mechanisms and functions of hedgehog signalling across the ...zider.free.fr/papers/paper...

A defining event in the emergence of multicellularity was the harnessing and refinement of mechanisms that facilitate interaction and communication between cells. Three decades of intensive research by developmental biologists have identified the key molecular players that underpin these mechanisms and have unveiled the pleth-ora of processes that they control. With the recent rapid growth in the availability of complete genome sequences of a wide array of organisms, we are now gaining new insights into the origins and evolution of this molecular machinery. One clear conclusion to be drawn from all these studies is that evolution has worked with a rather limited number of signalling pathways to generate the remarkable variety and complexity of form and function that we see today, both across phyla and within the indi-viduals of a given species. Understanding how these path-ways have evolved and function can thus illuminate the origin of morphological diversity, as well as the molecular and cellular basis of development and disease.

Amongst the central canon of developmental signal-ling pathways — transforming growth factor-β (TGFβ)–bone morphogenetic protein (BMP), the JAK–signal transducer and activator of transcription (STAT) pro-tein kinases, epidermal growth factor receptor (EGFR), NOTCH, fibroblast growth factor (FGF), WNT and Hedgehog (HH) — the HH pathway has been and per-haps remains the most enigmatic. In contrast to the other pathways, early studies of HH signalling were exclusively based on genetic analysis in Drosophila melanogaster1; by a twist of evolutionary fate the pathway has been lost from that rival workhorse of developmental geneticists,

the nematode Caenorhabditis elegans2. Moreover, unlike members of most of the other pathways, HH signalling components were not initially identified through their potential to transform cultured mammalian cells or to promote tumour formation in mice and humans. Perhaps partly for this reason, the characterization of the molecular and cellular basis of HH signalling has lagged behind that of its more mainstream counterparts. However, another impediment to progress has undoubt-edly been the Byzantine biochemical nature of the path-way: the coupling of the HH ligand to cholesterol3; the role of a large multipass transmembrane transporter-like protein, Patched (PTC), as the ligand receptor4–6; the lack of physical interaction between HH or PTC and the signal transducer Smoothened (SMO), which is a member of the G protein-coupled receptor (GPCR) superfamily that is neither a receptor nor obviously coupled to heterotrimeric G proteins7. These features have all conspired to make the elucidation of the HH pathway both intriguing but apparently intractable in equal measure. Add to this its dependence in vertebrates on an ancient organelle — the primary cilium — that has seemingly been dispensed with by Drosophila spp. and other insects, and you have a story that continues to retain the capacity to confound and surprise even the experts in the field.

In this Review we provide an updated overview of the HH pathway in the context of its evolutionary ori-gins. We address the origin of the HH ligands and how they are processed and distributed; describe the core ele-ments of the signal transduction pathway, as defined by

*Institute of Molecular and Cell Biology, 61 Biopolis Drive, Singapore 138673. ‡Department of Biological Science, National University of Singapore, 14 Science Drive 4, Singapore117543. §Department of Genetics, Hyogo College of Medicine,1‑1 Mukogawa‑cho, Nishinomiya, Hyogo 663‑8501, Japan.Correspondence to P.W.I. e‑mail: [email protected]‑star.edu.sgdoi:10.1038/nrg2984 Published online 19 April 2011

Mechanisms and functions of Hedgehog signalling across the metazoaPhilip W. Ingham*‡, Yoshiro Nakano*§ and Claudia Seger*

Abstract | Hedgehog proteins constitute one of a small number of families of secreted signals that have a central role in the development of metazoans. Genetic analyses in flies, fish and mice have uncovered the major components of the pathway that transduces Hedgehog signals, and recent genome sequence projects have provided clues about its evolutionary origins. In this Review we provide an updated overview of the mechanisms and functions of this signalling pathway, highlighting the conserved and divergent features of the pathway, as well as some of the common themes in its deployment that have emerged from recent studies.

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Cadherin repeatsExtracellular calcium-binding domains originally identified in the calcium-dependent adhesion proteins, cadherins.

TNFR repeatsCysteine-rich motifs originally defined in the extracellular domain of the tumour necrosis factor receptor but also found in laminins and other receptors.

InteinsProtein segments that can self-excise and rejoin the remaining portions of the protein with a peptide bond.

their functional conservation between species, and their modes of action; and consider the extent to which the mechanisms of transduction vary between species and how the pathway has evolved through the co-option of pre-existing components. Finally, we trace the deploy-ment of the pathway in animal development through evolutionary time.

Structure and properties of Hedgehog proteinsHedgehog proteins are synthesized as approximately 45 kDa pro-proteins that undergo autocleavage to yield two similarly sized fragments8: an amino-terminal

polypeptide (HH-N), which is characterized by a sig-nal sequence and a highly conserved ‘Hedge’ domain, and a carboxy-terminal polypeptide (HH-C), which contains the highly conserved ‘Hog’ domain9 (FIG. 1a). All of the signalling activity of HH proteins is vested in the secreted HH-N fragment, which has the unusual property of being covalently coupled to cholesterol at its C-terminal end3. The only known function of the Hog domain is to promote the autocleavage reaction, a process that resembles the protein-splicing activity of inteins and requires the ‘Hint’ region, which forms part of the Hog domain10. The cleavage reaction is driven

Figure 1 | Structure of Hedgehog and Hedgehog-related proteins. a | The full-length Hedgehog (HH) protein is comprised of two distinct domains: the amino-terminal ‘Hedge’ domain (shown in dark green) and the carboxy-terminal ‘Hog’ domain (shown in blue), both of which are also found in proteins other than members of the HH family. The Hedge domain is preceded by a signal peptide sequence (SS; shown in brown). The Hog domain itself can be separated into two regions; the first two-thirds share similarity to self-splicing inteins, and this module has been named ‘Hint’, whereas the C-terminal one-third binds cholesterol in HH proteins and has been named the sterol-recognition region (SRR). In other Hog domain-containing proteins this region is referred to as the adduct recognition region (ARR), as the nature of the adduct is not known. The Hog domain promotes autocleavage to release the N-terminal Hedge domain (HH-N). b | Hedge domain-containing proteins predate HH and are found in protozoa and metazoa. The choanoflagellate Monosiga brevicollis has a large Hedge domain-containing transmembrane protein that also contains a von Willebrand A domain (vWA), two cadherin repeats (CA repeats), multiple tumour necrosis factor receptor repeats (TNFR repeats), as well as single immunoglobulin (Ig), immunoglobulin I-set (I-set) and epidermal growth factor (EGF)-like repeats14; the Hedge domain is designated Nhh. Proteins with a similar domain organization, though with an expansion of the CA repeats at the expense of the TNFR repeats, are encoded by the so-called hedgling gene in the basal metazoan, Amphimedon queenslandica (sponge)15 and the Cnidarian, Nematostella vectensis15. Interestingly, the hedgling structure resembles that of the atypical proto-cadherin family members, including the Fat and Dachsous proteins, which have important roles in planar cell polarity in Drosophila melanogaster154. LAG, Laminin A-G motif; SH2, SH2 motif.

