您的当前位置：首页 12013The mechanisms of Hedgehog signalling and its roles in development and disease

12013The mechanisms of Hedgehog signalling and its roles in development and disease

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The mechanisms of Hedgehog

signalling and its roles in development and disease

James Briscoe1 and Pascal P. Thérond2,3

Abstract | The cloning of the founding member of the Hedgehog (HH) family of secreted proteins two decades ago inaugurated a field that has diversified to encompass embryonic development, stem cell biology and tissue homeostasis. Interest in HH signalling increased when the pathway was implicated in several cancers and congenital syndromes. The mechanism of HH signalling is complex and remains incompletely understood.

Nevertheless, studies have revealed novel biological insights into this system, including the function of HH lipidation in the secretion and transport of this ligand and details of the signal transduction pathway, which involves Patched 1, Smoothened and GLI proteins (Cubitus interruptus in Drosophila melanogaster), as well as, in vertebrates, primary cilia.

Morphogen

A protein that forms a gradient and directs tissue patterning.

Holoprosencephaly

Failure of the cephalic lobes to separate, which is associated with facial deformities.

Medical Research Council (MRC)-National Institute for Medical Research (NIMR), Mill Hill, London, NW7 1AA, UK. 2

Centre National de la Recherche Scientifique (CNRS) Unité Mixte

Recherche (UMR) 7277, Inserm UMR 1091, Institut de Biologie Valrose (IBV), France.3

Université de Nice-Sophia Antipolis, Centre de

Biochimie, Parc Valrose, F-06108 Nice Cedex 2, France.e-mails:

pascal.therond@unice.fr; jbrisco@nimr.mrc.ac.ukdoi:10.1038/nrm3598

Published online 30 May 2013

Hedgehog (Hh) was first identified by genetic screens in Drosophila melanogaster1. It earned its name from the appearance of embryos with null alleles of hh, which display a lawn of disorganized, hair-like bristles remini-scent of hedgehog spines. Its molecular identification occurred 12 years later, revealing it to be a secreted protein that directs pattern formation in adjacent cells2. Shortly thereafter, three mammalian counterparts, sonic hedge-hog (SHH), Indian hedgehog (IHH) and desert hedgehog (DHH), were found, and an evolutionarily conserved role for these molecules in body organization discovered2–7. SHH activity reproduces the actions of the zone of polar-izing activity in the limb bud and of the notochord and floor plate in the neural tube8,9, all of which are organizing centres that were described in classic grafting experiments decades ago. IHH regulates bone and cartilage develop-ment and is partly redundant with SHH, whereas DHH is essential for germ cell development in the testis and peripheral nerve sheath formation. The identification of these roles for HH proteins supported the textbook model of developmental biology, in which organizer tissue s secrete a morphogen, HH in this case, that diffuses to trigger differential cellular responses according to its concentration. Almost simultaneously, interest in HH sig-nalling came from another direction. Dysregulation of the HH pathway was found to be responsible for congenital syndromes, such as holoprosencephaly, and other devel-opmental malformations10. Moreover, HH signalling was subsequently shown to regulate stem cell homeostasis in adult tissues, and persistent HH pathway activity seems

to have pathological consequences in various cancers, including the skin cancer basal cell carcinoma and the brain tumour medulloblastoma11.

Our purpose here is to provide readers with an update on HH signalling. We discuss how studying this pathway has provided fundamental insights into diverse areas of cell and developmental biology. We depict the unexpected complexity in the molecular mechanism of HH signal production, including how lipidation affects the secre-tion and extracellular spread of this ligand. We highlight specific aspects of the variation and conservation of HH signalling between species (for a review on the evolution-ary origins of HH signalling, see REF. 12) and describe how the previously enigmatic primary cilia of vertebrate cells were found to function in HH signalling. We also review the multiple functions of HH and discuss how some of these diverse functions involve differential strength and duration of signalling, multiple feedback loops and non-canonica lsignalling.

HH maturation and secretion

Immature HH proteins undergo several post-translational modifications and cleavage events that modify their activity and regulate their spread from producing cells through tissues.

Dual lipidation of HH by cholesterol and palmitic acid. All HH proteins undergo signal sequence cleavage and enter the secretory pathway. Subsequently, HH mol-ecules undergo autoproteolytic cleavage to generate an

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Lipoprotein

Exovesicle

Soluble multimerApolipoproteinGlypicanHeparan sulphate chainPlasmamembraneDISP1HH-N23Notum4SCUBE2SKIPalmitic acidHH-NCholesterolHH-CProteasomaldegradationAutoproteolytic cleavageCytoplasmERHH-NHH-CFigure 1 | Hedgehog protein biogenesis and release. Hedgehog (HH) proteins are synthesized as precursors of about | Molecular Cell Biology45 kDa. Autoproteolytic cleavage probably occurs in the endoplasmic reticulum (ER)15. The sterol recognition region of the HH carboxy‑terminal peptide (HH‑C) recruits cholesterol, which then acts as a nucleophile to attack an intramolecular thioester intermediate of HH. This results in the covalent attachment of cholesterol to the amino‑terminal peptide (HH‑N) and the dissociation of HH‑C14. The acyltransferase skinny hedgehog (SKI) modifies HH‑N by attaching a stable amide‑linked palmitic acid group to the most N‑terminal Cys residue, whereas HH‑C translocates out of the ER and undergoes proteasomal degradation. Once at the outer surface of the plasma membrane, dually lipid‑modified HH‑N is associated with the lipid bilayer as a monomer until it is released by one of four key mechanisms. The cholesterol‑modified HH‑N monomer is released by the cooperative action of the transmembrane protein Dispatched (DISP) and the secreted SCUBE2 protein29,40 (1). Both proteins bind directly to a different part of the cholesterol moiety of HH‑N. Monomeric cholestero l‑modified HH‑N can also self‑associate to form large soluble multimers that are released from the

membrane31,36 (2). HH‑N oligomers can interact with the heparan sulphate chains of glypicans, which enables them to recruit lipophorin apolipoproteins and assemble into lipoprotein particles49,50. The glycosylphosphatidylinositol (GPI) anchor of glypican might be cleaved by the phospholipase C‑like protein Notum, promoting the release of the HH‑N‑associated lipoprotein particles214 (3). Alternatively, HH‑N may be released at the surface of exovesicles64 (4).

Inteins

Protein regions that are able to excise themselves and rejoin the remaining portions. This is the protein equivalent of an intron in a gene.

amino-terminal peptide that is linked to cholesterol at its carboxyl terminus (FIG. 1). All signalling activities are mediated by this N-terminal peptide, but the C-terminal region is necessary to catalyse the autoproteolytic cleavage event, a mechanism that is reminiscent of the protein splic-ing activity of inteins13,14. In vitro evidence suggests that the C-terminal fragment undergoes rapid degradation, a pro-cess that requires its translocation out of the endo plasmic reticulum (ER) to the proteasome15. HH cleavage is of importance, as mutations in human SHH that block the cleavage of the full-length protein cause the loss of SHH function, resulting in holoprosencephaly16–18. Notably, however, other studies have suggested that the C-terminal domain, independently of autoproteolytic cleavage, tar-gets Hh to axons and growth cones in D. melanogaster19. Further study will be required to pin down the function of the C-terminal domain of HH.

