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WO1999050385A2 - Regulation de la synthese de glycosaminoglycane, techniques apparentees et reactifs connexes - Google Patents

Regulation de la synthese de glycosaminoglycane, techniques apparentees et reactifs connexes Download PDF

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Publication number
WO1999050385A2
WO1999050385A2 PCT/US1999/006892 US9906892W WO9950385A2 WO 1999050385 A2 WO1999050385 A2 WO 1999050385A2 US 9906892 W US9906892 W US 9906892W WO 9950385 A2 WO9950385 A2 WO 9950385A2
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ttv
protein
signaling
cell
hedgehog
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PCT/US1999/006892
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WO1999050385A3 (fr
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Yohanns Bellaiche
Siu Inge The
Norbert Perrimon
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President And Fellows Of Harvard College
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Priority to AU33710/99A priority Critical patent/AU3371099A/en
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Publication of WO1999050385A3 publication Critical patent/WO1999050385A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase

Definitions

  • Growth factors and cytokines are important signaling molecules that control many physiological processes such as cell growth, division, differentiation, and migration. These molecules constitute some of the most important regulators of gene expression in eukaryotic systems. Much of the work to date focuses on the downstream effects of these molecules on cells, such as the initiation of signal transduction through the binding of the growth factor to its transmembrane receptor or the different second messenger pathways employed by certain signaling molecules. However, little attention has been paid to how the growth factors may be regulated through interactions with the ECM, from the time of their release until they form an active complex with their receptors at the cell surface.
  • ECM-growth factor interaction is especially critical in light of the fact that an increasing number of growth factors that have been found to form strong complexes with the ECM, specifically glycosaminoglycan-containing proteoglycans.
  • members of the fibroblast growth factor (FGF) family require the presence of glycosaminoglycan (GAG) chains to act as a cofactor for binding to, and activation of, FGF-receptors on the cell surface.
  • FGF fibroblast growth factor
  • GAG glycosaminoglycan
  • Platelet factor 4 binds heparin and is complexed to the missionoglycan serglycin upon secretion from platelet -alpha granules.
  • Membrane associated carbohydrate is exclusively in the form of oliogsaccharides covalently attached to proteins forming glycoproteins, and to a lesser extent covalently attached to lipid forming the glycolipids.
  • Glycoproteins consist of proteins covalently linked to carbohydrate. The predominant sugars found in glycoproteins are glucose, galactose, mannose, fucose, GalNAc, GlcNAc and NANA. The distinction between proteoglycans and glycoproteins resides in the level and types of carbohydrate modification. The carbohydrate modifications found in glycoproteins are rarely complex: carbohydrates are linked to the protein component through either O-glycosidic or N- glycosidic bonds.
  • the N-glycosidic linkage is through the amide group of asparagine.
  • the O-glycosidic linkage is to the hydroxyl of serine, threonine or hydroxylysine.
  • the linkage of carbohydrate to hydroxylysine is generally found only in the collagens.
  • the linkage of carbohydrate to 5 -hydroxylysine is either the single sugar galactose or the disaccharide glucosylgalactose.
  • the carbohydrate directly attached to the protein is GalNAc.
  • N-linked glycoproteins it is GlcNAc.
  • O-linked glycoproteins The synthesis of O-linked glycoproteins occurs via the stepwise addition of nucleotide-activated sugars directly onto the polypeptide.
  • the nucleotide-activated sugars are coupled to either UDP, GDP (as with mannose) or CMP (for instance, NANA).
  • the attachment of sugars is catalyzed by specific glycoprotein glycosyltransferases.
  • Evidence indicates that each specific type of carbohydrate linkage in O-linked glycoproteins is the result of a different glycosyltransferase.
  • the three major classes of N-linked carbohydrate modifications are high-mannose, hybrid and complex.
  • the major distinguishing feature of the complex class is the presence of sialic acid, whereas the hybrid class contains no sialic acid.
  • glycosaminoglycans The most abundant heteropolysaccharides in the body are the glycosaminoglycans (GAGs). These molecules are long unbranched polysaccharides containing a repeating disaccharide unit. The disaccharide units contain either of two modified sugars: N- acetylgalactosamine (GalNAc) or N-acetylglucosamine (GlcNAc), and a uronic acid such as glucuronate or iduronate. GAGs are highly negatively charged molecules, with extended conformation that imparts high viscosity to the solution. GAGs are located primarily on the surface of cells or in the extracellular matrix (ECM).
  • ECM extracellular matrix
  • GAGs Along with the high viscosity of GAGs comes low compressibility, which makes these molecules ideal for a lubricating fluid in the joints. At the same time, their rigidity provides structural integrity to cells and provides passageways between cells, allowing for cell migration.
  • the specific GAGs of physiological significance are hyaluronic acid, dermatan sulfate, chondroitin sulfate, heparin, heparan sulfate, and keratan sulfate. Although each of these GAGs has a predominant disaccharide component, heterogeneity does exist in the sugars present in the make-up of any given class of GAG.
  • proteoglycans and glycoproteins resides in the level and types of carbohydrate modification.
  • the carbohydrate modifications found in glycoproteins are rarely complex: carbohydrates are linked to the protein component through either O- glycosidic or N-glycosidic bonds.
  • the N-glycosidic linkage is through the amide group of asparagine.
  • the O-glycosidic linkage is to the hydroxyl of serine, threonine or hydroxylysine.
  • the linkage of carbohydrate to hydroxylysine is generally found only in the collagens.
  • the linkage of carbohydrate to 5 -hydroxylysine is either the single sugar galactose or the disaccharide glucosylgalactose.
  • the carbohydrate directly attached to the protein is GalNAc.
  • N-linked glycoproteins it is GlcNAc.
  • proteoglycans also called mucopolysaccharides
  • the GAGs extend perpendicularly from the core in a brush-like structure.
  • the linkage of GAGs to the protein core involves a specific trisacchari.de composed of two galactose residues and a xylulose residue (GAG- GalGalXyl-O-CH2 -protein).
  • the trisaccharide linker is coupled to the protein core through an O-glycosidic bond to a S residue in the protein.
  • Some forms of keratan sulfates are linked to the protein core through an N-asparaginyl bond.
  • the protein cores of proteoglycans are rich in S and T residues, which allows multiple GAG attachments.
  • HS-GAGs heparin-sulfate glycosaminoglycans
  • complex acidic polysaccharide polymers consisting of a disaccharide repeat unit of glucosamine and a uronic acid.
  • the carbohydrate backbone in heparin and HS consists of alternating hexuronic acid (D-glucuronic acid (GlcA) or L-iduronic acid (IdoA)) and D-glucosamine (GlcN) units.
  • the GlcN residues in heparin are predominantly N-sulfated, whereas those in HS show a more varied N-substitution pattern with appreciable proportions of both N-sulfated and N-acetylated and a smaller amount of N-unsubstituted GlcN units.
  • heparan sulfate proteoglycans are recognized as ubiquitous protein ligands. Binding of proteins to HS chains may serve a variety of functional purposes, from simple immobilization or protection against proteolytic degradation to distinct modulation of biological activity. Because of such interactions HS proteoglycans are critically involved in a variety of biological phenomena at various levels of complexity, including organogenesis in embryonic development, angiogenesis, regulation of blood coagulation and growth factor/cytokine action, cell adhesion, lipid metabolism, etc.
  • the present invention is directed to the discovery that there exists proteoglycans having specific glycosaminoglycan chains which provide extraordinar selectivity for binding to particular growth factors or cytokines, e.g., growth factor-specific GAG chains. Moreover, the specificity of the GAG chain structure, which gives rise to this binding specificity, is regulated by a biosynthetic pathway which can be selectively inhibited in order to antagonize the activity of a specific growth factor.
  • one aspect of the present invention provides drug discovery assays for identifying agents which can selectively alter the activity of a growth factor, cytokine or other serum proteins (collectively referred to hereinafter as "factors" for ease of reading).
  • the subject assay is designed to identify inhibitors of specific modifications of GAG chains, such as particular enzymes involved in, e.g. isomerization, sulfation, or acetylation which produce factor-specific binding by the resulting proteoglycan.
  • Such inhibitors can be used to alter the specificity of a proteoglycan for binding a particular factor (or family of related factors) by inhibiting specific modifications to the sugar sequence of the proteoglycan.
  • the assay identifies agents which selectively inhibit a sulfotransferase, preferably an N- deacetylase/N-sulfotransferase, involved in synthesis of a proteoglycan that binds a selected factor - so as to decrease the ability of the proteoglycan to bind the factor.
  • a sulfotransferase preferably an N- deacetylase/N-sulfotransferase
  • Another aspect of the present invention relates to a method for inhibiting the activity of a factor, in vitro or in vivo, by administering a compound which selectively inhibits synthesis of the factor-specific glycosaminoglycan in an amount sufficient to reduce the responsiveness of the treated cell(s) to the factor.
  • Such methods can be used to treat, e.g., unwanted cell proliferation or other unwanted effects of a factor.
  • Yet another aspect of the present invention provides a method for potentiating the activity of a selected factor, in vitro or in vivo, by administering a compound which selectively inhibits synthesis of GAG chains which bind other factors that compete with the selected factor, without substantially diminishing the synthesis of GAG chains which bind the selected factor.
  • the activity of the factor can be potentiated, e.g., its mitogenic, trophic or other activity enhanced.
  • this invention provides drug discovery assays for identifying agents which can modulate the biological function of selected factors, such as by (i) inhibiting the glycosyltransferase activity of an Ext protein, e.g. Extl; (ii) inhibiting a GAG-modifying activity of an enzyme associated with an Ext protein, e.g. a sulfotransferase; or (iii) inhibiting the interaction of an Ext protein with other enzymes involved in GAG synthesis, e.g. sulfotransferases.
  • the subject method can be used to inhibit hedgehog-mediated signal transduction by inhibiting synthesis of hedgehog specific GAG chains, e.g., by Ext-dependent pathways.
  • Such agents can be useful therapeutically to alter the growth, maintenance and/or differentiation of a tissue otherwise responsive to hedgehog proteins.
  • the subject inhibitors can be used to prevent hedgehog-dependent proliferation of cells, such as hedgehog-dependent basal cell carcinoma (BCC) or other hedgehog-dependent epithelia proliferative disorders.
  • the subject inhibitors can be used to alter the growth state of a mesodermally- derived tissue, such as cartilage.
  • this invention provides a method for identifying agents that are effective in amielorating at least one symptom of a disorder characterized by aberrant bone morphogenesis, such as multiple exostoses, chondrosarcomas, and or osteosarcomas.
  • the invention also helps in identifying agents useful therapeutically to inhibit hedgehog-mediated growth, maintenance and/or differentiation of a tissue involved in spermatogenesis and tissue derived from dorsal mesoderm; ectodermally-derived tissue, such as tissue derived from the epidermis, neural tube, neural crest, or head mesenchyme; endodermally-derived tissue, such as tissue derived from the primitive gut.
  • this invention provides drug discovery assays for identifying agents which can modulate the biological function of selected factors which bind to frizzled receptors, such as Wnt proteins.
  • the assays can be used to identify inhibitors of Wnt activity by (i) inhibiting Wnt-specfic modification of glypicans by a GAG-modifying activity, e.g. inhibiting a glypican-modifying N-deacetylase/N- sulfotransferase; (ii) inhibiting the interaction of a Wnt protein with a frizzled/glypican complex; or (iii) inhibiting formation of a a frizzled-glypican complex capable of binding to a Wnt protein.
  • the subject method can be used to inhibit Wnt-mediated signal transduction by inhibiting synthesis of Wnt-specific GAG chains, e.g., present on Wnt-specific glypicans.
  • Wnt-specific GAG chains e.g., present on Wnt-specific glypicans.
  • Such agents can be useful therapeutically to alter the growth, maintenance and/or differentiation of a tissue otherwise responsive to Wnt proteins.
  • the identification of modulators of Wnt activity are useful in treating disease states involving the Wnt activity.