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ChoanoflagellatesA group of protists that possess one flagellum at some stage of their life history.

EumetazoaAn animal sub-kingdom that includes Cnidarians, the ctenophorans and the bilaterians.

PoriferaA phylum of multicellular animals with only two cell layers, the ectoderm and the endoderm, which are separated by an acellular mesoglea. Commonly known as sponges.

CnidariansA simple and ancient phylum of multicellular animals, such as jelly fish or corals, found mainly in marine environments.

PalmitoylationThe covalent attachment of palmitic acid or other fatty acids to cysteine residues of proteins that promotes their association with membranes.

Imaginal discsThe undifferentiated epithelial sheets of cells that give rise to adult structures such as wings and legs.

LipophorinA family of high-density lipid-transporting lipoproteins found in the heamolymph of insects.

GlypicansA family of heparan sulphate proteoglycans that are localized to the cell surface via GPI anchors.

GPI anchorA glycolipid, glycosylphos-phatidylinositol, linked to the C-terminal amino acid of proteins anchoring them to the outer leaflet of the plasma membrane.

by a nucleophilic attack on a highly conserved GCF motif in which cholesterol, recruited by the sterol rec-ognition region (SRR) of the Hog domain, serves as the electron donor3.

The Hedge and Hog domains have ancient origins. Although Hedgehog proteins are the only proteins known to be conjugated to cholesterol in this way, the role of the Hog domain in processing secreted proteins seems to be an evolutionary ancient one: Hog domain proteins have been identified in a wide variety of organ-isms, including red algae, dinoflagellates and mosses, as well as throughout the metazoa (although not in higher plants)11. In the choanoflagellate Monosiga ovata, the protist that is most closely related to the metazoa, the ‘Hoglet’ gene encodes a protein with a C-terminal Hog domain that is coupled to an N-terminal domain with sequence similarity to cellulose-binding proteins12. However, in other organisms Hog domains have been found to be associated with an assortment of unrelated N-terminal domains. Only in the eumetazoa does the association of the Hedge and Hog domains in the form of Hedgehog proteins first appear11.

The Hedge domain, which has a striking structural similarity to the Streptomyces albus metalloprotein-ase d,d-carboxypeptidase13, is also found in Monosiga spp., in the N-terminal extracellular domain of a large transmembrane protein14 (FIG. 1b). A protein with a sim-ilar domain organization is encoded by the so-called Hedgling gene in the basal metazoan Amphimedon queenslandica (Porifera)15. The Cnidarian, Nematostella vectensis (the sea anenome) also has a Hedgling gene, as well as two true Hedgehog genes16; by contrast, no Hedgling genes have been reported in any bilaterian spe-cies. Thus, it seems likely that Hedgehog proteins first arose in the common ancestor of the Cnidarians and the bilateria more than 650 million years ago through the combination of domains that were present in pre-existing proteins, and that the Hedgling gene was sub-sequently lost from the bilateria11. Most bilaterians have been shown to possess at least one Hedgehog gene, with the genome expansions in vertebrates giving rise to three genes in amniotes (Desert Hedgehog (Dhh), Indian Hedgehog (Ihh) and Sonic Hedgehog (Shh)) and four or five (Dhh, Ihha, Ihhb, Shha and Shhb) in differ-ent teleost species17. A notable exception is C. elegans, which possesses multiple Hog domain-containing proteins but lacks a true Hedgehog gene2. By contrast, some other nematode species have retained a Hedgehog gene, though, like C. elegans and its closer relatives, they have lost some of the components of the core signal transduction pathway11 (described below).

Lipid modifications control the release and move-ment of HH. In addition to the covalent coupling of cholesterol to their C-termini, HH-N proteins also undergo palmitoylation at their N-termini, a modifica-tion that is promoted by an acyl transferase encoded in D. melanogaster by the skinny hedgehog (ski) gene18 and in vertebrates by its orthologue, HHAT19 (FIG. 2). This dual lipid modification of the HH signalling protein has

important effects on its properties, both enhancing its membrane association20 and potentiating its secretion and range of action. The secretion of lipidated HH-N requires the activity of a large multipass transmembrane protein, Dispatched (DISP)21. In mutant animals lack-ing DISP function, HH accumulates in producing cells and all HH-elicited responses are lost, except in cells that are immediately adjacent to signal producers21–24. Truncated proteins that are engineered to mimic HH-N but which lack the cholesterol moiety can override this requirement for DISP; however, the range and potency of such cholesterol-free HH-N is severely compromised compared with the normal protein, suggesting that the modification is crucial for the extracellular move-ment of the signal following secretion25,26. Consistent with this, the biochemical analysis of vertebrate SHH expressed in tissue culture cells indicates that lipid modification promotes the formation of freely diffusible multimeric complexes27.

In D. melanogaster, studies of HH signalling in imaginal discs have shown that the cholesterol adduct is essential for the incorporation of HH-N into lipoprotein particles that seem to mediate its long-range transport across epithelia28 (FIG. 2). The assembly of these HH sig-nalling entities is promoted by interaction with lipophorin, an apo-lipoprotein that circulates in the haemolymph of the fly29. Lipophorin is recruited to HH-N-secreting cells by its interaction with the heparin sulphate moieties of the glypicans Dally and Dally-like29. These proteoglycans, which can also interact with HH30,31, localize to the apical surfaces of epithelial cells via GPI anchors, the cleavage of which seems to be required for effective long-range HH signalling32. Although the properties of the Dally and Dally-like mutants can been subject to differing interpre-tations29,32,33, one appealing possibility is that glypicans promote the assembly of HH-N–lipophorin particles at the plasma membrane and that cleavage of their GPI anchor facilitates the release and dispersal of HH from producing cells (FIG. 2).

