Effects of Mutation or Deletion

Tout-velu is a Drosophila homologue of the putative tumour suppressor EXT-1 and is needed for Hh diffusion

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 (Bellaiche, 1998).

In the wing disc, Hh is expressed in cells of the posterior compartment and diffuses into the anterior compartment. In the anterior compartment, Hh signalling results in expression of Patched (Ptc), which can be detected as far as five cells away from the anterior/posterior boundary, and in stabilization of Cubitus interruptus (Ci), which is observed as far as 8-10 cells away. Ptc expression and Ci stabilization were used as reporters with which to examine the role of ttv in Hh signalling. Somatic mutant clones were generated with the ttvl(2)00681 mutation, which is either a null allele or a severe hypomorphic allele. In large anterior ttv mutant clones adjacent to the anterior/posterior border, Hh signalling is impaired, as indicated by the lack of Ptc staining in most of the clone. However, Ptc expression still occurs at the posterior edge of the clone next to wild-type cells. The level of signalling in these cells is nevertheless diminished, as shown by a reduction in their levels of Ptc staining compared with wild-type cells (Bellaiche, 1998).

A similar result was observed when Ci levels were used as a reporter for Hh signalling. In addition, the large region of Ci stabilization allowed the determination of a non-cell-autonomous effect of ttv mutant clones. When ttv mutant clones of only a few cells wide are located along the anterior/posterior boundary, wild-type cells anterior to the ttv mutant clone do not respond to the Hh signal, as shown by Ci staining. Since these cells are located within a domain to which Hh is normally able to diffuse, and in which Hh can normally induce the stabilization of Ci, it is concluded that ttv mutant clones have a directional, non-cell-autonomous effect on wild-type cells located anteriorly. Since the domain of Ci stabilization is under direct control of Hh signalling, this result is interpreted as an inability of Hh to reach wild-type cells located anterior to ttv mutant clones. Whether diffusion of Hh is impaired in the absence of ttv was then investigated (Bellaiche, 1998).

In a ptc mutant clone, Hh diffusion is observed as an ectopic induction of Ptc in wild-type cells localized anteriorly to a clone of ptc mutant cells. To determine whether Hh diffusion is modified by ttv in the ptc mutant clones, clones were generated with mutations in both ptc and ttv and the expression of Ptc was analyzed. In these double mutant clones, Ptc expression is not induced in wild-type cells anterior to the clone. To assess Hh diffusion in the ptc ttv mutant clones directly, the distribution of Hh was compared in ptc and ptc ttv clones. In ptc mutant cells, Hh protein was detected as diffuse membrane staining. When Hh reaches wild-type cells beyond a ptc clone, it can be seen in a punctate staining pattern that, for the most part, coincides with the punctate Ptc staining that reflects Ptc localization in vesicles. In ptc ttv double mutant clones, there is no Hh staining in mutant cells. On the basis of this result and the directional non-cell-autonomous effect of ttv mutant clones, it is proposed that Hh cannot diffuse in the absence of ttv activity (Bellaiche, 1998).

In order to diffuse, Hh must move from the sending to the receiving cell; therefore, ttv could function in the sending cell and/or the receiving cell. In the anterior cell, the sending of Hh depends on its reception from the previous cells; thus it is difficult to determine where Ttv functions by analyzing ttv mutant clones in the anterior compartment. Therefore mutant clones were generated in the posterior compartment next to the anterior/posterior boundary and their effects on Ci stabilization were analyzed. ttv mutant clones in the posterior compartment were found not to affect the diffusion of Hh. Thus, it is proposed that ttv functions in the receiving cells for the movement of Hh from sending to receiving cells. This observation also indicates that ttv is not required for Hh production. In a ttv clone located in the anterior compartment, Hh can still signal, although less efficiently, to the first receiving cells. It is suggested that this weakened signalling activity is mediated by Hh present on the membrane of the Hh-sending cells and that efficient Hh signalling, even to adjacent cells, requires diffusion of Hh (Bellaiche, 1998).

