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, meaning 'all hair,' has been identified: it is required for diffusion of Hedgehog. Ttv is involved in heparan sulfate proteoglycan (HSPG) biosynthesis, suggesting that HSPGs control Hh distribution (The, 1999). 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). Subsequent studies have shown striking defects in both Dpp signalling and the range of extracellular Wg protein distribution in ttv and the mutant sister of tout velu (sotv or Ext2), a gene that encodes a co-polymerase that synthesizes HSPG glycosaminoglycan (GAG) chains. Therefore, the previous view that Ttv is involved only in Hh signalling should be revised (Han, 2004b).

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. The interaction of Hh with HSPGs 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).

Heparan sulfate proteoglycans (HSPG) play important roles in signalling events controlled by secreted Wg, Hh and Dpp morphogens (Lander, 2000; Lin, 2000b; Nybakken, 2002; Perrimon, 2000). HSPGs consist of a protein core to which heparan sulfate (HS) glycosaminoglycan (GAG) chains are attached (Bernfield, 1999; Esko, 2002; Perrimon and Bernfield, 2000). The biosynthesis of HS GAG chains is initiated by the formation of a GAG-protein linkage region consisting of a tetrasaccharide (-GlcAß1-3Galß1-3Galß1-4Xylß-O-) attached to specific serine residues in a proteoglycan core protein (Bernfield, 1999; Esko, 2002). Following the transfer of alpha-GlcNAc as the first N-acetylhexosamine unit to this linkage region, heparin sulfate (HS) co-polymerases add alternating ß1-4-linked GlcA and alpha1-4-linked GlcNAc residues, generating HS GAG chains of 100 or more sugar units in length (Esko, 2002). Biochemical studies have demonstrated that both the attachment of the first alpha-GlcNAc to the GAG-protein linkage region and the subsequent polymer formation are catalyzed by members of the hereditary multiple exostoses (EXT) gene family of tumor suppressors (Esko, 2002; Zak, 2002). In vertebrates, the EXT gene family consists of EXT1, EXT2, and three EXT-like genes designated EXTL1, EXTL2 and EXTL3 (Zak, 2002). Human mutations in EXT1 and EXT2 are associated with hereditary multiple exostoses (HME), a benign bone tumor characterized by multiple cartilage-capped outgrowths of various bones. However, three EXT-like genes have not been demonstrated to be linked to genetic disorder(s). A number of biochemical studies have shown that EXT1 and EXT2 function as HS co-polymerases involved in HS polymerization. Recent biochemical studies also demonstrate that both EXTL2 and EXTL3 proteins possess enzymatic activities that can transfer alpha-GlcNAc to the GAG-protein linkage region and to intermediates of chain polymerization, suggesting roles for these proteins in initiation and polymerization reactions (Han, 2004b and references therein).

Thus studies have shown that Tout-velu 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, 2004a).

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, 2004a 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, 2004a).

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, 2004a).

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, 2004a).

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, 2004a).

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, 2004a).

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, 2004a).

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, 2004a).

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, 2004a).

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, 2004a).

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, 2004a).

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, 2004a).

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, 2004a).

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, 2004a).

GENE STRUCTURE

cDNA clone length - 3642 bases

Bases in 5' UTR - 262

Exons - 10

Bases in 3' UTR - 997

PROTEIN STRUCTURE

Amino Acids - 760

Structural Domains

Searches of annotated Drosophila genome databases identified a Drosophila EXT-like gene (CG15110) in 56A-56C and a Drosophila EXT2 (DEXT2; CG8433) in 52F. The Drosophila genome contains three EXT genes including ttv, CG15110 and CG8433. Based on the similarities of botv and sotv with ttv in both wing and embryonic cuticle defects, it was suspected that the CG15110 and CG8433 transcripts may encode Botv and Sotv, respectively. Two lines of evidence strongly suggest that this is indeed the case. First, the RNA interference (RNAi) method was used to perturb CG15110 and CG8433 transcripts. Embryos injected with either CG15110 or CG8433 double-stranded RNA showed segment-polarity defects. Second, all the sequenced alleles of botv and sotv have mutations in the CG15110 and CG8433 genes, respectively (Han, 2004b).

A phylogenetic tree of the EXT family members among humans, mouse and Drosophila was generated based on their amino acid sequences. Ttv is most similar to EXT1, whereas Sotv and Botv are more closely related with EXT2 and EXTL3, respectively. Ttv is 31.4% and 33.5% identical to Botv and Sotv proteins, respectively. In particular, all three Drosophila EXT members share high amino acid identity in their C-terminal regions (Han, 2004b).

date revised: 25 August 2004

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