HH is also modified by the attachment of a palmitic acid group, which greatly increases the activity of the pro-tein in cell-based assays20,21. This acylation event is cata-lysed by skinny hedgehog (SKI), which is a member of the membrane-bound O-acyltransferase (MBOAT) pro-tein family14,22–26. The subcellular compartment in which palmitoylation occurs has yet to be identified. However, engineered forms of vertebrate and D. melanogaster HH

that lack cholesterol are palmitoylated inefficiently.

This suggests that palmitoylation occurs after cholesterol modification21, although in vivo the addition of cholesterol to HH is not a prerequisite for palmitoylation27,28.

Spread of HH from producing cells. The distance HH travels to exert its effect seems to differ between tis-sues but can be up to 50 μm in the imaginal wing disc of D. melanogaster and up to 300 μm in the limb bud of vertebrates. The cholesterol modification of HH leads to its retention in the plasma membrane, restricting its free mobility29,30, and both cholesterol and palmitic acid promote the association of HH with sterol-rich mem-brane microdomains31,32. This might be important for interactions with proteins involved in the release and spread of HH, such as the cytoplasmic membrane scaf-folding protein Reggie 1, the D. melanogaster counter-part of verte brate flotillin 2 (REF. 33). In D. melanogaster, nanoscale and larger visible oligomers of Hh have been described at the cell surface and depend on both cho-lesterol and a conserved residue that is involved in Hh homo-electrostati cinteractions34–36. Disruption of these interactions adversely affects long-range activity, high-lighting the importance of HH oligomerization for the spread of this molecule.

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REVIEWSResistance-nodulation-division (RND) transporter familyA family of bacterial pumps that use a proton gradient to transport small lipophilic molecules across the membrane.

Glypicans

A family of heparan sulphate proteoglycans, members of which are anchored to cells via

glycosylphosphatidylinosi-tol (GPI).

Lipophorin

A scaffolding apolipoprotein that is highly enriched in Drosophila melanogaster haemolymph. Lipophorin is termed lipoprotein in vertebrates.

Dispatched (DISP), a multipass transmembrane protein from the resistance-nodulation-division (RND) transporter family37–39, is required for the secretion of lipidated HH proteins. DISP binds directly to the choles terol moiety of human SHH and acts in synergy with the vertebrate-specific SCUBE2, a secreted glyco-protein that binds to a different part of the cholesterol molecule, to promote the release of SHH from the cell surface29,40 (FIG. 1). It is unclear in which cellular com-partment DISP is required, but it is tempting to specu-late that DISP transfers cholesterol-modified HH to SCUBE2 at the plasma membrane, so that the choles-terol anchor of HH is shielded from the aqueous envi-ronment. Independently, in D. melanogaster, Disp has been shown to be necessary for apicobasal trafficking of Hh; whether this shuttling promotes HH exposure to SCUBE2 in vertebrates is currently unclear27. Other studies have suggested that secreted proteases dissoci-ate SHH from its two lipid moieties in vitro, resulting in SHH solubilization41. However, this mechanism has yet to be demonstrated in vivo.

Soluble cholesterol-modified HH ligands are found as monomers and as large multimers31,35,36,42,43 (FIG. 1). HH proteins lacking cholesterol or palmitate cannot form multimers, and this results in defects in the long-range spread and signalling of these molecules27,28,31,35,36,44,45. How the multimers form remains an open question.In D. melanogaster, glypicans stabilize and recruit Hh into visible clusters on Hh-producing cells34,46–48. Glypicans recruit circulating lipophorin to Hh-secreting cells and promote the association of Hh with lipo-proteins through interactions between their glycosa-minoglycan (GAG) chains49,50. Notably, removing circulating lipophorin reduces the range of Hh activity. The glypican glycosylphosphatidylinositol (GPI) anchor might be cleaved from the cell surface by a phospho-lipase C-like protein, allowing the release and spread of Hh in large soluble lipoprotein particles46,49,51. Another

Box 1 | Multiple carriers for Hedgehog transportIn addition to the soluble multimers and lipoproteins, there is compelling evidence for the existence of other carriers of Hedgehog (HH), perhaps reflecting the diverse functions of this morphogen. For example, several studies have suggested that HH is transported in extracellular vesicular particles (exovesicles) (FIG. 1). Exosomes, which are exovesicles originating from endocytic multivesicular bodies, may carry HH‑related peptides involved in cuticle formation in Caenorhabditis elegans212. Sonic hedgehog (SHH) has also been observed in exovesicles derived from apical microvilli budding at the surface of the mouse ventral node during embryonic development213. As other functional signalling molecules, such as Notch and Wingless (WG), have been detected on extracellular vesicles (reviewed in REF. 64), this type of transportation is an attractive hypothetical mechanism for the spread of HH that should be investigated further.Finally, modes of transportation that do not depend on the release of HH from producing cells have been reported. In Drosophila melanogaster, Hh has been shown to decorate long basal filopodia 48, dubbed cytonemes, and it has been suggested that these cellular extensions deliver Hh at some distance from niche cells for the maintenance of germline stem cells62. These extensions are highly dynamic and might extend the signalling range of Hh. Recently, similar cytoneme‑like structures carrying HH have also been reported in vertebrates214. Intracellular transport from the cell body to axons could also mediate the long‑range transport of HH. In the D. melanogaster photoreceptor neurons and in planaria, HH is transported along the axons to induce postsynaptic neuron differentiation and to stimulate regeneration, respectively19.secreted protein from D. melanogaster, Shifted (Shf), which shares sequence similarity with the vertebrate WNT inhibitory facto r (WIF), has also been shown to associate with both cholesterol-modified Hh and hep-aran sulphate proteo glycans (HSPGs). It remains unclear whether Shf is associated with the Hh-containing lipo-protein particles, but its loss decreases Hh stability and movement in D. melanogaste r, but not in vertebrates52,53.Glypicans are also important for the spread of Hh through tissue. Cholesterol-modified Hh cannot cross a field of cells depleted of HSPGs in D. melanogaster imagi-nal discs, raising the possibility that Hh and its carriers are transferred by planar diffusion on glypicans of adjoin-ing cells54,55. In vertebrates, the situation is complicated by the pleiotropic functions of glypicans (see below), but defects in the synthesis of the heparan sulphat e chains of proteoglycans affect the distribution and activity of IHH during endochondral ossification56, and HSPG sul-phation modulates SHH spread in vertebrates as it does in D. melanogaster57,58. Thus, glypicans seem to have a conserve d role in the spread of HH ligands.