To clone the ttv gene, the genomic region surrounding the inserted P-element, ttvl(2)00681, was isolated and two genes were identified in the vicinity of the P-element insertion site. One gene is lamin C, which is unlikely to correspond to ttv since it is not expressed maternally. Sequencing of the putative 3.8-kilobase (kb) complementary DNA encoding ttv shows that Ttv is a protein of 760 amino acids that is similar to the human multiple exostoses (EXT-1) protein. The 3.8-kb cDNA identifies a maternally expressed transcript on an RNA blot. A transgene of this transcript under the control of a heat-shock protein 70 gene (hsp70) promoter rescues ttv homozygous flies to viability (Bellaiche, 1998).

Ttv is 56% and 25% identical to the human EXT-1 and EXT-2 proteins, respectively. EXT-1 and EXT-2 probably form a new family of conserved molecules, since mouse and Caenorhabditis elegans homologs have been identified. However, the cellular compartment in which EXT molecules could function has remained unknown. 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. An in vitro translation assay was used to show that Ttv is indeed a membrane protein. In vitro-transcribed ttv messenger RNA is translated into a putative protein with a relative molecular mass (Mr) of 80K. In the presence of microsomes, the protein is glycosylated and fully protected from degradation by proteinase K, indicating that the protein is imported into microsomes. Furthermore, the Ttv protein remains associated with the membrane fraction after an alkaline wash; it is therefore a membrane-associated protein. Since the sequence surrounding the signal sequence does not closely match consensus cleavage sequences, it is proposed that the signal sequence is not cleaved and instead acts as an anchor region. It is concluded that Ttv is a type II integral membrane protein (Bellaiche, 1998).

Since the active form of Hh is tethered to the membrane by a cholesterol moiety, it is not known how Hh can diffuse from one cell to another. The movement of Hh does not depend on Ptc and Smoothened. Since Ttv is needed in the receiving cell to allow Hh diffusion, it is suggested that Ttv does not function in the dissociation of Hh from the membrane of the sending cells and is probably to be required to allow reassociation or maintenance of Hh on the surface of the receiving cells. The multiple exostoses syndrome is a dominantly inherited disease that is characterized by short stature, limb length inequalities, bone deformities and the presence of bone outgrowths, called exostoses, at the ends of long bones. The loss of any of the EXT-1, -2 or -3 loci is responsible for this syndrome. The EXT-1 and EXT-2 genes have been cloned and may be tumor-suppressor genes. During bone morphogenesis one of the established roles of Indian Hh is to limit the rate of chondrocyte differentiation. On the basis of the similarity between EXT-1 and Ttv, it is proposed that some aspects of the multiple exostoses syndrome are due to defects in diffusion and efficient signalling of Indian Hh. The characterization of Ttv is an important step toward the understanding of a cellular mechanism that allows Hh to diffuse and to exert its patterning activity (Bellaiche, 1998).

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).

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 (Bellaiche, 1998). In clones at the AP boundary, Ci and Ptc levels were maintained in only a single row of mutant cells along the posterior edge of the clone. Thus, it was proposed that cells lacking HSPGs are competent to receive, but are impaired in propagating, the Hh signal. Interestingly, ttv mutations only affect Hh-Np, the mature cholesterol-modified form of the ligand. In ttv mutant embryos, Hh-Np distribution is curtailed, while Hh-N, which lacks cholesterol, is unaffected. Based on these findings, it has been suggested that HSPGs may enhance the targeting of Hh-Np to 'lipid rafts' where the ligand can be then be transported from cell to cell (Bornemann, 2004).

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

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

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

A role for HSPGs in Wg signaling in Drosophila is well established. Addition of heparin sulfate (HS) or chondroitin sulfate (CS) GAGs to culture medium causes S2 cells to release secreted Wg protein, while heparinase treatment reduces their ability to respond to exogenous Wg. Furthermore, both Wg levels and signaling efficiency are sensitive to HSPG concentration; Wg activity is reduced when either HS synthesis or the core proteins for the glypicans Dally and Dlp are compromised. Severe disruption of extracellular Wg distribution and the altered expression of the downstream target Ac, in ttv and sotv mutant clones, demonstrates that HS chains are essential for establishing and/or maintaining the Wg gradient (Bornemann, 2004).

