8-associated retinal ciliopathiesin vivo, we utilized CRISPR/Cas9-mediated HDR to generate two knock-in mouse models carrying the corresponding mouse mutationsSdccag8-Y236X andSdccag8-E451GfsX467 (Figure 1A-C).

    The knock-in mouse mutations were verified by Sanger sequencing (Figure 1D, E).To facilitate knock-in mouse genotyping, we designed a BamHI restriction site (GGATCC)adjacent to the mutations in the HDR donor oligos and performed BamHI digestion of allele-specific PCR amplificons as a routine genotyping procedure.Thus, there were two mutant allele-specific bands (258 bp and 170 bp) and one wild-type allele-specific band (425 bp) inSdccag8-Y236X mouse genotyping (Figure 1F).Similarly, there were two mutant allele-specific bands (263 bp and 186 bp) and one wild-type allele-specific band (445 bp) inSdccag8-E451GfsX467 mouse genotyping (Figure 1G).All three bands were present in the heterozygous mice.

    Upon western blotting with anti-SDCCAG8 antibodies targeting the N-terminal epitope (1-360 aa) of SDCCAG8, we detected 27 kDa and 54 kDa truncated protein bands inSdccag8Y236X/Y236XandSdccag8E451GfsX467/E451GfsX467mouse retinas at P30, respectively, but the absence of the full-length SDCCAG8 protein at 83 kDa (Figure 2A).Molecular weights of the mutant proteins, SDCCAG8-Y236X and SDCCAG8-E451GfsX467, were consistent with the predicted truncations due to reading frame shift of theSdccag8mutations.The expression levels of both truncated proteins were significantly decreased in the knock-in mice compared to that of the fulllength protein in wild-type mice, indicating instability of the truncated proteins (Figure 2A).To confirm whether truncation and reduction of the SDCCAG8 mutant proteins could be attributed to the disease-causing mutations rather than other genetic engineering modifications via CRISPR/Cas9-mediated HDR, such as insertion of a BamHI restriction site, we expressed GFP-tagged SDCCAG8 wild-type and mutant proteins in HEK293T cells.Using immunoblotting analysis with anti-SDCCAG8 and anti-GFP antibodies, we confirmed the expression of truncated proteins in their corresponding transfected cells, consistent with theSdccag8mutants expressed inSdccag8knock-in mice at reduced levels(Figure 2B).To investigate the localization of truncated SDCCAG8 in mouse photoreceptors, we conducted immunochemical assays on mouse retinas at P30 (Figure 2C).SDCCAG8 was localized around the photoreceptor inner segment (IS) and CC in wild-type controls, as reported previously (Otto et al., 2010), whereas the truncated SDCCAG8 proteins were expressed in the same location in the mutant photoreceptors but with significantly deceased fluorescence signals (Figure 2C).Thus, we verified the two knock-in mouse lines carrying hypomorphicSdccag8mutant alleles,Sdccag8Y236XandSdccag8E451GfsX467.

    The two knock-in mouse lines displayed unexpected Mendelian ratios, with only 9.1% ofSdccag8Y236X/Y236Xmice(40/433) and 17.4% ofSdccag8E451GfsX467/E451GfsX467mice(83/476) surviving after birth, significantly lower than the expected ratio of 25%.Additionally, 45% ofSdccag8Y236X/Y236Xmice (18/40) and 42.2% ofSdccag8E451GfsX467/E451GfsX467mice(35/83) died within 24 h of birth.TheSdccag8knock-in mice had normal body size at birth but were significantly smaller than the age-matched controls at P30, indicating developmental retardation (Figure 2D).Compared to the controls (17.67±1.37 g), theSdccag8E451GfsX467/E451GfsX467mice(10.50±1.38 g) and especially theSdccag8Y236X/Y236Xmice(9.33±1.21 g) had significantly reduced body weight(Figure 2E).Notably, all mutant males were infertile.

    Development of early-onset and progressive rod-cone degeneration with varied severity in two Sdccag8 knockin mouse models

    Previous studies have reported that individuals withSDCCAG8-associated ciliopathies manifest early-onset and progressive retinal degeneration (Otto et al., 2010).To examine whether theSdccag8knock-in mice phenocopied human retinal degeneration caused bySDCCAG8mutations,we performed histological analysis on mouse retinas at ages P30, P90, and P180 (Figure 3A-C).In contrast to wild-type controls with 11-12 rows of photoreceptor nuclei lining the retinal ONL, theSdccag8Y236X/Y236XandSdccag8E451GfsX467/E451GfsX467mice had only around six and eight rows, respectively, at P30 (Figure 3A, D).In addition,their photoreceptor OSs were about half as short as that of the wild-type controls at P30 (Figure 3A).Shortening of the ONL and OSs progressed rapidly in the knock-in mice from P30 to P180.TheSdccag8E451GfsX467/E451GfsX467mice had only around six rows of photoreceptor nuclei remaining in the ONL at P90,and close to four rows at P180 (Figure 3B-F).TheSdccag8Y236X/Y236Xmice showed more severe retinal degeneration, with only four rows of photoreceptor nuclei left at P90, and two rows at P180 (Figure 3B-F).Correspondingly,their photoreceptor OSs were substantially shortened by P90 and almost completely diminished at P180, indicating rapid retinal degeneration between P30 and P180 in the knock-in mice.To examine whether theSdccag8mutations also caused degeneration of cone photoreceptors, we used PNA staining, a cone specific marker, on knock-in mouse retinas at P30, P90, and P180 (Figure 3G).There was no obviousreduction in cone photoreceptors, even by P90, in both knockin mice, althoughSdccag8Y236X/Y236Xmice showed significant loss of cones and reduced cone OS at P180, revealing lateonset cone generation inSdccag8knock-in mice.

