Cholesterol activates the G-protein coupled receptor Smoothened to promote Hedgehog signaling

Cholesterol activates the G-protein coupled receptor Smoothened to promote Hedgehog signaling

Cholesterol is necessary for the function of many G-protein coupled receptors (GPCRs). We find that cholesterol is not just necessary but also sufficient to activate signaling by the Hedgehog (Hh) pathway, a prominent cell-cell communication system in development. Cholesterol influences Hh signaling by directly activating Smoothened (SMO), an orphan GPCR that transmits the Hh signal across the membrane in all animals. Unlike many GPCRs, which are regulated by cholesterol through their heptahelical transmembrane domains, SMO is activated by cholesterol through its extracellular cysteine-rich domain (CRD). Residues shown to mediate cholesterol binding to the CRD in a recent structural analysis also dictate SMO activation, both in response to cholesterol and to native Hh ligands. These results show that cholesterol can initiate signaling from the cell surface by engaging the extracellular domain of a GPCR and suggest that SMO activity may be regulated by local changes in cholesterol abundance or accessibility (Luchetti, 2016).

To establish a causal or regulatory role for a component in a biological pathway, experiments should demonstrate that the component is both necessary and sufficient for activity. Cholesterol has been shown to be necessary for SMO activation, based on experiments using inhibitors of cholesterol biosynthesis and high concentrations of naked MβCD to strip the plasma membrane of cholesterol. Impaired SMO activation caused by cholesterol deficiency has also been noted in Smith-Lemli-Opitz syndrome (SLOS), a congenital malformation syndrome caused by defects in the enzyme that converts 7-dehydrocholesterol to cholesterol. In contrast to the current results, the SMO CRD is dispensable for this permissive role of cholesterol. The depletion of cholesterol reduces signaling by SMO mutants lacking the entire CRD or carrying mutations in the CRD binding-groove. By analogy with other GPCRs, these permissive effects are likely to be mediated by the SMO 7TMD (Luchetti, 2016).

This study found that cholesterol is also sufficient to activate Hh signalling in a dose-dependent manner. This instructive effect is mediated by the Class F GPCR SMO and maps to its extracellular CRD. Cholesterol engages a hydrophobic groove on the surface of the CRD, a groove that was previously shown to mediate the activating influence of oxysterols and represents an evolutionarily conserved mechanism for detecting hydrophobic small-molecule ligands. An analogous mechanism is present in the Frizzled family of Wnt receptors, where the Frizzled CRD binds to the palmitoleyl group of Wnt ligands, an interaction that is required for Wnt signaling. Thus, the instructive effects of cholesterol revealed in this study and the permissive effects of cholesterol reported previously map to distinct, separable SMO domains. The observation that Methyl-β-cyclodextrin(MβCD):cholesterol is sufficient to activate SMO through its CRD is in agreement with a second report (Huang, 2016) published recently (Luchetti, 2016).

There are many reasons why this activating effect of cholesterol on Hh signalling may not have been appreciated previously despite the fact that the activating effects of side-chain oxysterols have been known for a decade. First, the method of delivery, as an inclusion complex with MβCD, is critical to presenting cholesterol, a profoundly hydrophobic and insoluble lipid, in a bioavailable form capable of activating Smo. Even clear solutions of cholesterol in the absence of carriers like MβCD contain microcrystalline deposits or stable micelles that sequester cholesterol. In contrast, side-chain oxysterols, which harbor an additional hydroxyl group, are significantly more hydrophilic and soluble in aqueous solutions, shown by their ~ fold faster transfer rates between membranes. Second, cholesterol levels in the cell are difficult to manipulate because they are tightly controlled by elaborate homeostatic signalling mechanisms. MβCD:cholesterol inclusion complexes have been shown to be unique in their ability to increase the cholesterol content of the plasma membrane rapidly at timescales (~1- hr) at which cytoplasmic signaling pathways operate. Other methods of delivery using low density lipoprotein particles and lipid dispersions, or mutations in genes regulating cholesterol homeostasis, function on a much slower time scale and are thus more likely to be confounded by indirect effects given the myriad cellular processes affected by cholesterol. Finally, the bell-shaped Hh signal-response curve implies that MβCD:cholesterol must be delivered in a relatively narrow, intermediate concentration range to observe optimal activity, with higher concentrations commonly used to load cells with cholesterol producing markedly lower levels of signaling activity (Luchetti, 2016).

The current results are particularly informative in light of the recently solved crystal structure of a SMO protein containing the CRD, linker domain and entire 7TMD but lacking the cytoplasmic tail (hereafter called SMOΔC). SMOΔC was unexpectedly found to contain a cholesterol ligand in its CRD groove. Cholesterol also made key contacts with the linker domain and third extracellular loop of the 7TMD, and molecular dynamics simulations showed that cholesterol can stabilize these extracellular regions of SMO. However, the function of this bound cholesterol, whether it is an agonist, antagonist or co-factor, remains an important unresolved question in SMO regulation. Structure-guided point mutations in CRD residues that form hydrogen-bonding interactions with the 3β-hydroxyl of cholesterol, reduced signaling by cholesterol, making it likely that cholesterol activates SMO by binding to the CRD in the pose revealed in the structure. Thus, the cholesterol-bound SMO structure may very well represent an active-state conformation of the CRD. A comparison of this cholesterol-bound structure with a structure of inactive SMO bound to the potent 7TMD antagonist vismodegib (which lacks cholesterol in the CRD groove) revealed a conformational change that may drive SMO activation. Cholesterol binding is predicted to induce a clockwise rotation of the CRD on the 7TMD pedestal, perhaps driving SMO activation by a rearrangement of contacts between the CRD and the 7TMD. A caveat to this model is that it depends on a structural comparison with SMOΔC bound to a synthetic antagonist and not with un-liganded SMOΔC, which has thus far eluded crystallization (Luchetti, 2016).

Based on structures of the isolated Xenopus laevis CRD, either alone (apo-CRD) or in complex with 20(S)-OHC (but notably not cholesterol), a recently published report proposed that sterols drive SMO activation by inducing a conformational change within the CRD itself (Huang, 2016). The current study disagrees with this model for several reasons. First, these structures do not contain the linker domain and the entire 7TMD, both of which make critical contacts with cholesterol and the CRD, and hence cannot reveal changes in orientation between the CRD and the 7TMD that are essential to understanding how CRD ligands communicate with the 7TMD. Second, all structures of the SMO CRD (with the exception of the Xenopus apo-CRD) are conformationally identical, regardless of whether they contain a bound ligand (cholesterol-bound SMOΔC, cyclopamine-bound CRD or 20(S)-OHC-bound CRD) or not (Danio rerio apo-CRD or vismodegib-bound SMOΔC) and regardless of whether they were crystallized by standard vapor diffusion (the CRD structures) or lipidic cubic phase methods (the SMOΔC structures). Hence this conserved conformation, seen in both the isolated CRD and the more physiological SMOΔC molecule, is unlikely to be an artefact of crystal packing as suggested in the literature (Huang, 2016). Finally, a careful inspection of the crystal lattice contacts in the Xenopus apo-SMO structure (PDB ID 5KZZ) revealed that the region of the proposed conformational change is partially disordered and involved in the coordination of a zinc ion together with a symmetry-related molecule in the crystal, a very tight, near-covalent interaction that was likely driving crystal formation. Since the crystallization solution for the Xenopus apo-SMO, but not the solutions used to crystallize the sterol-bound CRDs, contained zinc acetate, the altered conformation observed may have been induced by a non-physiological, zinc-promoted crystal contact (Huang, 2016; Luchetti, 2016).

A surprising feature of the structure is that CRD-bound cholesterol is located at a considerable distance away from the membrane, which would require a cholesterol molecule to desolvate from the membrane and become exposed to water in order to access its CRD binding pocket. The kinetic barrier, or the activation energy for this transfer reaction is predicted to be high. The unique ability of MβCD to shield cholesterol from water while allowing its rapid transfer to acceptors would allow it to bypass this kinetically unfavorable step by delivering it to the CRD binding site. These considerations present a regulatory puzzle for future research: how does cholesterol gain access to the CRD-binding pocket without MβCD and is this process regulated by native Hh ligands? Indeed, the kinetic barrier for cholesterol transfer to the CRD pocket makes it an ideal candidate for a rate-limiting, regulated step controlling SMO activity in cells (Luchetti, 2016).

TMβCD:cholesterol was consistently less active than the native ligand SHH in these assays. Comparing the doses of MβCD:cholesterol to the doses of SHH delivered to cells is difficult. SHH was used at saturating concentrations; however, it was not possible to assess the effects of MβCD:cholesterol at saturating doses, because the downward phase of the bell-shaped dose-response curve (in cultured fibroblasts) and cell toxicity (in neural progenitors) proved to be dose-limiting. Aside from these technical considerations related to delivery, other possibilities for lower activity include the observation that MβCD:cholesterol did not induce the high-level accumulation of SMO in primary cilia and the possibility that a different ligand regulates high-level signaling by SMO. Mutations in the 7TMD binding-site do not alter the constitutive or SHH-induced signaling activity of SMO, which has led to view that this site does not regulate physiological signaling. In contrast, mutations in the cholesterol-binding site impaired responses to SHH. Hence, a putative alternate ligand would have to engage a third, undefined site. Lastly, the presence of active PTCH is a major difference between SHH- and MβCD:cholesterol-induced signaling. The biochemical activity of PTCH (which is inactivated by SHH) may oppose the effects of MβCD:cholesterol, limiting signaling responses. Interestingly, MβCD:cholesterol was able to restore maximal Hh responses in the absence of PTCH (Luchetti, 2016).

The results may have implications for understanding how PTCH inhibits SMO, a longstanding mystery in Hh signaling. The necessity and sufficiency of cholesterol for SMO activation, mediated through two different regions of the molecule, means that SMO activity is likely to be highly sensitive to both the abundance and the accessibility of cholesterol in its membrane environment. Furthermore, PTCH has homology to a lysosomal cholesterol transporter, the Niemann-Pick C (NPC1) protein, and PTCH has been purported to have cholesterol binding and transport activity. Thus, the current work supports a model where PTCH may inhibit SMO by reducing cholesterol content or cholesterol accessibility (or chemical activity) in a membrane compartment that also contains SMO, leading to alterations in SMO conformation or trafficking. Since cholesterol is a ubiquitous component of cellular membranes that affects many cellular processes, PTCH1-induced changes in cholesterol are likely to be confined to a specific membrane compartment. The base of the cilium is a good candidate for such a compartment because PTCH is localized most prominently at the ciliary base in Hh-responsive tissues in the mouse embryo and because most Hh pathway components, from PTCH to the GLI transcription factors, are found in and around primary cilia. Two key questions must be answered before endogenous cellular cholesterol can be considered the elusive second messenger that communicates the Hh signal from PTCH to SMO: Do Hh ligands alter cholesterol abundance or activity? and Is cholesterol a substrate for the predicted transporter activity of PTCH1? Answering these questions will require developing or adapting tools to measure and perturb cholesterol in specific cellular compartments and an assessment of the biochemical activities of purified SMO and PTCH reconstituted into cholesterol-containing membranes (Luchetti, 2016).

While cholesterol is an abundant lipid, clearly critical for maintaining membrane biophysical properties and for stabilizing membrane proteins, this work suggests that it may be also used as a second messenger to instruct signaling events at the cell surface through GPCRs and perhaps other cell-surface receptors (Luchetti, 2016).

Smoothened as a component of the Hedgehog signaling pathway

The Hedgehog (Hh) family of secreted proteins is involved in a number of developmental processes as well as in cancer. Genetic and biochemical data suggest that the Sonic hedgehog (Shh) receptor is composed of at least two proteins: the tumor suppressor protein Patched (Ptc) and the seven-transmembrane protein Smoothened (Smo). Using a biochemical assay for activation of the transcription factor Gli, a downstream component of the Hh pathway, it has been shown that Smo functions as the signaling component of the Shh receptor, and that this activity can be blocked by Ptc. The inhibition of Smo by Ptc can be relieved by the addition of Shh. Furthermore, oncogenic forms of Smo are insensitive to Ptc repression in this assay. Mapping of the Smo domains required for binding to Ptc and for signaling reveals that the Smo-Ptc interaction involves mainly the amino terminus of Smo, and that the third intracellular loop and the seventh transmembrane domain are required for signaling. This mapping was carried out by creating Smoothened-Frizzled chimeric proteins and assaying for Smo function. These data demonstrate that Smo is the signaling component of a multicomponent Hh receptor complex and that Ptc is a ligand-regulated inhibitor of Smo. Different domains of Smo are involved in Ptc binding and activation of a Gli reporter construct. The latter requires the third intracellular loop and the seventh transmembrane domain of Smo, regions often involved in coupling to G proteins. However, no changes in the levels of cyclic AMP or calcium associated with such pathways could be detected following receptor activation (Murone, 1999).

Sonic hedgehog (Shh) signal transduction involves the ligand binding Patched1 (Ptc1) protein and a signaling component, Smoothened (Smo). Combined genetic and biochemical studies have indicated that Ptc inhibits a latent, tonic signaling activity of Smo, and that Hh binding to Ptc releases the inhibition of Smo. A select group of compounds inhibits both Shh signaling, regulated by Ptc1, and late endosomal lipid sorting, regulated by the Ptc-related Niemann-Pick C1 (NPC1) protein. NPC1 functions in the sorting and recycling of cholesterol and glycosphingolipids in the late endosomal/lysosomal system. It is suggested that Ptc1 regulates Smo activity through a common late endosomal sorting pathway also utilized by NPC1. During signaling, Ptc accumulates in endosomal compartments, but it is unclear if Smo follows Ptc into the endocytic pathway. The dynamic subcellular distributions of Ptc1, Smo, and activated Smo mutants has been characterized individually and in combination. Ptc1 and Smo colocalize extensively in the absence of ligand and are internalized together after ligand binding, but Smo becomes segregated from Ptc1/Shh complexes destined for lysosomal degradation. In contrast, activated Smo mutants do not colocalize with nor are they cotransported with Ptc1. Agents that block late endosomal transport and protein sorting inhibit the ligand-induced segregation of Ptc1 and Smo. Like NPC1-regulated lipid sorting, Shh signal transduction is blocked by antibodies that specifically disrupt the internal membranes of late endosomes, which provide a platform for protein and lipid sorting. These data support a model in which Ptc1 inhibits Smo only when in the same compartment. Ligand-induced segregation allows Smo to signal independent of Ptc1 after becoming sorted from Ptc1/Shh complexes in the late endocytic pathway (Incardona, 2002).

