Primary cilia act as crucial regulators of embryo development and tissue homeostasis. They are instrumental for modulation of several signaling pathways, including Hedgehog, WNT, and TGF-β. However, gaps exist in our understanding of how cilia formation and function is regulated. Recent work has implicated WNT/β-catenin signaling pathway in the regulation of ciliogenesis, yet the results are conflicting. One model suggests that WNT/β-catenin signaling negatively regulates cilia formation, possibly via effects on cell cycle. In contrast, second model proposes a positive role of WNT/β-catenin signaling on cilia formation, mediated by the re-arrangement of centriolar satellites in response to phosphorylation of the key component of WNT/β-catenin pathway, β-catenin. To clarify these discrepancies, we investigated possible regulation of primary cilia by the WNT/β-catenin pathway in cell lines (RPE-1, NIH3T3, and HEK293) commonly used to study ciliogenesis. We used WNT3a to activate or LGK974 to block the pathway, and examined initiation of ciliogenesis, cilium length, and percentage of ciliated cells. We show that the treatment by WNT3a has no- or lesser inhibitory effect on cilia formation. Importantly, the inhibition of secretion of endogenous WNT ligands using LGK974 blocks WNT signaling but does not affect ciliogenesis. Finally, using knock-out cells for key WNT pathway components, namely DVL1/2/3, LRP5/6, or AXIN1/2 we show that neither activation nor deactivation of the WNT/β-catenin pathway affects the process of ciliogenesis. These results suggest that WNT/β-catenin-mediated signaling is not generally required for efficient cilia formation. In fact, activation of the WNT/β-catenin pathway in some systems seems to moderately suppress ciliogenesis.

Department of Histology and Embryology Faculty of Medicine Masaryk University Brno Czechia

Section of Animal Physiology and Immunology Department of Experimental Biology Faculty of Science Masaryk University Brno Czechia

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Angers S., Moon R. T. (2009). Proximal events in Wnt signal transduction. PubMed DOI

Anvarian Z., Mykytyn K., Mukhopadhyay S., Pedersen L. B., Christensen S. T. (2019). Cellular signalling by primary cilia in development, organ function and disease. PubMed DOI PMC

Badura L., Swanson T., Adamowicz W., Adams J., Cianfrogna J., Fisher K., et al. (2007). An inhibitor of casein kinase Iε induces phase delays in circadian rhythms under free-running and entrained conditions. PubMed DOI

Baker J. C., Beddington R. S. P., Harland R. M. (1999). Wnt signaling in PubMed DOI PMC

Balmer S., Dussert A., Collu G. M., Benitez E., Iomini C., Mlodzik M. (2015). Components of intraflagellar transport complex a function independently of the cilium to regulate canonical Wnt signaling in Drosophila. PubMed DOI PMC

Bangs F., Anderson K. V. (2017). Primary cilia and mammalian hedgehog signaling. PubMed DOI PMC

Barrow J. R., Thomas K. R., Boussadia-Zahui O., Moore R., Kemler R., Capecchi M. R., et al. (2003). Ectodermal Wnt3β-catenin signaling is required for the establishment and maintenance of the apical ectodermal ridge. PubMed DOI PMC

Behrens J., Von Kries J. P., Kühl M., Bruhn L., Wedlich D., Grosschedl R., et al. (1996). Functional interaction of β-catenin with the transcription factor LEF- 1. PubMed DOI

Berbari N. F., Sharma N., Malarkey E. B., Pieczynski J. N., Boddu R., Gaertig J., et al. (2013). Microtubule modifications and stability are altered by cilia perturbation and in cystic kidney disease. PubMed DOI PMC

Bernatik O., Pejskova P., Vyslouzil D., Hanakova K., Zdrahal Z., Cajanek L. (2020). Phosphorylation of multiple proteins involved in ciliogenesis by Tau Tubulin kinase 2. PubMed DOI PMC

Bernatik O., Sri Ganji R., Dijksterhuis J. P., Konik P., Cervenka I., Polonio T., et al. (2011). Sequential activation and inactivation of dishevelled in the Wnt/β-catenin pathway by casein kinases. PubMed DOI PMC