Membrane proteins involved in HH reception also regulate the spread of HH proteins. For example, Patched 1 (PTC), one of the HH receptors (see below), and the vertebrate-specific HH-interacting protein 1 (HIP1) sequester HH to limit its diffusion59–61. Both pro-teins are induced by HH signalling and, thus, participate in ligand-dependent negative feedback control and have a substantial impact on the range of ligand availability. By contrast, proteins acting as HH co-receptors (see below) can also limit the range of HH signalling, but their expression is downregulated by HH. These feed-back loops are probably necessary to refine the multiple thresholds of HH signalling within the target field.Taken together, the current view of HH spread sug-gests that HH proteins are assembled into specialized vehicles to escape the plasma membrane of producing cells and spread through tissue. Nevertheless, mecha-nisms that involve the spread of HH on filopodia-like extensions of cells have also been proposed62,63, and the various potential transportation mechanisms that HH might use, including transportation on extracellular vesicular particles (exovesicles), are outlined in BOX 1and FIG. 1. These highlight the importance of future studies to determine whether similar or distinct mechanisms are used in different tissues and whether this reflects the distance that HH needs to travel or a specific tissue sub-strate over which HH needs to act64. Moreover, several isoforms and/or pools of HH might be produced from the same cells. Recently, endogenously produced Hh was purified from D. melanogaster, and sterol and non-sterol modified Hh molecules with complementary functions were identified65. Furthermore, a recent study suggested that the Hh gradient in the D. melanogaster wing imaginal disc is a composite of pools secreted by different routes: an apically secreted pool with long-range activity and a more basolateral secreted pool with short-range activity46. The identification of the different mechanisms and carriers controlling the formation and routing of HH will represent a major challenge in the next few years.

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The HH signalling pathway

Genetic analysis in D. melanogaster has identified the major components of the Hh pathway66, which are con-served in diverse eumetazoa, including the non-bilate-rian cnidaria. The first HH receptor identified, PTC, is a transmembrane protein that constitutively represses HH signalling67,68 (FIG. 2). HH binding to PTC inhibits the repression of Smoothened (SMO), which is a member of the G protein-coupled receptor (GPCR) superfamily. This derepression results in the activation of the only known transcriptional mediators of the HH response, zinc-finger proteins of the GLI (also known as Cubitus interruptus (Ci) in D. melanogaster) family10. These are bifunctional transcription factors that can both activate or inhibit transcription. Regulation of the processing and nuclear translocation of GLI proteins has a key role in the HH signalling cascade (see below).

and with heparan sulphate chains on proteoglycans through two distinct domains; the loss of these inter-actions reduces HH signalling potency74–76. Moreover, heparin is required for the dimerization of Ihog and its high-affinity interaction with D. melanogaster Hh77. Nevertheless, there is no clear model for how glypicans regulate HH, although it is plausible that specific glypi-cans enhance HH–PTC binding and signalling, whereas others compete with PTC for HH binding and inhibit signalling73,78. In some cases, the heparan sulphate chains are not necessary for the glypican core protein to fully restore HH signalling78,79.

Other potential HH co-receptors have been identified, such as the low-density lipoprotein receptor-related pro-tein 2 (LRP2; also known as megalin), which is required in the forebrain epithelium for SHH signalling and might be involved in PTC trafficking80. In conclusion, HH is multivalent and does not bind exclusively to a single

HH receptor complexes. In addition to PTC, several cell receptor. The mechanism underlying the regulation of surface proteins that bind HH and promote signalling the multireceptor complex has yet to be elucidated and is have been identified69. These co-receptors form separate a matter of great importance for future research.multimolecular complexes with PTC and are required

for high-affinity HH binding. They include the con-Regulation of SMO by PTC. How PTC represses SMO served and structurally related Interference Hedgehog activity remains unclear. Like DISP, PTC contains a (Ihog) and Brother of Ihog (Boi) in D. melanogaster, and sterol-sensing domain (SSD) and has structural similar-their vertebrate orthologues CAM-related/downregu-ity to members of the RND transporter family81–83. lated by oncogenes (CDO) and brother of CDO (BOC), Mutations affecting the RND permease motif of PTC as well as growth arrest-specific 1 (GAS1), which is spe-abrogate the PTC-mediated repression of SMO, lead-cific to vertebrates. Ihog, Boi, CDO and BOC are single-ing to the suggestion that PTC controls the influx or the pass transmembrane proteins with immunoglobulin (Ig) efflux of a ligand that controls SMO84. SMO contains and fibronectin type III (FNIII) repeats, whereas GAS1 a highly conserved extracellular N-terminal Cys-rich is a GPI-linked protein with an extracellular domain domain (CRD), which is required for its dimerization homologous to glial cell-derived neurotrophic factor and function85, but unlike other closely related GPCRs86, (GDNF) receptors.no ligand-binding function has been assigned to it. HH binds directly to Ihog and CDO through different By contrast, several naturally occurring and synthetic non-orthologous FNIII repeat domains70. Curiously, the agonists and antagonists of vertebrate SMO have been crystal structures suggest that the mode of HH binding shown to bind to its membrane-integrated heptahelical by FNIII domains is not the same in D. melanogaster and domain87. Many of these are structurally related to ster-vertebrates, as the HH surfaces interacting with Ihog or ols, raising the possibility that an endogenous sterol-like CDO do not overlap and different cofactor dependencies molecule — an activating or repressing ligand — that have been identified: heparin is required for Hh–Ihog is transported across the membrane by PTC regulates interactions and Ca2+ is required for SHH–CDO inter-SMO. The most promising candidates for the activating actions70. GAS1, CDO and BOC also bind PTC to form ligand are oxysterol s, which directly bind SMO and pro-a multimolecular receptor complex (Ihog–Boi–Ptc in mote SHH signalling88–90. However, a sterol transporting D. melanogaster) that facilitates transduction. The binding function of PTC has yet to be demonstrated, and whether of Ihog to Ptc is essential for the presentation of Ptc at the PTC transports an activating ligand (such as oxysterols) cell surface. Surprisingly, in various mouse tissues, only away from SMO, or an inhibitory ligand to SMO, remains the absence of all three molecules, GAS1, BOC and CDO, to be clarified. Moreover, whether similar ligands also results in the complete loss of HH activity71,72. This fact, regulate D. melanogaste r Smo is unclear.along with biochemical findings, indicate that GAS1 binds A further clue to the mechanism of SMO regulation PTC separately from the BOC–PTC and CDO–PTC by PTC has come from studies in D. melanogaster. In dimers, suggesting that GAS1, BOC and CDO act col-flies, the inhibition of Ptc by Hh results in the accumula-lectively as co-receptors for SHH71,72. Nevertheless, despite tion of Smo at the plasma membrane as a consequence of their mutual contribution to the formation of a multimo-an increase in its trafficking from internal vesicles and/ or lecular complex, the GAS1, CDO and BOC co-receptors an increase in its stability91–93. Ptc has been shown to and PTC have opposing roles in pathway regulation — the inhibit this process and recruit internalized lipophorin

94,95

co-receptors promote HH signalling and PTC inhibits it. to Smo-enriched endosomes. This has led to the sug-HH signalling is also regulated by glypicans, which gestion that lipophorin may deliver sterol derivatives to enhance the stability of HH and promote its internali-Ptc and that these derivatives are responsible for negatively zation with PTC73. Consistent with this, SHH proteins regulating Smo activity94,95. Consistent with this hypoth-interact with the extracellular matrix component heparin esis, decreases in circulating lipophorin levels increase

Sterol-sensing domain

(SSD). A conserved portion of the resistance-nodulation-divi-sion (RND) domain. It was originally identified in proteins that have essential roles in the regulation of cholesterol homeostasis.

