hedgehog

In a process crucial for full biological activity, Hedgehog protein undergoes autoproteolysis to generate two biochemically distinct products: N, an 18K amino-terminal fragment, and C, a 25K carboxy-terminal fragment. Mutations that block autoproteolysis impair HH function. The site of autoproteolytic cleavage has been identified. It is broadly conserved throughout the Hedgehog family. The N product is the active species in both local and long-range signaling. Consistent with this, all twelve mapped hedgehog mutations either directly affect the structure of the N product or otherwise block the release of the biologically active N-terminal domain from the HH precursor, as a result of deletion or alteration of sequences in the C domain. Although the amino-terminal signaling domain expressed form a construct lacking all carboxy-terminal domain sequences sufficies to carry out hedgehog signaling activities in embryos, this truncated protein is not tightly cell-associated when expressed in cultured cells but, instead is found predominantly in the culture medium. This suggests that the autocatalytic processing activity of the C-terminal domain influences the cellular localization of the biologically active N-terminal domain (Porter, 1995).

The autocatalytic processing reaction which generates the biologically active HH amino-terminal product proceeds via an internal thioester intermediate which results in a covalent modification that increases the hydrophobic character of the signaling domain and influences its spatial and subcellular distribution. The hydrophobic character of the modified N-terminal domain suggests a lipid modification, but this modification appears to be novel as known lipid modifications of other proteins (myristoylation, palmitoylation, prenylation and GPI addition), are not apparent. The modified HH amino-terminal product is cell membrane associated, and this association affects the range of its activities (Porter, 1996a).

During biosynthesis HH undergoes an autocleavage reaction mediated by its carboxyl-terminal domain; this produces a lipid-modified amino-terminal fragment responsible for all known HH signaling activity. Cleavage of HH occurs between residues corresponding to Gly257 and Cys258 and likely proceeds through a labile thioester intermediate formed by the cysteine thiol and the glycine carbonyl carbon. In this study cholesterol is the lipophilic moiety covalently attached to the amino-terminal signaling domain during autoprocessing. The carboxyl-terminal domain acts as an intramolecular cholesterol transferase. This use of cholesterol to modify embryonic signaling proteins may account for some of the effects of perturbed cholesterol biosynthesis on animal development. For example treatment of rats in early pregnancy with inhibitors of cholesterol biosynthesis causes pronounced birth defects that include cyclopia, monorhinia, agenesis of the pituitary and other ventral neuronal cell types, and other variable manifestations of holoprosencephaly. These defects resemble the severe holoprosencephalic phenotype of embryos lacking a functional sonic hedgehog gene (Porter, 1996b).

Surprisingly, a vertebrate homolog of Smoothened (vSMO), shows no direct interaction with mouse Sonic hedgehog (SHH), and Sonic hedgehog binds mPtc, a murine homolog of Patched, with high affinity. For example, epitope-tagged SHH as well as IgG-Sonic HH, both hybrid proteins containing the N terminal region of Sonic HH attached covalently to second proteins, bind to cells expressing mPTC, and no change in the affinity between SHH and PTC is observed in the presence of vSMO. mPTC can be co-immunoprecipitated with epitope-tagged SHH. Nevertheless, the three proteins can form a physical complex. In cells coexpressing mPTC and vSMO, vSMO can be co-immunoprecipitated with antibody against epitope-tagged mPTC. Thus in the end, it appears that Patched, and not SMO is the protein that directly interacts with SHH (Stone, 1996).

Confirming evidence comes from experiments in which chicken ptc was expressed in Xenopus oocytes. Binding of labelled SHH is detected in ptc transduced oocytes but not in untransduced controls. Co-immunoprecipitation experiments reveal that when transduced cells are treated with SHH and extracted, SHH can be detected in immuno-preciptates carried out with antibody against epitope tagged PTC (Marigo, 1996b).

Genetic and expression evidence point to a role of both Patched and Smoothened in Hedgehog receptor function. In wings bearing large anterior compartment mutant smo clones there appears to be an anterior shift in the distribution of dpp-exressing cells. This shift is interpred as evidence that the loss of smo activity abolishes the ability of anterior cells to respond to HH and also allows HH to spread abnormally far into the anterior compartment until it reaches, and is transduced by smo+ cells. One mutant of ptc, ptcS2 gives rise to a PTC protein that appears indistinguishable from null alleles when assayed for its ability to repress inappropriate activity of the HH signal transduction pathway. Nevertheless, ptcS2 retains an activity that allows anterior compartment cells to sequester HH. Additionally, up-regulation of ptc by HH, a conserved feature of HH signaling, is required to limit the movement of HH from the posterior into the anterior compartment. Finally, PTC represses the HH signal transduction pathway by blocking the intrinsic activity of SMO. This suggests that PTC is positioned upstream of SMO in the HH signal transduction pathway, either as a factor that regulates SMO-transducting activity in response to HH or as a factor that facilitates the direct modulation of SMO activity by HH (Chen, 1996).

The segment-polarity gene smoothened is required for the response of cells to hedgehog signalling during the development of both the embryonic segments and imaginal discs. Sequence analysis of the smoothened transcription unit reveals a single open reading frame encoding a protein with seven putative transmembrane domains. This structure is typical of G-protein-coupled receptors, suggesting that the Smoothened protein may act as a receptor for the Hedgehog ligand (van den Heuvel, 1996).

Smoothened is a segment polarity gene required for the correct patterning of every segment in Drosophila. The earliest defect in smo mutant embryos is loss of expression of the Hedgehog-responsive gene wingless between 1 and 2 hr after gastrulation. Since smo mutant embryos cannot respond to exogenous Hedgehog (Hh) but can respond to exogenous Wingless, the smo product functions in Hh signaling. Smo acts downstream of or in parallel with Patched, an antagonist of the Hh signal. The smo gene encodes an integral membrane protein with characteristics of G protein-coupled receptors and shows homology to the Drosophila Frizzled protein. Based on its predicted physical characteristics and on its position in the Hh signaling pathway, it is suggested that smo encodes a receptor for the Hh signal (Alcedo, 1996).

During Drosophila development, cells belonging to the posterior compartment of each segment organize growth and patterning by secreting Hedgehog (Hh), a protein that induces a thin strip of adjacent cells in the anterior compartment to express the morphogens Decapentaplegic (Dpp) and Wingless (Wg). Hedgehog is bound and transduced by a receptor complex that includes Smoothened (Smo), a member of the Frizzled (Fz) family of seven-pass transmembrane receptors, as well as the multiple-pass transmembrane protein Patched (Ptc). Ptc is required for the binding of Hh to the complex as well as for the Hh-dependent activation of Smo within the complex. A likely null allele of the smo gene has been identified. It was used to determine whether Hh is bound by Ptc alone, or by Smo in concert with Ptc. Cells devoid of Smo can sequester Hh, but their ability to do so depends, as in wild-type cells, on the expression of high levels of Ptc protein. These results suggest that Ptc normally binds Hh without any help from Smo and hence favor a mechanism of signal transduction in which Hh binds specifically to Ptc and induces a conformational change leading to the release of latent Smo activity (Chen, 1998).

During wing development, cells in the posterior (P) compartment secrete Hh and cells in the anterior (A) compartment respond to Hh by turning on, or up-regulating, several genes, including those encoding Dpp and Ptc. These genes are expressed at high level only in a thin strip of A cells adjacent to the A/P compartment boundary, indicating that Hh normally moves only a short distance into the A compartment. Examined were clones of A compartment cells homozygous for the smo³ mutation, which codes for an altered protein that might not be null because it may retain the ability to insert into the cell membrane. These clones fail both to express dpp and to upregulate Ptc expression, even when adjacent to the A/P compartment boundary, indicating that they are unable to transduce Hh. Moreover, they appear unable to restrict the movement of Hh into the A compartment, as indicated by the response of wild-type cells positioned just anterior to large A compartment clones of smo³ cells that abut the A/P boundary. These smo+ cells express dpp and Ptc at high levels, even though they are located many cell diameters away from the A/P boundary, at positions that are normally too far from the boundary to be exposed to Hh. The same analysis was performed using the smo^D16 allele, certain to be a null allele because it gives rise to a truncated protein, in place of the smo³ allele, and the same result was obtained. Both Ptc expression as well as the expression of a dpp-lacZ transgene, placed in trans to the smo^D16 mutant allele, were monitored. A compartment cells lacking Smo protein not only fail to respond to Hh, but also fail to limit the further spread of Hh into the A compartment, indicating that they lack a Hh-sequestering activity (Chen, 1998).

In the case of the smo³ mutant allele, the failure of mutant cells to impede the movement of Hh into the A compartment can be attributed to their failure to up-regulate the expression of Ptc, which confers Hh-sequestering activity. Indeed, it is possible to restore the ability of smo³ mutant cells to restrict Hh movement by simultaneously eliminating the activity of Protein kinase A (PKA), a manipulation that constitutively activates the Hh signal transduction pathway causing smo³ mutant cells to express high levels of Ptc. However, it is not known whether the Hh-sequestering activity of smo³ PKA - cells is mediated solely by Ptc, or by smo³ mutant protein in conjunction with Ptc. Indeed, molecular analysis indicates the smo³ allele encodes a truncated form of Smo that includes the entire cysteine rich extracellular domain (CRD), as well as the first three transmembrane domains, consistent with the possibility that truncated Smo retains a Hh-binding activity. To resolve this uncertainty, the smo-;PKA- experiment was repeated using the smo^D16 allele in place of the smo³ allele. smo^D16;PKA- clones were generated using two genetic configurations. In the first configuration, both Ptc expression as well as the expression of a dpp-lacZ transgene were monitored. In contrast to smo^D16 clones, which express only low levels of Ptc protein, smo^D16;PKA- clones autonomously express high levels of Ptc protein throughout. Moreover, large smo^D16;PKA- clones that abut the A/P boundary differ from similarly positioned smo^D16 clones in that they appear to impede the movement of Hh through the clone. In the second genetic configuration, smo^D16;PKA- mutant cells were marked by the loss of a ubiquitously expressed arm-lacZ transgene. This configuration allows the smo PKA genotype to be assessed independently of Ptc expression. A compartment cells that are homozygous for the smo^D16 mutation and hence devoid of Smo protein are nevertheless able to sequester Hh, provided that they also express high levels of Ptc. These findings favor the proposal that Hh normally binds specifically to Ptc within the Hh receptor complex without any direct involvement of Smo (Chen, 1998).

Hedgehog signaling requires cholesterol in both signal-generating and -receiving cells, and it requires the tumor suppressor Patched (Ptc) in receiving cells in which it plays a negative role. Ptc both blocks the Hh pathway and limits the spread of Hh. Sequence analysis suggests that it has 12 transmembrane segments, 5 of which are homologous to a conserved region that has been identified in several proteins involved in cholesterol homeostasis and has been designated the sterol-sensing domain (SSD). In the present study, it is shown that a Ptc mutant with a single amino acid substitution in the SSD induces target gene activation in a ligand-independent manner. The mutant Ptc^SSD protein shows dominant-negative activity in blocking Hh signaling by preventing the downregulation of Smoothened (Smo), a positive effector of the Hh pathway. Despite its dominant-negative activity, the mutant Ptc protein functions like the wild-type protein in sequestering and internalizing Hh. In addition, Ptc^SSD preferentially accumulates in endosomes of the endocytic compartment. All these results suggest a role of the SSD of Ptc in mediating the vesicular trafficking of Ptc to regulate Smo activity (Martin, 2001).

The tumor suppressor gene patched (ptc) encodes an approximately 140 kDa polytopic transmembrane protein that binds members of the Hedgehog (Hh) family of signaling proteins and regulates the activity of Smoothened (Smo), a G protein-coupled receptor-like protein essential for Hh signal transduction. Ptc contains a sterol-sensing domain (SSD), a motif found in proteins implicated in the intracellular trafficking of cholesterol, and/or other cargoes. Cholesterol plays a critical role in Hedgehog (Hh) signaling by facilitating the regulated secretion and sequestration of the Hh protein, to which it is covalently coupled. In addition, cholesterol synthesis inhibitors block the ability of cells to respond to Hh, and this finding points to an additional requirement for the lipid in regulating downstream components of the Hh signaling pathway. Although the SSD of Ptc has been linked to both the sequestration of, and the cellular response to Hh, definitive evidence for its function has so far been lacking. The identification and characterization of two missense mutations in the SSD of Drosophila Ptc is described; strikingly, while both mutations abolish Smo repression, neither affects the ability of Ptc to interact with Hh. It is speculated that Ptc may control Smo activity by regulating an intracellular trafficking process dependent upon the integrity of the SSD (Strutt, 2001).

In vertebrates, the formation of raft lipid microdomains plays an important part in both polarized protein sorting and signal transduction. To establish a system in which raft-dependent processes could be studied genetically, the protein and lipid composition of these microdomains have been analyzed in Drosophila melanogaster. Using mass spectrometry, the phospholipids, sphingolipids, and sterols present in Drosophila membranes were identified. Despite chemical differences between Drosophila and mammalian lipids, their structure suggests that the biophysical properties that allow raft formation have been preserved. Consistent with this, a detergent-insoluble fraction of Drosophila membranes has been identified that, like mammalian rafts, is rich in sterol, sphingolipids, and glycosylphosphatidylinositol-linked proteins. The sterol-linked Hedgehog N-terminal fragment associates specifically with this detergent-insoluble membrane fraction. These findings demonstrate that raft formation is preserved across widely separated phyla in organisms with different lipid structures. They further suggest sterol modification as a novel mechanism for targeting proteins to raft membranes and raise the possibility that signaling and polarized intracellular transport of Hedgehog are based on raft association (Rietveld, 1999).

Hh cholesterol modification is important in stimulating the anterior target genes wg and ptc, while cholesterol is dispensable for posterior induction of ptc and rho

Hedgehog family members are secreted proteins involved in numerous patterning mechanisms. Different posttranslational modifications have been shown to modulate Hedgehog biological activity. The role of these modifications in regulating subcellular localization of Hedgehog has been investigated in the Drosophila embryonic epithelium. Cholesterol modification of Hedgehog is responsible for Hedgehog assembly in large punctate structures and apical sorting through the activity of the sterol-sensing domain-containing Dispatched protein. Movement of these specialized structures through the cellular field is contingent upon the activity of proteoglycans synthesized by the heparan sulfate polymerase Tout-Velu. Finally, the Hedgehog large punctate structures are necessary only for a subset of Hedgehog target genes across the parasegmental boundary, suggesting that presentation of Hedgehog from different membrane compartments is responsible for Hedgehog's functional diversity in epithelial cells (Gallet, 2003).

The repeated pattern of the Drosophila larval ectoderm (which secretes cuticle) has been used to follow Hh activity. Each abdominal segment is composed of two types of cuticle: the naked (or smooth) cuticle and the denticle belts, subdivided into six rows of denticles, easily identifiable by their orientation and shape. This cuticle pattern is under the control of several signaling pathways that are indirectly regulated by Hh. Engrailed (En) controls hh expression in the two rows of cells that define the posterior compartment of the segment. Across the parasegmental boundary (in cells anterior to the En/Hh domain), Hh maintains wingless (wg) transcription in one row of cells. The Wg signal then controls the specification of the naked cuticle. Posterior to the En/Hh domain, Hh initiates rhomboid (rho) transcription in one to two rows of cells. rho activation induces EGF signaling, allowing differentiation of denticles 1-4. Finally, Hh and Wg are required for serrate (ser) repression and restrict its expression in three rows of cells posterior to the rho-expressing cells. Ser initiates a third row of rho expression in adjacent cells. The Hh receptor Patched (Ptc) is also transcriptionally upregulated by the Hh pathway in cells on both sides of the En/Hh domain (Gallet, 2003).

Loss of hh results in loss of both naked cuticle and denticle diversity. This cuticle phenotype correlates with Hh target gene expression: loss of wg, extension of the ser expression domain (which now covers most of the segment) and absence of ptc upregulation. rho expression is strongly reduced, though some remains under the control of Ser. Conversely, ubiquitous expression of full-length hh (Hh^FL) in the ectoderm with the GAL4-UAS system induces an expansion over four to five cells of both wg and rho expression in the anterior and posterior directions, respectively, while ser expression is completely repressed. Accordingly, the denticle belts of these embryos contain several rows of type 2 denticles, reflecting a uniform level of rho expression in response to a uniform level of Hh. Thus, wg, rho, ser, and ptc expressions reflect direct Hh activity in cells anterior and posterior to en/hh-expressing cells (Gallet, 2003).

Two endogenous Hh isoforms are present in vivo: one bearing both posttranslational lipid modifications and another modified only by a cholesterol adduct. To address the role of these different modifications in Hh signaling, the biological activity of different Hh constructs that do not undergo all modifications was assessed (Gallet, 2003).

All Hh constructs used in this study have a similar level of expression. The reference transgenic strain expresses Hh^FL, which, after cleavage, yields cholesterol-modified Hh-Np that can also be palmitoylated. This construct, was ubiquitously expressed with the 69BGal4 driver and tested for its ability to rescue loss of hh function. Hh-Np restores hh-induced loss of naked cuticle and denticle diversity. Target gene expression reflectes this rescue: three to four rows of wg-expressing cells anterior to the En/Hh domain were observed. Posteriorly, four to five cell rows of rho-expressing cells are induced, and ser expression is totally repressed (Gallet, 2003).

A similar phenotype was observed for the C85S-Hh-Np construct that permits cholesterol modification, but not palmitoylation, of Hh. Nevertheless, target gene expression is less-potently rescued by this construct. wg expansion is developmentally delayed. Activation of rho and repression of ser are less efficient. It is concluded that absence of palmitoylation decreases the overall potency of Hh (Gallet, 2003).

Cholesterol-unmodified Hh-N rescues denticle diversity, but embryos still present abdominal denticle belt fusions (between two and three per embryo). Consistently, Hh-N is unable to fully rescue wg expression in all segments but can widen the domains of rho expression and ser repression. The behavior of another Hh construct in which the cholesterol adduct had been replaced by another membrane-anchored domain, was also analyzed. In this construct, the Hh-N coding sequence is fused to the transmembrane domain of the rat CD2 protein (Hh-N-CD2). Importantly, Hh-N-CD2 is unable to induce wg expression, while it still stimulated rho transcription. Consequently, rho-dependent denticle diversity is restored, while no naked cuticle was present. Most en cells are absent, but rho is evenly expressed, confirming that its expression is directly dependent on Hh activity and not on a secondary signal coming from en cells. Moreover, Ser-dependent rho expression is also excluded, since ser expression is fully repressed in these embryos. In another construct, the cholesterol binding site of Hh was replaced with another lipophilic linkage domain, the glycosyl-phosphatidylinositol (GPI) anchoring signal of Drosophila Fasciclin 1 (Hh-N-GPI). Similar phenotypes were observed with this protein (Gallet, 2003).

To assess whether the differences between wg and rho regulation could be accounted for by differential sensitivity to Hh levels, ptc, which is expressed on both sides of Hh-secreting cells, was analyzed. The different constructs were expressed in the endogenous Hh domain with the enGal4 driver in an hh null background. The initiation of en expression is independent of Wg activity and is sufficient to transiently express the Gal4 protein and, thus, the UAS transgene, independently of the Hh and Wg regulatory loop. In these embryos, ptc is always upregulated in cells posterior to the Hh source. However, anterior upregulation of ptc is always weaker, as observed for anterior ptc activation by endogenous Hh in wild-type embryos. Furthermore, anterior ptc activation is largely absent in embryos expressing non-cholesterol-modified Hh-N. This effect is enhanced when Hh-N-CD2 is expressed in en cells. This differential ptc upregulation reveals an asymmetric activation process of the Hh pathway on either side of the Hh source (Gallet, 2003).

On the basis of these data, it is hypothesized that the differences observed could be accounted for by differential activation mechanisms. These results outline the important role of the Hh cholesterol modification in stimulating the anterior target genes wg and ptc across the parasegmental boundary and, subsequently, naked cuticle differentiation, while cholesterol appears dispensable for posterior induction of ptc and rho and, thus, denticle diversity. Because some wg expression can still be activated by Hh-N, the presence of cholesterol modification on Hh might not be the only requirement for anterior target gene regulation. Hh-N-CD2 and Hh-N-GPI activities suggest that the differences observed could be a consequence of Hh differential sorting in the producing cells and/or access and presentation to the target cell surface (Gallet, 2003).

Evidence is presented for a functional role of cholesterol modification in the control of Hh subcellular localization in the embryonic epithelium. Cholesterol modification is required for Hh assembly into large punctate structures (LPSs) and its apical targeting in a Disp-dependent manner. Furthermore, LPS apical movement requires Ttv-dependent proteoglycans and this movement is necessary for adjacent anterior cells to receive Hh input and express wg. In contrast, basolateral Hh localization is sufficient for rho activation in adjacent posterior cells, independent of cholesterol modification of Hh (Gallet, 2003).

In certain developmental processes, palmitoylation increases Hh activity to reach the threshold necessary for target gene expression. Expressing C85S-Hh-Np lacking palmitic acid modification can rescue hh loss-of-function during embryogenesis, though less efficiently than a wild-type Hh molecule. Hence, as in vertebrates, palmitic acid modification potentiates Hh activity in Drosophila embryos. Interestingly, Hh-N is rendered inactive if not palmitoylated, suggesting that palmitoylation occurs independently of cholesterol modification (Gallet, 2003).

Hh is localized at the basolateral membrane of producing cells. Hh is also present in LPS, the formation of which is cholesterol dependent. Since no difference is observed in LPS formation with a nonpalmitoylated C85S-Hh-Np construct, the cholesterol modification on Hh appears to be the main requirement for Hh targeting to LPSs. Two different fractions of membrane-bound Hh have been identified in Drosophila: a detergent-insoluble fraction corresponding to lipid raft microdomains and a detergent-soluble fraction. Therefore, a potential hypothesis would be that the Hh LPSs correspond to cholesterol-enriched raft microdomains. Nevertheless, it was not possible to show any colocalization of Hh with Flo^Dm, the fly homolog of raft-associated caveolin. It was also not possible to see any Hh-related cuticle defects in embryos injected with drugs (methyl-ß-cyclodextrin and filipin) known to deplete cholesterol and, thus, lipid raft microdomains (Gallet, 2003).

The assembly and the apical sorting of Hh-Np LPSs are dependent upon both cholesterol and Disp activity. However, Disp is not necessary for cholesterol binding to Hh. In C. elegans, the disp homolog (CHE-14) is required for apical cuticle secretion. Therefore, one tempting possibility might be that the sterol-sensing domain (SSD) on Disp specifically recognizes cholesterol-modified Hh for its assembly into LPSs and apical sorting. Since Hh-Np is still present on basolateral membranes in disp mutants, formation of LPSs could start from these locations before apical sorting. Alternatively, two independent routes could be responsible for basolateral and apical targeting (Gallet, 2003).

Cholesterol-dependent Hh-Np LPSs require Ttv to diffuse in the cellular field. How can Hh-Np be released from cells if it is inserted in the lipid bilayers? The mechanism of release might involve either a displacement of the cholesterol tether on Hh-Np or the formation of membrane vesicles. So far, no evidence for vesiculation has been reported, but large soluble multimers of Shh-Np have been identified in conditioned media of vertebrate cells. Multimers of Hh-Np are also present in conditioned culture media from Hh-producing Drosophila Schneider cells. Furthermore, the fact that all Shh or Hh soluble molecules identified so far are cholesterol-modified, strongly suggests that Hh-Np cannot be released from its anchor by cleavage (Gallet, 2003).

How do Hh-Np LPSs move within the cellular field? At least two alternative mechanisms could explain this movement. Planar transcytosis and, thus, transit from cell to cell in an endocytic and recycling-dependent manner might be involved. Alternatively, Hh-Np LPSs could pinch off from membrane raft domains, spread in the extracellular space, and become internalized away from the source at different cell positions. The role of the Ttv-dependent heparan sulfates could either be to stabilize such structures or to transport them from cell to cell (Gallet, 2003).

Differential activation of wg and ptc in anterior cells and of rho and ptc in posterior cells is related to the membrane localization of Hh. Cholesterol-dependent LPS formation and apical targeting are shown to be necessary for proper anterior wg activation but dispensable for rho expression in posterior cells. Conversely, basolateral targeting of Hh in cells producing Hh-N-CD2 and Hh-N-GPI is sufficient to activate the posterior rho expression, independent of the presence of cholesterol (Gallet, 2003).

Interestingly, wg is expressed in adjacent cells located just anterior to the Hh-sending cells. Hence, long-range diffusion of Hh should not be required for wg activation. However, in the absence of Ttv function, Hh-Np LPSs are blocked apically in producing cells, and wg is not activated. Ttv-dependent heparan sulfate proteoglycans are required for long-range Hh-Np movement in the wing disc. Thus, these results suggest that, in the embryonic ectoderm, two different mechanisms of Hh pathway activation are present. wg activation requires all the events previously associated with long-range Hh target gene activation and thus depends on Hh secretion and transport mechanisms. However, rho does not require secretion of Hh and can be activated in a cell-cell contact-dependent manner, like a short-range target. This difference could be due to differential accessibility of Hh to anterior versus posterior cells caused by the presence of the parasegmental boundary between en and wg cells. Indeed, when Ttv is expressed exclusively in cells anterior to En cells, both wg- and rho-dependent cell differentiation are rescued. This indicates that a differential transport and/or presentation of Hh-Np could be responsible for the asymmetric cellular response to Hh (Gallet, 2003).