Bosakova M. K., Varecha M., Hampl M., Duran I., Nita A., Buchtova M., et al. (2018). Regulation of ciliary function by fibroblast growth factor signaling identifies FGFR3-related disorders achondroplasia and thanatophoric dysplasia as ciliopathies. PubMed DOI PMC

Bryja V., Červenka I., Čajánek L. (2017). The connections of Wnt pathway components with cell cycle and centrosome: side effects or a hidden logic? PubMed DOI PMC

Bryja V., Schulte G., Arenas E. (2007). Wnt-3a utilizes a novel low dose and rapid pathway that does not require casein kinase 1-mediated phosphorylation of Dvl to activate β-catenin. PubMed DOI

Butler M. T., Wallingford J. B. (2017). Planar cell polarity in development and disease. PubMed DOI PMC

Čajánek L., Nigg E. A. (2014). Cep164 triggers ciliogenesis by recruiting Tau tubulin kinase 2 to the mother centriole. PubMed DOI PMC

Cantagrel V., Silhavy J. L., Bielas S. L., Swistun D., Marsh S. E., Bertrand J. Y., et al. (2008). Mutations in the cilia gene ARL13B lead to the classical form of joubert syndrome. PubMed DOI PMC

Carvajal-Gonzalez J. M., Mulero-Navarro S., Mlodzik M. (2016). Centriole positioning in epithelial cells and its intimate relationship with planar cell polarity. PubMed DOI PMC

Caspary T., Larkins C. E., Anderson K. V. (2007). The graded response to sonic hedgehog depends on cilia architecture. PubMed DOI

Cevik S., Hori Y., Kaplan O. I., Kida K., Toivenon T., Foley-Fisher C., et al. (2010). Joubert syndrome Arl13b functions at ciliary membranes and stabilizes protein transport in Caenorhabditis elegans. PubMed DOI PMC

Chamorro M. N., Schwartz D. R., Vonica A., Brivanlou A. H., Cho K. R., Varmus H. E. (2005). FGF-20 and DKK1 are transcriptional targets of β-catenin and FGF-20 is implicated in cancer and development. PubMed DOI PMC

Cibois M., Luxardi G., Chevalier B., Thomé V., Mercey O., Zaragosi L. E., et al. (2015). BMP signalling controls the construction of vertebrate mucociliary epithelia. PubMed DOI

Conkar D., Firat-Karalar E. N. (2020). Microtubule-associated proteins and emerging links to primary cilium structure, assembly, maintenance, and disassembly. PubMed DOI

Corbit K. C., Aanstad P., Singla V., Norman A. R., Stainier D. Y. R., Reiter J. F. (2005). Vertebrate smoothened functions at the primary cilium. PubMed DOI

Corbit K. C., Shyer A. E., Dowdle W. E., Gaulden J., Singla V., Reiter J. F. (2008). Kif3a constrains β-catenin-dependent Wnt signalling through dual ciliary and non-ciliary mechanisms. PubMed DOI

Davidson G., Shen J., Huang Y. L., Su Y., Karaulanov E., Bartscherer K., et al. (2009). Cell cycle control of Wnt receptor activation. PubMed DOI

Duldulao N. A., Lee S., Sun Z. (2009). Cilia localization is essential for in vivo functions of the Joubert syndrome protein Arl13b/Scorpion. PubMed DOI PMC

Ford M. J., Yeyati P. L., Mali G. R., Keighren M. A., Waddell S. H., Mjoseng H. K., et al. (2018). A cell/cilia cycle biosensor for single-cell kinetics reveals persistence of cilia after G1/S transition is a general property in cells and mice. PubMed DOI PMC

Garcia-Gonzalo F. R., Reiter J. F. (2017). Open sesame: how transition fibers and the transition zone control ciliary composition. PubMed DOI PMC

Goetz S. C., Liem K. F., Anderson K. V. (2012). The spinocerebellar ataxia-associated gene tau tubulin kinase 2 controls the initiation of ciliogenesis. PubMed DOI PMC