  • Such compounds could be of use in the treatment of diseases in which activation or inactivation of the Wnt protein results in either cellular proliferation, cell death, nonproliferation, induction of cellular neoplastic transformations or metastatic tumor growth and hence could be used in the prevention and/or treatment of cancers such as bone and breast cancer for example.
  • the steps of the assay are repeated for a variegated library of at least 100 different test compounds, more preferably at least 10 ⁇ , 10 ⁇ or 10 ⁇ different test compounds.
  • the test compound can be, e.g., a peptide, a nucleic acid, a carbohydrate, a small organic molecule, or natural product extract (or fraction thereof).
  • the present invention provides diagnostic and therapeutic assays and reagents for detecting and treating disorders involving, for example, aberrant expression (or loss thereof) of an Ext glycosyltransferase gene, abberant expression of a dally N-deacetylase/N-sulfotransferase gene, or abberant expression of a glypican.
  • the present invention further contemplates the pharmaceutical formulation of one or more agents identified in such drug screening assays.
  • the present invention provides a molecule, preferably a small organic molecule, which is identified in the subject drug screening assays.
  • Yet another aspect of the present invention concerns a method for modulating one or more of growth, differentiation, or survival of a cell by treatment with a compound identify by the subject drug screening assay, e.g., by potentiating or inhibiting certain glycosyltransferase involved in factor-selective GAG synthesis.
  • the method comprises treating the cell with an effective amount of such an agent so as to alter, relative to the cell in the absence of treatment, at least one of (i) rate of growth, (ii) differentiation, or (iii) survival of the cell.
  • All discs are oriented as in a: A, anterior; P, posterior; V, ventral and D, dorsal. Blue lines indicate the A/P boundary. Dashed lines indicate the limit of the relevant mutant clones as determined by the absence of P-Galactosidase ( -Gal) staining (see below).
  • the ttv l(2)00681 allele which behaves as a genetic null and is a protein null (see figure 3d), was used to generate ttv somatic mutant clones.
  • a. Wild type Ptc expression, in red is detected with the Apal3 Mouse monoclonal antibody (Capdevila, J., Pariente, F., Sampedro, J., Alonso, J.L.
  • Ci is only expressed in the A compartment and it can be used as a marker for the A compartment.
  • the full length Ci protein is stabilized in response to Hh signaling as seen by a more intense staining. Note that the posterior limit of the domain of Ci stabilization does not correspond with the A/P boundary in late third instar wing discs due to the expression of Engrailed/Invected (En/Inv) in the A compartment.
  • the expression of En/Inv have been shown to be dependent on high level of Hh signaling (Strigini, M.
  • i shows the full disc where the relevant ttv clone is indicated by an arrow, j and k are magnifications of the clone, k, Wild type cells anterior to a ttv mutant clone do not stabilize Ci, although they are in a domain competent to respond to Hh signaling (see arrowheads).
  • the cell nonautonomous effect is directional because cells posterior to the clone respond to Hh signaling (see also h).
  • Within the clone the distance between the domain of Ci stabilization and the A/P boundary is reduced. This indicates that the level of Hh signaling is reduced since this distance is dependent on high levels of Hh signaling (Strigini, M. & Cohen, S.M.
  • Blue lines indicate A/P boundary. Dashed lines indicate the limits of the clones, unless they overlap with the A/P boundary. All discs are oriented as in Figure la. ttv l ( 2 ) 00681 and/?tc ⁇ alleles were used to generate the somatic mutant clones.
  • Hh is detected as a diffuse and as a punctate staining. When Hh reaches the wild type cells, its staining becomes more punctate and mostly coincides with punctate Ptc staining.
  • d A ptc ttv double mutant clone in the A compartment. Unlike what is observed in a ptc mutant clone alone, Hh staining cannot be detected in ptc ttv mutant clones.
  • the clone boundaries were identified by staining for P-Gal (not shown). e, f, g. Ci staining in green (e, g) and -Gal staining in red (e, f).
  • e shows the full disc with the relevant posterior clone indicated by an arrow, f and g are magnification of the clone.
  • the absence of -Gal staining is used as a marker for the clone.
  • Loss of ttv activity in the P compartment does not affect the diffusion of Hh in the A compartment since Ci is still stabilized in the A compartment in wild type cells adjacent to a ttv mutant clone induced in the P compartment.
  • Ci stabilization is not detected directly adjacent to the A/P boundary (Strigini, M. & Cohen, S.M. Development A Hedgehog activity gradient contributes to AP axial patterning of the Drosophila wing. 124, 4697-4705 (1997)). This shift (see bars
  • Figure 3 Cloning of the ttv locus.
  • a Genomic map of the ttv locus.
  • the P-element, ttv ⁇ 2)00681 maps to the 51 A polytene chromosome band which is deleted in Df(2R)Trix. It is inserted in the sixth intron of the ttv gene, which also includes the lamin C gene. Only introns of more than 1 kb are represented.
  • Dl, G10 and B 12 are three overlapping cosmids that were previously mapped to the region (Dickson, B.J., Dominguez, M., van der Straten, A. & Col, E.
  • Ttv is a type II integral membrane protein.
  • Ttv is detected as a higher migrating band (lane 3). This shift can be abolished by endoglycosydase H treatment, indicating that the Ttv protein is glycosylated in the presence of microsomes (lane 4). The full length Ttv protein enters microsomes since Ttv is protected from proteinase K digestion in the presence of intact microsomes (lanes 5 and 6). Ttv remains associated with the membrane fraction after alkaline wash (lane 7 and 8), indicating that Ttv is a membrane protein. c. Proposed structure for the Ttv protein. Note that the cytoplasmic tail is only six amino acids in length and is identical in Ext-1 and Ttv.
  • Ttv to the membrane by a glycoyl-phophatidylinositol (GPI) anchor since GPI anchor proteins are characterized by a large hydrophobic region at the C-terminus (Low, M.G. Biochim Biophys Acta. The glycosyl-phosphatidylinositol anchor of membrane proteins. 988, 427-454 (1989)) not present in Ttv.
  • GPI glycoyl-phophatidylinositol
  • Figure 5 Scheme illustrating the generation of the typical domain structure of a HS chain during polysaccharide biosynthesis.
  • the symbols used are defined by the structures illustrated below the scheme.
  • the open circle attached to the internal GlcNSO3 unit of the antithrombin-binding pentasaccharide sequence (AT) denotes a 3-O-sulfate group.
  • the target residues for the various enzymes (NDST, GlcA C-5 epimerase (EPI), 2-OST, 6-OST, and 3-OST) are indicated by arrows and by red residue symbols.
  • the arrows within parentheses indicate (arbitrary) sites of variable polymer modification, i.e. residues that satisfy the substrate specificity of the indicated reactions but nevertheless escape target selection.
  • Two NDST isoforms are indicated, along with their potential target residues.
  • isoforms of other enzymes see the text.
  • the reducing terminus is to the right.
  • Figures 6A-C Ttv is involved in HSPGs biosynthesis
  • the decrease staining is not due to variability in staining conditions because when ttv is ectopically expressed in a hairy pattern in ttv embryos, the strong 3G10 staining is recovered in the hairy striped pattern.
  • the predicted Molecular Weight of Ttv protein is 80 kD and a band comigrating with the 83 kD marker is detected in wild type and not in ttv embryos.
  • UAS-ttvwyc construct in the hairy domain In these panels, Myc staining is shown in green and Hh in red.
  • G Apical and (H) basolateral confocal section of wild type wing imaginal disc expressing UAS-ttvmyc in the Ptc-Gal4 domain. In these panels, Myc staining is shown in red, and Ptc in green.
  • Twi staining visualized by Twi staining in wild type (A and C) and ttv embryos (B and D) at stage 9.
  • a and C are ventral views of whole embryos
  • C and D are transverse sections.
  • Twi stained cells can be seen as a band with smooth edge on the ventral side of stage 10 embryos and cross sections through these embryos show a monolayer of Twi-stained cells.
  • Twi positive cells can be seen as a band with a rough edge on the ventral side and sections through these embryos will show the cells clustered near the ventral midline.
  • a and B are lateral views, C and D ventral views.
  • G Staining of HhN in ttv embryos expressing HhN under control of en promoter (en-GAL4 ; UAS-HhN).
  • H ttv embryos expressing en-HhN stained for both Hh in green and En in red.
  • HS-GAGs are synthesized by a complex that includes Ttv and specific GAG- modifying enzyme(s) such as a sulfotransferases and C-5 epimerases.
  • specific GAG- modifying enzyme(s) such as a sulfotransferases and C-5 epimerases.
  • Different Ext complexes might include specific sulfotransferases not found in complex with other Ext proteins.
  • induction a type of extracellular communication
  • chemical signals secreted by one cell population influence the developmental fate of a second cell population.
  • cells responding to the inductive signals are diverted from one cell fate to another, neither of which is the same as the fate of the signaling cells.
  • Glycosaminoglycans are sugars composed of long unbranched chains of repeating disaccharides, sometimes reaching hundreds of residues (reviewed in Kjellen, L. and Lindahl, U. (1991) Annu. Rev. Biochem. 60:443-475). These sugar chains are usually linked to a core protein forming a proteoglycan. There are two types of UDP-GlcDH-dependent glycosaminoglycans.
  • the glucosaminoglycans such as heparin and heparan sulfate, are synthesized as [GlcA ⁇ l,4- GlcNAc ⁇ l,4-] n ;
  • the galactosaminoglycans such as chondroitin sulfate, are synthesized as [GlcA ⁇ l,3- GalNAc ⁇ l,4-] n .
  • the sugars are modified and extensively sulfated, giving the chains a strong negative charge.
  • Different families within each chain type are generated by varying the kinds of modifications and the extent of sulfation.
  • proteoglycans can regulate growth factor and cytokine activity in vitro.
  • the ligand binds to the glycosaminoglycan chains on cell surface proteoglycans. This interaction can be quite strong; for example, the K_ for fibroblast growth factor (FGF) binding to syndecan, a heparan sulfated proteoglycan, is 10- 9 M (Moscatelli, D. (1987) J. Cell. Physiol. 131:123-130; Klagsbrun, M. And Baird, A. (1991) Cell 67:229-231). Given the affinity of many growth factors for proteoglycans and the relative abundance of the latter, it is likely that most secreted growth factors are not freely diffusible but are, instead, sequestered on proteoglycans.
  • FGF fibroblast growth factor
  • the present invention is directed to the discovery that there exists proteoglycans having specific glycosaminoglycan chains which provide extraordinar selectivity for binding to particular growth factors or cytokines, e.g., growth factor-specific GAG chains. That is, there exists in cells proteoglycans that,by virtue of the particular modifications to the GAG chains of the protein, selectively bind one factor (or family or related factors). Moreover, the specificity of the GAG chain structure, which gives rise to this binding specificity, is regulated by a biosynthetic pathway which can be selectively inhibited in order to antagonize the activity of a specific growth factor. To illustrate, one aspect of the present invention concerns the discovery that members of the Ext-family are required for Hh signaling.
  • the inventors analyzed the function of Ttv in the wing imaginal disc of Drosophila.
  • Hh is expressed in the cells of the posterior compartment and diffuse to the anterior compartment.
  • Such diffusion may be detected by detecting Patched (Ptc) expression or Cubitus interruptus (Ci) stabilization, which are known targets of Hh signaling. It was observed that Hh is unable to diffuse in the absence of Ttv activity.
  • the Ext genes encode a family of glycosyltransferases.
  • Ttv a Drosophila Ext homolog
  • hedgehog signaling suggests the existence of hedgehog-specific GAG chains.
  • GAG modifying enzymes such as sulfotransferases, deacetylases and the like. Each complex can generate qualitatively different GAG sequences with unique specificity for binding cytokines and other serum proteins.
  • Example 6 (below) further illustrates the invention. Characterization of the
  • Drosophila gene sulfateless which encodes a homolog of a vertebrate heparan sulfate N- deacetylase/N-sulfotransferase (a member of a class of enzymes essential for the modification of heparan sulfate), reveals that HS proteoglycans are necessary for Wg/Wnt signaling.
  • HS proteoglycans are necessary for Wg/Wnt signaling.
