Toriyama M , Lee C , Taylor SP , Duran I , Cohn DH , Bruel AL , Tabler JM , Drew K , Kelly MR , Kim S , Park TJ , Braun DA , Pierquin G , Biver A , Wagner K , Malfroot A , Panigrahi I , Franco B , Al-Lami HA , Yeung Y , Choi YJ , University of Washington Center for Mendelian Genomics , Duffourd Y , Faivre L , Rivière JB , Chen J , Liu KJ , Marcotte EM , Hildebrandt F , Thauvin-Robinet C , Krakow D , Jackson PK , Wallingford JB .

UC2 HL103010 NHLBI NIH HHS , UM1 HG006504 NHGRI NIH HHS , RC2 HL102926 NHLBI NIH HHS , DP1 GM106408 NIGMS NIH HHS , Howard Hughes Medical Institute , UL1 TR000124 NCATS NIH HHS , R01 GM086627 NIGMS NIH HHS , RC2 HL102924 NHLBI NIH HHS , R01 DK068306 NIDDK NIH HHS , R01 GM121565 NIGMS NIH HHS , R01 GM114276 NIGMS NIH HHS , UM1 HG006493 NHGRI NIH HHS , BB/K010492/1 Biotechnology and Biological Sciences Research Council , RC2 HL103010 NHLBI NIH HHS , R01 AR066124 NIAMS NIH HHS , R01 AR062651 NIAMS NIH HHS , MR/L017237/1 Medical Research Council , R01 GM104853 NIGMS NIH HHS , F32 GM112495 NIGMS NIH HHS , RC2 HL102923 NHLBI NIH HHS , U54 HG006493 NHGRI NIH HHS , UC2 HL102926 NHLBI NIH HHS , R01 AR061485 NIAMS NIH HHS , R01 HL117164 NHLBI NIH HHS , UC2 HL102923 NHLBI NIH HHS , UC2 HL102924 NHLBI NIH HHS , RC2 HL102925 NHLBI NIH HHS , UC2 HL102925 NHLBI NIH HHS

                Joubert syndrome 17

           JOUBERT SYNDROME 17; JBTS17

	Figure 2. (a) RT–PCR demonstrates disrupted jbts17 splicing after MO injection (Jbts17-KD). odc1 is the loading control. (b) In situ hybridization of the Sonic Hedgehog (SHH) direct target nkx2.2 in control embryos and ones with Jbts17 knockdown (stage 22). Scale bars, 2 mm. Fractions represent the fraction of embryos displaying the phenotype. (c) Expression of pitx2, which labels the left lateral plate, at stage 26. Arrows indicate signal in left lateral plate mesoderm (LPM); the graph represents pitx2 expression patterns in embryos. Scale bars, 1 mm. (d) Immunostaining for acetylated α-tubulin, which labels cilia, shows ciliogenesis defects after Jbts17 knockdown in the ventral neural tube (stage 22). Scale bars, 10 μm. (e) Immunostaining for acetylated α-tubulin shows that Jbts17 knockdown reduces cilia length; cilia numbers are unchanged. Scale bars, 10 μm; membrane-RFP labels membranes. The graphs in d and e each show pooled data from two independent experiments for cilia length (shown as means ± s.e.m.; ***P < 0.001). (f) MCCs from control embryos, embryos with Jbts17 knockdown showing disrupted cilia, and Jbts17-knockdown embryos rescued with untargeted jbts17 mRNA labeled by GFP-Cfap20 (green); membrane-RFP labels membranes. Scale bars, 10 μm. (g) GFP-tagged Jbts17 localizes near basal bodies (visualized with coexpressed Cetn4-RFP) in an MCC. Scale bar, 10 μm. (h) Super-resolution image of GFP-Jbts17 and mCherry-Cep164 at a single basal body; both form rings of ~260 nm in diameter around the basal body, visualized by Cetn4-BFP. Diameters are shown as means ± s.d. in each panel. The graph shows fluorescence intensities (in arbitrary units) for GFP-Jbts17, mCherry-Cep164 and Cetn4-BFP. Scale bar, 100 nm. (i) GFP-tagged CPLANE proteins (green) and basal bodies visualized by Cetn4-RFP (magenta) in control and Jbts17-knockdown MCCs. Scale bars, 1 μm. In box plots of CPLANE fluorescence intensities at basal bodies, boxes extend from the 25th to the 75th percentile, with a line at the median; whiskers indicate maximum and minimum values. **P = 0.0041, ***P < 0.001; NS, not significant. Con, control. (j) Table summarizing the localization of CPLANE proteins at basal bodies for each knockdown (Supplementary Fig. 2c–f).