How then is rho activated in cells posterior to Hh-producing cells? rho expression could depend on cell-cell contact activation with or without internalization of Hh. Although no detectable Hh in Hh-N-CD2 neighboring cells was observed, the possibility cannot be excluded that rho activation might depend on Hh internalization. It is worth mentioning that an Shh-CD4 transmembrane fusion protein has been shown to be internalized in adjacent cells through Ptc-1 activity in mammalian tissue culture cells and can induce formation of the most posterior digit of the chick limb. Moreover, expression of Hh-Np in disp mutant embryos that are defective in apical sorting induces rho expression in several rows of cells. In these embryos small dots of Hh-Np are seen outside the producing cells, confirming a possible internalization of Hh in posterior receiving cells through basolateral membrane interactions. This internalization could propagate at long range, since rho and ptc are activated in six to seven rows of cells when non-cholesterol-modified Hh-N is expressed in disp mutant embryos (Gallet, 2003).

In summary, these data suggests that some Hh/Shh targets can be activated through Hh trafficking in LPSs followed by apical secretion, whereas other targets might be activated by basolaterally targeted Hh. Hence, it is hypothesized that presentation of Hh from different cellular membrane compartments allows the receiving cells to differentially respond to the Hh input. This provides an interesting new paradigm regarding the mode of action of morphogens in all metazoans (Gallet, 2003).

Acylation of Hedgehog

The Drosophila Hedgehog protein and its vertebrate counterpart Sonic hedgehog are required for a wide variety of patterning events throughout development. Hedgehog proteins are secreted from cells and undergo autocatalytic cleavage and cholesterol modification to produce a mature signaling domain. This domain of Sonic hedgehog has recently been shown to acquire an N-terminal acyl group in cell culture. The in vivo role that such acylation might play in appendage patterning in mouse and Drosophila has been investigated; in both species Hedgehog proteins define a posterior domain of the limb or wing. A mutant form of Sonic hedgehog that cannot undergo acylation retains significant ability to repattern the mouse limb. However, the corresponding mutation in Drosophila Hedgehog renders it inactive in vivo, although it is normally processed. Furthermore, overexpression of the mutant form has dominant negative effects on Hedgehog signaling. These data suggest that the importance of the N-terminal cysteine of mature Hedgehog in patterning appendages differs between species (Lee, 2001a).

A mutant form of human Shh, which cannot be acylated because cysteine-24 is changed to serine (C24S-Shh-N), is at least 800-fold less active in inducing alkaline phosphatase activity in C3H10T1/2 cells than wild-type acylated Shh-N and can antagonize the effect of Shh-N on these cells. In addition, C24S-Shh has much less ability to ventralize the embryonic mouse forebrain than acylated Shh does. To further evaluate the impact of acylation on other aspects of Shh function during mouse development, retroviral vectors were used to misexpress full-length wild-type and C24S-Shh in developing limb buds. Shh and C24S-Shh sequences were inserted immediately upstream of an internal ribosome entry site (IRES) followed by a PLAP gene to allow easy detection of infected cells. Retroviral-mediated misexpression of wild-type Shh in the early mouse limb bud resulta in polydactyly and/or proximal deviation of the anterior digits resembling the hand signal of a hitchhiker. A strong correlation of such phenotypes with retroviral infection in the distal and anterior regions of the limb bud was observed. These results were consistent with previous studies demonstrating digit duplication and altered anterior-posterior patterning when ectopic Shh is delivered to the anterior limb mesenchyme and are consistent with the loss of anterior-posterior expansion of the handplate in the absence of Shh. In contrast, Shh overexpression by cells in the posterior limb where Shh is expressed endogenously or ectopic expression by cells in more proximal limb domains has no apparent impact on early limb patterning. Thus, as expected, the ability of Shh to influence limb development is dependent on its location along the anterior-posterior axis. The phenotypes, however, do not appear to require mesenchymal expression of Shh. Indeed, since retroviral injection after 9.0 dpc infects almost exclusively the surface ectoderm, the ectopic anterior outgrowths are most frequently induced by Shh-expressing cells located in close proximity to or within the apical ectodermal ridge (AER), an ectoderm-derived structure. Mutant C24S-Shh produces similar results in the limb bud. Ectopic anterior expression of C24S-Shh in the ectoderm induces polydactyly and deviation of the anterior digits. Also, similar to wild-type Shh, ectopic proximal or posterior C24S-Shh has no overt effects on limb patterning. Morphological analysis of 327 infected limbs indicates that both acylated and unacylated forms of Shh induces a similar spectrum of phenotypes, although C24S-Shh appears less active than wild type (Lee, 2001a).

In order to test whether acylation of the corresponding cysteine of Drosophila Hh could affect the activity of the protein, a mutant hh cDNA was constructed encoding serine instead of cysteine at position 84 (referred to hereafter as C84S-Hh). Cys-84 is the N-terminal residue of the mature Hh protein, corresponding to Cys-24 in Shh, and is followed by a conserved sequence of amino acids. The GAL4-UAS system was used to express wild-type and C84S-Hh in transgenic flies. To test whether C84S-Hh could replace wild-type Hh, C84S-Hh was expressed in the normal Hh pattern, under the control of engrailed-GAL4, in flies transheterozyogous for null and temperature-sensitive alleles of hh. At the restrictive temperature, embryos with temperature sensitive hh display a severe segment polarity phenotype in which the naked cuticle is lost. When a wild-type Hh transgene was expressed using en-GAL4 in hh^ts embryos, most of the cuticles examined are wild type or show a mild segment polarity phenotype. Thus this en-GAL4 line can direct the production of sufficient protein to rescue Hh signaling. In contrast, when C84S-Hh is similarly expressed, the mutant embryos fail to hatch and show a segment polarity phenotype that is indistinguishable from the hh^ts phenotype in the absence of any rescuing construct (Lee, 2001a).

The inability of C84S-Hh to rescue the hh mutant phenotype could be caused by an effect of the C84S mutation on protein stability or processing. To test this, a Western blot analysis was perfomed of protein extracts from embryos overexpressing the C84S-Hh or wild-type Hh constructs under the control of the ubiquitous daughterless-GAL4 driver. Using a polyclonal antiserum against Hh protein, it was found that the abundance of the 19-kDa N-terminal fragment of Hh is greatly increased in protein extracts from embryos expressing either wild-type or C84S Hh. Thus the C84S mutation abolishes the function of Hh without affecting its processing (Lee, 2001a).

To test whether any residual function of C84S-Hh could be detected in a gain of function assay, a series of GAL4 lines was used to express either C84S-Hh or wild-type Hh in a variety of embryonic and larval tissues. While misexpression of wild-type Hh causes a clear patterning defect often leading to lethality, C84S-Hh displays no Hh gain of function activity in any of the tissues tested. C84S-Hh misexpression caused a visible phenotype only in the developing wing and the ocelli (Lee, 2001a).

In the wing imaginal disc, Hh is produced by cells of the posterior compartment and signals to cells at the anterior-posterior (AP) border. Hh directly patterns the wing in the vicinity of the AP border, corresponding to the L3-L4 intervein region in the adult; Hh also indirectly patterns the remainder of the wing through the induction of dpp expression at the AP border. Overexpression of wild-type Hh in the posterior compartment of the wing disc using en-GAL4 results in an expansion of the anterior compartment. However, when C84S-Hh was overexpressed in the posterior compartment a loss of tissue was observed in the region surrounding the compartment boundary, between veins L3 and L4, and the partial fusion of these veins. This phenotype resembles loss of function mutations in the genes fused and cubitus interruptus, which are required for Hh signal transduction in the wing. Hh and C84S-Hh are also misexpressed with optomotor blind-GAL4, which is expressed in a broad domain of the wing disc centered on the AP compartment border. Expression of wild-type Hh using omb-GAL4 causes an expansion of the L3-L4 region; in contrast, C84S-Hh expression using omb-GAL4 results in an even stronger loss of L3-L4 tissue and partial or complete fusion of veins 3 and 4. These results suggest that, rather than mimicking wild-type Hh, C84S-Hh may interfere with the endogenous Hh signal. Likewise, when C84S-Hh is misexpressed in the ocellar region of the eye-antennal imaginal disc using eyeless-GAL4, formation of the ocelli is blocked; ocellar development requires Hh signaling (Lee, 2001a).

C84S-Hh may directly compete with wild-type Hh for binding to the Ptc receptor; alternatively, it could simply interfere with the production of wild-type Hh. To address this question, C84S-Hh was misexpressed in anterior wing disc cells that do not normally produce Hh protein, using ptc-GAL4. ptc is up-regulated in anterior cells responding to Hh; however, ptc is not expressed in the posterior cells that produce Hh. C84S-Hh expression under the control of ptc-GAL4 also reduces the amount of L3-L4 tissue produced. This result suggests that C84S-Hh interferes with Hh signaling in the extracellular environment and not at the level of intracellular processing (Lee, 2001a).

If C84S-Hh acts by competing with wild-type Hh, the severity of the phenotype should depend on the relative amounts of C84S-Hh and wild-type Hh present. This was tested by altering the ratio of wild type to mutant protein being expressed. When C84S-Hh is expressed in flies heterozygous for hh, the reduction of the L3-L4 region is enhanced, thus decreasing the dosage of wild-type Hh relative to C84S-Hh exacerbates the dominant negative phenotype (Lee, 2001a).

What role is played by the acylation? Fatty acid chains are frequently added to intracellular proteins in order to localize them to the plasma membrane. Acylation of extracellular proteins has very rarely been reported, but it would be expected to reduce their solubility and restrict their diffusion. Hh has already been shown to carry a C-terminal cholesterol modification that restricts its action to nearby cells; misexpression of the mature N-terminal signaling domain without cholesterol increases its range of activity, resulting in patterning defects. A single lipid modification is usually insufficient for membrane localization; thus acylation and cholesterol modification could both be required to tether Hh molecules. However, there is no evidence that C24S-Shh and C84S-Hh are less efficiently localized than wild-type Shh or Hh, since antibody staining shows no difference in the distribution of the two forms when misexpressed. Alternatively, the importance of acylation in addition to cholesterol modification could be to promote Hh association with raft membrane domains; this may be important for its cellular trafficking or signaling. Another possibility is that acylation might be required for Hh release from cells, perhaps localizing it to intracellular transport vesicles containing the Dispatched protein. However, the finding that C84S-Hh can inhibit the function of wild-type Hh even when expressed only in the responding cells using ptc-GAL4 suggests that it is able to exit the cell, rather than just interfering intracellularly with the release of wild-type Hh. Similarly, the ability of C24S-Shh expressed in the limb ectoderm to activate Hoxd13 expression in the underlying mesenchyme argues either that it can move freely between germ layers or that it activates a second signal with this ability. Thus it is likely that acylation is important for Hh signaling through Ptc, either because it causes more productive binding to Ptc itself or because it affects Hh interaction with other proteins (Lee, 2001a).

Hh proteins are synthesized as full-length precursors that are autocatalytically cleaved by their C-terminal domains to release the signaling N-terminal domains. The addition of a cholesterol molecule to the C terminus of the signaling domain is concomitant with cleavage. Vertebrate Sonic hedgehog (Shh) proteins have also been shown to acquire a fatty acid chain on the N-terminal cysteine of this domain, which is required for a subset of their in vivo functions. A mutation of the corresponding cysteine in Drosophila Hh transforms it into a dominant-negative protein. In a mosaic screen for novel genes required for Drosophila photoreceptor differentiation, two alleles were identified of a gene that has been named sightless (sit) for its effect on photoreceptor development. sit is required for the activity of Drosophila Hh in the eye and wing imaginal discs and in embryonic segmentation. sit acts in the cells that produce Hh, but does not affect hh transcription, Hh cleavage, or the accumulation of Hh protein. sit encodes a conserved transmembrane protein with homology to a family of membrane-bound acyltransferases. The Sit protein could act by acylating Hh or by promoting other modifications or trafficking events necessary for its function (Lee, 2001b).

Clones of sit mutant cells in the eye disc show a reduction in the number of Elav-expressing photoreceptors that is most pronounced near the center of large clones, suggesting that sit might act nonautonomously. Because both sit alleles cause pupal lethality, the eye discs of third instar larvae transheterozygous for two sit alleles could be examined. In these discs, only a few cells are able to differentiate as photoreceptors. Rescue by adjacent wild-type tissue may thus contribute to the differentiation observed in sit mutant clones (Lee, 2001b).

One of the critical signals triggering photoreceptor development is Hedgehog (Hh), which is expressed at the posterior margin of the disc prior to differentiation and subsequently in the differentiating photoreceptors. Hh activates the expression of decapentaplegic (dpp) in a stripe at the front of differentiation, or morphogenetic furrow; Dpp signaling also promotes photoreceptor formation. dpp expression is lost from the morphogenetic furrow in sit mutant eye discs. Another target of Hh signaling, the proneural gene atonal, also requires sit for its expression. Despite this lack of Hh target gene expression, a hh-lacZ enhancer trap is expressed at the posterior margin of sit mutant eye discs, indicating that hh expression is established normally. This suggests that the sit phenotype could be due to a defect in Hh signaling (Lee, 2001b).

Hh signaling has been extensively studied in the wing disc, where hh is expressed in the posterior compartment and signals to cells just anterior to the compartment boundary to upregulate the expression of dpp and patched (ptc). The Hh signal is mediated by the stabilization and activation of the full-length form of the transcription factor Cubitus interruptus (Ci). This stabilization can be detected with an antibody directed against the C-terminal region of Ci, which fails to recognize the cleaved form of Ci produced in the absence of Hh signaling. sit mutant wing discs show defects consistent with a lack of Hh pathway function; ptc expression is not upregulated at the compartment boundary, and dpp expression is almost completely absent. In addition, no stabilization of full-length Ci could be detected at the compartment boundary. However, hh-lacZ is expressed at wild-type levels in sit mutant discs, indicating that hh transcription is unaffected. This implicates Sit in the Hh pathway downstream of hh transcription and upstream of Ci stabilization (Lee, 2001b).

The defects in sit mutant discs appear to be specific to anterior-posterior patterning; wingless (wg), which marks the dorsal-ventral compartment boundary, and its target gene Distal-less are still expressed in sit mutant wing discs. In addition, the phenotype is not as severe as the complete loss of hh from early stages of larval development: although sit discs are smaller than wild-type discs, they are larger and more normally shaped than hh mutant discs. The sit alleles are therefore likely to cause an incomplete or late loss of Hh signaling; since they appear to be nulls at the molecular level, this may be due to the activity of maternally contributed sit (Lee, 2001b).

Hh signaling is also required for normal embryonic segmentation -- hh mutant embryos show a loss of naked cuticle and of wg expression. When the maternal contribution of sit is removed by making germline clones, a loss of naked cuticle strongly resembling the hh phenotype is observed, however, a wild-type copy of sit provided on the paternal chromosome is able to fully rescue the phenotype. In embryos lacking both maternal and paternal sit, stripes of Wg expression are lost from the ectoderm by stage 11. Thus, sit is required for the expression of Hh target genes in the embryo as well as in the eye and wing discs.

sit is required in the Hh-producing cells but does not affect the level of Hh protein. sit might affect Hh signaling by promoting the production of functional Hh or by allowing cells to respond to the Hh signal. To distinguish between these possibilities, mosaic analysis was used to determine in which cells sit function is required; in the wing disc, Hh-producing cells are restricted to the posterior compartment, and Hh-responding cells are restricted to the anterior compartment. Small clones of cells homozygous for sit have no effect on ptc or dpp expression in the wing disc, consistent with the nonautonomy of sit function in the eye disc. When the Minute technique was used to generate larger clones lacking sit, it was found that sit function is not required in the ptc-expressing cells or anywhere in the anterior compartment for ptc upregulation, provided that sit is present in the posterior compartment. The loss of sit from the posterior compartment prevents ptc upregulation in adjacent anterior cells even if they themselves are wild-type for sit. Thus, sit function in cells of the posterior compartment is both necessary and sufficient to upregulate ptc in anterior compartment cells. This suggests that sit may be required for the production, activity, or release of Hh protein (Lee, 2001b).

To determine whether Hh protein can be produced in the absence of sit function, sit mutant clones in the wing disc were stained with an antibody to the N-terminal domain of Hh. No change in the intensity of staining is apparent in sit mutant clones compared to adjacent wild-type tissue. Thus, sit is not required for Hh translation or stability. In clones that are mutant for dispatched (disp), which encodes a protein required for Hh release from the cell, Hh protein accumulates to high levels. No such accumulation of Hh is observed in sit mutant clones, suggesting that unlike Disp, Sit does not act at the level of Hh release (Lee, 2001b).

Hh is synthesized as a full-length precursor that is then cleaved by the autocatalytic activity of its C-terminal domain to release the N-terminal signaling domain. sit does not appear to be required for this cleavage, since similar proportions of full-length Hh and its cleaved N-terminal domain are detected on Western blots of extracts from sit mutant and wild-type third instar larvae. Whether the expression of an N-terminally truncated form of Hh (Hh-N) could rescue sit mutants was also tested; this form of the protein is not cholesterol-modified or restricted in its diffusion and does not require disp for its release from the cell. The expression of UAS-Hh-N with eyeless-GAL4 can induce premature photoreceptor differentiation in wild-type eye discs but does not alter the phenotype of sit mutant eye discs. These results suggest that sit is required for Hh activity, but not for its cleavage, cholesterol modification, or secretion (Lee, 2001b).

sit transcript is expressed uniformly at low levels in the imaginal discs and early embryo. The encoded protein has ten predicted transmembrane domains and shows homology to human, mouse, and C. elegans proteins present in the database. Its closest human homolog is BAA91772, to which it shows 28% identity and 45% similarity. In addition, the Sit protein shows more distant homology to a family of proteins that have been shown to transfer acyl chains onto hydroxyl groups of membrane-bound lipid or protein targets. An invariant histidine that has been suggested as a possible active-site residue is conserved in the Sit sequence, and both sit mutations truncate the protein prior to the region of acyltransferase homology (Lee, 2001b).

Since sit does not alter the level of Hh protein present in the cell, it is unlikely to affect Hh translation or release. The cleavage of Hh to release the N-terminal signaling domain also does not require sit, and an exogenously provided Hh-N domain is inactive in the absence of sit. sit is unlikely to be required for cholesterol addition to the C terminus of the signaling domain; bacterially produced Hh protein becomes cholesterol-modified in vitro, and this modification restricts Hh localization, but does not increase its activity in vivo. Human and rodent Sonic hedgehog (Shh) proteins have been shown to acquire a palmitoyl modification on the N-terminal cysteine of the signaling domain in cell culture. Mutation of this cysteine to serine in human Shh prevents its palmitoylation and greatly reduces its ability to ventralize the mouse forebrain. The corresponding cysteine to serine mutation in Drosophila Hh (Hh-C84S) completely abolishes its activity; however, the mutant protein appears to be secreted and appears to block the effects of wild-type Hh in the extracellular space. Together with the homology of Sit to acyltransferases, this raises the intriguing possibility that Sit might be the enzyme responsible for the palmitoylation of Hh. However, this would represent a difference in specificity between Sit and the other acyltransferases of this family, which acylate hydroxyl groups; the inactivity of Hh-C84S indicates that a hydroxyl group cannot act as a substrate in this case. Further biochemical analysis will be needed to determine whether Drosophila Hh is in fact palmitoylated and whether this palmitoylation requires sit function. Alternatively, Sit could be required for another modification or trafficking event required for Hh activity. Sit is distantly related to the Porcupine (Porc) protein, which is required in Wg-producing cells for Wg activity. Porc is localized to the endoplasmic reticulum and alters the glycosylation state of Wg. Interestingly, Porc also has homology to the membrane-bound O-acyltransferase family. The requirement of Porc for Wg function and Sit for Hh function suggests that modifications that are essential for the activity of signaling proteins may be more widespread than previously believed (Lee, 2001b).

A critical step in maturation of the Hh protein is autoproteolytic cleavage at an internal site, during which the bioactive NH₂-terminal product of cleavage acquires a COOH-terminally attached cholesterol. This modification can be bypassed by the expression of truncated or chimeric Hh proteins, such as HhN, that are produced in bioactive form without cleavage or cholesterol addition. Neither of these proteins is active in the absence of ski function, indicating that Ski must control a different property of the Hh signal, one that is shared by HhNp (N-palmitoylated Hh), HhN, and HhCD2, a Hh/Cd2 fusion protein (Chamoun, 2001).

It has recently been demonstrated that in addition to COOH-terminal addition of cholesterol, the human Sonic hedgehog (Shh) protein is further modified by NH₂-terminal attachment of a palmitoyl adduct in a manner dependent upon the NH₂-terminal cysteine residue (Pepinski, 1998). The finding that ski encodes a putative acyl transferase raises the possibility that it could function as an enzyme for the palmitoylation of Hh protein. To determine whether Drosophila Hh is NH₂-terminally acylated, the protein was purified from Drosophila tissue culture cells and subjected to mass spectrometric analysis. The experimentally determined mass of HhNp (20,235 daltons) exceeds by 238 daltons the average mass expected for the amino-terminal signaling domain linked to cholesterol alone (19,996.8 daltons). The difference between these values corresponds closely to the expected mass of a palmitoyl adduct in ester or amide linkage (238.4 daltons). Furthermore, substitution of a serine in place of the NH₂-terminal cysteine results in a protein modified by cholesterol alone. These results support the conclusion that Drosophila HhNp undergoes NH₂-terminal palmitoylation (Chamoun, 2001).

If Ski functions as the Hh palmitoyl-transferase, then the absence of Ski should result in Hh protein that is not NH₂-terminally modified, with a consequent loss or reduction of Hh signaling activity. Genetic evidence for this possibility derives from the functional equivalence between mutations that abolish the enzymatic activity of Ski and alterations in Hh proteins that prevent their NH₂-terminal acylation. If the wild-type ski alleles are replaced by a ski transgene in which two presumptive active-site residues of Ski are mutated, Hh signaling activity is greatly reduced. The same phenotypes are observed when posterior wing cells express the NH₂-terminally mutant form of Hh (Hh^C85S) instead of wild-type Hh protein. Interestingly, although mouse Shh protein also depends on ski function for maximal activity, it retains some signaling activity if secreted from ski mutant cells. The same partial reduction in Shh activity is observed with a mutant form of Shh in which the NH₂-terminal cysteine residue; mutated Shh is not further reduced when secreted from ski mutant cells. These results suggest that Ski is specifically required for the NH₂-terminal addition of palmitate to Hh (Chamoun, 2001).

Biochemical evidence for Ski's role in Hh palmitoylation was obtained by examination of Hh protein produced from cells with reduced ski function. Hh proteins were analyzed by reversed-phase high pressure liquid chromatography (RP-HPLC), which resolves doubly-modified HhNp from proteins lacking the NH₂-terminal palmitate adduct. A small proportion of HhNp in normal cells and tissues displays the earlier elution profile characteristic of the HhNp^C85Sprotein, suggesting that autoprocessing and cholesterol modification precede palmitoylation during Hh biogenesis. Drosophila cultured cells expressing HhNp were treated with double-stranded RNA corresponding to two regions of the ski coding sequence. A significantly increased proportion of the protein from these cells eluted at a position indicative of absence of the NH₂-terminal palmitate. A similar shift in elution was observed for HhNp from tissues of mutant third instar larvae, except that all of the HhNp eluted earlier, as would be expected for a complete loss of palmitate transfer. Reduction or loss of Ski function thus reduces the hydrophobicity of HhNp to that of HhNp^C85S, consistent with a loss of NH₂-terminal palmitoylation and with a role for Ski function in palmitate transfer (Chamoun, 2001).

It has been proposed that the linkage of palmitate to the NH₂-terminus of ShhNp is via an amide bond (Pepinsky, 1998), but the mechanism of amide formation is not clear. The membership of Ski in a family of enzymes catalyzing O-linked acyltransfers strongly argues for a mechanism in which a thioester intermediate is formed with the side chain of the NH₂-terminal cysteine, followed by a rearrangement through a five-membered cyclic intermediate to form the amide. It is curious that addition of cholesterol, the other lipid modification of the mature Hh signal, also involves a cyclic intermediate and thioester chemistry but proceeds in reverse, from amide to ester (Chamoun, 2001).

Distinct roles of Central missing and Dispatched in sending the Hedgehog signal

Secreted Hedgehog (Hh) proteins control many aspects of growth and patterning in animal development. The mechanism by which the Hh signal is sent and transduced is still not well understood. A genetic screen is described that aimed at identifying positive regulators in the hh pathway. Multiple new alleles of hh and dispatched (disp) were recovered. In addition, a novel component in the hh pathway, named central missing (cmn), was identified. Central missing is an alternative name for Sightless, the transmembrane acyltransferase required to generate active Hedgehog protein (Lee, 2001b). Loss-of-function mutations in cmn cause patterning defects similar to those caused by hh or dispatched (disp) mutations. Moreover, cmn affects the expression of hh responsive genes but not the expression of hh itself. Like disp, cmn acts upstream of patched (ptc) and its activity is required only in the Hh secreting cells. However, unlike disp, which is required for the release of the cholesterol-modified form of Hh, cmn regulates the activity of Hh in a manner that is independent of cholesterol modification. cmn mutations bear molecular lesions in CG11495, which encodes a putative membrane bound acyltransferase related to Porcupine, a protein implicated in regulating the secretion of Wingless (Wg) signal (Amanai, 2001).