  • a GPI-linked Glypican as the HSPG molecule involved in Wg signaling. Loss of dally activity, both in the embryo and imaginal dies, generates phenotypes reminiscent to loss of Wg activity.
  • glypicans serve as co-receptors for the Wnt receptor, e.g., frizzled proteins, and together with frizzled proteins, modulate both short and long-range activities of Wnt proteins.
  • one aspect of the present invention provides drug discovery assays for identifying agents which can selectively alter the activity of a growth factor, cytokine or other serum proteins (collectively referred to hereinafter as "factors" for ease of reading).
  • the subject assay is designed to identify inhibitors of specific modifications of GAG chains, such as particular enzymes involved in, e.g. isomerization, sulfation, or acetylation which produce factor-specific binding by the resulting proteoglycan.
  • Such inhibitors can be used to alter the specificity of a proteoglycan for binding a particular factor (or family of related factors) by inhibiting specific modifications to the sugar sequence of the proteoglycan.
  • the assay identifies agents which selectively inhibit a sulfofransferase, preferably an N- deacetylase/N-sulfotransferase, involved in synthesis of a proteoglycan that binds a selected factor - so as to decrease the ability of the proteoglycan to bind the factor.
  • Another aspect of the present invention relates to a method for inhibiting the activity of a factor, in vitro or in vivo, by administering a compound which selectively inhibits synthesis of the factor-specific glycosaminoglycan in an amount sufficient to reduce the responsiveness of the treated cell(s) to the factor.
  • Such methods can be used to treat, e.g., unwanted cell proliferation or other unwanted effects of a factor.
  • Yet another aspect of the present invention provides a method for potentiating the activity of a selected factor, in vitro or in vivo, by administering a compound which selectively inhibits synthesis of GAG chains which bind other factors that compete with the selected factor, without substantially diminishing the synthesis of GAG chains which bind the selected factor.
  • the activity of the factor can be potentiated, e.g., its mitogenic, trophic or other activity enhanced.
  • this invention provides drug discovery assays for identifying agents which can modulate the biological function of selected factors, such as by (i) inhibiting the glycosyltransferase activity of an Ext protein, e.g. Extl; (ii) inhibiting a GAG-modifying activity of an enzyme associated with an Ext protein, e.g. a sulfofransferase; or (iii) inhibiting the interaction of an Ext protein with other enzymes involved in GAG synthesis, e.g. sulfotransferases.
  • the subject method can be used to inhibit hedgehog-mediated signal transduction by inhibiting synthesis of hedgehog specific GAG chains, e.g., by Ext-dependent pathways.
  • Such agents can be useful therapeutically to alter the growth, maintenance and/or differentiation of a tissue otherwise responsive to hedgehog proteins.
  • the subject inhibitors can be used to prevent hedgehog-dependent proliferation of cells, such as hedgehog-dependent basal cell carcinoma (BCC) or other hedgehog-dependent epithelia proliferative disorders.
  • the subject inhibitors can be used to alter the growth state of a mesodermally- derived tissue, such as cartilage.
  • this invention provides a method for identifying agents that are effective in amielorating at least one symptom of a disorder characterized by aberrant bone morphogenesis, such as multiple exostoses, chondrosarcomas, and/or osteosarcomas.
  • the invention also helps in identifying agents useful therapeutically to inhibit hedgehog-mediated growth, maintenance and/or differentiation of a tissue involved in spermatogenesis and tissue derived from dorsal mesoderm; ectodermally-derived tissue, such as tissue derived from the epidermis, neural tube, neural crest, or head mesenchyme; endodermally-derived tissue, such as tissue derived from the primitive gut.
  • this invention provides drug discovery assays for identifying agents which can modulate the biological function of selected factors which bind to frizzled receptors, such as Wnt proteins.
  • the assays can be used to identify inhibitors of Wnt activity by (i) inhibiting Wnt-specfic modification of glypicans by a GAG-modifying activity, e.g. inhibiting a glypican-modifying N-deacetylase/N- sulfofransferase; (ii) inhibiting the interaction of a Wnt protein with a frizzled/glypican complex; or (iii) inhibiting formation of a a frizzled-glypican complex capable of binding to a Wnt protein.
  • a GAG-modifying activity e.g. inhibiting a glypican-modifying N-deacetylase/N- sulfofransferase
  • inhibiting the interaction of a Wnt protein with a frizzled/glypican complex e.g. inhibiting a glypican-modifying N-deacetylase/N- sulfofransferase
  • the subject method can be used to inhibit Wnt-mediated signal transduction by inhibiting synthesis of Wnt-specific GAG chains, e.g., present on Wnt-specific glypicans.
  • Wnt-specific GAG chains e.g., present on Wnt-specific glypicans.
  • Such agents can be useful therapeutically to alter the growth, maintenance and/or differentiation of a tissue otherwise responsive to Wnt proteins.
  • the identification of modulators of Wnt activity are useful in treating disease states involving the Wnt activity.
  • Such compounds could be of use in the treatment of diseases in which activation or inactivation of the Wnt protein results in either cellular proliferation, cell death, nonproliferation, induction of cellular neoplastic transformations or metastatic tumor growth and hence could be used in the prevention and/or treatment of cancers such as bone and breast cancer for example.
  • the present invention provides diagnostic and therapeutic assays and reagents for detecting and treating disorders involving, for example, aberrant expression (or loss thereof) of an Ext glycosyltransferase gene, abberant expression of a dally N-deacetylase/N-sulfotransferase gene, or abberant expression of a glypican.
  • nucleic acid refers to polynucleotides such as deoxyribonucleic acid (DNA), and, where appropriate, ribonucleic acid (RNA).
  • DNA deoxyribonucleic acid
  • RNA ribonucleic acid
  • the term should also be understood to include, as equivalents, analogs of either RNA or DNA made from nucleotide analogs, and, as applicable to the embodiment being described, single (sense or antisense) and double-stranded polynucleotides.
  • the term “gene” or “recombinant gene” refers to a nucleic acid comprising an open reading frame encoding a polypeptide, including both exon and (optionally) intron sequences.
  • a “recombinant gene” refers to nucleic acid encoding a polypeptide, e.g., comprising coding exon sequences and (optionally) intron sequences.
  • the term “intron” refers to a DNA sequence present in a given gene which is not translated into protein and is generally found between exons.
  • the term “transfection” means the introduction of a nucleic acid, e.g., an expression vector, into a recipient cell by nucleic acid-mediated gene transfer.
  • "Transformation” refers to a process in which a cell's genotype is changed as a result of the cellular uptake of exogenous DNA or RNA, and, for example, the transformed cell expresses a recombinant form of a polypeptide or, where anti-sense expression occurs from the transferred gene, the expression of a naturally-occurring form of the protein is disrupted.
  • the term “specifically hybridizes” refers to the ability of a nucleic acid probe/primer of the invention to hybridize to at least 15 consecutive nucleotides of a gene, such as a Ext sequence designated in any one or more of S ⁇ Q ID Nos: 1-3 or a sequence complementary thereto, or naturally occurring mutants thereof, such that it has less than 15%, preferably less than 10%, and more preferably less than 5% background hybridization to a cellular nucleic acid (e.g., mRNA or genomic DNA) encoding a protein other than a protein, as defined herein.
  • a cellular nucleic acid e.g., mRNA or genomic DNA
  • vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • One type of preferred vector is an episome, i.e., a nucleic acid capable of extra-chromosomal replication.
  • Preferred vectors are those capable of autonomous replication and/expression of nucleic acids to which they are linked.
  • Vectors capable of directing the expression of genes to which they are operatively linked are referred to herein as "expression vectors”.
  • expression vectors of utility in recombinant DNA techniques are often in the form of "plasmids" which refer generally to circular double stranded DNA loops which, in their vector form are not bound to the chromosome.
  • plasmid and "vector” are used interchangeably as the plasmid is the most commonly used form of vector.
  • vector is intended to include such other forms of expression vectors which serve equivalent functions and which become known in the art subsequently hereto.
  • Transcriptional regulatory sequence is a generic term used throughout the specification to refer to DNA sequences, such as initiation signals, enhancers, and promoters, which induce or control transcription of protein coding sequences with which they are operably linked.
  • transcription of a recombinant Ext gene is under the control of a promoter sequence (or other transcriptional regulatory sequence) which controls the expression of the recombinant gene in a cell-type in which expression is intended. It will also be understood that the recombinant gene can be under the control of transcriptional regulatory sequences which are the same or which are different from those sequences which control transcription of the naturally-occurring forms of Ext genes.
  • tissue-specific promoter means a DNA sequence that serves as a promoter, i.e., regulates expression of a selected DNA sequence operably linked to the promoter, and which effects expression of the selected DNA sequence in specific cells of a tissue, such as cells of neuronal or hematopoietic origin.
  • tissue-specific promoter means a DNA sequence that serves as a promoter, i.e., regulates expression of a selected DNA sequence operably linked to the promoter, and which effects expression of the selected DNA sequence in specific cells of a tissue, such as cells of neuronal or hematopoietic origin.
  • the term also covers so-called “leaky” promoters, which regulate expression of a selected DNA primarily in one tissue, but can cause at least low level expression in other tissues as well.
  • a “chimeric protein” or “fusion protein” is a fusion of a first amino acid sequence encoding a first polypeptide with a second amino acid sequence defining a domain (e.g. polypeptide portion) foreign to and not substantially homologous with any domain of the firsst protein.
  • a chimeric protein may present a foreign domain which is found (albeit in a different protein) in an organism which also expresses the first protein, or it may be an "interspecies", “intergenic”, etc. fusion of protein structures expressed by different kinds of organisms.
  • a fusion protein in general, can be represented by the general formula X-P-Y, wherein P represents a portion of the fusion protein which is derived from a first protein, and X and Y are, independently, absent or represent amino acid sequences which are not related to the P sequences in an organism.
  • substantially homologous when used in connection with amino acid sequences, refers to sequences which are substantially identical to or similar in sequence, giving rise to a homology in conformation and thus to similar biological activity. The term is not intended to imply a common evolution of the sequences.
  • percent identical refers to sequence identity between two amino acid sequences or between two nucleotide sequences. Identity can each be determined by comparing a position in each sequence which may be aligned for purposes of comparison.
  • the molecules are identical at that position; when the equivalent site occupied by the same or a similar amino acid residue (e.g., similar in steric and/or electronic nature), then the molecules can be referred to as homologous (similar) at that position.
  • homologous similar
  • Expression as a percentage of homo logy/similarity or identity refers to a function of the number of identical or similar amino acids at positions shared by the compared sequences.
  • Various alignment algorithms and/or programs may be used, including FASTA, BLAST or ENTREZ.
  • FASTA and BLAST are available as a part of the GCG sequence analysis package (University of Wisconsin, Madison, Wis.), and can be used with, e.g., default settings. ENTREZ is available through the National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, Md.
  • the percent identity of two sequences can be determined by the GCG program with a gap weight of 1, e.g., each amino acid gap is weighted as if it were a single amino acid or nucleotide mismatch between the two sequences.
  • ortholog refers to genes or proteins which are homologs via speciation, e.g., closely related and assumed to have common descent based on structural and functional considerations. Orthologous proteins function as recognizably the same activity in different species.
  • paralog refers to genes or proteins which are homologs via gene duplication, e.g., duplicated variants of a gene within a genome. See also, Fritch, WM (1970) Syst Zool 19:99-113.
  • Ext-family protein or "Ext-family polypeptide” refers to a family of polypeptides characterized at least in part by being identical or sharing a degree of sequence homology with all or a portion of the Ext-family polypeptides represented in any of SEQ ID Nos: 4-6.
  • the Ext-family polypeptides can be cloned or purified from any of a number of invertebrate or vertebrate organisms, especially vertebrates, and particularly mammals.
  • other Ext-family polypeptides can be generated according to the present invention, which polypeptides do not ordinarily exist in nature, but rather are generated by non-natural mutagenic techniques.
  • vertebrate hedgehog protein refers to vertebrate intercellular signaling molecules related to the drosophila hedgehog protein.
  • Three of the vertebrate hedgehog proteins Desert hedgehog (Dhh), Sonic hedgehog (Shh) and Indian hedgehog (Ihh), apparently exist in all vertebrates, including amphibians, fish, birds, and mammals.