In humans, dysfunctions of the Hedgehog receptors Patched and Smoothened are responsible for numerous pathologies. However, signaling mechanisms involving these receptors are less well characterized in mammals than in Drosophila. To obtain structure-function relationship information on human Patched and Smoothened, these human receptors were expressed in Drosophila Schneider 2 cells. As its Drosophila counterpart, human Patched is able to repress the signaling pathway in the absence of Hedgehog ligand. In response to Hedgehog, human Patched is able to release Drosophila Smoothened inhibition, suggesting that human Patched is expressed in a functional state in Drosophila cells. Human Smo, when expressed in Schneider cells, is able to bind the alkaloid cyclopamine, suggesting that it is expressed in a native conformational state. Furthermore, contrary to Drosophila Smoothened, human Smoothened does not interact with the kinesin Costal 2 and thus is unable to transduce the Hedgehog signal. Moreover, cell surface fluorescent labeling suggest that human Smoothened is enriched at the Schneider 2 plasma membrane in response to Hedgehog. These results suggest that human Smoothened is expressed in a functional state in Drosophila cells, where it undergoes a regulation of its localization comparable with its Drosophila homologue. Thus, it is proposed that the upstream part of the Hedgehog pathway involving Hedgehog interaction with Patched, regulation of Smoothened by Patched, and Smoothened enrichment at the plasma membrane is highly conserved between Drosophila and humans; in contrast, signaling downstream of Smoothened is different (De Rivoyre, 2006; full text of article).

Functional divergence in the role of N-linked glycosylation in Smoothened signaling

Functional divergence in the role of N-linked glycosylation in Smoothened signaling

The G protein-coupled receptor (GPCR) Smoothened (Smo) is the requisite signal transducer of the evolutionarily conserved Hedgehog (Hh) pathway. Although aspects of Smo signaling are conserved from Drosophila to vertebrates, significant differences have evolved. These include changes in its active sub-cellular localization, and the ability of vertebrate Smo to induce distinct G protein-dependent and independent signals in response to ligand. Whereas the canonical Smo signal to Gli transcriptional effectors occurs in a G protein-independent manner, its non-canonical signal employs Galphai. Whether vertebrate Smo can selectively bias its signal between these routes is not yet known. N-linked glycosylation is a post-translational modification that can influence GPCR trafficking, ligand responsiveness and signal output. Smo proteins in Drosophila and vertebrate systems harbor N-linked glycans, but their role in Smo signaling has not been established. This study presents a comprehensive analysis of Drosophila and murine Smo glycosylation that supports a functional divergence in the contribution of N-linked glycans to signaling. Of the seven predicted glycan acceptor sites in Drosophila Smo, one is essential. Loss of N-glycosylation at this site disrupted Smo trafficking and attenuated its signaling capability. In stark contrast, all four predicted N-glycosylation sites on murine Smo were dispensable for proper trafficking, agonist binding and canonical signal induction. However, the under-glycosylated protein was compromised in its ability to induce a non-canonical signal through Galphai, providing for the first time evidence that Smo can bias its signal and that a post-translational modification can impact this process. As such, a profound shift is postulated in N-glycan function from affecting Smo ER exit in flies to influencing its signal output in mice (Marada, 2015).

Smoothened is a target of cyclopamine

Cellular responses to the Hh signal are controlled by two transmembrane proteins, Smo and Ptch, which are predicted to have seven and twelve transmembrane spans, respectively. Genetic and biochemical evidence indicates that Ptch suppresses the activity of Smo, and that binding of Hh to Ptch relieves this suppression, allowing activation of downstream targets through the Ci/Gli family of transcriptional effectors. Because a Hh signaling assay using with Drosophila cultured cells is not sensitive to cyclopamine, several vertebrate cell lines were screened for a sensitive transcriptional response to palmitoyl- and cholesteryl-modified ShhN polypeptide (ShhNp) using a Gli-dependent luciferase reporter. Among several responsive fibroblast cell lines, NIH-3T3 mouse embryonic fibroblasts, which respond with a 20- to 150-fold induction of luciferase activity, were selected for all further studies except those requiring particular genetic backgrounds. Treatment of the cells with cyclopamine completely abolishes the response to ShhNp. To confirm the validity of this assay, the effects of overexpression of known pathway components, treatment with known pathway inhibitors, or both were analysed. The main findings of Drosophila and mouse genetic analyses were confirmed, indicating that NIH-3T3 cells provide a faithful and physiologically meaningful model for analysis of the Shh signaling pathway. Interestingly, for a full response to ShhNp cells had to be assayed after reaching saturation density (Taipale, 2000).

Basal cell carcinoma, medulloblastoma, rhabdomyosarcoma and other human tumors are associated with mutations that activate the proto-oncogene Smoothened (SMO) or that inactivate the tumor suppressor Patched (PTCH). Smoothened and Patched mediate the cellular response to the Hedgehog (Hh) secreted protein signal, and oncogenic mutations affecting these proteins cause excess activity of the Hh response pathway. The plant-derived teratogen cyclopamine, which inhibits the Hh response, is a potential 'mechanism-based' therapeutic agent for treatment of these tumors. Cyclopamine or synthetic derivatives with improved potency block activation of the Hh response pathway and abnormal cell growth associated with both types of oncogenic mutation. These results also indicate that cyclopamine may act by influencing the balance between active and inactive forms of Smoothened (Taipale, 2000).

Whereas embryonic loss of Sonic hedgehog (Shh) signaling can result in cyclopia and other developmental defects, inappropriate activation of the Shh response pathway is associated with several types of human tumor. Current approaches to treatment of such neoplastic disorders are limited by the cytotoxic effects of therapeutic agents on proliferating tissues. Alternative 'mechanism-based' approaches specifically targeting abnormally active signaling pathways in defined types of cancer might avoid such toxicity, particularly if the pathways in question functioned primarily in embryonic development and were not required for survival in adults. Cyclopamine, a plant steroidal alkaloid, induces cyclopia in vertebrate embryos and has been shown to act by inhibiting the cellular response to the Shh signal. To evaluate the therapeutic potential of cyclopamine for the treatment of Hh-pathway-associated disorders, the mechanism by which cyclopamine acts was investigated (Taipale, 2000).

The steroidal nature of cyclopamine and its ability to disrupt cholesterol synthesis or transport indicates that it might affect the action of Ptch, which contains an apparent sterol-sensing domain. Having established the general characteristics of Shh response and cyclopamine inhibition in mouse embryonic fibroblasts, fibroblasts derived from Ptch-/- mouse embryos were assayed for cyclopamine sensitivity. Mice lacking functional Ptch show widespread transcriptional activation of targets of Shh signaling, including Ptch itself. As beta-galactosidase is expressed under the control of the Ptch promoter in these cells, its expression can be used to assay the state of Shh pathway activity. Addition of cyclopamine to Ptch-/- cells significantly suppresses beta-galactosidase expression and the activity of the Gli-luc reporter, indicating that cyclopamine can inhibit Shh pathway activity in the absence of Ptch function. In contrast, cyclopamine fails to prevent pathway activation induced by Gli2 overexpression (Taipale, 2000).

These results suggest that Hh-pathway-related tumors associated with loss of Ptch function might respond to treatment with cyclopamine, and that the target of cyclopamine action is likely to be a pathway component that functions between Ptch and the Gli proteins. It is unlikely that Ptch2 is the target of cyclopamine action in Ptch-/- cells, since Ptch2 is the main regulator of Shh pathway activation in embryos and Ptch2 activity appears not to be expressed in Ptch-/- cells (Taipale, 2000).

To investigate further the mechanism of cyclopamine action, NIH-3T3 cells were transiently transfected with both luciferase reporter and Smo complementary DNA, and it was found that overexpression of Smo in the absence of Shh induces reporter expression about tenfold. This Shh-independent activation of the response pathway can be suppressed by 5 µM cyclopamine, consistent with a target of cyclopamine action downstream of Ptch and with a mechanism that does not involve direct interference with Shh binding. Cyclopamine at this concentration has little effect on reporter expression induced by the oncogenic Smo mutants W539L (SmoA1) and S537N (SmoA2); cells from Ptch mutant embryos give similar results (Taipale, 2000).

These results indicate that cyclopamine may act upon Smo, and that activating mutations render Smo proteins resistant. An alternative interpretation would be that activated Smo proteins produce a high abundance of a downstream component and that a high cyclopamine level is required to suppress the increased concentration of this component. This alternative model, however, would predict that intermediate or low levels of pathway activation by oncogenic Smo proteins expressed at low levels should be subject to cyclopamine inhibition; on the contrary, it is observed that cyclopamine resistance is sustained under these conditions. In addition, cyclopamine resistance is not observed in cells expressing high levels of wild-type Smo activated by maximal Shh stimulation, again suggesting that cyclopamine does not act upon a component downstream of Smo (Taipale, 2000).

The oncogenic SmoA1 protein has been reported to resist suppression by Ptch, indicating that oncogenic Smo proteins may not be subject to normal regulation. It was found, however, that this resistance is partial. The activating effects of SmoA1 or SmoA2 can be completely inhibited by transfection of a 9-to-1 ratio of a Ptch construct or of Ptch-CTD, which encodes a carboxy-terminally deleted protein expressed at a higher level. It was also found that SmoA1, thus inhibited by Ptch-CTD, responds well to stimulation by ShhNp. Under these circumstances, induction of the Gli-responsive reporter is resistant to 5 µM cyclopamine, which would normally abolish Shh signaling. These results indicate that activated Smo molecules in the presence of sufficient Ptch can contribute to an essentially normal, albeit cyclopamine-resistant, response to the Shh signal (Taipale, 2000).

The finding that oncogenic Smo is regulated by high levels of Ptch indicates that it might also be subject to regulation by high levels of cyclopamine. To circumvent the cytotoxic effects of cyclopamine concentrations greater than 10 µM, several chemically synthesized cyclopamine derivatives were tested. The cyclopamine derivative 3-keto, N-aminoethyl aminocaproyl dihydrocinnamoyl cyclopamine (KAAD-cyclopamine), has 10-20-fold higher potency than cyclopamine in inhibition of beta-galactosidase expression in p2Ptch-/-cells, with similar or lower toxicity. This compound also has greater potency in suppression of ShhNp-induced pathway activity. Importantly, it completely suppresses SmoA1-induced reporter activity at a concentration around tenfold higher than that required for suppression of pathway activation induced by ShhNp (Taipale, 2000).

Since activation of the Hh response pathway is associated with neoplastic transformation in various types of tumor, the growth properties of response-activated cells were investigated in low serum or soft agar, conditions generally considered to reveal neoplastic transformation. p2Ptch-/- cell growth in low serum is markedly inhibited by addition of KAAD-cyclopamine, with 50% of maximal inhibition at ~50 nM. SmoA1-LIGHT cells, a cell line expressing SmoA1 clonally derived from NIH-3T3 cells, can form colonies in soft agar medium. Addition of KAAD-cyclopamine markedly inhibits colony growth, although ~500 nM is required for 50% inhibition. This concentration is higher than that required for 50% maximal inhibition of p2Ptch-/- cell growth, consistent with the higher amount of KAAD-cyclopamine required to block activation of the Hh response pathway by oncogenic Smo (Taipale, 2000).

The transition of G-protein-coupled receptors with seven transmembrane (TM) domains from the inactive to the active state is thought to involve a conformational shift in which the cytoplasmic ends of the TM6 and TM7 helices tilt outwards, exposing a binding pocket for a downstream signaling molecule, the Galpha subunit. Mutational analysis of Smo is consistent with such a conformational shift, since eight of the Smo-activating mutations introduce bulkier side chains at G533 and S537. Assuming an alpha-helical conformation for TM7, these substitutions all protrude from the same face of the helix. Furthermore, because multiple distinct substitutions at each of these two residues result in activation, Smo would appear to be activated by alteration of helix-packing interactions rather than by creation or disruption of a single critical interaction. A conformational shift of the TM7 helix with respect to other TM helices is suggestive of the type of conformational transition postulated for activation of G-protein-coupled receptors and raises the possibility that conformation-based transduction has been conserved in the evolution of seven-transmembrane-domain receptors (Taipale, 2000).

Activation of the Hh response pathway has been linked to several types of human tumor. For example, patients with basal cell nevus syndrome (also termed Gorlin syndrome), an autosomal dominant disorder associated with heterozygous loss-of-function mutations in PTCH, display increased incidence of many tumors, most notably basal cell carcinoma (BCC), medulloblastoma, rhabdomyosarcoma and fibrosarcoma. In addition, loss-of-function mutations in PTCH or activating mutations in SMO are found in around 40% of sporadic BCC and 25% of primitive neuroectodermal tumors. The results indicate that cyclopamine inhibits the Shh pathway by antagonizing Smo and that the activation of the Hh response pathway by either type of oncogenic mutation is blocked by cyclopamine or its derivatives. Levels of cyclopamine or the related compound jervine required to phenocopy the embryonic malformations in Shh-/- embryos are tolerated by pregnant females of various species, indicating that it might be possible to use these compounds or their derivatives without severe toxicity in non-pregnant adults to reverse activation of the Shh response pathway for therapeutic purposes (Taipale, 2000).