The effects of HSPGs on signaling are further modulated by secondary modifications to the GAG chains. For example in Drosophila, loss of either sfl or slalom (sll) compromises both Wg and Hh signaling. Sfl is an N-deacetyl/N-sulfotransferase, and Sll is required to transport a high-energy sulfate donor molecule to the golgi, where HSPGs such as dally and dlp are sulfate modified. Loss of GAG sulfation results in signaling defects, despite the fact that unsulfated HS-GAGs are still present in sfl mutants. Consequently, not only are HS-GAG chains required for signaling, but the extent of sulfation is also crucial. In vertebrates, Qsulf1 promotes Wg signaling by catalyzing removal of 6-O sulfate groups from HS chains of HSPGs, including the Dally homolog glypican 1. It has been proposed that in the absence of Qsulf1, Wg stays tightly bound to HSPGs, which prevents the ligand from interacting with receptors. However, in Qsulf1-expressing cells, selective 6-O desulfation reduces the binding affinity sufficiently to permit ligand-receptor interaction. In this context, the altered HS disaccharide distribution and abnormal sulfation patterns encountered in hypomorphic sotv alleles is intriguing. If partial loss of function EXT2 mutations in individuals with HME causes similar disruption of GAG modifications, they could also have complex and allele-specific effects on signaling (Bornemann, 2004).

The data also provide direct evidence that HS chains are required for Dpp signaling in vivo. The core protein of the glypican Dally has been implicated in Dpp signaling based on genetic interactions and recent studies demonstrating its role in regulating the Dpp morphogen gradient in the wing. However, the contribution of HS chains to Dpp signaling has remained unclear. Dpp signaling in the wing disc is reduced in ttv or sotv mutant clones independent of effects on Hh signaling, establishing that HS GAG chains are required for optimal activity of the Dpp pathway. Although Dpp activity is clearly compromised in mutant tissue, signaling is still detectable in mutant clones located where ligand levels are the highest, such as near the AP compartment boundary. These results imply that, like Wg, Dpp can signal in the absence of HSPGs, albeit at lower efficiency (Bornemann, 2004).

Initial reports that ttv was required for Hh, but not Wg or FGF, signaling, prompted speculation that Ttv might generate a Hh-specific HSPG, and that, unlike its mammalian orthologs, Drosophila Sotv might retain significant functional activity in the absence of its partner. However, the demonstration that Hh, Wg and Dpp signaling are affected in single mutants for ttv and for sotv, together with the biochemical analysis presented in this study, suggest that the mammalian model of EXT1 and EXT2 as obligate co-polymerases and applies equally well to Drosophila. The severe and comparable reductions in HS disaccharides observed in ttv and sotv null mutant larvae lend additional support to the co-polymerase model. Moreover, the fact that the phenotype of both single and ttv, sotv double mutants is indistinguishable, strongly suggests that any residual partner activity is not biologically significant (Bornemann, 2004).

In contrast to the null alleles, hypomorphic mutations of sotv that result in C-terminal truncations retain some biosynthetic activity that varies directly with the predicted length of the protein. Thus, sotv1.4.1, which encodes the longest mutant product, generates HS at 3.1% the wild-type level compared with 0.21% in the null allele sotv1.8.1. Retention of similar levels of function by truncated human EXT2 proteins could explain why all 27 of the disease-causing dominant mutations in EXT2 are clustered within the N-terminal two-thirds of the protein, rather than spread throughout its length. Perhaps C-terminal truncations retain sufficient function to supplement the HSPG levels from the wild-type copy and achieve the critical threshold required for normal bone growth. Alternatively, reduced levels or altered ratios of proteoglycans bearing specific sulfate modifications may contribute to the clinical manifestation of the disease. It is noted, for example, that the residual HS produced from null and sotv8.2 alleles contain little or no UA-GlcNAc6S. However, that species constitutes a substantial fraction of the HS produced by the less truncated alleles sotv18.2 and sotv1.4.1 (Bornemann, 2004).

