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. 2009 May 7;459(7243):98-102.
doi: 10.1038/nature07883. Epub 2009 Mar 22.

Fused has evolved divergent roles in vertebrate Hedgehog signalling and motile ciliogenesis

Christopher W Wilson  1 , Catherine T Nguyen, Miao-Hsueh Chen, Jehn-Hsiahn Yang, Rhodora Gacayan, Jie Huang, Jau-Nian Chen, Pao-Tien Chuang
Affiliations

Fused has evolved divergent roles in vertebrate Hedgehog signalling and motile ciliogenesis

Christopher W Wilson et al. Nature. .

Abstract

Hedgehog (Hh) signalling is essential for several aspects of embryogenesis. In Drosophila, Hh transduction is mediated by a cytoplasmic signalling complex 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 mammals. 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 orthologue, 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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Figures

Figure 1
Figure 1. Mouse Fu is required for central pair (CP) apparatus construction
a-c, Expression of Fu (pink signal) in the mouse tracheal epithelium (a), ependyma of the lateral ventricles (b), and testis (c) at postnatal (p) day 14 by section in situ hybridization to Fu. Arrows indicate sites of Fu expression. d, e, Transmission electron micrographs (TEM) of motile cilia from wild-type (wt) and Fu-/- tracheae. Arrows denote the CP microtubules. f, Quantification of ultrastructural defects from p1 (wt, n = 4 animals, mean 88 cilia/animal analyzed; Fu-/-, n = 4 animals, mean 64 cilia/animal analyzed) and p14 (wt, n = 7, mean 113 cilia/animal analyzed; Fu-/-, n = 6 animals, mean 93 cilia/animal analyzed) tracheae. Error bars indicate standard deviation (s.d.). g, h, Traces of fluorescent bead movement over tracheal explants. i, Mean particle velocity in p14 wt (n = 5) and Fu-/- (n = 5) tracheae. Error bars indicate s.d. j, k, Traces of cilia beat path overlaid on still DIC images of tracheal cilia (upper), and lateral traces of cilia waveform (lower). Mean ciliary beat frequency (CBF) was calculated from 30 cilia (n = 3 animals for wt and Fu-/-). Arrows indicate directions of the forward effective strokes. l, Quantification of circular standard deviation (CSD) of basal feet from p1 (wt, n = 24 cells from 4 animals; Fu-/-, n = 38 cells from 4 animals; P < 3.4 × 10−6; unpaired Student’s t-test) and p14 (wt, n = 31 cells from 4 animals; Fu-/-, n = 36 cells from 3 animals; P < 2.6 × 10−7; unpaired Student’s t-test). Error bars indicate s.d. m, n, Representative TEM images of basal foot polarity (arrows) in p14 wt and Fu-/- tracheae.
Figure 2
Figure 2. Mouse Fu is capable of rescuing Hh-related phenotypes in zebrafish fu morphants
a, Whole-mount in situ hybridization to fkd4 (purple signal) in both medial and lateral floor plate of wt zebrafish embryos at 24 hours post fertilization (hpf). b, c, fkd4 expression is lost in the lateral floor plate of fu morphants (b) and is restored when mouse Fu is expressed (c). d, Whole-mount in situ hybridization to nkx2.2b (purple signal) in lateral floor plate of wt zebrafish embryos at 24 hpf. e, f, nkx2.2b expression is lost in the lateral floor plate of fu morphants (d) and is restored when mouse Fu is expressed (e). View is dorsal. mf, medial floor plate; lf, lateral floor plate. g, Whole-mount in situ hybridization to ptc1 (purple signal) in somites of wt zebrafish embryos at 10-somite stage. View is dorsal. h, i, ptc1 expression is greatly reduced in somites of fu morphants (h) and is restored when mouse Fu is expressed (i). j, Immunohistochemistry against En (arrow), which labels the muscle pioneer population in wt zebrafish somites at 24 hpf. View is lateral. k, l, Rescue of En expression in fu morphant somites (k) by co-injection with mouse Fu (l). m, Lateral view of chevron-shaped somites in wt zebrafish embryos at 24 hpf. n, o, Rescue of U-shaped somites in fu morphants (n) by co-injection with mouse Fu (o). Dotted lines delineate the boundaries of somites. p, Whole-mount in situ hybridization to ptc1 (purple signal) in somites of wt zebrafish embryos at 10-somite stage. View is dorsal. q, r, Upregulation of ptc1 expression in ptc1 morphants (q) is abolished by knocking down fu (r).
Figure 3
Figure 3. Zebrafish fu has a Hh-independent role in L-R asymmetry and generation of 9+2 cilia
a, b, Section in situ hybridization to Fu (pink signal) in chick trachea (a) and X. tropicalis testis (b). Arrow indicates sites of Fu expression. c, Whole mount in situ hybridization to cmlc2 (purple signal) in smohi1640Tg fish embryos at 24 hpf. View is dorsal. d, e, Whole-mount in situ hybridization to cmlc2 in wt (d) and fu morphants at 24 hpf. View is dorsal. (e). f, Summary of cardiac laterality defects in wt (n = 206), fu morphants (n = 130), and fu morphants rescued with mouse Fu (n = 134). L, left; R, right; M, medial. g, h, Whole mount in situ hybridization to spaw at 15-somite stage. View is dorsal. i, Summary of spaw expression in the lateral plate mesoderm (LPM) in wt (n = 116), fu morphants (n = 92), and fu morphants rescued with mouse Fu (n = 73). L, left; R, right; A, absent; B, bilateral. j, k, Electron micrograph of Kupffer’s vesicle (KV) cilia from wt (j) and fu morphant (k). l, Quantification of ultrastructural defects in KV cilia from wt and fu morphants. Error bars indicate standard deviation (s.d.). m, n, Whole mount in situ hybridization to cmlc2 in wt and kif7 morphants at 24 hpf. View is dorsal. o, Summary of cardiac laterality defects in wt (n = 87) and kif7 morphants (n = 109). p, Summary of essential Fu and Cos2/Kif27 functions in metazoan model organisms.
Figure 4
Figure 4. Mouse Fu interacts with the CP protein SPAG16L and the Cos2 ortholog Kif27
a, Western blot of immunoprecipitated mouse Fu-FLAG to detect its physical interaction with mouse SPAG6-HA or SPAG16L-HA from HEK 293T lysates. WB, western blot; in, input; IP, immunoprecipitation. b, Western blot of immunoprecipitated mouse Fu-FLAG to determine its physical association with mouse Kif7-myc or Kif27-myc from HEK 293T lysates. c-h, Confocal images of fully differentiated mouse tracheal epithelial cells (MTECs) to visualize localization of Kif27-GFP to the basal body (marked by anti-γ-tubulin) of motile cilia labeled with acetylated tubulin (ac-tubulin). j, Model of Fu, Kif27, and SPAG16 function in motile cilia construction.
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