Structure and function of the Smoothened extracellular domain in vertebrate Hedgehog signaling

The Hedgehog (Hh) signal is transduced across the membrane by the heptahelical protein Smoothened (Smo), a developmental regulator, oncoprotein and drug target in oncology. This study presents the 2.3 Å crystal structure of the extracellular cysteine rich domain (CRD) of vertebrate Smo and show that it binds to oxysterols, endogenous lipids that activate Hh signaling. The oxysterol-binding groove in the Smo CRD is analogous to that used by Frizzled 8 to bind to the palmitoleyl group of Wnt ligands and to similar pockets used by other Frizzled-like CRDs to bind hydrophobic ligands. The CRD is required for signaling in response to native Hh ligands, showing that it is an important regulatory module for Smo activation. Indeed, targeting of the Smo CRD by oxysterol-inspired small molecules can block signaling by all known classes of Hh activators and by clinically relevant Smo mutants (Nachtergaele, 2013).

Structural insights into the role of the Smoothened cysteine-rich domain in Hedgehog signalling

Smoothened (Smo) is a member of the Frizzled (FzD) class of G-protein-coupled receptors (GPCRs), and functions as the key transducer in the Hedgehog (Hh) signalling pathway. Smo has an extracellular cysteine-rich domain (CRD), indispensable for its function and downstream Hh signalling. Despite its essential role, the functional contribution of the CRD to Smo signalling has not been clearly elucidated. However, given that the FzD CRD binds to the endogenous Wnt ligand, it has been proposed that the Smo CRD may bind its own endogenous ligand. This study presents the NMR solution structure of the Drosophila Smo CRD and describes interactions between the glucocorticoid budesonide (Bud) and the Smo CRDs from both Drosophila and human. These results highlight a function of the Smo CRD, demonstrating its role in binding to small-molecule modulators (Rana, 2013).

Cellular Cholesterol Directly Activates Smoothened in Hedgehog Signaling

In vertebrates, sterols are necessary for Hedgehog signaling, a pathway critical in embryogenesis and cancer. Sterols activate the membrane protein Smoothened by binding its extracellular, cysteine-rich domain (CRD). Major unanswered questions concern the nature of the endogenous, activating sterol and the mechanism by which it regulates Smoothened. This study reports crystal structures of CRD complexed with sterols and alone, revealing that sterols induce a dramatic conformational change of the binding site, which is sufficient for Smoothened activation and is unique among CRD-containing receptors. Hedgehog signaling was shown to require sterol binding to Smoothened, and key residues for sterol recognition and activity were defined. Cholesterol itself was shown to bind and activate Smoothened. Furthermore, the effect of oxysterols is abolished in Smoothened mutants that retain activation by cholesterol and Hedgehog. It is proposed that the endogenous Smoothened activator is cholesterol, not oxysterols, and that vertebrate Hedgehog signaling controls Smoothened by regulating its access to cholesterol (Huang, 2016).

Neuropilins are positive regulators of Hedgehog signal transduction

The Hedgehog (Hh) pathway is essential for vertebrate embryogenesis, and excessive Hh target gene activation can cause cancer in humans. This study shows that Neuropilin 1 (Nrp1) and Nrp2, transmembrane proteins with roles in axon guidance and vascular endothelial growth factor (VEGF) signaling, are important positive regulators of Hh signal transduction. Nrps are expressed at times and locations of active Hh signal transduction during mouse development. Using cell lines lacking key Hh pathway components, it was shown that Nrps mediate Hh transduction between activated Smoothened (Smo) protein and the negative regulator Suppressor of Fused (SuFu). Nrp1 transcription is induced by Hh signaling, and Nrp1 overexpression increases maximal Hh target gene activation, indicating the existence of a positive feedback circuit. The regulation of Hh signal transduction by Nrps is conserved between mammals and bony fish; morpholinos targeting the Nrp zebrafish ortholog nrp1a produce a specific and highly penetrant Hh pathway loss-of-function phenotype. These findings enhance knowledge of Hh pathway regulation and provide evidence for a conserved nexus between Nrps and this important developmental signaling system (Hillman, 2011).

This study investigated the mechanism of Nrp action in the Hh pathway to the extent allowed by contemporary understanding of pathway biology. The molecular mechanisms of well-studied pathway components such as Smo and SuFu are only beginning to be understood, making it likely that a more detailed understanding of Nrp function will emerge concomitantly with increases in understanding of Hh pathway biology. Nrps could affect Hh signal transduction in one or more of several distinct ways: action as a coreceptor for ligand or as a downstream transducer and integrator of external stimuli, or by affecting a basic cell property such as cilium formation, adhesion, or intracellular trafficking. The experiments narrow the possibilities. First, it is thought unlikely that Nrps control Hh reception by acting in a coreceptor capacity for Hh ligands, since activation of Hh signal transduction by ligand-independent methods (Ptc1 mutation, SAG stimulation) is sensitive to Nrp loss of function. Similarly, the inhibition of Hh signaling caused by Nrp RNAi is not simply due to generalized cellular derangement in fibroblasts, since canonical Wnt signaling is intact in the absence of Nrp function (Hillman, 2011).

No evidence was found that Nrps act as signal integrators, because there appears to be no convergence between VEGF or Sema signals and the Hh pathway. In the cultured fibroblasts where Hh signal transduction was observed to require Nrp function, the Semas that interact with Nrp had no effect on Hh transduction and the VEGF receptor is not expressed. Possible interactions between Hh and these other pathways remain an open question for other cell types, but they do not explain the requirement for Nrps in cultured fibroblasts (Hillman, 2011).

Primary cilia are required for Hh transduction, based on two combined lines of evidence. Mutations in components of cilia interfere with Hh transduction, and several pathway components are found located in cilia, some of them dynamically in response to ligand or to drugs that affect Hh transduction. Mutations that alter cilia lead to altered Hh transduction, so Nrp inhibition could affect cilia and, thus, Hh signals. Therefore cilia structure were monitored after Nrp inhibition and no change was seen in frequency or size of cilia. As a more precise measure of cilia function, the trafficking of Smo and Gli2, two proteins whose concentration in cilia is a reflection of Hh ligand received, were examined. Both were unchanged following Nrp1+2 RNAi. Therefore, gross changes in cilia function or Hh pathway component localization are not responsible for the connection of Nrps to Hh transduction. As more is learned about how cilia process and transmit Hh transduction steps, additional tests of Nrp effects will be important. It is always possible that Nrps affect a subtle post-translational modification of a Hh pathway component that awaits elucidation (Hillman, 2011).

Last, the mechanism of Nrp action could be to influence the Hh pathway by altering general cell properties. Overexpression of Nrp1 in fibroblasts can result in increased cell-cell adhesion, likely involving an interaction between Nrp1 and a second, unknown cell surface protein. Subsequent work clarified the region of Nrp1 that mediates this adhesion but did not identify the putative interacting partner. This study found that Nrp1 protein in NIH3T3 fibroblasts is present on the cell surface and exists predominantly outside the primary cilium. Nrp-mediated cell-cell adhesion could contribute to a cytoskeletal scaffold important for an as-yet-uncharacterized step in Hh signal transduction (Hillman, 2011).

Cell migration that orients the dorsoventral axis is coordinated with anteroposterior patterning mediated by Hedgehog signaling in the early spider embryo

The early embryo of the spider Achaearanea tepidariorum is emerging as a model for the simultaneous study of cell migration and pattern formation. A cell cluster internalized at the center of the radially symmetric germ disc expresses the evolutionarily conserved dorsal signal Decapentaplegic. This cell cluster migrates away from the germ disc center along the basal side of the epithelium to the germ disc rim. This cell migration is thought to be the symmetry-breaking event that establishes the orientation of the dorsoventral axis. In this study, knockdown of a patched homolog, At-ptc, that encodes a putative negative regulator of Hedgehog (Hh) signaling, prevents initiation of the symmetry-breaking cell migration (see The formation and migration of CM cells during early Achaearanea embryogenesis and cumulus-shift defects caused by At-ptc1 dsRNA injection). Knockdown of a smoothened homolog, At-smo, shows that Hh signaling inactivation also arrests the cells at the germ disc center, whereas moderate inactivation results in sporadic failure of cell migration termination at the germ disc rim. hh transcript expression patterns indicated that the rim and outside of the germ disc are the source of the Hh ligand. Analyses of patterning events suggests that in the germ disc, short-range Hh signal promotes anterior specification and long-range Hh signal represses caudal specification. Moreover, negative regulation of Hh signaling by At-ptc appears to be required for progressive derepression of caudal specification from the germ disc center. Cell migration defects caused by At-ptc and At-smo knockdown correlated with patterning defects in the germ disc epithelium. It is proposed that the cell migration crucial for dorsoventral axis orientation in Achaearanea is coordinated with anteroposterior patterning mediated by Hh signaling (Akiyama-Oda, 2010).

Primary cilia act as specialized signal transduction organelles required for coupling Smo activity to the biochemical processing of Gli

The unanticipated involvement of several intraflagellar transport proteins in the mammalian Hedgehog (Hh) pathway has hinted at a functional connection between cilia and Hh signal transduction. Mammalian Smoothened (Smo), a seven-transmembrane protein essential for Hh signalling, is expressed on the primary cilium. This ciliary expression is regulated by Hh pathway activity; Sonic hedgehog or activating mutations in Smo promote ciliary localization, whereas the Smo antagonist cyclopamine inhibits ciliary localization. The translocation of Smo to primary cilia depends upon a conserved hydrophobic and basic residue sequence homologous to a domain shown to be required for the ciliary localization of seven-transmembrane proteins in Caenorhabditis elegans. Mutation of this domain not only prevents ciliary localization but also eliminates Smo activity both in cultured cells and in zebrafish embryos. Thus, Hh-dependent translocation to cilia is essential for Smo activity, suggesting that Smo acts at the primary cilium (Corbit, 2005).

Nearly all interphase vertebrate cells possess a primary cilium that extends into the extracellular environment. Recent studies have pointed to a role for primary cilia as sensors capable of transmitting diverse types of information from the environment. One example of this is the photoreceptor outer segment, a derivative of the primary cilium and the site at which light activates the seven-transmembrane (7TM) protein rhodopsin. This connection between 7TM proteins and cilia seems to be an ancient one; odorant receptors function on the cilia of C. elegans sensory neurons (Corbit, 2005).

Mutations in genes required for cilia formation cause defects in mouse neural tube patterning indicative of altered Hh signalling. The mechanism by which these genes participate in Hh signalling is unclear, but one possibility is that their involvement signifies a requirement for cilia in Hh signal transduction (Corbit, 2005).

Hh signalling in Drosophila, zebrafish and mice is transduced through Smo, a 7TM protein. Two polyclonal antibodies were used to investigate the expression of endogenous Smo during mouse development two polyclonal anti-Smo antibodies. The antibodies are highly specific, since no protein is detected in Smo-/- samples by immunofluorescence and Western blot analyses. During early somite stages, Smo protein is modestly upregulated in the cells of the node, an organizer involved in several developmental processes including left-right axis determination and floor plate induction (Corbit, 2005).

One structural characteristic that distinguishes ventral node cells from other cells of the early embryo is the presence of a primary cilium. Examination of Smo localization in the nodes of early headfold, late headfold, one-somite, three-somite and five-somite-stage embryos revealed that Smo is located predominantly on primary cilia. Although Smo does not localize uniformly along the entire length of cilia, average expression on cilia is greater than threefold higher than elsewhere on nodal cells (Corbit, 2005).

The increased level of Smo protein expression specifically in the node is notable given that Smo messenger RNA is present throughout the embryo. Because Sonic hedgehog and Indian hedgehog genes (Shh and Ihh) are expressed in and near the node, respectively, it was hypothesized that, as in Drosophila, mouse Smo might be stabilized in regions of Hh signalling. Indeed, Smo is upregulated specifically in and around the Shh-producing cells of the node, as demonstrated by the co-expression of Smo and Shh in early headfold-stage embryos (Corbit, 2005).

To test whether Smo localization to cilia is regulated by Hh signalling, a renal epithelial MDCK (Madin-Darby canine kidney) cell line was created that constitutively expresses Myc-tagged murine Smo. Smo is detectable on the plasma cell membrane and intracellular vesicles of MDCK cells, as described for Drosophila salivary gland and imaginal disc cells and rat KNRK (Kirsten murine sarcoma virus-transformed normal rat kidney) cells. Smo distribution was examined in MDCK cells cultured in the presence of Shh or cyclopamine, a Smo antagonist. After culture for 1h in Shh-conditioned medium, the amount of Smo present on the primary cilium is markedly upregulated. Similar localization of endogenous Smo is seen in Shh-treated mouse embryonic fibroblasts (MEFs) and IMCD-3 (inner medullary collecting duct) kidney cells. Addition of cyclopamine to Shh-conditioned medium eliminates detectable Smo from the primary cilium of MDCK cells (Corbit, 2005).

The mouse homologue of an activated Smo mutant, SmoA1, has been demonstrated to affect Smo distribution. Unlike wild-type Smo and unlike its behaviour in unciliated cells, SmoA1 strongly localizes to the primary cilium of MDCK cells even in the absence of Shh. High concentrations of cyclopamine sufficient to prevent SmoA1 activation of the Hh pathway are able to eliminate SmoA1 from the cilium. To assess whether the presence of Smo on nodal cilia is also regulated by Hedgehog signalling, mouse embryos were cultured in cyclopamine. Comparison of the treated and untreated embryos reveals that cyclopamine markedly diminishes Smo localization to cilia, demonstrating that inhibition of Hh signalling in vivo correlates with the absence of Smo from the cilium (Corbit, 2005).