  • Other members of this family such as Banded hedgehog, Cephalic hedgehog, tiggy-winkle hedgehog, and echidna hedgehog have been so far identified in fish and/or amphibians.
  • Exemplary hedgehog polypeptides are described in PCT applications WO96/17924, WO96/16668, WO95/18856.
  • Wnts or "Wnt gene product” or " Wnt polypeptide” when used herein encompass native sequence Wnt polypeptides, Wnt polypeptide variants, Wnt polypeptide fragments and chimeric Wnt polypeptides.
  • the definition specifically includes human Wnt polypeptides, Wnt -1, 2, 3a, 3b, , 4, 5a, 5b, 6, 7a, 7b, 8a, 8b, 10a, 10b, 11, and 12.
  • transforming growth factor-beta and "TGF-" denote a family of structurally related paracrine polypeptides found ubiquitously in vertebrates, and prototypic of a large family of metazoan growth, differentiation, and morphogenesis factors (see, for review, Massaque et al. (1990) Ann Rev Cell Biol 6:597-641; and Sporn et al. (1992) J Cell Biol 119:1017-1021). Included in this family are the "bone morphogenetic proteins” or "BMPs”, which refers to proteins isolated from bone, and fragments thereof and synthetic peptides which are capable of inducing bone deposition alone or when combined with appropriate cofactors.
  • BMPs bone morphogenetic proteins
  • BMPs such as BMP-1, -2, -3, and -4
  • BMP-7 Preparation of BMPs, such as BMP-1, -2, -3, and -4, is described in, for example, PCT publication WO 88/00205.
  • Wozney (1989) Growth Fact Res 1:267-280 describes additional BMP proteins closely related to BMP-2, and which have been designated BMP-5, -6, and -7.
  • PCT publications WO89/09787 and WO89/09788 describe a protein called "OP-1," now known to be BMP-7.
  • Other BMPs are known in the art.
  • Cells “host cells” or “recombinant host cells” are terms used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.
  • phenotype refers to the entire physical, biochemical, and physiological makeup of a cell, e.g., having any one trait or any group of traits.
  • enhancing differentiation of a cell is meant the act of increasing the extent of the acquisition or possession of one or more characteristics or functions which differ from that of the original cell (i.e., cell specialization). This can be detected by screening for a change in the phenotype of the cell (e.g., identifying morphological changes in the cell and/or surface markers on the cell).
  • enhancing survival or maintenance of a cell encompasses the step of increasing the extent of the possession of one or more characteristics or functions which are the same as that of the original cell (i.e., cell phenotype maintenance).
  • induction refers generally to the process or act of causing to occur a specific effect on the phenotype of cell.
  • Such effect can be in the form of causing a change in the phenotype, e.g., proliferation, differentiation to another cell phenotype, secretion, or can be in the form of maintaining the cell, e.g., preventing dedifferentation or promoting survival of a cell.
  • target tissue refers to connective tissue, cartilage, bone tissue or limb tissue, which is either present in an animal, e.g., a mammal, e.g., a human or is present in in vitro culture, e.g., a cell culture.
  • an "effective amount" of an agent refers to an amount of agonist or antagonist in a preparation which, when applied as part of a desired dosage regimen, provides modulation of growth, differentiation or survival of cells, e.g., modulation of skeletogenesis, e.g., osteogenesis, chondrogenesis, or limb patterning, spermatogenesis, and neuronal differentiation.
  • modulation of skeletogenesis e.g., osteogenesis, chondrogenesis, or limb patterning, spermatogenesis, and neuronal differentiation.
  • a “patient” or “subject” to be treated can mean either a human or non-human animal.
  • small organic molecule refers to a non-peptide, non-nucleotide organic compound having a molecular weight less than 7500amu, more preferably less than 2500amu, and even more preferably less than 750amu.
  • a "reporter gene construct” is a nucleic acid that includes a “reporter gene” operatively linked to a transcriptional regulatory sequences. Transcription of the reporter gene is controlled by these sequences. The activity of at least one or more of these control sequences is directly or indirectly regulated by a signal transduction pathway involving a phospholipase, e.g., is directly or indirectly regulated by a second messenger produced by the phospholipase activity.
  • the transcriptional regulatory sequences can include a promoter and other regulatory regions, such as enhancer sequences, that modulate the activity of the promoter, or regulatory sequences that modulate the activity or efficiency of the RNA polymerase that recognizes the promoter, or regulatory sequences that are recognized by effector molecules, including those that are specifically induced upon activation of a phospholipase.
  • modulation of the activity of the promoter may be effected by altering the RNA polymerase binding to the promoter region, or, alternatively, by interfering with initiation of transcription or elongation of the mRNA.
  • Such sequences are herein collectively referred to as transcriptional regulatory elements or sequences.
  • the construct may include sequences of nucleotides that alter the stability or rate of translation of the resulting mRNA in response to second messages, thereby altering the amount of reporter gene product.
  • an isolated nucleic acid encoding a polypeptide preferably includes no more than 10 kilobases (kb) of nucleic acid sequence which naturally immediately flanks the gene in genomic DNA, more preferably no more than 5kb of such naturally occurring flanking sequences, and most preferably less than 1.5kb of such naturally occurring flanking sequence.
  • kb kilobases
  • isolated also refers to a nucleic acid or peptide that is substantially free of cellular material, or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized.
  • isolated nucleic acid is meant to include nucleic acid fragments which are not naturally occurring as fragments and would not be found in the natural state.
  • the present invention provides drug screening assays for identifying pharmaceutically effective compounds that specifically potentiate or inhibit the activity of a growth factor, cytokine or other serum protein.
  • One aspect of the present invention is directed to methods for screening compounds which inhibit the synthesis of factor- selective glycosaminoglycan chains, e.g., by inhibiting one or more glycosyltransferases which are essential and/or specific for synthesis of a factor selective GAG.
  • a "factor- selective GAG” is one which confers a selective binding activity to a proteoglycan for a selected factor.
  • a factor-selective GAG can be characterisexed by it ability to confer a Kj for binding to a selected factor (or family of related factors) which is at least one order of magnitude, more preferably two, three or four orders of magnitude less than the K ⁇ for other factors which are unrelated to the selected factor.
  • the factor-selectivity of the GAG chain may be characterized by its ability to confer an IC50 or EC50 for an activity of the selected factor which is at least one order of magnitude, more preferably two, three or four orders of magnitude less than the IC 5Q or
  • an "unrelated factor” is one which does not share sufficient homology or biological activity to be considered an ortholog or paralog of the selected factor, e.g., it may be less than 50, 60, 70, 80 or 90 percent identical to the selected factor.
  • the assay can be formated to identify an inhibitor of a glycosyltransferase(s) such as, for example: an N- acetylgalactosaminyltransferase (GalNAc transferase) such as EC 2.4.1.40, EC 2.4.1.92, EC 2.4.1.41; an N-acetylglucosaminyltransferase (GlcNAc transferase) such as EC 2.4.1.101, EC 2.4.1.143, EC 2.4.1.144, EC 2.4.1.155; an N-acetylglucosaminyl-1- phosphate transferase (GlcNAc- 1-P transferase) such as EC 2.7.8.15; a Fucosyltransferase (Fuc transferase) such as EC 2.4.1.69, EC 2.4.1.65; a galactosyltransferase (Gal transferase) such as EC 2.
  • the assay is formated to identify inhibitors of one or more glycosaminoglycan sulfotransferases or glycosaminoglycan deacetylases.
  • Glycosaminoglycan N-deacetylases/N-sulfotransferases are structurally related enzymes that play an important role in the biosynthesis of heparan sulfate and heparin. They are dual catalytic, single membrane-spanning polypeptides of approximately 850-880 amino acids that catalyze the N-deacetylation of N-acetylglucosamine of glycosaminoglycans followed by N-sulfation of the same sugar.
  • the sulfofransferase is selected from amongst the EC (Enzyme Commission) sulfofransferase sub-subclass 2.8.2.-, e.g., an aryl sulfofransferase (EC 2.8.2.1), an arylamine sulfofransferase (EC 2.8.2.3), a chondroitin 4-sulfotransferase (EC 2.8.2.5), a UDP-N-acetylgalactosamine-4-sulfate sulfofransferase (EC 2.8.2.7), a desulfoheparin sulfofransferase (EC 2.8.2.8), a galactosylceramide sulfofransferase (EC 2.8.2.11), a heparitin sulfofransferase (EC 2.8.2.12), a chondroitin
  • the glycosyltransferase is an N-deacetylase (EC 3.1.1.-) / N-sulfotransferase (EC 2.8.2.-).
  • the glycosyltransferase is sulfofransferase which interacts with an Ext protein and/or is essential for hedgehog-specific GAG chains.
  • the glycosyltransferase is sulfofransferase which is essential for Wnt-specific GAG chains, e.g., is essential for synthesis of glypicans which selectively interact with Wnt proteins.
  • sulfotransferases which can be used in the subject assay are described in Tables 1 and 2.
  • the subject assay can also be carried out using related homologs, e.g., which are at least 75, 85, 90 or 95 percent identical to one of the sulfotransferases enumerated herein, or which hybridize under under stringent to highly stringent hybridization and wash conditions (e.g., 2 x SSC at 50°C to 0.2 x SSC at 50°C).
  • the subject assay can be carried out by any of a variety of drug screening techniques. Such assays can be used to identify agents, especially small organic molecules (preferably ⁇ 750 amu) which either potentiate or inhibit the glycosyltransferase activity of one or more enzymes involved in synthesis of a factor-selective GAG chain.
  • the enzymes, or fragments thereof may either be free in solution, affixed to a solid support, or provided in a host cell.
  • the assay is preferably repeated for a variegated library of at least 100 different test compounds, though preferably libraries of at least 10 ⁇ , 10 ⁇ , 10 ⁇ , and 10 ⁇ compunds are tested.
  • the test compound can be, for example, small organic molecules, and/or natural product extracts.
  • a selected glycosyltransferase is isolated in solution, e.g., as a reconsitituted (or otherwise purified) protein preparation or from a cell- lysate.
  • the enzyme is contacted with an appopriate substrate and a test compound, and rate of the enzyme-mediated conversion of the substrate to product is assessed and compared to the rate of conversion in the absense of the test compound.
  • an N-deacetylase EC 3.1.1.-
  • N-sulfotransferase a variety of assays have been developed in the art to detect such activities, and can be readily adapted for use in the subject methods.
  • N-deacetylase activity can be measured by determining the release of [ ⁇ HJacetate from N-[3H]acetylated-polysaccharide, e.g., isolated from E. coli K5-derived capsular polysaccharide as described (Bame et al., (1991) J. Biol. Chem. 266:12461-12468,).
  • N-sulfotransferase and 0-sulfofransferase activities can be measured by determining 35sc>4-incorporation into N-desulfated heparin and completely desulfated,
  • N-resulfated heparin respectively, from 35s-labeled adenosine 3 'phosphate phosphosulfate (PAPS) as described (Ishihara et al., (1992) Anal. Biochem. 206:400-407).
  • PAPS 35s-labeled adenosine 3 'phosphate phosphosulfate
  • Cocktail Buffer (for one reaction only): 25 ⁇ l of 50 mM potassium phosphate buffer, pH 6.5, 25 ⁇ l of dithiothreitol (7.4 mg/ml) and 1.28 mM 35S-PAPS (NEN, NEG-
  • Stop Mixture a 1 : 1 vol/vol mixture of 0.1 M Ba(OH)2 and 0.1 M barium acetate made fresh each day.
  • Sulfotransferases can show some degree of overlapping substrate specificity. It is therefore important to perform experiments under linear conditions and define the kinetic parameters for substrate conjugation before making conclusions about isozyme specificity. Also these enzymes can show significant substrate inhibition, and it may be necessary to try a variety of substrate concentrations.
  • a cell-free assay for UDP-glucuronic acid (Singh et al. (1980) Biochem. J. 189:369) can be utilized for determining UDP-glucose dehydrogenase activity.
