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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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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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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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  • DOI: https://doi.org/10.1038/ng1892

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