The upregulation of Smo on the primary cilium in response to Shh or activating mutations suggests that the regulation of mammalian Smo trafficking may be more similar to that of Drosophila Smo than previously recognized. In both a Drosophila cell line and in imaginal discs, Hh induces the accumulation of Smo at the cell surface, whereas in rat KNRK cells, Shh stimulation induces the internalization of cell-surface Smo. One reason KNRK cells process Smo differently may be that they lack cilia. The data suggest that Hh-dependent cell-surface enrichment of Smo may be common to both Drosophila cells and ciliated mammalian cells (Corbit, 2005).

Ciliary localization of the C. elegans 7TM proteins ODR-10 and STR-1 depends on a hydrophobic and basic residue motif immediately carboxy-terminal to the seventh transmembrane segmen. This ciliary localization motif is also present in other 7TM proteins known to localize to mammalian primary cilia, such as somatostatin receptor 3 and serotonin receptor 6. Sequence inspection reveals that Smo proteins also contain a hydrophobic and basic residue motif carboxy-terminal to the seventh transmembrane segment (Corbit, 2005).

To test whether this motif is required for ciliary localization, Trp549 and Arg550 of mouse Smo-Myc were replaced with alanines. The resultant mutant version of Smo is expressed on the cell plasma membrane and intracellular vesicles of MDCK cells, suggesting that it is folded and at least partly released from the endoplasmic reticulum. However, unlike wild-type Smo, this mutant Smo is absent from primary cilia, even in the presence of Shh. Consequently, this mutant was named CLDSmo for ciliary localization defective (Corbit, 2005).

These results indicate that the conserved motif carboxy-terminal to the last transmembrane segment of mouse Smo is a motif essential for ciliary localization, providing evidence that, like C. elegans 7TM proteins, mammalian 7TM proteins can utilize a juxtamembrane hydrophobic and basic residue sequence for proper localization to cilia. Drosophila Smo contains a similar motif, although cilia do not seem to be required for Drosophila Hh signal transduction. It is tempting to speculate that this domain may participate in the movement of Drosophila Smo to the plasma membrane in response to Hh signalling (Corbit, 2005).

To test whether localization to primary cilia is required for Smo function, the activities of wild-type Smo and CLDSmo were compared in two established read-outs of Hh pathway activation. (1) Hh responsiveness was determined in the presence of wild-type or CLDSmo using a Gli-dependent luciferase reporter in NIH-3T3 cells. In the absence of transfected Smo, Shh-conditioned medium stimulates a threefold induction of normalized luciferase activity. Cells transfected with wild-type Smo exhibit a robust increase in luciferase activity. In contrast, CLDSmo does not stimulate Hh pathway activity above control levels (Corbit, 2005). (2) The function of wild-type Smo and CLDSmo was compared in zebrafish. Hh signalling is required for specification of the zebrafish slow muscle lineage. Within the slow muscle lineage, Prox1-expressing superficial slow fibres (SSFs) require low to medium levels of Hh pathway activity, whereas Engrailed-expressing muscle pioneers require high levels of Hh pathway activity. Disruption of the zebrafish Smo homologue, slow muscle omitted (smu), prevents formation of both Prox1-positive SSFs and Engrailed-positive muscle pioneers, resulting in embryos that exhibit characteristic U-shaped somites. Injection of wild-type murine Smo mRNA into smu mutant embryos rescues this morphological defect and restores formation of both SSFs and muscle pioneers. In contrast, injection of CLDSmo mRNA into smu mutant embryos does not rescue shape, SSF formation or muscle pioneer formation. These results demonstrate that the ciliary localization motif is required for Smo function. Whether this motif might have roles in addition to ciliary localization remains to be determined (Corbit, 2005).

Thus, not only does Smo ciliary localization depend upon Hh signalling, but Hh signalling depends upon a Smo ciliary localization motif. Taken together, these findings strongly suggest that Smo acts at the primary cilium to transduce Hh signalling and that Smo localization to cilia is a key regulated step in Hh pathway activation. These studies provide an example of a 7TM protein translocating to the primary cilium in response to pathway activation, and it will be interesting to determine whether the localization of other ciliary 7TM proteins is similarly regulated (Corbit, 2005).

Previous studies have suggested that redistribution of Smo to the appropriate subcellular location might be the main determinant of Smo activity. The current findings indicate that in mammals, the primary cilium may be that subcellular location. However, whether Smo functions at the cilium in all cell types remains to be determined. In addition, how Smo activates the Hh pathway at the cilium remains unclear. One possibility is that Smo must translocate to the cilium to encounter an activator. An alternative possibility is that the cilium structurally coordinates Smo with downstream Hh pathway components to permit productive interactions. In support of this second possibility, beta-arrestin 2, a component of the zebrafish Hh signal transduction apparatus and a known Smo interactor is concentrated in cilia (Corbit, 2005).

Sonic Hedgehog (Shh) signals are transduced into nuclear ratios of Gli transcriptional activator versus repressor. The initial part of this process is accomplished by Shh acting through Patched (Ptc) to regulate Smoothened (Smo) activity. The mechanisms by which Ptc regulates Smo, and Smo activity is transduced to processing of Gli proteins remain unclear. Recently, a forward genetic approach in mice identified a role for intraflagellar transport (IFT) genes in Shh signal transduction, downstream of Patched (Ptc) and Rab23. This study shows that the retrograde motor for IFT is required in the mouse for the phenotypic expression of both Gli activator and repressor function and for effective proteolytic processing of Gli3. Furthermore, the localization of Smo to primary cilia is disrupted in mutants. These data indicate that primary cilia act as specialized signal transduction organelles required for coupling Smo activity to the biochemical processing of Gli3 protein (May, 2005).

Patched1 regulates hedgehog signaling at the primary cilium

Primary cilia are essential for transduction of the Hedgehog (Hh) signal in mammals. This study investigated the role of primary cilia in regulation of Patched1 (Ptc1), the receptor for Sonic Hedgehog (Shh). Ptc1 localizes to cilia and inhibites Smoothened (Smo) by preventing its accumulation within cilia. When Shh binds to Ptc1, Ptc1 leaves the cilia, leading to accumulation of Smo and activation of signaling. Thus, primary cilia sense Shh and transduce signals that play critical roles in development, carcinogenesis, and stem cell function (Rohatgi, 2007).

In Drosophila, Ptc inhibits the movement of Smo to the plasma membrane. Binding of Hh causes the internalization of Ptc from the plasma membrane to vesicles, allowing Smo to translocate to the plasma membrane and activate downstream signaling. The discovery that protein components of primary cilia are required for Hh signaling suggested that subcellular localization has an important role in mammalian Hh signaling. Primary cilia are cell surface projections found on most vertebrate cells that function as sensory 'antennae' for signals. Several components of the Hh pathway, including Smo and the Gli proteins, accumulate in primary cilia, and Smo is enriched in cilia upon stimulation with Shh (Rohatgi, 2007).

The dynamic subcellular localization of Ptc1 and Smo in mammalian cells was studied with the use of novel antibodies to the two proteins. These antibodies allowed detection of endogenous Ptc1 and Smo in cultured mouse fibroblasts (NIH 3T3 cells) and mouse embryonic fibroblasts (MEFs), two Hh-responsive cell types. Hh signaling was activated in NIH 3T3 cells by treatment with either Shh or SAG (Shh-agonist), a small molecule that directly binds and activates Smo. Because ptc1 is itself a transcriptional target of Hh signaling, increases in Ptc1 protein levels can serve as a metric for pathway activation. Ptc1 protein levels began to rise by 4 hours and continued to increase until 24 hours after addition of Shh. After stimulation of cells with Shh or SAG, endogenous Smo was enriched in primary cilia. The mean fluorescence intensity of Smo in cilia began to increase as early as 1 hour after stimulation of cells with Shh or SAG. This likely represented relocalization from a cytoplasmic pool, because the total amount of Smo protein did not increase at this time point (Rohatgi, 2007).

To determine whether Ptc1 regulates the localization of Smo, Smo localization was examined in MEFs from ptc1-/- mice. These cells showed constitutive activation of Hh target gene transcription. Consistent with a role of Ptc1 in regulating Smo trafficking, Smo was constitutively localized to primary cilia in these cells even in the absence of Shh or SAG. Reintroduction of Ptc1 into these cells by means of a retrovirus suppressed Hh-pathway activity and prevented Smo accumulation in primary cilia. Thus, the regulation of Smo localization by Ptc1 is conserved from flies to mammals (Rohatgi, 2007).

To understand how Ptc1 may regulate entry of Smo into the cilium, the localization of Ptc1 was examined in MEFs and mouse embryos. Endogenous Ptc1 was present in small amounts in MEFs, near the limit of detection by immunofluorescence. Therefore the amounts of Ptc1 protein was increased by stimulating cells with SAG. Under these conditions, Ptc1 was highly enriched in primary cilia. The ciliary localization of Ptc1 was confirmed in three additional ways. First, Ptc1 fused to yellow fluorescent protein (Ptc1-YFP) was found around the base and in the shaft of cilia in unstimulated ptc1-/- cells infected with a retrovirus encoding Ptc1-YFP. Second, Ptc1-YFP overproduced in ptc1-/- cells by transfection showed clear ciliary localization in both live and fixed cells. Third and most important, endogenous Ptc1 was found in the cilia of mouse embryo mesoderm cells responsive to Shh (Rohatgi, 2007).

Ptc1 staining in cross sections of embryonic day 9.5 (E9.5) embryos was detected in cells of the ventral neural tube, notochord, splanchnic mesoderm, and paraxial mesoderm, precisely the regions where Hh signaling is known to be active and Shh target genes such as ptc1 are highly expressed. Focus was placed on mesoderm cells because they are likely the cells that gave rise to the MEFs that were analyzed in culture. Endogenous Ptc1 showed asymmetric localization to a domain surrounding the base of the cilium and in particles along the shaft of the cilium. This localization pattern around the base and in a particulate pattern along the shaft of the cilium is similar to that seen in cultured fibroblasts. In embryo cells, there was more variability in the amount of Ptc1 in the shaft of cilia, a finding likely related to differences in the amount of Shh signal received by cells in the complex milieu of embryonic tissue. The concentration of Ptc1 at the base of primary cilia suggests a mechanism for how it may inhibit Smo activation. Transport of proteins in and out of primary cilia is thought to be regulated at their base, and Ptc1 could function at this location to inhibit a protein-trafficking step critical for Smo activation (Rohatgi, 2007).

Shh could inactivate Ptc1 by binding to it at the cilium and inducing its internalization, degradation, or movement to other regions of the plasma membrane. To determine whether Ptc1 at the cilium can bind to Shh, a fluorescently labeled version of the N-terminal signaling fragment of Shh (ShhN-A594) was produced. Minute amounts of ShhN-A594, one-hundredth of those required to activate signaling, were added to live ptc1-/- cells transfected with Ptc1-YFP and a marker for cilia, inversin fused to cyan fluorescent protein (inversin-CFP). Live cells were used because the interaction between Shh and Ptc1 does not survive fixation. ShhN-A594 concentrated at cilia containing Ptc1-YFP and colocalized with puncta of Ptc1-YFP. Ptc1-/- cells expressing inversin-CFP alone did not bind ShhN-A594, and an excess of unlabeled ShhN prevented binding of ShhN-A594 (Rohatgi, 2007).

It was next asked whether the interaction of Shh with Ptc1 influences the localization of Ptc1. Ptc1 was concentrated at cilia after treatment of cells with SAG alone but not after treatment with Shh or a combination of Shh and SAG. This suggested that Shh binding might trigger the removal of the Ptc1-Shh complex from the cilium, or that new Ptc1 produced in response to Shh was not localized in the cilium. To distinguish these possibilities, the production of large amounts of Ptc1 was induced in the cilia of NIH 3T3 cells with SAG treatment and then the cells were switched to control medium or medium containing Shh. Ptc1 levels in the cilium remained stable in the control, but Shh treatment caused a time-dependent disappearance of Ptc1 from the primary cilium. The loss of Ptc1 from cilia was not associated with a decrease in total Ptc1 protein levels and thus implied movement of Ptc1 from cilia to another location in the cell. This delocalization was only evident with the endogenous protein and not upon examination of transfected Ptc1-YFP, a far more abundant protein (Rohatgi, 2007).

Ptc1 and Smo localization were examined in the same experiment. Because the localization changes for Ptc1 and Smo described above were each seen in >80% of the cilia visualized, the levels of Ptc1 and Smo in cilia were inversely correlated. The reciprocal time courses of Ptc1 disappearance and Smo appearance at cilia after Shh addition support a model in which Shh triggers the removal of Ptc1 from the cilium, allowing Smo to enter and activate signaling. Consistent with this idea, cells of the ventral neural tube and floor plate, which receive large amounts of Shh, showed high levels of Smo and low levels of Ptc1 in cilia. The movement of Ptc1 and Smo at the cilium is analogous to the situation in Drosophila, where pathway activation is associated with Smo movement to the plasma membrane and movement of Ptc away (Rohatgi, 2007).

Ptc1 may regulate Smo localization through a small molecule. Because Smo translocation to the primary cilium appears to be a critical step in its activation, a regulatory small molecule would be predicted to control this step. Naturally occurring oxysterols are good candidates for endogenous small molecules that regulate Smo function. Cellular sterol concentrations are important determinants of a cell's responsiveness to Shh, and oxysterols can activate Hh signaling. When NIH 3T3 cells were treated with activating concentrations of the oxysterol 20alpha-hydroxycholesterol, Smo rapidly translocated to the primary cilium with kinetics that were identical to those seen in cells treated with SAG or Shh. Treatment with 7alpha-hydroxycholesterol, an oxysterol that does not activate the Hh pathway, did not induce translocation of Smo. This result provides a specific molecular mechanism -- Smo translocation to cilia -- to explain how oxysterols regulate Hh signaling (Rohatgi, 2007).