Gonçalves J., Pelletier L. (2017). The ciliary transition zone: finding the pieces and assembling the gate. PubMed DOI PMC

González-Sancho J. M., Greer Y. E., Abrahams C. L., Takigawa Y., Baljinnyam B., Lee K. H., et al. (2013). Functional consequences of Wnt-induced dishevelled 2 phosphorylation in canonical and noncanonical Wnt signaling. PubMed DOI PMC

Han Y. G., Kim H. J., Dlugosz A. A., Ellison D. W., Gilbertson R. J., Alvarez-Buylla A. (2009). Dual and opposing roles of primary cilia in medulloblastoma development. PubMed DOI PMC

Hanáková K., Bernatík O., Kravec M., Micka M., Kumar J., Harnoš J., et al. (2019). Comparative phosphorylation map of Dishevelled 3 links phospho-signatures to biological outputs. PubMed DOI PMC

He K., Ma X., Xu T., Li Y., Hodge A., Zhang Q., et al. (2018). Axoneme polyglutamylation regulated by Joubert syndrome protein ARL13B controls ciliary targeting of signaling molecules. PubMed DOI PMC

Hendrix N. D., Wu R., Kuick R., Schwartz D. R., Fearon E. R., Cho K. R. (2006). Fibroblast growth factor 9 has oncogenic activity and is a downstream target of Wnt signaling in ovarian endometrioid adenocarcinomas. PubMed DOI

Hildebrandt F., Benzing T., Katsanis N. (2011). Ciliopathies. PubMed DOI PMC

Hori Y., Kobayashi T., Kikko Y., Kontani K., Katada T. (2008). Domain architecture of the atypical Arf-family GTPase Arl13b involved in cilia formation. PubMed DOI

Huang P., Schier A. F. (2009). Dampened Hedgehog signaling but normal Wnt signaling in zebrafish without cilia. PubMed DOI PMC

Huangfu D., Liu A., Rakeman A. S., Murcia N. S., Niswander L., Anderson K. V. (2003). Hedgehog signalling in the mouse requires intraflagellar transport proteins. PubMed DOI

Humphries A. C., Mlodzik M. (2018). From instruction to output: Wnt/PCP signaling in development and cancer. PubMed DOI PMC

Ishikawa H., Marshall W. F. (2017). Intraflagellar transport and ciliary dynamics. PubMed DOI PMC

Janovska P., Verner J., Kohoutek J., Bryjova L., Gregorova M., Dzimkova M., et al. (2018). Casein kinase 1 is a therapeutic target in chronic lymphocytic leukemia. PubMed DOI

Jenks A. D., Vyse S., Wong J. P., Kostaras E., Keller D., Burgoyne T., et al. (2018). Primary cilia mediate diverse kinase inhibitor resistance mechanisms in cancer. PubMed DOI PMC

Jiang X., Hao H. X., Growney J. D., Woolfenden S., Bottiglio C., Ng N., et al. (2013). Inactivating mutations of RNF43 confer Wnt dependency in pancreatic ductal adenocarcinoma. PubMed DOI PMC

Kim J., Jo H., Hong H., Kim M. H., Kim J. M., Lee J. K., et al. (2015). Actin remodelling factors control ciliogenesis by regulating YAP/TAZ activity and vesicle trafficking. PubMed DOI

Kim J. S., Crooks H., Dracheva T., Nishanian T. G., Singh B., Jen J., et al. (2002). Oncogenic β-catenin is required for bone morphogenetic protein 4 expression in human cancer cells. PubMed

Kim M., Suh Y. A., Oh J. H., Lee B. R., Kim J., Jang S. J. (2016). KIF3A binds to β-arrestin for suppressing Wnt/β-catenin signalling independently of primary cilia in lung cancer. PubMed DOI PMC

Komatsu Y., Kaartinen V., Mishina Y. (2011). Cell cycle arrest in node cells governs ciliogenesis at the node to break left-right symmetry. PubMed DOI PMC