    Figure 1 Generation of Sdccag8Y236X/Y236X and Sdccag8E451GfsX467/E451GfsX467 knock-in mice

    Figure 2 Sdccag8 knock-in mice carrying a hypomorphic allele with growth retardation

    We next conducted scotopic and photopic ERGs on the knock-in mice to evaluate visual function of their rod and cone photoreceptors, respectively.For the scotopic ERGs, the awave amplitudes under 1.30 log cd·s/m2light stimuli significantly decreased by 53% inSdccag8Y236X/Y236Xmice(177.90±27.96 μV) and by 49% inSdccag8E451GfsX467/E451GfsX467mice (192.90±31.55 μV) at P30 compared to the wild-type controls (375.50±36.36 μV) (Figure 4A-C).The scotopic ERG responses recorded from both knock-in mice declined by 78%and 69% at P90 and by 93% and 85% by P180, respectively,(Figure 4A-C).For the photopic ERGs, the b-wave amplitudes under 1.30 log cd·s/m2light stimuli decreased by 33% inSdccag8E451GfsX467/E451GfsX467mice (116.40±20.58 μV) and by 38% inSdccag8Y236X/Y236Xmice (109.30±37.69 μV) at P30compared to the wild-type controls (174.10±20.93 μV)(Figure 4A, B, D).Progressively, the photopic ERG responses decreased by 71% inSdccag8Y236X/Y236Xmice (49.33±15.34 μV) and 60% inSdccag8E451GfsX467/E451GfsX467mice(66.75±12.36 μV) by P90, and further significantly declined in both knock-in mice (91% (15.33±6.01 μV) and 85%(26.67±7.17 μV), respectively) by P180 (Figure 4A, B, D),indicating that visual dysfunction caused by theSdccag8mutations was more severe inSdccag8Y236X/Y236Xmice than inSdccag8E451GfsX467/E451GfsX467mice, and was initiated earlier in rods than in cones.

    Figure 3 Retinal morphology of rod-cone photoreceptor degeneration in Sdccag8Y236X/Y236X and Sdccag8E451GfsX467/E451GfsX467 knock-in mice

    Figure 4 Scotopic and photopic electroretinograms (ERG) of rod-cone photoreceptor degeneration in Sdccag8Y236X/Y236X and Sdccag8E451GfsX467/E451GfsX467 mice

    Taken together, the retinal morphological and visual functional data of the knock-in mouse models demonstrated thatSdccag8mutations caused early-onset and rapidly progressive photoreceptor degeneration, which was more severe inSdccag8Y236X/Y236Xmice.In addition, rods degenerated earlier and faster than cones in both models,presenting as rod-cone degeneration.

    Mislocalization of phototransduction proteins in Sdccag8 mutant mouse photoreceptors

    Most retinal ciliopathy-causing mutations disrupt photoreceptor morphology and function through abrogation of phototransduction cascade components, trafficking defects of the photoreceptor OS proteins, and impaired cilium biogenesis or maintenance (Bujakowska et al., 2017; Reiter & Leroux,2017).Thus, to determine whether theSdccag8truncating mutations impaired ciliary protein trafficking in photoreceptors,we first examined the localization of several membrane and membrane-associated phototransduction proteins in the knock-in mice at P30 and P90 by immunohistochemical analysis.The transmembrane protein rhodopsin was observed in the photoreceptor OS inSdccag8E451GfsX467/E451GfsX467mice at P30 and P90, as in wild-type controls (Figure 5A, B).However, rhodopsin was mislocalized in the photoreceptor IS inSdccag8Y236X/Y236Xmice at P90, when the OS was significantly shortened (Figure 5A, B).Rhodopsin kinase(GRK1), a peripheral membrane protein involved in the phototransduction cascade, was localized in the shortened photoreceptor OS, with very little mistrafficking in the IS, ONL,and synaptic terminal inSdccag8Y236X/Y236XandSdccag8E451GfsX467/E451GfsX467retinas at P30 (Figure 5C).At P90, decreased mistrafficking of GRK1 was observed as the mutant photoreceptors were significantly decreased and their OSs were extensively shortened in both knock-in mice(Figure 5D).Likewise, PDE6b, another rod peripheral membrane protein, showed mistrafficking in the photoreceptors of the knock-in mice at both P30 and P90(Figure 5E, F).Subsequently, we examined the localization of two cone phototransduction proteins in the knock-in mouse retinas, namely membrane protein S-opsin and membraneassociated protein cone arrestin, which trafficked to the cone OS in the wild-type controls.We observed remarkable mislocalization of S-opsin and cone arrestin in the cone IS and synaptic terminal in the knock-in mice at P30 and P90(Figure 6A-D).Thus, our data revealed progressive mislocalization of OS-specific membrane proteins and membrane-associated proteins in the mutant photoreceptors,which was more obvious in the cones than in the rods.

    Figure 5 lmmunolocalization of rod phototransduction-related proteins in Sdccag8Y236X/Y236X and Sdccag8E451GfsX467/E451GfsX467 mouse retinas

    Figure 6 lmmunolocalization of cone phototransduction-related proteins in Sdccag8Y236X/Y236X and Sdccag8E451GfsX467/E451GfsX467 mouse retinas

    We subsequently tested whether protein mislocalization occurred before or after photoreceptor cell death by TUNEL staining of the knock-in mouse retinas at P21, before protein mislocalization occurred.TUNEL signals represent apoptosis,a predominant cell death mode of photoreceptor degeneration(Wright et al., 2010).We detected TUNEL signals in the ONL of bothSdccag8Y236X/Y236XandSdccag8E451GfsX467/E451GfsX467mice at P21, but not in wild-type controls (Figure 6E),revealing that OS protein mislocalization occurred after photoreceptor cell death in the knock-in mice, probably due to shortening of the photoreceptor OS, a sink of phototransduction proteins.

    lmpaired photoreceptor cilia drive retinal degeneration in Sdccag8 knock-in mice

    To test whether theSdccag8truncating mutations impaired photoreceptor cilia in the knock-in mice, we examined the photoreceptor ultrastructures of mutant mice at P60 by TEM,specifically focusing on the photoreceptor ciliary compartments, including BB, CC, and OS.The wild-type mice developed robust photoreceptor BB, CC, and OS compartments, with evenly stacked disk membranes(Figure 7A).In contrast, despite BB docking to photoreceptor apical membranes as usual, bothSdccag8Y236X/Y236XandSdccag8E451GfsX467/E451GfsX467mice exhibited shortened CC and disorganized OS compartments, with significantly deteriorated disk membranes (Figure 7A).These results revealed that the structure of the photoreceptor cilia in theSdccag8mutant mice was destroyed, suggesting that SDCCAG8 may function in photoreceptor cilium formation and/or maintenance.