Cells treated with 20alpha-hydroxycholesterol also retained Ptc1 in cilia in a pattern similar to that seen in cells treated with SAG. Thus, oxysterols appear to function not like Shh, by causing the removal of Ptc1 from cilia, but at a more downstream step to make Smo insensitive to the inhibitory effects of Ptc1. However, oxysterols function differently from SAG because they likely do not directly bind to Smo (Rohatgi, 2007).

These results suggest that Ptc1 localization to primary cilia inhibits the Hh pathway by excluding Smo and also allows cilia to function as chemosensors for the detection of extracellular Shh. Binding of Shh to Ptc1 at primary cilia is coupled to pathway activation by the reciprocal movement of Ptc1 out of the cilia and Smo into the cilia, a process that may be mediated by oxysterols. Elucidating the molecular machinery that controls Ptc1 and Smo trafficking at primary cilia will likely provide new targets for modulation of this important pathway (Rohatgi, 2007).

Smoothened transduces Hedgehog signal by forming a complex with Evc/Evc2

Hedgehog (Hh) signaling plays pivotal roles in embryonic development and adult tissue homeostasis in species ranging from Drosophila to mammals. The Hh signal is transduced by Smoothened (Smo), a seven-transmembrane protein related to G protein coupled receptors. Despite a conserved mechanism by which Hh activates Smo in Drosophila and mammals, how mammalian Hh signal is transduced from Smo to the Gli transcription factors is poorly understood. This study provides evidence that two ciliary proteins, Evc and Evc2, the products of human disease genes responsible for the Ellis-van Creveld syndrome, act downstream of Smo to transduce the Hh signal. Loss of ciliary protein complex Evc/Evc2 does not affect Sonic Hedgehog-induced Smo phosphorylation and ciliary localization but impedes Hh pathway activation mediated by constitutively active forms of Smo. Evc/Evc2 are dispensable for the constitutive Gli activity in Sufu(-/-) cells, suggesting that Evc/Evc2 act upstream of Sufu to promote Gli activation. Furthermore, it was demonstrated that Hh stimulates binding of Evc/Evc2 to Smo depending on phosphorylation of the Smo C-terminal intracellular tail and that the binding is abolished in Kif3a(-/-) cilium-deficient cells. It is propose that Hh activates Smo by inducing its phosphorylation, which recruits Evc/Evc2 to activate Gli proteins by antagonizing Sufu in the primary cilia (Yang, 2012).

This study has obtained evidence that Hh stimulates the binding of Kif7 to Smo, which is facilitated by Evc/Evc2. Increased binding of Cos2 to activated Smo has also been observed in Drosophila. It has been shown that binding of Cos2 to activated Smo promotes Fu dimerization, phosphorylation and activation, and activated Fu then regulates Ci by inhibiting its repressor form and stimulating its activator form. The mammalian Fu homolog is dispensable for Hh signaling; however, it is possible that another kinase(s) may substitute for the Fu function in the mammalian Hh pathway. It has been shown that Hh stimulates Gli3 phosphorylation, which correlates with formation of Gli3. Therefore, it would be interesting to determine whether Hh-induced Gli3 phosphorylation is affected by loss of Evc/Evc2. It is tempting to speculate that the activated Smo/Evc/Evc2 complex may recruit one or more kinases to phosphorylate Gli proteins and promote their activation (Yang, 2012).

Smoothened signal transduction is promoted by G protein-coupled receptor kinase 2

Deregulation of the Sonic hedgehog pathway has been implicated in an increasing number of human cancers. In this pathway, the seven-transmembrane (7TM) signaling protein Smoothened regulates cellular proliferation and differentiation through activation of the transcription factor Gli. The activity of mammalian Smoothened is controlled by three different hedgehog proteins, Indian, Desert, and Sonic hedgehog, through their interaction with the Smoothened inhibitor Patched. However, the mechanisms of signal transduction from Smoothened are poorly understood. This study shows that a kinase which regulates signaling by many 'conventional' 7TM G-protein-coupled receptors, G protein-coupled receptor kinase 2 (GRK2), participates in Smoothened signaling. Expression of GRK2, but not catalytically inactive GRK2, synergizes with active Smoothened to mediate Gli-dependent transcription. Moreover, knockdown of endogenous GRK2 by short hairpin RNA (shRNA) significantly reduces signaling in response to the Smoothened agonist SAG and also inhibits signaling induced by an oncogenic Smoothened mutant, Smo M2. GRK2 promotes the association between active Smoothened and beta-arrestin 2. Indeed, Gli-dependent signaling, mediated by coexpression of Smoothened and GRK2, is diminished by beta-arrestin 2 knockdown with shRNA. Together, these data suggest that GRK2 plays a positive role in Smoothened signaling, at least in part, through the promotion of an association between beta-arrestin 2 and Smoothened (Meloni, 2006).

Patched1 and Patched2 inhibit Smoothened non-cell autonomously

Smoothened (Smo) inhibition by Patched (Ptch) is central to Hedgehog (Hh) signaling. Ptch, a proton driven antiporter, is required for Smo inhibition via an unknown mechanism. Hh ligand binding to Ptch reverses this inhibition and activated Smo initiates the Hh response. To determine whether Ptch inhibits Smo strictly in the same cell or also mediates non cell-autonomous Smo inhibition, genetically mosaic neuralized embryoid bodies (nEBs) were generated from mouse embryonic stem cells (mESCs). These experiments utilized novel mESC lines in which Ptch1, Ptch2, Smo, Shh and 7dhcr were inactivated via gene editing in multiple combinations, allowing measurement of non-cell autonomous interactions between cells with differing Ptch1/2 status. In several independent assays the Hh response was repressed by Ptch1/2 in nearby cells. When 7dhcr was targeted, cells displayed elevated non-cell autonomous inhibition. These findings support a model in which Ptch1/2 mediate secretion of a Smo-inhibitory cholesterol precursor (Roberts, 2016).

Developmental roles of Smoothened homologs

Hedgehog ligands interact with receptor complexes containing Patched (PTC) and Smoothened (SMO) proteins to regulate many aspects of development. The mutation W535L (SmoM2) in human Smo is associated with basal cell skin cancers, causes constitutive, ligand-independent signaling through the Hedgehog pathway, and provides a powerful means to test effects of unregulated Hedgehog signaling. Expression of SmoM2 in Xenopus embryos leads to developmental anomalies that are consistent with known requirements for regulated Hedgehog signaling in the eye and pancreas. Additionally, it results in failure of midgut epithelial cytodifferentiation and of the intestine to lengthen and coil. The midgut mesenchyme shows increased cell numbers and attenuated expression of the differentiation marker smooth muscle actin. With the exception of the pancreas, differentiation of foregut and hindgut derivatives is unaffected. The intestinal epithelial abnormalities are reproduced in embryos or organ explants treated directly with active recombinant hedgehog protein. Ptc mRNA, a principal target of Hedgehog signaling, is maximally expressed at stages corresponding to the onset of the intestinal defects. In advanced embryos expressing SmoM2, Ptc expression is remarkably confined to the intestinal wall. Considered together, these findings suggest that the splanchnic mesoderm responds to endodermal Hedgehog signals by inhibiting the transition of midgut endoderm into intestinal epithelium and that attenuation of this feedback is required for normal development of the vertebrate intestine (Y. Zhang, 2001).

These observations suggest that the response to Hh signaling in the developing gut is manifested in part in the same cells that produce Hh ligands, the gut endoderm. Early in gut development, it is inferred that the splanchnic mesoderm responds to Hh signals by actively inhibiting epithelial differentiation. Later, Hh signaling must be attenuated to allow proper midgut development, including elongation, coiling, radial intercalation of ventral endodermal cells, and ordered differentiation of both the epithelium and the surrounding mesenchyme. Constitutive signaling through SmoM2 is principally activated in the gut mesoderm, where morphologic correlates include patchy thickening and reduced expression of the differentiation marker smooth muscle actin. Although failure of the gut tube to elongate and coil is also likely related to mesenchymal defects, the major consequence is absence of midgut epithelial cytodifferentiation. Since effectors of Hh signaling localize to the gut mesoderm and not the endoderm, this is interpreted to represent a secondary effect reflecting a requirement for regulated attenuation of Hh activity in the differentiation of the intestinal epithelium. The decline in Hh mRNA levels after Stage 40 in Xenopus development is consistent with this model. However, the identity of the reverse signal is not known. BMP-4 is one important target of Hh signaling in the hindgut of chick embryos, and the Drosophila homolog decapentaplegic mediates signaling between splanchnic mesoderm and endoderm in flies. Factors of this class are hence candidate mediators of the isolated midgut phenotype observed in SmoM2- expressing Xenopus embryos. Treatment of Xenopus embryos with retinoic acid or injection of a truncated, dominant inhibitory FGF receptor results in intestinal developmental abnormalities partially resembling those seen with expression of SmoM2. In particular, FGF receptor signaling is required for synchronized differentiation of intestinal smooth muscle. Thus, a number of known signaling pathways appear to function in concert to effect intestinal organogenesis during vertebrate development (Y. Zhang, 2001).

Despite extensive studies, there are still many unanswered questions regarding the mechanism of hedgehog signaling and the phylogenic conservation of hedgehog function in vertebrates. For example, whether hedgehog signaling in vertebrates requires smoothened is unclear, and the role of hedgehog activity in zebrafish is controversial. Inactivation of smoothened by retroviral insertions in zebrafish results in defects that are characteristic of hedgehog deficiencies, including abnormalities in body size, the central nervous system, adaxial mesoderm, cartilage and pectoral fins. As in Drosophila, vertebrate smoothened is essential for hedgehog signaling, and functions upstream of protein kinase A. Further analysis of neural tube defects reveals the absence of lateral floor plate and secondary motoneurons (SMN), but the presence of medial floor plate and primary motoneurons (PMN) in smoothened mutant embryos. Blocking maternal hedgehog signaling by cyclopamine eliminates primary motoneurons, but not medial floor plate. Interestingly, even after inhibition of maternal hedgehog activity, the midbrain dopaminergic neurons still form, and looping of the heart does not randomize in the mutants. Decreased proliferation and increased apoptosis are also found in the mutants. Taken together, these data demonstrate the conserved role of vertebrate smoothened in the hedgehog signaling pathway, and reveal similarities and differences in hedgehog function between teleosts and amniotes (Chen, 2001).

The Hh pathway plays an important role in the development of motor neurons (MN) in amniotes. MN development was examined in smo mutants. Unlike amniotes, teleosts and amphibians have two types of MN: PMNs and SMNs. The latter are thought to be the equivalent of the lateral MN column in amniotes. In smo mutant embryos, SMNs are absent, as indicated by the lack of Islet-1 and Zn-5 antibody staining in ventral spinal cord at 36 or 48 hpf. Therefore, the smo gene is essential for the development of SMNs (Chen, 2001).

The lack of Islet-1 staining at 36 hpf also suggests that the function of the smo gene is required for PMN development. However, irregular spontaneous local contractions and occasional body 'wiggles' have been observed from 20 to 26 hours of development in the mutant embryos. This prompted an examination of PMNs at earlier stages. At 20 hpf, wild-type embryos have three to four PMNs in each hemisegment. However, mutant embryos only have two or three PMNs per hemisegment in the anterior half of the spinal cord and one or none in the posterior spinal cord. In addition, unlike PMNs in wild-type embryos, which each extends an axon along a specific path, axons of these PMNs in mutant embryos are disorganized. Thus, zygotic function of the smo gene is not essential for the specification of early-born PMN in the anterior spinal cord. However, it is important for guiding axons of those PMNs to the correct targets, and for the formation of the late-born PMN in the posterior spinal cord (Chen, 2001).

The induction of PMNs in the anterior spinal cord in the smo mutants could be a function of maternal smo mRNA. The development of PMNs was examined in cyclopamine-treated embryos. Neither wild-type nor mutant embryos treated from 4-10 hpf displayed any muscle contraction. Few PMNs or SMNs were detectable in these embryos using Islet-1 antibody. Thus, the Hh pathway is essential for both PMN and SMN development in zebrafish, and maternal smo mRNA is responsible for the formation of the early-born PMNs. This also explains the increasing severity of PMN defects from rostral to caudal. As development proceeds and maternal mRNA is depleted, more defects might be expected to occur in the later-developing caudal neural tube (Chen, 2001).

Genetic analyses in Drosophila have demonstrated that the multipass membrane protein Smoothened (Smo) is essential for all Hedgehog signaling. Smo acts epistatic to Ptc1 to mediate Shh and Ihh signaling in the early mouse embryo. Smo and Shh/Ihh compound mutants have identical phenotypes: embryos fail to turn, arresting at somite stages with a small, linear heart tube, an open gut and cyclopia. The absence of visible left/right (L/R) asymmetry led to an examination of the pathways controlling L/R situs. Hedgehog signaling within the node is thought to be required for activation of Gdf1 (a TGFbeta family member that acts upstream of Nodal, Lefty1/2, and Pitx2), and induction of left-side determinants. Further, an absolute requirement is demonstrated for Hedgehog signaling in sclerotomal development and a role in cardiac morphogenesis (X. Zhang, 2001).