Notably, HH-N also localizes to the basolateral mem-brane of secreting and receiving cells in imaginal discs, leading to the proposal by some authors that this repre-sents the major route of signal secretion and spread34,35. Experimental generation of HH-N ‘traps’ in groups of responding cells, however, suggests that the range of the basally secreted protein is fairly limited32: this raises the interesting possibility that basal HH-N principally acts as a short-range signal, whereas apically released HH-N acts as a long-range signal. In line with this, api-cally localized HH-N can be observed across the imaginal disc epithelium in a graded distribution that mirrors the functionally defined gradient of signalling activity32.

Whether the secretion and movement of HH proteins is regulated in a similar way in vertebrates is currently unclear. Certainly, lipid modification has an important role in the release of SHH25, and a graded distribution that mirrors its dose-dependent effects can be observed in the developing neural tube. But whether this distribu-tion reflects the movement of the protein itself or the movement of the cells that have taken up the protein remains to be determined36.

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GLI protein familyZinc finger domain-containing transcription factors mediating HH activity, so-called because the gene encoding the founding member GLI1 is frequently amplified in glioblastoma cells.

Another important parameter in regulating HH protein distribution is its sequestration by various trans-membrane proteins. The best characterized of these is PTC, which also has a pivotal role in the reception and transduction of the HH signal (see below). Activation of the HH pathway leads to the upregulation of PTC expression, which in turn enhances HH sequestration and thus attenuates its signalling activity. Recent studies in D. melanogaster have suggested that the Brother of interference hedgehog (BOI) protein, which functions as a co-receptor for the HH signal, also regulates HH sig-nalling by ligand sequestration37. Hedgehog-interacting protein (HHIP), which was first identified in vertebrates but which was subsequently found to be encoded by the sponge genome, has a similar role38. Expression of HHIP is also upregulated in response to HH signalling but, in contrast to PTC and BOI, HHIP seems to act exclusively to modulate HH ligand distribution and has no role in signal reception or transduction. Interestingly, D. mela-nogaster lack both an HHIP-encoding gene, as well as the zinc-binding site in HH that is crucial for its interaction with HHIP39.

The Hedgehog signal transduction pathwayOriginally defined through genetic analysis in D. mela-nogaster 1, the components of the HH signalling path-way have subsequently been functionally characterized in a number of vertebrate species — principally, mouse, zebrafish and human — and have also been identified through genome sequence analyses in species from a

wide range of phyla (FIG. 3). These studies have revealed a high level of conservation of the ‘core’ components of the signal transduction pathway that is likely to extend across the eumetazoa, as well as some variation in their deployment that has arisen over the course of evolution. First, we outline the highly conserved core aspects of the pathway and then consider its divergence between species.

The GLI transcription factors are key effectors of HH activity. Although recent studies have suggested a role for HH in modulating the cytoskeleton via SRC family kinases40, the most widespread and best-characterized response of cells to HH signalling is the upregulation of transcription of target genes. Only a few such targets have been described in detail41–44, but recent genome-wide analyses suggest that there are several hundred45–47. Some targets (for example, the gene encoding PTC) are shared across different phyla44,48–50, whereas others are almost certainly specific to particular orders, families or even species.

The principal mediators of the transcriptional response to HH are members of the zinc finger-containing GLI protein family43,44,51. Genes encoding GLI proteins are found in the most primitive metazoa, the sponges52, and so predate the probable origin of the HH pathway itself (FIG. 3). D. melanogaster has a single GLI protein that is encoded by the cubitus interruptus (ci) gene44. The CI protein and its vertebrate counterparts, GLI2 and GLI3, are bi-functional transcription factors: their

Figure 2 | Lipid modification and release of the Hedgehog ligand. Following its translation, full-length Hedgehog (HH) undergoes autoproteolysis in the endoplasmic reticulum (ER)155, resulting in its covalent coupling to cholesterol. The amino-terminal ‘Hedge’ domain (HH-N) fragment is further modified through N-terminal palmitoylation that is promoted by the transmembrane acyl transferase Skinny hedgehog (SKI). Release of this doubly lipid-conjugated form of HH requires the activity of the multipass transmembrane protein Dispatched (DISP), which probably transports the protein across the plasma membrane. Once on the outer surface of the cell, HH remains associated with the lipid bilayer and interacts with the heparan sulphate moieties of the glypicans that are encoded by the dally and dally-like genes (in Drosophila melanogaster). These are also thought to recruit the apolipoprotein lipophorin, which, together with HH, becomes assembled into lipoprotein particles. Release of these particles might be mediated by the phospholipase C-like Notum, which cleaves the GPI anchors from the glypicans (indicated by scissors).

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Figure 3 | Conservation of individual molecules across species with phylogenetic representation. Genomic inventory of Hedgehog (HH) pathway components in species representative of different phyla. The components are colour-coded to indicate their likely origin and are distributed between receiving cells (shaded beige, nuclei shown as ovals) and signalling cells (shaded purple, top of panels). Interactions established on the basis of genetic and/or biochemical data are indicated by the clustering of components. CKI, casein kinase 1; COS, Costal; DISP, Dispatched; FU, Fused; GSK3, glycogen synthase kinase 3; HHIP, Hedgehog-interacting protein; HLING, hedgling; IHOG, Interference hedgehog; PKA, protein kinase A; PTC, Patched; SKI, Skinny hedgehog; SMO, Smoothened; SUFU, Suppressor of fused. Left panels are modified, with permission, from REF. 38 © (2009) CSHL Press. Upper right panel is modified, with permission, from REF. 52 © (2010) Macmillan Publishers Ltd. All rights reserved.

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RND proteinsA superfamily of transmembrane proteins, originally defined by bacterial proteins that function as transporters in drug resistance, legume nodulation and cell division (hence RND), but now including various eukaryotic proteins with diverse functions.

ProteasomeLarge protein complex that contains proteases that regulate the concentration of particular cellular proteins and degrade misfolded proteins by proteolysis.

full-length forms function as transcriptional activators, but they can be converted into lower molecular mass transcriptional repressors by removal of their C-terminal activation domains51,53. It is principally by modulating the modification, processing and nuclear trafficking of the GLI proteins that HH signals elicit their transcrip-tional responses (FIG. 4). In vertebrates, these responses are amplified by a third member of the family, GLI1, which lacks the N-terminal repressor domain and functions exclusively as a transcriptional activator54.