    We further studied whether theSdccag8truncating mutations affected global cilium formation and maintenance by examining primary cilium formation in MEFs derived from knock-in mice (Figure 7B).We assessed cilium occurrence and length of mutant MEFs by immunofluorescence staining with an antibody against the ciliary marker acetylated tubulin after serum starvation (Figure 7B-D).Primary cilium formationwas only found in 8% (8/100) ofSdccag8Y236X/Y236XMEFs and 39% (39/100) ofSdccag8E451GfsX467/E451GfsX467MEFs, in contrast to 80% (80/100) of wild-type control MEFs (Figure 7C).Additionally, the average lengths of cilia in theSdccag8Y236X/Y236XandSdccag8E451GfsX467/E451GfsX467MEFs were 0.87 μm and 2.10 μm, respectively, significantly shorter than that of the controls (Figure 7B, D).These results suggest that SDCCAG8 is essential for global cilium formation.

    Figure 7 lmpaired cilia formation in photoreceptors and MEFs from Sdccag8Y236X/Y236X and Sdccag8E451GfsX467/E451GfsX467 mice

    Our results revealed that impaired photoreceptor ciliary structure is a primary pathogenic force driving photoreceptor degeneration in the knock-in mice, with SDCCAG8 implicated to play a critical role in photoreceptor ciliary formation and maintenance, rather than ciliary protein trafficking.

    Nephronophthisis and defective epithelial cilia in Sdccag8 knock-in mice

    Patients with SDCCAG8-associated ciliopathies andSdccag8gt/gtgene-trap mice with loss ofSdccag8function exhibit NPHP, a renal cystic disease characterized by corticomedullary cysts, tubular basement membrane disruption, and tubulointerstitial nephropathy in renal histology(Stokman et al., 2016).Thus, we investigated whether the knock-in mice developed NPHP.

    We observed kidney enlargement inSdccag8Y236X/Y236Xmice as early as P30, and the mutant kidneys became progressively larger at P90 and deformed at P180 (Figure 8A).Correspondingly, small cysts formed in the cortical region of theSdccag8Y236X/Y236Xmice at P30, as visualized by renal histological assay (Figure 8B).From P90 to P180, theSdccag8Y236X/Y236Xmouse kidneys progressively deteriorated,with renal cysts spreading beyond the cortical region to the corticomedullary junction, cortical cysts becoming enlarged,and renal parenchyma replaced by interstitial infiltrates(Figure 8B).However, different from early-onset NPHP inSdccag8Y236X/Y236Xmice, no shape change or cyst formation was detected in theSdccag8E451GfsX467/E451GfsX467mouse kidneys at P30 (Figure 8A, B).Relatively mild kidney enlargement and cyst formation in the kidney cortical region were observed in theSdccag8E451GfsX467/E451GfsX467mice at P90 to P180 (Figure 8A, B).To examine renal fibrosis in the knockin mice, we performed Masson trichrome staining of renal sections.Consistent with histology, theSdccag8Y236X/Y236Xmice showed rapidly progressive renal fibrosis, from mild fibrosis surrounding the dilated tubules at P30 and P90 to extensive collagen deposits distributed across the renal tissue at P180 (Figure 8C).Renal fibrosis was not detected in theSdccag8E451GfsX467/E451GfsX467mouse kidneys at P30 but was observed minimally at P90 and obviously at P180 (Figure 8C).Thus, our data demonstrated that NPHP occurred in both knock-in mouse models but with different severities andprogressed in concert with photoreceptor degeneration.

    Figure 8 Nephronophthisis in Sdccag8Y236X/Y236X and Sdccag8E451GfsX467/E451GfsX467 mice accompanied by defective renal cilia

    We next tested the uACR to investigate whether the knockin mice developed CKD, a typical clinical feature of NPHP and precursor to end-stage renal disease (ESRD).The uACR is a sensitive and specific surrogate marker for proteinuria,indicating progression of CKD (Hildebrandt et al., 2009).We collected 24 h urine samples from three P180 mice for each knock-in model and analyzed the uACR with an automatic biochemical analyzer.The 24 h uACRs inSdccag8Y236X/Y236X(132.5±7.0 μg/mg) andSdccag8E451GfsX467/E451GfsX467(59.4±7.0 μg/mg) mice were significantly elevated (3.3 and 1.5 times,respectively) compared to the wild-type controls (39.6±5.4 μg/mg).The uACR results indicated CKD progression in both knock-in mice by P180, with increased severity inSdccag8Y236X/Y236Xmice.

    We next investigated whether theSdccag8mutations affected biogenesis of cilia in the mouse kidneys, similar to their effects on photoreceptors and MEFs.Renal cilia were examined by staining kidney sections with an antibody against the cilium marker acetylated tubulin.The number and length of renal epithelial cilia were significantly decreased in the distal convoluted tubules and cortical collecting ducts of theSdccag8E451GfsX467/E451GfsX467kidneys and were completely absent in theSdccag8Y236X/Y236Xkidneys (Figure 8D).

    Taken together, our data demonstrate that the knock-in mice carryingSdccag8truncating mutations displayed NPHP and developed CKD, with earlier onset and increased severity inSdccag8Y236X/Y236Xmice.The ciliary defects detected in the kidney epithelial cells may be a driving force of renal cyst formation inSdccag8knock-in mice.

    Hind limb preaxial polydactyly in Sdccag8 knock-in mice

    Postaxial polydactyly is a major clinical feature of BBS in humans.Thus, we examined whether this phenotype was present in the knock-in mouse models.Interestingly, the mutant mice exhibited preaxial polydactyly of the hind limbs,with 100% (40/40) penetrance in theSdccag8Y236X/Y236Xmice and 95% (79/83) penetrance in theSdccag8E451GfsX467/E451GfsX467mice (Figure 9A, B).Bilateral polydactyly was predominant, accounting for 80% (32/40) of theSdccag8Y236X/Y236Xmice and 58% (48/83) of theSdccag8E451GfsX467/E451GfsX467mice (Figure 9B).Among the mice with unilateral polydactyly, 75% (6/8) of theSdccag8Y236X/Y236Xmice and 65% (20/31) of theSdccag8E451GfsX467/E451GfsX467mice presented with right-sided polydactyly, significantly more than left-sided polydactyly (Figure 9B).The development of polydactyly as a ciliopathy phenotype in the mouse models is consistent with previous findings in three gene-trap mouse models (Airik et al., 2016; Insolera et al., 2014; Weihbrecht et al., 2018), supporting the hypothesis that SDCCAG8 participates in digit development in mice.

