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

Abstract

Hedgehog (Hh) signaling is essential for multiple aspects of embryogenesis1 , 2. In Drosophila, Hh transduction is mediated by a cytoplasmic signaling complex3 -5 that includes the putative serinethreonine kinase Fused (Fu) and the kinesin Costal 2 (Cos2), yet Fu does not play a conserved role in Hh signaling in mammals6 , 7. Mouse Fu mutants are viable and appear to respond normally to Hh signaling. Here we show that mouse Fu is essential for construction of the central pair (CP) apparatus of motile, 9+2 cilia and offers a novel model of human primary ciliary dyskinesia. We found that mouse Fu physically interacts with Kif27, a mammalian Cos2 ortholog8, and linked Fu to known structural components of the CP apparatus, providing evidence for the first regulatory component involved in CP construction. We also demonstrated that zebrafish Fu is required both for Hh signaling and cilia biogenesis in Kupffer's vesicle. Mouse Fu rescued both Hh-dependent and independent defects in zebrafish. Our results delineate a novel pathway for CP apparatus assembly, identify common regulators of Hh signaling and motile ciliogenesis, and add insight into evolution of the Hh cascade.To further investigate the role of Fu in mammalian Hh signaling, we asked whether Hhdependent Smo localization to the primary cilium is affected in the absence of Fu. Primary cilia, which have a "9+0" arrangement of 9 outer doublet microtubules (MTs), are required for Hh responses and contain several Hh pathway components2 , 9. We found that Fu -/-mouse embryonic fibroblasts (MEFs) formed primary cilia normally, trafficked Smo to the primary cilium in response to Hh ligand, and exhibited a typical Gli transcriptional response (Supplementary Fig. 1 , 2; data not shown). This suggests that the single mammalian Fu ortholog is dispensable for Hh signaling. To explore the function of Fu in mice, we examined its expression in postnatal tissues by in situ hybridization. Fu transcript was expressed strongly in the respiratory epithelium, the ependymal lining of the ventricles in the brain, and in oviduct and testis ( Fig. 1a-c; data not shown). These expression patterns are reminiscent of genes involved in biogenesis of motile cilia, which function in these tissues to propel mucus, fluid and cells. In contrast to the primary cilium, the classical "9+2" motile cilium consists of 9 outer doublet MTs and two singlet central pair (CP) MTs10. The CP apparatus plays a key role in regulating ciliary motility but its formation is poorly understood since the centriole-derived basal body, from which the cilia axoneme extends, does not provide a template for CP outgrowth. Disruption of human motile cilia function leads to primary ciliary dyskinesia (PCD), which is associated with recurrent respiratory infection, hydrocephalus and infertility11 -13. To determine whether motile cilia function is * These authors contributed equally to this work.

Dual Phosphorylation of Suppressor of Fused (Sufu) by PKA and GSK3β Regulates Its Stability and Localization in the Primary Cilium

“…2D). The difference between these two types of phosphorylation is another example of design shift of the mammalian Hedgehog pathway during evolution and, perhaps, a reflection of the functional divergence of the mammalian Fu kinase, which is involved in the ciliogenesis rather than directly participating in Shh signal transduction (39). The four consensus PKA recognition sites are not conserved in the sequence of Drosophila Sufu.…”

Protein diversity in discrete structures at the distal tip of the trypanosome flagellum

“…They belong to ULK families already implicated in flagellum-related functions in animals (35,36) with the mammalian ULK Fused demonstrated to be essential for construction of motile cilia (35). Plant orthologs of both kinases were shown to be critical for the phragmoplast organization, resembling the function of plant kinesin-15s (37,38).…”

Congenital Hydrocephalus in Genetically Engineered Mice

“…Stk36 (serine/threonine kinase 36; fused homolog, Drosophila): 65,106 Due to ill health, the majority of the mutants were euthanized between 3 and 6 weeks of age, although several survived up to 12 weeks. All mutant mice exhibited hydrocephalus, craniofacial abnormalities, and growth retardation (reduced body weight and mean body length in males and females).…”