Cep290 disruption causes ciliopathy in zebrafish. (A) RT-PCR from representative single wild-type (WT) and cep290ex25 morphant embryos. Cep290 morpholino oligonucleotide targeted to the exon 25 splice donor caused mispliced RNAs and intron retention. (B) Chromatogram of a CRISPR/Cas9-generated single representative genetic cep290 mutant showing the 10-bp deletion in exon 16 that results in a premature stop codon. (C) Morphology of an uninjected embryo compared to cep290ex25 and cep290ATG morphants at 2 dpf. (D) Morphology of wild-type and MZcep290fb208/fb208 mutants (hereafter called MZcep290−/− mutants) at 3 dpf. (E) Western blot showing loss of Cep290CT immunoreactivity in cep290ATG and cep290ex25 morphants (pool of 40 48-hpf embryos). The position of the 250-kDa molecular weight marker is indicated in red. A tubulin antibody was used as a loading control (bottom). (F) Western blot showing loss of Cep290CT and Cep290NT immunoreactivity in MZcep290−/− mutants. A tubulin antibody was used as a loading control. Experiments were replicated a minimum of three times.

Functional loss of Cep290 in zebrafish morphants and mutant embryos. (A-C) Cep290CT antibody staining was lost in pronephric tubule multicilia bundles (A), at the base of nose multicilia (B) and in photoreceptor, outer and inner plexiform retina layers (C) upon injection of the cep290ex25 morpholino or in MZcep290^−/− mutants. Loss of Cep290 expression caused an alteration of retina lamination. No effect was observed on pronephros and olfactory placode cilia structure. (D) Kupffer's vesicle cilia were significantly shorter in 10ss cep290ex25 morphant embryos compared to stage-matched embryos injected with control morpholino. By contrast, 10ss MZcep290 mutant embryos did not show shorter Kupffer's vesicle cilia compared with heterozygous sibling embryos. Images are representative of >8 embryos/biological replicates. Experiments were replicated a minimum of three times. Scale bars: 10 µm (A); 5 µm (B); 20 µm (C); 2 µm (D).

Cep290 morphants and mutants exhibit photoreceptor outer segment length defects. (A-D) BodipyTR (red) staining showed a reduction of photoreceptor outer segment length of 4-dpf cep290ex25 morphants compared with age-matched control morpholino-injected embryos (A,B). MZcep290 mutants also showed a disorganization of photoreceptor outer segment structure at 6 dpf (C,D). (E,F) Electron micrographs of 3-dpf wild-type and cep290ex25 morphant photoreceptors. Although outer segments were found in wild-type retinas at this stage, very few outer segments were observed in cep290ex25 morphants. In some cases, basal bodies and short axonemes could be seen projecting from the cell surface (arrow in F). (G,H) Electron micrographs of 6-dpf heterozygous sibling and MZcep290 mutant photoreceptors showed an abnormal accumulation of cytoplasmic vesicles at the base of the connecting cilium (arrows in H). (I-L) Photoreceptor structure alteration persists until adulthood. Methylene Blue (I,K) and BodipyTR (J,L) staining showed an alteration of photoreceptor outer segment structure and retina lamination in MZcep290^−/− adult (1 year old) retinas. Images are representative of >8 embryos/biological replicates. INL, inner nuclear layer; ONL, outer nuclear layer; PR, photoreceptor. Experiments were replicated a minimum of three times. Scale bars: 5 µm (A-D); 50 nm (E-H).

MZcep290^−/− mutants present milder phenotypes that recover after 4-5 dpf. (A) Clutch of cep290 maternal zygotic cep290 mutants. Body shape curvature seen at 3 dpf was lost at 7 dpf (n=100). (B) Maternal zygotic cep290 mutants reach adulthood and can present scoliosis (15-40% of adult fish; n=31). (C) RT-PCR from MZcep290^−/− and wild-type embryos. No exon skipping around exon 16 was detected in mutant embryos. (D) RT-qPCR from adult eye and kidney samples (n=3). Specific wild-type (WT) and mutant primers for the targeted exon 16 were used to quantify wild-type and mutated cep290 mRNA. As expected, wild-type primers did not generate an RT-PCR product with mutant mRNA samples relative to wild-type mRNA, whereas mutant primers generated significant product with mutant mRNA samples relative to wild-type mRNA samples. Primers for ex3-ex5, ex19-ex20 and ex50-52 were used to quantify nonsense-mediated decay. No changes in cep290 mRNA levels were detected in MZcep290^−/− mutant samples compared with controls. n=3 biological replicates. Data are mean±s.d. Experiments were replicated at a minimum three times. *P<0.05; ****P<0.0001; ns, not significant (unpaired t-test).