	Figure 3: Jbts17 is necessary for recruitment of peripheral IFT-A proteins to basal bodies.(a) Ift43 localization at basal bodies in Xenopus MCCs, as marked by Cetn4-RFP, is lost in MCCs after Jbts17 knockdown. Scale bars, 10 μm. (b) Peripheral IFT-A components are not recruited to Cetn4-RFP-labeled basal bodies after Jbts17 knockdown. IFT-A components are fused to GFP. Scale bars, 1 μm. (c) Quantification of IFT protein localization to basal bodies from two independent experiments. Box plots show fluorescence intensities of GFP fusions to indicated IFT proteins normalized against the intensity of Cetn4-RFP (Online Methods). Peripheral IFT-A proteins are specifically lost after Jbts17 knockdown. **P = 0.0013, ***P < 0.001; NS, not significant.
	Figure 6: CPLANE gene mutations in human ciliopathies. (a) Pedigree showing WDPCP mutations in a patient with OFD. (b) The patient displays tongue hamartomas and dental anomalies. (c) When expressed in Xenopus embryos, the allele of human WDPCP encoding Asp54Ala produces less protein than the wild-type allele; the allele encoding Leu176Phefs*23 produces no protein. (d) Pedigree showing INTU mutations in an individual with SRPS. (e) X-ray of the affected individual. (f) Wild-type Xenopus Intu localizes to basal bodies, but the Xenopus cognate of human INTU Glu355* (Gln361*) fails to localize to basal bodies. Scale bars, 10 μm. (g,h) Expression of Xenopus Intu rescues Ift43 localization to basal bodies after Intu knockdown, but the Xenopus cognate of human INTU Glu550Ala (Xenopus Intu Glu506Ala; see Supplementary Fig. 6f) does not. Scale bars, 1 μm. Data shown in h are pooled from three independent experiments. *P < 0.05, ***P < 0.001; NS, not significant.
	Figure 3. Jbts17 is necessary for recruitment of peripheral IFT-A proteins to basal bodies(a) Ift43 localization at basal bodies in Xenopus MCCs as marked by centrin4-RFP is lost in MCCs after Jbts17 knockdown. (b) Peripheral IFT-A components are not recruited to centrin4-RFP labeled basal bodies after Jbts17 knockdown. (c) Quantification of IFT protein localization to basal bodies from two independent experiments. Graphs show fluorescence intensity of GFP fusions to indicated IFT proteins normalized against that of centrin4-RFP (see methods). Peripheral IFT-A proteins are specifically lost after Jbts17 knockdown.
	Figure 4. Jbts17 is required for bi-directional axonemal transport of IFT-B particles, but not the core IFT-AStill images from high-speed time-lapse movies of IFT using GFP fusion to IFT proteins (green) and membrane-RFP (magenta). Cluap1-GFP in control embryo (a) and Jbts17 morphant (b). GFP-IFT144 in control (c) and Jbts17 morphant (d). Insets show high magnification views of localization of IFT particles in a single axoneme in the boxed regions. Scale bars = 10 μm. Associated kymograph representing movements of IFT particles are shown in panels a’-d’.