Whether cmn affects the secretion of Hh into the anterior compartment was investigated by examining Hh distribution in cmn mutant discs. To facilitate the detection of the Hh signal, Hh was overexpressed in P-compartment cells using the hh-gal4 driver line to activate a UAS-Hh transgene. Wild-type wing discs overexpressing Hh in P-compartment cells exhibit Hh staining in A-compartment cells near the AP compartment boundary. In these cells, Hh colocalizes with Ptc in intracellular vesicles. In contrast, cmn mutant discs that overexpress Hh in P-compartment cells exhibit little if any Hh signal in neighboring A-compartment cells. This observation suggests that secretion of Hh into the anterior compartment might be impeded in cmn mutant discs. cmn mutant discs exhibit lower levels of cell surface staining of Hh in the P-compartment. Moreover, cmn mutant P-compartment cells appear to accumulate more punctate intracellular staining of Hh than wild-type P-compartment cells. These observations suggest that cmn may affect Hh trafficking (Amanai, 2001).

It is possible that normal levels of active Hh are produced in P-compartment cells of cmn mutant discs but somehow Hh fails to be released into the anterior compartment. If this is true, one would expect that P-compartment cells should activate Hh responsive genes if provided with Ci. To test this possibility, an uncleavable form of Ci (CiU) was used that requires Hh for its activation. A wing-specific Gal4 driver (MS1096) was used to express UAS-CiU in wild-type, disp or cmn discs and these discs were examined for the expression of a Hh-responsive gene collier (col, a.k.a. knot). In wild-type discs, Hh induces col expression in a stripe of cells in the A-compartment abutting the AP compartment boundary. This col expression is reduced or abolished in disp and cmn mutant discs. Consistent with the previous finding that the activity of CiU depends on Hh, expressing CiU in wild-type wing discs ectopically activates col only in P-compartment cells. Misexpressing CiU in disp mutant discs activates col in P-compartment cells at levels comparable to those in wild-type discs. In contrast, P-compartment cells of cmn discs expressing CiU express diminished levels of col. Smo stabilization was also examined as a readout for Hh activity. Wild-type and disp mutant discs stabilize Smo in P-compartment cells at comparable levels. In contrast, cmn mutant discs stabilize Smo at levels much lower than wild-type or disp mutant discs. Taken together, these observations demonstrate that disp mutant discs produce normal levels of active Hh in P-compartment cells whereas cmn mutant P-compartment cells produce reduced levels of active Hh (Amanai, 2001).

Hh is produced as a full-length precursor, which undergoes an auto-processing event to generate a cholesterol-modified N-terminal fragment that functions as a ligand. To determine if cmn affects Hh processing, a test was made to determine whether a pre-cleaved form of Hh (HhN) could rescue cmn mutant phenotypes. The actin>CD2>Gal4 driver line was used to express UAS-HhN uniformly in wild-type, disp or cmn mutant discs and ptc upregulation was examined as a readout for the Hh signaling activity. Indiscriminately expressing HhN in either wild-type or disp mutant discs causes ectopic ptc upregulation in the entire A-compartment. In contrast, uniformly expressing HhN in cmn mutant discs fails to induce upregulation of ptc. These results suggest that Cmn does not regulate the cleavage of the Hh precursor into the mature form of Hh. Since HhN is no longer modified by cholesterol, this result also suggests that cmn is required for the activity of Hh, independent of cholesterol modification (Amanai, 2001).

These experiments suggest that cmn acts upstream of ptc and its function is required in the Hh sending cells but not in cells that receive the Hh signal. Thus, cmn represents a second gene after disp that regulates sending of the Hh signal. In cmn mutant discs, no Hh signal is detected in anterior compartment cells near the AP compartment boundary. However, unlike the case of disp, no accumulation of Hh staining is observed in P-compartment cmn mutant cells. In contrast, cmn mutant P-compartment cells consistently exhibited lower levels of cell surface staining of Hh with concomitant increase in the number and size of intracellular Hh aggregates as compared with wild-type cells. This observation implies that cmn might affect cellular trafficking of Hh. Consistent with lower levels of cell surface Hh staining, it was found that cmn mutant P-compartment cells produce lower levels of active Hh as compared with wild-type or disp mutant cells (Amanai, 2001).

Patched controls the Hedgehog gradient by endocytosis in a dynamin-dependent manner, but this internalization does not play a major role in signal transduction

The Hedgehog (Hh) morphogenetic gradient controls multiple developmental patterning events in Drosophila and vertebrates. Patched (Ptc), the Hh receptor, restrains both Hh spreading and Hh signaling. Endocytosis regulates the concentration and activity of Hh in the wing imaginal disc. Ptc limits the Hh gradient by internalizing Hh through endosomes in a dynamin-dependent manner, and both Hh and Ptc are targeted to lysosomal degradation. The ptc¹⁴ mutant does not block Hh spreading, because it has a failure in endocytosis. However, this mutant protein is able to control the expression of Hh target genes as does the wild-type protein, indicating that the internalization mediated by Ptc is not required for signal transduction. In addition, both in this mutant and in those not producing Ptc protein, Hh still occurs in the endocytic vesicles of Hh-receiving cells, suggesting the existence of a second, Ptc-independent, mechanism of Hh internalization (Torrioja, 2004).

Through the analysis of ptc¹⁴ (a mutant that does not internalize Hh but is able to perform Hh signal transduction) this study shows that both proposed Ptc functions are genetically uncoupled. Ptc limits the Hh gradient by internalizing Hh in a dynamin-dependent manner, and this Hh-Ptc complex is targeted to the degradation pathway. These findings strongly suggest that internalization mediated by Ptc shapes the Hh gradient and also leads to the challenging suggestion that Hh signaling can occur in the absence of Ptc-mediated Hh internalization. The two functions of Ptc in Hh signal transduction are discussed in the light of these results (Torrioja, 2004).

Hh and Ptc sorting to the endocytic membrane-bound compartment plays a crucial role in modulating Hh levels during development. A strong support of the conclusions in this work comes from the analysis of the ptc¹⁴ allele. Although ptc¹⁴ mutants are lethal with a strong ptc^- embryonic phenotype, ptc¹⁴ mutant cells in the imaginal discs show an effect only when the clone touches the AP compartment border but not in any other part of the disc. This result indicates that the presence of Hh is required to reveal a defect in Ptc¹⁴ function. This Hh requirement has been probed by the lack of activation of the Hh targets in ptc¹⁴ cells in the absence of Hh, either in the embryos or in the imaginal discs. The complementation of ptc¹⁴ with ptc^S2 allele, which is considered as null for blocking Hh signal transduction and acts as dominant negative, indicates that Ptc¹⁴ does not have a greater sensibility to Hh than the Ptc wild-type protein. Conversely, it has been shown in this study that there is a decrease of internalization of Hh in ptc¹⁴ mutant clones compared with wild-type Ptc territory and an extension of the range of Hh gradient. Therefore, it can be concluded that Ptc¹⁴ is unable to sequester Hh efficiently in either the embryo or imaginal discs and that the ptc¹⁴ embryonic phenotype would be the result of greater spreading of Hh and not due to the constitutive activation of the Hh pathway (Torrioja, 2004).

Ptc¹⁴ responds to Hh as does the wild-type Ptc protein and activates the signaling pathway indicating that the interaction of Ptc¹⁴ and Hh is probably normal. However, this Hh-Ptc interaction does not necessarily imply sequestration. Although Ptc¹⁴ occurs at the plasma membrane, no internalization of Hh or extracellular Hh accumulation occurs in ptc¹⁴ mutant clones. These results, therefore, suggest that Hh-Ptc interaction is not sufficient to sequester Hh and that an active internalization process of Hh mediated by Ptc to control Hh gradient is required. This Hh internalization mediated by Ptc is Dynamin-dependent, based on the membrane accumulation of Hh and Ptc in shi mutant clones and the lack of accumulation of Hh in shi^ts1; ptc¹⁶ double mutant clones. However, the initiation of the internalization process is not blocked in shi mutants because Dynamin is required for fission of clathrin-coated vesicles after the internalization process has already started. This fact would explain why Hh gradient and signaling is not extended when endocytosis is blocked in shi mutant cells. Since Ptc¹⁴ seems to have a problem in entering the endocytic compartment and no Hh accumulation is found in shi^ts1; ptc¹⁴ double mutant clones, it is concluded that the initiation of the internalization process does not occur in Ptc¹⁴. Taken together, these data indicate that only when Ptc forces Hh to the endocytic pathway Hh is sequestered in the receiving cells (Torrioja, 2004).

To block the degradative pathway, deep orange (dor) mutants were used. dor, one of the mutations that affects eye pigmentation in Drosophila, is required for normal delivery of proteins to lysosomes. The behavior of Hh and Ptc in dor^- cells indicates that after sequestration, Ptc internalizes Hh, and both Hh and Ptc are degraded. Thus, controlling both endocytosis and degradation of Hh modulates its gradient. Similar mechanisms have been described for controlling the asymmetric gradient of Wg in embryonic segments. It is possible that additional factors may contribute to shaping the Hh gradient, because in large ptc^- clones close to the AP border, which lack Ptc protein to sequester Hh, an Hh gradient in endocytic vesicles is also observed, although the range of this gradient is more extended than in wild-type cells. This is consistent with two mechanisms of Hh internalization in Hh receiving, one mediated by Ptc and another not mediated by Ptc (Torrioja, 2004).

From studies in both vertebrates and Drosophila, it was thought that Hh protein binds to Ptc. Ptc is then internalized and traffics Hh to endosomal compartments where both are degraded, the entire process triggering activation of the Hh pathway. It is shown in this study that Ptc¹⁴ responds to Hh as would the wild-type Ptc protein in activating the pathway. However, Ptc¹⁴ does not internalize Hh to the endocytic compartment because it is defective in endocytosis. It is therefore suggested that the massive Hh internalization by Ptc to control the gradient is not a requirement for Hh pathway signal transduction (Torrioja, 2004).

In Hh signal transduction, the cellular mechanisms that regulate Smo function remain unclear, although the distribution of Ptc/Smo suggests that Ptc destabilizes Smo levels. It has also been proposed that Ptc-mediated Hh internalization changes the subcellular localization of Ptc preventing Smo downregulation. Furthermore, in cultured cells, Shh induces the segregation of Ptc and Smo in endosomes, allowing Smo signaling, independently of Ptc. It is known, however, that binding of Shh to Ptc is not sufficient to relieve the repression of the Hh pathway (Torrioja, 2004).

As in wild-type cells, in the absence of Hh, Ptc¹⁴ downregulates both Smo levels and Smo activity, while in the presence of Hh, the normal upregulation of Smo occurs. Consequently, Ptc¹⁴ levels are high at the AP border because upregulation of Ptc by Hh occurs in the absence of internalization of Hh to the degradative pathway. It might then be expected that the high levels of Ptc¹⁴ not targeted to the degradative pathway would block Smo activity. However, against all predictions, the presence of Hh is still able to release Smo activity in mutant ptc¹⁴ cells. Thus, there must be a positive mediator of Smo activity to overcome the repressive effect of Ptc¹⁴ and allow Hh pathway activation in response to Hh. Alternatively, if Ptc¹⁴ is located at the plasma membrane, it could control Smo activity without entering the endocytic compartment by regulating the entrance of small molecules, as has been proposed. In fact, Ptc is similar to a family of bacterial proton-gradient-driven transmembrane molecule transporters known as RND proteins. Accordingly, as a membrane transporter, Ptc could indirectly inhibit Smo through translocation of a small molecule that conformationally regulates the active state of Smo. The inter-allelic complementation of Ptc suggests that Ptc has the oligomeric structure needed for this type of transporter (Torrioja, 2004).

Although one of the normal functions of Ptc is to mediate Hh internalization, the data demonstrate the presence of internalized Hh vesicles in the absence of Ptc protein. It is therefore suggested that another receptor mediates Hh internalization in Hh-receiving cells. This molecule could act as a positive mediator of Hh signaling. Several observations have been published that cannot easily be reconciled with the idea of Ptc acting as the only receptor for Hh. For example, it was found that Hh activates signal transduction in both A and P compartment cells of wing imaginal discs, despite the absence of Ptc in P cells. Furthermore, it has been reported that some neuroblasts in Drosophila embryos, the maturation of which is dependent on Hh, do not express or require Ptc. This suggests that a receptor other than Ptc mediates Hh signaling. Recently, the glypican protein Dally-like, which belongs to the heparan sulfate proteoglycan protein family, was found to be required for Hh signal transduction and probably for the reception of the Hh signal in Drosophila tissue culture cells. Dally-like could act as co-receptor for Hh and it would be interesting to know if Dally-like is required for Hh endocytosis. In addition, the large glycoprotein 'Megalin' has recently been identified as a Shh-binding protein. Megalin is a multi-ligand-binding protein of the low-density lipoprotein (LDL) receptor family whose function is to mediate the endocytosis of ligands. The finding that megalin-mediated endocytosed N-Shh is not efficiently targeted to lysosomes for degradation suggests that N-Shh may also traffic in complexes with Megalin and thus be recycled and/or transcytosed. In the Wg pathway, specific LDL receptor-related proteins are essential co-receptors for Wnt ligands. Further investigation will determine whether LDL receptor-related proteins could function as co-receptors that internalize Hh in the absence of Ptc. Alternatively, these proteins could be required for endocytosis and further delivery of Hh to Ptc in intracellular vesicles, perhaps facilitating the transcytosis of Hh. A future challenge will be to find other molecules that internalize Hh and to understand how Hh interacts with Smo to activate the Hh pathway (Torrioja, 2004).

The extracellular matrix, glycosaminoglycan and Hedgehog

Hedgehog (Hh) proteins act through both short-range and long-range signaling to pattern tissues during invertebrate and vertebrate development. The mechanisms allowing Hedgehog to diffuse over a long distance and to exert its long-range effects are not understood. A new Drosophila gene, named tout-velu and meaning "all hair" has been identified; it is required for diffusion of Hedgehog. ttv was identified in a screen for maternal-effect mutations associated with segment polarity. ttv clones prove to have a non-cell-autonomous effect. Hh signal is shown to be unable to reach wild-type cells located anterior to ttv mutant cells. It is proposed that ttv functions in the receiving cells for the movement of Hh from sending to receiving cells. Characterization of tout-velu shows that it encodes an integral membrane protein that belongs to the EXT gene family. Members of this family are involved in the human multiple exostoses syndrome, which affects bone morphogenesis. Analysis of the Ttv sequence shows that there is a hydrophobic stretch at the amino terminus of the Ttv protein, indicating that Ttv might be a transmembrane protein. Ttv appears to be a type II integral protein, with the C-terminal region displayed extracellularly. These results, together with the previous characterization of the role of Indian Hedgehog in bone morphogenesis, have led to a proposal that the multiple exostoses syndrome is associated with abnormal diffusion of Hedgehog proteins. These results show the existence of a new conserved mechanism required for diffusion of Hedgehog. EXT-1 has been found in the extracellular reticulum, where it helps to regulate the synthesis and display of cell-surface heparin sulphate glycosaminoglycans (GAGs). Because GAGs have been implicated in receiving signaling molecules, it is plausible to speculate that Ttv is involved in the synthesis of a GAG that specifically interacts with Hh at the cell surface. Such an interaction might result in the endocytosis of Hh or, alternatively, it may facilitate the movement of Hh from one cell to the next by translocating Hh around the surface of the cell (Bellaiche, 1998).

A sensitive method has been devised for the isolation and structural analysis of glycosaminoglycans from two genetically tractable model organisms, the fruit fly, and the nematode. Chondroitin/chondroitin sulfate-derived and heparan sulfate-derived disaccharides were detected in both organisms. Chondroitinase digestion of glycosaminoglycans from adult Drosophila produces both nonsulfated and 4-O-sulfated unsaturated disaccharides, whereas only unsulfated forms are detected in C. elegans. Heparin lyases releases disaccharides bearing N-, 2-O-, and 6-O-sulfated species, including mono-, di-, and tri-sulfated forms. Tissue- and stage-specific differences were observed in both chondroitin sulfate and heparan sulfate composition in Drosophila. These methods were applied toward the analysis of tout-velu, an EXT-related gene in Drosophila that controls the tissue distribution of the growth factor Hedgehog. The proteins encoded by the vertebrate tumor suppressor genes EXT1 and 2, show heparan sulfate co-polymerase activity, and it has been proposed that tout-velu affects Hedgehog activity via its role in heparan sulfate biosynthesis. Analysis of total glycosaminoglycans from tout-velu mutant larvae show marked reductions in heparan sulfate but not chondroitin sulfate, consistent with Tout-velu's proposed function as a heparan sulfate co-polymerase (Toyada, 2000).

Classical genetic screens can be limited by the selectivity of mutational targeting, the complexities of anatomically based phenotypic analysis, or difficulties in subsequent gene identification. Focusing on signaling response to the secreted morphogen Hedgehog (Hh), RNA interference (RNAi) and a quantitative cultured cell assay were used to systematically screen functional roles of all kinases and phosphatases, and subsequently 43% of predicted Drosophila genes. Two gene products reported to function in Wingless (Wg) signaling were identified as Hh pathway components: a cell surface protein (Dally-like protein) required for Hh signal reception, and casein kinase 1alpha, a candidate tumor suppressor that regulates basal activities of both Hh and Wg pathways. This type of cultured cell-based functional genomics approach may be useful in the systematic analysis of other biological processes (Lum, 2002).

Shifted controls the distribution and movement of Hedgehog

The Hedgehog (Hh) family of morphogenetic proteins has important instructional roles in metazoan development and human diseases. Lipid modified Hedgehog is able to migrate to and program cells far away from its site of production despite being associated with membranes. To investigate the Hh spreading mechanism, Shifted (Shf) was characterized as a component in the Drosophila Hh pathway. Shf, discovered by Calvin B. Bridges in 1913, is the ortholog of the human Wnt inhibitory factor (WIF), a secreted antagonist of the Wingless pathway. In contrast, Shf is required for Hh stability and for lipid-modified Hh diffusion. Shf colocalizes with Hh in the extracellular matrix and interacts with the heparan sulfate proteoglycans (HSPG), leading to the suggestion that Shf could provide HSPG specificity for Hh. Shifted acts over a long distance and is required for the normal accumulation of Hh protein and its movement in the wing. Human WIF inhibits Wg signaling in Drosophila without affecting the Hh pathway, indicating that different WIF family members might have divergent functions in each pathway (Gorfinkiel, 2005; Glise, 2005).

The role of glypicans in Wnt inhibitory factor-1 activity and the structural basis of Wif1's effects on Wnt and Hedgehog signaling

Proper assignment of cellular fates relies on correct interpretation of Wnt and Hedgehog (Hh) signals. Members of the Wnt Inhibitory Factor-1 (WIF1) family are secreted modulators of these extracellular signaling pathways. Vertebrate WIF1 binds Wnts and inhibits their signaling, but its Drosophila melanogaster ortholog Shifted (Shf) binds Hh and extends the range of Hh activity in the developing wing. Shf activity is thought to depend on reinforcing interactions between Hh and glypican HSPGs. Using zebrafish embryos and the heterologous system provided by D. melanogaster wing, this study reports on the contribution of glypican HSPGs to the Wnt-inhibiting activity of zebrafish Wif1 and on the protein domains responsible for the differences in Wif1 and Shf specificity. Wif1 strengthens interactions between Wnt and glypicans, modulating the biphasic action of glypicans towards Wnt inhibition; conversely, glypicans and the glypican-binding 'EGF-like' domains of Wif1 are required for Wif1's full Wnt-inhibiting activity. Chimeric constructs between Wif1 and Shf were used to investigate their specificities for Wnt and Hh signaling. Full Wnt inhibition required the 'WIF' domain of Wif1, and the HSPG-binding EGF-like domains of either Wif1 or Shf. Full promotion of Hh signaling requires both the EGF-like domains of Shf and the WIF domains of either Wif1 or Shf. That the Wif1 WIF domain can increase the Hh promoting activity of Shf's EGF domains suggests it is capable of interacting with Hh. In fact, full-length Wif1 affected distribution and signaling of Hh in D. melanogaster, albeit weakly, suggesting a possible role for Wif1 as a modulator of vertebrate Hh signaling (Avanesov, 2012; full text of article). The 'WIF' domain of WIF1 does not bind HS sidechains, but is sufficient for Wnt binding; the 'EGF-like' domains show only weak binding to Wnts on their own, but appear to strengthen Wnt binding to the 'WIF' domain. But while the Drososphila WIF1 homolog Shf contains both 'WIF' and 'EGF-like' domains, it does not inhibit Wg signaling; instead, it increases the levels or range of Hh signaling. This study found that a construct containing Shf's 'WIF' domain and the zebrafish Wif1's 'EGF-like' domains also cannot inhibit Wnt signaling, while the reciprocal construct with Wif1's 'WIF' domain and Shf's 'EGF-like' domain can. Similar results have been obtained with constructs made from Shifted and human WIF1. Thus, the ability to inhibit Wg activity, and likely to bind significant levels of Wg, resides in the different 'WIF' domains of Wif1 and Shf (Avanesov, 2012).

Surprisingly, Shf did show a weak ability to improve Wg signaling in sensitized backgrounds expressing either Wif1 or the dominant negative DFz2-GPI construct. While no obvious effect was ever detected of Shf on ex-Wg levels, it may weakly interact with Wg in a manner that reduces the levels bound to Wif1 or DFz2-GPI and increases the levels available for the Wg receptors. Consistent with this interpretation, UAS-shf did not alleviate margin defects caused by expression of UAS-wg RNAi, even though UAS-Dfz2-GPI and UAS-wg RNAi show a very comparable impact on Wg activity. Alternatively, Shf's effect on Wnt signaling might be due to interactions with the Wnt4 or Wnt6 expressed along the wing margin, which may have redundant roles in wing margin development that are only obvious in a sensitized background. Indirect effects via Hh signaling are unlikely, as Shf overexpression does not further increase Hh signaling (Avanesov, 2012).

The situation with Hh signaling is more complex. First, vertebrate WIF1's are not known to regulate vertebrate Hh signaling, but this study found that zebrafish Wif1 can weakly affect the reduced movement or accumulation of Hh normally observed in shf mutant wing discs. The Hh-GFP accumulation is abnormal, however, appearing more punctuate than in normal wing discs, perhaps accounting for its ability to reduce the expression of Hh targets (Avanesov, 2012).

Placing WIF domain of zebrafish Wif1 in the context of Shf's 'EGF-like' domains in a chimeric WIFWif1-EGFShf construct almost fully rescues loss of shf function, something not observed after expression of the Shf 'EGF-like' domains alone. Together, these data suggest that the 'WIF' domains of both Shf and zebrafish Wif1 are capable of interacting with Hh. Like Wnts, Hh is palmitoylated, and it has been suggested that these palmitates might bind a hydrophobic pocket found in the WIF domain, although this has been recently questioned. The activity of 'WIF' domains in Hh signaling may also vary between different vertebrates, since unlike the WIFWif1-EGFShf construct made using zebrafish 'WIF' domains, a similar construct made using the 'WIF' domain from human WIF1 does not rescue loss of shf function (Avanesov, 2012).

The Shf 'EGF-like' domains are necessary to confer a Shf-like level of Hh-promoting activity to the 'WIF' domains of zebrafish Wif1. The Hh-promoting activity of Wif1's 'WIF' domain is increased by placing it in the context of Shf's 'EGF-like' domains, and the low Hh-promoting activity of Shf's 'WIF' domain is not changed by placing it in the context of Wif1's 'EGF-like' domains. It is unlikely that the 'EGF-like' domains of Shf and Wif1 differ significantly in their HSPG-binding activities, since Wif1 and WIFWif1-EGFShf differ only slightly in their ability to inhibit Wnt signaling and interact genetically with Dlp. Therefore the alternative hypothesis is favored that Shf's 'EGF-like' domains contribute to Hh signaling through a mechanism independent of glypican binding. While the Shf 'EGF-like' domains alone (ShfδWIF) cannot increase Hh signaling, it was found that they can increase the levels of extracellular Hh, suggesting that they contribute to Hh binding, much as the 'EGF-like' domains of WIF1 do to Wnt binding (Avanesov, 2012).

Since Wif1 can alter Hh distribution and, more weakly, signaling in Drosophila, an important question is whether it can also do so in vertebrates. Because of its strong effects on Wnt signaling, vertebrate WIF1 family proteins have rarely been assayed for their effects on other pathways, so a weak modulation of one of the vertebrate Hhs remains a possibility (Avanesov, 2012).

