Fig 2: Loss of TALPID3 (KIAA0586) causes abnormal tissue and cell polarity and abnormal intracellular organization.(A, A', A'') ß-catenin expression is localized anteriorly within feather buds of the wildtype chicken at day 9.5 and (B, B', B'') in the talpid3 chicken at day 9.5. Black circles indicate feather buds with correct polarity; dashed black circles represent no polarity; blue circles represent abnormal polarity (Schematic C). The talpid3 chicken (B'') demonstrates feather buds which lack polarity (blue circles) which is not seen in the wildtype chicken (A''). Asterisks represent merged feather buds. (C') Quantification of the percentage of feather buds with correct, abnormal or no polarity in wildtype and talpid3 (D, E) SEM of the basilar papilla in wildtype (D) and talpid3 (E) chickens. Arrows indicate cilia. Curved lines represent the base of stereocilia hair bundles. (F–K) Actin bundles identified by phalloidin (green) and centriolar localization identified by ? tubulin (red). (F', G') overview of wildtype and talpid3 basilar papilla, higher magnification in (F, G), red circles with line represent orientation of polarized actin bundles in basilar papilla; dashed red circles represent unpolarized actin bundles (Schematic L, M). (L') Quantification of polarized haircells. (M') Quantification of the angle of polarised hair cells. (H–K) Dashed white circles represent magnified images (Ji–Kii'). (Ji–Kii) White arrows indicate aligned centrosomes; blue arrows indicate unaligned centrosomes (Schematic N). (N') Quantification of cells with aligned centrosomes. (O, P) Orientation based on placement of Golgi (TGN46, red) in comparison to actin indicating the leading edge (phalloidin, green) and nucleus (Dapi, blue, schematic in Q) in MEFs. Asterisks represent areas of higher magnification (not all represented at lower magnification). (Q') Quantification of the angle of orientation of MEF cells in scratch assay. Scale Bars: A, B 5 mm; A', A'', B', B'' 1 mm; D, E 1 µm; F, G, H, I, J, K 20 µm; F', G' 100 nm; Ji, Ji', Jii, Jii', Ki, Ki', Kii, Kii' 10 µm; O, P 100 µm; Oi, Oii, Oiii, Oiv, Ov, Ovi, Pi, Pii, Piii, Piv, Pv, Pvi 25 µm.DOI: http://dx.doi.org/10.7554/eLife.08077.006

Fig 3: Analysis of potential interactions between Talpid3/TALPID3, Kif7/KIF7 and IFT57 in the mouse and in chicken.Biallelic KIF7 mutations cause JBTS type 12 in human (Dafinger et al., 2011). Although both the KIAA0586 mutation c.428delG and the KIF7 mutation c.811delG were paternally inherited in patient G2, we sought to test for subtle abnormalities resulting from this double heterozygosity. In addition, we had previously found through a microarray analysis of talpid3 limb buds that IFT57, a protein associated with ciliopathy phenotypes in mice and zebrafish, is downregulated in talpid3 embryos (Bangs et al., 2011). Using in ovo complementation of the talpid3 neural tube, we could not detect induction of ISLET1 expression or ventralized PAX7 in the wildtype or talpid3 neural tube by overexpression of KIF7 or IFT57 (Figure 1—figure supplement 1A,B). We then used siRNA constructs against KIF7 to model a heterozygous loss of KIF7 in the TALPID3+/- neural tube. Knock-down with two siRNA constructs had a weak effect on neural tube patterning compared to the mouse KIF7-/- knockout (Cheung et al., 2009, Liem et al., 2009). Although the NKX2.2 expression domain could be marginally expanded in wildtype embryos (not shown), there was no expansion of ISLET1-positive motorneuron progenitors in wildtype or TALPID3+/- embryos. PAX7, however, was weakly dorsalized in both wildtype and talpid3+/- embryos (Figure 1FC). These results suggested that some KIF7 function may be cilia-independent as has been suggested (Liem et al., 2009). To more precisely investigate for a possible epistatic relationship between Kif7 and Talpid3, particularly in the organs primarily affected in JBTS, such as the cerebellum, we undertook a Talpid3+/- × Kif7+/- mouse cross in order to determine if double Talpid3+/-/Kif7+/- heterozygous animals had brain patterning malformations. We first dissected embryos at E15.5, 16.5, and 17.5 and found that Talpid3+/-/Kif7+/- embryos were morphologically normal, including size, situs and limb patterning. MEFs derived from E12.5 embryos were normally ciliated, with the percentage of ciliated cells and cilia length comparable to those seen in wildtype, Talpid3+/+/Kif7+/- and Talpid3+/-/Kif7+/+cells (Figure 1—figure supplement 1F,G). MRI and sectioning of the brain also showed no brain patterning abnormalities (Figure 1—figure supplement 1D,E). Subsequently, Talpid3+/-/Kif7+/- animals were born and grew normally compared to their litter mates and showed no abnormal brain morphology (Figure 1—figure supplement 1). We conclude that KIAA0586 (TALPID3) and KIF7 do not act epistatically and hypothesize that additional genetic alterations in ciliopathy genes of patient G2, eventually including those identified in CEP41 (JBTS15), KIF14, and WDPCP, may contribute to a mutational load that is sufficient to elicit a JBTS phenotype on a KIAA0586+/-; KIF7+/- background. (A) Overexpression of IFT57 does not have an effect on patterning of the neural tube in the talpid3 chicken. (B) Overexpression of KIF7 does not rescue or alter neural tube patterning in the talpid3 chicken. (C) siRNA knockdown of KIF7 resulted in a weak dorsalization of PAX7 but no expansion of ISLET1. (D) Talpid3+/- Kif7+/- mice showed no gross anatomical abnormalities, neither were developmental brain defects identified through MRI (D) or histology (E). (F, G) No abnormalities were identified in either the percentage of ciliated cells (F), nor the length of cilia (G) in MEFs derived from wildtype, Talpid3+/+Kif7+/-, Talpid3+/- Kif7+/+ or Talpid3+/- Kif7+/- mice.DOI: http://dx.doi.org/10.7554/eLife.08077.004