	Figure 5. CPLANE mutant mice display diagnostic features of Oral-Facial-Digital Syndrome Type 6(a and b) Frontal sections at E14.5 reveal that Fuz mutant mice display high arched palate (arrow) and lobulation of the tongue (arrowheads). (c and d) Fuz mutant mice display develop polydactyly with Y-shaped metacarpals. (e) Wdpcp mutant mice display develop polydactyly with Y-shaped metacarpals. (f, g, h) Frontal sections of E13.5 Wildtype, Wdpcp and Fuz mutant embryos. DAPI labels nuclei (cyan). (f’, g’, h’) Illustrations highlighting the corresponding palatal condensations (purple) and tongue (pink) in F-H. Mutant palatal condensations form more medially than do controls and fail to extend into the mouth (See Tabler et al., 2013).
	Figure 6. CPLANE mutations in human ciliopathies(a) Pedigree showing WDPCP mutations in an OFD patient. (b) The patient displays tongue hamartomas and dental anomalies. (c) When expressed in Xenopus embryos, the D54A allele of human WDPCP produces less protein compared to wild-type; the L176F-fs26* allele produces no protein. (d) Pedigree showing INTU mutations in an SRPS phenotype. (e) X-ray of the patient. (f) The E355* allele of INTU disrupts basal body localization. (g and h) Wild-type Intu rescues Ift43 localization to basal bodies after Intu knockdown; the E500A allele of Intu does not. Data shown are pooled from three independent experiments.
	Figure 7. Models for CPLANE function and structure(a) Schematic of normal IFT. Peripheral proteins are assembled onto the IFT-A core in the cytoplasm and injected together with IFT-B for bi-direction transport in axonemes. (b) In the absence of CPLANE, IFT-A core particles lacking peripheral proteins are injected into axonemes and traffic normally; IFT-B enters axonemes but fails to move in a retrograde direction and accumulates.
	Supplementary Figure 1 CPLANE pulldowns. (a) Tandem affinity purification of Inturned interacting proteins. Lysates from murine IMCD3 collecting duct cells stably expressing LAP-Intu were subjected to tandem affinity purification and silver stain to visualize major interacting species. Molecular mass markers are indicated on the left. (b) Tandem affinity purification of interacting proteins for Inturned, Fuz and Wdpcp. “s” indicates the tagged proteins. Molecular mass markers are indicated on the left. The calculated molecular masses for these proteins are: mIntu, 104.8 kDa; mFuz, 45.5 kDa; mWdpcp, 81.7 kDa. (c-e) Tables showing an extracted subset of data from Supplementary Data Set 1 and highlighting key findings reported here. Numbers indicate peptide spectral matches for preys retrieved after pulldowns with indicated baits. The “Other Ciliopathy” column reflects combined data from pulldowns of Ahi1, Cep290, Invs, Iqcb1 and Nphp4.
	Supplementary Figure 2 CPLANE interactors. (a) Venn diagram showing overlap between the interactomes of Intu, Fuz, and Wdpcp. The intersection of the three (“combined CPLANE interactome”) contains ~250 proteins. (b) Additional components of the extended CPLANE protein network built using data from tandem affinity purification of Intu, Fuz, Wdpcp, IFT-A, and the published NPHP network and thresholded for most likely network members. (c) The CPLANE proteins associate with IFT-A proteins and poorly characterized cilia-related proteins (arrows indicates baitprey in Fuz, Wdpcp and Intu pulldowns). (d) The core CPLANE complex was identified by reciprocal pulldowns. (e) Co-IP with in vitro translated proteins confirms CPLANE interactions with Jbts17 (a fragment of Jbts17 was used, as the very large size of this protein made in vitro translation intractable).