Distinct and collaborative roles of Drosophila EXT family proteins in morphogen signalling and gradient formation

Heparan sulfate proteoglycans (HSPG) have been implicated in regulating the signalling activities of secreted morphogen molecules including Wingless (Wg), Hedgehog (Hh) and Decapentaplegic (Dpp). HSPG consists of a protein core to which heparan sulfate (HS) glycosaminoglycan (GAG) chains are attached. The formation of HS GAG chains is catalyzed by glycosyltransferases encoded by members of the EXT family of putative tumor suppressors linked to hereditary multiple exostoses. Previous studies in Drosophila demonstrated that tout-velu (ttv), the Drosophila EXT1, is required for Hh movement. However, the functions of other EXT family members are unknown. The other two members of the Drosophila EXT family genes, named sister of tout-velu (sotv) and brother of tout-velu (botv), have been identified and isolated. They encode Drosophila homologs of vertebrate EXT2 and EXT-like 3 (EXTL3), respectively. Both Hh and Dpp signalling activities, as well as their morphogen distributions, are defective in cells mutant for ttv, sotv or botv in the wing disc. Surprisingly, although Wg morphogen distribution is abnormal in ttv, sotv and botv, Wg signalling is only defective in botv mutants or ttv-sotv double mutants, and not in ttv nor sotv alone, suggesting that Ttv and Sotv are redundant in Wg signalling. It is demonstrated further that Ttv and Sotv form a complex and co-localize in vivo. These results, along with previous studies on Ttv, provide evidence that all three Drosophila EXT proteins are required for the biosynthesis of HSPGs, and for the gradient formation of the Wg, Hh and Dpp morphogens. These results also suggest that HSPGs have two distinct roles in Wg morphogen distribution and signalling (Han, 2004b).

These results demonstrate essential functions of all three Drosophila EXT family proteins in signalling events mediated by the Wg, Hh, and Dpp morphogens. Strong evidence that all three Drosophila EXT proteins are involved in HS biosynthesis and are required for the proper distributions of the morphogen molecules Wg, Hh, and Dpp. Interestingly, Wg signalling was found to be defective only in botv mutant or ttv-sotv double mutant cells, but not in ttv nor sotv mutant cells, which suggests partially redundant roles for Ttv and Sotv in Wg signalling. These results are consistent with a model in which Ttv and Sotv collectively function as a co-polymerase required for the biosynthesis of HS GAG chains, whereas Botv is likely to be involved in distinct step(s), possibly in the initiation of HS GAG biosynthesis. The results also suggest that HSPGs have two distinct roles in regulating Wg morphogen activity: a function in maintaining the extracellular Wg protein and a co-receptor role in Wg signalling (Han, 2004b).

Previous studies have demonstrated that Ttv is involved in Hh movement (Bellaiche, 1998; Gallet, 2003; The, 1999). Like Ttv, both Sotv and Botv are also required for Hh movement. Interestingly, Hh is detectable in the first row of mutant cells immediately adjacent to its posterior-producing cells. It is proposed that specific HSPGs modified by EXT family proteins are required for the movement of Hh from its expressing cells into the anterior-receiving cells. In the absence of EXT activities, Hh can be carried into the first row of mutant cells, but fails to move further. These results are consistent with previous work that demonstrated that the first row of ttv mutant cells can still transduce Hh signalling and can activate the expression of its downstream target gene patched (ptc) (Bellaiche, 1998). It is important to note that, while HSPGs modified by EXT family members are likely to be required for the movement of Hh, they may also be involved in preventing Hh from being degraded on the cell surface. In the absence of EXT proteins, Hh may be degraded and therefore it cannot reach the wild-type cells anterior to the clones of EXT mutant cells. It remains to be determined whether both mechanisms are involved in Hh transport (Han, 2004b).