Reception and transduction of the HH signal by PTC and SMO. The transcriptional response to HH depends on the activity of a heptahelical transmem-brane protein, SMO, which has been shown by genetic analysis to be indispensable for HH signalling both in D. melanogaster and in vertebrates55–59. Genome sequence analyses have identified SMO orthologues across the bilateria, as well as in Cnidaria, but not in Amphimedon queenslandica, which is consistent with the proposed eumetazoan origin of the HH pathway38. Despite being a member of the superfamily of GPCRs, there is no evidence that SMO acts as a receptor for HH proteins. Rather, its activity is regulated by PTC, ortho-logues of which are present in many bilaterian species38 and in Cnidaria16 but, like SMO, not in Amphimedon queenslandica52 (FIG. 3).

Genetic analyses in D. melanogaster and vertebrates have shown that PTC inhibits SMO activity, so the acti-vation of HH-target genes by the GLI proteins ultimately depends on the inhibition of PTC (FIG. 4). Inhibition is effected by HH binding to the two large extracellular domains of PTC60, an interaction that is promoted in D. melanogaster by the transmembrane proteins Interference hedgehog (IHOG) and BOI6, and in ver-tebrates by their orthologues CDO and brother of CDO (BOC)61. Genes encoding these proteins are present in the genomes of Amphimedon queenslandica38,52 and the placazoan Trichoplax adhaerens62 and thus predate the origins of the HH pathway — but they have been recruited into the pathway to function as co-receptors for the HH proteins. Interestingly, the fibronectin domain that mediates binding to HH differs between the orthologous D. melanogaster and vertebrate proteins63.

A major unresolved issue in HH signalling is the biochemical mechanism by which PTC inhibits SMO activity. Early suggestions that the two proteins form a HH receptor complex64 were refuted by the demonstra-tion that PTC inhibits SMO non-stoichiometrically65. The similarity of PTC to bacterial RND proteins66 has led to the suggestion that it may act as a transporter, possibly of a lipid activator or inhibitor of SMO65. This is supported by the finding that the steroidal alkaloid cyclopamine binds to and inhibits SMO activity67. Recent studies in D. melanogaster have implicated the phospholipid phosphatidylinositol-4-phosphate (PI4P; also known as PtdIns4P) in the regulatory relationship between the two proteins68. These findings, based on the genetic modulation of PI4P synthesis and turnover and supported by data from experiments using mam-malian fibroblasts, suggest that SMO is activated by an

increase in intracellular PI4P levels and that PTC modu-lates these levels by inhibiting the activity of the kinase that is responsible for PI4P synthesis68. Exactly how PI4P levels could regulate the hypothetical lipid modulator of SMO remains unclear, but one suggestion is that it might mediate the intracellular trafficking of such a molecule.

Downstream of SMO. Despite this major gap in our knowledge, it is well established that SMO transduces the HH signal into the cell and attenuates the processing of the CI (GLI in vertebrates) proteins: but how is this accomplished? Cleavage of CI (GLI) occurs in the absence of HH stimulation — and hence of SMO activation — and is promoted by the phosphorylation of motifs within the C-terminal domain of CI (GLI)69. These motifs are phosphorylated, first by protein kinase A (PKA), which primes them for further phosphorylation at adjacent residues by two other serine/threonine kinases, glycogen synthase kinase 3 (GSK3) and casein kinase 1 (CKI)69–71. The phosphorylated motifs are recognized by the F-box protein BTRCP (known as SLMB in D. melanogaster), a component of the SCF complex, which in turn cataly-ses the ubiquitylation of the C terminus, targeting it for degradation by the proteasome to yield the truncated N-terminal repressor forms of CI (GLI)72,73. Such kinase recognition motifs are found in GLI proteins across the bilateria, which is consistent with the modulation of their phosphorylation being fundamental to the response of cells to HH signalling. Exactly how SMO activity effects this modulation, however, is still far from fully understood (BOX 1; see Supplementary information S1 (figure)).

The most complete picture that is currently available comes from studies in D. melanogaster in which genetic analysis has identified costal 2 (cos2) as a key negative regulator of the HH pathway74. The cos2 gene encodes the D. melanogaster orthologue of KIF7 (REFS 75,76), which is a highly conserved member of the kinesin family of motor proteins with roles that predate the emergence of the HH pathway; in the slime mould Dictyostelium discoideum for instance, KIF7 is required for the trans-port of cellular organelles77. Despite the integrity of the motor domain in D. melanogaster COS2 not being fully conserved76, studies in tissue culture cells have indi-cated that it retains considerable motor activity and can mediate the movement of tagged forms of CI within the cytoplasm in an ATP- and microtubule-dependent manner78. Although the importance of this movement remains unclear, many lines of evidence point to a role for COS2 in recruiting CI, PKA, GSK3 and CKI to form the HH signalling complex (HSC)75,79–81. Activation of SMO causes the partial dissociation of this HSC, thus relieving CI from phosphorylation and promoting the accumulation of its full-length form (FIG. 4b).

The HSC contains another ancient serine/threonine kinase, Fused (FU), orthologues of which control cyto-kinesis in higher plants82 and cell polarization and chemotaxis in Dictyostelium discoideum83. In D. mela-nogaster, FU is a positive regulator of the HH pathway: translocation of FU to the plasma membrane induces the phosphorylation of COS2, which promotes the dissocia-tion of CI from the HSC79,84. In addition, FU promotes

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the phosphorylation of suppressor of fused (SUFU)30, a protein that is highly conserved throughout the metazoa and shares a strong structural similarity to several bacte-rial proteins85. In D. melanogaster, SUFU binds to and stabilizes CI in the cytoplasm86,87, attenuating its nuclear import once released from the HSC88,89; phosphorylation of SUFU abrogates this interaction, thereby promoting target gene activation by CI. Thus, in unstimulated cells the absence of SUFU has no effect, but its removal is sufficient to restore pathway activity in the absence of FU activity.

Pathway divergence and the primary cilium. In contrast to the situation in D. melanogaster, loss-of-function muta-tions of the mouse SUFU orthologue cause widespread ligand-independent activation of HH target genes90,91, a phenotype which is similar to that seen in PTC loss-of-function mutants92. Interaction between SUFU and GLI proteins seems to have a key role in promoting the processing of GLIs into their truncated repressor forms in the absence of HH signalling93,94. Strikingly, functional studies have revealed that SUFU has a similarly crucial role in HH signalling in planarians95,96, suggesting that

the PTC–SMO–SUFU–GLI axis represents the core components of the HH pathway, and implying that the role of SUFU has been largely subsumed by COS2 in D. melanogaster.