arl3, arl13b and unc119b are upregulated in MZcep290 mutants. (A) RNAseq workflow for the identification of candidate cep290 compensating genes. RNAseq gene expression analysis was performed with 3-dpf cep290ex25 morphants, cep290 mutants and sibling wild-type (WT) embryos (NCBI GEO accession GSE175491), with three biological replicates. Upregulated genes in each group were identified, and genes specifically upregulated in MZcep290−/− mutants were selected for further analysis. MZcep290 upregulated genes were compared to a cilia proteome database to identify candidate cep290 compensating cilia-related genes. (B) RNAseq results showing upregulation of cilia-related genes arl3, arl13b and unc119b in 72-hpf embryos. (C) RT-qPCR quantification of arl3, arl13b and unc119b from MZcep290 mutant adult eye samples. n=3 biological replicates per condition; technical triplicates. (D) The upregulation of arl3, arl13b and unc119b was not detected at 3 dpf in cep290ex25 morphants embryos. n=20 embryos pooled per condition; technical triplicates. (E) The upregulation of arl3, arl13b and unc119b also was not detected at 3 dpf in cep290 zygotic mutants embryos. n=20 embryos pooled per condition; technical triplicates. (F) The ciliary mutants smh (ccdc103) and oval (ift88) did not show upregulation of arl3, arl13b and unc119b at 3 dpf. n=20 embryos pooled per condition; technical triplicates. All RT-qPCR data are expressed as fold change (2−ΔΔCt) compared with age-matched control embryos. Data are mean±s.d.

Joubert patient-derived cells also show upregulation of UNC119b. (A) RT-qPCR analysis from hURECs from JBTS CEP290 patients showed upregulation of UNC119B expression compared with control patient samples. No change in ARL3 and ARL13B gene expression level was observed. Values (dots) represent arithmetic means of −ΔΔCt values between experimental replicates (three controls, four CEP290 patients). Bars represent 95% c.i. *P<0.05; **P<0.01; ns, not significant (unpaired t-test, two-tailed). (B) In zebrafish MZcep290−/− adult kidney samples also only unc119b induction was observed. n=3 biological replicates per condition, technical triplicates. RT-qPCR data are expressed as fold change (2−ΔΔCt) compared with age-matched control samples. Data are mean±s.d.

Upregulation of small GTPases/chaperones that mediate cilia lipidated protein delivery rescue cep290ex25 morphant axoneme defects. (A) Overexpression of cilia GTPases/chaperone (arl3, arl13b and unc119b) in cep290ex25 morphant embryos partially rescued the curly axis cilia defect related phenotype. (B) arl13b overexpression was sufficient to rescue Kupffer's vesicle (KV) cilia length defects observed in stage-matched cep290ex25 morphant embryos. (C) Kupffer's vesicle cilia length was also rescued by overexpression of unc119b mRNA. (D) Injection of arl3 mRNA only partially rescued Kupffer's vesicle cilia length in cep290ex25 morphant embryos. (E) The injection of arl3, arl13b and unc119b mRNAs also was sufficient to rescue the photoreceptor outer segment length defect on cep290ex25 morphants. *P<0.01; ***P<0.001; ****P<0.0001; ns, not significant (Dunnett's or Tukey's test). y-axis values represent the average length of >70 Kupffer's vesicle cilia from 3-6 embryos per condition or of >200 OS from four embryos per condition. Data are mean±s.d.

ZFIN is incorporating published figure images and captions as part of an ongoing project. Figures from some publications have not yet been curated, or are not available for display because of copyright restrictions.

ZFIN is incorporating published figure images and captions as part of an ongoing project. Figures from some publications have not yet been curated, or are not available for display because of copyright restrictions.