  • the assay for UDP-glucuronic acid, a product of UDP-glucose dehydrogenase, is based on the fluorometric determination of D-glucuronosyl benzo(a)pyrene. This compound is formed from UDP-glucuronic acid and 3- hydroxybenzo(a)pyrene in a reaction catalyzed by the glycuronosyl transferase of guinea pig microsomes.
  • a radiochemical assay for UDP-glucose dehydrogenase which can be used in the subject drug screening assays.
  • the drug screening assay can be carried out in a cell- based format.
  • a cell e.g., a eukaryotic cell
  • the cell is preferably engineered to express one or more of, a proteoglycan which can specifically interact with the selected factor, and/or a glycosyltransferase which is essential for generating a factor-selective GAG chain.
  • the cell can be engineered to express a recombinant EXT or sulfofransferase which interacts therewith, or a glypican or sulfofransferase which modifies the GAG chain(s) of the glypican.
  • the cell is contacted with a test agent(s), and the level of expression of the reporter gene is assessed. An change in the level of expression of the reporter gene, relative to the control of no test agent, indicates that the test agent is able to modulate (potentiate or inhibit) the activity of selected factor.
  • the invention provides drug screening assays which are capable of identifying agents that can disrupt the glypican-dependent interaction between a Wnt protein it cognate receptor, e.g., with a frizzled protein, or which inhibit the formation of glypican-frizzled receptor complexes.
  • An exemplary drug screening assay of the present invention includes the steps of (a) forming a reaction mixture including: (i) a Wnt polypeptide, (ii) glypican-frizzled complex, and (iii) a test compound; and (b) detecting interaction of the Wnt polypeptide with the complex.
  • the reaction mixture can be a cell-free protein preparation, e.g., a reconstituted protein mixture or a cell lysate, or it can be a recombinant cell including a heterologous nucleic acid recombinantly expressing the glypican polypeptide.
  • the drug screening assay is a cell-free system
  • it can be, e.g., a cell membrane preparation, a reconstituted protein mixture, or a liposome reconstituting the receptor subunits as a Wnt receptor.
  • the protein subunits of a Wnt receptor complex can be purified from detergent extracts from both authentic and recombinant origins can be reconstituted in artificial lipid vesicles (e.g. phosphatidylcholine liposomes) or in cell membrane-derived vesicles (see, for example, Bear et al. (1992) CeU 68:809-818; Newton et al.
  • the lamellar structure and size of the resulting liposomes can be characterized using electron microscopy. External orientation of the receptor in the reconstituted membranes can be demonstrated, for example, by immunoelectron microscopy.
  • the interaction of a Wnt protein with liposomes containing such glypican-frizzled complexes and liposomes without the protein, in the presence of candidate agents, can be compared in order to identify potential modulators of the interaction.
  • the drug screening assay is derived to include a whole cell expressing a recombinant glypican polypeptide.
  • the ability of a test agent to alter the activity of the glypican-frizzled complex can be detected by analysis of the recombinant cell.
  • agonists and antagonists of the receptors biological activity can by detected by scoring for alterations in growth or differentiation (phenotype) of the cell.
  • General techniques for detecting each are well known, and will vary with respect to the source of the particular reagent cell utilized in any given assay.
  • the recombinant cell is preferably a metazoan cell, e.g., a mammalian cell, e.g., an insect cell, e.g., a xenopus cell, e.g., an oocyte.
  • the receptor can be reconstituted in a yeast cell.
  • a cell which expresses the receombinant receptor can be contacted with a Wnt protein which is capable of inducing signal transduction from the receptor, and the resulting signaling detected either at various points in the pathway, or on the basis of a phenotypic change to the reagent cell.
  • a test compound which modulates that pathway e.g., potentiates or inhibits, can be detected by comparison with control experiments which either lack the receptor or lack the test compound. For example, visual inspection of the mo ⁇ hology of the reagent cell can be used to determine whether the biological activity of the glypican-dependent Wnt pathway has been affected by the added agent.
  • change(s) in the level of an intracellular second messenger responsive to signaling by the glypican complex can be detected.
  • the assay may assess the ability of test agent to cause changes in phosphorylation patterns, adenylate cyclase activity (cAMP production), GTP hydrolysis, calcium mobilization, and/or phospholipid hydrolysis (IP3, DAG production) upon receptor stimulation.
  • a reporter gene can also be provided in the cell, being selected to include a transcriptional regulatory element which is responsive to glypican-dependent Wnt signal transduction.
  • Simple competitive binding assays can also be used to assess the ability of a test compound to inhibit formation of glypican- Wnt complexes in cell- free mixtures.
  • agents identified in the subject assay can be formulated in pharmaceutical preparations for in vivo administration to an animal, preferably a human.
  • the cloned Ext-family genes set out in the appended sequence listing are also each part of an infra-species family. That is, it is anticipated that other paralogs of the human and Drosophila Ext-family proteins exist in those animals, and orthologs of each Ext-family gene are conserved amongst other animals.
  • human Ext-1 is a polypeptide encoded by a nucleic acid represented by SEQ ID No:l; human Ext-2 is encoded by the nucleic acid SEQ ID No: 2; Ttv polypeptide is encoded by SEQ ID No: 3.
  • Table 3
  • Ext-1 gene has been localized to chromosomal position 8q24.1
  • human Ext-2 (Gen. Bank Accession No. U62740) has been localized to 1 lpl 1-13
  • human Ext- 3 has been localized to 19p .
  • Other Ext family members include, but are not limited to ExtRl (Gen. Bank Accession No. AB007042), ExtR2 (Gen. Bank Accession No. AB009284), Ext2.I (Gen. Bank Accession No. U72263), ExtL (Gen. Bank Accession No. U67191), ExtL3 (Gen. Bank Accession No. U76188) and ExtL2 (Gen. Bank Accession No. U76189).
  • the coding sequence for the Drosophile dally protein can found as GenBank accession U31985, and is also described by Nakato et al. (1995) Development 121:3687.
  • the drosophila dally protein is closely related to the mammalian glypican proteins, particularly human glypican 5.
  • the coding sequence for human glypican 5 is provided herein as SEQ IS No. 7, and the corresponding protein sequence as SEQ ID No. 8.
  • Other exemplary mammalian glypicans, or related proteins, which can be used in the subject assays are set forth in Table 4
  • antisense therapy refers to administration or in situ generation of oligonucleotide probes or their derivatives which specifically hybridize (e.g. binds) under cellular conditions, with the cellular mRNA and/or genomic DNA encoding a Ext, a glypican, or a glycosyltransferase essential to synthesis of a factor-selective GAG chain, so as to inhibit expression of that protein, e.g. by inhibiting transcription and/or translation.
  • binding may be by conventional base pair complementarity, or, for example, in the case of binding to DNA duplexes, through specific interactions in the major groove of the double helix.
  • antisense therapy refers to the range of techniques generally employed in the art, and includes any therapy which relies on specific binding to oligonucleotide sequences.
  • an antisense construct of the present invention can be delivered, for example, as an expression plasmid which, when transcribed in the cell, produces RNA which is complementary to at least a unique portion of the cellular mRNA which encodes a the targeted protein.
  • the antisense construct is an oligonucleotide probe which is generated ex vivo and which, when introduced into the cell causes inhibition of expression by hybridizing with the mRNA and/or genomic sequences.
  • oligonucleotide probes are preferably modified oligonucleotides which are resistant to endogenous nucleases, e.g. exonucleases and/or endonucleases, and are therefore stable in vivo.
  • nucleic acid molecules for use as antisense oligonucleotides are phosphoramidate, phosphothioate and methylphosphonate analogs of DNA (see also U.S. Patents 5,176,996; 5,264,564; and 5,256,775), or peptide nucleic acids (PNAs). Additionally, general approaches to constructing oligomers useful in antisense therapy have been reviewed, for example, by Van der Krol et al. (1988) Biotechniques 6:958-976; and Stein et al. (1988) Cancer Res 48:2659-2668.
  • the present method also provides a method for determining if a subject is at risk for a disorder characterized by aberrant apoptosis, cell proliferation and/or differentiation.
  • method can be generally characterized as comprising detecting, in a sample of cells from the subject, the presence or absence of a genetic lesion characterized by at least one of (i) an alteration affecting the integrity of a gene encoding a Ext-protein, or (ii) the mis-expression of the Ext gene or glypican gene.
  • such genetic lesions can be detected by ascertaining the existence of at least one of (i) a deletion of one or more nucleotides from a Ext gene or glypican gene, (ii) an addition of one or more nucleotides to a Ext gene or glypican gene, (iii) a substitution of one or more nucleotides of a Ext gene or glypican gene, (iv) a gross chromosomal rearrangement of a Ext gene or glypican gene, (v) a gross alteration in the level of a messenger RNA transcript of a Ext gene or glypican gene, (vii) aberrant modification of a Ext gene or glypican gene, such as of the methylation pattern of the genomic DNA, (vii) the presence of a non-wild type splicing pattern of a messenger RNA transcript of a Ext gene or glypican gene, (viii) a non- wild type level of
  • the present invention provides a large number of assay techniques for detecting lesions in a Ext gene or glypican gene, and importantly, provides the ability to discern between different molecular causes underlying Ext-dependent aberrant cell growth, proliferation and/or differentiation.
  • a nucleic acid composition comprising a (purified) oligonucleotide probe including a region of nucleotide sequence which is capable of hybridizing to a sense or antisense sequence of a Ext gene or glypican gene, such as represented by any one of S ⁇ Q ID Nos: 1-4 or 7, or naturally occurring mutants thereof, or 5' or 3' flanking sequences or intronic sequences naturally associated with the subject Ext gene or glypican genes or naturally occurring mutants thereof.
  • the nucleic acid of a cell is rendered accessible for hybridization, the probe is exposed to nucleic acid of the sample, and the hybridization of the probe to the sample nucleic acid is detected.
  • Such techniques can be used to detect lesions at either the genomic or mRNA level, including deletions, substitutions, etc., as well as to determine mRNA transcript levels.
  • detection of the lesion comprises utilizing the probe/primer in a polymerase chain reaction (PCR) (see, e.g. U.S. Patent Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or, alternatively, in a ligation chain reaction (LCR) (see, e.g., Landegran et al. (1988) Science 241:1077-1080; and Nakazawa et al. (1944) PNAS 91:360-364), the later of which can be particularly useful for detecting point mutations in the Ext gene or glypican gene.
  • PCR polymerase chain reaction
  • LCR ligation chain reaction
  • the method includes the steps of (i) collecting a sample of cells from a patient, (ii) isolating nucleic acid (e.g., genomic, mRNA or both) from the cells of the sample, (iii) contacting the nucleic acid sample with one or more primers which specifically hybridize to a Ext gene or glypican gene under conditions such that hybridization and amplification of the Ext gene or glypican gene (if present) occurs, and (iv) detecting the presence or absence of an amplification product, or detecting the size of the amplification product and comparing the length to a control sample.
  • nucleic acid e.g., genomic, mRNA or both
  • such assays as described above can be used to detect mutations, e.g., loss-of-function mutations, to a gene encoding a glycoyltransferase which is essential to signal transduction by a growth factor, cytokine or the like.
  • the human homolog of sulfateless, see example, 6, is a heparan sulfate-N-deacetylase/N- sulfofransferase.
  • the human gene maps to 5q32-q33.3. That locus is associated with such developmental disorders as Treacher Collins syndrome.
  • Treacher Collins syndrome is an autosomal dominant disorder of craniofacial development, which has been localized to chromosome 5q32-33.1. The symptoms of the disorder are pronounced of abberant signal transduction by a Wnt or hedgehog protein.
  • Another aspect of the present invention relates to a method of inducing and/or maintaining a differentiated state, enhancing survival, and/or inhibiting (or alternatively potentiating) proliferation of a cell, by contacting the cells with an agent which modulates Hedgehog- or Wnt-dependent signal transduction pathways.
  • the subject method could be used to generate and/or maintain an array of different tissue both in vitro and in vivo.