Kratochwil K., Galceran J., Tontsch S., Roth W., Grosschedl R. (2002). FGF4, a direct target of LEF1 and Wnt signaling, can rescue the arrest of tooth organogenesis in Lef1-/- mice. PubMed DOI PMC

Kyun M. L., Kim S. O., Lee H. G., Hwang J. A., Hwang J., Soung N. K., et al. (2020). Wnt3a stimulation promotes primary ciliogenesis through β-catenin phosphorylation-induced reorganization of centriolar satellites. PubMed DOI

Lancaster M. A., Schroth J., Gleeson J. G. (2011). Subcellular spatial regulation of canonical Wnt signalling at the primary cilium. PubMed DOI PMC

Lauring M. C., Zhu T., Luo W., Wu W., Yu F., Toomre D. (2019). New software for automated cilia detection in cells (ACDC). PubMed DOI PMC

Li Y., Wei Q., Zhang Y., Ling K., Hu J. (2010). The small GTPases ARL-13 and ARL-3 coordinate intraflagellar transport and ciliogenesis. PubMed DOI PMC

Liu B., Chen S., Cheng D., Jing W., Helms J. A. (2014). Primary cilia integrate hedgehog and Wnt signaling during tooth development. PubMed DOI PMC

Liu C., Li Y., Semenov M., Han C., Baeg G. H., Tan Y., et al. (2002). Control of β-catenin phosphorylation/degradation by a dual-kinase mechanism. PubMed DOI

Lo C. H., Lin I. H., Yang T. T., Huang Y. C., Tanos B. E., Chou P. C., et al. (2019). Phosphorylation of CEP83 by TTBK2 is necessary for cilia initiation. PubMed DOI PMC

Löber C., Lenz-Stöppler C., Dobbelstein M. (2002). Adenovirus E1-transformed cells grow despite the continuous presence of transcriptionally active p53. PubMed DOI

Lu Q., Insinna C., Ott C., Stauffer J., Pintado P. A., Rahajeng J., et al. (2015). Early steps in primary cilium assembly require EHD1/EHD3-dependent ciliary vesicle formation. PubMed DOI PMC

May-Simera H., Kelley M. W. (2012). Planar cell polarity in the inner ear. PubMed DOI

McDermott K. M., Liu B. Y., Tlsty T. D., Pazour G. J. (2010). Primary cilia regulate branching morphogenesis during mammary gland development. PubMed DOI PMC

Mirvis M., Stearns T., Nelson W. J. (2018). Cilium structure, assembly, and disassembly regulated by the cytoskeleton. PubMed DOI PMC

Mitchison H. M., Valente E. M. (2017). Motile and non-motile cilia in human pathology: from function to phenotypes. PubMed DOI

Molenaar M., Van De Wetering M., Oosterwegel M., Peterson-Maduro J., Godsave S., Korinek V., et al. (1996). XTcf-3 transcription factor mediates β-catenin-induced axis formation in xenopus embryos. PubMed DOI

Nachury M. V. (2018). The molecular machines that traffic signaling receptors into and out of cilia. PubMed DOI PMC

Nachury M. V., Mick D. U. (2019). Establishing and regulating the composition of cilia for signal transduction. PubMed DOI PMC

Naik S., Piwnica-Worms D. (2007). Real-time imaging of β-catenin dynamics in cells and living mice. PubMed DOI PMC

Nakagawa N., Li J., Yabuno-Nakagawa K., Eom T. Y., Cowles M., Mapp T., et al. (2017). APC sets the Wnt tone necessary for cerebral cortical progenitor development. PubMed DOI PMC

Neugebauer J. M., Amack J. D., Peterson A. G., Bisgrove B. W., Yost H. J. (2009). FGF signalling during embryo development regulates cilia length in diverse epithelia. PubMed DOI PMC

Niehrs C., Acebron S. P. (2012). Mitotic and mitogenic Wnt signalling. PubMed DOI PMC

Nusse R., Clevers H. (2017). Wnt/β-catenin signaling, disease, and emerging therapeutic modalities. PubMed DOI

Ocbina P. J. R., Tuson M., Anderson K. V. (2009). Primary cilia are not required for normal canonical Wnt signaling in the mouse embryo. PubMed DOI PMC