Slalom encodes an adenosine 3'-phosphate 5'-phosphosulfate transporter essential for development in Drosophila

Sulfation of all macromolecules entering the secretory pathway in higher organisms occurs in the Golgi and requires the high-energy sulfate donor adenosine 3'-phosphate 5'-phosphosulfate. A gene has been identified that encodes a transmembrane protein required to transport adenosine 3'-phosphate 5'-phosphosulfate from the cytosol into the Golgi lumen. Mutations in this gene, which has been called slalom, display defects in Wg and Hh signaling; these defects are likely due to the lack of sulfation of glycosaminoglycans by the sulfotransferase sulfateless. Analysis of mosaic mutant ovaries shows that sll function is also essential for dorsal-ventral axis determination, suggesting that sll transports the sulfate donor required for sulfotransferase activity of the dorsal-ventral determinant Pipe (Lüders, 2003).

Secreted signaling molecules of the FGF, Hh, TGF-ß and WNT families rely on proteoglycans (PGs) for efficient activation of their respective signaling pathways. PGs consist of secreted or transmembrane core proteins to which glycosaminoglycan (GAG) side chains are attached at specific consensus sites. In Drosophila, the secreted PG perlecan, the transmembrane PG syndecan and two members of the glypican family of glycosylphosphatidylinositol (GPI) anchored PGs have been identified. Phenotypes associated with loss of function mutations of the glypican-encoding genes division abnormally delayed and dally-like (dlp) have revealed the requirement of these PGs for efficient activation of several signal transduction pathways (Lüders, 2003).

The function of PGs is critically dependent on the integrity of the attached GAGs. GAGs are unbranched polysaccharide chains, which are synthesized on proteoglycan core proteins in the Golgi and undergo complex modification reactions before the PG to which they are attached is transported to the cell surface. In the case of glypican, heparan sulfate (HS) chains, consisting of a sugar backbone of alternating units of N-acetylglucosamine (GlcNAc) and glucuronic acid (GlcA), are synthesized on the core protein. The nucleotide sugar substrates for this reaction are synthesized in the cytoplasm and must be transported into the Golgi. In Drosophila, the activated precursor UDP-GlcA is synthesized from UDP-glucose by the enzymatic activity of the sugarless (sgl) gene product, a homolog of mammalian UDP-glucose dehydrogenases. The gene product of fringe connection (frc), a predicted ER/Golgi multipass transmembrane protein, has been shown to transport UDP-GlcA and UDP-GlcNAc from the cytosol into the Golgi. Mutations in either gene severely affect the Wg and FGF signaling pathways. Elongation of the HS chains requires the activity of HS polymerases. tout-velu (ttv) encodes a protein with homology to the mammalian HS co-polymerase EXT1 and has been demonstrated to be required specifically for Hh signaling. Subsequent to their synthesis, GAGs undergo multiple modifications such as epimerization and sulfation. Mutations in sulfateless (sfl), a homolog of vertebrate N-deacetylase/N-sulfotransferases, lead to a severe reduction in the activity of the Wg, Hh and FGF signaling pathways. A characteristic feature of all mutations in genes involved in GAG biosynthesis is that their segment polarity phenotypes can be rescued by ectopic expression of wg or hh, suggesting that GAGs are not essential components of the respective signaling cascades but accessory factors most likely required for the proper distribution of extracellular signaling molecules throughout morphogenetically active tissues in vivo (Lüders, 2003).

GAGs have also been proposed to play a role in the determination of the dorsal-ventral (D/V) axis of the Drosophila embryo. The D/V polarity of the embryo is established during oogenesis by asymmetric expression of the key D/V determinant pipe (pip) in the follicle cell epithelium. pip expression in the ventral follicle cell layer is necessary and sufficient to trigger a serine-protease cascade in the perivitelline space; this leads to the generation of an active ligand for the transmembrane receptor Toll (Tl). Activation of Tl on the ventral side of the embryo results in a gradient of nuclear localization of the transcription factor Dorsal, which patterns the D/V axis. Based on sequence similarity to a family of vertebrate enzymes and its localization in the Golgi apparatus, pip has been hypothesized to encode a heparan sulfate 2-O-sulfotransferase. However, neither the enzymatic activity nor the substrate specificity of pip have been demonstrated directly (Lüders, 2003).

Sulfation of secreted molecules occurs in the Golgi and requires the high-energy sulfate donor adenosine 3'-phosphate 5'-phosphosulfate (PAPS) to be present within that organelle. In Drosophila, PAPS is synthesized in the cytoplasm by PAPS-synthetase, which incorporates both ATP-sulfurylase and adenosine 5'-phosphosulfate-kinase (APS-kinase) activity. PAPS must be transported into the Golgi thereafter to serve as a substrate for sulfotransferases. This study reports the molecular identification and functional characterization of a PAPS transporter. Mutations in this gene, called slalom (sll), are associated with defects in multiple signaling pathways, including Wg and Hh signaling. A phenotypic analysis suggests that the effects of sll on signal transduction are caused by its requirement for GAG modification. Evidence is presented that sll is also required to supply PAPS to the machinery initiating the establishment of embryonic D/V polarity, supporting the view that Pipe protein is a sulfotransferase (Lüders, 2003).

A hydrophobicity analysis of the putative Sll protein predicts a hydrophobic polypeptide with at least 10 transmembrane regions, a structural characteristic of many nucleotide-sugar transporters. A BLAST search of non-redundant protein databases with the Sll sequence revealed that Sll is conserved throughout the animal kingdom, as well as in plants, and shares nearly 40% of amino acid sequence identity with predicted mouse and human proteins of unknown function. Sll is also similar to mammalian proteins that have been classified as nucleotide-sugar transporters on the basis of their homology to the human UDP-galactose transporter hUGT. However, Sll itself has no significant sequence similarity to hUGT. While these data suggest that Sll encodes a transmembrane transporter, they leave open what substrate Sll may be transporting (Lüders, 2003).

In order to demonstrate directly that Sll has transporter activity, and to determine its substrate specificity, an in vitro vesicle transport assay was used to examine the ability of Sll to facilitate the transport of defined nucleotide substrates into Saccharomyces cerevisae microsomes. Heterologous expression of sll in yeast was favored over other systems since procedures in this organism have been successfully used for transfection of nucleotide-sugar genes, high-level preparation of sealed microsomal vesicles and nucleotide-sugar assays. In vesicles derived from cells transfected with a sll-expression construct, a 5-fold increase in the uptake of PAPS was detected. Import of several other nucleotide-sugars did not increase after sll expression. Importantly, the sll-dependent PAPS transport activity could be efficiently competed by structurally similar substrate analogs but not by more distantly related nucleotide monophosphates, confirming that the transport was specific (Lüders, 2003).

PAPS is synthesized in the cytoplasm and serves as a substrate for sulfotransferases during the post-translational modification of macromolecules in the Golgi. Therefore, the result that sll encodes a PAPS transporter suggests that Sll should be localized to the Golgi membranes. To address this question, peptide antibodies were raised against Sll. Localization of Sll in salivary glands of wild-type embryos, using Sll antiserum, showed a punctate staining pattern in the perinuclear region of cells. Co-staining of salivary glands with antibodies against an endogenous Golgi protein revealed extensive co-localization of the two proteins, indicating that Sll is a resident Golgi protein. Together, these data suggest that Sll encodes a nucleotide transporter required to translocate the high-energy sulfate donor PAPS into the lumen of the Golgi (Lüders, 2003).

The phenotypical analysis of sll mutants shows that sll function is required for Wg and Hh signaling, and suggests that sll may also play a role in the activation of other signaling pathways, such as the TGF-ß pathway and possibly others. The broad requirement for sll raises the question of how sulfation influences the function of multiple secreted signaling factors. Proteins entering the secretory pathway are ubiquitously sulfated on specific tyrosine residues. However, according to sulfation consensus prediction algorithms, neither Wg nor Hh is predicted to be a substrate for tyrosine sulfation (see 'Sulfinator' at http://www.expasy.org/tools/sulfinator/). In addition, overexpression experiments show that wg and hh have the ability to at least partially activate their respective signaling cascades in sll mutants, which would be unlikely if the signaling factors themselves were non-functional due to lack of sulfate modification. Therefore, it is unlikely that lack of tyrosine sulfation of either Wg or Hh proteins is responsible for the defects in Wg or Hh signaling pathways in sll mutants (Lüders, 2003).

The Wg and Hh signaling pathways are activated through interaction of secreted ligands with their respective cell surface receptors. This activation relies on the action of GAGs attached to proteoglycan core proteins of the glypican family. In the case of Wg, the proteoglycans are thought to act as coreceptors, retaining the ligand at the cell surface in the vicinity of its receptor. Hh has been proposed to depend on GAGs for efficient transport to its target cells. In the absence of functional GAGs, the signaling molecules are not present in sufficiently high concentrations at the receptor to activate the signal transduction pathway. This defect can be compensated by overexpression of the signaling molecule. Therefore, a characteristic feature of mutations in genes involved in GAG metabolism is that their segment polarity defects can be rescued by overexpression of the ligand. These data show that Wg protein levels and Hh signaling activity are reduced in sll mutant clones. The segment polarity defects of sll mutants can be rescued by overexpression of wg or hh. In addition sll and dally mutants affect Wg signaling synergistically. The data also suggest that the ability of cells to sulfate GAGs is dramatically reduced in sll mutants because the sulfate donor necessary for sfl to modify the polysaccharide chains cannot be transported into the Golgi in sufficient amounts. Taken together, these observations strongly suggest that lack of functional GAGs is responsible for the segment polarity defects in sll mutants (Lüders, 2003).

Interestingly, the overall size of sugar chains attached to the glypican Dally does not appear to be affected in sll mutants. Consistent with this result, it has been observed that the overall level of HS, which is reduced to trace amounts in the sgl mutant that affects GAG biosynthesis, is not markedly changed in sfl mutants, which should affect sulfate modification but not synthesis of GAG chains. However, it cannot be excluded that residual sulfation of GAGs occurs in the cell culture assay owing to incomplete inhibition of the PAPS transport activity of sll by dsRNA interference. However, the phenotypic analysis of sll suggests that sll function is essential for at least two sulfotransferases, sfl and pip, and that the GAG chains present in sll mutants are not able to fulfill their normal function owing to altered sulfation patterns (Lüders, 2003).

The central position of sll in sulfate metabolism along the secretory pathway makes it an interesting tool for the identification of developmental pathways sensitive to sulfate modification. The results demonstrate that several cell-cell communication pathways are critically dependent on the sulfation of macromolecules, and highlight the importance of sulfation during pattern formation and development (Lüders, 2003).

Dally and Dally-like, two Drosophila glypican members of the heparan sulphate proteoglycan family, are the substrates of Ttv and are essential for Hh movement

The signalling molecule Hedgehog (Hh) functions as a morphogen to pattern a field of cells in animal development. Previous studies in Drosophila have demonstrated that Tout-velu (Ttv), a heparan sulphate polymerase, is required for Hh movement across receiving cells. However, the molecular mechanism of Ttv- mediated Hh movement is poorly defined. Dally and Dally-like (Dly), two Drosophila glypican members of the heparan sulphate proteoglycan (HSPG) family, are shown to be the substrates of Ttv and are essential for Hh movement. Embryos lacking dly activity exhibit defects in Hh distribution and its subsequent signalling. However, both Dally and Dly are involved and are functionally redundant in Hh movement during wing development. Hh movement in its receiving cells is regulated by a cell-to-cell mechanism that is independent of dynamin-mediated endocytosis. It is proposed that glypicans transfer Hh along the cell membrane to pattern a field of cells (Han, 2004).

To dissect the molecular mechanism(s) by which HSPG(s) regulates Hh signalling, attempts were made to identify specific proteoglycan(s) involved in Hh signalling during embryonic patterning. During embryogenesis, Hh and Wingless (Wg) are expressed in adjacent cells and are required for patterning of epidermis. In stage 10 embryos, Hh is expressed in two rows of cells in the posterior compartment of each parasegment, while Wg is expressed in one row of cells anterior to Hh expression cells. The expression of Hh is controlled by Engrailed (En) whose expression is maintained by Wg signalling through a paracrine regulatory loop. Hh signalling in turn is required for maintaining the expression of wg whose activity controls the production of the naked cuticles. Loss of either Hh or Wg signalling leads to a loss of naked cuticle, which is defined as segment polarity phenotype (Han, 2004 and references therein).

Disruption of dly in embryos by RNA interference (RNAi) leads to a strong segment polarity defect, suggesting that Dly is likely to be involved in Hh and/or Wingless (Wg) signalling in embryonic epidermis. To explore the potential role of Dly in Hh signalling, a number of dly mutant alleles were isolated using EMS mutagenesis. dly^A187 is a null allele and is used for further analyses. Animals zygotically mutant for dly appears to have normal cuticle patterning and survive until third instar larvae. However, homozygous mutant embryos derived from females lacking maternal dly activity (referred to as dly embryos hereafter) die with a strong segment-polarity phenotype resembling those of mutants of the segment polarity genes hh and wg. In dly embryos, both En expression and wg transcription fade by stage 10, suggesting further that dly is involved in the Hh and/or Wg pathways (Han, 2004).

To further determine whether Dly activity is required for Hh signalling in embryogenesis, Hh signalling activity was examined in dly embryos during mesoderm development. Hh and Wg signalling have distinct roles in patterning embryonic mesoderm. Hh signalling activates the expression of a mesodermal specific gene bagpipe (bap) in the anterior region of each parasegment, whereas Wg signalling inhibits bap expression in the posterior region. bap expression is diminished in the hh mutant, but is expanded to the posterior parasegment in the wg mutant. Consistent with a role of Dly in Hh signalling, it was found that bap expression was strikingly reduced in dly embryos. Together with the segment polarity phenotype, these results strongly argue that Dly is required for Hh signalling during embryogenesis (Han, 2004).

The role of Dly in Hh signalling was further examined during wing development in which Hh and Wg signalling function independently of each other. In the wing disc, Hh signalling induces the expression of its target genes in a narrow stripe of tissue in the A compartment abutting the AP boundary. Hh signalling patterns the central domain of wing blade and controls the positioning of longitudinal veins L3 and L4. The roles of Dly in Hh signalling were examined by analyzing adult wing defects using 'directed mosaic' technique. Surprisingly, no detectable phenotypes were observed in adult wings bearing dly mutant clones. It was reasoned that Hh signalling may be mediated by other HSPGs in the wing. One candidate is the glypican dally that has been shown to be involved in Wg and Dpp signalling. Because available dally alleles used previously were hypomorphic, several dally null alleles were generated by P-element mediated mutagenesis. dally⁸⁰ is a null allele and was used for analysis. However, similar to other dally alleles, homozygous dally⁸⁰animals are viable. The wing bearing dally⁸⁰ clones exhibits a partial loss of the L5 vein with a high penetrance, but no detectable defects in the central domain of wing blade. To determine whether dally and dly have overlapping roles in Hh signalling in wing development, clones mutant for both dally⁸⁰ and dly^A187 (referred as dally-dly hereafter) were generated. Interestingly, the adult wings bearing clones mutant for dally-dly show L3-L4 fusion. This phenotype is typical of loss of Hh function, suggesting that Dally and Dly play redundant roles in Hh signalling in wing development (Han, 2004).

This study demonstrates that Dly is the main HSPG involved in Hh signalling during embryogenesis, at least in epidermis and mesoderm, the two tissues that were carefully examined. Three lines of evidence strongly support this conclusion. (1) Embryos lacking both maternal and zygotic dly activities develop a strong segment polarity defect and exhibit diminished expression of En and Wg. (2) Hh can be detected as punctate particles at least one cell diameter from its producing cells and these punctate particles are absent in dly-null embryos. (3) A reduced expression of bap was observed in dly mutant embryos, a phenotype specifically attributed to the Hh signalling rather than Wg signalling defect. Previously, it was shown that the punctate particles of Hh staining are absent in ttv null embryos. The formation of such Hh staining particles, referred to as large punctate structures (LPS), requires cholesterol modification, and movement of these large punctate structures across cells is dependent on Ttv activity. The current results are consistent with these observations and suggest that Dly is the main HSPG involved in the movement of these LPS across cells. It is conceivable that the punctate particles of Hh staining that were observed may represent Hh-Dly complexes. In this regard, Dly may either prevent secreted Hh from being degraded and/or facilitate Hh movement from its expression cells to adjacent receiving cells. These two mechanisms are not mutually exclusive. In the absence of Dly function, secreted Hh is either degraded or fails to move to the adjacent cells (Han, 2004).

In addition to dly, three other HSPGs, including Dally, Dsyndecan and Trol, are also expressed in various tissues during embryogenesis. In particular, dally is expressed in epidermis and has been shown to be involved in Wg signalling. Removal of Dally activity in embryos either by dally hypomorphic mutants or by RNA interference (RNAi) generates denticle fusions. Further studies demonstrate that the cuticle defect associated with dally embryos by RNAi is weaker than that of dly. The results in this study suggest that Dly plays more profound roles in embryonic patterning than Dally. It remains to be determined whether Dally and other two Drosophila HSPGs are involved in Hh signalling in other developmental processes during embryogenesis (Han, 2004).

Dally and Dly are involved and are redundant in Hh signalling in the wing disc. Consistent with this, the GAG chains of Dally and Dly are shown to be altered in the absence of Ttv activity, suggesting that both Dally and Dly are indeed the substrates for Ttv. Redundant roles of cell membrane proteins have been demonstrated in many other signalling systems. For example, both Frizzled (Fz) and Drosophila Frizzled 2 (Fz2) are redundant receptors for Wg, although Fz2 has relative high affinity in binding to Wg protein. Dly protein is distributed throughout the entire wing disc. Previous studies have demonstrated that dally is highly expressed at the AP border. Interestingly, Dally expression at the AP border is overlapped with the ptc expression domain and is under the control of Hh signalling. It is likely that both Dally and Dly are capable of binding to Hh and facilitating the movement of the Hh protein. In the absence of one of them, another member is probably sufficient to facilitate Hh movement (Han, 2004).

dally-dly double mutant clones have relatively weaker defects in Hh signalling in the wing disc than those of the ttv and sfl mutants. One possible explanation is the perdurance of Dally and Dly proteins. Alternatively, two other HSPGs, Dsyndecan and Trol, may also participate in Hh signalling in the absence of Dally and Dly in the wing disc. These issues remain to be examined using both dsyndecan and trol null mutants (Han, 2004).

Do HSPGs act as co-receptors in Hh signal transduction? Hh is a heparin-binding protein and is likely to interact with HSPGs through their HS GAG chains. In support of this, Dly was shown to colocalize with Hh punctate particles. It is conceivable that Dally and Dly could either transfer Hh to its receptor Ptc or form a Hh-Dally/Dly-Ptc ternary complex in which Dally and Dly may function to facilitate Hh-Ptc interaction or stabilize a Hh-Ptc complex. In this regard, Dally and Dly may function both in transporting Hh protein and acting as co-receptors in Hh signalling. Consistent with this view, a recent report using RNAi in tissue culture based assays identified Dly as a new component of the Hh pathway (Lum, 2003). It was shown that Dly plays a cell-autonomous role upstream or at the level of Ptc in activating the expression of Hh responsive-reporter, suggesting a role of Dly in the delivery of Hh to Ptc (Han, 2004).

It is important to note that some of results obtained from tissue culture based assays (Lum, 2003) are not consistent with in vivo results reported in this study as well as previous studies on Ttv. Cl-8 cells were originally derived from the wing disc. However, it was found that removal of dly activity alone has no detectable effect on Hh signalling in the wing disc. This apparent discrepancy may due to several factors: (1) Hh-N, instead of Hh-Np was used as a source for Hh in their work; (2) Cl-8 cell may have altered the proteoglycan expression pattern, which can be significantly different from Hh-responding wing cells in which Dally expression is upregulated by Hh signalling; (3) it is possible that Dly may have a higher capacity than Dally to bind Hh, as in the case for Wg. In this regard, removal of Dly will probably lead to more profound effects than removal of other HSPGs on binding of Hh-N to the cell surface, perhaps in the delivery of Hh-N to Ptc (Han, 2004).

Within sfl, or ttv or dally-dly mutant clones, the posterior-most cells adjacent to wild-type cells are still capable of transducing Hh signalling. It is most likely that Hh proteins bound by Dally and Dly in wild-type cells can directly interact with Ptc located on the cell surface of the adjacent mutant cells to transduce its signalling. In support of this view, a Hh-CD2 membrane fusion protein has the ability to activate Hh signalling in its adjacent cells. Furthermore, studies on Dispatched (Disp), a novel sterol-sensing domain protein dedicated to the release of cholesterol-modified hedgehog from signaling cells, have shown that the first row of anterior cells adjacent to posterior Hh-producing cells have significant Hh signalling activity in disp mutant wing discx, in which Hh is retained on the cell surface of Hh producing cells. Interestingly, Hh punctate particles were observed in the posterior-most HSPG mutant cells adjacent to wild-type cells. These Hh punctate particles are most likely intracellular Hh proteins internalized through Ptc mediated endocytosis process. In this regard, HSPGs may not be required for Ptc-mediated Hh internalization (Han, 2004).

Recent biochemical studies from vertebrate cells have shown that Shh-Np is secreted from cells and can be readily detected in conditioned culture medium. It was also shown that overexpression of Disp can increase the yield of Hh protein in the culture medium. These experiments suggest that Hh can be directly secreted from Hh expressing cells. Can secreted Hh proteins freely diffuse to Hh receiving cells through extracellular spaces? To address this issue, detailed analyses for Hh signalling have been carried out in the complete absence of HS GAG using sfl and ttv or absence of glypicans using dally-dly. A narrow strip (one cell diameter in width) of sulfateless (sfl) or ttv, or dally-dly mutant cells prevents the transpassing of the Hh signal. Hh staining disappears in sfl mutant clones, except at a residual level in the posterior-most row of cells. Based on these observations, a model is favored in which Hh movement is regulated by a cell-to-cell mechanism rather than by free diffusion (Han, 2004).

The results of this study further suggest that Hh movement is independent of dynamin-mediated endocytosis, which has been shown to be involved in the transportation of morphogen molecules such as Dpp and Wg. A blockage of dynamin function does not eliminate Hh movement and its subsequent signalling; instead, it leads to a striking reduction of punctate particles of Hh staining and an accumulation of cell-surface Hh protein. Expanded Ptc expression domain is observed when dynamin-mediated endocytosis is blocked. These new findings provide compelling evidence that dynamin-mediated endocytosis is not required for Hh movement and its subsequent signalling, but is involved in Ptc-mediated internalization of the Hh protein (Han, 2004).

Several mechanisms have been proposed to explain morphogen transport across a field of cells. These mechanisms include (1) free diffusion, (2) active transport by planar transcytosis, (3) cytonemes, (4) argosomes. The results of this study suggest that Hh moves through a cell-to-cell mechanism rather than free diffusion. Furthermore, dynamin-mediated endocytosis is unlikely to be involved in Hh movement. On the basis of these findings, the following model is proposed by which the HSPGs Dally and Dly may regulate the cell-to-cell movement of the Hh protein across a field of cells. In this model, Hh is released by Disp from its producing cells and is immediately captured by the GAG chains of glypicans on the cell surface. The differential concentration of Hh proteins on the surface of producing cells and receiving cells drives the unidirectional displacement of Hh from one GAG chain to another towards more distant receiving cells. Within the same cell, the transport of Hh may be facilitated by the lateral movement of glypicans on the cell membrane. On the receiving cells, glypicans may present Hh to Ptc, which then mediates the internalization of Hh. Glypican mutant cells can not relay Hh proteins further because they lack HS GAG on the surface. However, they are able to respond to the Hh signal because Ptc may contact the Hh on the membrane of the adjacent wild-type cells. Further studies are needed to determine whether other mechanism(s) including cytonemes and argosomes are also involved in Hh movement (Han, 2004).

Three Drosophila EXT genes shape morphogen gradients through synthesis of heparan sulfate proteoglycans

The signaling molecules Hedgehog (Hh), Decapentaplegic (Dpp) and Wingless (Wg) function as morphogens and organize wing patterning in Drosophila. In the screen for mutations that alter the morphogen activity, novel mutants of two Drosophila genes, sister of tout-velu (sotv) and brother of tout-velu (botv), and new alleles of toutvelu (ttv), were identified. The encoded proteins of these genes belong to an EXT family of proteins that have or are closely related to glycosyltransferase activities required for biosynthesis of heparan sulfate proteoglycans (HSPGs). Mutation in any of these genes impairs biosynthesis of HSPGs in vivo, indicating that, despite their structural similarity, they are not redundant in the HSPG biosynthesis. Protein levels and signaling activities of Hh, Dpp and Wg were reduced in the cells mutant for any of these EXT genes to a various degree, Wg signaling being the least sensitive. Moreover, all three morphogens were accumulated in the front of EXT mutant cells, suggesting that these morphogens require HSPGs to move efficiently. In contrast to previous reports that ttv is involved exclusively in Hh signaling, ttv mutations were also found to affect Dpp and Wg. These data led to the conclusion that each of three EXT genes studied contributes to Hh, Dpp and Wg morphogen signaling. It is proposed that HSPGs facilitate the spreading of morphogens and therefore, function to generate morphogen concentration gradients (Takei, 2004).

