Fig 2: Treatment with Ex41 skip ASO induces the expression of a near full-length CEP290 protein that localises at the TZ. (a) Immunofluorescence shows that CEP290 protein is not detectable in untreated P1 and P2 cells. Treatment with 5 µM Ex41 skip ASO in serum-free medium for 48 h rescues CEP290 protein expression which localises to the base of the cilium, in P1 and P2 cells. Notably, skipping of exon 41 does not alter CEP290 protein localisation in control lines C1 and C2. Green - CEP290, Red - ARL13B, Violet - pericentrin. Scale bar 5 µm. (b) Immunofluorescence shows that treatment with 5 µM Ex41 skip ASO in serum-free medium for 48 h induces the expression of near full-length CEP290 protein that localises at the same position along the ciliary proximal-distal axis as the TZ component TMEM67. Green - CEP290, Violet - TMEM67, Red - ARL13B. Scale bar 5 µm. (c) Immunofluorescence shows that treatment with 5 µM Ex41 skip ASO in serum-free medium for 48 h induces the expression of near full-length CEP290 protein that localises at the same position along the ciliary proximal-distal axis as the TZ component AHI1. Green - CEP290, Violet - AHI1, Red - ARL13B. Scale bar 5 µm. (d) Representative immunofluorescence micrographs of control fibroblasts C1, C2 and JBTS5 cells P1 and P2. PCM-1 localisation at centriolar satellites is not altered in the absence of full-length CEP290. Green - CEP290, Violet - PCM-1, Red - ARL13B. Scale bar 5 µm.

Fig 3: Validation of C. elegans predictions of TMEM67 VUS pathogenicity in cell culture. (A) Schematic summarizes the genetic complementation assay in hTERT-RPE1 TMEM67 knock-out cells. Left: in the presence of TMEM67, phosphorylation of the co-receptor ROR2 is stimulated by exogenous treatment with the non-canonical ligand Wnt5a in comparison to control treatment. Right: if TMEM67 is lost or disrupted, ROR2 phosphorylation is not stimulated by this treatment. (B) Western blots of ROR2, with upper phosphorylated isoform indicated (top panel), following transfection and expression (bottom panel) of TMEM67 constructs (wild-type, empty vector negative control, known benign variant control, known pathogenic variant control, and a selection of VUS alleles). Transfected TMEM67-/- knock-out cells were treated with control conditioned medium (-) or Wnt5a-conditioned medium (+). Loading control for normalization is \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} }{}$\beta$\end{document}-actin. (C) Densitometry scans of the phosphorylated ROR2 isoform (for n = 3 biological replicates) were quantitated in the bar graph for percentage induction of Wnt5a-stimulated response compared to control response, normalized against responses to the wild-type TMEM67 construct. Statistical significance was determined in pairwise t-tests with wild-type (#) for a minimum of n = 3 biological replicates. P-values are listed, n.s., not significant. Error bars indicate standard error of the mean. Statistical significance is not included for Benign(D359Q) and Pathogenic(Q376P) because these values were derived from a single biological replicate.

Fig 4: Immunohistochemical (IHC) and transcriptional analysis of rod and cone photoreceptors for embryonic and adult retina sections of WT and cilia-related mutants. (A) IHC staining of retinal cryosections of WT, and ift88, ift172, b9d2, mks1, tmem67, rpgrip1l, tctn1, tmem216, cc2d2a, cep290, nphp1 and nphp4 mutant embryos at 5 dpf. Dorsal views are shown. Images of IHC staining of cone photoreceptors with Zpr1 (green; inner segment), PNA (red; outer segment) and DAPI (blue; nuclei) in column 1. Higher-magnification images are in column 2. Images of IHC staining of rod photoreceptors with Zpr3 (red; rhodopsin – outer segment if trafficked properly) and nuclei with DAPI (blue) are in column 3. Higher-magnification images are in column 4. Scale bars: 50 µm (columns 1 and 3); 25 µm (columns 2 and 4). (B) The table depicting retinal defects observed in mutant embryos. ift172-/- and ift88-/- results are highlighted by a red box. (C) K-means clustering of log2 normalized counts of the 65 retinal genes. The experiment workflow for how we defined these 65 genes is shown in Fig. S9A. Colored sidebars represent different clustering groups (yellow, blue, red and orange). (D) Representative cryosectioned 3-month-old adult retinae of WT, and viable b9d2, cep290, tmem67, rpgrip1l, tctn1, nphp1 and nphp4 mutants stained with Zpr1/PNA (green/red; column 1), Zpr3 (red; column 2), Pcna (proliferating cells; red; column 3) and L-plastin/4C4 (green; pan-leukocyte marker/red; microglia marker; column 4). Scale bars: 100 µM.

Fig 5: The maintenance of Cep290 by FSD1 at CS is required for TZ assembly. a Non-ciliated (+Serum) or ciliated (-Serum) RPE-1 cells were stained with indicated antibodies as shown. Scale bars, 5 µm (main image) and 1 µm (magnified region). b Effects of FSD1 depletion on the localization of Cep290 at centriolar satellites in cycling cells. RPE-1 cells transfected with control or FSD1 siRNAs were stained with anti-Cep290 (green) and anti-Centrin2 (red) antibodies. Scale bars, 5 µm (main image) and 1 µm (magnified region). c Effects of FSD1 depletion on the PCM1 localization at centriolar satellites in cycling RPE-1 cells. RPE-1 cells transfected with control or FSD1 siRNAs were stained with indicated antibodies. Scale bars, 5 µm (main image) and 1 µm (magnified region). d Effects of FSD1 or PCM1 depletion on the Cep290 localization at centriolar satellites in cycling RPE-1 cells. e, f Effects of FSD1 or PCM1 depletion on the relative intensity of the transition zone components TMEM67 (e) and NPHP8 (f) at mother centrioles in quiescent cells. g Model accounting for the TZ assembly defects caused by FSD1 depletion due to the disruption of CS Cep290. TZ transition zone, CS centriolar satellite. Data are presented as mean ± s.d. of three independent experiments. n number of cells. In all panels, statistical comparisons between two groups were carried out by two-tailed t-test. **P < 0.01, ***P < 0.001