A further notable interspecies difference, which was initially uncovered in mice, is the dependence of HH signalling in some species on the primary cilium97 — an ancient organelle that is found in many eumetazoa but that is conspicuously absent from most D. melanogaster cells98. In mammalian cells, exposure to HH ligand causes SMO to localize to the axoneme of the primary cilium99, quickly followed by the accumulation of the SUFU–GLI complex at the cilium tip100,101. This translocation seems to facilitate the dissociation of the complex in response to SMO activity102, thereby abrogating cleavage of GLI proteins and promoting the import of their full-length forms into the nucleus where they are converted, by an as yet uncharacterized mechanism, into labile transcrip-tional activators93. How this dissociation is effected by SMO and how the GLI proteins move from the cilium to the nucleus is currently unclear.

Paradoxically, despite the importance of SUFU, the murine orthologue of FU has no role in HH signalling

Figure 4 | The Hedgehog signalling pathway in Drosophila melanogaster. Schematic representation of the subcellular localization and interactions of the core components of the Hedgehog (HH) signalling pathway in Drosophila melanogaster, in the absence (part a) or presence (part b) of HH ligand. In uninduced cells (part a), Patched (PTC) inhibits Smoothened (SMO) (dashed inhibitory arrow), which is located predominantly in intracellular vesicles. Cubitus interuptus (CI) is recruited to the Costal 2 (COS2) scaffold protein, which also recruits a number of serine/threonine kinases to form the HH signalling complex (HSC) (represented by the cluster of proteins; centre); phosphorylation (P) (dashed arrow) of full-length CI (CI-FL) by protein kinase A (PKA), glycogen synthase kinase 3 (GSK3) and casein kinase 1 (CKI) generates recognition signals for the F-box protein Slimb (SLMB), which catalyses the ubiquitylation of the carboxyl terminus of the protein, targeting it for degradation by the proteasome. The resulting truncated, repressor form of CI (CI-R) dissociates from the complex (solid arrow) and translocates to the nucleus where it represses transcription of HH target genes. Secreted HH binds to the transmembrane proteins Interference hedgehog (IHOG)and Brother of interference hedgehog (BOI; not shown) (part b), which present HH to PTC. Binding of HH to PTC blocks the activity of PTC, thereby releasing SMO from inhibition; SMO translocates to the plasma membrane where it is phosphorylated by PKA, GSK3 and CKI. This phosphorylation causes a conformational change in the SMO C-terminal domain, enhancing its interaction with COS2. Phosphorylation of COS2 by Fused (FU; dashed arrow) causes CI to be released from the HSC, abrogating its phosphorylation and so promoting the accumulation of its full-length156 form. FU-dependent phosphorylation of Suppressor of fused (SUFU; dashed arrow) promotes its dissociation from CI-FL, allowing CI-FL to translocate to the nucleus where it undergoes further modification to its activated form (CI-A) and thus promotes the transcriptional activation of HH target genes.

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in mice103,104, and although another kinase, CDC2L1, has been implicated as a negative regulator of mammalian SUFU105, there is currently no evidence that SUFU is phosphorylated in response to HH activity102. By contrast, a requirement for KIF7, the murine COS2 orthologue, is conserved101,106,107. Like its D. melanogaster counterpart,

KIF7 can physically interact with GLI proteins, and its motor activity seems to be essential for the transport of GLI3 to the tip of the primary cilium in response to HH stimulation101,107. In the absence of HH signalling, KIF7 predominantly localizes to the basal body of the primary cilium101,107, a structure that is enriched in pro-teasomes108,109 and PKA110. Thus, like COS2 in D. mela-nogaster, KIF7 may control the processing of GLI proteins by regulating their intracellular localization.

Recent studies have shown that the primary cilium is also required for HH signalling in zebrafish111–113, suggesting that it is a conserved feature of the pathway throughout the vertebrates. By contrast, disruption of ciliogenesis has no discernible effect on HH signalling in Planaria95,96,113, implying that the association may be vertebrate specific. Intriguingly, although FU is dis-pensable for HH signalling in planarians and mice, it is essential for motile ciliogenesis in both species95,104, as is KIF27, the closest planarian homologue of COS2 (KIF7). These findings suggest that the association between HH signalling and ciliogenesis may be ancestral, with cilia-associated proteins, such as FU and KIF7, having been co-opted into the HH pathway at an early stage in its evo-lution. In this view, the function of cilia-associated genes has been retained by the HH pathway in D. melanogaster despite the loss of primary cilia; in Planaria their activi-ties have somehow been dispensed with for HH pathway activity, but retained for ciliogenesis. Clearly, a compre-hensive understanding of the evolution of the pathway requires functional analyses in a wider range of species than is currently available. However, several ideas about its origins have been proposed (BOX 2).

Diversification of HH function and deploymentHH signalling has a multitude of functions in embryonic and adult tissues: it acts as a key mediator of fundamen-tal processes, such as cell fate specification, prolifera-tion and patterning, as well tissue morphogenesis and homeostasis — roles that have been extensively reviewed elsewhere114–116. We focus on recent findings that shed light on the evolutionary origins of HH deployment and its roles in body segmentation, tissue regeneration and homeostasis.

Hedgehog and body segmentation in arthropods and annelids. The segmental subdivision of the D. mela-nogaster embryo along its anteroposterior axis is con-trolled by a cascade of transcription factor interactions that culminates in the establishment of domains of expression of hh and wingless (wg) (the D. melanogaster orthologue of vertebrate WNT1, which encodes another secreted signalling protein). The interface between these cell populations defines each parasgemental boundary and their mutual interaction, mediated by the HH and WNT proteins, is crucial for boundary maintenance (FIG. 5Aa). Absence of HH activity results in the loss of wg transcription and the breakdown of segmental bounda-ries; by contrast, derepression of the HH pathway causes wg to be misexpressed and segmental boundaries to be duplicated117,118 (FIG. 5Ab). In addition, HH and WNT pattern the parasegment along its anteroposterior axis,

Box 1 | Smoothened: sequence conservation and divergence across phyla

Positioned at the nexus of the extracellular and intracellular sections of the Hedgehog (HH) pathway, Smoothened (SMO) holds the key to a comprehensive understanding of the mode of pathway action. Inhibition of SMO activity by Patched (PTC) and the modulation of its subcellular localization in response to HH activity are common themes across species, but the mechanisms and importance of these processes remain poorly understood, as does the means by which SMO engages with the intracellular HH signalling complex.

Sequence comparisons and structure–function analyses provide some clues about SMO function, but also highlight perplexing differences between species (see also Supplementary Information S1 (figure)). SMO is a divergent member of the frizzled (FZ) family of G protein-coupled receptors (GPCRs), sharing with them the amino-terminal extracellular cysteine-rich domain (CRD) that is crucial for binding of secreted WNT proteins by FZ proteins. Although no similar ligand-binding role is known for SMO, the CRD is highly conserved from Cnidaria to humans, and missense mutations in Drosophila melanogaster and zebrafish have shown that its integrity is crucial for SMO function143,144.