    Figure 9 Preaxial polydactyly phenotype in Sdccag8Y236X/Y236X and Sdccag8E451GfsX467/E451GfsX467 mice

    DlSCUSSlON

    In this study, two novelSdccag8knock-in mouse linesSdccag8Y236X/Y236XandSdccag8E451GfsX467/E451GfsX467, each carrying a distinct hypomorphic allele, were generated by CRISPR/Cas9-HDR.They faithfully recapitulated human SDCCAG8-associated BBS phenotypes with varied phenotypic age of onset and severity, which were directly proportional to the hypomorphic strength of theSdccag8mutations, Y236X and E451GfsX467.To the best of our knowledge, these knock-in mice were the first BBS mouse models to present with polydactyly.Impaired cilia were observed in the mutant photoreceptors, renal epithelial cells,and MEFs derived from the knock-in mouse embryos.Therefore, we propose that cilium defects are the primary driving force of SDCCAG8-associcated BBS in humans.

    Due to a pivotal role of cilia in diverse cellular processes of embryonic and postnatal development (Reiter & Leroux,2017), most ciliopathy-associated mutations identified in humans are hypomorphic, includingSdccag8mutations,resulting in prenatal developmental defects, as well as clinical manifestations arising after birth.Thus, it is unsurprising that complete loss of a ciliopathy-associated gene is devastating to mice and humans (Norris & Grimes, 2012).Two out of threeSdccag8mutant mice previously generated by gene trap show embryonic lethality, with developmental defects in the central nervous system, limbs, and lungs, but without primary features of SDCCAG8-assciated ciliopathies, i.e., retinal and renal defects (Insolera et al., 2014; Weihbrecht et al., 2018).Therefore, conventional genetic modification approaches to generate null alleles may not be appropriate for modeling ciliopathy-associated diseases.The generation of hypomorphic alleles with a range of strengths for diseases that explicitly model human ciliopathies can be technically demanding (Rix et al., 2011).Among current genome engineering technologies, the CRISPR/Cas9 system is a programmable nuclease-based genome-editing technology that enables highly efficient and precise modification of avariety of eukaryotic and mammalian species (Banan, 2020;Hsu et al., 2014).Employing CRISPR/Cas9-HDR in this study,we successfully developed two mouse models carrying hypomorphic alleles ofSdccag8, which closely mimicked human BBS with phenotypic variation.

    The mouseSdccag8mutationsSdccag8-Y236X andSdccag8-E451GfsX467 respectively correspond to BBS- and SLS-causing mutations in humans (Halbritter et al., 2013a;Otto et al., 2010).The corresponding knock-in mice generated in this study lacked the full-length SDCCAG8 protein and showed significantly decreased expression of the specific truncated proteins, in contrast to the null alleles described in three previously generatedSdccag8gene-trap mice (Airik et al., 2014; Insolera et al., 2014; Weihbrecht et al., 2018).The fewer truncated proteins could be attributed to nonsense mediated decay due to the inclusion of an early stop codon in theSdccag8mutant alleles.Previous genetic studies have suggested that only the full-length isoform SDCCAG8-a is relevant for the retinal-renal phenotype of SDCCAG8-associated ciliopathies, with specific expression in the photoreceptor CC and ISs of mouse retinas (Otto et al., 2010).The mutantsSDCCAG8-Y236X andSDCCAG8-E451GfsX467 caused different-sized truncations of the C-terminal CCD.Notably, based on immunochemical analysis of the knock-in mouse retinas, SDCCAG8-Y236X exhibited weaker fluorescence signals than SDCCAG8-E451GfsX467 in the mutant photoreceptor CC and ISs, suggesting that the size of the truncated CCD was directly proportional to the hypomorphic strength of the mutations.

    Histological and ERG analysis of mouse retinas showed early-onset and rapidly progressive rod-cone photoreceptor degeneration in theSdccag8knock-in mice.Retinal degenerative changes, including photoreceptor ONL and OS shortening, were observed in theSdccag8Y236X/Y236Xmice as early as P30, and rapidly progressed to almost complete deterioration within 6 months.TheSdccag8E451GfsX467/E451GfsX467mice showed less severe morphological and functional changes in photoreceptor degeneration, progressing about one month later than theSdccag8Y236X/Y236Xmice.In addition,loss of cone photoreceptors was first observed in mutant retinas at P180, and ERG responses from cones decreased by ~20% in the knock-in mice at P30.In contrast, a 35%-50%loss of rods and 50% decrease in ERG responses from rods were observed in the knock-in mice even at P30.Thus, lateonset cone death and dysfunction revealed rod-cone photoreceptor degeneration in SDCCAG8-associated retinal ciliopathies.

    In addition, early- and late-onset NPHP developed in the two knock-in mouse models, respectively, with formation of renal cysts inSdccag8Y236X/Y236Xmice as early as P30, but at P90 inSdccag8E451GfsX467/E451GfsX467mice.However, among the three previously generatedSdccag8gene-trap mouse models,onlySdccag8gt/gtmice exhibited late-onset NPHP, with embryonic lethality reported in the other mice (Airik et al.,2014; Insolera et al., 2014; Weihbrecht et al., 2018).NPHP is a cystic renal disease that constitutes the most frequent genetic cause of ESRD in children and young adults, and is characterized by corticomedullary cysts, tubular basement membrane disruption, and tubulointerstitial nephropathy in renal histology (Stokman et al., 2016).TheSdccag8knock-in mice displayed moderately enlarged and/or deformed kidneys,not unlike infantile NPHP in humans (Airik et al., 2014; Braun& Hildebrandt, 2017; Kang et al., 2016; Otto et al., 2010).Previous mouse models of cystic renal disease have shown renal cysts in the cortical region during the early stages, with spread to the corticomedullary junction and interstitial infiltrates at the end stage (Atala et al., 1993; Attanasio et al.,2007).Similarly, our mouse models demonstrated renal fibrosis surrounding dilated tubules during early disease, and later widespread deterioration of renal tissue.Here, mouse renal function was evaluated by 24 h uACR testing, which indicated excessive proteinuria inSdccag8Y236X/Y236Xmice at P180, consistent with ESRD.The different severities of NPHP in the two knock-in mouse models further demonstrated the different hypomorphic strengths of theSdccag8-Y236X andSdccag8-E451GfsX467 mutant alleles.