Oxysterols

Natural molecules derived from cholesterol oxidation.

NATURE REVIEWS | MOLECULAR CELL BIOLOGY

REVIEWSaOﬀPlasmamembraneIhogBoiGlypicanPtcMicrotubulesPkaSmoCos2FuSuFuPDegradationCiPProteolyticprocessingCiRNucleusDegradationCiACkIαGsk3βHhSmoPFuPSuFuCiCos2IhogBoiGlypicanHhHhPtcPPPkaCkIαOnGprk2NucleusOnbOﬀMicrotubulesGLI2,3SUFUKIF7EVC2GPR161SHH↑cAMPKIF7GAS1CDOandBOCSMOPTCLRP2SUFUGLI2,3BasalbodyPKACKIGSK3βPlasmamembraneSHHPTCSMOEVCSUFUGLI2,3APPGPRK2CKIPGLI2,3ProteolyticprocessingGPR161SUFUGLI2,3Non-canonicalsignalling(G protein-coupled)SHHGLI2,3RSMONucleusDegradationNucleusFigure 2 | Reception of Hedgehog and initiation of signal transduction. a | In Drosophila melanogaster , the transmembrane proteins Interference Hedgehog (Ihog) and Brother of Ihog (Boi) promote Hedgehog (Hh)–Patched 1 (Ptc) binding. Hh also interacts with glypicans. Ligand‑free Ptc represses Smo by triggering its rapid degradation and/or its confinement to an intracellular compartment (left panel, ‘off’). Furthermore, in the absence of Hh, the Hh signalling complex (HSC), which includes Costal 2 (Cos2), Fused (Fu), Suppressor of Fu (SuFu) and Cubitus interruptus (Ci), is associated with microtubules. This complex promotes, through the activity of protein kinase A (Pka), casein kinase Iα (CkIα) and glycogen synthase kinase 3β (Gsk3β) , the formation of the Ci repressor form (CiR). Binding of Hh to Ptc relieves Smo repression. Smo translocates to the membrane and is activated by phosphorylation on its carboxy terminal tail, by PKA, CkIα and G protein‑coupled receptor (Gpcr) kinase 2 (Gprk2), which induces a conformational change85. This promotes its association with the HSC and the sequential activation of Fu and Cos2, which releases uncleaved Ci from the HSC to result in the activation of Ci (CiA)46,118. b | In vertebrates

CAM‑related/downregulated by oncogenes (CDO), brother of CDO (BOC), growth arrest‑specific 1 (GAS1) and low‑density lipoprotein

receptor‑related protein 2 (LRP2) promote HH–PTC binding69. PTC is enriched in and around the primary cilium141, where it acts via a poorly | Molecular Cell Biologycharacterized mechanism that might involve lipid transport to inhibit SMO activity (left panel, ‘off’). In the absence of sonic hedgehog (SHH), at the base of cilia, the GLI proteins GLI2 and GLI3 are phosphorylated by PKA, CKI and GSK3β (PKA might be activated by an increase in cAMP generated by GPR161). This leads to their proteolytic cleavage to generate the repressor forms (GLI2R and GLI3R, respectively). In the presence of SHH ligand, PTC and GPR161 exit the cilia, and SMO is phosphorylated by GPRK2 and CKI and gated into the primary cilium in association

with β-arrestin and the microtubule motor KIF3A155,156 (right panel, ‘on’; β-arrestin and KIF3A not shown). Within the cilium, SMO is enriched proximally in association with EVC (Ellis‑van Creveld syndrome protein) and EVC2 (REFS 157,158). The activation of SMO results in an increased cilia dwell time for SUFU and GLI2 and GLI3, the dissociation of the GLI–SUFU complex within the cilia and the transport of full‑length, activated GLI2 and GLI3 proteins from the cilia to the nucleus bypassing proteolytic processing. The movement of GLI2 and GLI3 through cilia is dependent in part on KIF7, the vertebrate Cos2 orthologue140. Activated SMO has also been proposed to initiate non‑canonical HH signalling outside cilia in the form of GPCR signalling.

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Smo stability at the plasma membrane independently of Ptc in D. melanogaster wing imaginal disc. Thus, Ptc could be responsible for modifying the lipid composition of the endosomes through which Smo is trafficked, block-ing its trafficking to the plasma membrane. Alongside these studies, increases in the level of the phospholipid phosphatidylinositol-4-phosphate (PtdIns(4) P) in D. melanogaster and mammalian fibroblasts have recently been shown to stabilize SMO proteins at the plasma membrane and increase HH signalling96. On the basis of genetic interactions it has been suggested that PTC maintains low levels of PtdIns(4)P in the cell cortex by downregulating the STT4 kinase activity responsible for converting PtdIns into PtdIns(4)P. As PtdIns(4) P is a pre-cursor of signalling PtdIns, further studies are required to determine the link between PtdInsP, any lipid regulator of SMO and intracellular trafficking.

Activation of SMO also involves a conformational switch. In the absence of HH ligand, SMO is found as a dimer in which the cytoplasmic tails are in a closed (inac-tive) conformation. This conformation is maintained by electrostatic interactions in the C-terminal domain between positively charged Arg and negatively charged Asp clusters85. HH activation neutralizes the Arg cluster by triggering the sequential phosphorylation of a domain adjacent to it, promoting the conversion of SMO to an open conformation. This switch seems to be essential for the cell surface accumulation of SMO and signal trans-duction85,97. In D. melanogaster, Smo is phosphorylated successively by protein kinase A (Pka), casein kinase Iα (CkIα), CkII and Gpcr kinase 2 (Gprk2)97–99. Moreover, the various states of Smo phosphorylation are regu-lated by a set of protein phosphatases that act on these phosphorylated residues, including Pp1, Pp2a and Pp4 (REFS. 100,101). Genetic studies have shown that differ-ences in the strength of Hh signalling are generated by the gradual phosphorylation of Smo, with a greater degree of phosphorylation corresponding to stronger signalling, probably as a result of a gradual change in the conforma-tion of the cytoplasmic tail99. Strikingly, the cytoplasmic tail of vertebrate SMO has diverged substantially from that of D. melanogaster Smo, and lacks the cluster of Pka phosphorylation sites responsible for D. melanogaster Smo activation. Pharmacological inhibitors of PKA block SMO activation in vertebrate cells102,103, but there is no evidence that PKA phosphorylates SMO directly, and genetic ablation of PKA does not seem to affect SMO104. Despite this divergence, however, mouse SMO is regu-lated by an analogous mechanism involving CKIα and GPRK2 phosphorylation105. It will be important to deter-mine whether HH signalling regulates the activity or the distribution of these kinases and phosphatases.HH signalling from SMO to Ci and GLI. Although SMO is a member of the GPCR family, whether SMO couples to G proteins remains a matter of debate. In D. melanogaster, Xenopus laevis and cultured fibroblasts, the transduc-tion of SMO activation has been suggested to depend on the Gαi subunit106, which inhibits adenylyl cyclase and, thus, the formation of cyclic AMP (cAMP) and the activation of PKA. However, these findings are at odds

NATURE REVIEWS | MOLECULAR CELL BIOLOGY

with previous studies in D. melanogaster showing that a cAMP-insensitiv e form of Pka does not interfere with normal Hh signalling and studies indicating that Smo is activated by Pka-dependent phosphorylation102,103; further investigatio n is needed to resolve this discrepancy.