The central region of the protein, containing the seven membrane-spanning domains, also shows a high level of conservation across phyla. Notably, the seventh transmembrane domain (TM7) and flanking regions are encoded by a single exon, the boundaries of which are absolutely conserved from Cnidaria to humans. Just N-terminal to TM6 are several highly conserved residues, which in classical GPCRs are crucial for coupling to G proteins; mutation of one of these, R474, has been show to inactivate SMO in D. melanogaster143. There is evidence from functional analyses in D. melanogaster that SMO can modulate cyclic AMP levels via interaction with Gαi145.

By contrast, the intracellular carboxy-terminal domain (CTD) of SMO is highly divergent across phyla. The CTD of Planaria, spider and D. melanogaster SMO are substantially longer than those in other studied species. In D. melanogaster, the last 59 amino acids have been implicated in binding the serine/threonine kinase Fused (FU)146; notably there is little conservation of this sequence even between insects and none between D. melanogaster and zebrafish, the only other organism in which FU has been reported to function in HH signalling147. The involvement of FU in HH signalling in other species awaits further investigation.

Direct interaction between SMO and the Costal 2 (COS2) protein has also been shown to be crucial for HH signalling in D. melanogaster, and a COS2-binding site has been identified in the SMO CTD. This sequence shows over 50% conservation within insects and also in the crustacean, Daphnia pulex, but is not found in vertebrates, sea urchin, annelids or even spiders, despite the fact that the function of KIF7 (the COS2 orthologue) is conserved, at least in vertebrates. COS2 preferentially binds to phosphorylated SMO30 and the CTD includes a number of K/R-K/R-X-T/S sites that are phosphorylated by protein kinase A (PKA)87, neutralizing electrostatic interactions that are mediated by adjacent clusters of positively charged Arg residues, thereby inducing a conformational change in the CTD148. The same electrostatic interactions between clusters of Arg residues have been shown to play an important part in regulating conformational changes in the CTD in vertebrate SMO. However, whereas the PKA sites are conserved in insects, crustaceans and arachnids, they are absent from vertebrate SMO, raising the question of how the conformation is regulated in these species. GPCR kinase 2 (GRK2) has been implicated in HH signalling in both D. melanogaster and vertebrates149,150, and the SMO CTD contains potential consensus sites for GRK2; however, the functional importance of these has not been tested in vertebrates.

An important feature of vertebrate SMO is its localization to the primary cilium in response to HH activity. A putative localization motif has been identified at the juxtamembrane region of the CTD151 that is conserved in all known vertebrate SMO. Remarkably, however, this motif is also conserved in D. melanogaster and Planaria, neither of which requires primary cilia for HH signalling, but it is absent from other invertebrates, including mosquito, beetle, bee, wasp and aphid, raising questions as to its functional importance.

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DeuterostomiaAnimals characterized by the formation of distinct mouth and anal openings during embryonic development.

SANT1A potent HH pathway antagonist that directly binds and inhibits the activity of both wild-type and oncogenic forms of SMO.

ProtostomesAnimals the embryonic development of which is characterized by the formation of a single opening.

in part by regulating the expression of ligands for EGFR and NOTCH119.

Orthologues of the hh and wg genes in the flour beetle, Tribolium castaneum, exhibit an almost identical pattern of expression in the developing embryo, and functional studies using RNAi have confirmed a conserved role for both genes in the maintenance and patterning of seg-ments in this insect120. Moreover, expression studies in species from two other arthropod sub-phyla, the cheli-cerata Euscorpius flavicaudis and the crustacean Artemia franciscana, have revealed a similar spatial deployment of HH, implying a conserved role for HH (and WNT) in segmental maintenance and patterning throughout the arthropods121.

Like the arthropods, two other bilaterian phyla — the annelids and the vertebrates — are characterized by their segmented body plans. Arthropods, annelids and verte-brates belong to divergent superclades — the Ecdysozoa, Lophotrochozoa and Deuterostomia, respectively — all three of which include phyla that lack overt body seg-mentation. This could mean that segmentation evolved independently within these three lineages; alternatively, it is possible that Urbilateria, the last common ances-tor of bilaterian animals, was segmented and that this condition has subsequently been lost from some phyla. Although the lack of evidence of segmental expression of Hedgehog genes in vertebrates argues against the latter

hypothesis, the patterns of expression of the hh and wg (WNT1) genes in the embryo of the annelid Platynereis dumerilii display a striking similarity to those of their arthropod counterparts122. These recent findings con-trast with earlier studies in other annelid species that failed to identify clear homologies in the expression patterns of wg and hh orthologues123,124. However, their importance seems quite compelling in the light of func-tional manipulations using two small-molecule inhibi-tors of the HH pathway, cyclopamine and SANT1, which revealed an essential role for HH in maintaining both the expression of wg (wnt1) and the segmental boundaries in Platynereis spp. embryos122. Taken together, these find-ings suggest a common origin of segmentation, at least within the protostomes.

Hedgehog and regeneration in planarians. Recent studies of regeneration in the unsegmented flatworm, Planaria have uncovered a role for HH signalling in the anteroposterior patterning of these animals that bears striking analogies to its function in annelid and arthro-pod segmentation. Remarkably, derepression of the HH pathway (achieved by RNAi knock down of PTC activ-ity) causes the suppression of head regeneration follow-ing amputation; instead, the posterior trunk fragments form double abdomens95,96. Conversely, knock down of HH activity has the opposite effect, causing anterior

