Hedgehog Signalling

Javier Capdevila, The Salk Institute for Biological Studies, La Jolla, California, USA
Hedgehog genes encode a family of secreted proteins that control the development of many organs and structures in vertebrates and invertebrates.

Secondary article
Article Contents
. Introduction . Hedgehog and Anterior posterior (AP) compartment boundaries in Drosophila . Vertebrate spinal cord

Introduction
Hedgehog (hh) genes encode a family of secreted proteins that control the development of many organs and structures in vertebrates and invertebrates. The interaction of Hedgehog (HH) proteins with their membrane receptors triggers a signalling pathway that activates or represses a number of gene targets in the responding cells. HH signalling pathways control growth and patterning in structures as diverse as the limbs, neural tube, lungs, skin, teeth and many others. In humans, mutations in several members of the HH pathway have been shown to be involved in a variety of developmental defects and in tumour formation. The rst hh gene was discovered in the fruity, Drosophila melanogaster, as the gene altered in a previously known mutation that disrupts the segmental polarity of the Drosophila embryo. From that moment on, genetic screens and functional assays have allowed researchers to identify additional elements of the HH signalling pathway. After the discovery of hh in Drosophila, many hh and hh-related genes were identied in vertebrates. Three hh genes exist in mammals: Sonic hedgehog (Shh), Indian hedgehog (Ihh) and Desert hedgehog (Dhh). Shh has been shown to control patterning of the vertebrate limb, lung, neural tube, eye, somites, gut, hair follicles, and many other structures, besides acting as a key player in the mechanism that establishes the leftright embryonic axis. Ihh is expressed in developing cartilage and it appears to regulate the rate of chondrocyte maturation during endochondral bone growth, and it is also expressed in the gut. Dhh is expressed in the testis, but not the ovary, and it regulates both early and late stages of spermatogenesis. Also, Dhh produced by Schwann cells controls the development of peripheral nerve sheaths. Additional hh and hh-related genes have also been identied in other vertebrates, such as Xenopus and zebrash, where they are also involved in leftright determination and patterning of the neural tube, somites and eyes. A combination of hh genes in zebrash also appears to control the specication of dierent muscle cell types. Here, we will briey review two of the best characterized patterning activities of HH proteins, namely, the role of HH in patterning the Drosophila embryo and imaginal discs, and the role of SHH in patterning the vertebrate ventral neural tube. We will also comment on the

. Autocatalytic processing and role of cholesterol . Receptors and Signal Transduction . Conclusions and Future Prospects

extracellular mechanisms that modulate hh activity, and we will briey mention what is known about the transduction of HH signals.

Hedgehog and Anterior posterior (AP) compartment boundaries in Drosophila

After the identication of the Drosophila hh gene, expression studies revealed that it was transcribed in stripes of cells corresponding to the most posterior cells in each embryonic segment. hh activity was shown to be required to maintain the expression of the gene wingless (wg) in the adjacent anterior cells of each embryonic segment. The wg gene encodes another secreted factor that has many patterning roles in Drosophila, and it is now known that a complex interaction between the wg and hh pathways controls patterning of all embryonic segments (reviewed by Placzek and Ingham, 2000). The posterior localization of hh transcription was also observed in the precursors of the adult structures of the y, the so-called imaginal discs, which are divided into anterior and posterior cell lineage compartments, so that cells in each compartment do not mingle with cells in the other. For example, in the wing imaginal disc (which gives rise to the wing and thorax of the adult y), the anteriorposterior (AP) compartment boundary (which does not correspond to any specic morphological feature) runs through the middle of the imaginal disc. hh is transcribed exclusively in the posterior cells, under the control of the homeotic selector gene engrailed (en), which also controls hh in the embryo. In the wing imaginal disc, HH signalling has been shown to be required for the establishment of the AP compartmental boundary. Moreover, HH protein diuses a few cell diameters into anterior compartment cells, where it activates the decapentaplegic (dpp) gene, which encodes a member of the transforming growth factor b (TGFb) superfamily of secreted factors, also involved in many developmental decisions in vertebrates and invertebrates. Clonal analysis techniques allowed researchers to create patches of cells in the developing wing that lacked either hh or dpp function, and these experiments revealed that both
1

ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing Group / www.els.net

Hedgehog Signalling

genes were required for the normal development of the wing. Specically, a variety of functional analyses revealed that HH acts as the organizer of the anteriorposterior patterning of the y wing through two activities: rst, HH itself acts as a short-range signal, patterning the central portion of the wing (specically, the region between veins three and four); second, HH induces DPP, which in turn acts as a long-range signal that patterns the rest of the wing. There is considerable evidence that shows that both HH and DPP proteins act as classical morphogens, that is, as secreted factors that provide positional information to their responding cells in a concentration-dependent manner. In the Drosophila eye primordia, however, hh expression is not under compartmental control, and hh plays an interesting role related to the function of the so-called morphogenetic furrow (see Greenwood and Struhl, 1999). Drosophila eyes and antennae derive from primordia called eyeantenna imaginal discs. In these discs, the precursors of the photoreceptors of the eye develop in a posterior-to-anterior wave of dierentiation that sweeps through the eye portion of the imaginal disc. Immediately anterior to this wave of dierentiation, there is a physical indentation called the morphogenetic furrow, which separates developing photoreceptors (posterior to the furrow) from undierentiated cells (anterior to the furrow). The HH protein is produced by the maturing photoreceptors, posterior to the morphogenetic furrow, and it is absolutely required to direct its anterior progression, since HH deciency slows or stops the furrow, and ectopic HH anterior to the furrow triggers ectopic furrow (and photoreceptor) formation. Interestingly, however, cells that are unable to transduce the HH signal (because they lack the HH receptor, for example), still can form photoreceptors, which suggests that a secondary signal triggered by HH may be involved in the role of inducing photoreceptor dierentiation. Actually, DPP is expressed in and ahead of the morphogenetic furrow in response to HH, and DPP has been shown to act as a longrange signal that confers a pre-proneural state to cells anterior to the furrow, which subsequently achieve a proneural state by the activity of another yet unknown (but hh-dependent) signal. Thus, hh expression in the eye imaginal disc controls both the progression of the morphogenetic furrow and the dierentiation of photoreceptors through the induction of DPP. Interestingly, even in the absence of DPP, HH is able to confer preproneural state to anterior cells, but only at a very short range, which indicates that DPP acts as a sort of HHdependent accelerator of morphogenetic furrow progression, which ends up determining the size of the Drosophila eye. Remarkably, and underscoring the evolutionary conservation of hh patterning activities, one of the vertebrate hh genes, Shh, is also responsible for controlling anterior posterior patterning in the vertebrate limb. Shh is
2

expressed in a group of posterior mesenchymal cells in the primordia of the limbs, the limb buds. This posterior region (which is not a cell lineage compartment) has been shown to organize the AP patterning of the vertebrate limb, and it is known as the zone of polarizing activity (ZPA), because it can inuence neighbouring cells to form a specic sequence of pattern elements. For example, when a ZPA is implanted into the anterior margin of a host limb bud, a mirror-image duplication of skeletal elements develops. In 1993, it was shown that Shh is able to mediate the polarizing activity of the ZPA in the chick limb. Also, Shh-decient mice have limbs, but they are severely truncated, lacking the most distal parts. This indicates that Shh is not required for the initiation of the limb bud in vertebrates or for establishing the pattern of the most proximal structures of the limb, but it is absolutely required for distal outgrowth. Both in Drosophila and in vertebrates, the lack of hh is incompatible with distal development of the appendages, and ectopic Hh is able to induce mirror-image duplications of pattern elements, which indicates that hh genes coordinate growth and patterning along the anteriorposterior and proximaldistal axes of the limb primordia both in vertebrates and invertebrates nchez-Herrero, 1999). (reviewed by Morata and Sa

Vertebrate spinal cord

The correct patterning of the ventral portion of the vertebrate neural tube is regulated by extracellular signals produced by the underlying notochord and the oor plate. SHH is expressed in both the notochord and the oor plate, and it has been shown to function in a dose-dependent manner to induce dierent cell types (motor neurons and interneurons), and, in general, to control specic patterns of gene expression in the ventral neural tube (reviewed by Briscoe and Ericson, 1999). For example, SHH can induce spinal motor neurons and midbrain dopaminergic neurons in experiments involving explants of neural tissue. SHH is also expressed in the ventral domain of the forebrain, where it induces the dierentiation of ventral neuronal cell types (Ericson et al., 1995). Thus, SHH controls ventral patterning along the entire rostrocaudal length of the central nervous system. The vital role of SHH in this process has also been substantiated by the fact that Shhdecient mice have severe deciencies of ventral patterning of the central nervous system (Chiang et al., 1996). The expression of Shh in the notochord is required for the induction of the oor plate, but the induction of motor neurons in the spinal cord does not depend on oor plate dierentiation (Tanabe et al., 1995), which indicates that SHH can act both in a contact-dependent way (to induce oor plate) and a contact-independent way (at a distance, to induce motor neurons). Recently, it has been shown that an activated form of the SHH receptor is able to activate

ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing Group / www.els.net

Hedgehog Signalling

specic, Shh-dependent ventral genes in an autonomous way (Hynes et al., 2000). This means that the activation of the targets only occurs in the cells that express the activated form of the receptor, which suggests that SHH patterns the ventral neural tube directly, and not through the induction of a secondary signal. Recently, it has been shown that SHH interacts with the extracellular matrix protein vitronectin to induce spinal motor neuron dierentiation, which indicates that the presentation of SHH to the responding cells in the ventral neural tube may be regulated by interactions with a number of extracellular factors. It is still not entirely clear how the SHH signal is translated into specic neuronal identities, but it appears that SHH acts in a graded manner to establish dierent populations of neural progenitor cells, which are characterized by the expression of specic homeodomain transcription factors. Subsequently, these factors control the activity of genes that confer neuronal subtype identity to postmitotic neurons. Recently several transcription factors (such as Nkx2.2, Nkx6.1 and Nkx6.2) have been shown to act as targets of the SHH pathway in the ventral neural tube.

Autocatalytic processing and role of cholesterol

HH proteins suer interesting extracellular modications that are required for their normal signalling functions (reviewed by Murone et al., 1999). They are synthesized as precursors that undergo an autoproteolytic cleavage that renders two biochemically distinct products: a 19 kDa Nterminal fragment (N-Hh), and a 2528 kDa C-terminal fragment (C-Hh). C-Hh mediates the autoproteolytic reaction, and the N-Hh fragment is the active portion of Hh, which is sucient to perform all known Hh activities. During autoproteolysis, a cholesterol moiety is attached to the C-end of N-Hh, and a palmitic acid is attached to the Nend. These lipid-modied forms of HH proteins have two important characteristics: rst, they are more potent than nonmodied forms of Hh, and second, they are limited in their diusion through the extracellular space, being closely associated with the producing cells (nonmodied forms of Hh are freely diusible). The lipidic moieties may mediate the association of Hedgehog proteins with lipid rafts, which are cell membrane microdomains that act as platforms for the assembly of receptor complexes for several signalling factors, including immunoglobulin E (IgE), ephrins and others. In Drosophila, HH proteins have actually been detected associated to these lipid rafts. Thus, lipid modication of HH proteins appears to be a novel mechanism to concentrate signalling factors, which could also be involved in the movement of Hh from one cell to another. The discovery of these lipid modications of HH proteins has provided insights into a variety of perturba-

tions of cholesterol metabolism that result in developmental defects similar to the lack of HH signalling. For example, alterations in cholesterol metabolism can lead to holoprosencephaly (HPE), which is the most common brain anomaly observed in humans, and it consists of abnormal septation of the central nervous system, which results in cyclopia and other craniofacial anomalies. Interestingly, mutations in the Shh gene have been detected in HPE-aected individuals, and Shh-decient mice also have HPE. It has also been shown that cell surface heparan sulfate glycosaminoglycans (GAGs) are required in the receiving cells for the extracellular movement of HH proteins. Also recently, a novel protein called Dispatched (Disp) has been discovered in Drosophila. This novel protein appears to have the dedicated role of releasing the cholesterolmodied forms of N-Hh from the producing cells, so that they can aect neighbouring cells, although the exact mechanism of action of this novel protein is still unknown (see Figure 1c). Still another extracellular protein, HHinteracting protein (Hip), is a target of Hh which appears to limit its range of activity through direct contact. In conclusion, the activity of HH proteins is regulated by a variety of extracellular modications and interactions that contributes to ne-tune the amount and the location of HH protein available for signalling.