Ultimately, the HH transduction pathway culminates in the regulation of Ci and GLI protein activity10. Three genes comprise this family in amniotes (GLI1, GLI2 and GLI3), whereas a single ancestral gene encoding Ci is present in amphioxus, ciona and Drosophila species12. All GLI and Ci proteins seem to have similar DNA-binding specificities, imparted by five tandem C2H2 zinc-fingers that comprise the DNA-binding domain. They all contain a C-terminal activation domain, whereas only Ci, GLI2 and GLI3 contain an N-terminal repressor domain (FIG. 3).HH signalling results in a change in the balance between the activator and repressor forms of the Ci and GLI proteins by regulating their post-translational pro-teolytic processing. In the absence of HH signalling, the C-terminal domain of Ci undergoes sequential phospho-rylation on multiple sites, first by PKA, which primes sites for additional phosphorylation at nearby residues by glycogen synthase kinase 3β (GSK3β) and different members of the CKI family10,107. This generates a bind-ing site for the F-box-containing protein β-transducin repeat-containing protein (βTrCP; also known as Slimb in D. melanogaster), which recruits the S phase associated protein kinase 1 (SKP1)–cullin 1 (CUL1)–F-box protein (SCF) E3 ubiquitin ligase complex108,109. Ubiquitylation of specific residues then targets the modified protein to the proteasome, where the C-terminal transactiva-tion domain is removed by partial degradation (FIG. 3a). The remaining protein, which comprises the N-terminal and DNA-binding domains, translocates to the nucleus, binds genomic target sites and represses transcription. Activation of Smo inhibits the phosphorylation and par-tial proteolytic processing of Ci, allowing full length Ci to act as a transcriptional activator.

The mechanism of GLI2 and GLI3 regulation in verte-brates seems broadly similar to Ci. Nevertheless, the roles of GLI2 and GLI3 have become more specialized. Mouse mutant studies indicate that GLI2 is chiefly responsible for the activator function in response to HH signalling, and GLI3 is responsible for the repressor activity110,111. Whether this reflects the greater efficiency of GLI3 processing to its repressor form compared with GLI2 pro-cessing112 or the relative strength of the transcriptional activator and inhibitor domains of the two proteins, remains to be determined. GLI1 has diverged evolution-arily, lacking a transcriptional repressor domain and, in mammals, it seems to have only a minor role in ampli-fying the transcriptional response113. Different phos-phorylation events have been found to either inhibit or potentiate GLI1 transcriptional activator function114,115, but the details of these mechanisms remain to be resolved. In contrast to mammals, Gli1 is essential for normal Hh responses in zebrafish116, suggesting some divergence of function within vertebrates. Moreover, amphioxus has a single Gli gene but, unlike D. melanogaster ci, this gene is alternatively spliced to produce gene products that act as either activators or inhibitors117.

VOLUME 14 | JULY 2013 | 421REVIEWSaPlasma membraneSMOPartialdegradationCiR and GLIRTranscriptionalrepressor Ci and GLICos2 and KIF7PKACKIGSK3βSlimb and βTrCPCos2 and KIF7Fu(D. melanogaster only)SUFUPost-translationalmodiﬁcationPhosphorylationAcetylationSumoylationCiA and GLIATranscriptionalactivator Ci and GLICi and GLIProcessingRepressionActivationdomaindomainPhosphorylation,Zinc-ﬁngerubiquitylationNuclearimportNuclearimportProteasomaldegradationHibCUL3SPOP+ + +Target geneNucleus– – –Homeostasis, tissue repairAdult neural stem cells,several epithelial tissuesDevelopment patterningD. melanogaster embryo,wing, vertebrate limb,neural tubeTissue growth, mitogenesisCerebellum, retinaOtherAxon guidance, cellularmetabolism, pain perceptionb GLI2 and GLI3 structureDeterminesproteolyticprocessingTranscriptionalactivation domainsTranscriptionalrepressor domainDegronZinc-ﬁngerDNA-bindingdomainDegronPKA, GSK3β, CKI clusteredphosphorylation sites SUFU-bindingMain proteolyticprocessing eventdomainFigure 3 | Schematic of Ci and GLI regulation. a | In the absence of signal, Drosophila melanogaster Cubitus

interruptus (Ci) and vertebrate GLI proteins are converted into a transcriptional repressor (CiR and GLIR, respectively). | Molecular Cell BiologySpecifically, the sequential phosphorylation of Ci and GLI proteins by protein kinase A (PKA), CKI and glycogen

synthase kinase 3β (GSK3β), which is in part facilitated by Costal 2 (Cos2) and KIF7 in D. melanogaster and vertebrates, respectively, promotes the binding of the E3 ubiquitin ligase Slimb and β‑transducin repeat‑containing protein (βTrCP) and the ubiquitylation of Ci and GLI107. The modified protein is then partially proteolytically processed by the

proteasome. The truncated protein, which retains its DNA‑binding domain, then translocates to the nucleus to repress target gene expression. Activation of Smoothened (SMO) promotes the formation of a transcriptional activator (CiA and GLIA) by blocking Ci and GLI processing and inhibiting the activity of Suppressor of Fu (SUFU; which is a

cytoplasmic protein that sequesters Ci and GLI) and Cos2 and KIF7. In D. melanogaster, Ci activation depends on the kinase Fused (Fu). In addition to the blockade of Ci and GLI processing, further post‑translational modifications, which might include phosphorylation (by unc‑51‑like kinase 3 (ULK3) or CDC2L1), acetylation or sumoylation, generate the transcriptionally active forms of Ci and GLI proteins (CiA and GliA)134–137,139. Inhibition of SUFU activity allows CiA and GLIA to translocate to the nucleus, where they replace CiR and GLIR, respectively, on target genes to activate transcription. Simultaneously, CiA and GLIA become a substrate for proteasomal degradation mediated by Hib (Hedgehog (Hh)‑induced BTB protein, in D. melanogaster) and SPOP (Speckle‑type PDZ protein, in vertebrates)

together with cullin 3 (CUL3)128,130. HH signalling has pleiotropic functions that include regulating pattern formation in developing tissues, controlling cell proliferation, supporting tissue homeostasis and repair of adult tissues.

b | A schematic of the domains of GLI2 and GLI3 highlighting the main structural features. These include a DNA‑binding domain comprising five C2H2 zinc‑fingers, an amino‑terminal transcriptional repressor domain and a carboxy‑terminal activation domain. In addition, a region C‑terminal to the DNA‑binding domain contains clusters of phosphorylation sites that are essential for the proteolytic processing event that removes the C‑terminal region of the proteins to yield a transcriptional repressor.