    The three previously reportedSdccag8gene-trap mouse lines exhibit preaxial polydactyly penetrance of 65%-100%(Airik et al., 2016; Insolera et al., 2014; Weihbrecht et al.,2018), similar to the two knock-in mouse lines at 95% and 100%, respectively.As an embryonically developed phenotype, polydactyly is a primary feature of BBS and other ciliopathies, such as MKS, JBTS, orofaciodigital syndrome(OFD), and McKusick-Kaufman syndrome (MKKS), but not SLS (Zaghloul & Katsanis, 2009).About 79% of BBS patients present with postaxial polydactyly, but the polydactyly phenotype is absent in human SDCCAG8-assciated ciliopathies (Beales et al., 1999; Forsythe & Beales, 2013;Otto et al., 2010; Schaefer et al., 2011).To the best of our knowledge, theSdccag8-associated mutant mice, including the gene-trap and knock-in mice, are the first BBS mouse models characterized by digital malformation.In addition,instead of presenting postaxial polydactyly on any limb, as seen in BBS patients, theSdccag8-associated mouse models exhibited preaxial polydactyly on hind limbs only.Many ciliary gene mutations causing polydactyly are attributed to disruption of the Sonic Hedgehog (Shh) signaling pathway, which requires the presence of intact primary cilia for activation(Zaghloul & Katsanis, 2009).The presence of polydactyly in the knock-in mice suggests that SDCCAG8 is necessary for cilium biogenesis and/or maintenance of the developing limb bud in mice, but not in humans.Furthermore, this suggests species-specific differences in Shh-dependent determination of digit number and identity.Therefore, further studies are needed to improve our understanding of the underlying pathogenesis of polydactyly in human BBS.

    The twoSdccag8knock-in mouse lines were characterized as BBS animal models, displaying four out of the six BBS primary manifestations, including rod-cone dystrophy, cystic renal disorder, polydactyly, and male infertility, as well as secondary developmental delay.Phenotypic severity and age of onset were dependent on the hypomorphic strength of theSdccag8mutations, which was directly correlated to truncation size and expression level of the mutant alleles.

    We also explored the pathogenesis of SDCCAG8 mutationcausing retinal ciliopathies by dissecting OS protein trafficking and ciliary structure of the mutant photoreceptors in theSdccag8knock- in mice.We discovered rodphototransduction-related proteins, including rhodopsin,GRK1, and PDE6, localized not only in the OS, but also in the photoreceptor IS, ONL, and/or synaptic terminals of the knockin mice at P90, when rod photoreceptors were dying and their OSs were significantly shortened.In addition, cone phototransduction-related proteins S-opsin and cone arrestin were significantly mislocalized in the IS, ONL, and synaptic terminals at P30, which was probably due to differences in OS biogenesis or protein trafficking between cones and rods(Anderson et al., 1978; Eckmiller, 1987).However, based on TUNEL staining, we found that photoreceptor cell death occurred earlier than OS protein mislocalization in the mutant photoreceptors, suggesting thatSdccag8mutation-causing protein mislocalization occurred after photoreceptor cell death.Impaired ciliary structures, including shortened CC and disrupted OS, were detected in the knock-in mice by TEM analysis of retinas.Impaired renal epithelial cilia were observed in the distal convoluted tubules and cortical collecting ducts of the mutant kidneys.In addition,Sdccag8mutation-causing global cilium defects were verified in MEFs derived from the knock-in mice.Thus, our data demonstrated that cilium defects were a primary force driving BBS phenotypes in the knock-in mice, suggesting that SDCCAG8 plays an essential role in biogenesis and maintenance of cilia in multiple systems during embryonic development and after birth.Notably, we found that SDCCAG8 is a ciliary protein involved in retinal ciliopathies and plays a key role in cilium assembly and/or maintenance.Further analysis of its role should provide insights into ciliary function and increase our understanding of the signaling, physiological, and developmental functions of cilia.

    COMPETlNG lNTERESTS

    The authors declare that they have no competing interests.

    AUTHORS’ CONTRlBUTlONS

    L.J.and Z.L.Y.designed the study and wrote and revised the manuscript.Z.L.R.performed the experiments, analyzed the data, and drafted the manuscript.H.B.Z.and L.L.helped analyze the data and reviewed and edited the manuscript.All authors read and approved the final version of the manuscript.

    ACKNOWLEDGEMENTS

    We thank Shu-Jin Li, Mu Yang, and Shan-Shan Zhang for assistance in the immunochemical experiments and Jia-Liang Yang and Zhi-Lin Jiang for assistance in the ERG experiment.We also thank Lin Wang for help in breeding mice and Tian-Ge Song and Ke-Cheng Li for help in obtaining tail clips from mice.