Box 2 | Origins and evolution of the Hedgehog pathway

The Hedgehog (HH) pathway has a number of unusual, if not unique, features that make speculation about its origins particularly intriguing. Prominent among these are the coupling of the signalling moiety of HH to cholesterol and the close homology between Patched (PTC) and proteins involved in cholesterol homeostasis. For example, PTC has a sterol- sensing domain that it shares with proteins such as HMG-CoA reductase, sterol regulatory element-binding protein cleavage-activating protein (SCAP) and niemann-pick C1 protein (NPC1)152. Consideration of these features led Hausmann and colleagues153 to propose that the HH pathway evolved from a pre-existing system that controlled lipid homeostasis in single-celled eukaryotes, in which Smoothened (SMO) acted as a sensor of extracellular lipid levels and PTC functioned as a transporter of this lipid. A key element of this model is the transcriptional regulation of Ptc by SMO; accordingly, activation of SMO by elevated lipid levels would lead to an upregulation of PTC expression, resulting in increased transport of the lipid out of the membrane153. In this view, the crucial event in the evolution of the HH pathway would have been the fortuitous fusion of DNA sequences encoding the ‘Hedge’ domain and the ‘Hog’ domain. This fusion would have facilitated the coupling of the SMO-activating lipid to the Hedge domain protein through the processing reaction that is promoted by the Hog domain, thus creating a secreted form of the lipid normally transported by PTC. Also, attachment of the lipid to the Hedge domain would have transformed it from a small molecule that was easily transported by PTC to a large potentially inhibitory molecule that is capable of blocking the membrane pore that is formed by PTC trimers. In this way a reciprocal regulatory interaction between PTC and SMO could have been established, through which inhibition of PTC activity allowed intracellular accumulation of SMO ligand, resulting in SMO activation. Subsequently, a specific protein–protein interaction interface evolved between HH and PTC that became sufficient for their binding, so that the regulation of PTC — but not the secretion and transport of HH — became independent of the lipid moiety153.

One assumption of this model is that a SMO-like protein predates the origin of multicellular organisms and, although not stated explicitly, that this SMO prototype probably already regulated GLI activity, presumably via some form of HH signalling complex (HSC). In this regard, it is notable that true GLI orthologues have not been found outside the metazoa and that Cnidaria seem to be the most basal eukaryotes to possess a SMO protein. Notably, whereas the Amphimedon queenslandica genome encodes the full complement of HSC proteins — namely, GLI, KIF7, Fused (FU), Suppressor of fused (SUFU), protein kinase A (PKA), glycogen synthase kinase 3 (GSK3) and casein kinase 1 (CKI) — it lacks not only SMO but also PTC and HH15. Conversely, this primitive metazoan does possess genes that encode a hedgling protein, as well as Interference hedgehog (IHOG) (and its vertebrate orthologue CDO) and Hedgehog-interacting protein (HHIP), leading to speculation that a functional interaction between these proteins might predate the origin of the HH pathway38. In this view, interaction between the cadherin-like hedgling protein and the calmodulin-like IHOG (CDO) protein may have contributed to the direct cell–cell interactions that underpinned the emergence of multicellular organization. Subsequent fusion of the Hedge and Hog domains would have created a secreted form of the IHOG (CDO) ligand, the prototype of the HH protein that subsequently evolved an interaction with PTC.

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Figure 5 | Conserved roles of Hedgehog signalling between phyla. The Hedgehog (hh) and wingless (wg) (which encodes WNT), genes are expressed in series of segmentally repeating stripes along the anteroposterior body axes of arthropod and annelid embryos. In the Drosophila melanogaster embryo (part Aa), the interface between cells expressing HH and WNT defines the parasegment boundary (indicated by a grey line), which is maintained by a positive feedback loop between HH and WNT (black lines indicate segmental boundaries). In a wild-type D. melanogaster larva (part Ab), each abdominal segment is decorated on its ventral surface with a trapezoidal belt of denticles (blue with v marks), which are mostly of anterior provenance (A), and flanked by a posterior (P) naked cuticle: the parasegment boundary (PS; indicated by a grey line) separates these two regions. In hh mutants, each parasegment boundary is lost and each posterior naked cuticle is replaced with a mirror image duplicated denticle belt. In ptc mutants, by contrast, most of the anterior region of each segment is replaced by a duplicated posterior naked cuticle with mirror symmetry, resulting in the duplication of segmental boundaries (S; indicated a black line). HH signalling specifies anteroposterior polarity in regenerating planarians (part Ba). Cells expressing hh are distributed along the ventral nerve cord (VNC; indicated in green) of planarians; HH protein is thought to be transported (arrows) along the axons to the posterior tip of the animal where it is secreted and stimulates transcription of the wntp1 gene in a small population of cells (shown by red dots). According to this model, amputation (indicated by line I or line II) leads to the establishment of a new source of wntp1 expression at the caudal edge of the head (left of figure) and trunk (middle of figure) fragments, leading to the re-establishment of the full-body axis in each regenerate. In the absence of HH activity (part Bb), wntp1 cannot be re-activated in the head fragments following amputation, resulting in tailless regenerates and, in extreme cases, the development of mirror image head structures. Conversely, ubiquitous activation of the HH pathway (through RNAi-mediated knockdown of ptc) results in widespread wntp1 expression that suppresses head formation and promotes the development of mirror image tails regenerating trunk fragments (part Bc). In the mammalian small intestine (part Ca), WNT signalling is restricted to the stem cell population (shown in red) at the base of the crypts, where it promotes their proliferation and blocks apoptosis. HH that is secreted by adjacent cells (shown in blue) signals to the gut mesenchyme (indicated by yellow cells), which signals back to the epithelium via bone morphogenetic protein (BMP) signalling, to suppress crypt formation. In the D. melanogaster hindgut (part Cb), WNT is expressed in a stem cell population at the anterior, whereas HH is expressed in adjacent transit-amplifying cells and promotes their exit from the cell cycle and differentiation. In the mammalian bladder (part D), Sonic Hedgehog (SHH) expression is upregulated in basal stem cells in response to infection-induced tissue damage and signals to the underlying stromal cells to induce WNT expression; WNT signals back to the stem cells, promoting their proliferation. Part B is modified, with permission, from REF. 96 © (2009) National Academy of Sciences. Part C is modified, with permission, from REF. 157 © (2008) Macmillan Publishers Ltd. All rights reserved. Part D is modified, with permission, from REF. 136 © (2011) Macmillan Publishers Ltd. All rights reserved.

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fragment regenerates to lack tail structures and to form duplicate head structures96 (FIG. 5B). Notably, earlier experiments using a similar RNAi approach had shown a requirement for WNT signalling in the regeneration of the tail: the manipulated animals in this case also formed duplicated head structures125,126 and, as in the D. melanogaster segment, the duplications caused by HH derepression are dependent on WNT activity. Consistent with this, expression of wntP1 transcription is upregu-lated in the absence of PTC activity and lost when HH activity is knocked down, implying a role for HH as an activator of wntP1 expression96. Intriguingly, despite the lack of conservation of this HH–WNT interaction in vertebrate segmentation, the role of WNT in promot-ing posterior identity in Planaria recalls its function as a posteriorizing signal in vertebrate embryos127.

