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Regulation of primary cilia formation and left-right patterning in zebrafish by a noncanonical Wnt signaling mediator, duboraya

Abstract

Primary cilia are microtubule-based organelles that project from the surface of nearly every animal cell1. Although important functions of primary cilia in morphogenesis and tissue homeostasis have been identified2,3, the mechanisms that control the formation of primary cilia are not understood. Here we characterize a zebrafish gene, termed duboraya (dub), that is essential for ciliogenesis. Knockdown of dub in zebrafish embryos results in both defects in primary cilia formation in Kupffer's vesicle and randomization of left-right organ asymmetries. We show that, at the molecular level, the function of dub in ciliogenesis is regulated by phosphorylation, which in turn depends on Frizzled-2–mediated noncanonical Wnt signaling. We also provide evidence that, at the cellular level, dub function is essential for actin organization in the cells lining Kupffer's vesicle. Taken together, our findings identify a molecular factor that links noncanonical Wnt signaling with the control of left-right axis specification, and provide an entry point for analyzing the mechanisms that regulate primary cilia formation.

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Figure 1: dub is necessary for proper left-right patterning in zebrafish embryos.
Figure 2: dub is essential for cilia development in Kupffer's vesicle and pronephric duct.
Figure 3: Ser71 and Ser94 are required for dub function.
Figure 4: frizzled-2 morphants show phenotypic similarities to dub morphants.
Figure 5: Frizzled-2–mediated noncanonical Wnt signaling regulates dub phosphorylation.
Figure 6: dub is required for actin cytoskeletal organization in Kupffer's vesicle and pronephric epithelial cells.

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References

  1. Wheatley, D.N. Primary cilia in normal and pathological tissues. Pathobiology 63, 222–238 (1995).

    Article  CAS  PubMed  Google Scholar 

  2. Rosenbaum, J.L. & Witman, G.B. Intraflagellar transport. Nat. Rev. Mol. Cell Biol. 3, 813–825 (2002).

    Article  CAS  PubMed  Google Scholar 

  3. Praetorius, H.A. & Spring, K.R. A physiological view of the primary cilium. Annu. Rev. Physiol. 67, 515–529 (2005).

    Article  CAS  PubMed  Google Scholar 

  4. Eyers, C.E. et al. The phosphorylation of CapZ-interacting protein (CapZIP) by stress-activated protein kinases triggers its dissociation from CapZ. Biochem. J. 389, 127–135 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Hammerschmidt, M. et al. Mutations affecting morphogenesis during gastrulation and tail formation in the zebrafish, Danio rerio. Development 123, 143–151 (1996).

    CAS  PubMed  Google Scholar 

  6. Veeman, M.T., Axelrod, J.D. & Moon, R.T. A second canon. Functions and mechanisms of β-catenin–independent Wnt signaling. Dev. Cell 5, 367–377 (2003).

    Article  CAS  PubMed  Google Scholar 

  7. Capdevila, J., Vogan, K.J., Tabin, C.J. & Izpisua Belmonte, J.C. Mechanisms of left-right determination in vertebrates. Cell 101, 9–21 (2000).

    Article  CAS  PubMed  Google Scholar 

  8. Raya, A. & Belmonte, J.C. Left-right asymmetry in the vertebrate embryo: from early information to higher-level integration. Nat. Rev. Genet. 7, 283–293 (2006).

    Article  CAS  PubMed  Google Scholar 

  9. Hamada, H., Meno, C., Watanabe, D. & Saijoh, Y. Establishment of vertebrate left-right asymmetry. Nat. Rev. Genet. 3, 103–113 (2002).

    Article  CAS  PubMed  Google Scholar 

  10. Bisgrove, B.W., Morelli, S.H. & Yost, H.J. Genetics of human laterality disorders: insights from vertebrate model systems. Annu. Rev. Genomics Hum. Genet. 4, 1–32 (2003).

    Article  CAS  PubMed  Google Scholar 

  11. Kawakami, Y., Raya, A., Raya, R.M., Rodriguez-Esteban, C. & Belmonte, J.C. Retinoic acid signaling links left-right asymmetric patterning and bilaterally symmetric somitogenesis in the zebrafish embryo. Nature 435, 165–171 (2005).

    Article  CAS  PubMed  Google Scholar 

  12. Essner, J.J. et al. Conserved function for embryonic nodal cilia. Nature 418, 37–38 (2002).

    Article  CAS  PubMed  Google Scholar 

  13. Essner, J.J., Amack, J.D., Nyholm, M.K., Harris, E.B. & Yost, H.J. Kupffer's vesicle is a ciliated organ of asymmetry in the zebrafish embryo that initiates left-right development of the brain, heart and gut. Development 132, 1247–1260 (2005).

    Article  CAS  PubMed  Google Scholar 

  14. Kramer-Zucker, A.G. et al. Cilia-driven fluid flow in the zebrafish pronephros, brain and Kupffer's vesicle is required for normal organogenesis. Development 132, 1907–1921 (2005).

    Article  CAS  PubMed  Google Scholar 

  15. Piperno, G. & Fuller, M.T. Monoclonal antibodies specific for an acetylated form of α-tubulin recognize the antigen in cilia and flagella from a variety of organisms. J. Cell Biol. 101, 2085–2094 (1985).

    Article  CAS  PubMed  Google Scholar 

  16. Sumanas, S., Kim, H.J., Hermanson, S. & Ekker, S.C. Zebrafish frizzled-2 morphant displays defects in body axis elongation. Genesis 30, 114–118 (2001).