    在线观看午夜福利视频| 国内少妇人妻偷人精品xxx网站| 精品久久久久久,| 五月玫瑰六月丁香| 99国产精品一区二区蜜桃av| 成年女人毛片免费观看观看9| 精品久久久久久久人妻蜜臀av| 亚洲 欧美 日韩 在线 免费| 亚洲,欧美精品.| 亚洲aⅴ乱码一区二区在线播放| 国内精品久久久久久久电影| 午夜日韩欧美国产| 亚洲专区中文字幕在线| 国产精品爽爽va在线观看网站| 青草久久国产| 九色国产91popny在线| 国语自产精品视频在线第100页| 欧美+亚洲+日韩+国产| 天堂动漫精品| 色视频www国产| av专区在线播放| 国产亚洲精品久久久久久毛片| 色噜噜av男人的天堂激情| 香蕉丝袜av| 国产成人av教育| 一级毛片女人18水好多| 日韩免费av在线播放| 国产欧美日韩精品亚洲av| 十八禁人妻一区二区| 欧美一级毛片孕妇| 中文资源天堂在线| 精品人妻偷拍中文字幕| 男女床上黄色一级片免费看| 国产成人影院久久av| 亚洲国产精品久久男人天堂| www日本在线高清视频| 午夜免费成人在线视频| 久久香蕉国产精品| 国产精华一区二区三区| 亚洲avbb在线观看| 国内精品一区二区在线观看| 在线视频色国产色| 俄罗斯特黄特色一大片| 欧美色欧美亚洲另类二区| 成人欧美大片| 国内精品美女久久久久久| 日本精品一区二区三区蜜桃| 国产成人影院久久av| 最新美女视频免费是黄的| 国产老妇女一区| 啦啦啦免费观看视频1| 我要搜黄色片| e午夜精品久久久久久久| 亚洲国产欧美网| 欧美又色又爽又黄视频| 少妇的逼好多水| 亚洲国产色片| 免费观看精品视频网站| 亚洲一区二区三区色噜噜| 亚洲av免费在线观看| 久久天躁狠狠躁夜夜2o2o| 欧美国产日韩亚洲一区| 老司机在亚洲福利影院| 欧美bdsm另类| 亚洲av熟女| 欧美成人免费av一区二区三区| 在线十欧美十亚洲十日本专区| 精华霜和精华液先用哪个| 亚洲七黄色美女视频| 久久久久性生活片| 久久精品国产综合久久久| 人人妻,人人澡人人爽秒播| 综合色av麻豆| 中文字幕精品亚洲无线码一区| 小蜜桃在线观看免费完整版高清| 久久精品人妻少妇| 噜噜噜噜噜久久久久久91| 午夜激情福利司机影院| 国产亚洲精品av在线| 人人妻,人人澡人人爽秒播| 欧美性猛交╳xxx乱大交人| 国产日本99.免费观看| 国产伦在线观看视频一区| 亚洲美女黄片视频| 搡老妇女老女人老熟妇| 婷婷丁香在线五月| 757午夜福利合集在线观看| 色噜噜av男人的天堂激情| 天天躁日日操中文字幕| 国产av一区在线观看免费| 日韩中文字幕欧美一区二区| 99热只有精品国产| 亚洲成av人片免费观看| 啦啦啦观看免费观看视频高清| 亚洲一区二区三区色噜噜| 亚洲精品国产精品久久久不卡| 国产成人av教育| 欧美日韩国产亚洲二区| 啦啦啦免费观看视频1| 夜夜爽天天搞| 老师上课跳d突然被开到最大视频 久久午夜综合久久蜜桃 | 中文字幕人妻熟人妻熟丝袜美 | 午夜激情福利司机影院| 中文资源天堂在线| 美女免费视频网站| 亚洲黑人精品在线| 国产av一区在线观看免费| 精品国产超薄肉色丝袜足j| 欧美黄色片欧美黄色片| 亚洲av免费高清在线观看| 国产伦精品一区二区三区四那| 91久久精品电影网| 欧美一区二区国产精品久久精品| 很黄的视频免费| 一区二区三区国产精品乱码| 日韩欧美国产一区二区入口| 黄色视频,在线免费观看| 少妇的丰满在线观看| 欧美激情久久久久久爽电影| 丰满的人妻完整版| 毛片女人毛片| 国产伦精品一区二区三区四那| 人人妻人人澡欧美一区二区| 观看美女的网站| 欧美中文综合在线视频| 亚洲成人免费电影在线观看| 国产国拍精品亚洲av在线观看 | 麻豆久久精品国产亚洲av| 亚洲精品粉嫩美女一区| e午夜精品久久久久久久| 日韩精品中文字幕看吧| 18禁国产床啪视频网站| 亚洲欧美日韩高清专用| 一级毛片高清免费大全| 精品人妻1区二区| 久久6这里有精品| 国产综合懂色| 午夜福利高清视频| 偷拍熟女少妇极品色| 欧美日韩亚洲国产一区二区在线观看| 亚洲中文字幕日韩| 九色成人免费人妻av| 国产97色在线日韩免费| 手机成人av网站| 美女黄网站色视频| 国产美女午夜福利| 一本综合久久免费| 国内揄拍国产精品人妻在线| 亚洲五月婷婷丁香| 小蜜桃在线观看免费完整版高清| 亚洲国产精品999在线| 欧美xxxx黑人xx丫x性爽| 1024手机看黄色片| 欧美高清成人免费视频www| 啦啦啦免费观看视频1| 俺也久久电影网| 99热这里只有是精品50| 日韩欧美国产一区二区入口| 久久久久久久精品吃奶| 国产aⅴ精品一区二区三区波| 91久久精品电影网| 国产黄色小视频在线观看| 99久久成人亚洲精品观看| 成人av一区二区三区在线看| 国产精品精品国产色婷婷| 欧美日韩亚洲国产一区二区在线观看| 日本黄色视频三级网站网址| 九九热线精品视视频播放| 亚洲av电影不卡..在线观看| 熟女电影av网| 九九热线精品视视频播放| 亚洲av不卡在线观看| 亚洲国产高清在线一区二区三| 亚洲精品国产精品久久久不卡| 国产精品久久久久久久久免 | 国产乱人视频| 午夜亚洲福利在线播放| 午夜久久久久精精品| ponron亚洲| 性色av乱码一区二区三区2| 精品人妻一区二区三区麻豆 | 久久性视频一级片| 亚洲中文日韩欧美视频| 在线观看一区二区三区| 久久性视频一级片| 99久久99久久久精品蜜桃| 国产一区二区在线观看日韩 | 老司机在亚洲福利影院| 哪里可以看免费的av片| 国产成人欧美在线观看| 午夜两性在线视频| 亚洲性夜色夜夜综合| 99久久成人亚洲精品观看| 日韩人妻高清精品专区| 亚洲av熟女| 看免费av毛片| 日本与韩国留学比较| 一个人观看的视频www高清免费观看| 99热这里只有是精品50| 人人妻人人澡欧美一区二区| 日韩高清综合在线| 精品久久久久久久人妻蜜臀av| 国产精品香港三级国产av潘金莲| 欧美成狂野欧美在线观看| 