  • a "Hedgehog or Wnt therapeutic,” whether inhibitory or potentiating with respect to modulating the activity of a Hedgehog or Wnt protein respectively, can be, as appropriate, any of the preparations described above, including agents identified in the subject assays, and antisense molecules which selectively inhibits expression of a glycosltransferase enzyme, such as an Ext or a sulfofransferase, essential to the synthesis of hedgehog- selective or Wnt-selective GAG chains, or an antisense molecule which selectively inhibits expression of a glypican.
  • a glycosltransferase enzyme such as an Ext or a sulfofransferase, essential to the synthesis of hedgehog- selective or Wnt-selective GAG chains, or an antisense molecule which selectively inhibits expression of a glypican.
  • compositions comprising Hedgehog or Wnt therapeutics can be used in the in vitro generation of skeletal tissue, such as from skeletogenic stem cells, as well as the in vivo treatment of skeletal tissue deficiencies.
  • Hedgehog therapeutics are also expected to play an important role in regulating uncontrolled cellular proliferation leading to exostoses, and in severe cases progression of the exostoses to Chondrosarcomas and Osteosarcomas.
  • Ihh Indian hedgehog
  • PTHrP parathyroid hormone-related protein
  • chondrocytes As proliferating chondrocytes decide to undergo hypertrophy, they express high levels of the PTH/PTHrP receptor. When they subsequently become committed to this pathway, they transiently express Ihh, until they become fully hypertrophic. The Ihh signal acts on the perichondrium adjacent to the prehypertrophic zone (Ptc/Gli-expressing cells) and (directly or indirectly) on the more distant periarticular perichondrium. ultimately inducing the expression of PTHrP. PTHrP then signals back to chondrocytes expressing the PTH/PTHrP receptor, thereby preventing non-differentiated chondrocytes from moving down the hypertrophic pathway.
  • the present invention particularly contemplates the use of Hedgehog therapeutics which agonize and/or antagonize a hedgehog, e.g., Ihh activity as the case may be.
  • this invention contemplates the use of Hedgehog therapeutics, in alleviating the symptoms associated with multiple exostoses.
  • Hereditary multiple exostoses is an autosomal dominant condition characterized by multiple cartilage- capped projections originating from the epiphyseal plates of endochondral bones (Jaffe 1943; Solomon 1961). The prevalence of this disorder has been estimated at between 1/50,000 and 1/100,000 (Hennekam 1991; Cook et al. 1993; Schmale et al. 1994). The development of exostoses appears to be correlated with growth of the epiphyseal plate, with new exostoses rarely appearing after epiphyseal plate closure (Hennekam 1991).
  • Exostoses usually form at the ends of long bones but can also occur on the hands, feet, pelvis, ribs, and scapula. Shortened stature is sometimes seen in this disorder and is possibly attributable to these benign tumors interfering with the epiphyseal growth plates and reducing overall bone growth (Hennekam 1991). Exostoses also frequently impinge on adjacent nerves, vessels, tendons, and joints and thus require surgical removal. A serious complication of this disorder is the occasional transformation to chondrosarcoma or osteosarcoma that occurs in 0.9%-2.9% of cases (Voutsinas and Wynne-Davies 1983; Wicklund et al. 1995).
  • Hedgehog therapeutics that agonize Ihh activity and modulate diffusion of Ihh would therefore be useful in alleviating the symptoms associated with Multiple exostoses.
  • the present invention contemplates the use of Hedgehog therapeutics which agonize a hedgehog skeletogenic activity, such as an ability to induce chondrogenesis and/or osteogenesis.
  • skeletal tissue deficiency it is meant a deficiency in bone or other skeletal connective tissue at any site where it is desired to restore the bone or connective tissue, no matter how the deficiency originated, e.g. whether as a result of surgical intervention, removal of tumor, ulceration, implant, fracture, or other traumatic or degenerative conditions.
  • the present invention makes available effective therapeutic methods and compositions for restoring cartilage function to a connective tissue.
  • Such methods are useful in, for example, the repair of defects or lesions in cartilage tissue which is the result of degenerative wear such as that which results in arthritis, as well as other mechamcal derangements which may be caused by trauma to the tissue, such as a displacement of torn meniscus tissue, meniscectomy, a laxation of a joint by a torn ligament, malignment of joints, bone fracture, or by hereditary disease.
  • the present reparative method is also useful for remodeling cartilage matrix, such as in plastic or reconstructive surgery, as well as periodontal surgery.
  • the present method may also be applied to improving a previous reparative procedure, for example, following surgical repair of a meniscus, ligament, or cartilage. Furthermore, it may prevent the onset or exacerbation of degenerative disease if applied early enough after trauma.
  • the subject method comprises treating the afflicted connective tissue with a therapeutically sufficient amount of a hedgehog agonist, particularly Hedgehog therapeutic which agonizes Ihh activity, to generate a cartilage repair response in the connective tissue by stimulating the differentiation and/or proliferation of chondrocytes embedded in the tissue.
  • a hedgehog agonist particularly Hedgehog therapeutic which agonizes Ihh activity
  • Induction of chondrocytes by treatment with a hedgehog agonist can subsequently result in the synthesis of new cartilage matrix by the treated cells.
  • Such connective tissues as articular cartilage, interarticular cartilage (menisci), costal cartilage (connecting the true ribs and the sternum), ligaments, and tendons are particularly amenable to treatment in reconstructive and/or regenerative therapies using the subject method.
  • regenerative therapies include treatment of degenerative states which have progressed to the point of which impairment of the tissue is obviously manifest, as well as preventive treatments of tissue where degeneration is in its earliest stages or imminent.
  • the subject method can further be used to prevent the spread of mineralisation into fibrotic tissue by maintaining a constant production of new cartilage.
  • the subject method can be used to treat cartilage of a diarthroidal joint, such as a knee, an ankle, an elbow, a wrist, a knuckle of either a fmger or toe, or a temperomandibular joint.
  • the treatment can be directed to the meniscus of the joint, to the articular cartilage of the joint, or both.
  • the subject method can be used to treat a degenerative disorder of a knee, such as which might be the result of traumatic injury (e.g., a sports injury or excessive wear) or osteoarthritis.
  • An injection of a Hedgehog therapeutic into the joint with, for instance, an arthroscopic needle can be used to treat the afflicted cartilage.
  • the injected agent can be in the form of a hydrogel or other slow release vehicle described above in order to permit a more hedgehog or extended and regular contact of the agent with the treated tissue.
  • the present invention further contemplates the use of the subject method in the field of cartilage transplantation and prosthetic device therapies.
  • the growth of new cartilage from either transplantation of autologous or allogenic cartilage has been largely unsuccessful.
  • the zonal arrangement of these tissues may reflect a gradual change in mechanical properties, and failure occurs when implanted tissue, which has not differentiated under those conditions, lacks the ability to appropriately respond.
  • the tissue undergoes a metaplasia to pure fibrous tissue.
  • the subject method can be used to particularly addresses this problem, by causing the implanted cells to become more adaptive to the new environment and effectively resemble hypertrophic chondrocytes of an earlier developmental stage of the tissue.
  • the action of chondrogenesis in the implanted tissue, as provided by the subject method, and the mechanical forces on the actively remodeling tissue can synergize to produce an improved implant more suitable for the new function to which it is to be put.
  • the subject method can be applied to enhancing both the generation of prosthetic cartilage devices and to their implantation.
  • the need for improved treatment has motivated research aimed at creating new cartilage that is based on collagen- glycosaminoglycan templates (Stone et al. (1990) Clin Orthop Relat Red 252:129), isolated chondrocytes (Grande et al. (1989) J Orthop Res 7:208; and Takigawa et al. (1987) Bone Miner 2:449), and chondrocytes attached to natural or synthetic polymers (Walitani et al. (1989) J Bone Jt Surg 71B:74; Vacanti et al.
  • chondrocytes can be grown in culture on biodegradable, biocompatible highly porous scaffolds formed from polymers such as polyglycolic acid, polylactic acid, agarose gel, or other polymers which degrade over time as function of hydrolysis of the polymer backbone into innocuous monomers.
  • the matrices are designed to allow adequate nutrient and gas exchange to the cells until engraftment occurs.
  • the cells can be cultured in vitro until adequate cell volume and density has developed for the cells to be implanted.
  • One advantage of the matrices is that they can be cast or molded into a desired shape on an individual basis, so that the final product closely resembles the patient's own ear or nose (by way of example), or flexible matrices can be used which allow for manipulation at the time of implantation, as in a joint.
  • the implants are contacted with a Hedgehog therapeutic during the culturing process, such as an Ihh agonist, in order to induce and/or maintain differentiated chondrocytes in the culture in order as to further stimulate cartilage matrix production within the implant.
  • a Hedgehog therapeutic such as an Ihh agonist
  • the cultured cells can be caused to maintain a phenotype typical of a chondrogenic cell (i.e. hypertrophic), and hence continue the population of the matrix and production of cartilage tissue.
  • the implanted device is treated with a Hedgehog therapeutic in order to actively remodel the implanted matrix and to make it more suitable for its intended function.
  • a Hedgehog therapeutic in order to actively remodel the implanted matrix and to make it more suitable for its intended function.
  • the artificial transplants suffer from the same deficiency of not being derived in a setting which is comparable to the actual mechamcal environment in which the matrix is implanted.
  • the activation of the chondrocytes in the matrix by the subject method can allow the implant to acquire characteristics similar to the tissue for which it is intended to replace.
  • the subject method is used to enhance attachment of prosthetic devices.
  • the subject method can be used in the implantation of a periodontal prosthesis, wherein the treatment of the surrounding connective tissue stimulates formation of periodontal ligament about the prosthesis, as well as inhibits formation of fibrotic tissue proximate the prosthetic device.
  • the subject method can be employed for the generation of bone (osteogenesis) at a site in the animal where such skeletal tissue is deficient.
  • Indian hedgehog is particularly associated with the hypertrophic chondrocytes that are ultimately replaced by osteoblasts.
  • administration of a Hedgehog or Wnt therapeutic of the present invention can be employed as part of a method for treating bone loss in a subject, e.g. to prevent and/or reverse osteoporosis and other osteopenic disorders, as well as to regulate bone growth and maturation.
  • preparations comprising hedgehog agonists can be employed, for example, to induce endochondral ossification, at least so far as to facilitate the formation of cartilaginous tissue precursors to form the "model" for ossification.
  • Therapeutic compositions of Hedgehog or Wnt therapeutics can be supplemented, if required, with other osteoinductive factors, such as bone growth factors (e.g. TGF- factors, such as the bone mo ⁇ hogenetic factors BMP-2 and BMP-4, as well as activin), and may also include, or be administered in combination with, an inhibitor of bone reso ⁇ tion such as estrogen, bisphosphonate, sodium fluoride, calcitonin, or tamoxifen, or related compounds.
  • TGF- factors such as the bone mo ⁇ hogenetic factors BMP-2 and BMP-4, as well as activin
  • an inhibitor of bone reso ⁇ tion such as estrogen, bisphosphonate, sodium fluoride, calcitonin, or tamoxifen,
  • Hedgehog activity in vivo reformation of tissue can be accomplished, e.g. in the development and maintenance of organs such as ectodermal patterning, as well as certain mesodermal and endodermal differentiation processes.
  • organs such as ectodermal patterning, as well as certain mesodermal and endodermal differentiation processes.
  • the subject Hedgehog therapeutics can be used to reform injured tissue, or to improve grafting and mo ⁇ hology of transplanted tissue.
  • Hedgehog antagonists and agonists can be employed in a differential manner to regulate different stages of organ repair after physical, chemical or pathological insult.
  • the present method is also applicable to cell culture techniques.
  • the subject hedgehog inhibitors of the present invention can be used to inhibit hedgehog-mediated proliferation of cells, e.g., to treat unwanted proliferation.
  • the subject inhibitors can be used to treat hedgehog-dependent basal cell carcinoma (BCC) or other cancer resulting from over-activation of hedgehog signaling.
  • BCC hedgehog- dependent basal cell carcinoma
  • the subject invention provides inhibitors of Wnt signal transduction. Such compounds could be of use in the treatment of diseases in which activation or inactivation of the Wnt protein results in either cellular proliferation, cell death, nonproliferation, induction of cellular neoplastic transformations or metastatic tumor growth and hence could be used in the prevention and/or treatment of cancers such as bone and breast cancer for example.