In addition to monitoring signaling in EXT mutant cells, antibodies that recognize Hh, Dpp and Wg, and a GFP-tagged version of Dpp were used to analyze whether the levels or distribution of these morphogens had been affected. Levels of each of these proteins were significantly reduced in the mutant, both in the morphogen-expressing region and in the receiving region. For Hh, Dpp and Wg, similar results were observed in cells mutant singly for any of the EXT genes. Single mutation was not tested for the distribution of Dpp-GFP. In the morphogen-expressing region, hh expression was not downregulated, however levels of Hh protein were significantly decreased. This may indicate that Hh protein is destabilized and/or not retained efficiently on the cell surface in the absence of HSPGs. In contrast to hh, expression of the wg and dpp and levels of Wg and Dpp were decreased in the EXT clones. The decrease in dpp expression is easily accountable because Hh signaling is impaired in the absence of HSPGs. In contrast, the decrease in wg expression is not as readily explainable: cut and wg are both targets of Notch signaling, however the protein level of Cut was not altered in EXT clones. This suggests that wg is also regulated by an unknown mechanism dependent on HSPGs (Takei, 2004).

In the morphogen-receiving region, each of these proteins was significantly decreased in the clones of cells mutant for EXT genes, although a little leakage of morphogen molecules was seen even in the clones doubly mutant for ttv and botv. This suggests two possible mechanisms that do not exclude each other: in the absence of HSPGs these three morphogens are (1) destabilized and/or are not retained efficiently on the cell surface, like Hh in morphogen-expressing region, or (2) prevented from diffusing efficiently into the region consisting of EXT mutant cells. Intriguingly, close observation of the distribution of Hh strongly suggested a function for HSPGs in morphogen movement. In the wild-type discs, Hh protein synthesized in the posterior compartment appears to flow into the anterior compartment, with a moderate concentration gradient starting from the middle of the posterior compartment. However, Hh abnormally accumulates in the posterior compartment when the EXT mutant clone is in the anterior compartment along the A/P boundary. This effect is seen both in the ventral region and in the dorsal region. This suggests that Hh fails to move into the mutant cells and as a consequence accumulates in posterior cells instead. Dpp-GFP and Wg accumulation in front of the mutant clones was also apparent, however less pronounced compared with the case of Hh. Therefore it is concluded that the HSPG-dependent diffusion is the common mechanism for the movement of these three morphogens (Takei, 2004).

The glypican Dally-like is required for Hedgehog signalling in the embryonic epidermis of Drosophila

The Drosophila genes dally and dally-like encode glypicans, which are heparan sulphate proteoglycans anchored to the cell membrane by a glycosylphosphatidylinositol link. Genetic studies have implicated Dally and Dally-like in Wingless signalling in embryos and imaginal discs. The signalling properties of these molecules in the embryonic epidermis have been tested. RNA interference silencing of dally-like, but not dally, gives a segment polarity phenotype identical to that of null mutations in wingless or hedgehog. Using heterologous expression in embryos, the Hedgehog and Wingless signalling pathways were uncoupled; Dally-like and Dally, separately or together, were found to be unnecessary for Wingless signalling. Dally-like, however, is strictly necessary for Hedgehog signal transduction. Epistatic experiments show that Dally-like is required for the reception of the Hedgehog signal, upstream or at the level of the Patched receptor (Desbordes, 2003).

Although heparan sulphate modifications have been implicated in several signalling pathways, it remains unclear which proteins are modified by these enzymes, and how the modifications affect a given signalling event. Since most heparan sulphate chains at the cell surface are thought to be carried by proteoglycans of the syndecan or glypican families, this study has examined the function of the two Drosophila homologs of glypicans, dally and dally-like (dlp), in the embryonic epidermis. Unexpectedly, this study has found a restricted and specific role for the fly glypicans. RNAi silencing shows that Dlp is a segment polarity gene that is absolutely required for Hh signalling. This requirement is specific to the Hh pathway; RNAi silencing of dlp does not affect Wg signalling in embryos. In contrast, RNAi silencing of dally, the other homolog of glypicans in Drosophila, does not produce a segment polarity phenotype, suggesting that Dally is dispensable for Wg or Hh signalling in embryos. Furthermore, RNAi silencing of both dally and dlp does not affect Wg signalling, suggesting that they do not function redundantly in this pathway (Desbordes, 2003).

dlp is a bona fide segment polarity gene since dlp RNAi generates embryos that fail to maintain en and wg expression at mid-embryogenesis, and exhibit a full segment polarity phenotype in the cuticle at the end of embryogenesis. The late disappearance of en expression and the single stripe of rho expression in dlp embryos suggest a loss of Hh activity. This is confirmed by the fact that when hh expression is under heterologous control, ectopic wg transcription is lost in dlp RNAi embryos, whether Hh is provided autonomously (armGal4 experiments) or non-autonomously (simGal4 experiments). These experiments demonstrate unambiguously that dlp is required for Hh signalling and rule out a requirement for hh transcription (Desbordes, 2003).

Dlp is a GPI-anchored protein and is likely to be localised at the cell surface. This leaves two plausible roles for Dlp: either it is required for the release of active Hh from the secreting cells, or it is required for the interpretation of the Hh signal on the receiving cells. Several possibilities have been eliminated. First, Dlp is required for the activity of Hh-N, an engineered form of Hh which is pre-processed and unmodified by cholesterol. This suggests that Dlp is necessary downstream of Hh processing and cholesterol modification. Downstream of these events, Hh undergoes another lipid modification, the addition of a palmitoyl moiety. The segment polarity gene rasp codes for an acyltransferase which is thought to be needed for Hh palmitoylation. Thus, Dlp could be required for the function of rasp in the signalling cells. However, whereas palmitolylation is essential for Hh-N activity, a recent report shows that it is not strictly required for the activity of wild-type Hh in Drosophila embryos. This suggests that the cholesterol and palmitoylate modifications might be partially redundant for the activity of wild-type Hh, at least in embryos. Thus, although Dlp could still act at the level of rasp on another function, loss of palmitoylation alone cannot account for the complete loss of Hh signalling seen in dlp RNAi embryos. It seems therefore more likely that dlp functions in the responding cells (Desbordes, 2003).

ptc is epistatic to dlp, indicating that Dlp acts upstream or at the level of the Ptc receptor. One possibility is that Dlp binds Hh and facilitates its interaction with Ptc. Increasing the concentration of Hh in receiving cells in either armGal4/UAShh or armGal4/UAShh-N experiments, does not abolish the requirement for Dlp. This argues against a role of Dlp in merely increasing the concentration of Hh ligand at the cell surface, and suggests a more specific role. Recent evidence supports a model in which, upon Hh binding, Ptc is endocytosed and inactivated by degradation, and this in turn indirectly activates Smoothened and the Hh intracellular pathway. Dlp may localize Hh and Ptc in membrane microdomains required for Ptc endocytosis and subsequent degradation (Desbordes, 2003).

Abrogation of heparan sulfate synthesis in Drosophila disrupts the Wingless, Hedgehog and Decapentaplegic signaling pathways

Studies in Drosophila and vertebrate systems have demonstrated that heparan sulfate proteoglycans (HSPGs) play crucial roles in modulating growth factor signaling. Mutations have been isolated in sister of tout velu (sotv), a gene that encodes a co-polymerase that synthesizes HSPG glycosaminoglycan (GAG) chains. Phenotypic and biochemical analyses reveal that HS levels are dramatically reduced in the absence of Sotv or its partner co-polymerase Tout velu (Ttv), suggesting that both copolymerases are essential for GAG synthesis. Furthermore, mutations in sotv and ttv impair Hh, Wg and Decapentaplegic (Dpp) signaling. This contrasts with previous studies that suggested loss of ttv compromises only Hh signaling. These results may contribute to understanding the biological basis of hereditary multiple exostoses (HME), a disease associated with bone overgrowth that results from mutations in EXT1 and EXT2, the human orthologs of ttv and sotv (Bornemann, 2004).

The involvement of HSPGs in Hh signaling was first demonstrated when Ci stabilization and Ptc expression were shown to be reduced in ttv mutant clones in the wing disc. In clones at the AP boundary, Ci and Ptc levels were maintained in only a single row of mutant cells along the posterior edge of the clone. Thus, it was proposed that cells lacking HSPGs are competent to receive, but are impaired in propagating, the Hh signal. Interestingly, ttv mutations only affect Hh-Np, the mature cholesterol-modified form of the ligand. In ttv mutant embryos, Hh-Np distribution is curtailed, while Hh-N, which lacks cholesterol, is unaffected. Based on these findings, it has been suggested that HSPGs may enhance the targeting of Hh-Np to 'lipid rafts' where the ligand can be then be transported from cell to cell (Bornemann, 2004).

Like ttv clones, sotv and ttv, sotv double mutant clones limit the domain of Ci stabilization, indicating that the range of Hh signaling is impaired (Bellaiche, 1998). This reduction is consistent with a requirement for HSPGs in Hh transport. However, the observations that Hh levels are reduced in posterior compartment clones suggest an alternative (or additional) mechanism by which HSPGs could affect Hh signaling: by altering ligand stability. In wild type, hh is transcribed and expressed uniformly throughout the posterior compartment. Therefore, the weaker staining in posterior clones is unlikely to be due to failure of concentration-dependent transport mechanisms. Moreover, lower Hh levels are not caused by reduced expression, since hh transcription (monitored through expression of a hh-lacZ transgene) is unaffected in sotv mutant clones. By extension, Hh ligand instability in cells lacking HSPGs could also contribute to the reduced effectiveness of signaling in clones along the AP boundary. HSPGs could bind to and stabilize the ligand directly, or alternatively may act indirectly to reduce the activity of extracellular proteases. Consistent with the latter model, heparin is known to promote inhibition of thrombin by acting through the protease inhibitor serpin AT III. Reduced Hh ligand stability would lower the distance over which the growth factor can signal in a manner difficult to distinguish from compromised ligand transport. Although these models presume that loss of HS impacts growth factor signaling by disrupting protein interaction with GAG chains, it is also possible that without GAG synthesis HSPG core proteins are mislocalized or less stable, contributing to the observed phenotypes (Bornemann, 2004).

It has been suggested that disruption of intracellular transport should lead to a diagnostic accumulation of ligand on the side of a clone closest to the morphogen source. Although no ligand accumulation at the boundaries of ttv or sotv clones, consistent with a failure to affect transport, these results are interpreted cautiously. Since Hh stability is compromised in mutant cells, it is possible that the rate of ligand degradation simply exceeds the rate of accumulation. Additionally, it has been argued that ligand accumulation may not be a reliable indicator of a blockage in transport, and instead could reflect increased expression of ligand-binding factors (such as receptors or HSPGs) (Bornemann, 2004).

A recent cell culture-based screen to identify novel components in the Hh pathway demonstrated that RNAi-based degradation of Ttv, Sotv and Botv did not reduce the transcriptional response to exogenously added Hh ligand. These results are consistent with the current findings, since addition of exogenous ligand bypasses any requirement for ligand stabilization or transport. These data underscore the idea that GAG chain synthesis in receiving cells is not essential for transduction of the Hh signal. Loss of Hh in posterior compartment clones lacking ttv or sotv also argues against a current model that implicates HSPGs and the transmembrane protein Dispatched (Disp) in promoting ligand transport. Disp is required to release Hh from the membrane of expressing cells. Thus, disp mutant cells show high levels of Hh accumulation. A Ttv-modified HSPG has been proposed to aid in releasing Hh-Np from Disp and prevent reinsertion of the ligand into the membrane, thereby enhancing its diffusion. However, if a Ttv-modified HSPG were essential for Hh-Np release from Disp, then ttv and sotv mutant clones in the posterior compartment should accumulate high levels of Hh, similar to disp clones. Finally, the current results conflict with the proposal that Ttv could synthesize HSPGs that promote Hh signaling specifically (Bellaiche, 1998; The, 1999), since the Dpp and Wg pathways are also affected by loss of Ttv or Sotv. Wg and Dpp signaling has been examined only in wing discs, and it remains possible that these ligands have differential requirements for HS during embryogenesis (Bornemann, 2004).

Temporal modulation of the Hedgehog morphogen gradient by a patched-dependent targeting to lysosomal compartment

The morphogenetic gradient of Hh is tightly regulated for correct patterning in Drosophila and vertebrates. The Patched (Ptc) receptor is required for restricting Hh long-range activity in the imaginal discs. In this study, the different types of Hh accretion that can be observed in the Drosophila embryonic epithelial cells were investigated. In receiving cells, large apical punctate structures of Hh (Hh-LPSs) are not depending on the Ptc receptor-dependent internalization of Hh but rather reflect Hh gradient. By analyzing the dynamic of the Hh-LPS gradient formation, it was demonstrated that Hh distribution is strongly restricted during late embryonic stages compared to earlier stages. The up-regulation of Ptc is required for the temporal regulation of the Hh gradient. Dynamin-dependent internalization of Hh does not regulate Hh spreading but is involved in shaping Hh gradient. Hh gradient modulation is directly related to the dynamic expression of the ventral Hh target gene serrate (ser) and with the Hh-dependent dorsal cell fate determination. Finally, this study shows that, in vivo, the Hh/Ptc complex is internalized in the Rab7-enriched lysosomal compartment in a Ptc-dependent manner without the co-receptor Smoothened (Smo). It is proposed that controlled degradation is an active mechanism important for Hh gradient formation (Gallet, 2005).

To distinguish the several types of Hh accretion that can be observed in the ectodermal cells, different markers were used. In the secretory cells [engrailed (en) expressing cells], cytosolic punctate staining of Hh colocalizes with a secreted Green Fluorescent Protein at least initially in the cell secretory machinery. Hh accretions are also distributed very apically in expressing cells. Indeed, their position is more apical to Crumbs, which is localized in the subapical complex close to the top of the cells. These accretions are referred to as Hh-LPSs. Hh-LPS formation depends on Dispatched (Disp) activity and on the presence of the cholesterol modification on Hh and that Hh-LPS movement depends on specific proteoglycans at the cell surface. Similar apical localization of Hh-LPS is also observed in the receiving cells. Additionally, more basal cytosolic Hh accretions are observed in receiving cells and are frequently associated with Ptc. In ptc mutant embryos, these accretions disappear, while apical accumulation of Hh-LPS is observed. This suggests that large cytoplasmic Hh accretions reflect internalization of the Hh/Ptc complex after ligand-receptor binding. It also suggests that the presence of Hh-LPS is not consequent to Ptc binding or to internalization with Ptc. Also, one has to notice that small cytosolic Hh staining is still visible in the absence of Ptc in receiving cells (Gallet, 2005).

Altogether, the data show that it is possible to distinguish three different types of Hh accretions: (1) apical Hh-LPSs representing the state of Hh before internalization; (2) more basal cytosolic accretions that are strictly depending on the presence of Ptc and represent internalization of the ligand/receptor complex; (3) Ptc-independent cytosolic Hh particles that might correspond to internalization of Hh independently of its known receptor. It is proposed that LPSs reflect Hh functional gradient since their range correlates with the expression of target genes and because their assembly and movement depends, respectively, on two genes, disp and tout velu, necessary for Hh activity (Gallet, 2005).

During Drosophila embryonic development, hh is activated by the selector gene en in two rows of cells in the posterior compartment of each segment. In the embryonic ectoderm, Hh is thought to elicit pattern formation through short-range activity by inducing two different signal relays anterior and posterior to its source of expression. In addition, Hh movement encompasses more than one or two cells from the source of Hh. In order to study Hh gradient, the distribution of the apical Hh-LPS was analyzed. Secreted Hh-LPSs are easily detectable at a distance from its source and cover apically the entire segmental field during stages 9 and 10 of embryogenesis. Interestingly, it was observed that although the level of hh mRNA expression is similar in stages 10 and 11, the Hh gradient appears to be spatially restricted from stage 11 onward, Hh-LPSs being more restricted to the vicinity of the secreting cells. This observation was confirmed with a plot analysis of Hh-LPS apical distribution within the segment. At stage 11 and later, the slope of the Hh-LPS gradient is steeper compared to earlier stages suggesting that Hh movement becomes restricted. Alternatively, this could be due to a decrease in Hh protein stability. Moreover, it is not thought that the restriction of Hh distribution observed at stage 11 could be consequent to a decrease in the rate of Hh-LPS formation nor to a different apicobasal localization of Hh-LPS at this stage. Indeed, Hh-LPSs in the secreting cells at stages 10 and 11 seem similar in their density and subcellular localization (Gallet, 2005).

To confirm that Ptc is involved in the temporal restriction of Hh movement, Hh distribution was analyzed in ptc mutant embryos and in embryos expressing ptc in en/hh cells. In ptc null embryos, the Hh gradient is impaired and Hh-LPSs distribution was found to be extended throughout the entire segment without restriction. In such embryos, ser expression is totally repressed in a manner similar to that seen under ubiquitous Hh expression. Note that the ectopic hh expressing source present in ptc mutant might also contribute to the broad distribution of Hh. When Ptc is expressed in en/hh expressing cells, the range of Hh-LPSs movement is limited to the vicinity of the producing cells. This effect is not due to a diminution of hh expression since it has been shown that Ptc does not affect hh transcription. Hence, Ptc might directly affect Hh-LPS range of action. Indeed, the slope of the Hh-LPSs gradient decreased sharply compared to wild-type stage 11 embryos. Interestingly, in these embryos, ser expression was extended correlating with the absence of Hh-LPS away from the source (Gallet, 2005).

Temporal regulation of Hh gradient is necessary because signaling requirements for Hh change in a time-dependent manner. One can suggest that, during early development, Hh acts at long range due to moderate levels of Ptc. Hh would easily overcome repression by the low concentrations of Ptc protein to prime a subset of ectodermal cells at a long distance to make them competent to respond to other signals. At later stages, Hh distribution is restricted and allows expression of ser and acts over a short range to induce rhomboid, both genes being necessary for ventral denticle specification (Gallet, 2005).

It appears that temporal regulation of Hh movement is an evolutionarily conserved mechanism since similar observations to these have been reported for vertebrate development. Altogether these data suggest that endocytosis of Hh and Ptc is needed to restrict Hh distribution in the ectodermal field and to shape Hh gradient. It is hypothesized that regulation of Ptc turn-over at the plasma membrane is important in this process (Gallet, 2005).

From these data, several observations were made that are worthy of mention. (1) It is interesting to note that even in the absence of Ptc, some Hh staining is still detected in receiving cells, suggesting that some Hh internalization still occurs in the absence of Ptc. These and the few percentage of Ptc/Hh colocalizations suggest that another Hh receptor might be involved in Hh internalization. (2) The presence of Ptc was observed in Rab7 vesicles without Hh. These Ptc-Rab7 vesicles were present in cells located close or away from Hh source, suggesting that Ptc might be internalized and targeted for degradation independently of Hh. Accordingly, analysis of the subcellular localization of Ptc showed that Ptc was mostly present in internal punctate structures, suggesting that turnover of Ptc at the plasma membrane is very high (Gallet, 2005).

Taken together these results suggest that after Hh binding to Ptc, the Hh/Ptc complexes are targeted to a lysosomal compartment. Moreover Ptc appears to be constitutively internalized and degraded independently of its binding to Hh. Likewise, in vertebrate cultured cell, Ptc is constitutively transported to lysosomes and degraded. In conclusion, the data suggest that, as in the case of Wingless gradient, controlled degradation is an active mechanism for Hh gradient formation and Ptc plays a major role in this process (Gallet, 2005).

It has been reported that in Hh receiving cells, the co-receptor Smo is stabilized at the plasma membrane while, in the absence of Hh, Ptc exerts its repressive activity on Smo by destabilizing it. However, no clear mechanism has been demonstrated for Smo destabilization. The possibility that Ptc might target Smo to lysosomal compartments was explored. Thus, the possible presence of Smo in Rab7-containing vesicles was analyzed. Very little colocalization was found between Rab7 and Smo in cells close to hh source, or further away. Thus, these data suggest that the Hh/Ptc-dependent regulation of Smo stability does not principally involve a lysosomal targeting mechanism (Gallet, 2005).

Cholesterol modification is necessary for controlled planar long-range activity of Hedgehog in Drosophila epithelia

The Hedgehog morphogen is a major developmental regulator that acts at short and long range to direct cell fate decisions in invertebrate and vertebrate tissues. Hedgehog is the only known metazoan protein to possess a covalently linked cholesterol moiety. Although the role of the cholesterol group of Hedgehog remains unclear, it has been suggested to be dispensable for the its long-range activity in Drosophila. This study provides data in three different epithelia -- ventral and dorsal embryonic ectoderm, and larval imaginal disc tissue -- showing that cholesterol modification is in fact necessary for the controlled long-range activity of Drosophila Hedgehog. An explanation is provided for the discrepancy between the current results and previous reports by showing that unmodified Hh can act at long range, albeit in an uncontrolled manner, only when expressed in squamous cells. These data show that cholesterol modification controls long-range Hh activity at multiple levels. Initially, cholesterol increases the affinity of Hh for the plasma membrane, and consequently enhances its apparent intrinsic activity, both in vitro and in vivo. In addition, multimerisation of active Hh requires the presence of cholesterol. These multimers are correlated with the assembly of Hh into apically located, large punctate structures present in active Hh gradients in vivo. By comparing the activity of cholesterol-modified Hh in columnar epithelial cells and peripodial squamous cells, this study shows that epithelial cells provide the machinery necessary for the controlled planar movement of Hh, thereby preventing the unrestricted spreading of the protein within the three-dimensional space of the epithelium. It is concluded that, as in vertebrates, cholesterol modification is essential for controlled long-range Hh signalling in Drosophila (Gallet, 2006).

A particularly intriguing issue that is likely to be key to understanding the developmental function of Hh is the role that its lipophilic anchors play in controlling its long-range activity through tissues. Hh is synthesised as a precursor that, following autocleavage yields an N-terminal signalling secreted peptide that is covalently linked to a cholesterol molecule at its C terminus. This signalling Hh peptide (termed Hh-Np, with 'p' standing for 'processed') is further modified by palmitoylation on the first cysteine, both on Drosophila Hh and on its vertebrate orthologue sonic hedgehog (Shh). Hh is the only known metazoan protein with a covalently linked cholesterol moiety. Earlier studies have shown that the cholesterol group plays a role in the membrane retention of the protein (Gallet, 2006).

The role of cholesterol in the control of Hh long-range activity appears to be different in Drosophila and in vertebrates. In flies, a Hh peptide that is devoid of cholesterol (named Hh-N) has been described as being able to fulfill all Hh-Np functions. Moreover, it can induce the expansion of Hh-responsive cells and bypass the requirement for Disp function in its secretion and long-range activity through the Drosophila wing imaginal disc. By contrast, in vertebrates, the non-cholesterol-modified Hh form (Sonic Hh-N or Shh-N) is unable to act at a distance from its source and is thus unable to rescue Shh-dependent long-range activity in the limb bud (Gallet, 2006).

It is unclear how Hh-Np, which is membrane-tethered through its cholesterol adduct, can reach distant cells. One possible answer may involve large punctate structures (LPSs), which are formed by Hh-Np, but not Hh-N, in Drosophila. Because their formation and movement require Disp and Ttv activity respectively, it has been proposed that LPSs provide a vehicle for Hh long-range activity. Interestingly, in cultured cells, Shh-Np multimerises, but Shh-N does not, suggesting that Hh-LPSs might also depend on Hh-Np multimerisation in flies. Multimerisation of Shh-Np has been proposed to be required for its solubility and long range activity. Alternative mechanisms are currently being explored and several types of vehicles for Hh/Shh have been proposed. For example, a few percent of total Drosophila Hh have been identified in lipoprotein particles that might resemble vertebrate low-density lipoprotein or LDL. It has also been shown that Shh, on the surface of mouse ventral node, is packed in membrane vesicles called nodal-vesicular-parcels (Gallet, 2006).

How can the contradictory data in Drosophila and vertebrates concerning the requirement of cholesterol for the long-range activity of Hedgehog be reconciled? In three different assays this study clearly demonstrates the existence of Hh-Np long-range activity in the ventral and dorsal embryonic ectoderm and in the imaginal discs of Drosophila. These assays provide evidences that, in the absence of cholesterol modification, Hh is devoid of controlled long-range activity in embryonic or in imaginal disc cells. Furthermore, whereas Hh-Np dimerises and forms multimers, Hh behaves as a monomer in the absence of its cholesterol moiety. Fractionated Hh-Np multimers aggregate at the plasma membrane similar to Hh-LPSs and have full Hh activity. It is also demonstrated that the lack of cholesterol affects both the intrinsic activity of Hh and the routing of its secretion. By expressing cholesterol-modified Hh in columnar epithelial cells or in peripodial squamous cells, it is shown that epithelial cells provide the machinery necessary for the controlled planar movement of Hh. Based on these data, it is proposed that the cholesterol adduct is necessary to prevent the unrestricted spreading of Hh within the three-dimensional space of the epithelium (Gallet, 2006).

Hedgehog lipid modifications are required for Hedgehog stabilization in the extracellular matrix

The Hedgehog (Hh) family of morphogenetic proteins has important instructional roles in metazoan development. Despite Hh being modified by Ct-cholesterol and Nt-palmitate adducts, Hh migrates far from its site of synthesis and programs cellular outcomes, depending on its local concentrations. In the receiving cells of the Drosophila wing imaginal disc, lipid-unmodified Hh spreads across many more cell diameters than the wild type and this spreading leads to the activation of low but not high threshold responses. Unlipidated Hh forms become internalized through the apical plasma membrane, while wild-type Hh enters through the basolateral cell surface -- in all cases via a dynamin-dependent mechanism. Full activation of the Hh pathway and the spread of Hh throughout the extracellular matrix depend on the ability of lipid-modified Hh to interact with heparan sulfate proteoglycans (HSPG). However, neither Hh-lipid modifications nor HSPG function are required to activate the targets that respond to low levels of Hh. All these data show that the interaction of lipid-modified Hh with HSPG is important both for precise Hh spreading through the epithelium surface and for correct Hh reception (Callejo, 2006).