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Ci regulation in D. melanogaster

Insight into the way in which Smo activity regulates Ci was provided by the identification of a Hh signal-ling complex (HSC), which interacts with the cytoplas-mic C-terminal domain of Smo in D. melanogaster and contains Ci and other signalling components118. The k inesin-like protein Costal 2 (Cos2)119 in the HSC serves as a cytosolic scaffold that retains Ci, connecting it to its positive (that is, the Ser/Thr kinase Fused (Fu)) and negative (that is, Pka, Gsk3β and CkI) regulators (FIG. 2a). The kinesin motor function of Cos2 mediates the move-ment of the HSC along microtubules in cultured cells, at a velocity similar to that achieved with classic kinesins120. The role of this movement is not understood, but it may be required to transport Ci and Smo to specialized cellu-lar compartments. In the absence of Hh, HSC-associated kinases promote the partial degradation of Ci to form the transcriptional repressor (CiR)121. In the presence of Hh, stabilization and dimerization of phosphoryl-ated Smo at the plasma membrane promotes its associa-tion with the HSC and subsequently the autoactivation of Fu, possibly through its dimerization, which in turn phosphorylates Cos2 (REFS 122–125). Different magni-tudes of Smo activation are translated into differences in Fu activation, which manifests as different degrees of Cos2 phosphorylation126. As a consequence of Cos2 phosphorylation, the uncleaved form of Ci dissociates from the HSC and is able to translocate to the nucleus. Translocation of Ci can also be restrained by its bind-ing to the cytoplasmic protein Sufu (Suppressor of Fu), which is present in large excess127. Genetic analysis has shown that the absence of Sufu compensates partially for the absence of Fu, suggesting that Fu is required to antagonize Sufu. How this antagonism operates is unclear as none of the Fu-dependent phosphorylation sites on Sufu identified to date have a significant role123. Thus, Fu and other proteins in the HSC may modify Ci directly to prevent its association with Sufu, but this needs to be investigated further. In D. melanogaster, the absence of Sufu has no effect on Hh signalling, whereas the absence of Cos2 leads to the ectopic activation of the pathway, suggesting that Cos2 and not Sufu is the major inhibitory regulator of Ci. Once released from Cos2 and Sufu, Ci translocates to the nucleus, however the regulation of the nuclear import of Ci (and GLI proteins) has not been investigated in any detail. In the nucleus, Ci binds to target sites, presumably replacing CiR, and activates gene expression. Notable targets include ptc, the upregulation of which confers negative feedback to the pathway. Ultimately, in both D. mela-nogaster and vertebrates, activated Ci or GLI proteins become substrates for CUL3-based E3 ubiquitin ligase SPOP (Speckle-type POZ; the vertebrate homologue of D. melanogaster Hib), leading to their degradation128–131.Vertebrate GLI proteins and primary cilia

The mechanism of signal transmission between SMO and GLI proteins has diverged between mammals and D. melanogaster. In mouse, loss of SUFU results in constitutive activation of the HH pathway132,133, but the loss of FU (also known as STK36) has no effect on

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HH signalling12. Other kinases and additional post-translational modifications have been suggested to have a role in regulating GLI activity134–139. The finding that vertebrate, but not D. melanogaster, HH signal-ling requires the primary cilia of a cell might explain some of the molecular differences between Ci and GLI regulation140. Genetic screens and targeted mutations have implicated a host of cilia-associated proteins in vertebrate HH signalling. In addition, several human diseases, collectively termed ciliopathies, that have symptoms characteristic of defective HH signalling, are caused by mutations in components of cilia.

Scrutiny of the HH signalling defects caused by muta-tions in ciliary proteins revealed that cilia are essential for keeping the pathway inactive, as well as transducing the activating signal. Clues as to why this is have come from studies of the subcellular location of components of the SHH signalling pathway. In unstimulated cells, PTC is observed in cilia and around the ciliary base141 and, although SMO, GLI2 and GLI3 are not detectable in cilia in the absence of ligand, they seem to transit through it129,142–144. The flux of GLI2 and GLI3 through cilia, which depends on the Cos2 orthologue KIF7, is essential for their proteolytic processing into the repres-sor forms143,145. The three kinases —PKA, GSK3β and CKI — implicated in GLI processing are associated with the basal body of primary cilia104,146–148 (FIG. 2b). Moreover, ACIII and other adenylyl cyclases149,150, which generate cAMP to activate PKA, are trafficked into cilia by a mechanism that depends on TULP3 (Tubby-related pro-tein 3) and Tectonic 1. These two proteins have also been implicated in HH signalling. Mice lacking either gene exhibit HH signalling defects reminiscent of the defects observed in embryos lacking cilia, and both proteins are observed at the base of cilia, a position that would allow them to gate proteins into the cilium shaft151. Recently, GPR161, which increases cAMP levels, has been found to localize to primary cilia in a TULP3-dependent manner. Moreover, loss of GPR161 increases SHH signal-ling, suggesting a link between PKA activity and SHH signalling152. Together with the observation that pro-teasomes are enriched at basal bodies153, this suggests that GLI proteins are phosphorylated and processed into their repressor forms as they exit cilia.

HH pathway activation induces changes in cilia com-position. PTC141 and GPR161 (REF. 152) exit the cilia and SMO enters, either via lateral transport from the plasma membrane102 or directly from an intracellular vesicle154 (FIG. 2b). The phosphorylated, active form of SMO inter-acts with β-arrestin and KIF3A, which facilitates its ciliary accumulation155,156, and SMO forms a complex with the ciliary proteins EVC (Ellis-van Creveld syn-drome protein) and EVC2 (REFS 157,158). This results in the accumulation of SMO in a region just distal to the basal body and enables signalling. HH signalling also increases the levels of GLI2, GLI3 and SUFU in cilia, particularly at the tip129,143,159. The end result of pathway activation is the dissociation of the GLI–SUFU complex, within the cilia142,144, and the transport of the newly liber-ated full-length, activated GLI2 and GLI3 proteins from the cilia to the nucleus bypassing proteolytic processing.

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Despite this wealth of observations, a coherent molecular mechanism by which a signal is transduced within the cilium remains elusive. Accumulation of SMO in the cilium is not sufficient to transduce the normal intra cellular signal, as cyclopamine (a small-molecule antagonist of SMO) promotes the translocation of SMO to cilia but blocks GLI activation160. Phosphorylation and a conformational change in the C-terminal tail of SMO is important105, and this seems to promote an interaction with EVC and EVC2 (REF. 158), but how this is coupled to post-translational changes in GLI proteins is unclear. Furthermore, how components of the signalling pathway are gated into the cilium is unclear, and what modifica-tions or interactions are responsible for regulating their function has not been deciphered. Equally intriguingly, the evolutionary origin of the role of cilia remains unclear. In planarians, cilia do not seem to be required for HH signalling, nevertheless, orthologues of FU and COS2 are present161. Thus, the most parsimonious interpretation is that the link between cilia and HH signalling was present in the bilaterian ancestor and this was then adapted and remodelled in distinct lineages.

acts as a component of the gene regulatory networks responsible for patterning the particular tissue, and this allows both the level and the timing of Ci and GLI activity to influence when and where genes are activated174.