  • ttv is required for Hh signaling (I.T., Y.B. and N.P., in preparation). Consistent with this hypothesis, ttv somatic clones are associated with wing phenotypes similar to the phenotypes of clones of mutations in Hh signaling components (data not shown). In this paper we analyze the function of ttv in detail in the wing imaginal disc to determine its role in the Hh signaling pathway.
  • Hh is expressed in cells of the posterior (P) compartment and has been proposed to diffuse into the anterior (A) compartment.
  • Ptc receptor Patched
  • binding of Hh to its receptor Patched (Ptc) activates the Hh signaling pathway that results in the stabilization of the full length Cubitus interruptus (Ci) protein, a transcription factor of the GLI family.
  • Ci Cubitus interruptus
  • Ci stabilization allowed us to determine that a cell non autonomous effect was associated with ttv mutant clones.
  • ttv mutant clones of only a few cells wide are located along the A/P boundary, wild type cells anterior to the ttv mutant clone do not respond to the Hh signal as shown by Ci staining ( Figures li, Ik). Because these cells are located within a domain where Hh is normally able to diffuse to and induce stabilization of Ci, we conclude that ttv mutant clones have a directional non cell autonomous effect on wild type cells located anteriorly.
  • Ci stabilization Since the domain of Ci stabilization is under the direct control of Hh signaling (Aza-Blanc, P., Ramirez- Weber, F.A., Rheint, M.P., Schwartz, C. & Kornberg, T.B. Cell Proteolysis that is inhibited by hedgehog targets Cubitus interruptus protein to the nucleus and converts it to a repressor. 89, 1043-1053 (1997)); (Strigini, M. & Cohen, S.M. Development A Hedgehog activity gradient contributes to AP axial patterning of the Drosophila wing.
  • Hh In the ptc mutant cells, we detect the Hh protein as a diffuse membrane staining. When Hh reaches the 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 ( Figure 2c). In ptc ttv double mutant clones, we do not detect any Hh staining in mutant cells ( Figure 2d). Based on this result and the directional cell non- autonomous effect of ttv mutant clones, we propose that Hh is unable to diffuse in the absence of ttv activity.
  • ttv could function in the sending cell and/or the receiving cell.
  • the posterior cells that produce and send Hh and the anterior cells which send previously received Hh. Since in the anterior cell the sending of Hh depends on its reception from the previous cells, it is difficult to address where ttv functions by generating ttv mutant clones in the anterior compartment. We therefore generated clones in the posterior compartment adjacent to the A/P boundary and analyzed their effects on Ci stabilization. We observed that ttv mutant clones in the posterior compartment do not affect the diffusion of Hh since the stabilization of Ci is not affected in the anterior compartment ( Figures 2e, 2and see also 2b). Therefore, we propose that ttv functions in the receiving cells and is probably required for the movement of Hh from sending to receiving cells. This observation also indicates that ttv is not required for Hh production.
  • Hh in a ttv clone located in the anterior compartment, Hh can still signal although less efficiently to the first receiving cells.
  • Hh we hypothesize that this weakened signaling activity is mediated by the Hh present on the membrane of the Hh sending cells and that therefore efficient Hh signaling even to adjacent cells requires Hh diffusion.
  • Ttv is 56% and 25% identical to the human Ext-1 and Ext-2 protein, respectively.
  • Ext-i and Ext-2 genes have implicated in the human Multiple Exostoses syndrome.. (Ahn, J., et al. Nat Genet Cloning of the putative tumor suppressor gene for hereditary multiple exostoses (EXT1). 11, 137-143 (1995)); (Stickens, D., et al. Nat Genet The EXT2 multiple exostoses gene defines a family of putative tumor suppressor genes. 14, 25- 32 (1996)) and (Wicklund, C.L., Pauli, R.M., Johnston, D. & Hecht, J.T. Amj Med Genet. Natural history study of hereditary multiple exostoses. 55, 43-46 (1995)).
  • Ext proteins appear to form a novel family of conserved molecules in Metazoans, since mouse and C. elegans homologues have been cloned recently.
  • no studies have determined the cellular compartment where Ext molecules could function.
  • EXT1 hereditary multiple exostoses
  • Ttv is an integral membrane protein
  • Ttv is an integral membrane protein
  • the active form of Hh is tethered to the membrane by a cholesterol moiety, (Porter, J.A., Young, K.E. & Beachy, P.A. Science Cholesterol modification of hedgehog signaling proteins in animal development. 274, 255-259 (1996)) and (Porter, J.A., et al. Cell Hedgehog patterning activity: role of a lipophilic modification mediated by the carboxy- terminal autoprocessing domain.
  • Hh should first be dissociated from the sending cell membrane and then re-associated with the receiving cell membrane.
  • the movement of Hh cannot be dependent on the Hh receptor Ptc and the membrane signaling component Smoothened (Smo), since Hh diffuses in ptc or smo mutant clones (Chen, Y. & Struhl, G. Cell Dual roles for patched in sequestering and transducing Hedgehog. 87, 553-563 (1996)).
  • Ttv appears to play a important role in this process. Since Ttv is required in the receiving cell to allow Hh diffusion, we speculate that Ttv does not function in the dissociation of Hh from the membrane of the sending cells but more likely to permit the reassociation or the maintenance of Hh on the surface of the receiving cells.
  • the Multiple Exostoses syndrome is a dominantly inherited disease characterized by short stature, limb length inequalities, bone deformities and the presence of bone outgrowths, called exostoses, at the ends of long bones (Wicklund, C.L., Pauli, R.M., Johnston, D. & Hecht, J.T. Am j Med Genet. Natural history study of hereditary multiple exostoses. 55, 43-46 (1995)).
  • the ⁇ XT2 multiple exostoses gene defines a family of putative tumor suppressor genes. 14, 25-32 (1996)) and are homologous to each other. Ext- 7 and Ext-2 genes have been proposed to act as putative tumor suppressor genes due to the slightly elevated incidence of chondrosarcomas and osteosarcomas in ⁇ xt patients (Ahn, J., et al. Nat Genet Cloning of the putative tumor suppressor gene for hereditary multiple exostoses ( ⁇ XT1). 11, 137-143 (1995)); (Stickens, D., et al. Nat Genet The ⁇ XT2 multiple exostoses gene defines a family of putative tumor suppressor genes.
  • Ttv is required for the diffusion of Hh molecules and therefore increases the level of Hh far from its source.
  • ttv affects the diffusion of Hh to the first receiving cells and at further distance, suggesting that both the mechanisms of Hh short and long range diffusion depends on ttv, and are most likely identical.
  • 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.
  • Antibodies were diluted in PBT as follows: rabbit anti- -Gal (Cappel) 1 :4000; rat anti- -Gal 1 :100; mouse anti-Ptc 1 : 100; rabbit anti-Hh 1 :2000 (TS A amplification was used to enhance the signal, NEN); rat anti-Ci 1 :5. Secondary antibodies coupled to FITC, Texas Red or Cy5 (Jackson) were used at 1 :200 dilution. Secondary coupled to HRP for TSA amplification (Vector) was used at 1 :2000 dilution. Stainings were visualized using a Leica TCS/NP confocal microscope.
  • Antibody production the ttv cDNA sequence encoding amino acid 30 to 376 of the Ttv protein was cloned into the pQE 10 vector (Qiagen) and expressed in the E.coli Ml 5 strain. The purified protein was injected into rabbit and rat. Antisera were affinity purified against GST-Ttv (amino acid 30 to 376) linked on a HiTrap NHS-activated column (Pharmacia). No specific stainings could be detected with these affinity purified sera in embryos or imaginal discs.
  • Western Blot Wild type and ttv unfertilized embryos derived from GLCs
  • ttv RNA were produced by in vitro transcription using the SP6 RNA Machine kit (Ambion); rabbit reticulocyte lysates, microsomes and endoglycosydase H were purchased from Boehringer.
  • sample buffer was then resuspended in sample buffer and analyzed by SDS PAGE.
  • the alkaline wash was performed by adding 12 reaction volumes of. 0.1 M Na 2 C0 3 (pH 12) to the sample and incubating it for 5 minutes at 4"C.
  • the sample was then spinned at 75 000 ⁇ m for 15 minutes. The supernatant was removed and the pellet was dissolved in loading buffer and analyzed by SDS PAGE.
  • Standard molecular biology protocols were used for Northern blot, library screening and molecular cloning.
  • Example 5 Specific Heparan Sulfate Proteoglycans regulate the movement of Hedgehog in Drosophila tissues
  • ttv glycosaminoglycan chains
  • HSPGs Heparan Sulfate Proteoglycans
  • Wnt proteins In the case of Wnt proteins there is evidence that these molecules can be secreted in the extracellular space as well as transported through cells. Wnt proteins are poorly secreted in the extracellular space and bind the extracellular matrix tightly (Bradley and Brown, 1990; Gonzalez et al., 1991; Reichsman et al., 1996). These features suggest that these proteins may not be able to freely diffuse and thus raise the questions of how many cells away they are able to act. Studies in the Drosophila wing imaginal discs have provided compelling evidence that Wingless (Wg), the homolog of mammalian Wnt-1, is required directly for patterning a field of cells such as the wing blade.
  • Wg Wingless
  • Wg PE4 protein is competent to generate both short and long-range Wg effects when provided ectopically, suggesting that Wg PE4 is specifically defective in transport.
  • Wg CE7 Another mutant Wg protein, Wg CE7 , is distributed over many cell diameters on either side of wg-expressing cells, and is internalized into cells without transducing any of the known signaling activites of Wg.
  • Hh proteins can travel long range.
  • Drosophila Hh induces the expression of its target genes patched (ptc) and decapentaplegic (dpp) directly in a broad domain of 8-10 cell diameters along the anterior-posterior (A/P) boundary (Mullor et al, 1997; Strigini and Cohen, 1997).
  • Hh is made as a precursor protein, which is autocatalytically cleaved to produce an
  • Hh-N N-terminal
  • Hh-C C-terminal fragment
  • Hh cholesterol-modified Hh molecules may be released from the plasma membrane, or that some mehanism exists that releases HhN from HhNp.
  • Another model is that the cholesterol-modified form of Hh may be transported to target cells by a specific mechanism.
  • Biochemical analyses have indicated that Wnts and Hh are Heparin-binding proteins (Bradley and Brown, 1990) (Reichsman et al, 1996) (Bumcrot et al, 1995).
  • HSPGs Heparan Sulfate Proteoglycans
  • HS can be covalently linked to a variety of cell surface proteins but is found consistently on two major families of proteoglycans, Syndecans and Glypicans (David, 1993) .
  • the biological roles of HSPGs are highly diversified, ranging from simple mechanical support to as yet poorly understood effects on various cellular processes such as cell adhesion, motility, proliferation, differentiation and mo ⁇ hogenesis.
  • HSPGs have been implicated in a number of events that include co-receptors for insoluble ligands, co-receptors for soluble ligands, internalization of receptors, transport of molecules or as soluble paracrine effectors (Salmivirta et al., 1996).
  • GAG chains consists of alternating sugar chains of D-glucuronic (GlcA) and N-acetyl-D- glucosamine (GlcNAc) units, which are joined together by a glucosyltransferase.
  • the final GAG structures are formed by a series of polymer-modification reactions that are initiated by N-deacetylation and N-sulfation of GlcNAc units, followed by C-5 epimerization of D- glucuronic acid (GlcA) to L-iduronic acid (IdoA) and finally by inco ⁇ oration of O-sulfate groups at various positions.
  • the reactions involved in this process are generally incomplete such that a fraction of the potential substrate residues are not modified. Therefore, GAGs display considerable sequence heterogeneity both within and between different chains.
  • the number of GAG chains on a protein core may also vary in length and number.
  • Hh specific HS-GAGs are synthesized by a complex that includes Ttv and at least one specific sulfofransferase.
  • Tout velu is involved in HSPGs biosynthesis ttv is 56% homologous to Exti, and 26% homologous to Ext2, ttv is a zygotic lethal mutation and animals derived from heterozygous ttv females die at the pupal stage.
