Hedgehog (Hh) signalling is essential for several aspects of embryogenesis1,2. In Drosophila, Hh transduction is mediated by a cytoplasmic signalling complex3,4,5 that includes the putative serine-threonine kinase Fused (Fu) and the kinesin Costal 2 (Cos2, also known as Cos), yet Fu does not have a conserved role in Hh signalling in mammals6,7. Mouse Fu (also known as Stk36) mutants are viable and seem to respond normally to Hh signalling. Here we show that mouse Fu is essential for construction of the central pair apparatus of motile, 9+2 cilia and offers a new model of human primary ciliary dyskinesia. We found that mouse Fu physically interacts with Kif27, a mammalian Cos2 orthologue8, and linked Fu to known structural components of the central pair apparatus, providing evidence for the first regulatory component involved in central pair construction. We also demonstrated that zebrafish Fu is required both for Hh signalling and cilia biogenesis in Kupffer’s vesicle. Mouse Fu rescued both Hh-dependent and -independent defects in zebrafish. Our results delineate a new pathway for central pair apparatus assembly, identify common regulators of Hh signalling and motile ciliogenesis, and provide insights into the evolution of the Hh cascade.
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We thank H. Bourne, C. C. Hui, Z. Zhang, J. Strauss III and W. Hwang for constructs, antibodies and sharing of unpublished results; R. Harland and T. Mikawa for X. tropicalis and chicken tissue; K. Thorn and S. Dandekar for assistance with microscopy and ciliary beat frequency analysis; M.-L. Cheong and Y. Nozawa for technical assistance; and D. Casso, S. Coughlin, T. Kornberg, W. Marshall, T. Mikawa, K. Wemmer and members of the Chen and Chuang laboratories for discussion and critical reading of the manuscript. Some data for this study were acquired at the Nikon Imaging Center at UCSF/QB3. This work was supported by grants from the National Institutes of Health to J.-N.C. and P.-T.C., and a Career Investigator Award from the American Lung Association to P.-T.C.
This movie shows tracheal flow (visualized using Fluospheres at 40x magnification) in a wild-type mouse tracheal explant (from postnatal day (p) 14). Movie was acquired at 26 frames per second (fps) and is played back at the same speed. Left is proximal and right is distal.
This movie shows tracheal flow (visualized and acquired as per Supplementary Movie 1) in a Fu-/- tracheal explant. In contrast to the wild-type trachea, Fu-/- mutants do not generate a coordinated, directional flow.
This movie shows a lateral view of motile, 9+2 cilia in a wild-type mouse tracheal explant (p14). Movie was acquired using 90x magnification at 60 fps and is played back at both 60 fps and 10 fps.
This movie shows a lateral view of cilia in a Fu-/- tracheal explant (acquired and visualized as per Supplementary Movie 3). Cilia in the Fu-/- trachea do not beat in a coordinated manner; many are immotile or move slowly and stiffly.
This movie shows a top-down view of motile, 9+2 cilia tips in a wild-type tracheal explant (acquired and visualized as per Supplementary Movie 3). Movies are shown without and with traces of individual cilia paths.
This movie shows a top-down view of cilia tips in a Fu-/- tracheal explant (acquired and visualized as per Supplementary Movie 3). Many cilia in the Fu-/- trachea have a limited range of motion, collide with their neighbours, and do not beat in a coordinated fashion.
This movie shows a ventral view of counterclockwise fluid flow (visualized by fluorescent beads at 40x magnification) in Kupffer's vesicle (KV) from wild-type zebrafish embryos at the 8-10 somite stage.
This movie shows fluid flow (visualized as per Supplementary Movie 7) in Kupffer's vesicle (KV) from a fu morphant at the 8-10 somite stage. Counterclockwise fluid flow is disrupted in fu morphants.
This movie shows fluid flow (visualized as per Supplementary Movie 7) in Kupffer's vesicle (KV) from a fu morphant co-injected with mouse Fu mRNA at the 8-10 somite stage. Counterclockwise flow is restored in fu morphants by co-injection of mouse Fu.