Chromatin immunoprecipitation studies in neural and limb cells, using epitope-tagged versions of GLI1 or GLI3 in cells responding to SHH signalling, have revealed several thousand genomic binding sites for these pro-teins162–164,175. Most, but not all, of these contain a motif similar to the previously defined Ci- and GLI-binding consensus sequence GACCACCCA176. As expected, GLI binding is observed around the genomic areas encoding core targets of the pathway, such as PTC, in cells from each of the tissues analysed. More strikingly, many of the targets that are regulated exclusively in one tissue are also bound by GLI proteins in the other tissue, suggest-ing that GLI binding alone does not explain specificity of regulation162. In addition, functional tests in the neural tube identified GLI-binding enhancer elements acting as positive regulators of gene expression that require SHH signalling for their activation, whereas other GLI-binding elements act as inhibitors of gene expression and require the removal of GLI repressor activity for gene induction170. This is in line with the differential sensitivity of genes to the ratio of repressor Ci and GLI proteins to activator Ci and GLI proteins. It is also consistent with the observa-tion that, in mouse embryos lacking SHH, the removal of GLI3, which provides most of the transcriptional repressor activity, recovers the expression of some target genes111. Thus, some SHH target genes require positive input from a GLI transactivator to initiate transcription, whereas others are activated by removing the GLI repres-sor protein from the enhancer. The mechanism that deter-mines these different responses to GLI proteins remains to be determined.

The role of HH signalling in various developing tissues explains why genetic defects in the pathway lead to several severe congenital abnormalities10. In addition to ciliopa-thies, HH pathway defects can cause holoprosencephaly, which results from an incomplete cleavage of the forebrain along the ventral midline, or complex genetic diseases, such as Pallister–Hall syndrome, that involve polydac-tyly (additional fingers or toes) and other organ defects. Moreover, the involvement of IHH in the development of the endochondral skeleton of vertebrates can explain the relationship between HH signalling and the regulation of adult height177 and why point mutations in IHH cause brachydactyly (truncated limb digits)178.

HH acts in networks of secreted factors. HH signal-ling frequently induces the expression of additional secreted molecules to produce interacting signalling networks. For example, the hedgehog like appearance of D. melanogaster embryos lacking Hh is a consequence of a loss of reciprocal feedback between adjacent stripes of cells that normally express Wingless (Wg) and Hh179. This reciprocal positive feedback between Wg and Hh main-tains the expression of the two ligands in stripes along the anterior–posterior axis and elaborates the segmental development of the embryo. Similarly, during bladder regeneration in vertebrates, cells within the urothelium

www.nature.com/reviews/molcellbioFunctions of HH signalling

HH signalling has vital and diverse roles throughout animal development and adult tissue homeostasis. Here, we summarize some of the themes that connect these diverse functions.

Graded HH signalling and developmental pattern for-mation. HH acts as a long-range morphogen to control cell patterning and differentiation in several embryonic tissues107. Examples include the pattern of venation in D. melanogaster wing discs, the digit pattern in vertebrate limbs and the organization of neuronal subtype identity in the vertebrate central nervous system (CNS). In each case, Ci and GLI activity results in the induction of a very similar core set of target genes162–164, including genes encoding Ptc in D. melanogaster and in vertebrates PTC, Patched 2, HIP1 and GLI1, all of which participate directly in the pathway, either influencing the spread of the ligand or downstream signalling. However, diverse cellular responses are seen in different tissues owing to the regu-lation of different sets of target genes in response to gra-dients of HH ligands. Thus, in the wing disc, a gradient of Hh induces several genes, including decapentaplegic (dpp), collier and engrailed, at different thresholds165, whereas in the neural tube the genes encoding the transcription fac-tors NK6 homeo box 1 (NKX6.1), oligo dendrocyte tran-scription factor 2 (OLIG2) and NKX2.2 are induced by progressively higher concentrations of SHH166. There is evidence that different concentrations of ligand are trans-formed into different levels of intracellular signalling99,126, resulting in a Ci and GLI activity gradient167. As the acti-vation of some genes requires a greater change in the repressor to activator ratios of Ci and GLI proteins than others168–170, a gradient of Ci and GLI activity could con-tribute to tissue patterning activity. However, studies in several tissues indicate that the duration of HH signalling is also important171–173. This has led to a model in which Ci and GLI activity, which is controlled by HH signalling,

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secrete SHH to induce the expression of WNT proteins in the adjacent stromal cell layer, which in turn signal back to the urothelium180.

In addition to WNT, HH partners with other signal-ling molecules. In the D. melanogaster wing disc, Hh secreted from the posterior half of the disc induces the bone morphogenetic protein (BMP) family protein Dpp, in a stripe in the middle of the disc, which then controls patterning and growth of the nascent wing blade181. During vertebrate limb development, a set of linked feedback loops, involving SHH, BMP and the BMP antagonist gremlin 1 (GREM1), are responsible for limb outgrowth and patterning182. In this case, SHH signalling maintains the expression of GREM1 to limit BMP sig-nalling. An analogous signalling loop between SHH and BMP antagonists has been proposed to contribute to the elaboration of hair follicles in the skin183. Just as for SHH and WNT in the bladder, this involves reciprocal feedback between SHH-expressing cells in the epithelial layer and BMP-expressing cells in the mesenchymal layer. Similar examples of HH proteins participating in networks of signalling molecules that maintain and elaborate tissue architecture can be found in many tissues. Moreover, evi-dence of intracellular crosstalk between the HH pathway and other signalling pathways also adds to the complexity in understanding the interactions between the pathways (for a review, see REF. 184).

HH signalling in stem cells and cancer. Together with its function as a developmental morphogen, HH regulates the survival and proliferation of several tissue progenitor and stem populations11. During cerebellum development, SHH secreted by Purkinje cells supports the proliferation of granule cell precursors in the external granular layer185 by promoting the expression of several well-known stem cell and proliferative genes, including genes encoding MYC, cyclin D1, insulin-like growth factor 2 (IGF2) and BMI1 (REF. 186). In the adult brain, HH signalling maintains neural stem cells that continuously supply new neurons187,188. HH signalling is also required for the main-tenance of stem cells in a range of tissues, from the hair follicle to the haemato poetic system, and it is also involved in the injury-dependent regeneration of many organs, including the exocrine pancreas, prostate and bladder11. Whether there are common molecular mechanisms that link these mitogenic and survival roles of HH remains to be seen.