Evidence is provided for the involvement of the three EXT proteins in Dpp signalling and its morphogen distribution. The results suggest that specific HSPG(s) modified by the three EXT proteins may promote Dpp morphogen signalling by modulating levels of Dpp morphogen ligands in Dpp-receiving cells. Previous studies have implicated a role for Dally, a Drosophila glypican member of HSPGs, in Dpp signalling in the development of imaginal discs. Elevated expression of Dally in the wing disc can promote Dpp signalling, as assayed by p-Mad levels. It has been proposed that Dally may act as a co-receptor for Dpp in activating its signalling. The results in this work suggest that HS GAG chains control Dpp signalling by modulating levels of Dpp morphogen in its receiving cells. It is proposed that the three EXT proteins are required for the biosynthesis of HS GAG chains on the Dally protein and that at least one of the roles of Dally in Dpp signalling is to modulate levels of the Dpp morphogen on its receiving cells (Han, 2004b).

Perhaps one of the most interesting findings in this work is the differential roles of the three EXT proteins in Wg morphogen signalling. Although mutations in any of the three EXT genes lead to reduced ranges of extracellular Wg distribution, it was found that Wg signalling is defective only in botv mutant cells, and not in either ttv or sotv mutant cells. However, ttv-sotv double mutants show virtually identical defects in Wg signalling to those of botv mutants. This qualitative difference between Botv and Ttv or Sotv in Wg signalling is unexpected since mutations in all three genes led to striking reductions in Hh and Dpp signalling (Han, 2004b).

Previous studies have implicated HSPGs in both Wg signalling and its morphogen gradient distribution. Embryos null for either sugarless or sulfateless, exhibit defects in Wg signalling in various tissues. The Drosophila glypican Dally has been shown to be required for Wg signalling. A reduction of dally activity can block the Wg signalling activity induced by overexpression of the Wg receptor Drosophila frizzled 2 (fz2) in the wing disc. Interestingly, recent studies have demonstrated that Notum (Wingful), a putative Drosophila pectin acetylesterase, can inhibit Wg signalling activity by modulating the activity of the heparin sulfate proteoglycans Dally-like (Dly) and Dally (Gerlitz, 2002; Giraldez, 2002). Overexpression of Notum can block the signalling activity of the Wg protein tethered to the cell surface by a transmembrane domain from Neurotactin, suggesting that Notum inhibits the function of proteoglycans that are involved in Wg signalling (Gerlitz, 2002). The function of HSPGs in Wg morphogen gradient distribution has also been demonstrated. In the wing disc, cells mutant for sulfateless showed a reduction in the levels of extracellular Wg. It has been shown that ectopic expression of Dly leads to the accumulation of extracellular Wg protein. Furthermore, loss of Notum function (Giraldez, 2002) leads to increased Wingless activity by altering the shape of the Wingless protein gradient (Han, 2004b).

Taken together, these results, along with previous studies, suggest that HSPGs are involved in both Wg signalling reception and in extracellular Wg morphogen distribution in the wing disc. It is suggested that HSPGs have at least two distinct functions in the wing disc: (1) in the distribution of the extracellular Wg protein, and (2) as a co-receptor for Wg signalling. In this regard, Botv is required both for Wg signalling and for its morphogen gradient formation, whereas Ttv and Sotv are only required for the distribution of extracellular Wg protein: they are functionally redundant in Wg signalling. Consistent with these observations, a previous report on ttv has shown that, during embryogenesis, Wg signalling in the stomatogastric nervous system (SNS) is not defective in ttv mutant embryos (The, 1999). It remains to be determined whether botv mutants and sotv-ttv double mutants exhibit Wg signalling defects in the SNS and in other tissues as well (Han, 2004b).