The node is thought to play dual roles in the control of laterality in the mouse through the production of lateralizing signals and directing their asymmetric distribution by cilial movement within the node. Several lateralizing factors are uniformly expressed in the node. In addition to Shh and Ihh, the mouse node expresses Nodal, its cofactor Cryptic, Fgf8, and Gdf1. All of these genes also have expression domains outside of the node. Therefore, in the absence of genetic tools that specifically remove node-derived signals, there is some ambiguity as to the actual source of the signals controlling L/R situs. Interestingly, Gdf1 and Fgf8 mutants both fail to activate Nodal in the left lateral plate mesoderm, suggesting that either, or both, of these factors may be targets of HH signaling. Expression of Gdf1, but not Fgf8, in the mouse node is HH dependent. Gdf1 is downregulated in Smo mutants and upregulated in Ptc1 mutants. Thus, HH signaling in the node appears to lie upstream of Gdf1, suggesting that at least part of the laterality defects observed can be explained by loss of Gdf1 activity. However, the failure of Nodal induction in the left lateral plate mesoderm is fully penetrant in Smo, but not Gdf1, mutants. Thus, there are likely to be mechanisms other than the control of Gdf1 expression by which HH signaling regulates activity of the node. In support of this view, reduced expression of Nodal and Cryptic is observed in Smo mutants. At the gross structural level, the node appears morphologically normal and expresses several genes that are not implicated directly in the regulation of situs (for example Brachyury). Further, the notochord, which is derived from the node, forms in Smo mutants, indicating that this aspect of node function is intact. Preliminary scanning electron microscopy indicates that node cilia are present and appear similar to those of wild-type embryos. However, no attempt was made to measure cilial-based nodal flow in Smo mutants. Thus either Shh or Ihh signaling is required for activation of Gdf1 and for establishing normal levels of expression of Nodal in the node. As a result of defective node signaling, no left-side pathway of development is initiated. Whether there is also a right-sided pathway and, if so, what happens to expression of these genes in the absence of HH signaling remains an open question. Unfortunately, no genes have been reported that are only expressed on the right side of the mouse embryo at equivalent stages (X. Zhang, 2001).

Sonic hedgehog (Shh) signaling patterns many vertebrate tissues. shh mutations dramatically affect mouse ventral forebrain and floor plate but produce minor defects in zebrafish. Zebrafish have two mammalian Shh orthologs, sonic hedgehog and tiggy-winkle hedgehog, and another gene, echidna hedgehog, that could have overlapping functions. To examine the role of Hedgehog signaling in zebrafish, slow muscle omitted (smu) mutants were characterized. smu encodes a zebrafish ortholog of Smoothened that transduces Hedgehog signals. Zebrafish smoothened is expressed maternally and zygotically and supports specification of motoneurons, pituitary cells and ventral forebrain. It is proposed that smoothened is required for induction of lateral floor plate and a subpopulation of hypothalamic cells and for maintenance of medial floor plate and hypothalamic cells (Varga, 2001).

Lateral floor plate and parts of ventral forebrain fail to form in smu mutants. Patterning defects in anterior neural plate of smu mutants at the end of gastrulation correlate with reduced hypothalamic tissue and interocular distance at 24 h. In comparison, nodal pathway mutant cyclops (ndr2) lack medial floor plate and ventral forebrain and form cyclopic eyes. Recent studies have suggested that in hypothalamus, both Nodal and Hh signaling are required for normal ventral forebrain specification. The results support this interpretation, because Nodal is thought to act upstream of Hh, and Hh expression is lost from the anterior neural plate in Nodal pathway mutants such as cyclops (ndr2) and one-eyed-pinhead (oep). Thus, in Nodal mutants, both medial floorplate, as well as its presumptive anterior extension into hypothalamus, is lost, whereas in the Hh pathway mutants, only lateral floor plate is affected and the hypothalamus initially forms. It is therefore suggested that the less severe forebrain defects in smu mutants are due to loss of the anterior extension of lateral floor plate in the forebrain. Furthermore, although medial floor plate may initially be induced in a Hh-independent manner by Nodal signals, Hh signaling is required much later for its maintenance (Varga, 2001).

The data are consistent with the idea that, as in mouse, Hh signaling is required for zebrafish motoneuron formation. In smu mutants, primary motoneurons form in anterior spinal cord, but not posteriorly, and later-developing secondary motoneurons completely fail to form. smoothened is expressed both maternally and zygotically, suggesting a dual role: maternal expression supports anterior primary motoneuron formation, whereas zygotic expression is required for primary motoneuron induction in posterior trunk and tail, as well as for later-developing secondary motoneurons. The results also suggest that induction of primary motoneurons and secondary motoneurons in syu (shh) mutants probably results from other Hh proteins acting in place of Shh, reinforcing the idea that these signals have overlapping functions (Varga, 2001).

Dorsoventral forebrain patterning is disrupted in smu mutants. The analysis suggests that diencephalon and telencephalon lose ventral character. Hh signaling leads to induction of genes expressed ventrally, such as nkx and dlx. In smu mutants, reduced expression of genes that normally demarcate ventral regions was found, like nkx2.2, dlx2 and pax6a, and expansion of dorsally expressed emx1 into ventral regions, consistent with studies indicating a role of Hh signaling for specification of ventral forebrain fates. Hh signaling antagonizes Gli3, a cubitus interruptus homolog that suppresses ventral fates. Gli3 could mediate dorsal restriction of telencephalic genes such as emx1 and, in the absence of Hh signaling, dorsal telencephalic genes may be de-repressed ventrally (Varga, 2001).

In addition to gene expression defects, axon tract formation is severely affected in the smu mutant ventral forebrain. In wild type, anterior commissure axons project to ventral telencephalon and not diencephalon. In smu mutants, however, anterior commissure axons grow aberrantly into diencephalon, which correlates with the expansion of emx1 expression into ventral telencephalon and anterior diencephalon. Aberrant growth of anterior commissure axons into diencephalon in smu mutants may result from expression of dorsal genes like emx1 in ventral telencephalic and anterior diencephalic cells that redirects these cells to adopt fates of more dorsal cells, including normal targets of these axons. Additionally, Hh signaling may regulate directly expression of Netrin1 and other extracellular guidance cues in ventral neural tube. Thus, changes in the distributions of these extracellular cues in Hh pathway mutants, such as has been demonstrated for Netrin1 in smu mutants, may lead to the aberrant growth of axons, including loss of commissures (Varga, 2001).

The hedgehog signaling pathway organizes the developing ventral neural tube by establishing distinct neural progenitor fates along the dorsoventral axis. In the embryonic spinal unique and partially overlapping patterns of expression of several families of homeodomain-containing transcriptional regulators define five neural progenitor populations in the ventral half of the neural tube. From ventral to dorsal, these are the p3, pMN, p2, p1, and p0 progenitors. pMN progenitors later give rise to motorneurons, and p3, p2, p1, and p0 progenitors give rise to v3, v2, v1, and v0 interneurons, respectively. Smoothened (Smo) is essential for all Hedgehog (Hh) signaling, and genetic inactivation of Smo cells autonomously blocks the ability of cells to transduce the Hh signal. Using a chimeric approach, the behavior of Smo null mutant neural progenitor cells was examined in the developing vertebrate spinal cord; direct Hh signaling is essential for the specification of all ventral progenitor populations. Further, Hh signaling extends into the dorsal half of the spinal cord including the intermediate Dbx expression domain. Surprisingly, in the absence of Sonic hedgehog (Shh), the presence of a Smo-dependent Hh signaling activity operating in the ventral half of the spinal cord is observed that most likely reflects Indian hedgehog (Ihh) signaling originating from the underlying gut endoderm. Comparative studies of Shh, Smo, and Gli3 single and compound mutants reveal that Hh signaling acts in part to specify neural cell identity by counteracting the repressive action of Gli3 on p0, p1, p2, and pMN formation. However, whereas these cell identities are restored in Gli3/Smo compound mutants, correct stratification of the rescued ventral cell types is lost. Thus, Hh signaling is essential for organizing ventral cell pattern, possibly through the control of differential cell affinities (Wijgerde, 2002).

Further, although the data indicate that there is a direct Hh input for the establishment of all ventral cell identities, they do not address whether Hedgehog action is concentration-dependent. For example, Hedgehog signaling might define a domain of ventral competence, whereas other factors might play a more direct role in the induction of each individual cell fate. Repression of Pax7 in ventral cells is thought to be a critical first step in the induction of ventral cell identities, and the ventral limit of Pax7-expressing cells has long been seen as the limit for Shh signaling. Thus, repression of Pax7 might define a ventral competence domain. All Smo-/- cells that lie ventral to the normal ventral limit of Pax7 expression at 10.5 dpc maintain Pax7 expression, consistent with there being an absolute requirement for a Hh input to repress Pax7 (Wijgerde, 2002).

The results also demonstrate that the presence of v0 and v1 progenitors observed in Shh mutants reflects the presence of low-level Hh signaling as indicated by the absence of Pax7 expression and the upregulation of Ptch1 at the ventral midline of the Shh mutant neural tube, where these ventral interneuron precursors arise. Since Ihh is expressed in both the node and the gut endoderm that underlies the notochord, it is speculated that Ihh signaling is responsible for the limited ventralization in Shh mutants. Interestingly, Shh and Ihh play semiredundant roles in patterning somite domains that lie adjacent to the neural tube. Whether Ihh plays any normal role in patterning the ventral neural tube in the context of an active Shh signal is doubtful (Wijgerde, 2002).

Examining the subsequent fate of Pax7-producing cells provides an insight into the assignment of ventral cell fates in the neural tube. Based on the expression profile of ventral Smo-/- neural progenitor cells (Pax7on, Dbx1on, Dbx2on, Irx3on, and Pax6on where 'on refers to cells expressing the particular transcription factor) and the neuron types generated by Smo-/- progenitors (Lim1/2on, Pax2on), it appears that many ventral Smo-/- cells adopt a dorsal identity, possibly dorsal p5. However, a few cells also express Evx1/2, indicating that at least some Smo-/- progenitors give rise to v0 precursors. In contrast, as discussed above, Smo-/- cells are unable to generate Dbx-producing neural progenitor cells or v0 precursors where these cells normally arise, in the intermediate domains of the spinal cord. How can these paradoxical results be explained (Wijgerde, 2002)?

Interestingly, whereas v0 precursors are appropriately positioned in the neural tube of Smo/Gli3 compound mutants, v1, v2, and MN precursors, which normally do not overlap, now extend over much of the ventral half of the neural tube. Thus, a direct Hh signaling input is required for the normal stratification of ventral progenitor populations within separate domains of the ventral neural tube. This indicates that a central role of the hedgehog-Gli3 signaling axis is to refine the size and position of ventral progenitor pools rather than specifying the individual identity of MN, v2, v1, and v0 precursors. During normal DV patterning, each precursor population forms a sharp boundary with its neighbor, suggesting that there may be some Hh-dependent mechanism that prevents their mixing. In Drosophila, Hh signaling maintains a sharp anterior-posterior (AP) compartment boundary, preventing the mixing of cells at the AP interface. Further, analysis of Smo-/- cells in the abdomen of the fly suggests that a gradient of Hedgehog signaling specifies graded levels of cell affinity. Thus, it is tempting to speculate that Hh signaling in the mammalian neural tube might regulate the precise separation of precursor domain boundaries along the DV axis through the control of cell affinities (Wijgerde, 2002).

If a DV gradient of affinities normally contributes to the segregation of progenitors, then dorsalized Smo-/- cells in ventral positions might be expected to cluster with each other, minimizing contacts with their ventral neighbors, similar to the behavior of clones of Smo-/- clones in the anterior compartment of the fly wing or abdomen. In this regard it is noted that whereas Smo+/- cells show a fine-grained mosaicism in the neural tube of chimeras, Smo-/- cells tend to cluster in patches, a finding observed in other Hh target fields. Functionally, modifying differences in cell affinities could prevent cells from mixing freely within a morphogenetic field as cell identities are being specified, a mechanism that may contribute to precision and stability in the induction of different progenitor domains. Presumably, once neurons are generated from progenitor cells their identity is fixed. At this time they may mix 'freely' to participate in the formation of appropriate neural circuits (Wijgerde, 2002).

Sonic hedgehog plays a key role during embryogenesis and organogenesis. Tooth development (odontogenesis) is governed by sequential and reciprocal epithelial-mesenchymal interactions. The first visible indication of the initiation of odontogenesis is the appearance of a local thickening of the oral ectoderm, which subsequently grows into the underlying neural crest-derived mesenchyme of the first branchial arch to form epithelial buds. Ectomesenchymal cells condense around the epithelial buds to form the dental mesenchyme. The primary enamel knot, a tooth signaling center, becomes prominent at the cap stage. At the bell stage, the tooth consists of an epithelial enamel organ (EEO) and a dental mesenchyme. The EEO components include proliferating preameloblasts and their progenitors, the cells of the inner dental epithelium (IDE), the secondary enamel knots at the tip of nascent cusps, the stellate reticulum (SR), the outer dental epithelium (ODE) and the stratum intermedium (SI). The latter consists of squamous cells adjacent to the IDE and preameloblasts. Later-arising secondary enamel knots, which form in the developing molars, are thought to control cuspal morphogenesis and terminal differentiation of odontoblasts. Mesenchymal cells of the dental papilla that lie adjacent to the IDE form the preodontoblast layer of proliferating cells; the rest of the dental papilla cells contribute to the later development of the dental pulp. Finally, dental mesenchyme that surrounds the tooth germ forms the dental sac, which gives rise to periodontal tissues (Gritli-Linde, 2002).

During the cytodifferentiation stage, the terminal differentiation of odontoblasts is accomplished by their withdrawal from the cell cycle, elongation, polarization and secretion of a predentin matrix. This, in turn, triggers terminal differentiation of ameloblasts. Differentiation of the ameloblast into a highly-polarized complex secretory cell involves considerable growth, elongation of the cytoplasm, a change in nuclear polarity, a sequential development and change in polarity of organelles, and the appearance of a complex cytoskeleton. During this stage, there are other progressive changes within the enamel organ. Cells from the SI that are adjacent to polarizing post-mitotic ameloblasts become cuboidal in shape, except in the future enamel-free areas in rodent molars. In addition, the SR is invaded by blood vessels and fibroblasts emanating from the dental sac (Gritli-Linde, 2002 and references therein).