One of the first observations made when expressing the lipid-unmodified forms of Hh is an extended gradient compared with that elicited by wild-type Hh. It is known that Hh gradient formation depends on the presence of its receptor Ptc, which is responsible for the internalization and degradation of Hh. Thus, the internalization and degradation of lipid-unmodified forms of Hh were studied, in the search for an explanation for the vast expansion of the gradient. These mutant forms of Hh are efficiently internalized and degraded throughout the disc. So, why is there an extended gradient? One clue emerged from an analysis of Hh internalization in shi mutant cells. It was found that the lipid-unmodified forms of Hh are internalized through the apical side of the epithelium, while wild-type Hh is internalized mainly through the basolateral surface. This differential internalization matches the preferential localization of lipid-unmodified Hh at the apical surface plasma membrane of A compartment cells. It was also found that the lipid-unmodified forms of Hh can be internalized and degraded through a Ptc-independent mechanism. Thus, it is possible that this Ptc-independent mechanism would have no positive feedback from Hh, and would therefore not work as efficiently as when internalization was mediated by Ptc or by just a lower rate of internalization and degradation of Ptc. Alternatively or in addition, a reduced restriction of spreading through the extracellular matrix could explain why the gradient is extended. In agreement with this, the localization of lipid-unmodified Hh is not affected in mutants for HSPGs. Furthermore, Hh is less represented at the basolateral membrane in the absence of HSPGs, indicating an active role of HSPGs in anchoring lipid-modified Hh in the lateral cell region. This conclusion is further supported by the localization of the glypican Dally-like at the basolateral membrane of wing imaginal disc cells (Callejo, 2006).

If the interaction of dual lipid modified Hh with HSPGs is important for Hh retention in the extracellular matrix, then one would expect to see the same phenotype if the lipids were removed from Hh as ttv mutants, i.e. the extended movement of Hh. This possibility could only be demonstrated using a whole disc mutant for ttv, which is difficult to obtain because ttv mutants are larval lethal. A further possibility would be to use a hypomorphic mutation of ttv, which is not available for Drosophila. In mice, in which an Ext1 hypomorphic mutant already exists, it has been demonstrated that this is indeed the case and Ihh signaling during embryonic chondrocyte differentiation shows an extended range (Callejo, 2006).

In the formation of the Hh morphogenetic gradient throughout the extracellular matrix, not only the function of HSPGs, rendering a restricted space suitable for the spreading of Hh, has to be considered but also a possible role of HSPGs in signal reception. Thus, if a specific HSPG acts as a co-receptor for Hh together with Ptc, binding the ligand to the HSPG and to the receptor, it could limit the range of ligand movement and, at the same time, boost the signaling process. In effect, compelling evidence in the fly embryo and in fly tissue culture cells indicates that the presentation of Hh to Ptc might require a specific HSPG such as Dally-like. In this sense, HSPG function is also required for Hh reception in the imaginal disc, but only to trigger Hh high-threshold response genes. Thus, it is plausible that Hh coupled to a specific HSPG could form a high-affinity complex together with Ptc to allow these high-threshold responses. However, in the wing imaginal disc, Hh lacking cholesterol is able to induce the same low responses as fully lipid-modified Hh, in both cases in an HSPG-independent manner. This suggests that the low-level response of the pathway might involve a different reception complex in which HSPGs are not necessary (Callejo, 2006).

Two different mechanisms of Hh target gene activation have been also proposed in the ectoderm of Drosophila embryos. These mechanisms correlate with asymmetric cellular responses to Hh signaling. Hh requires the cholesterol modification for a maximal response of the pathway, i.e. for the activation of Wingless (Wg) and Ptc in anteriorly located cells (compartment border), but cholesterol is dispensable for the activation of Rhomboid (Rho) and Ptc in posteriorly located cells (segmental border). Moreover, ttv function is needed only for activating target genes in anterior cells (Wg) and not for target genes posterior to the Hh source (Rho). This result was interpreted as a requirement for Hh movement towards anteriorly located cells to activate Wg. Based on the requirement of ttv for Hh high threshold responses in wing imaginal disc cells, it is anticipated that ttv is required in the embryonic epidermis for the reception of Hh in the activation of specific Hh target genes but not others. This differential activation of Hh targets in the embryonic ventral epidermis and wing imaginal disc cells is known to be dependent on fused (fu) activity. Hence, it is possible that this differential activation of the Hh pathway could be exerted through different structures of the Hh receptor complex. Thus, Ptc plus a glypican, possibly Dlp, could act as a high-affinity receptor whose activation would then be mediated by Fu kinase. Ptc without Dlp could act as a low-affinity receptor whose activation was independent of Fu (Callejo, 2006).

Also in the embryonic epidermis, it has been proposed that apically distributed Hh LPSs are needed to activate Wg in anterior cells. However, in the wing disc, all forms of Hh produced punctate structures, both apical and basolateral, which are also observed using different Hh antibodies. These structures are endocytic vesicles and are the result of the accumulation of Hh in the endocytic compartment rather than the visualization of multimeric lipid-modified Hh complexes moving from one cell to another. The high accretion of Hh observed in the deep orange dor^- clones indicates that these large endocytic vesicles are targeted to the degradation pathway (Callejo, 2006). Dor functions in the fusion of Golgi-derived vesicles with large Rab7-positive endocytic compartments (Sriram, 2003)

Hh lipidation seems to confer a specific conformation to the Hh molecule so that it is targeted to specific locations in the receiving cells for signaling. Hence, the Ptc receptor might be located in sterol-rich membrane microdomains or lipid rafts in Drosophila, which function as platforms for intracellular sorting and signal transduction. Hh without lipids might not recognize these platforms, resulting in less efficient signaling. Interestingly, it was observed that the internalization of the lipid-unmodified Hh was not mediated by Ptc; this was more striking in the case of Hhc85s (lacking the palmitate modification). HhN (lacking the cholesterol moiety) has less potency to activate the Hh pathway than wild-type Hh. Hhc85s is much less potent than HhN and Hhc85sN is even less potent. This low response in terms of target activation of unlipidated forms of Hh could be explained by their diminished access to Ptc (Callejo, 2006).

The literature contains several contradictory conclusions regarding the signaling functions of Hh lipids in the mouse and Drosophila. Thus, the elimination of cholesterol has been reported to have a major effect on Hh signaling in vertebrates but only minor effects in Drosophila. However, the diffusion of Drosophila HhN and Hhc85s differs from that of their vertebrate counterparts. In vertebrates, forms of Shh lacking cholesterol or palmitic acid show restricted signaling and diffusion, while in Drosophila, Hh without lipids is able to diffuse longer distances. It is likely that the different structural characteristics of the target tissues where Hh acts could account for differences in the lipid requirements of signaling and diffusion between Drosophila and vertebrates. The wing imaginal disc consists of a single-layered sac of polarized epithelial cells with their apical surfaces oriented towards the disc lumen. If the extracellular matrix were not able to retain lipid-unmodified Hh, this molecule would be delivered to the disc lumen. The peculiar structure of the wing disc concentrates the lipid-unmodified forms of Hh in the lumen and this is likely to promote the activation of low-threshold target genes in cells far away from the Hh-producing cells. A role for the luminal transmission of ligands has already been described for Dpp signaling in Drosophila wing disc. By contrast, only lipid-unmodified Hh can travel from the peripodial membrane to the disc proper cells. These results indicate that wild-type Hh is not traveling from the peripodial membrane towards the wing disc. In other systems, Hh without lipids might be lost because it is not retained and stabilised by the extracellular matrix; therefore, it would be expected that very restricted diffusion and signaling of non lipid-modified forms of Hh occurs. The current results indicate that the role of lipids in Hh signaling is similar in Drosophila and vertebrates, and corroborate the previous notion that lipid-modified forms of Hh are predominantly membrane associated and that Hh mutated forms lacking lipid adducts dissociate from cells after secretion. In summary, it is concluded that dual lipid modification, by cholesterol and palmitic acid, appears to be crucial for interaction between Hh and HSPGs, as well as the Ptc receptor, and that these interactions are important both for a precise Hh spreading through the epithelium surface and for proper Hh reception (Callejo, 2006).

The ihog cell-surface proteins bind Hedgehog and mediate pathway activation

The ihog gene (interference hedgehog), identified by RNA interference in Drosophila cultured cells, encodes a type 1 membrane protein shown in this study to bind and to mediate response to the active Hedgehog (Hh) protein signal. ihog mutations produce defects characteristic of Hh signaling loss in embryos and imaginal discs, and epistasis analysis places ihog action at or upstream of the negatively acting receptor component, Patched (Ptc). The first of two extracellular fibronectin type III (FNIII) domains of the Ihog protein mediates a specific interaction with Hh protein in vitro, but the second FNIII domain is additionally required for in vivo signaling activity and for Ihog-enhanced binding of Hh protein to cells coexpressing Ptc. Other members of the Ihog family, including Drosophila Boi and mammalian CDO and BOC, also interact with Hh ligands via a specific FNIII domain, thus identifying an evolutionarily conserved family of membrane proteins that function in Hh signal response (Yao, 2006; full text of article).

Dispatched mediates Hedgehog basolateral release to form the long-range morphogenetic gradient in the Drosophila wing disk epithelium

Hedgehog (Hh) moves from the producing cells to regulate the growth and development of distant cells in a variety of tissues. This study has investigated the mechanism of Hh release from the producing cells to form a morphogenetic gradient in the Drosophila wing imaginal disk epithelium. Hh reaches both apical and basolateral plasma membranes, but the apical Hh is subsequently internalized in the producing cells and routed to the basolateral surface, where Hh is released to form a long-range gradient. Functional analysis of the 12-transmembrane protein Dispatched, the glypican Dally-like (Dlp) protein, and the Ig-like and FNNIII domains of protein Interference Hh (Ihog) revealed that Dispatched could be involved in the regulation of vesicular trafficking necessary for basolateral release of Hh, Dlp, and Ihog. It was also shown that Dlp is needed in Hh-producing cells to allow for Hh release and that Ihog, which has been previously described as an Hh coreceptor, anchors Hh to the basolateral part of the disk epithelium (Callejo, 2011).

By using shi^ts1 mutant disks, it is possible to freeze Hh internalization and visualize on which side of the anterior compartment (A) wing disk epithelium, apical or basal, Hh gradient is being formed. Thus, an extended basolateral accumulation of Hh was observed in receiving cells, whereas, at the apical plane, Hh accumulation was only evident in the first row of A cells, indicating that the long-range Hh gradient is formed mainly basolaterally. Accordingly, Ptc accumulated in shi^ts1 disks equally apically and basolaterally but mainly colocalized with Hh at the basolateral sections, suggesting a specific mechanism to deliver Hh to Ptc in this surface of the disk epithelium. To analyze the mechanism of Hh release in the P cells, an apical accumulation of Hh was noticed in the producing cells in shi^ts1 mutant disks that was also observed in P shi^ts1 clones and in Rab5^DN ectopic clones, suggesting that the apically secreted Hh is also internalized in P cells and probably is recycling to other membranes. Accordingly, when blocking recycling endosomes (using the dominant-negative form of Rab8 or Rab4), Hh accumulation could be detected in producing cells. This Hh recycling in P cells is probably necessary to form a proper Hh gradient in the receiving cells. In agreement with the above, Hh signaling is compromised when endocytosis is blocked by expressing either Rab5^DN or Shi^DN in the P compartment. Interestingly, and in agreement with these results, Ayers (2011) suggested that apical Hh internalization in the Hh-producing cells is necessary to process or route Hh to activate the responses that require high levels of Hh. However, in contrast to the interpretation provided in this study, Ayers also proposed that the apical Hh pool is responsible for the long-range Hh gradient formation. It is not necessary to envision two Hh gradients, an apical long-range Hh and another basolateral short-range Hh, when all responses can be produced by means of a single gradient. Because only the recycled Hh is capable of activating the high-threshold Hh targets, there is no reason to believe that this processed Hh would not be efficient enough to induce the low-threshold targets basolaterally (Callejo, 2011).

Also in contrast to a previous report proposing a function for Disp in regulating the apical secretion of Hh in Drosophila epithelia, this study demonstrates that Disp is required for the basolateral release of Hh in the wing imaginal disk epithelium. The subcellular localization of Disp, and its function in the basolateral release of Hh, is in agreement with a recent report in vertebrates (Etheridge, 2010). The cellular phenotype of the loss of Disp function, such as the increase in the amount of Hh found in endocytic vesicles, which are supernumerary and disorganized, can be interpreted as a failure in Hh trafficking that subsequently affects its proper release. In this sorting process, Hh would interact with Disp either in the recycling endosome or in MVBs. Disp, a member of the RND family of proton-driven transporters, is likely to function only in compartments where a transmembrane proton gradient exists, such as in early and late endosomes, trans-Golgi, and MVBs. In support of this view, this study showed by confocal and EM studies that Disp protein is located not only at the basolateral plasma membrane but in vesicles and MVBs, where it colocalizes with Hh. Based on the disp^-/- phenotypes and the localization of Disp protein in MVBs, it is proposed that Disp might have a function in redirecting the apically internalized Hh toward the basal domain. Interestingly, and in agreement with the above, a form of Disp, mutant for the proton-driven transporter function (Disp^AAA), does not localize at the basolateral plasma membrane but in supernumerary cytoplasmic vesicles that do not colocalize with Hh puncta, implying that Disp^AAA may not participate in Hh vesicular trafficking to the basolateral plasma membrane (Callejo, 2011).

In noticeable contrast but also supporting the above, after freezing Hh internalization in shi^ts1 mutant disks, Hh accumulates apically in the first row of A compartment cells, suggesting that paracrine signaling could also occur through the apical plasma membrane. Interestingly, in P mutant cells for Dlp, Hh is able to signal to the abutting A cells but long-range signaling does not occur. A suggestive possibility is that the capacity for apical signaling is not affected in these mutant conditions; however, for long-range signaling to occur, a basolateral release implicating the coordinated actions of Dlp and Ihog together with Disp would be required. During Hh sorting in the producing cells, Disp may interact with Dlp and Ihog; in fact, the interaction of Disp with Dlp and Ihog may be important for the apical-to-basal transcytosis of these proteins, because the ectopic expression of Disp but not of mutant Disp^AAA increases Dlp and Ihog levels at the basolateral membranes. As in disp^-/- cells, dlp^-/- cells in the P compartment showed an accumulation of Hh at both the apical and basolateral plasma membranes, suggesting that Dlp might cooperate with Disp during Hh release. In agreement with the proposed mechanism, transcytosis of Dlp has previously been suggested to be important for Wingless (Wg) release and spreadin (Callejo, 2011).

Although the data cannot support that the total amount of synthesized Hh has to undergo this apical-to-basal transcytosis, an intriguing question is why Hh is placed and internalized apically and then shuttled to the basolateral part of the cell. One possibility is that the newly synthesized Hh protein, because of its unusual modifications with cholesterol and palmitate, uses the apical surface, which is enriched in cholesterol and glycosphingolipids, for primarily plasma membrane localization. Alternatively, it is also possible that the apical internalization of Hh allows its interaction with Disp, glypicans, and Ihog. Therefore, Hh that reaches the apical plasma membrane needs to be internalized to recycle to the basolateral plasma membrane, where the machinery for secretion and gradient formation is found. As has already been discussed, transcytosis of Dlp and Ihog together with Hh from the apical membrane to the basolateral membrane may also occur. Cholesterol and triglycerides also undergo apical-to-basolateral transcytosis across intestinal epithelial barriers to reach the blood. Cholesterol and palmitic acid modifications could attribute lipid-like properties to the Hh protein, such as the ability to be anchored to the plasma membrane, and could thus affect Hh intracellular trafficking. In agreement with the above, it has been described that lipid-unmodified Hh in the wing disk epithelium is not able to form a proper Hh gradien. As previously reported, it was observed that Hh mutant forms that lack lipid modifications are released and do not accumulate in disp^-/- clones. Interestingly, this study shows that lipid-unmodified Hh does not colocalize with Ihog-labeled basal cell extensions, indicating that lipid modifications are necessary to interact with Disp, Dlp, and Ihog, and therefore for proper Hh trafficking from the apical to basolateral plasma membrane regulated by Disp function. Reinforcing these findings, it has been reported that disp and ttv functions are not required for either release or transport of lipid-unmodified Hh, strongly suggesting that Disp and glypicans are needed for the appropriate basolateral release of Hh (Callejo, 2011).

This work shows that Hh has a more complicated mechanism for release than has been previously anticipated. The finding of a basolateral route for Hh release and gradient formation will help to understand Hh interaction with different Hh pathway components, such as Disp, Dally, Dlp, Ihog, Boi, and Ptc, during the process of Hh gradient formation. Related to this issue, it is quite intriguing to find Disp, Dlp, Ihog, and Hh decorating long basal cellular extensions in disk cells expressing Ihog ectopically. Some of the long filaments labeled with IhogYFP extend up to several cell diameters and are reminiscent of the 'cytonemes', with a function in the transport of morphogens. In contrast to the previously described apical cytonemes, the extensions this study visualize are mainly found at the basal part of the disk epithelium. Interestingly, in the context of Notch signaling, basal actin-based filopodia are important for lateral inhibition between nonneighboring cells. However, further investigation will be necessary to demonstrate the implication of cytonemes in Hh gradient formation (Callejo, 2011).

Genetic and biochemical definition of the Hedgehog receptor

Although the transporter-like protein Patched (Ptc) is genetically implicated in reception of the extracellular Hedgehog (Hh) protein signal, a clear definition of the Hh receptor is complicated by the existence of additional Hh-binding proteins and, in Drosophila, by the lack of physical evidence for direct binding of Hh to Ptc. This study shows that activity of Ihog (Interference hedgehog), or of its close relative Boi (Brother of Ihog), is absolutely required for Hh biological response and for sequestration of the Hh protein to limit long-range signaling. This study shows that Ihog interacts directly with Ptc, is required for presentation of Ptc on the cell surface, and that Ihog and Ptc are both required for high-affinity Hh binding. On the basis of their joint roles in ligand binding, signal transduction, and receptor trafficking, it is concluded that Ihog and Ptc together constitute the Drosophila Hh receptor (Zheng, 2010).

Using the targeted alleles of ihog and boi developed in this study, evidence is provided that Ihog proteins are an essential component required for all biological responses to the Hh signal, including target gene induction and patterning in the embryonic segment and in the wing imaginal disc. The central role of Ihog proteins in Hh response was not noted previously because of the functionally overlapping expression of Ihog and Boi in embryos and imaginal discs, which complicates genetic screens and analysis and accounts for the observation that neither the ihog nor boi targeted alleles are lethal in homozygous form. The cl-8 cells used in the genome-scale RNAi screen, in contrast, do not express Boi, and this exposed a critical role for Ihog and facilitated initial discovery of this essential component. In addition to functional overlap, analysis of these functions has been complicated by the required removal of all maternal function for fully penetrant expression of embryonic phenotypes, although maternal expression is neither necessary nor sufficient for Hh response (Zheng, 2010).

The interaction of Ihog Fn2 (the second FNIII domain) with Ptc is essential for presentation of wild-type Ptc on the cell surface. It is not possible, at present, to distinguish between the possibilities that Ihog-mediated surface presentation of Ptc is due to an increased rate of transport to the surface or to an increased duration of residence on the surface. Whatever the mechanism, Fn2 can interact with Ptc in vitro and promote surface presentation of Ptc in cells, even in the absence of the first FNIII domain (Fn1). Similarly, Fn1 alone can interact with the cleavage and cholesterol modified Hh protein HhN in vitro, and Fn1 and Fn2 thus have demonstrably independent functions. Neither domain alone, however, suffices for formation of a high-affinity complex, and the presence of both domains is required for Hh signal reception and transduction and participation in signaling in vivo (Zheng, 2010).

In addition to surface presentation of Ptcour evidence indicates that Ihog proteins also play a direct role in binding the Hh ligand in a multimolecular receptor complex that is critical for transduction. It was thus found that Hh ligand is bound to the surface of cultured cells expressing a variant of Ptc (Ptc1130) with increased localization on the surface. It was also found, with the use of quantitative assays, that endogenous Ihog expressed in these cultured cells contributes critically to binding, and that additional Ihog expression can dramatically enhance binding. In addition, expression of Ptc1130 in the wing imaginal disc clearly produces visible accumulation of the Hh protein on what appears to be the surface of anterior cells at the compartment boundary; this accumulation depends critically on the expression of Ihog/Boi (Zheng, 2010).

Consistent with the role of Ihog in binding, a striking contribution was noted of Ihog to binding in membrane vesicle preparations when present in combination with Ptc. In addition, purified, immobilized HhN and detergent-solubilized extracts containing Ptc and Ihog could be used to demonstrate Ihog-dependent, enhanced precipitation of Ptc. In these biochemical experiments, it was observed that immobilized HhN fails to precipitate detergent-solubilized Ptc alone, but does so in the presence of detergent-solubilized Ihog, and that Ihog alone precipitates Ptc much less efficiently than when HhN is present. This enhancement of Ptc precipitation was dependent on the presence of both the HhN-binding Fn1 domain and the Ptc-binding Fn2 domain of Ihog, consistent with the formation of a multimolecular complex involving HhN, Ptc, and Ihog. Similar results were noted for (Zheng, 2010).

It is interesting to note that little interaction between HhN and Ptc was observed in the absence of Ihog. Formally, it is possible that the interaction of Ptc with HhN is indirect and mediated through enhanced Ihog interaction due to Ptc-induced multimerization or allosteric effects on Ihog. This is thought to be unlikely, however, because Ihog is capable of dimerization in the absence of Ptc, and because the HhN-interacting surface of Ihog is located on the Fn1 domain, which folds independently and is quite distinct from the Ptc-interacting Fn2 domain, thus making allostery unlikely. Thus the interpretation is favored that favorable energetic contributions in the multimolecular receptor/ligand complex derive from Ptc-HhN contacts as well as contacts between Ihog-Ptc and Ihog-HhN (Zheng, 2010).

It is important to note that, despite a direct physical interaction of Ihog and Ptc and their mutual contributions to formation and surface presentation of receptor, and to ligand binding, these two pathway components have opposing roles in pathway regulation. Ihog proteins are thus absolutely required for pathway activation in response to Hh ligand, whereas Ptc alone suffices for suppression of Smo activity in the absence of ligands (Zheng, 2010).

Functional genetic analyses of the mammalian Ihog proteins Cdo and Boc have revealed roles in Hh signaling. Cdo mutant mice thus display mild to intermediate forms of holoprosencephaly, a classic manifestation of Hh signaling deficiency, with the severity of the effect depending on strain background and subject to modifying effects of mutations in other Hh pathway components. Boc mutant mice also show defects in Hh signal-dependent axonal pathfinding by dorsal neurons with ventral commissural projections in the developing neural tube. Neither of these mutants displays phenotypes as severe as those seen in the Shh mutant mouse, or in the Smo mutant, which affects all aspects of Hh signaling. It is possible, however, that a systematic analysis of the double mutant Cdo; Boc animals might reveal more severe phenotypes, as is noted in this study for ihog; boi in Drosophila. In addition, phenotypic characterization of ihog and boi mutants was not designed to reveal defects in axonal pathfinding functions like that of murine Boc, and the possibility of such a function in Drosophila remains to be explored (Zheng, 2010).

Hedgehog regulates smoothened activity by inducing a conformational switch

Hedgehog (HH) morphogen is essential for metazoan development. The seven-transmembrane protein smoothened (SMO) transduces the HH signal across the plasma membrane, but how SMO is activated remains poorly understood. In Drosophila, HH induces phosphorylation at multiple Ser/Thr residues in the SMO carboxy-terminal cytoplasmic tail, leading to its cell surface accumulation and activation. This study provides evidence that phosphorylation activates SMO by inducing a conformational switch. This occurs by antagonizing multiple Arg clusters in the SMO cytoplasmic tail. The Arg clusters inhibit SMO by blocking its cell surface expression and keeping it in an inactive conformation that is maintained by intramolecular electrostatic interactions. HH-induced phosphorylation disrupts the interaction, and induces a conformational switch and dimerization of SMO cytoplasmic tails, which is essential for pathway activation. Increasing the number of mutations in the Arg clusters progressively activates SMO. Hence, by employing multiple Arg clusters as inhibitory elements counteracted by differential phosphorylation, SMO acts as a rheostat to translate graded HH signals into distinct responses (Zhao, 2007; full text of article).

The prevalent view regarding SMO regulation is that SMO is activated as a result of subcellular compartmentation. This study provides substantial evidence that SMO activity is also regulated by a conformational switch. In particular, an autoinhibitory domain (SAID) was identified in the Drosophila SMO cytoplasmic tail, containing multiple Arg clusters that keep SMO in a closed inactive conformation through intracellular electrostatic interaction. HH-induced phosphorylation disrupts such interaction and triggers a conformational switch and increased proximity of SMO cytoplasmic tails, which may further promote recruitment and interaction of intracellular signalling complexes. The results also indicate that the Arg clusters may promote endocytosis and degradation of SMO, whereas multiple phosphorylation events neutralize the negative effect of the Arg clusters either by inhibiting endocytosis and/or promoting recycling of SMO (Zhao, 2007).

