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).

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