The rodent incisor is unique in its tissue organization and consists of stem cells, differentiating cells and mature cells organized in defined regions along its anteroposterior and labial-lingual axes. The rodent incisor is asymmetrical, as the labial or amelogenic IDE gives rise to ameloblasts and enamel, whereas the IDE on the lingual side does not produce enamel. The posterior-most aspect of the incisor has been postulated to contain stem cells which give rise to the different dental cell populations. A posteroanterior gradient of cytodifferentiation is thus present in the rodent incisor throughout life, with the less differentiated cells located posteriorly and the most mature cells anteriorly. Odontoblasts differentiate all along the epitheliomesenchymal interface of the incisor (Gritli-Linde, 2002 and references therein).

Like many organs, morphogenesis and cytodifferentiation of the tooth is governed by sequential and reciprocal epithelialmesenchymal interactions mediated by several soluble bioactive proteins. In addition, cell-matrix interactions and cell-cell junctional complexes and cytoskeletal components have been implicated in the regulation of histomorphogenesis and proliferation. Sonic hedgehog signals are received within a target tissue by the general Hedgehog receptor Patched 1 (Ptc1). Transduction of the signal within a responding cell absolutely requires the activity of a second, multi-pass, membrane protein Smoothened (Smo). Smoothened activity leads to a conserved transcriptional response: up-regulation of Ptc1 and Gli1 in the target tissue (Gritli-Linde, 2002).

Shh is expressed exclusively in the epithelial component of the murine tooth from the dental lamina stage until cytodifferentiation. At the cap stage, Shh is confined to the primary enamel knot. Expression spreads thereafter to the rest of the IDE laterally, the stratum intermedium and the stellate reticulum. At the cap stage, general transcriptional targets and effectors of Shh signaling, including Ptc1, Gli1 and Smoothened (Smo) are, however, expressed in both dental epithelium and mesenchyme, but are excluded from the enamel knot. In contrast, Ptc2, while appearing to bind all mammalian Hedgehog proteins similarly to the related Hedgehog receptor Ptc1, is expressed in the enamel knot and IDE at the cap and early bell stages, respectively (Gritli-Linde, 2002).

Shh protein produced by the enamel knot and IDE moves many cell diameters to reach the rest of the dental epithelium and the dental papilla, indicating that Shh has a long-range activity, consistent with the broad expression of Shh target genes. Together, the above observations suggest that Shh signaling may be operative intra-epithelially as well as in mediating epithelial-mesenchymal interactions during tooth development. Finally, genetic removal of Shh activity from the tooth leads to alterations in growth and morphogenesis and results in tissue disorganization, affecting both the dental epithelium and mesenchyme derivatives. It has not been possible to determine clearly whether the alterations in the dental mesenchyme and its derivatives are solely generated by lack of Shh signaling by the dental epithelium, or whether they are secondary to the lack of proper signaling (via other bioactive molecules) in the abnormal dental epithelium. Conversely, this approach has left unanswered the question of whether abnormal development of the epithelial enamel organ in Shh mutant teeth is a result of a loss of intra-epithelial Shh signaling, or a secondary consequence of altered signaling by the underlying dysplastic dental mesenchyme. In order to distinguish between these alternatives, and further define the roles of Shh in regulating morphogenesis of the tooth, Shh signal transduction was abrogated by genetically removing the activity of Smo from the dental epithelium and its derivatives while maintaining Shh responsiveness in the dental mesenchyme (Gritli-Linde, 2002).

Genetic removal of Shh activity from the dental epithelium, the sole source of Shh during tooth development, alters tooth growth and cytological organization within both the dental epithelium and mesenchyme of the tooth. As explained, it has not been clear which aspects of the phenotype are the result of the direct action of Shh on a target tissue and which are indirect effects due to deficiencies in reciprocal signalings between the epithelial and mesenchymal components. To distinguish between these two alternatives and extend understanding of Shh's actions in odontogenesis, the Cre-loxP system was used to remove Smoothened (Smo) activity in the dental epithelium. Smo, a seven-pass membrane protein is essential for the transduction of all Hh signals. Hence, removal of Smo activity from the dental epithelium should block Shh signaling within dental epithelial derivatives while preserving normal mesenchymal signaling. This study shows that Shh-dependent interactions occur within the dental epithelium itself. The dental mesenchyme develops normally up until birth. In contrast, dental epithelial derivatives show altered proliferation, growth, differentiation and polarization. This approach uncovers roles for Shh in controlling epithelial cell size, organelle development and polarization. Furthermore, evidence is provided that Shh signaling between ameloblasts and the overlying stratum intermedium may involve subcellular localization of Patched 2 and Gli1 mRNAs, both of which are targets of Shh signaling in these cells (Gritli-Linde, 2002).

Developing axons are guided to their targets by attractive and repulsive guidance cues. In the embryonic spinal cord, the floor plate chemoattractant Netrin-1 is required to guide commissural neuron axons to the midline. However, genetic evidence suggests that other chemoattractant(s) are also involved. The morphogen Sonic hedgehog (Shh) can mimic the additional chemoattractant activity of the floor plate in vitro and can act directly as a chemoattractant on isolated axons. Cyclopamine-mediated inhibition of the Shh signaling mediator Smoothened (Smo) or conditional inactivation of Smo in commissural neurons indicates that Smo activity is important for the additional chemoattractant activity of the floor plate in vitro and for the normal projection of commissural axons to the floor plate in vivo. These results provide evidence that Shh, acting via Smo, is a midline-derived chemoattractant for commissural axons and shows that a morphogen can also act as an axonal chemoattractant (Charron, 2003).

Indian hedgehog (Ihh) is indispensable for development of the osteoblast lineage in the endochondral skeleton. In order to determine whether Ihh is directly required for osteoblast differentiation, smoothened (Smo), which encodes a transmembrane protein that is essential for transducing all Hedgehog (Hh) signals, was genetically manipulated. Removal of Smo from perichondrial cells by the Cre-LoxP approach prevents formation of a normal bone collar and also abolishes development of the primary spongiosa. Analysis of chimeric embryos composed of wild-type and Smon/n cells indicates that Smon/n cells fail to contribute to osteoblasts in either the bone collar or the primary spongiosa but generate ectopic chondrocytes. In order to assess whether Ihh is sufficient to induce bone formation in vivo, the bone collar was analyzed in the long bones of embryos in which Ihh was artificially expressed in all chondrocytes by the UAS-GAL4 bigenic system. Although ectopic Ihh does not induce overt ossification along the entire cartilage anlage, it promotes progression of the bone collar toward the epiphysis, suggesting a synergistic effect between ectopic Ihh and endogenous factors such as the bone morphogenetic proteins (BMPs). In keeping with this model, Hh signaling is further found to be required in BMP-induced osteogenesis in cultures of a limb-bud cell line. Taken together, these results demonstrate that Ihh signaling is directly required for the osteoblast lineage in the developing long bones and that Ihh functions in conjunction with other factors such as BMPs to induce osteoblast differentiation. It is suggested that Ihh acts in vivo on a potential progenitor cell to promote osteoblast and prevent chondrocyte differentiation (Long, 2004).

Hedgehog signaling is required for multiple aspects of brain development, including growth, the establishment of both dorsal and ventral midline patterning and the generation of specific cell types such as oligodendrocytes and interneurons. To identify more precisely when during development hedgehog signaling mediates these events, the removal of hedgehog signaling within the brain was directed by embryonic day 9 of development, using a FoxG1Cre driver line to mediate the removal of a conditional smoothened null allele. A loss of ventral telencephalic patterning was observed that appears to result from an initial lack of specification of these structures rather than by changes in proliferation or cell death. A further consequence of the removal of smoothened in these mice is the near absence of both oligodendrocytes and interneurons. Surprisingly, the dorsal midline appears to be patterned normally in these mutants. Together with previous analyses, the present results demonstrate that hedgehog signaling in the period between E9.0 and E12 is essential for the patterning of ventral regions and the generation of cell types that are thought to largely arise from them (Puccillo, 2004).

In the developing spinal cord, early progenitor cells of the oligodendrocyte lineage are induced in the motor neuron progenitor (pMN) domain of the ventral neuroepithelium by the ventral midline signal Sonic hedgehog (Shh). The ventral generation of oligodendrocytes requires Nkx6-regulated expression of the bHLH gene Olig2 in this domain. In the absence of Nkx6 genes or Shh signaling, the initial expression of Olig2 in the pMN domain is completely abolished. In vivo evidence is provided for a late phase of Olig gene expression independent of Nkx6 and Shh gene activities and reveal a brief second wave of oligodendrogenesis in the dorsal spinal cord. In addition, genetic evidence is provided that oligodendrogenesis can occur in the absence of hedgehog receptor Smoothened, which is essential for all hedgehog signaling (Cai, 2005).

In mouse embryos lacking sonic hedgehog (Shh), dorsoventral polarity within the otic vesicle is disrupted. Consequently, ventral otic derivatives, including the cochlear duct and saccule, fail to form, and dorsal otic derivatives, including the semicircular canals, endolymphatic duct and utricle, are malformed or absent. Since inner ear patterning and morphogenesis are heavily dependent on extracellular signals derived from tissues that are also compromised by the loss of Shh, the extent to which Shh signaling acts directly on the inner ear for its development is unclear. To address this question, embryos were generated in which smoothened (Smo), an essential transducer of Hedgehog (Hh) signaling, was conditionally inactivated in the otic epithelium (Smoecko). Ventral otic derivatives failed to form in Smoecko embryos, whereas vestibular structures developed properly. Consistent with these findings, ventral, but not dorsal, otic identity was found to be directly dependent on Hh. The role of Hh in cochlear-vestibular ganglion (cvg) formation is more complex, as both direct and indirect signaling mechanisms are implicated. The data suggest that the loss of cvg neurons in Shh-/- animals is due, in part, to an increase in Wnt responsiveness in the otic vesicle, resulting in the ectopic expression of Tbx1 in the neurogenic domain and subsequent repression of Ngn1 transcription. A mitogenic role for Shh in cvg progenitor proliferation was also revealed in analysis of Smoecko embryos. Taken together, these data contribute to a better understanding of the intrinsic and extrinsic signaling properties of Shh during inner ear development (Brown, 2011).

Hedgehog and retinoic acid signaling cooperate to promote motoneurogenesis in zebrafish

The precise requirements of Hedgehog (Hh) pathway activity in vertebrate central nervous system development remain unclear, particularly in organisms with both maternally and zygotically derived signaling. This study describes the motoneural phenotype of zebrafish that lack maternal and zygotic contributions of the Hh signaling transducer Smoothened (MZsmo mutants) and therefore are completely devoid of ligand-dependent pathway activation. Some functional primary motoneurons (PMNs) persist in the absence of Hh signaling, and it was found that their induction requires both basal Gli transcription factor activity and retinoic acid (RA) signaling. Evidence is provided that RA pathway activation can modulate Gli function in a Hh ligand-independent manner. These findings support a model in which Hh and RA signaling cooperate to promote PMN cell fates in zebrafish (Mich, 2011).

Constitutive activation of smoothened (SMO) in mammary glands of transgenic mice leads to increased proliferation, altered differentiation and ductal dysplasia

The hedgehog signaling network regulates pattern formation, proliferation, cell fate and stem/progenitor cell self-renewal in many organs. Altered hedgehog signaling is implicated in 20-25% of all cancers, including breast cancer. Heterozygous disruption of the gene encoding the patched-1 (PTCH1) hedgehog receptor, a negative regulator of smoothened (Smo) in the absence of ligand, leads to mammary ductal dysplasia in virgin mice. Expression of activated human SMO (SmoM2) under the mouse mammary tumor virus (MMTV) promoter in transgenic mice leads to increased proliferation, altered differentiation, and ductal dysplasias distinct from those caused by Ptch1 heterozygosity. SMO activation also increased the mammosphere-forming efficiency of primary mammary epithelial cells. However, limiting-dilution transplantation showed a decrease in the frequency of regenerative stem cells in MMTV-SmoM2 epithelium relative to wild type, suggesting enhanced mammosphere-forming efficiency was due to increased survival or activity of division-competent cell types under anchorage-independent growth conditions, rather than an increase in the proportion of regenerative stem cells per se. In human clinical samples, altered hedgehog signaling occurs early in breast cancer development, with PTCH1 expression reduced in ~50% of ductal carcinoma in situ (DCIS) and invasive breast cancers (IBC). Conversely, SMO is ectopically expressed in 70% of DCIS and 30% of IBC. Surprisingly, in both human tumors and MMTV-SmoM2 mice, SMO rarely colocalized with the Ki67 proliferation marker. These data suggest that altered hedgehog signaling may contribute to breast cancer development by stimulating proliferation, and by increasing the pool of division-competent cells capable of anchorage-independent growth (Moraes, 2007).

Epithelial and non-epithelial Ptch1 play opposing roles to regulate proliferation and morphogenesis of the mouse mammary gland

Patched 1 (Ptch1; see Drosophila Patched) has epithelial, stromal and systemic roles in murine mammary gland organogenesis, yet specific functions remain undefined. Cre-recombinase-mediated Ptch1 ablation in mammary epithelium increased proliferation and branching, but did not phenocopy transgenic expression of activated smoothened (SmoM2; see Drosophila Smoothened). The epithelium showed no evidence of canonical hedgehog signaling, and hyperproliferation was not blocked by smoothened (SMO) inhibition, suggesting a non-canonical function of PTCH1. Consistent with this possibility, nuclear localization of cyclin B1 was increased. In non-epithelial cells, heterozygous Fsp-Cre-mediated Ptch1 ablation increased proliferation and branching, with dysplastic terminal end buds (TEB) and ducts. By contrast, homozygous Ptch1 ablation decreased proliferation and branching, producing stunted ducts filled with luminal cells showing altered ovarian hormone receptor expression. Ducts of Fsp-Cre;Ptch1fl/fl mice were similar to Fsp-Cre;SmoM2 ducts, but Fsp-Cre;SmoM2 outgrowths were not stunted, suggesting that the histology might be mediated by Smo in the local stroma, with systemic Ptch1 required for ductal outgrowth and proper hormone receptor expression in the mammary epithelium (Monkkonen, 2017).