Oda T., Chiba S., Nagai T., Mizuno K. (2014). Binding to Cep164, but not EB1, is essential for centriolar localization of TTBK2 and its function in ciliogenesis. PubMed DOI

Paclíková P., Bernatík O., Radaszkiewicz T. W., Bryja V. (2017). The N-terminal part of the dishevelled DEP domain is required for Wnt/β-catenin signaling in mammalian cells. PubMed DOI PMC

Park T. J., Mitchell B. J., Abitua P. B., Kintner C., Wallingford J. B. (2008). Dishevelled controls apical docking and planar polarization of basal bodies in ciliated epithelial cells. PubMed DOI PMC

Patnaik S. R., Kretschmer V., Brücker L., Schneider S., Volz A. K., Oancea-Castillo L., et al. (2019). Bardet–Biedl Syndrome proteins regulate cilia disassembly during tissue maturation. PubMed DOI PMC

Pejskova P., Reilly M. L., Bino L., Bernatik O., Dolanska L., Ganji R. S., et al. (2020). KIF14 controls ciliogenesis via regulation of Aurora A and is important for Hedgehog signaling. PubMed DOI PMC

Pinson K. I., Brennan J., Monkley S., Avery B. J., Skarnes W. C. (2000). An LDL-receptor-related protein mediates Wnt signalling in mice. PubMed DOI

Piperno G., LeDizet M., Chang X. J. (1987). Microtubules containing acetylated alpha-tubulin in mammalian cells in culture. PubMed DOI PMC

Pitaval A., Tseng Q., Bornens M., Théry M. (2010). Cell shape and contractility regulate ciliogenesis in cell cycle-arrested cells. PubMed DOI PMC

Pugacheva E. N., Jablonski S. A., Hartman T. R., Henske E. P., Golemis E. A. (2007). HEF1-dependent aurora a activation induces disassembly of the primary cilium. PubMed DOI PMC

Reiter J. F., Leroux M. R. (2017). Genes and molecular pathways underpinning ciliopathies. PubMed DOI PMC

Rieder C. L., Jensen C. G., Jensen L. C. W. (1979). The resorption of primary cilia during mitosis in a vertebrate (PtK1) cell line. PubMed DOI

Rohatgi R., Milenkovic L., Scott M. P. (2007). Patched1 regulates hedgehog signaling at the primary cilium. PubMed DOI

Sampilo N. F., Stepicheva N. A., Zaidi S. A. M., Wang L., Wu W., Wikramanayake A., et al. (2018). Inhibition of microRNA suppression of dishevelled results in Wnt pathway-associated developmental defects in sea urchin. PubMed DOI PMC

Schindelin J., Arganda-Carreras I., Frise E., Kaynig V., Longair M., Pietzsch T., et al. (2012). Fiji: an open-source platform for biological-image analysis. PubMed DOI PMC

Schmidt K. N., Kuhns S., Neuner A., Hub B., Zentgraf H., Pereira G. (2012). Cep164 mediates vesicular docking to the mother centriole during early steps of ciliogenesis. PubMed DOI PMC

Seeley E. S., Nachury M. V. (2010). The perennial organelle: assembly and disassembly of the primary cilium. PubMed DOI PMC

Shimokawa T., Furukawa Y., Sakai M., Li M., Miwa N., Lin Y. M., et al. (2003). Involvement of the FGF18 gene in colorectal carcinogenesis, as a novel downstream target of the β-catenin/T-cell factor complex. PubMed

Shnitsar I., Bashkurov M., Masson G. R., Ogunjimi A. A., Mosessian S., Cabeza E. A., et al. (2015). PTEN regulates cilia through dishevelled. PubMed DOI PMC

Shu W., Guttentag S., Wang Z., Andl T., Ballard P., Lu M. M., et al. (2005). Wnt/β-catenin signaling acts upstream of N-myc, BMP4, and FGF signaling to regulate proximal-distal patterning in the lung. PubMed DOI

Sokol S. Y. (1996). Analysis of dishevelled signalling pathways during PubMed DOI