Fig 4: The TALPID3-ANKRD26-FBF1 module plays a conserved role in mammalian cells.RPE cells were treated with negative control siRNA (siNC) or ANKRD26/TALPID3-specific siRNAs (siANKRD26 and siTALPID3, respectively) for 48 h and then subjected to indirect IF with the indicated antibodies after 24 h of serum starvation. a Compared with control cells and cells depleted of only TALPID3 or ANKRD26, cells in which ANKRD26 and TALPID3 were both depleted showed significantly enhanced ciliogenesis defects and the compromised ciliary entry of the IFT component IFT140. The percentage of ciliated cells and the relative intensity of IFT140 in the cilia are shown in the right panel. For ciliated cell ratio, n = 300 cells over three independent experiments. For IFT140 intensity, numbers of cell examined are indicated in the bars. b ANKRD26 and TALPID3 co-depletion impairs FBF1 localization on the distal centriole, but does not affect the localization of CEP164 and CEP89. Quantitative data are shown in the right panel. Numbers of cell examined are indicated in the bars or above each dataset. c ANKRD26 and TALPID3 co-depletion disrupts the recruitment of FBF1 to the distal centriole during centriole mutation but does not affect the recruitment of CEP164 and CEP89. Quantitative data are shown in the right panel. n = 300 cells over three independent experiments. All data are presented as the mean ± s.e.m. Significant differences were identified by two-tailed unpaired Student’s t test. NS, P > 0.05; ***, P < 0.001. Scale bars, 2 µm. Source data are provided as a Source Data file.

Fig 5: ORF10 downregulates cilium-related proteins. (A) Volcano plots of differentially expressed proteins (DEPs) were detected in a quantitative proteomic analysis of ORF10-expressing and empty vector control HEK293T cells. The red spots and blue spots indicate significantly upregulated and downregulated proteins, respectively. (B) Functional classification analysis of DEPs according to enrichment analysis of the biological process (BP) and cellular component (CC) terms and KEGG pathways. (C) The network of DEPs and enriched cilium-related gene ontology terms. (D) Cells expressing ORF10 display reduced accumulation of multiple cilium-related proteins. Immunoblotting analysis of TALPID3, TTBK2, BBS4, SEPTIN2, IFT46, and ORF10 levels in ORF10-expressing and empty vector (NC) control HEK293T cells. GAPDH served as a loading control. (E) Odyssey-based quantification of protein levels in D (n = 3 independent experiments). Data are presented as the mean ± SEM. ** indicated P < 0.01, *** indicated P < 0.001. Statistical analysis was performed with two-tailed unpaired Student’s t test. Source data are available for this figure: SourceData F2.