Previous analysis of Ttv has demonstrated its specificity in cell signalling (The, 1999). Although Hh signalling is defective in the ttv null mutant, neither Wg nor Fgf signalling is altered (The, 1999). It was proposed that Ttv is required for the synthesis of an Hh-specific HSPG. Consistent with the previous report, the current results demonstrate that Wg signalling is defective only in the ttv-sotv double mutant, and is not altered in either ttv or sotv single mutants. However, striking defects are also observed in both Dpp signalling and the range of extracellular Wg protein distribution in ttv and sotv mutants. Therefore, the previous view that Ttv is involved only in Hh signalling should be revised (Han, 2004b).

On the basis of genetic evidence and previous biochemical studies, it is proposed that Ttv and Sotv are likely to function as co-polymerases required for HS GAG polymerization, whereas Botv is likely to be involved in the initiation of HS GAG and is possibly involved in HS GAG polymerization as well. In the absence of Botv, no HS GAG chains are initiated and therefore mutations in Botv disrupt all the functions of HS GAG chains. However, in the absence of either Ttv or Sotv, initiation of HS GAG biosynthesis occurs, and the residual activity of HS GAG polymerase(s), carried out by another member (either Sotv or Ttv) together with Botv, may synthesize relatively short HS GAG chains that could act as co-receptors for Wg signalling, but have less capacity for maintaining the levels of secreted Wg, Hh and Dpp morphogen proteins. When the activities of both Ttv and Sotv are removed in the ttv-sotv double mutant, HS GAG polymerization may not occur because of the lack of GlcA transferase activity, even though Botv is present. In support of this view, previous biochemical studies have demonstrated that short HS GAG oligosaccharides have the capacity to form Fgf-Fgfr-HS complexes and to stimulate Fgf signalling (Pellegrini, 2001). In this regard, in the absence of Ttv, both Wg and Fgf signalling may occur. Consistent with this view, a previous study demonstrated that Fgf signalling is not affected in ttv mutant embryos (The, 1999). Biochemical studies on the activities of Ttv and Sotv as HS co-polymerases will further validate this view. An alternative model is that fewer intact HS GAG chains are synthesized in the absence of either Ttv or Sotv. Although this is less likely to be the case, the current results can not exclude this possibility (Han, 2004b).

Human mutations in EXT1 and EXT2 are associated with hereditary multiple exostoses (HME), which is an autosomal dominant disorder characterized by the formation of multiple cartilage-capped tumors (exostoses) of various bones. The results demonstrate the essential functions of both Ttv (EXT1) and Sotv (EXT2) in regulating the activities of the three secreted morphogen molecules Hh, Wg and Dpp. Wg and Dpp are the homologues of human WNT and bone morphogen protein (BMP) molecules, respectively. Since both WNT and BMP family proteins have been shown to be essential for bone growth and differentiation, the results suggest possible roles for WNT and BMP signalling in the generation of HME diseases associated with EXT1 and EXT2. The new findings together with previous work on the role of Ttv in Hh movement may provide new insights into the molecular mechanism(s) associated with HME disease (Han, 2004b).


Reference names in red indicate recommended papers.

Ahn, J., et al. (1995). Cloning of the putative tumor suppressor gene for hereditary multiple exostoses (EXT1). Nature Genet. 11: 137-143. 7550340 .

Bellaiche, Y., The, I. and Perrimon, N. (1998). Tout-velu is a Drosophila homologue of the putative tumour suppressor EXT-1 and is needed for Hh diffusion. Nature 394(6688): 85-88. 9665133

Bernfield, M., Gotte, M., Park, P. W., Reizes, O., Fitzgerald, M. L., Lincecum, J. and Zako, M. (1999). Functions of cell surface heparan sulfate proteoglycans. Annu. Rev. Biochem. 68: 729-777. 10872465

Blanchette, C. R., Thackeray, A., Perrat, P. N., Hekimi, S. and Benard, C. Y. (2017). Functional Requirements for Heparan Sulfate Biosynthesis in Morphogenesis and Nervous System Development in C. elegans. PLoS Genet 13(1): e1006525. PubMed ID: 28068429

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tout-velu: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation

date revised: 20 February 2017

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