国产熟女xx| 色综合亚洲欧美另类图片| 国产美女午夜福利| 国产黄片美女视频| 十八禁网站免费在线| 又爽又黄无遮挡网站| 欧美三级亚洲精品| 亚洲国产欧美人成| 色av中文字幕| 人妻夜夜爽99麻豆av| 成人午夜高清在线视频| 两个人看的免费小视频| 日韩欧美精品v在线| a在线观看视频网站| 真人做人爱边吃奶动态| 热99在线观看视频| 女警被强在线播放| 免费无遮挡裸体视频| 国产亚洲欧美在线一区二区| 特大巨黑吊av在线直播| av在线蜜桃| 69人妻影院| 一级毛片女人18水好多| 91av网一区二区| 高清毛片免费观看视频网站| 搡老熟女国产l中国老女人| 亚洲激情在线av| 免费在线观看成人毛片| 国产精品98久久久久久宅男小说| 露出奶头的视频| 亚洲国产中文字幕在线视频| 成人av一区二区三区在线看| 91久久精品国产一区二区成人 | 熟女电影av网| 午夜亚洲福利在线播放| 亚洲天堂国产精品一区在线| av中文乱码字幕在线| 欧美黑人巨大hd| 亚洲成av人片在线播放无| 色噜噜av男人的天堂激情| 天美传媒精品一区二区| 精品久久久久久成人av| 精品国产超薄肉色丝袜足j| 深夜精品福利| 国产精品久久久久久久久免 | 丁香欧美五月| 最近在线观看免费完整版| 国产精品国产高清国产av| 女生性感内裤真人,穿戴方法视频| 国产成人啪精品午夜网站| 人人妻,人人澡人人爽秒播| 禁无遮挡网站| 婷婷亚洲欧美| 舔av片在线| 天堂网av新在线| 精品国产三级普通话版| 热99re8久久精品国产| 精品不卡国产一区二区三区| 欧美一区二区亚洲| 脱女人内裤的视频| 色综合亚洲欧美另类图片| 国产精品亚洲av一区麻豆| 一区二区三区高清视频在线| 在线观看午夜福利视频| 两性午夜刺激爽爽歪歪视频在线观看| 亚洲精品粉嫩美女一区| 日本在线视频免费播放| 欧美又色又爽又黄视频| 精品欧美国产一区二区三| 网址你懂的国产日韩在线| 偷拍熟女少妇极品色| 成人一区二区视频在线观看| 99久久精品热视频| 美女高潮的动态| 悠悠久久av| 欧美成人一区二区免费高清观看| 国产成+人综合+亚洲专区| 天堂√8在线中文| 精品久久久久久久久久久久久| 色在线成人网| 亚洲人与动物交配视频| 日韩中文字幕欧美一区二区| 免费搜索国产男女视频| 国产精品亚洲美女久久久| 国产一区二区激情短视频| 最新美女视频免费是黄的| 国产精品香港三级国产av潘金莲| 午夜激情欧美在线| 久久国产精品影院| 亚洲欧美日韩卡通动漫| 国产午夜精品论理片| 国产亚洲欧美在线一区二区| 亚洲av成人精品一区久久| 婷婷六月久久综合丁香| 91麻豆av在线| 婷婷精品国产亚洲av在线| 男人和女人高潮做爰伦理| 黄色日韩在线| 一个人免费在线观看电影| 色综合婷婷激情| 变态另类丝袜制服| 国产精品一区二区免费欧美| 精品国产美女av久久久久小说| 亚洲国产精品久久男人天堂| 国产av在哪里看| 日韩有码中文字幕| 国产一级毛片七仙女欲春2| 久久天躁狠狠躁夜夜2o2o| 亚洲最大成人中文| 叶爱在线成人免费视频播放| 天堂av国产一区二区熟女人妻| 国产精品av视频在线免费观看| 桃色一区二区三区在线观看| 亚洲 国产 在线| 好男人在线观看高清免费视频| 99视频精品全部免费 在线| 免费搜索国产男女视频| 亚洲欧美日韩东京热| 99国产综合亚洲精品| 亚洲中文日韩欧美视频| 变态另类丝袜制服| 欧美一级a爱片免费观看看| 色综合亚洲欧美另类图片| 欧美日韩亚洲国产一区二区在线观看| 国产成人系列免费观看| 两人在一起打扑克的视频| 欧美+亚洲+日韩+国产| 麻豆成人午夜福利视频| 亚洲精品在线美女| 午夜老司机福利剧场| 国产精品99久久99久久久不卡| 日日干狠狠操夜夜爽| 欧美+日韩+精品| or卡值多少钱| 午夜日韩欧美国产| 成人特级av手机在线观看| 国产激情欧美一区二区| 日本精品一区二区三区蜜桃| 老师上课跳d突然被开到最大视频 久久午夜综合久久蜜桃 | 国产欧美日韩一区二区三| 亚洲国产中文字幕在线视频| 国产一区在线观看成人免费| 又爽又黄无遮挡网站| 2021天堂中文幕一二区在线观| 女警被强在线播放| 日本免费一区二区三区高清不卡| 久9热在线精品视频| 国产一级毛片七仙女欲春2| 黄片大片在线免费观看| 又紧又爽又黄一区二区| 亚洲精品影视一区二区三区av| 手机成人av网站| 69av精品久久久久久| 国产免费男女视频| 香蕉丝袜av| 好看av亚洲va欧美ⅴa在| 听说在线观看完整版免费高清| 美女被艹到高潮喷水动态| 国产成人福利小说| 窝窝影院91人妻| 天堂动漫精品| 久久久久久久亚洲中文字幕 | 好男人在线观看高清免费视频| 亚洲中文字幕一区二区三区有码在线看| 成人无遮挡网站| 国产精品98久久久久久宅男小说| 老鸭窝网址在线观看| 一个人看的www免费观看视频| 男女视频在线观看网站免费| 黄色日韩在线| 成人无遮挡网站| 欧美区成人在线视频| www.www免费av| 三级男女做爰猛烈吃奶摸视频| 久久亚洲真实| 在线观看66精品国产| 啪啪无遮挡十八禁网站| 亚洲av五月六月丁香网| 欧美在线一区亚洲| 成年版毛片免费区| 母亲3免费完整高清在线观看| 亚洲色图av天堂| 最近最新免费中文字幕在线| 国产精品1区2区在线观看.