A striking feature of the SAID domain is that it contains multiple regulatory modules each of which consists of an Arg cluster linked to a phosphorylation cluster. The pairing of positive and negative regulatory elements may offer precise regulation, because phosphorylation at a given cluster may only neutralize adjacent negative element(s), leading to an incremental change in SMO activity. It is proposed that increasing phosphorylation gradually neutralizes the negative effect of multiple Arg clusters, leading to a progressive increase in SMO cell surface expression and activity. Thus, by employing multiple Arg clusters as inhibitory elements that are counteracted by differential phosphorylation, SMO acts as a rheostat to translate graded HH signals into distinct responses (Zhao, 2007).

Cellular trafficking of the glypican Dally-like is required for full-strength Hedgehog signaling and Wingless transcytosis

Hedgehog (Hh) and Wingless (Wg) morphogens specify cell fate in a concentration-dependent manner in the Drosophila wing imaginal disc. Proteoglycans, components of the extracellular matrix, are involved in Hh and Wg stability, spreading, and reception. This study demonstrates that the glycosyl-phosphatidyl-inositol (GPI) anchor of the glypican Dally-like (Dlp) is required for its apical internalization and its subsequent targeting to the basolateral compartment of the epithelium. Dlp endocytosis from the apical surface of Hh-receiving cells catalyzes the internalization of Hh bound to its receptor Patched (Ptc). The cointernalization of Dlp with the Hh/Ptc complex is dynamin dependent and necessary for full-strength Hh signaling. Wg is secreted apically in the disc epithelium and apicobasal trafficking of Dlp allows Wg transcytosis to favor Wg spreading along the basolateral compartment. Thus, Dlp endocytosis is a common regulatory mechanism of both Hh and Wg morphogen action (Gallet, 2008).

Previous studies failed to observe Dlp at the apical surface of the wing disc epithelium despite the fact that it has been extensively shown in vertebrates that GPI-linked proteins are mainly targeted to the apical surface of epithelial cells. By performing extracellular labeling and kinetic experiments, this study demonstrated that Dlp is targeted to the apical surface before being endocytosed and readdressed to the basolateral compartment. Blocking endocytosis allowed demonstration that apical surface accumulation takes place at the expense of its basolateral location, showing that Dlp is first targeted to the apical domain of the epithelium before being sent to the basolateral compartment. The GPI anchor of Dlp is essential for its internalization, as Dlp tethering by a transmembrane domain (e.g., GFP-Dlp-CD2) abolishes its capacity to be internalized. It is also concluded that Dlp apical targeting followed by its rapid internalization is essential for full Hh signal transduction and for shaping the Wg gradient because when Dlp internalization is blocked, both processes are impaired (Gallet, 2008).

Interestingly, a rescue was observed of the first row of dlp mutant cells by the wild-type surrounding cells. Two mechanisms could explain this: (1) GPI-linked proteins are inserted in the outer leaflet of the plasma membrane and are exposed to the extracellular space. Dlp could interact with extracellular proteins located in nearby cells that could aid in flipping Dlp from the outer leaflet of the plasma membrane of one cell to the next, and/or (2) Dlp might be carried by argosomes, which are large extracellular particles resembling low-density lipoproteins containing lipophorins, esterified cholesterol, and triglycerides surrounded by a phospholipid monolayer. These argosomes not only bear GPI-linked proteins such as HSPGs but also morphogens such as Wg or Hh and are able to travel over several rows of cells (Gallet, 2008).

Whereas numerous data have clearly demonstrated the role of the HSPG in Hh signaling through regulation of Hh stabilization, movement, and reception, the function of Dlp in Hh signaling remained unclear. This study clearly demonstrates that Dlp is exclusively necessary in Hh-receiving cells for full-strength Hh signaling. Strong colocalization of Ptc, Hh, and Dlp is observed in endocytic vesicles in Hh-receiving cells. Ptc is probably responsible for the Hh/Dlp internalization, because it was not possible to detect Dlp-Hh-containing endocytic vesicles in the absence of Ptc. Overexpressing Dlp increased the number of Ptc-Hh-internalized vesicles, whereas absence or tethering Dlp at the cell surface (e.g., GFP-Dlp-CD2) lowered the number of Ptc/Hh vesicles. Therefore, it can be imagined that the presence of Dlp increases Ptc-Hh internalization to elicit a high level of pathway activation. Dlp could also stabilize Ptc/Hh within the intracellular compartment, allowing a stronger and/or a longer signaling. The role of Dlp is clearly different from other Hh coreceptors. Indeed, already identified Hh coreceptors such as Ihog and Brother of Ihog (Boi) (Cdo and Boc, respectively, in vertebrates) stabilize Hh at the cell surface, whereas Dlp overexpression does not increase Hh binding on receiving cells in vivo or in vitro (Gallet, 2008).

It has been demonstrated that Hh is apically secreted by wing disc epithelial cells; however, the compartment by which target cells receive Hh signal (e.g., apical, lateral, or basal) remains controversial. Nevertheless, the data suggest that endocytosis from the apical surface is necessary to sustain full-strength Hh signaling because blocking endocytosis inhibits Dlp internalization from the apical surface and decreases Hh signaling activation. Moreover, a colocalization between Hh and extracellular Dlp is observed at the apical side of Hh-receiving cells but not at the basolateral part of the cell. It has also showed previously that in ptc mutant embryos, Hh accumulates at the apical side of receiving cells. Nevertheless, although blocking endocytosis strongly stabilized Dlp at the apical cell surface, it also stabilized Ptc at both poles of the epithelial cells, raising the possibility that part of the signal transduction occurs basally. Unfortunately, it was not possible to see any Hh accumulation at either pole of the receiving cells when endocytosis was blocked. Hence, a model is favored in which Hh can trigger its signal both basally and apically, where the apical signaling is amplified by Dlp and is necessary for a full-strength Hh signal (Gallet, 2008).

Blocking endocytosis impairs Hh signaling both in embryos and in wing discs. However, it has been previously published that inhibiting endocytosis in imaginal discs using a thermosensitive allele of shi (shi^ts) does not impaired Hh signaling: Ci and collier (an Hh target gene) are unaffected but Ptc is stabilized. How can the differences with these data be explained? It is important to note that the shi null allele is cell lethal; therefore, either a dominant-negative or a shi^ts allele, which may not fully inhibit endocytosis, must be used. Accordingly, Ptc stabilization is observed in only 30% of discs, but each time such a stabilization is observed, a decrease of dpp expression was observed. Increasing the level of Shi^DN expression increased the penetrance of the phenotype but triggered lethality, making analysis difficult (Gallet, 2008).

Wg is mainly secreted via the apical pole of producing cells. Strikingly, in those cells, Wg is strongly localized in endocytic vesicles that are abundant apically but also in multivesicular endosomes. Dlp overexpression decreases the level of Wg at the apical surface of cells while increasing Wg stability along their lateral compartment. Therefore, it is proposed that Wg is secreted apically and is then endocytosed with the help of Dlp. Once internalized, Dlp targets Wg by transcytosis to the lateral compartment, where it is stabilized and can spread farther away to activate long-range target genes (Gallet, 2008).

Intriguingly, Dlp seems to play antagonistic roles in Wg signaling. Although it inhibits Wg activity near the Wg source, it is also necessary for Wg pathway activation far from the Wg source. This functional duality is directly related to its pattern of expression. Indeed, Dlp is expressed at low levels along the Wg source (e.g., the D/V axis) owing to repression by the Wg pathway itself, whereas it is expressed at higher levels far from the Wg source. The results could explain how Dlp functions antagonistically in Wg signaling. The two Wg receptors DFrizzled2 (Dfz2) and Arrow are involved in both the signal transduction and the internalization of Wg, mainly through the apical surface to shape its gradient by targeting Wg to the lysosome. Therefore, it is proposed that low Dlp-dependent transcytosis of Wg in producing cells and neighboring ones allows a high level of Wg at the apical surface and hence a strong activation of the pathway. On the contrary, higher Dlp-dependent transcytosis of Wg in more distant cells reduces both Wg pathway activation and degradation, promoting Wg movement along the basolateral compartment. Accordingly, it is observed that, in absence of dlp, extracellular Wg is absent from the lateral compartment of distant cells (Gallet, 2008).

This model supposes that Dlp is able to internalize Wg independently of the receptors DFz2 and Arrow. Interestingly, several groups found some internalized Wg in the absence of the Wg receptors Dfz2 and Arrow. Moreover, Dlp overexpression stabilizes Wg at the cell surface at the expense of short-range signaling activity, in accord with the fact that Wg must be endocytosed by Dfz2/Arrow to promote strong signaling. The results further support the view that Wg may form two different complexes: on the one hand a Dlp-Wg complex involved in Wg transcytosis and stabilization, and on the other hand a DFz2-Arrow-Wg signaling complex that shapes the Wg morphogen gradient and signals. Interestingly, when GFP-Dlp-CD2 is overexpressed, although Wg is stabilized at the apical surface over a very long range, where it should activate its pathway, a much stronger inhibition of Wg signaling is observed and an absence of internalized Wg. Therefore, GFP-Dlp-CD2 may titrate Wg from its receptors and prevent internalization, giving rise to both the stabilization of Wg and the inhibition of the pathway. Under physiological conditions, Dlp targeting of Wg to the lateral compartment supports its stabilization and spreading at the expense of its internalization/degradation from the apical surface by its receptors (Gallet, 2008).

Patched, the receptor of Hedgehog, is a lipoprotein receptor

The Hedgehog (Hh) family of secreted signaling proteins has a broad variety of functions during metazoan development and implications in human disease. Despite Hh being modified by two lipophilic adducts, Hh migrates far from its site of synthesis and programs cellular outcomes depending on its local concentrations. Recently, lipoproteins were suggested to act as carriers to mediate Hh transport in Drosophila. This study examined the role of lipophorins (Lp), the Drosophila lipoproteins, in Hh signaling in the wing imaginal disk, a tissue that does not express Lp but obtains it through the hemolymph. The up-regulation of the Lp receptor 2 (LpR2), the main Lp receptor expressed in the imaginal disk cells, was used to increase Lp endocytosis and locally reduce the amount of available free extracellular Lp in the wing disk epithelium. Under this condition, secreted Hh is not stabilized in the extracellular matrix. Similar results were obtained after a generalized knock-down of hemolymph Lp levels. These data suggest that Hh must be packaged with Lp in the producing cells for proper spreading. Interestingly, it was also shown that Patched (Ptc), the Hh receptor, is a lipoprotein receptor; Ptc actively internalizes Lp into the endocytic compartment in a Hh-independent manner and physically interacts with Lp. Ptc, as a lipoprotein receptor, can affect intracellular lipid homeostasis in imaginal disk cells. However, by using different Ptc mutants, it was shown that Lp internalization does not play a major role in Hh signal transduction but does in Hh gradient formation (Callejo, 2008).

At least two models have been proposed to explain how the lipophilic Hh can spread through an aqueous tissue. Fractionation studies of the supernatant of Hh-expressing cells showed that Hh participates in high molecular weight structures that probably represent multimeric complexes, and cholesterol and palmitic acid seems to mediate this multimerization. The lipid moieties are thought to be embedded in the core of these complexes, in analogy to micelles. Recently, a second model was proposed: it suggests that lipoprotein particles could carry lipid-modified ligands such as Hh and Wingless, acting as vehicles for long-range transport. Vertebrate lipoprotein particles are scaffolded by apolipoproteins and consist of a phospholipid monolayer surrounding a core of esterified cholesterol and triglycerides. Insects form similar particles that are called Lipophorins (Lp) and contain Apolipophorins I and II (ApoLI and ApoLII). These proteins are produced in the fat body by cleavage of the precursor pro-Apolipophorin, and are not synthesized by imaginal disk cells but receive them through the hemolymph. Panakova (2005) described that a systemic reduction of lipoprotein levels in the hemolymph, by expression of Lp (ApoLI-II) RNAi in the fat body, affects long-range but not short-range Hh signaling. That study also found that Wnt and Hh proteins copurify with lipoproteins from tissue homogenates and colocalize with lipoprotein particles in the developing wing epithelium. More recently, an interaction between Lp and the glypicans, Dally and Dally-like, has been found (Callejo, 2008).

This study has tested the role of lipoproteins in Hh signaling. To this aim, the lipoprotein gene was knocked down by RNA interference, reducing Lp supply in the hemolymph. In addition, the amount of extracellular Lp was locally reduced in the wing imaginal disk cells by overexpressing Lipophorin receptor 2 (LpR2), which increases Lp endocytosis. Under both experimental conditions a decrease was observed in extracellular Hh. These results suggest an important role of lipoproteins in Hh anchoring and spreading through the extracellular matrix. Moreover, this study has observed that Ptc actively internalizes Lipophorins, effectively acting as a Lipoprotein receptor, and that its over-expression can alter intracellular lipid homeostasis. Collectively, these results are consistent with the model of lipoprotein particles acting as vehicles for Hh transport (Callejo, 2008).

The long-range activity of Hedgehog is regulated in the apical extracellular space by the glypican Dally and the hydrolase Notum

Cell fate determination during developmental patterning is often controlled by concentration gradients of morphogens. In the epithelial field, morphogens like the Hedgehog (Hh) peptides diffuse both apically and basolaterally; however, whether both pools of Hh are sensed at the cellular level is unclear. This study shows that interfering with the amount of apical Hh causes a dramatic change in the long-range activation of low-threshold Hh target genes, without similar effect on short-range, high-threshold targets. Genetic evidence is provided that the glypican Dally upregulates apical Hh levels, and that the release of Dally by the hydrolase Notum promotes apical Hh long-range activity. The data suggest that several pools of Hh are perceived in epithelial tissues. Thus, it is proposed that the overall gradient of Hh is a composite of pools secreted by different routes (apical and basolateral), and that a cellular summation of these components is required for appropriate developmental patterning (Ayers, 2010).

Morphogens form long-range concentration gradients to signal positional information to cells within a complex tissue. Within an epithelium, the exact apicobasal cellular position of long-range gradient formation is not well understood. The morphogen Hh appears to be released both apically and basolaterally; however, which pool of Hh represents the functional long-range Hh was previously unknown. These studies have provided firm evidence that the wing imaginal disc uses its apical space to form a long-range gradient of Hh responsible for target gene activation. These data also suggest that short-range activity of Hh may be regulated by Hh released into the basolateral space. This implies that morphogen-receiving cells must integrate the apicobasal value of the extracellular Hh gradient present at different planes (Ayers, 2010).

In addition, it was found that morphogenic long-range gradients are shaped by extracellular matrix proteins. In the case of Hh, the glypican Dally positively regulates apical Hh levels in the producing cells, and Dally release promotes long-range spreading in this plane. Finallywe show direct genetic evidence is shown that the enzyme Notum regulates Dally release, thereby controlling the formation of a functional long-range gradient of Hh. A model is therefore proposed in which Dally controls the apical accumulation of Hh in the Hh-producing cells, and cleavage of Dally by Notum helps to shape the long-range gradient (Ayers, 2010).

It is believed, based on various observations in other organisms, that apical release and formation of a functional long-range Hh gradient may be a conserved mechanism. First, studies in Caenorhabditis elegans and Drosophila embryos have hinted at a role of apical secretion for Hh. Indeed, active apical secretion of exosomes containing Hh-like peptides in C. elegans epithelial cells has been demonstrated. In accordance, this study shows that the protein Dispatched (Disp), which is essential for Hh release in invertebrates and vertebrates, regulates apical Hh release in Drosophila embryos. Moreover, the formation of C. elegans cuticle requires the apical secretion of exosomes by Che14, the homolog of Disp. Shh aggregates in the apical lumen of the chick neural tube have been described, whereas an endogenously tagged Shh:GFP has been used that enabled visualization of the Hh gradient in mouse embryos. At embryonic day 8.5 (E8.5), when Shh is produced solely in the notochord, Shh:GFP (in Nodal Vesicular Particles [NVPs]) was found mostly apically in the neural tube (although the notochord has direct contact with the basal membrane of the neural tube). This apically concentrated Shh:GFP forms a gradient that drops exponentially along the dorsal-ventral axis (i.e., farther from the source). Similar observations have been seen when using antibodies against Shh. Thus, although apical gradients have been described in several organisms, these gradients need to be functionally challenged, something that was developed in this study (Ayers, 2010).

A tradeoff has often been observed between the long-range and the short-range Hh pathway targets; for example, increased range of dpp is often coupled with a decreased En range. Several different explanations for this could be suggested. First, increasing the spreading of Hh apically (for example, in overexpression of Sec:Dally) may result in a lower level of apical Hh close to the source, perhaps below the level required for En expression, while increasing the range of the low level needed for dpp expression farther from the Hh source. However, other data presented in this study do not fit this model. Indeed, a secreted form of Dally (Sec:Dally) did not reduce Hh apical levels at or close to the source, but seemed to just increase and elongate the levels of spreading Hh, especially far in the A compartment, whereas basolateral levels of Hh were reduced. Also, when levels of lumenal Hh were reduced (by blocking Hh on the PPM with Ptc^1130x), only a reduction in expression of long-range targets (dpp) was observed, not a change in En or Ptc expression. Furthermore, along with short-range signaling, the basolateral Hh gradient was unchanged in this genotype. Thus, two different secretion/signaling mechanisms of Hh are thought to occur from the Hh-secreting cells to the receiving cells. This tradeoff is often observed between increased dpp expression and decreased En expression when trafficking of Hh in its secreting cells is perturbed (e.g., in Shi^DN expression), suggesting that apical and basolateral pools of Hh in the producing cells are not independent. Furthermore, because this tradeoff is also observed when Dally in the P compartment is modified, it seems that Dally partially controls the balance between apical and basolateral Hh gradient formation. The data also indicate that basolateral Hh activity is formed independently of Notum, as short-range signaling is not affected in mutants of this protein. Having said this, it is not ruled out that transcription of short-range targets, such as En, may be a response to reception of both apical and basolateral Hh pools (Ayers, 2010).

Although often decreased, En expression was never lost completely; it is often unaffected in the first row of cells. Therefore, it is proposed that Hh may be secreted to the basolateral membrane, and here may signal to the adjacent receiving cells by both cell-cell contact (resulting in high En expression in the first row of receiving cells) and very limited diffusion (to activate En in up to three cells away from the A-P boundary) (Ayers, 2010).

This study suggests that the Hh that forms a basolateral gradient may be loaded in a different form to that released into the apical lumen. This is because at the lateral position, the Hh trapped by expression of Ptc^1130X in the disc proper illustrated a very different staining (highly membranous and nonpunctuate) than that found apically (which was highly punctuate). Also, the presence of basolateral Hh was found in only up to 2-3 cells away from the source, indicating a very limited ability to disperse; this could also be due to a different extracellular environment compared with the apical lumen, where Hh dispersal is higher. Although it may be basolaterally targeted, poorly diffusing Hh that is responsible for En expression in the three rows of cells, this is extremely difficult to prove. Indeed, little is known about polarized trafficking in wing imaginal discs. Although several methods have been described with which it was possible to modify apical Hh levels, no way has been found to specifically enrich or block basolateral Hh in the P compartment without affecting the apical Hh pool. Therefore, it has not been possible to directly test whether it is this gradient of Hh responsible for the pathway activation in the first row of cells in the A compartment (Ayers, 2010).

Evidence has been found for the involvement of the glypican Dally and the hydrolase Notum in the long-range apical spreading of Hh. Reduction of either of these proteins in Hh-producing cells reduces the range within which dpp is expressed, whereas short-range target En is untouched. Furthermore, Dally has been found to positively regulate apical Hh levels, and Dally release aids in long-range spreading of Hh, whereas Notum appears to be essential just for the latter. But what could the exact role of GPI-tethered Dally be in the Hh-secreting cells? It has been shown that the Heparan Sulfate of Glycosaminoglycan chains can bind and sequester both Hh and Lipophorins, which are thought to carry Hh. Therefore, GAG chain binding of Hh at the apical surface may work as a platform by which to stabilize and associate Hh with components necessary for its release and long-range spreading (Ayers, 2010).

In addition to regulating apical Hh accumulation, secretion of Dally appears to play a role in the long-range spreading and activity of Hh. It has been suggested that the secreted form of Dally increased dpp expression due to its mediation of Hh binding to the receptor complex. It cannot be ruled out that secreted Dally could have a role in stabilizing the Hh-receptor complex interaction and signaling, but it is believed that Dally shed from the P compartment is mainly involved in the formation of a long-range Hh gradient, due to changes seen in the apical Hh gradient profile. In addition, if secreted Dally was necessary for Hh-receptor interaction and signaling, then the absence of Dally in the P compartment should affect all Hh target genes. On the contrary, it was found that dally mutant clones in the P cells only affect the expression of the long-range target dpp. If promotion of ligand-receptor interaction was the only role of Dally released from the P compartment, then one would expect expression of secreted Dally to induce a higher level of signaling in the A compartment close to the Hh source, as Hh would be more highly sequestered to its receptors. On the contrary, a decrease is seen in En expression. Therefore, it is proposed that secreted Dally augments movement and extension of the Hh gradient, as opposed to solely increasing signaling (Ayers, 2010).

Lastly, analysis of Notum, a protein described as a GPI anchor cleaver (Traister, 2008), indicates that Notum in the P compartment promotes Hh long movement through its regulation of Dally. Therefore, it is proposed that after apical accumulation and clustering of Hh by GPI-anchored Dally, Notum cleaves and releases Dally, preparing Hh for its long apical voyage (Ayers, 2010).

The cell-surface proteins Dally-like and Ihog differentially regulate Hedgehog signaling strength and range during development

Hedgehog (Hh) acts as a morphogen in various developmental contexts to specify distinct cell fates in a concentration-dependent manner. Hh signaling is regulated by two conserved cell-surface proteins: Ig/fibronectin superfamily member Interference hedgehog (Ihog) and Dally-like (Dlp), a glypican that comprises a core protein and heparan sulfate glycosaminoglycan (GAG) chains. Dlp core protein can interact with Hh and is essential for its function in Hh signaling. In wing discs, overexpression of Dlp increases short-range Hh signaling while reducing long-range signaling. By contrast, Ihog has biphasic activity in Hh signaling in cultured cells: low levels of Ihog increase Hh signaling, whereas high levels decrease it. In wing discs, overexpression of Ihog represses high-threshold targets, while extending the range of low-threshold targets, thus showing opposite effects to Dlp. It was further shown that Ihog and its family member Boi are required to maintain Hh on the cell surface. Finally, Ihog and Dlp have complementary expression patterns in discs. These data have led to a proposal that Dlp acts as a signaling co-receptor. However, Ihog might not act as a classic co-receptor; rather, it may act as an exchange factor by retaining Hh on the cell surface, but also compete with the receptor for Hh binding (Yan, 2010).

Previous studies have shown that Dlp is specifically required for Hh signaling in cell-based assays and in embryos. However, the molecular basis of this specificity was unknown. This study shows that overexpression of Hh can restore naked cuticle in sugarless (sgl) and sulfateless (sfl) embryos, but not in dlp embryos. It was further demonstrated that the specificity of Dlp in Hh signaling results from its core protein. The Dlp core protein can restore Hh signaling autonomously in dlp embryos and in dlp-RNAi cells. In addition, the Dlp core protein interacts with Hh and promotes Hh signaling in the disc. Overexpression of Dlp increases Hh signaling strength, but reduces signaling range as well as Hh gradient range. These data suggest that the Dlp core protein could act as a classic co-receptor in Hh signaling by facilitating Hh-Ptc interaction (Yan, 2010).

Recent studies have shown that the vertebrate glypican-3 core protein directly promotes Wnt signaling in cancer cells, but inhibits sonic hedghog signaling during development. That the same glypican has opposite effects on Wnt and Hh is interesting because Dlp can inhibit high-threshold Wg signaling when overexpressed in discs. In addition to the essential role of the core protein, the attached GAG chains are important for the non-cell-autonomous functions of Dlp. The current results demonstrated that wild-type Dlp can rescue non-cell-autonomously in dlp embryos, whereas the core protein mainly acts in its expression domains. Interestingly, the CD2 forms of Dlp also lose the non-autonomous activity in dlp embryos. Several studies suggest that the GPI anchor of Dlp can be cleaved by the hydrolase Notum and that the GAG chains of Dlp can recruit lipoprotein particles. Thus, it will be interesting to determine the mechanism of Dlp non-autonomous activity (Yan, 2010).