Sorokin S. (1962). Centrioles and the formation of rudimentary cilia by fibroblasts and smooth muscle cells. PubMed DOI PMC

Spektor A., Tsang W. Y., Khoo D., Dynlacht B. D. (2007). Cep97 and CP110 suppress a cilia assembly program. PubMed DOI

Steinhart Z., Angers S. (2018). Wnt signaling in development and tissue homeostasis. PubMed DOI

Stepanenko A. A., Dmitrenko V. V. (2015). HEK293 in cell biology and cancer research: phenotype, karyotype, tumorigenicity, and stress-induced genome-phenotype evolution. PubMed DOI

Tamai K., Semenov M., Kato Y., Spokony R., Liu C., Katsuyama Y., et al. (2000). LDL-receptor-related proteins in Wnt signal transduction. PubMed DOI

Tamai K., Zeng X., Liu C., Zhang X., Harada Y., Chang Z., et al. (2004). A Mechanism for Wnt coreceptor activation. PubMed DOI

Tucker R. W., Pardee A. B., Fujiwara K. (1979). Centriole ciliation is related to quiescence and DNA synthesis in 3T3 cells. PubMed DOI

Vertii A., Bright A., Delaval B., Hehnly H., Doxsey S. (2015). New frontiers: discovering cilia-independent functions of cilia proteins. PubMed DOI PMC

Vora S. M., Fassler J. S., Phillips B. T. (2020). Centrosomes are required for proper β-catenin processing and Wnt response. PubMed DOI PMC

Wallingford J. B., Mitchell B. (2011). Strange as it may seem: the many links between Wnt signaling, planar cell polarity, and cilia. PubMed DOI PMC

Wallingford J. B., Rowning B. A., Vogell K. M., Rothbächer U., Fraser S. E., Harland R. M. (2000). Dishevelled controls cell polarity during PubMed DOI

Wang L., Dynlacht B. D. (2018). The regulation of cilium assembly and disassembly in development and disease. PubMed DOI PMC

Wehrli M., Dougan S. T., Caldwell K., O’Keefe L., Schwartz S., Valzel-Ohayon D., et al. (2000). Arrow encodes an LDL-receptor-related protein essential for Wingless signalling. PubMed DOI

Westlake C. J., Baye L. M., Nachury M. V., Wright K. J., Ervin K. E., Phu L., et al. (2011). Primary cilia membrane assembly is initiated by Rab11 and transport protein particle II (TRAPPII) complex-dependent trafficking of Rabin8 to the centrosome. PubMed DOI PMC

Wiens C. J., Tong Y., Esmail M. A., Oh E., Gerdes J. M., Wang J., et al. (2010). Bardet-biedl syndrome-associated small GTPase ARL6 (BBS3) functions at or near the ciliary gate and modulates Wnt signaling. PubMed DOI PMC

Willert K., Brown J. D., Danenberg E., Duncan A. W., Weissman I. L., Reya T., et al. (2003). Wnt proteins are lipid-modified and can act as stem cell growth factors. PubMed DOI

Wong S. Y., Seol A. D., So P. L., Ermilov A. N., Bichakjian C. K., Epstein E. H., et al. (2009). Primary cilia can both mediate and suppress Hedgehog pathway-dependent tumorigenesis. PubMed DOI PMC

Wu C. T., Chen H. Y., Tang T. K. (2018). Myosin-Va is required for preciliary vesicle transportation to the mother centriole during ciliogenesis. PubMed DOI

Zhai L., Chaturvedi D., Cumberledge S. (2004). Drosophila Wnt-1 undergoes a hydrophobic modification and is targeted to lipid rafts, a process that requires porcupine. PubMed DOI

Zhan T., Rindtorff N., Boutros M. (2017). Wnt signaling in cancer. PubMed DOI PMC

Zingg D., Debbache J., Peña-Hernández R., Antunes A. T., Schaefer S. M., Cheng P. F., et al. (2018). EZH2-mediated primary cilium deconstruction drives metastatic melanoma formation. PubMed DOI

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