| 欧美黑人巨大hd| 麻豆成人av在线观看| 高清毛片免费观看视频网站| 欧美日韩黄片免| 欧美成狂野欧美在线观看| 麻豆久久精品国产亚洲av| 成人av在线播放网站| 国产精品综合久久久久久久免费| 黄色片一级片一级黄色片| 午夜福利在线在线| 日本撒尿小便嘘嘘汇集6| 婷婷精品国产亚洲av在线| 美女高潮的动态| 一级a爱片免费观看的视频| 欧美极品一区二区三区四区| 男女做爰动态图高潮gif福利片| 日本在线视频免费播放| 色吧在线观看| 中文字幕久久专区| 色综合站精品国产| 搡老熟女国产l中国老女人| 欧美日韩乱码在线| 婷婷亚洲欧美| 日韩欧美精品免费久久 | 国产高清视频在线观看网站| 窝窝影院91人妻| 露出奶头的视频| 国产成+人综合+亚洲专区| 欧美成人性av电影在线观看| 国产精品一区二区三区四区免费观看 | 亚洲精品乱码久久久v下载方式 | 久久久久久久久中文| 18禁在线播放成人免费| www日本在线高清视频| 亚洲精品影视一区二区三区av| 全区人妻精品视频| 国产欧美日韩一区二区精品| 亚洲av成人不卡在线观看播放网| 成人鲁丝片一二三区免费| 一级a爱片免费观看的视频| 日韩欧美在线二视频| 国产精品免费一区二区三区在线| 亚洲欧美日韩高清在线视频| 90打野战视频偷拍视频| 国产伦在线观看视频一区| 成人午夜高清在线视频| 国产老妇女一区| 国产欧美日韩一区二区精品| 波多野结衣高清无吗| 最新在线观看一区二区三区| 中文字幕人成人乱码亚洲影| 国内久久婷婷六月综合欲色啪| 欧美黄色片欧美黄色片| 精品久久久久久久人妻蜜臀av| 免费看美女性在线毛片视频| 国产精品国产高清国产av| 性色avwww在线观看| 999久久久精品免费观看国产| 亚洲人与动物交配视频| 少妇人妻一区二区三区视频| 亚洲五月天丁香| 免费无遮挡裸体视频| 桃红色精品国产亚洲av| 欧美+亚洲+日韩+国产| 国产单亲对白刺激| 亚洲激情在线av| 99精品在免费线老司机午夜| 国产一区二区三区在线臀色熟女| 婷婷亚洲欧美| 日韩亚洲欧美综合| 人人妻人人澡欧美一区二区| 久久伊人香网站| 国产高清视频在线观看网站| 在线观看舔阴道视频| 99国产精品一区二区蜜桃av| 国产一级毛片七仙女欲春2| 热99re8久久精品国产| 国产成人系列免费观看| 国产 一区 欧美 日韩| 国产三级黄色录像| 久久精品国产亚洲av涩爱 | 久久久久精品国产欧美久久久| 亚洲精品美女久久久久99蜜臀| 人人妻人人看人人澡| 男女之事视频高清在线观看| 全区人妻精品视频| 国内精品久久久久久久电影| 久久亚洲精品不卡| 国产精品电影一区二区三区| 少妇裸体淫交视频免费看高清| av福利片在线观看| 国产美女午夜福利| 国产不卡一卡二| 最近最新中文字幕大全电影3| 成年女人永久免费观看视频| 亚洲国产精品合色在线| 久久久色成人| 亚洲成人精品中文字幕电影| 高清在线国产一区| 亚洲精品国产精品久久久不卡| 老司机深夜福利视频在线观看| 久久久久国内视频| 久久欧美精品欧美久久欧美| 法律面前人人平等表现在哪些方面| 亚洲熟妇中文字幕五十中出| 国产免费男女视频| 国产亚洲精品久久久com| 午夜免费成人在线视频| 高清在线国产一区| 久久香蕉国产精品| 欧美日韩瑟瑟在线播放| 亚洲欧美日韩高清专用| 成人三级黄色视频| 国产成人av激情在线播放| 俄罗斯特黄特色一大片| 99视频精品全部免费 在线| 国产成人av教育| 国内揄拍国产精品人妻在线| 窝窝影院91人妻| 狠狠狠狠99中文字幕| 999久久久精品免费观看国产| 色视频www国产| 午夜免费观看网址| 国产亚洲精品av在线| av视频在线观看入口| 色哟哟哟哟哟哟| 国产成人欧美在线观看| 99精品欧美一区二区三区四区| 搡老熟女国产l中国老女人| 在线观看午夜福利视频| av天堂在线播放| 黄色丝袜av网址大全| 国产三级黄色录像| 一个人看的www免费观看视频| 日韩有码中文字幕| 亚洲美女视频黄频| 中文字幕av在线有码专区| 嫩草影院入口| 91字幕亚洲| 精品久久久久久成人av| 欧美一区二区国产精品久久精品| 黄色日韩在线| 少妇人妻精品综合一区二区 | 国产三级黄色录像| 老司机在亚洲福利影院| 51国产日韩欧美| 久久精品国产亚洲av香蕉五月| 少妇的逼水好多| 嫩草影院入口| 99国产极品粉嫩在线观看| 级片在线观看| 欧美一区二区亚洲| 999久久久精品免费观看国产| 久久精品国产99精品国产亚洲性色| 久久精品国产清高在天天线| 亚洲国产日韩欧美精品在线观看 | 亚洲 国产 在线| 久久伊人香网站| 精品久久久久久久毛片微露脸| 精品不卡国产一区二区三区| 国产亚洲欧美98| 88av欧美| 岛国在线观看网站| 国产真实乱freesex| 午夜a级毛片| av专区在线播放| 日本 欧美在线| 午夜福利在线在线| xxx96com| 搡老妇女老女人老熟妇| 国产高清视频在线播放一区| 欧美另类亚洲清纯唯美| 久久久久久国产a免费观看| 免费av不卡在线播放| 欧美色欧美亚洲另类二区| 97超视频在线观看视频| 在线十欧美十亚洲十日本专区| 成人av在线播放网站| 国产伦精品一区二区三区视频9 | 亚洲精品美女久久久久99蜜臀| 岛国在线观看网站| 51国产日韩欧美| 91麻豆精品激情在线观看国产| 成人永久免费在线观看视频| 1024手机看黄色片| 久久这里只有精品中国| 欧美高清成人免费视频www| 久久久久九九精品影院| 亚洲精品日韩av片在线观看 | 丰满人妻一区二区三区视频av | 淫秽高清视频在线观看| 全区人妻精品视频| 亚洲欧美精品综合久久99| 国产三级黄色录像| 国产视频一区二区在线看| 国产视频内射| 88av欧美| 白带黄色成豆腐渣| 中文字幕精品亚洲无线码一区| 黄片小视频在线播放| 757午夜福利合集在线观看| 狂野欧美白嫩少妇大欣赏| 国产免费av片在线观看野外av| 亚洲无线观看免费| 久9热在线精品视频| 黄色女人牲交| 久久久久免费精品人妻一区二区| 毛片女人毛片| 国内揄拍国产精品人妻在线| 中文资源天堂在线| 99在线视频只有这里精品首页| 九九久久精品国产亚洲av麻豆| 国产伦在线观看视频一区| 别揉我奶头~嗯~啊~动态视频| www.色视频.com| 两性午夜刺激爽爽歪歪视频在线观看| 国产精品美女特级片免费视频播放器| 久久久久久久久大av| 国产精品乱码一区二三区的特点| 18禁黄网站禁片午夜丰满| 黄片小视频在线播放| 国产精品一区二区三区四区免费观看 | 国产黄片美女视频| 夜夜夜夜夜久久久久| 国产成人福利小说| 九九在线视频观看精品| 亚洲欧美精品综合久久99|