Hedgehog and the development of the endoderm. Extensive analyses of the role of HH signalling in pat-terning the neural tube has highlighted the notochord as a major source of SHH in vertebrates128, but a sec-ond prominent site of Hedgehog gene expression in vertebrate embryos is the endoderm. Notably, both Shh and Ihh are expressed in overlapping patterns along the developing gastrointestinal tract, prompting suggestions that this represents an ancestral site of HH function129. In line with this, the patterns of expression of the Hedgehog genes and their target genes Ptc and Gli in the embryo of the sea anenome, Nematostella vectensis, implicate the pathway in the development of the Cnidarian gut16. Expression of one of the two Nematostella Hedgehog genes, NvHh1, initiates early in gastrulation in the pha-ryngeal and mesenteric ectoderm, whereas Ptc and Gli are expressed in the flanking pharyngeal and oral body wall endoderm, implying that HH mediates interactions between the ectoderm and the endoderm16. The embryo of the basal deuterostome, the sea urchin Lytechinus vari-egatus, shows a similarly complementary pattern of Hh and Ptc expression in its developing gut, though in this case Hh is expressed in the endoderm and Ptc in the adja-cent mesoderm130,131. Functional studies have revealed a widespread requirement for endodermally derived HH in this species; inactivation of the pathway causes defects in mesodermal derivatives, including disruption of the skeleton and disorganization of the muscles that surround the oesophagus131. In vertebrates, reciprocal interactions between the endoderm and the overlying mesoderm have essential roles in the specification and differentiation of these layers125,126. As in the sea urchin, expression of Hedgehog genes in the endodermal epithe-lium is accompanied by Ptc1 expression in the adjacent mesoderm129, indicative of its response to HH signalling, which promotes its proliferation and differentiation into smooth muscle132.

HH in endodermal tissue homeostasis and regenera-tion. In vertebrates, the intestinal epithelium is renewed throughout adult life, with mature enterocytes replaced by cells that are derived from stem cells in invagina-tions of the epithelium, known as crypts. These cells express genes that encode several WNT proteins and

their receptors, the functions of which are essential to maintain the stem cell population. Strikingly, expression of both IHH and SHH become restricted to the crypts as they develop. In contrast to the WNTs, however, these proteins signal exclusively to the surrounding mesoderm to induce the expression of BMP proteins, which sig-nal back to the endodermal epithelium to inhibit crypt formation132,133. In this way, HH signalling restricts the stem cell population to the crypts and promotes the differentiation of their progeny as they exit the crypt134 (FIG. 5Ca). Strikingly, HH and WNT signalling is also deployed in D. melanogaster hindgut homeostasis: as in the vertebrate crypts, expression of a WNT pro-tein, in this case WG, is restricted to the stem cell niche at the anterior end of the hindgut, where it promotes stem cell proliferation and self-renewal135. As cells move away from the niche they undergo rapid proliferation before entering a zone in which they activate HH expres-sion, and this promotes their exit from the cell cycle and differentiation135 (FIG. 5Cb).

Recent studies have also implicated HH signalling in driving regenerative proliferation in a mitotically qui-escent endodermal tissue — the bladder urothelium — in mammals136. SHH is expressed in stem cells located in the basal layer of the urothelium that proliferate and regenerate damaged tissue in response to infection. Interestingly, the proliferative response requires WNT signals that emanate from the adjacent stromal layer, expression of which is induced by the stem cell-derived SHH (FIG. 5D).

A common theme in most of these instances is the involvement of HH signalling in mediating reciprocal interactions between adjacent cell populations, either between different lineage compartments derived from the same germ layer or between tissues that originate from different germ layers. Remarkably, the positive feedback loop between HH and WNT signalling, which was first identified in the maintenance of the paraseg-ment boundary in D. melanogaster, seems to be similar to the regenerative response of the mammalian blad-der. It will be interesting to determine whether a simi-lar regulatory relationship exists in Cnidaria, a phylum that diverged from the bilateria more than 650 million years ago.

Concluding commentsA great deal has been learned about HH signalling in the past two decades, but much remains to be discov-ered. Key unresolved questions include the molecular basis of SMO repression by PTC and how the subcel-lular localization of SMO is regulated in response to PTC activity. The mechanism or mechanisms of action of SMO in promoting the dissociation of SUFU from GLI proteins, the nature of the modifications required for maximal activation of GLI proteins, and the precise role of the cilia in regulating GLI processing and acti-vation also remain to be elucidated. Nevertheless, the analysis of the roles of HH in different model organisms has shed light on the molecular and cellular mechanisms underlying a plethora of processes, from the segmen-tation of the bodies of arthropods and annelids to the

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specification of vertebrate neuronal identity. This knowl-edge has already provided us with new insights into the molecular basis of morphological diversity, highlighting variation in HH signalling as a key factor in both micro-evolution and macro-evolution. Developmental analyses of the blind cave-dwelling variety of the teleost Astyanax mexicanus, for example, have pointed to an expansion of Shh expression in the ventral brain as a major driver of the anatomical adaptations that distinguish this vari-ety from its surface river-dwelling relatives137; and at a macro-evolutionary level, the acquisition of a forebrain enhancer by the Shh gene seems to have played a crucial part in the emergence of the vertebrates138,139. Similarly, changes in the transcriptional regulation of hh in the primordial wings of butterflies have been implicated

in the development of their characteristic eyespots140, a morphological adaptation that has a key role in predator avoidance.

Concurrent with these advances, the elucidation of the pathway components and their involvement in human disease has provided a major stimulus for the dis-covery of small-molecule antagonists (and agonists)141, providing new tools for experimental manipulation of the pathway and new leads for the development of novel targeted therapeutic agents142. The application of these reagents to the analysis of HH function in a wider range of species, together with increasing knowledge of the interspecies variation in HH pathway gene structure, is set to provide further insights into the contribution of this fascinating pathway to metazoan evolution.

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AcknowledgementsThe authors are grateful to A. McMahon for his input into earlier drafts of the manuscript and to the anonymous Reviewers for their helpful suggestions for improvements. The authors would like to apologize to those authors whose work they have failed to cite owing to journal space constraints. Y.N.’s sabbatical visit to Institute of Molecular and Cell Biology was supported by a Naito Foundation Fellowship (Japan) and by the IMCB.

Competing interests statementThe authors declare competing financial interests: see Web version for details.

FURTHER INFORMATIONPhilip W. Ingham’s homepage: http://www.imcb.a-star.edu.sg/php/philipingham.php

SUPPLEMENTARY INFORMATIONSee online article: S1 (figure)

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