A central question in Hedgehog (Hh) signaling is how evolutionarily conserved components of the pathway might use the primary cilium in mammals but not fly. We focus on Suppressor of fused (Sufu), a major Hh regulator in mammals, and reveal that Sufu controls protein levels of full-length Gli transcription factors, thus affecting the production of Gli activators and repressors essential for graded Hh responses. Surprisingly, despite ciliary localization of most Hh pathway components, regulation of Gli protein levels by Sufu is cilium-independent. We propose that Sufu-dependent processes in Hh signaling are evolutionarily conserved. Consistent with this, Sufu regulates Gli protein levels by antagonizing the activity of Spop, a conserved Gli-degrading factor. Furthermore, addition of zebrafish or fly Sufu restores Gli protein function in Sufu-deficient mammalian cells. In contrast, fly Smo is unable to translocate to the primary cilium and activate the mammalian Hh pathway. We also uncover a novel positive role of Sufu in regulating Hh signaling, resulting from its control of both Gli activator and repressor function. Taken together, these studies delineate important aspects of cilium-dependent and ciliumindependent Hh signal transduction and provide significant mechanistic insight into Hh signaling in diverse species.[Keywords: Hedgehog; signal transduction; evolution; primary cilium; Sufu; Gli] Supplemental material is available at http://www.genesdev.org. In particular, the primary cilium, an ancient and evolutionarily conserved organelle, is essential for mammalian Hh signal transduction but dispensable for Hh signaling in Drosophila. The extent of Hh pathway divergence in different organisms is a major unresolved issue. Delineating cilium-dependent and cilium-independent processes of Hh signal transduction is crucial to understanding how the mammalian Hh pathway has evolved. Insight into this question will not only advance our mechanistic understanding of Hh signaling but also serve as a paradigm for investigating the evolution of signal transduction pathways.Most vertebrate cells possess a nonmotile primary cilium (Huangfu and Anderson 2005). Primary cilia contain a long microtubular axoneme that extends from the basal body and is surrounded by an external membrane that is continuous with the plasma membrane (Rosenbaum and Witman 2002). Assembly and maintenance of the primary cilium are mediated by a process called intraflagellar transport (IFT), which involves bidirectional movement of IFT particles powered by anterograde kinesin (Kif3a, Kif3b, and Kif3c) and retrograde dynein motors (Rosenbaum and Witman 2002). Mouse ethylnitrosourea (ENU) mutants in genes encoding IFT proteins, or the respective motors, have defective Hh signaling (Huangfu et al. 2003), providing strong evidence that the primary cilium plays a key role in mammalian Hh signaling. Moreover, most core mammalian 3 These authors contributed equally to this work. 4 Corresponding author. E-MAIL pao-tien.chuang@ucsf.edu; FAX (415) 476-8173. Arti...

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.

SummaryHedgehog (Hh) signaling is required for embryonic patterning and postnatal physiology in invertebrates and vertebrates. With the revelation that the primary cilium is crucial for mammalian Hh signaling, the prevailing view that Hh signal transduction mechanisms are conserved across species has been challenged. However, more recent progress on elucidating the function of core Hh pathway cytosolic regulators in Drosophila, zebrafish and mice has confirmed that the essential logic of Hh transduction is similar between species. Here, we review Hh signaling events at the membrane and in the cytosol, and focus on parallel and divergent functions of cytosolic Hh regulators in Drosophila and mammals.

*Truncating mutations in Gli3, an intracellular effector in the SHH-SMO-GLI signaling pathway, cause renal aplasia/dysplasia in humans and mice. Yet, the pathogenic mechanisms are undefined. Here, we report the effect of decreased SHH-SMO signaling on renal morphogenesis, the expression of SHH target genes and GLI binding to Shh target genes. Shh deficiency or cyclopaminemediated SMO inhibition disrupted renal organogenesis, decreased expression of GLI1 and GLI2 proteins, but increased expression of GLI3 repressor relative to GLI3 activator. Shh deficiency decreased expression of kidney patterning genes (Pax2 and Sall1) and cell cycle regulators (cyclin D1 and MYCN). Elimination of Gli3 in Shh -/-mice rescued kidney malformation and restored expression of Pax2, Sall1, cyclin D1, MYCN, Gli1 and Gli2. To define mechanisms by which SHH-SMO signaling controls gene expression, we determined the binding of GLI proteins to 5Ј flanking regions containing GLI consensus binding sequences in Shh target genes using chromatin immunoprecipitation. In normal embryonic kidney tissue, GLI1 and/or GLI2 were bound to each target gene. By contrast, treatment of embryonic kidney explants with cyclopamine decreased GLI1 and/or GLI2 binding, and induced binding of GLI3. However, cyclopamine failed to decrease Gli1 and Gli2 expression and branching morphogenesis in Gli3-deficient embryonic kidney tissue. Together, these results demonstrate that SHH-SMO signaling controls renal morphogenesis via transcriptional control of Gli, renal patterning and cell cycle regulator genes in a manner that is opposed by GLI3.

Activation of Hedgehog (Hh) signaling requires the transmembrane protein Smoothened (Smo), a member of the G-protein coupled receptor superfamily. In mammals, Smo translocates to the primary cilium upon binding of Hh ligands to their receptor, Patched (Ptch1), but it is unclear if ciliary trafficking of Smo is sufficient for pathway activation. Here, we demonstrate that cyclopamine and jervine, two structurally related inhibitors of Smo, force ciliary translocation of Smo. Treatment with SANT-1, an unrelated Smo antagonist, abrogates cyclopamine- and jervine-mediated Smo translocation. Further, activation of protein kinase A, either directly or through activation of Gαs, causes Smo to translocate to a proximal region of the primary cilium. We propose that Smo adopts multiple inactive and active conformations, which influence its localization and trafficking on the primary cilium.