This study suggests that the GPI anchor of Dlp is not essential for its activity in Hh signaling. Most importantly, two CD2 forms of Dlp, Dlp(-HS)-CD2 and GFP-Dlp-CD2, can effectively rescue Hh signaling in dlp embryos. In addition, CD2 forms of Dlp can also signal in cultured cells and discs. It is important to note that although the GPI, but not the CD2, form of Dlp is colocalized with Ptc in intracellular vesicles, both forms have similar activities in promoting Hh signaling in the disc. These data argue that colocalization of Dlp with Ptc in endocytic vesicles is not essential for Dlp function in Hh signaling. Consistent with this view, several studies have shown that endocytosis is not essential for Hh signaling in Drosophila wing discs. The current conclusion differs from a recent publication arguing that the GPI anchor of Dlp is required for its function in Hh signaling (Gallet, 2008). In that study, it was shown that overexpression of GFP-Dlp-CD2 can reduce Hh signaling in the wing discs. However, this study did not observe any dominant-negative effect of GFP-Dlp-CD2, which can rescue dlp mutant embryos and enhance Hh signaling strength in cells and discs. A possible explanation for the discrepancy is that expression of Dlp enhances signaling strength but also reduces signaling range. ap-Gal4 was always used, allowing use of the ventral disc as an internal control. The observed dominant-negative effect might reflect the reduced signaling range rather than signaling strength (Yan, 2010).

Previous studies in both Drosophila and vertebrates have demonstrated positive roles of the Ihog family proteins in Hh signaling. Ihog and Ptc synergize in mediating Hh binding to cells. These observations suggest that Ihog functions as a co-receptor for Hh. However, the new data argue that Ihog does not simply act as a classic co-receptor that only increases the binding of ligand to the signaling receptor. By altering Ihog levels in vivo and in vitro, it was shown that Ihog has biphasic activity in Hh signaling, with too much or too little Ihog leading to reduced signaling. Overexpression of Ihog leads to the accumulation of Hh, and knockdown of Ihog results in reduced Hh levels, suggesting that one major activity of Ihog is to retain Hh on the cell surface. Moreover, knockdown of both Ihog and Boi dramatically reduces Hh levels and signaling. This reduction of Hh signaling activity is likely to be due to an absence of Hh on the cell surface of the double-mutant tissue. However, a high level of Ihog causes a large amount of Hh to accumulate on the cell surface, but also reduces signaling strength. One explanation for this result is that Ihog can compete with Ptc for Hh binding. Thus, a low level of Ihog is required to maintain Hh on the cell surface, whereas a high level of Ihog can sequester Hh from its receptor. In other words, depending on the context, Ihog can either provide Hh for the receptor by retaining Hh on the cell surface, or compete with the receptor for Hh binding. This activity of Ihog is very similar to the recently proposed 'exchange factor' model, which allows the exchange of Hh between Ptc and Ihog. A recent study has demonstrated that Ihog also interacts with Ptc and that Ihog, Ptc and Hh form a triple complex (Zheng, 2010). The close association between the Ihog and Ptc receptors may thus allow them to exchange Hh ligand. It will be important to determine whether the triple complex has a greatly reduced ability to signal, a prediction from the mathematical exchange factor model (Yan, 2010).

Although this is the first demonstration of a biphasic co-factor in Hh signaling, similar biphasic co-factors have been reported. Drosophila Cv-2 enhances BMP signaling at low concentrations, but inhibits signaling at high concentrations. Syndecan-1 shows a similar concentration-dependent activation or inhibition of BMP signaling in Xenopus. Interestingly, Dlp has biphasic activity in Wg signaling, depending on its protein levels. All these co-factors are likely to act by a similar mechanism, suggesting that the biphasic co-factor is a recurring motif in different morphogen systems (Yan, 2010).

This study has shown that Dlp and Ihog play distinct roles in Hh signaling. Expression of Dlp enhances Hh signaling strength, but reduces signaling range. By contrast, expression of Ihog reduces Hh signaling strength, but extends signaling range. In addition, the Dlp level is elevated in the high Hh signaling area, whereas the Ihog level is reduced in that region. It is important to consider from a system point of view what these two co-factors provide for the Hh morphogen. For morphogens to work, they should be able to generate sharp boundaries between target genes with different thresholds. They also need to diffuse over a certain range without being lost in the extracellular space. The positive feedback of Dlp expression in the high Hh signaling areas helps to sharpen the boundaries between high- and low-threshold target genes. The negative-feedback regulation of Ihog might ensure that a strong Hh signal is attained in the areas close to the Hh source and also allow the Hh gradient to diffuse to those areas distant from the source (Yan, 2010).

Structure of the protein core of the glypican Dally-like and localization of a region important for hedgehog signaling

Glypicans are heparan sulfate proteoglycans that modulate the signaling of multiple growth factors active during animal development, and loss of glypican function is associated with widespread developmental abnormalities. Glypicans consist of a conserved, approximately 45-kDa N-terminal protein core region followed by a stalk region that is tethered to the cell membrane by a glycosyl-phosphatidylinositol anchor. The stalk regions are predicted to be random coil but contain a variable number of attachment sites for heparan sulfate chains. Both the N-terminal protein core and the heparan sulfate attachments are important for glypican function. This paper reports the 2.4-Å crystal structure of the N-terminal protein core region of the Drosophila glypican Dally-like (Dlp). This structure reveals an elongated, α-helical fold for glypican core regions that does not appear homologous to any known structure. The Dlp core protein is required for normal responsiveness to Hedgehog (Hh) signals, and a localized region on the Dlp surface important for mediating its function in Hh signaling was identified. Purified Dlp protein core does not, however, interact appreciably with either Hh or an Hh:Ihog complex (Kim, 2011).

Glypicans modulate the activity of multiple growth factors active during development, and defects in glypican function lead to widespread and diverse developmental malformations. Much of the activity of glypicans can be attributed to interactions between their heparan sulfate attachments and heparan-binding growth factors, but recent work has demonstrated important functional roles for the protein cores of glypicans, notably in Hh and Wnt signaling. In particular, the protein cores seem likely to mediate functions that are specific to particular glypicans. The crystal structure of the N-terminal globular region of a glypican, Dlp_δNCF, adopts an elongated, all α-helical fold with no evident homology to previously determined structures. The high level of sequence conservation among the N-terminal protein cores of glypicans (greater than 40% sequence identity exists between Drosophila and human glypicans in this region) indicates that the Dlp_δNCF structure provides a sound basis for the design and interpretation of experiments with other glypicans. The absence of any apparent active site-like cavities or sources of conformational flexibility in the Dlp_δNCF structure suggests that the protein regions of glypicans exert their effects by serving as binding proteins, consistent with proposed roles as coreceptors or in targeting ligands to specific subcellular compartments (Kim, 2011).

One reason Dlp has been proposed as an Hh coreceptor is the ability of Dlp lacking heparan sulfate to coimmunoprecipitate with Hh. The inability to detect a high-affinity interaction between Dlp_δNCF and HhN using purified proteins suggests that Dlp may interact with Hh as part of a larger complex and additional factors are needed to promote Dlp/Hh interactions. One candidate for such a factor is the Hh coreceptor Ihog, which is an essential component of the Hh receptor complex, but this study shows that purified Dlp_δNCF does not form a high-affinity complex with either an active fragment of Ihog (IhogFn12) or an HhN:IhogFn12 complex. This result does not rule out Ihog as important for mediating Hh:Dlp interactions but suggests that an additional factor or factors may be needed. An obvious candidate for such a factor is Patched, a key cell-surface component of the Hh signaling pathway, but assessing its role in Hh-containing complexes awaits purification of suitable amounts of this 12-pass integral membrane protein. An additional element complicating interpretation of results of studies of receptor-ligand interactions in solution arises from the absence of membrane tethering and ligand multivalency. Restricting components to a membrane surface orients them and greatly enhances their local concentration, and a multivalent ligand greatly increases the avidity of binding. Interactions between a monovalent ligand and cell-surface components may thus not be strong enough to be observed with soluble components in solution (Kim, 2011).

The inability to observe an interaction between ShhN and the N-terminal domain of glypican-3 is puzzling, however, given that the protein region of glypican-3 has been reported to bind to ShhN with nanomolar affinity. Several possibilities may explain this discrepancy: (1) ShhN may interact with the glypican- 3 stalk region, which was present in the earlier study but not in the current experiment; (2) attachment of histidine-tagged ShhN to Ni-NTA agarose may have blocked a glypican interaction site in these studies; or (3) an unidentified cofactor that promotes high-affinity interaction was present in the earlier studies but absent in the earlier studies. Calcium ions, for example, are required to promote high-affinity interactions between ShhN and CDO, the mammalian homolog of Ihog (Kim, 2011).

The ability to identify a localized region on the C lobe of the Dlp surface important for proper Dlp function in Hh signaling is further consistent with Dlp participating in Hh signaling primarily as a binding protein, although the nature and number of binding partners remains to be determined. Curiously, the identified surface is composed largely of hydrophilic residues, which is unusual for protein-protein interfaces. This surface also occurs on the opposite surface of Dlp relative to the disordered Dlp-specific insertion that follows the furin-like processing site, suggesting that interactions mediated by this region are likely independent of furin-like processing and this insertion. Glypican mutations previously associated with functional impairments, for example those in glypican-3 that cause Simpson-Gohlabi-Behmel syndrome and those in glypican-6 that cause omodysplasia, appear to result in severe truncations or complete loss of expression of the affected glypican. Whether the C-lobe region identified on Dlp is generally involved in glypican function or is specific to Dlp or positive regulators of Hh signaling is an interesting question for future investigation. The results presented in this study establish a molecular foundation to guide design and interpretation of studies investigating the molecular bases of glypican function (Kim, 2011).

Balancing Hedgehog, a retention and release equilibrium given by Dally, Ihog, Boi and shifted/DmWif

Hedgehog can signal both at a short and long-range, and acts as a morphogen during development in various systems. The mechanisms of Hh release and spread were studied using the Drosophila wing imaginal disc as a model system for polarized epithelium. The cooperative role of the glypican Dally, the extracellular factor Shifted (Shf, also known as DmWif), and the Immunoglobulin-like (Ig-like) and Fibronectin III (FNNIII) domain-containing transmembrane proteins, Interference hedgehog (Ihog) and its related protein Brother of Ihog (Boi), was analyzed in the stability, release and spread of Hh. Dally and Boi were shown to be required to prevent apical dispersion of Hh; they also aid Hh recycling for its release along the basolateral part of the epithelium to form a long-range gradient. Shf/DmWif on the other hand facilitates Hh movement restrained by Ihog, Boi and Dally, establishing equilibrium between membrane attachment and release of Hh. Furthermore, this protein complex is part of thin filopodia-like structures or cytonemes, suggesting that the interaction between Dally, Ihog, Boi and Shf/DmWif is required for cytoneme-mediated Hh distribution during gradient formation (Bilioni, 2013).

This study has approached the functional interaction of the ECM components Shf/dWif and Dally, and of the Hh coreceptors Ihog and Boi in Hh release and/or spreading to form a gradient. Two major findings are described: one is an unpredicted role of Dally and Boi in the apical retention and subsequent internalization of Hh in producing cells, and the other the interaction of Dally and Ihog/ Boi with Shf/dWif, facilitating Hh movement in the basolateral part of the disc epithelium. Interactions between these components allow retention at the apical plasma membranes of producing cells necessary to prevent apical Hh spreading and facilitate subsequent recycling to basolateral side, as well as Hh release and movement in this side of the epithelium (Bilioni, 2013).

Apical Hh levels in the Hh producing cells are affected in both dally and boi (but not in ihog) null mutant conditions. Moreover, ectopic Dally or Boi (but not Ihog) cause an increase in Hh retention at the apical plane of the disc. Accordingly, Dally and Boi have an Ihog-independent function in maintaining Hh concentration at the apical part of the disc epithelium. As has been previously demonstrated, apically externalized Hh does not form a gradient (Callejo, 2011); thus, this might be accounted as a mechanism preventing the spread of apically externalized Hh. In agreement with an Ihog-independent role of Boi in apical Hh retention, a recently published study (Hartman, 2010) demonstrates that Boi is expressed in apical cells of the ovary and suppresses follicular stem cells (FSC) proliferation by binding to and sequestering Hh on the apical cell surface, thereby inhibiting Hh long range distribution (Bilioni, 2013).

In addition, it has been observed that the apically externalized Hh is subsequently internalized and recycled to the basolateral membranes of the wing disc epithelium (Callejo, 2011). In strong support of this Hh recycling scheme, overexpression of Dally in the P compartment was shown to enhance Hh apical retention, decreasing Hh levels in the most basal side and reducing Hh target activation in the A compartment. Therefore, it is likely that Dally and Boi not only prevent apical Hh spreading but also mediate the apical Hh internalization in the Hh producing cells. The observation that Hh, Dally and Boi accumulate in the apical surface when internalization is blocked by a dynamin mutation reinforces this possibility (Bilioni, 2013).

The increment of apical spreading of Hh in the A compartment cells caused by overexpression of a secretable form of Dally in the P compartment also supports Dally's function in Hh apical retention in the P compartment cells. Given that the enhanced apical spreading of Hh correlates with a reduced basolateral Hh gradient, it is proposed that in normal conditions recycling of the apical Hh pool results in the formation of a basolateral Hh gradient. In contrast, it has been propose that the hydrolase, Notum, is implicated in the release of Dally and the abnormal increase in the apical spreading of Hh in the A compartment cells by overexpression of a DallySec has been interpreted as a direct evidence of a long-range apical Hh gradient. However, notum mutants show a Wg (not a Hh) signaling phenotype, which argues against this hypothesis. Furthermore, the cell-autonomous requirement of wild type Dally for keeping Hh in the ECM suggests that Dally may not be released from its GPI anchor for this function. In addition, no non-autonomous effects of Dally were observed on Shf/dWif stability, which implies that Dally remains membrane-anchored (Bilioni, 2013).

In conclusion, these data show that Dally has a cell-autonomous role in Hh attachment to the ECM, with a double purpose: in the producing cells Dally facilitates Hh retention necessary to prevent Hh spreading, and in receiving cells it supports Hh presentation to the receptor. An autonomous Dally requirement for Hh signaling has been recently proposed. This cell-autonomous requirement for Dally in maintaining extracellular Hh concentration is in agreement with the previously described role of Dally in Wg and Dpp signaling (Bilioni, 2013).

It has been suggested that Shf/dWif mediates the function of the HSPGs in Hh stability in the ECM. This study finds that Shf/dWif stabilization also depends on Dally, Ihog and Boi because Shf/dWif levels vary accordingly in both loss and gain of function of these genes. In these mutants, Shf/dWif levels are reduced at the basolateral side of the disc epithelium and, as a consequence, Hh levels also decrease. Thus, Dally together with Shf/dWif, Boi and Ihog is implicated in Hh stability in the ECM. On the other hand, despite an increment in Hh levels when overexpressing Dally in a shf mutant background, the target expression remains severely impaired. Thus, an excess of Dally or Ihog/Boi can offset the effects of Shf/dWif mutation in terms of Hh concentration but not in terms of Hh movement. Taken together, these results lead to the conclusion that Shf/dWif is an ECM factor that counteracts the impact of Dally and Ihog/Boi on Hh attachment at the membranes. Interestingly, ectopic expression of Ihog, Boi or Dally stabilizes Shf/dWif mainly in the basolateral domain where most of Shf/dWif protein is located (Bilioni, 2013).

Shf/dWif is then required for Hh movement even when overexpressing Hh. Counteracting this effect, Ihog and Boi mediate the attachment of extracellular Hh to plasma membranes in Hh producing cells. In ihog and boi mutant cells Hh levels, mainly at the basolateral plane, are very low, and overexpression of Ihog or Boi not only causes Hh accumulation at the plasma membrane but also a restriction in Hh movement. Moreover, Shf/dWif can rescue the phenotype of restricted Hh movement imposed by ectopic Ihog or Boi, and is necessary to allow the increment of Hh spreading when knocking down Boi and Ihog in the P compartment, demonstrating that proper gradient formation requires equilibrium between these proteins. This is further confirmed when Ihog overexpression in the P compartment restricts Hh movement and decreases Hh signaling in the region anterior to a smo⁻ clone located at the A/P compartment border; and then again simultaneous overexpression of Shf/dWif reestablishes the equilibrium so Hh can now reach the wild type territory and signal across the clone (Bilioni, 2013).

It has been proposed that Boi and Ihog are not required in P compartment cells because double boi and ihog mutant clones had no effect on Hh signaling. However, this conclusion did not take account of Hh non-autonomy. Indeed, it has been reported that wings develop normally even with P compartments that have large hh mutant clones. This non-autonomy of Hh is also supported by the lack of an effect on Hh signaling of disp^−/− clones or by long-range spreading of Hh through large smo mutant clones. Because of Hh non-autonomy, the function of Boi and Ihog in Hh-producing cells was only reveled when Boi and Ihog were knocked down in the whole P compartment. Interestingly, despite of the low Hh levels in the P compartment in the absence of Boi and Ihog functio, an increase of long-range Hh gradient was observed. It is thought is that a low Hh retention at the plasma membrane of P compartment cells causes an increase of Hh release, so the bulk of Hh that reaches the A compartment is higher than in the wild type condition. Supporting this hypothesis, it was shown that Ihog and Boi from A cells have indeed the capacity to 'capture' Hh from P compartment cells (Bilioni, 2013).

In addition, the ectopic expression of Ihog increases not only the endogenous levels of Hh but also Shf/dWif, Dally along cytonemes located at the basolateral side of the disc epithelium, as previously described for Dlp (Callejo, 2011). Some of these long filaments extend up to several cell diameters and are reminiscent of 'cytonemes'. In the context of Notch signaling, basal actin-based filopodia are important for lateral inhibition between non-neighboring cells. In Hh signaling, it was have observed that cytonemes act as vectors for Hh movement in the ECM, contributing to Hh gradient formation. Since Boi and Ihog are absolutely required for reception, Ptc, Ihog- and Boi-labeled cytonemes emanating from A compartment cells are probably essential for sequestering Hh from P cells. Although this work does not provide the molecular mechanism by which Shf/dWif, Dally, Ihog and Boi proteins affect cytoneme-mediated Hh transport, it is suggested that Shf/dWif might be responsible for maintaining the equilibrium between Hh attachment to cytonemes -- mediated by Dally, Ihog and Boi -- and Hh release or movement (Bilioni, 2013).

Previous analysis on Hh release in the wing imaginal disc epithelium indicates that although Hh is initially externalized through all plasma membranes, the apical Hh pool is internalized and recycled to basolateral plasma membranes where the long-range Hh gradient is formed (Callejo, 2011). This article has provided a compressive genetic analysis that confirms the hypothesis. A novel role is described of the glypican Dally and of the transmembrane protein Boi in the process of the apical internalization of Hh in P compartment cells, which is essential to guarantee that the bulk of Hh protein produced in the P compartment cells is redirected towards the basolateral domain. A role is also described of the Hh coreceptors Ihog and Boi, and the diffusible Shf/dWif factor at the basolateral plane of the epithelium. These proteins interact physically and together with Dally act to establish a balance between Hh attachment to membranes and movement across the ECM to promote gradient formation and signaling. Moreover, all these proteins associate to cytonemes in the basolateral part of the disc epithelium. Thus, the interplay of all these proteins creates an environment supporting Hh transport along cytonemes to shape a proper gradient (Bilioni, 2013).

Ihog and Boi elicit Hh signaling via Ptc but do not aid Ptc in sequestering the Hh ligand

Hedgehog (Hh) proteins are secreted molecules essential for tissue development in vertebrates and invertebrates. Hh reception via the 12-pass transmembrane protein Patched (Ptc) elicits intracellular signaling through Smoothened (Smo). Hh binding to Ptc is also proposed to sequester the ligand, limiting its spatial range of activity. In Drosophila, Interference hedgehog (Ihog) and Brother of ihog (Boi) are two conserved and redundant transmembrane proteins that are essential for Hh pathway activation. How Ihog and Boi activate signaling in response to Hh remains unknown; each can bind both Hh and Ptc and so it has been proposed that they are essential for both Hh reception and sequestration. Using genetic epistasis this study established that Ihog and Boi, and their orthologs in mice, act upstream or at the level of Ptc to allow Hh signal transduction. In the Drosophila developing wing model it was found that through Hh pathway activation Ihog and Boi maintain the boundary between the anterior and posterior compartments. The contributions of Ptc was dissociated from those of Ihog/Boi, and, surprisingly, it was found that cells expressing Ptc can retain and sequester the Hh ligand without Ihog and Boi, but that Ihog and Boi cannot do so without Ptc. Together, these results reinforce the central role for Ptc in Hh binding in vivo and demonstrate that, although Ihog and Boi are dispensable for Hh sequestration, they are essential for pathway activation because they allow Hh to inhibit Ptc and thereby relieve its repression of Smo (Camp, 2014).

Ihog and Boi have been shown to be absolutely essential within Hh-responding cells for activation of the Hh signaling pathway, acting upstream of Smo. The current experiments advance understanding of Ihog and Boi function by drawing three major conclusions: First, in genetic epistasis experiments it was found that Ihog and Boi also act upstream or at the level of Ptc, supporting the idea that they function through Ptc to relieve suppression of Smo. This epistatic relationship appears conserved in evolution, as it was found that Cdon and Boc also function upstream or at the level of Ptch1 for Hh signal transduction in mice. These genetic findings establish this relationship unequivocally, and so have profound implications for future studies to further clarify how these co-receptors participate in Hh reception and pathway activation (Camp, 2014).

Second, based on experiments to dissect the relative contributions of Ihog and Boi in processes involving Ptc, it is concluded that it is through their essential roles in Hh signal transduction that Ihog, Boi and Ptc contribute to anterior-posterior compartment segregation: once the pathway is activated, all three proteins are dispensable for maintenance of the compartment boundary, implicating other, yet unidentified, cell surface recognition molecules in compartment-specific cell affinity and adhesion (Camp, 2014).

Third, it is concluded that Ihog and Boi, unlike Ptc, are completely dispensable for the sequestration and retention of Hh. Cells lacking Ihog and Boi can sequester and retain the Hh signal if the pathway is activated and Ptc is upregulated. They do so via physiological levels of endogenous Ptc induced either by pathway activation in ^ptcS2 mutants or by expression of Smo^SD123. Incidentally, it was also found that Hh sequestration was rescued in boi;ihog double mutant clones overexpressing Ptc¹¹³⁰, a dominant-negative that fully activates the Hh pathway and upregulates endogenous, wild-type Ptc. This third conclusion is not consistent with the view that Ihog and Boi aid in addressing Ptc to the cell surface and that, once there, they are required for Ptc to bind and sequester Hh. This view is based primarily on Ptc and Ihog overexpression in cultured cells, and on an experiment that failed to restore Hh sequestration to boi;ihog double mutant cells with mutation of cAMP-dependent protein kinase 1 (Pka-C1), which upregulates Ptc and other target genes because loss of Pka-C1 disinhibits the activity of the transcription factor Ci. It is unclear why Ptc upregulation in Pka-C1 mutants was unable to rescue Hh sequestration in boi;ihog double mutants, whereas Ptc upregulation in the current experiments was able to do so. In cells lacking Ihog and Boi, perhaps the level to which Ptc is upregulated in Pka-C1 mutants is inadequate. Regardless, the current data indicate that Ptc has a central role in the binding and sequestration of Hh, whereas Ihog and Boi are dispensable, despite their requirement for Hh signal transduction (Camp, 2014).

The results are consistent with vertebrate systems, in which current models strongly favor direct contacts between Hh and Ptc, primarily because: (1) expression of the Ptc ortholog Ptch1 promotes binding of Shh to transfected cells, (2) radiolabeled Shh can be chemically cross-linked to Ptch1 expressed on the cell surface and (3) Ptch1 can reach the cell surface in the absence of the Ihog/Boi-related proteins Cdon and Boc. Whether Ptc is sufficient on its own to bind Hh remains an important question that awaits technically challenging studies using purified proteins. An alternative possibility is that Hh could have additional receptor(s), with candidates including the proteoglycans Dally and Dally-like (Dlp), and Shifted, a secreted protein of the Wnt inhibitory factor 1 (WIF1) family (Camp, 2014).

The results clearly distinguish a role for Ptc that relies on Ihog/Boi (Hh reception/signal transduction) from one that does not (Hh sequestration), and so they contribute to an emerging view of the function of Ihog, Boi and related proteins. In non-responding cells, others have shown that Ihog and Boi are involved in restricting the movement of Hh, and so may contribute to its overall distribution. Within Hh-responding cells, where Ptc is co-expressed with Ihog and Boi, it was found that Ihog and Boi are essential for Hh signal transduction, but not Hh sequestration and retention. As Ihog and Boi act upstream or at the level of Ptc, they must mediate a crucial, rate-limiting step in the inhibition of Ptc in response to Hh. However, as they are not essential for Ptc to bind Hh, how they affect Ptc function remains to be elucidated. Whereas the precise molecular mechanism remains elusive, several lines of evidence provide important clues. First, an Ihog variant lacking the cytoplasmic tail can rescue boi;ihog double mutants. Second, the second Fn3 domain of Ihog or Boi interacts physically with Ptc and is quite distinct from the first Fn3 domain that harbors the Hh-interacting surface. Third, the presence of Ihog or Boi potentiates co-immunoprecipitation of Hh and Ptc. Together, these results suggest that the primary role of Ihog and Boi in Hh signaling involves the ability of their ectodomains to form favorable protein complexes with Ptc or Hh, or with both simultaneously. Although the data indicate that Ptc does not need Ihog and Boi to bind Hh in vivo, it is surmised that it is through these multimolecular complexes that Ihog and Boi allow Hh to inhibit Ptc and thereby relieve its suppression of Smo and the Hh signaling cascade (Camp, 2014).

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