Smoothened homologs and eye development

Sonic hedgehog is involved in eye field separation along the proximodistal axis. As the optic vesicle and optic cup mature, Hh signalling continues to be important in defining aspects of the proximodistal axis. Two other Hedgehog proteins, Banded hedgehog and Cephalic hedgehog, related to the mouse Indian hedgehog and Desert hedgehog, respectively, are strongly expressed in the central retinal pigment epithelium but excluded from the peripheral pigment epithelium surrounding the ciliary marginal zone. By contrast, downstream components of the Hedgehog signalling pathway, Gli2, Gli3 and X-Smoothened, are expressed in this narrow peripheral epithelium. This zone contains cells that are in the proliferative state. This equivalent region in the adult mammalian eye, the pigmented ciliary epithelium, has been identified as a zone in which retinal stem cells reside. These data, combined with double labelling and the use of other retinal pigment epithelium markers, show that the retinal pigment epithelium of tadpole embryos has a molecularly distinct peripheral to central axis. In addition, Gli2, Gli3 and X-Smoothened are also expressed in the neural retina, in the most peripheral region of the ciliary marginal zone, where retinal stem cells are found in Xenopus, suggesting that they are good markers for retinal stem cells. To test the role of the Hedgehog pathway at different stages of retinogenesis, the pathway was activated by injecting a dominant-negative form of PKA or blocking it by treating embryos with cyclopamine. Embryos injected or treated at early stages display clear proximodistal defects in the retina. Interestingly, the main phenotype of embryos treated with cyclopamine at late stages is a severe defect in RPE differentiation. This study thus provides new insights into the role of Hedgehog signalling in the formation of the proximodistal axis of the eye and the differentiation of retinal pigment epithelium (Perron, 2003).

Hedgehog (Hh) signaling is required for eye development in vertebrates; known roles in the zebrafish include regulation of eye morphogenesis and ganglion cell and photoreceptor differentiation. A temporally selective Hh signaling knockdown strategy was used (either antisense morpholino oligonucleotides or the teratogenic alkaloid cyclopamine) in order to dissect the separate roles of Hh signaling arising from specific sources. Also, the eye phenotype was examined of zebrafish slow muscle-omitted (smu) mutants, which lack a functional smoothened gene which encodes a component of the Hh signal transduction pathway. Hh signaling from extraretinal sources is found to be required for the initiation of retinal differentiation, but this involvement may be independent of the effects of Hh signaling on optic stalk development. Hh signals from ganglion cells participate in propagating expression of ath5. It is suggested that the effects of Hh signals from the retinal pigmented epithelium on photoreceptor differentiation may be mediated by the transcription factor rx1 (Stenkamp, 2003).

The results of this study suggest that the effects of Hh signaling on photoreceptor development may involve the transcription factor rx1, and further confirm that Hh signaling from the retinal pigment epithelium (RPE) is primarily implicated. Antisense injections delivered at 51 hpf generated some of the same retinal phenotypes as antisense injections delivered at earlier time points, indicating that interference with Hh signaling rather late in development is sufficient to interfere with photoreceptor differentiation. Knockdown of Hh signaling with antisense-MO consistently resulted in failed rx1 expression in the outer nuclear layer (ONL), while crx expression was unaffected, further supporting the hypothesis that Hh signaling may influence photoreceptor differentiation via the transcription factor rx1. The rx gene product has been shown to participate in regulating photoreceptor-specific gene expression in cell-free systems. The chicken homolog of zebrafish rx1/2, RaxL, is involved in the early stages of photoreceptor differentiation. To confirm the proposed interaction in zebrafish it will be important to demonstrate that rx1 expression regulates photoreceptor differentiation (opsin expression) in vivo. One alternative to this hypothesis is that effects of reduced Hh signaling on rx and opsin genes are related manifestations of a photoreceptor maturation defect (Stenkamp, 2003).

The smu-/- embryos similarly show reduced expression of photoreceptor markers, and lack of rx1 in the photoreceptor layer. Interestingly, many of the smu-/- embryos develop normally laminated retinas. It is suspected that these mutants are those that had sufficient maternal smoothened expression to initiate retinal retinal differentiation, but lacked functional (zygotic) Smoothened at the time of Hh signaling from the RPE. In these mutants, it would be predicted that the only notable retinal defects would be those related to Hh signaling from ganglion cells and RPE. A fraction of the mutants showed a small patch of rx1 and rod opsin expression in the ventronasal ONL, suggesting that this region of retina may have requirements for cell differentiation that are distinct from the rest of the retina. This is consistent with the proposal that the ventral retina of the embryonic zebrafish comprises a discrete domain, influenced primarily by signals originating outside the eye, while the differentiation of the remainder of the retina requires the propagation of additional Hh, and other signals, from within the eye (Stenkamp, 2003).

Accumulating evidence suggests that Sonic hedgehog (Shh) signaling plays a crucial role in eye vesicle patterning in vertebrates. Shh promotes expression of Pax2 in the optic stalk and represses expression of Pax6 in the optic cup. Shh signaling contributes to establishment of both proximal-distal and dorsal-ventral axes by activating Vax1, Vax2, and Pax2. In the dorsal part of the developing retina, Bmp4 is expressed and antagonizes the ventralizing effects of Shh signaling through the activation of Tbx5 expression in chick and Xenopus. To examine the roles of Shh signaling in optic cup formation and optic stalk development, the Smoothened (Smo) conditional knockout (CKO) mouse line was used. Smo is a membrane protein which mediates Shh signaling into inside of cells. Cre expression was driven by Fgf15 enhancer. The ventral evagination of the optic cup deteriorated from E10 in the Smo-CKO, whereas the dorsal optic cup and optic stalk develop normally until E11. Expression was examined of various genes, such as Pax family (Pax2/Pax6), Vax family (Vax1/Vax2) and Bmp4. Bmp4 expression was greatly upregulated in the optic vesicle by the 21-somite stage. Then Vax1/2 expression was decreased at the 20- to 24-somite stages. Pax2/6 expression was affected at the 27- to 32-somite stages. These data suggest that the effects of the absence of Shh signaling on Vax1/Vax2 are mediated through increased Bmp4 expression throughout the optic cup. Also unchanged patterns of Raldh2 and Raldh3 suggest that retinoic acid is not the downstream to Shh signaling to control the ventral optic cup morphology (Zhao, 2010).

Smoothened and neural stem cells

To directly test the requirement for hedgehog signaling in the telencephalon from early neurogenesis, conditional null alleles of both the Sonic hedgehog and Smoothened genes were examined. While the removal of Shh signaling in these animals results in only minor patterning abnormalities, the number of neural progenitors in both the postnatal subventricular zone and hippocampus is dramatically reduced. In the subventricular zone, this was partially attributable to a marked increase in programmed cell death. Consistent with Hedgehog signaling being required for the maintenance of stem cell niches in the adult brain, progenitors from the subventricular zone of floxed Smo animals form significantly fewer neurospheres. The loss of hedgehog signaling also results in abnormalities in the dentate gyrus and olfactory bulb. Furthermore, stimulation of the hedgehog pathway in the mature brain resulted in elevated proliferation in telencephalic progenitors. These results suggest that hedgehog signaling is required to maintain progenitor cells in the postnatal telencephalon (Machold, 2003).

In order to explore which populations in the postnatal (P15) brain might be affected by the loss of hedgehog signaling, the distribution of Shh and its transcriptional targets Ptch1 and Gli1 was examined. Immunostaining using a Shh-specific antibody revealed that Shh is broadly distributed in the P15 telencephalon. The most prominent areas of Shh immunoreactivity are in the ventral telencephalon where embryonic Shh expression is observed. Specifically, Shh-positive cells are seen within the ventral accumbens, entorhinal cortex, and ventral septum, along the path transited by cells in the rostral migratory stream. Furthermore, within layer 3 of the cortex and along the corpus callosum Shh immunoreactivity is observed. Finally, a population of axons in the medial septum is also immunopositive for Shh. This observation is consistent with the observation that the septal (i.e., fimbria) fibers projecting to the hippocampus are a source of Shh within this structure. Shh staining is seen in the white matter tracks in the CA3 and hilus regions of the hippocampus. Mice heterozygous for a LacZ reporter under the control of the endogenous Shh promoter display a similar pattern of cellular Shh expression. In addition, these mice revealed the existence of Shh-expressing cells within the hilus of the dentate gyrus, adjacent to the hippocampal stem cell population. Since this staining was not evident in antibody visualization of Shh, it seems likely that these cells only express low levels of Shh and require the enzymatic amplification provided by LacZ staining to visualize. The expression of LacZ directed through either the Ptch1 or Gli1 loci is generally consistent in indicating in which populations the hedgehog pathway is activated. The expression of LacZ directed by the Ptch1 loci is broader than that seen from the Gli1 loci. The observation that Gli1 expression more closely matched sources of endogenous Shh suggests that Gli1 is the more accurate readout of Shh activation. Furthermore, embryonic studies indicate that all Gli1 expression is Shh dependent, whereas Ptch1, the Shh receptor, is expressed at basal levels in the absence of any direct Hedgehog signaling input (Machold, 2003).

The data suggests that Shh signaling has only modest effects on the general growth of ventral telencephalic regions after E12.5. In contrast, it is essential in the dentate gyrus and the olfactory bulb, regions in which neurogenesis continues in the adult. Specifically, Smon/c;Ncre mice display a marked decrease in the levels of proliferation in both these areas. In addition, in the SVZ, apoptotic cell death is dramatically increased. Within the hippocampus Shh signaling acts as a mitogen for adult progenitors residing in the dentate gyrus. While the data support the contention that Shh is required for the maintenance of hippocampal and SVZ progenitors, it is less clear that within the SVZ Shh acts directly as a mitogen. Although both the SVZ and dentate gyrus of Smon/c;Ncre mutants appear to have fewer progenitors, Shh alone is unable to support neurosphere formation or expansion from either wild-type or Smon/c;Ncre SVZ progenitors, whereas Shh appears to be able to substitute for FGF in the expansion of dentate gyrus precursors in rat. Similarly, while almost all hippocampal cells labeled by a Brdu pulse 2 hr prior to sacrifice at P15 expressed Gli1, only a small subpopulation of similarly labeled cells in the SVZ express this direct target of Shh signaling. Nonetheless, in both the SVZ and hippocampus, increasing levels of Shh signaling increase cell proliferation. Hence, it will require further work to determine the precise actions of hedgehog signaling in maintaining telencephalic progenitor regions (Machold, 2003).

Smoothened homologs and cancer

Basal-cell carcinomas (BCCs) are the commonest human cancer. Insight into their genesis came from identification of mutations in the patched gene (PTCH) in patients with the basal-cell nevus syndrome, a hereditary disease characterized by multiple BCCs and by developmental abnormalities. The binding of Sonic hedgehog (SHH) to its receptor, PTCH, is thought to prevent normal inhibition by PTCH of Smoothened (SMO), a seven-span transmembrane protein. According to this model, the inhibition of SMO signaling is relieved following mutational inactivation of PTCH in basal-cell nevus syndrome. Activating somatic missense mutations have been identified in the SMO gene itself in sporadic BCCs from three patients. Mutant SMO, unlike wild type, can cooperate with adenovirus E1A to transform rat embryonic fibroblast cells in culture. Skin abnormalities similar to BCCs develop in transgenic murine skin overexpressing mutant SMO. These findings support the role of SMO as a signaling component of the SHH-receptor complex and provide direct evidence that mutated SMO can function as an oncogene in BCCs. The increased PTCH mRNA observed in BCC can be ascribed to missense mutations in SMO (Xie, 1998).

About one-third of sporadic basal cell carcinomas (BCCs) of the skin and 10%-15% of primitive neuroectodermal tumors (PNETs) of the central nervous system show mutations in the PTCH tumor suppressor gene. The PTCH gene product (Ptch) functions as a transmembrane receptor for the Sonic hedgehog protein (Shh) and interacts with another transmembrane protein called Smoh. To further elucidate the significance of alterations in the Shh signaling pathway, thirty one sporadic BCCs and fifteen PNETs were investigated for the mutation and/or expression of SMOH, PTCH, SHH, and GL11. In addition, the SMOH gene locus was fine-mapped by fluorescence in situ hybridization to chromosomal band 7q32. Mutational analysis has identified four BCCs with somatic missense mutations in SMOH affecting codon 535 (TGG to TTG: Trp to Leu) in three tumors and codon 199 (CGG to TGG: Arg to Trp) in one tumor. A missense mutation at codon 533 (AGC to AAC: Ser to Asn) was found in one PNET. PTCH mutations were detected in eight BCCs and one PNET. Two BCCs demonstrate mutations in both SMOH and PTCH. The majority of tumors show an increased expression of SMOH, PTCH, and GL11 transcripts as compared with that of normal skin and nonneoplastic brain tissue, respectively. In contrast, only one BCC and one PNET express SHH mRNA at levels detectable by reverse transcription-PCR, and no SHH gene mutations were found. In summary, these results indicate that both PTCH and SMOH represent important targets for genetic alterations in sporadic BCCs and PNETs (Reifenberger, 1998).

smoothened: Regulation | Developmental Biology | Effects of Mutation | References

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