Receptors and Signal Transduction

Genetic studies in Drosophila have been invaluable in dissecting the molecular pathway that transduces the HH signal (reviewed by Placzek and Ingham, 2000). A variety of genetic screenings and functional assays have allowed researchers to identify genes that modify or interact in some way with hh mutations, thus opening the door for the identication of proteins that operate in the HH pathway. From these and other studies, a model has emerged that describes how the HH signal is received and interpreted by the responding cells (Figure 1; reviewed by Ingham, 1998 and Murone et al., 1999). In the cell membrane, HH proteins interact with a reception complex that includes two multipass transmembrane proteins that are absolutely required for transducing the HH signal: Patched (Ptc) and Smoothened (Smo). Ptc, a 12-pass transmembrane protein, interacts directly with HH; Smo, a seven-pass transmembrane protein, appears to be the transducing component of the reception complex. In the absence of Hh, Smo is maintained in an inactive state by Ptc (Figure 1a), but when Hh binds Ptc, a conformational change in Smo is induced that results in transduction of the signal by a still unknown mechanism (Figure 1b). Specic mutations in Smo result in constitutively active forms of this protein, which signals independently of the presence or absence of Hh. Interestingly, the interaction of Hh with Ptc
3

ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing Group / www.els.net

Hedgehog Signalling

appears to sequester Hh, thus contributing to limit its range of activity. This was demonstrated by elegant experiments in Drosophila wing imaginal discs, where HH protein was found to diuse very long distances when cells that normally receive Hh were engineered to lack the Ptc protein, thus showing that Ptc is required both for the transduction of the HH signal (along with Smo) and for restricting its range of action. The ptc gene happens to be a transcriptional target of HH so that the HH protein actually controls its own range of activity through the induction of Ptc. But how is the HH signal actually transduced? SMO has homology to G protein-coupled receptors, and there is some evidence that G proteins or their associated second messengers participate in signalling. HH signalling regulates the activity of a family of zinc-nger transcription factors called Cubitus interruptus/Gli (Ci/Gli), which bind DNA and regulate the transcription of Hh-dependent targets. Thus, Ci/Gli proteins are thought to be the main nal eectors of HH signalling, directly controlling the transcriptional response to HH signals. Interestingly, the activity of Ci/Gli transcription factors is regulated at the level of proteolytic modication, coupled to subcellular localization. In Drosophila, in the absence of Hh (Figure 1a), cytoplasmatic Ci is proteolytically processed to render a Nterminal portion that acts as a repressor in the nucleus, in a process triggered (at least in part) by protein kinase A (PKA)-mediated phosphorylation. This process also in-

volves the ubiquitin targeting protein Slimb. In response to HH signalling (Figure 1b), cleavage of Ci is inhibited, and the full-length protein appears to act as an activator in the nucleus, interacting with other nuclear factors, such as CREB-binding protein (CBP). Thus, dierent levels of hedgehog signalling may result in specic combinations of repressor and activator forms of Ci proteins, which in turn would activate or repress target genes. Although only one ci gene has been discovered in Drosophila, at least three Gli genes are present in vertebrates (Gli-1, 2 and 3). It appears that the regulation of Hh-dependent targets in vertebrates may result from the combined activities of these proteins, although more experiments are needed in order to assign specic activities to each Gli protein. Quite unexpectedly, the Ci protein was found to be associated to microtubules. In the absence of Hh, fulllength Ci forms a complex associated to microtubules with at least three other proteins: Costal2 (Cos2, a kinesinrelated protein), Fused (Fu, a protein-serine/threonine kinase) and Suppressor of Fused (Su(fu), a novel protein) and Ci is cleaved to produce the repressor form, which keeps downstream targets repressed (Figure 1a). In the presence of Hh, however, the protein complex dissociates from microtubules, and the cleavage of Ci is inhibited, leaving the full-length form available to activate targets in the nucleus (Figure 1b). Although the interactions between these proteins are not entirely clear, it is thought that Su(Fu) binds and inactivates Ci/Gli proteins in the absence
HH HH

D i sp

at c h

e
d

PTC

SMO

PTC HH

SMO HH HH HH

N u

CI

CI

HH-dependent targets repressed (a)

HH-dependent targets activated (c) (b)

Figure 1 A simplified view of the Hedgehog (HH) signalling pathway. In cells that do not receive the HH signal (a), Patched (PTC) acts by repressing Smoothened (SMO), and the proteins Fused (FU), Suppressor of Fused (SU(FU)), Costal2 (COS2) and Cubitus interruptus (CI) (GLI in vertebrates) form a complex associated to microtubules. CI is proteolytically cleaved in a process involving phosphorylation by protein kinase A (PKA) and the activity of SLIMB, rendering a form of CI that keeps HH-dependent targets repressed. In cells that do receive the HH signal (b), interaction of HH with PTC relieves the repressive effect on SMO, which leads to the dissociation of the protein complex from microtubules and inhibition of CI processing. This results in the full-length form of CI acting as an activator of HH-dependent targets, interacting with Cre-Binding Protein (CBP) and other nuclear proteins. It is still not known whether activation of SMO antagonizes PKA activity and SLIMB. In cells that produce the HH signal (c), activity of the protein Dispatched is required for releasing the lipid-modified active form of the HH protein, so that it can signal to adjacent cells. Lipid-modified HH proteins are found associated to lipid rafts, where interaction with glycosaminoglycans (GAGs) may also assist in the diffusion and/or interaction of HH with its receptor.

ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing Group / www.els.net

N u

M i c r o t u b u l e s

FU COS2

PKA SLIMB?
SU (FU)

P CI P

CI

P P

cle

us

M i c r o t u b u l e s

? FU COS2
SU (FU)

? PKA

? SLIMB HH source

CI

u cle

CB

u cle

Hedgehog Signalling

of Hh. When Hh is present, however, Fu counteracts Su(fu), resulting in the activation of Ci/Gli. The role of Cos2 protein could be to tether Fu, Su(Fu) and Ci/Gli to microtubules, so that inactivation of Cos2 by the Hh signal would result in dissociation of the protein complex from microtubules, an event linked to phosphorylation of both Fu and Cos2 proteins (not shown in the gure).

the proper restriction of HH signalling to the right places in the embryo. Deregulation of the HH pathway may result in severe abnormalities, including the formation of tumours such as basal cell carcinomas (BCC), medulloblastomas, rhabdomyosarcomas and gliomas. Thus, it is of particular importance to identify and characterize additional components of the HH pathway, which could provide additional entry points to develop new antitumoral therapies.

Conclusions and Future Prospects

Ever since the identication in 1992 of the founding member of the hh gene family, it has become evident that the HH signalling pathway constitutes a genetic module that is used time and again in many dierent developmental processes. Besides the few examples described above, HH proteins also control an astonishing variety of growth and patterning processes in many other organs and structures, through a signalling mechanism that appears to be evolutionarily conserved. Many questions remain unanswered about the mechanism of action of HH proteins. Specically, HH proteins have dierent eects on cells depending on the molecular context. For example, SHH has been shown to act as a survival factor for several cell types in the embryo, but it is also involved in inducing programmed cell death in other cell types. Clearly, HH proteins cooperate with additional factors to promote specic responses, and to appreciate the full complexity of the patterning events directed by HH signals, one has to consider the whole network of interacting factors that modulate them. It is generally accepted that HH proteins act as morphogens during embryonic development. HH proteins provide positional information both by acting directly on neighbouring cells and by inducing the expression of secondary signals, such as DPP. HH proteins are extremely potent signalling factors, and their spatial and temporal transcriptional patterns and range of activity are tightly regulated by a complex network of interactions that ensure

References
Briscoe J and Ericson J (1999) The specication of neuronal identity by graded Sonic hedgehog signalling. Seminars in Cell and Developmental Biology 10: 353362. Chiang C, Litingtung Y, Lee E et al. (1996) Cyclopia and defective axial patterning in mice lacking Sonic hedgehog gene function. Nature 383: 407413. Ericson J, Muhr J, Placzek M, Lints T, Jessell TM and Edlund T (1995) Sonic hedgehog induces the dierentiation of ventral forebrain neurons: a common signal for ventral patterning within the neural tube. Cell 81: 747756. Greenwood S and Struhl G (1999) Progression of the morphogenetic furrow in the Drosophila eye: the roles of Hedgehog, Decapentaplegic and the Raf pathway. Development 126: 57955808. Hynes M, Ye W, Wang K et al. (2000) The seven-transmembrane receptor smoothened cell-autonomously induces multiple ventral cell types. Nature Neuroscience 3: 4146. Ingham PW (1998) Transducing Hedgehog: the story so far. EMBO Journal 17: 35053511. nchez-Herrero E (1999) Patterning mechanisms in the Morata G and Sa body trunk and the appendages of Drosophila. Development 126: 28232828. Murone M, Rosenthal A and de Sauvage FJ (1999) Hedgehog signal transduction: from ies to vertebrates. Experimental Cell Research 253: 2533. Placzek M and Ingham PW (2000) Developmental genetics in Sheeld: a meeting point for Hedgehog researchers. International Journal of Developmental Biology 44: 6572. Tanabe Y, Roelink H and Jessell TM (1995) Induction of motor neurons by Sonic hedgehog is independent of oor plate dierentiation. Current Biology 5: 651658.

ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing Group / www.els.net