	Supplementary Figure 3 Basal body localization of CPLANE proteins. (a) Dorsal views of stage 19 Xenopus embryos show neural tube closure defects after Jbts17-knockdown that are rescued by expression of Jbst17-wild-type but not the Joubert-associated truncation (R1569*). The graph shows average distance between neural folds. (b) In situ hybridization to stage 30 Xenopus embryos shows loss of vax1, a gene downstream of sonic hedgehog, after Jbts17 knockdown; numbers in each panel indicate the number of embryos showing the phenotype. (c-f) Fluorescence micrographs show the localization of CPLANE proteins at basal bodies in Inturned (c), Fuzzy (d), Wdpcp (e) or Rsg1 (f) knockdown multi-ciliated cells. Green and red signals indicate GFP-tagged CPLANE proteins and centrin4-RFP, respectively. The graphs to the right in each figure show fluorescence intensity of GFP signals normalized against Centrin4-RFP (ns, non-significant; **P < 0.01; *** P < 0.001). (g) Fluorescence micrographs show the localization GFP-tagged Cep164, Ofd1, Hook2 and Mks1 in multi-ciliated cells in controls and after Jbts17 knockdown. The graph at right shows the normalized fluorescence intensity for the indicated proteins, as described for c-f. (h) CRISPR disruption of Jbts17 phenocopies MO knockdown. Gels at left demonstrate targeting of Jbts17 (see methods for details); images at right show disruption of ciliogenesis in MCCs.
	Supplementary Figure 4 Effect of a Joubert-associated CPLANE allele. (a) Jbts17 truncated mutants and fragments construct used in this study. (b) Fluorescence images in multi-ciliated cells expressing GFP-tagged Jbts17 wild-type, R1569*, R2406* and amino acids 1770-2318. (c,d) Fluorescence images GFP-tagged Inturned (c) and GFP-Ift43 (d) in multi-ciliated cells in control, Jbts17 knockdown, Jbst17-wild-type or truncated mutant (R1569*) expressing cells in Jbts17 knockdown embryos. The graph shows the normalized fluorescence intensity of GFP-Inturned (c) and GFP-IFT43 (d) at basal bodies, as described in legend to Supplementary Figure 2. Scale bars, 10 m.
	Supplementary Figure 5 CPLANE and IFT. (a,b) Fluorescence micrographs show the localization of indicated, GFP-tagged IFT-A (a) and IFT-B (b) proteins at basal bodies in multi-ciliated cells. Bottom panels in each figure show high-magnification images of basal bodies. (c-e) Live images of multi-ciliated cells expressing GFP-tagged IFTs (green) and membrane-RFP (magenta) in control and Jbts17 morphants. (f,g) Kymographs representing movements of IFT particles in control, Jbts17 and WDPCP morphant. (f) GFP-IFT20. (g) GFP-IFT122. (h) Plot showing mean intensity of IFT protein fusions in axonemes. Scale bars, 10 m.
	Supplementary Figure 6 CPLANE mutations in human patients. (a) The D54 residue of WDPCP that is mutated in OFD is invariant from human to fish. (b) Y-shaped metacarpals in a human OFD patient with a mutation in INTU; this is patient #2 from Panigrahi et al., 2013. (c) An INTU mutation segregates with the OFD phenotype. (d) An INTU missense mutation segregates with NPHP in another family. (e) The A452 residue of INTU that is mutated in NPHP is not well conserved and may be hypomorphic, consistent with the more restricted phenotype in this patient. (f) The E500 residue of INTU that is mutated in SRP is invariant from human to fish. (g,h) Radiographs showing the phenotype of an SRP patient transheterozygous for mutations in INTU and WDR35 (Ift121), as indicated by the pedigree in panel i. (j) The W311 residue of WDR35 that is mutated in SRP is invariant from human to fish.
	Supplementary Figure 7 Models of CPLANE protein structures. (a) Fuz. (b) Wdpcp. (c) Intu. (d) Rsg1. (e) Jbts17. As outlined in the Discussion, modeling predicts similarities between CPLANE proteins and vesicle trafficking machinery.

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