The influence of HH signalling on stem cells suggests why activation of the pathway has been found in vari-ous human tumours189. The heritable condition Gorlin’s syndrome is caused by heterozygousity in PTC190,191 and is characterized by a high incidence of the skin cancer basal cell carcinoma (BCC) and the cerebellum cancer medulloblastoma. Moreover, sporadic occurrences of BCC and a subset of medulloblastomas are the result of de novo mutational activation of the pathway189. In both BCCs and medulloblastomas, dysregulation of HH signalling in a stem cell and precursor cell popula-tion, respectively, seems to explain tumour formation. For medulloblastomas, the granular cell precursors in the neonatal cerebellum are the cells of origin and this

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explains the childhood bias of this tumour192. For BCCs the origin seems to be either stem cells within the hair follicle193 or in the interfollicular epidermis194. The recent regulatory approval of a drug that blocks SMO activity has raised the prospect that these tumours may now be amenable to treatment195.

In contrast to these ligand-independent tumours, many of the other tumours associated with HH pathway activation do not contain mutations in pathway com-ponents. Instead, pathway activation depends on ligand production, either by the tumour cells themselves or the surrounding stroma189,196. Two hypotheses, which are not mutually exclusive, have been suggested to explain the role of HH signalling in these tumours. First, the survival or proliferation of tumour cells directly requires HH signal-ling. In support of this, in vitro studies have demonstrated that HH signalling supports and enhances cancer cell growth197. In vivo evidence in support of this autocrine and juxtacrine mechanism is currently more limited and in some cases contentious198, although the recent observa-tion that HH signalling promotes Warburg-like glycolytic metabolism199, which is found in many tumours, suggests a possible role for signalling within the tumour cells. As an alternative, however, HH production has been hypo-thesized to promote the tumour microenvironment in a paracrine manner by signalling to the stroma, which then signals back to the tumour198. This could be analogous to the reciprocal signalling networks that HH establishes during embryonic development. The exact function of HH signalling in these tumours, and whether it represents a dysregulation of the normal stem cell maintenance and regeneration functions of HH signalling, remains to be determined. Disappointingly, however, recent clinical tri-als of drugs that target the HH pathway in patients with tumours classed as ligand-dependent have been discour-aging, suggesting that we still have much to learn about these cancers. Resolving the importance of HH signalling, and whether it acts autonomously or non-autonomously in ligand-dependent tumours, is crucial as these cancers affect many more individuals than those with BCCs and medulloblastomas.

Non-canonical HH signalling. The functions described above represent only a few examples of the roles ascribed to HH signalling. The list continues to grow, for instance, HH signalling has recently been shown to regulate pain perception200 and cellular metabolism199. Several of the non-canonical roles of HH signalling seem to depend on HH signalling via alternative, non-transcriptional mechanisms. Among these, HH signalling in axon and cell chemotaxis are the best described. The projection of spinal commissural axons towards the ventral mid-line requires SHH that is produced in the floor plate and depends on SHH signalling through PTC and SMO but not on a transcriptional response201,202. Instead, SMO-dependent activation of SRC family kinases has been implicated203. Once across the midline, the expression of HIP1 in commissural spinal neurons has been proposed to participate in their anterior routing, independently of SMO202. SHH also guides axons of retinal ganglion cells in the visual system204,205.

VOLUME 14 | JULY 2013 | 425REVIEWSBox 2 | Ten unanswered questions on Hedgehog signalling and its physiological roles•󰀂In what form(s), and by what route, does Hedgehog (HH) spread through tissues?•󰀂What is the composition of the HH receptor complex(es)?•󰀂How does Patched 1 (PTC) regulate Smoothened (SMO)?•󰀂How is the signal transduced from SMO to GLI and what controls the subcellular localization of GLI proteins and D. melanogaster Cubitus interruptus (Ci)?•󰀂What is the function of cilia and proteins localized at the cilia in vertebrate HH signalling?•󰀂What is the evolutionary origin of the role of cilia in the HH pathway and/or how was this function lost?•󰀂What non‑canonical mechanisms of HH signalling are activated by the pathway?•󰀂What functions does HH signalling have in addition to its roles in tissue development and homeostasis?•󰀂What role does HH signalling have in cancers other than basal cell carcinoma and medulloblastoma?•󰀂What is the mechanism of gene regulation by GLI proteins and how are cell type‑specific responses determined?Chemotaxis of fibroblasts induced by SHH signalling also seems to be independent of transcription206,207. Recent studies have suggested that this process does not require cilia. Instead, the small RHO GTPases RAC1 and RHOA, which are activated by SMO in a Gi-dependent fashion, have been implicated208. The activation of Gi proteins and downstream phospho lipase Cγ (PLCγ) might also

account for the Ca spikes observed in neural progenitors in response to SHH209. Intriguingly, a recent report indi-cates that cyclopamine, which blocks canonical signalling from SMO to GLI proteins, is sufficient to mobilize Ca2+, suggesting that cyclopamine acts as a partial and selective agonist for SMO signalling199. Finally, PTC has been pro-posed to act as a ‘dependence receptor’ that induces apop-tosis in the absence of SHH. This seems to be the result of PTC interacting with a protein complex, including down-regulated in rhabdomyosarcoma LIM (DRAL) and caspase 9, which triggers a caspase-dependent cell death pathway210,211. Clearly more work is required to elucidate the various proposed non-canonical signalling pathways and to understand their contribution to HH responses.

different branches of the bilaterian family emphasizes its importance and deep evolutionary origin. The conserved but unusual way in which HH ligands are generated and released from cells represented a great surprise. Moreover, the relatively recent finding that cilia are involved in verte-brate HH signalling but not in D. melanogaster has added a new twist to the field and revitalized interest in this pre-viously ignored organelle. Despite the advances, however, there are still large gaps in our knowledge of HH signalling and many unanswered questions (BOX 2). In some cases it seems likely that new tools and reagents will provide new insight, perhaps the increasing power of proteomic and metabolomic techniques will help address how PTC regulates SMO and the role of cilia in this process and in the downstream transduction of the signal. For other questions, such as the function of HH signalling in tissue development and stem cells, dissecting the complex gene networks that are involved and searching for commonali-ties between the networks in different cell types is likely to yield conceptual insight. It is perhaps the importance of these outstanding issues that say most about the pathway. Addressing these issues is crucial for understanding this

Outlookfascinating signalling pathway and for developing thera-Taken as a whole, the past 20 years of study has revealed a peutic strategies to target the medical consequences of its wealth of detail and an unexpected diversity in the mecha-dysregulation. Furthermore, given the surprises this path-nisms and functions of HH signalling. The identification way has delivered over the past two decades, who knows of the ligands and core components of the pathway in what secrets are yet to be revealed.

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Acknowledgements

The authors apologize to their colleagues whose work has gone unmentioned owing to space limitations. J.B is sup-ported by the Medical Research Council (MRC, UK) and the Wellcome Trust, and P.P.T. by the Ligue National Contre le Cancer Program ‘Equipe labellisée 2012’.

Competing interests statement

The authors declare no competing financial interests.

FURTHER INFORMATIONJames Briscoe’s homepage:http://www.nimr.mrc.ac.uk/research/james‑briscoePascal P. Thérond’s homepage:http://ibv.unice.fr/EN/equipe/therond.phpALL LINKS ARE ACTIVE IN THE ONLINE PDFNATURE REVIEWS | MOLECULAR CELL BIOLOGY

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