Mutations in genes encoding ciliary components cause ciliopathies, but how many of these mutations disrupt ciliary function is unclear. We investigated Tectonic1 (Tctn1), a regulator of mouse Hedgehog signaling, and found that it is essential for ciliogenesis in some, but not all, tissues. Cell types that do not require Tctn1 for ciliogenesis require it to localize select membrane-associated proteins to the cilium, including Arl13b, AC3, Smoothened and Pkd2. Tctn1 forms a complex with multiple ciliopathy proteins associated with Meckel (MKS) and Joubert (JBTS) syndromes, including Mks1, Tmem216, Tmem67, Cep290, B9d1, Tctn2, and Cc2d2a. Components of the Tectonic ciliopathy complex colocalize at the transition zone, a region between the basal body and ciliary axoneme. Like Tctn1, loss of complex components Tctn2, Tmem67 or Cc2d2a causes tissue-specific defects in ciliogenesis and ciliary membrane composition. Consistent with a shared function for complex components, we identified a mutation in TCTN1 that causes JBTS. Thus, a transition zone complex of MKS and JBTS proteins regulates ciliary assembly and trafficking, suggesting that transition zone dysfunction is the cause of these ciliopathies.

Nephronophthisis (NPHP), Joubert (JBTS) and Meckel-Gruber (MKS) syndromes are autosomal-recessive ciliopathies presenting with cystic kidneys, retinal degeneration, and cerebellar/neural tube malformation. Whether defects in kidney, retinal, or neural disease primarily involve ciliary, Hedgehog, or cell polarity pathways remains unclear. Using high-confidence proteomics, we identified 850 interactors copurifying with nine NPHP/JBTS/MKS proteins, and discovered three connected modules: “NPHP1-4-8” functioning at the apical surface; “NPHP5-6” at centrosomes; and “MKS” linked to Hedgehog signaling. Assays for ciliogenesis and epithelial morphogenesis in 3D renal cultures link renal cystic disease to apical organization defects, whereas ciliary and Hedgehog pathway defects lead to retinal or neural deficits. Using 38 interactors as candidates, linkage and sequencing analysis of 250 patients identified ATXN10 and TCTN2 as new NPHP-JBTS genes and our Tctn2 mouse knockout shows neural tube and Hedgehog signaling defects. Our study further illustrates the power of linking proteomic networks and human genetics to uncover critical disease pathways.

Primary cilia are present on most mammalian cells and are implicated in transducing Hedgehog (Hh) signals during development; however, the prevalence of cilia on human tumors remains unclear, and the role of cilia in cancer has not been examined. Here we show that human basal cell carcinomas (BCCs) are frequently ciliated, and we test the role of cilia in BCC by conditionally deleting Kif3a (encoding kinesin family member 3A) or Ift88 (encoding intraflagellar transport protein 88), genes required for ciliogenesis, in two Hh pathway-dependent mouse tumor models. Ciliary ablation strongly inhibited BCC-like tumors induced by an activated form of Smoothened. In contrast, removal of cilia accelerated tumors induced by activated Gli2, a transcriptional effector of Hh signaling. These seemingly paradoxical effects are consistent with a dual role for cilia in mediating both the activation and the repression of the Hh signaling pathway. Our findings demonstrate that cilia function as unique signaling organelles that can either mediate or suppress tumorigenesis depending on the nature of the oncogenic initiating event.Elevated Hh pathway activity is associated with diverse tumors, including basal cell carcinoma, the most commonly diagnosed cancer in North America 1,2 . Hh signaling is normally restrained by the tumor suppressor Patched (Ptch1), which inhibits the function of the protooncogene Smoothened (Smo), a central activator of the pathway 3 . Binding of Hh ligand to Ptch1 relieves its inhibition of Smo, allowing Smo to induce downstream Gli activators and inhibit the formation of Gli repressors. In BCCs, loss of function mutations in PTCH1, gain of function mutations in SMO, and upregulation of GLI1 and GLI2 are frequently observed, suggesting that dysregulated Hh signaling is the underlying cause of this disease [4][5][6][7][8] .Recent studies have shown that the primary cilium has a prominent role in modulating mammalian Hh signaling 9 . Ptch1 suppresses the Hh pathway, at least in part, by preventing the trafficking of Smo into the primary cilium 10 In addition to its essential role in transducing Hh signals, the cilium also negatively regulates the pathway. Genetic analyses have shown that the cilium is required for the proteolytic processing of Gli3 into a form that represses the Hh transcriptional program (Gli3-R) [12][13][14][15] . Cilium-dependent formation of Gli3-R occurs in the absence of Hh and is suppressed upon Smo movement to the cilium. Thus, the cilium exerts both positive and negative control over the Hh pathway.Although cilia are present on many vertebrate cells, their involvement in cancer has not been explored. Early ultrastructural studies have indicated that individual cells from human BCCs can be ciliated 16 ; however, the prevalence of these ciliated cells has remained unclear. To determine whether cancer cells frequently possess cilia, we examined clinical biopsies from eight human BCCs. We observed that five BCCs contained numerous ciliated cells ( Fig. 1a and Supplementary Fig. ...

Formation of cilia, microtubule‐based structures that function in propulsion and sensation, requires Kif3a, a subunit of Kinesin II essential for intraflagellar transport (IFT). We have found that, Kif3a is also required to organize centrioles. In the absence of Kif3a, the subdistal appendages of centrioles are disorganized and lack p150Glued and Ninein. Consequently, microtubule anchoring, centriole cohesion and basal foot formation are abrogated by loss of Kif3a. Kif3a localizes to the mother centriole and interacts with the Dynactin subunit p150Glued. Depletion of p150Glued phenocopies the effects of loss of Kif3a, indicating that Kif3a recruitment of p150Glued is critical for subdistal appendage formation. The transport functions of Kif3a are dispensable for subdistal appendage organization as mutant forms of Kif3a lacking motor activity or the motor domain can restore p150Glued localization. Comparison to cells lacking Ift88 reveals that the centriolar functions of Kif3a are independent of IFT. Thus, in addition to its ciliogenic roles, Kif3a recruits p150Glued to the subdistal appendages of mother centrioles, critical for centrosomes to function as microtubule‐organizing centres.

We describe a method for the highly efficient and precise targeted modification of gene trap loci in mouse embryonic stem cells (ESCs). Through the Floxin method, gene trap mutations are reverted and new DNA sequences inserted using Cre recombinase and a shuttle vector, pFloxin. Floxin technology is applicable to the existing collection of 24,149 compatible gene trap cell lines, which should enable the high-throughput modification of many genes in mouse ESCs.