    Article  CAS  PubMed  Google Scholar 

  17. Kuhl, M., Sheldahl, L.C., Park, M., Miller, J.R. & Moon, R.T. The Wnt/Ca2+ pathway: a new vertebrate Wnt signaling pathway takes shape. Trends Genet. 16, 279–283 (2000).

    Article  CAS  PubMed  Google Scholar 

  18. Tada, M. & Smith, J.C. Xwnt11 is a target of Xenopus Brachyury: regulation of gastrulation movements via Dishevelled, but not through the canonical Wnt pathway. Development 127, 2227–2238 (2000).

    CAS  PubMed  Google Scholar 

  19. Habas, R., Kato, Y. & He, X. Wnt/Frizzled activation of Rho regulates vertebrate gastrulation and requires a novel Formin homology protein Daam1. Cell 107, 843–854 (2001).

    Article  CAS  PubMed  Google Scholar 

  20. Liu, X. et al. Activation of a frizzled-2/β-adrenergic receptor chimera promotes Wnt signaling and differentiation of mouse F9 teratocarcinoma cells via Galphao and Galphat. Proc. Natl. Acad. Sci. USA 96, 14383–14388 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Ahumada, A. et al. Signaling of rat Frizzled-2 through phosphodiesterase and cyclic GMP. Science 298, 2006–2010 (2002).

    Article  CAS  PubMed  Google Scholar 

  22. Kuhl, M., Sheldahl, L.C., Malbon, C.C. & Moon, R.T. Ca2+/calmodulin-dependent protein kinase II is stimulated by Wnt and Frizzled homologs and promotes ventral cell fates in Xenopus. J. Biol. Chem. 275, 12701–12711 (2000).

    Article  CAS  PubMed  Google Scholar 

  23. Ross, A.J. et al. Disruption of Bardet-Biedl syndrome ciliary proteins perturbs planar cell polarity in vertebrates. Nat. Genet. 37, 1135–1140 (2005).

    Article  CAS  PubMed  Google Scholar 

  24. Park, T.J., Haigo, S.L. & Wallingford, J.B. Ciliogenesis defects in embryos lacking inturned or fuzzy function are associated with failure of planar cell polarity and Hedgehog signaling. Nat. Genet. 38, 303–311 (2006).

    Article  CAS  PubMed  Google Scholar 

  25. Simons, M. et al. Inversin, the gene product mutated in nephronophthisis type II, functions as a molecular switch between Wnt signaling pathways. Nat. Genet. 37, 537–543 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Germino, G.G. Linking cilia to Wnts. Nat. Genet. 37, 455–457 (2005).

    Article  CAS  PubMed  Google Scholar 

  27. Raya, A. et al. Notch activity induces Nodal expression and mediates the establishment of left-right asymmetry in vertebrate embryos. Genes Dev. 17, 1213–1218 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Huang, C.J., Tu, C.T., Hsiao, C.D., Hsieh, F.J. & Tsai, H.J. Germ-line transmission of a myocardium-specific GFP transgene reveals critical regulatory elements in the cardiac myosin light chain 2 promoter of zebrafish. Dev. Dyn. 228, 30–40 (2003).

    Article  CAS  PubMed  Google Scholar 

  29. Ng, J.K. et al. The limb identity gene Tbx5 promotes limb initiation by interacting with Wnt2b and Fgf10. Development 129, 5161–5170 (2002).

    CAS  PubMed  Google Scholar 

  30. Matsui, T. et al. Noncanonical Wnt signaling regulates midline convergence of organ primordia during zebrafish development. Genes Dev. 19, 164–175 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We thank M. Schwarz for help in preparing the manuscript; M. Marti Gaudes for help with confocal microscopy and T. Matsui and A. Rojas for discussion. I.O. was initially partially supported by fellowships from The Cell Science Research Foundation and the Japan Heart Foundation. Work in the laboratory of J.C.I.B. was supported by the US National Institutes of Health and the Cellex, Biobide and G. Harold and Leila Y. Mathers charitable foundations.

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Authors

Contributions

This study was designed by I.O., Y.K., Á.R. and J.C.I.B; research was performed by I.O., Y.K. and C.C.-M.; phenotype assessment was performed by C.C.-M; data were analyzed by I.O.; I.O., Y.K., Á.R.; and J.C.I.B. wrote the paper.

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Supplementary information

Supplementary Fig. 1

Structure of dub protein and expression of dub during development of zebrafish. (PDF 912 kb)

Supplementary Fig. 2

Morphological phenotypes in dub morphant embryos. (PDF 920 kb)

Supplementary Fig. 3

Expression pattern of frizzled-2 and subcellular distribution of dub-eGFP fusion protein. (PDF 747 kb)

Supplementary Table 1

Defects in left-right patterning and Kupffer's vesicle cilia formation in dub morphants. (PDF 48 kb)

Supplementary Video 1

Counterclockwise fluid flow in control Kupffer's vesicle. (MOV 2793 kb)

Supplementary Video 2

Injection of dub-MO disrupts Kupffer's vesicle fluid flow. (MOV 1354 kb)

Supplementary Video 3

Renal cilia beating in 2.5-dpf control embryo. (AVI 4110 kb)

Supplementary Video 4

Pronephric duct in 2.5-dpf dub morphant embryo. (AVI 4921 kb)

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Oishi, I., Kawakami, Y., Raya, Á. et al. Regulation of primary cilia formation and left-right patterning in zebrafish by a noncanonical Wnt signaling mediator, duboraya. Nat Genet 38, 1316–1322 (2006). https://doi.org/10.1038/ng1892

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