  • ttv embryos die during embryogenesis with a segment polarity phenotype (see below). We refer to these embryos as "ttv embryos" in the text.
  • Ttv Ttv encodes a type II protein (Bellaiche et al., 1998) which could be localized at the plasma membrane or in the membranes of the secretory apparatus.
  • Ttv a polyclonal antibody against Ttv (see Experimental Procedures). Although the antibody recognizes Ttv on Western Blots (Fig. 6H), we could not obtain a convincing staining pattern in embryos or in imaginal discs.
  • ttvmyc This ttvmyc gene when expressed under the control of the UAS promoter using the Gal4-UAS system (Brand and Perrimon, 1993) is able to rescue the segment polarity phenotype of ttv embryos (data not shown).
  • Ttv affects Hh signaling
  • Hh Fig. 8E and F
  • Ptc Fig. 8G and H
  • FGF and Wg signaling is not affected in ttv embryos HSPGs have been implicated in the signaling pathways of several growth factors.
  • the ventral mesoderm invaginates at stage 6 and at stage 9 mesoderm cells rearrange and form a monolayer. Mutants with defects in Htl signaling show aberrant mesodermal cell migration and mesodermal cells do not form a monolayer. Similarly, mutations in sgl and sfl also exhibit a mesoderm migration defect consistent with the role of HSPGs in FGF/Htl signaling (Lin et al., 1999). However, su ⁇ risingly for a mutation involved in HS biosynthesis, we could not detect any defects in mesoderm migration in ttv embryos (Fig. 9A-D). Migration of Twist (Twi) positive mesodermal cells is normal as well as the domain of Twi staining which indicates that there is no D/V patterning defect in ttv embryos.
  • Wg signaling is affected in the absence of Ttv activity.
  • Wg signaling is involved in a number of developmental processes that include the maintenance of En expression
  • ttv embryos display a segment polarity phenotype similar to loss of Wg signaling, as shown by both the absence of naked cuticle (Fig 6H ) and disapearance of both wg and en expression (Fig. 10A-D).
  • ttv mutant embryos appear wild type (Fig 10E-G), suggesting that Wg signaling is not affected by loss of Ttv activity. Similarly, we could not detect a requirement for Ttv activity in formation of the RP2 neurons. In ttv embryos, the correct number of RP2 neurons, detected using an Even-skipped (Eve) antibody, are found (not shown). Thus, several Wg dependent processes are not affected in ttv embryos.
  • Ttv is required for Hh diffusion
  • the expression pattern of the gene bagpipe also provides evidence that Hh signaling and not Wg is affected in ttv embryos.
  • the bap segmental expression at stage 10 during the formation of the mesoderm is dependent upon both Wg and Hh signaling.
  • Wg is required for repression of bap expression between the segments, and in wg mutant embryos bap expression becomes continuous.
  • Hh is required for maintenance of bap expression and in hh mutant embryos bap expression fades.
  • bap staining disappears providing further evidence that ttvis involved in Hh and not Wg signaling in the embryo (Fig. 11A-D).
  • HhNp N-terminal part of Hh
  • HhNp N-terminal part of Hh
  • Ttv is specific to Hh signaling
  • Wg and FGF signaling may not be affected because the HS-GAGs that these factors bind to are present.
  • HS-GAGs consist of an alternative chain of GlcA and GlcNAc residues which are linked together by the glycosyltransferase/Ext enzymes.
  • a puzzle is why there are several Ext-like genes in Drosophila and vertebrates if their functions are completely redundant. This diversity in Ext genes does not reflect the tissue specific expression pattern of the Ext genes because both ttv and DExt2 are uniformly expressed during embryogenesis as assayed by RNA in situ.
  • Ttv to Hh signaling we propose that different ⁇ xt glycosyltransferases form specific complexes in the golgi with different GAG modifying enzymes.
  • GAG sequences which will specifically bind certain growth factors.
  • Ext proteins may have the same biochemical activities.
  • specificity of the GAG sequences is generated by specific sulfotransferases. Interactions between HS and proteins depend on the presence of sulfate groups. Binding of the different members of the FGF family, for example, may require different combinations of sulfate groups, hence different saccharide sequences. Such sequences may be represented in the same GAG chains, they may also be differentially expressed in separate GAG chains of the HS. The generation of such specific sugar chains would require selective polymer modification.
  • Ttv might form a complex with enzymes which generate a GAG sequence required for Hh binding only, which would explain why only Hh signaling is affected in ttv embryos.
  • enzyme complex it has been proposed that chain elongation and modification occur simultaneously (Lidholt and Lindahl, 1992).
  • a prediction of this model would be that there is a specific sulfofransferase which is associated in a complex with Ttv, to generate Hh specific binding sites on GAGs.
  • mice lacking a HS 2-O-sulfotransferase undergo normal embryogenesis, but die around birth. They exhibit a fully penetrant defect in kidney development. In addition, the mice also have defects in the eye, skeleton, a cleft palate and polydactily of the limb (Bullock et al., 1998). Role of HSPGs involved in Hh signaling
  • HSPGs are involved in the ability of Hh to reach target cells. Although there is only a very low concentration of Hh detected outside Hh-producing cells, Hh can have its effect a few cell diameters wide. Therefore, either the concentration of Hh required to signal is very low and the low amount of diffusible Hh is sufficient for signaling or the membrane tethered Hh can be transported from cells to cells.
  • One model in which HSPGs could influence Hh distribution is by concentrating Hh and perhaps presenting it to its receptor (Fig 12B1). Such a function has been proposed for HSPGs in FGF signaling.
  • Hh has been reported to localize into the raft fraction after separation of cell extracts (Rietveld et al., 1999). Perhaps a GPI-anchored HSPGs is required to localize Hh in these rafts.
  • Hh Transfer of GPI anchored proteins between cells have been observed and Hh might be transferred from cell to cell in this way (Kooyman et al., 1995).
  • the cholesterol modification on Hh might also facilitate Hh localization into the rafts after which transport of Hh can occur.
  • HSPGs play a role in a process which releases Hh from its cholesterol anchor and generates a non-membrane bound Hh (Fig 12B3). Since the HS 2- O-sulfotransferase encoded by pipe affects processing of Spatzle it is conceivable that HSPGs have a similar role in Hh processing. Our previous data in the wing disc, however, argues against a role for ttv in Hh processing. The processing of Hh presumably takes place in the Hh-producing cell. However, removing ttv in the Hh producing cells in the posterior compartment did not abolish Hh signaling in the anterior compartment (Bellaiche et al., 1998).
  • the first phenotype described for Dally division abnormally delayed in the proliferation centers of the brain, is a phenotype which could be attributed to Hh as well (Nakato et al., 1995). Hh has been shown to be transported through the retinal axons and trigger proliferation in the brain (Huang and Kunes, 1996). Thus, it would be interesting to see if transport of Hh is affected in dally mutants.
  • growth factors like FGFs, BMPs, and also Indian Hh are important for skeletal growth regulation (Erlebacher et al., 1995; Lanske et al, 1996; Vortkamp et al, 1996). Mutations in these growth factors or their receptors have been linked to several bone malformity diseases. These growth factors are secreted into the extracellular matrix, where they diffuse to their target cells.
  • GAGs and PGs One of the major component of extracellular matrix.
  • the macromolecules of the extracellular matrix in cartilage is secreted by chondroblasts and by osteoblasts and the matrix can become calcified to form a hard structure.
  • FRi ttv /FRi Pfovo ] females which carry homozygous ttv germline clones were selected. These females were mated to ttv/CyO, ftz-LacZ males and maternal/zygotic null embryos were identified by the absence of beta-galactosidase expression.
  • the UAS-ttv construct is an Spel/Notl fragment of the ttv gene which consist of the whole gene cloned into the XXX site of pUAST (Brand and Perrimon, 1993).
  • the UAS- ttvmyc construct is the same construct with 6 myc epitope tags at its C-terminus.
  • RNA in situ hybridization was performed as described in (Tautz and Pfeifle 1989)
  • Ttv For Western blot analyses, embryos were dechorionated with 50% bleach, dounced in 20 mM Tris HCL PH 7.5, 150 mM NaCl, 5mM EDTA, 1% Triton X-100 at 4 C with protease inhibitors and centrifuged for 20 min at 14 OOOxg at 4°C. Amount of protein was measured using Biorads protein assay (Biorad) and 100 ⁇ g of protein was loaded in each lane, immunoblotting was performed as described in Harlow and Lane.
  • Embryonic cuticles were prepared by the Hoyer's mountant method (van der Meer, 1977). Fixation of embryos, antibody staining were performed as described in Patel, 1994. Treatment of embryos to expose the epitope recognized by the 3G10 monoclonal antibody (Seikagaku) was done with 500 mU/ml heparinaselll (Sigma) in 50 mM Tris-HCL PH 7.2, 100 mM NaCl, ImM Ca C12, 0.1% Triton-XlOO, 5 ⁇ g/ml BSA. Embryos were incubated for 16 hrs at 37° C and stained with 3G10 antibody. Secondary antibodies for histochemical staining were from Vectorlabs, fluorescent secondary antibodies from Jackson Immunoresearch. Embryos sections were performed as described in Gisselbrecht, 1996.
  • Tout-velu is a Drosophila homologue of the putative tumour suppressor EXT-1 and is needed for Hh diffusion [see comments]. Nature 394, 85-8.
  • the proto-oncogene int-1 encodes a secreted protein associated with the extracellular matrix. Embo J 9, 1569-75.
  • Hedgehog transmitted along retinal axons, triggers neurogenesis in the developing visual centers of the Drosophila brain. Cell 86, 411-22.
  • Heparan sulfate proteoglycans are essential for FGF receptor signaling during Drosophila embryonic development, submitted. Lind, T., Tufaro, F., McCormick, C, Lindahl, U., and Lidholt, K. (1998).
  • the putative tumor suppressors EXT1 and EXT2 are glycosyltransferases required for the biosynthesis of heparan sulfate. J Biol Chem 273, 26265-8. Lindahl, U., Kusche-Gullberg, M., and Kjellen, L. (1998). Regulated diversity of heparan sulfate. J Biol Chem 273, 24979-82.
  • the division abnormally delayed (dally) gene a putative integral membrane proteoglycan required for cell division patterning during postembryonic development of the nervous system in Drosophila. Development 121, 3687-702.
  • Hedgehog patterning activity role of a lipophilic modification mediated by the carboxy-terminal autoprocessing domain. Cell 86, 21-34.
  • Example 6 Dally, a member of the glypican family of Heparan sulfate proteoglycans. regulates Wingless signaling in Drosophila
  • HS proteoglycans HSPGs
  • Dally a GPI-linked Glypican, as the HSPG molecule involved in Wg signaling. Loss of dally activity, both in the embryo and imaginal dies, generates phenotypes reminiscent to loss of Wg activity. Interestingly, dally is co- expressed with the Wg receptor Dfz2.
  • Dally serves as a co-receptor for Dfz2, and together with Dfz2 modulates both short and long-range activities of Wg.
  • HSPGs are ubiquitous macromolecules associated with both the cell surface and the extracellular matrix (ECM).
  • ECM extracellular matrix
  • HSPGs consist of a protein core to which Heparin/HS glycosaminoglycans (HS GAGs) are attached (reviewed by Bernfield et al., 1992; David, 1993; Kjellen and Lindahl, 1991; Yanagishita and Hascall, 1992).
  • GPI glycosyl phosphatidylinositol linked Glypicans and the transmembrane Syndecans represent the two major cell surface HSPGs.
  • HSPGs play critical roles in various cellular processes such as cell adhesion, neurite outgrowth, angiogenesis, tumorigenesis and tissue repair mechanisms (reviewed by Bernfield et al., 1992; David, 1993; Kjellen and Lindahl, 1991; Yanagishita and Hascall, 1992).
  • signal transduction HSPGs have been implicated to function as co-receptors for a number of growth factors, internalization of receptors and transport of signaling molecules (Salmivirta et al., 1996).
  • the importance of these molecules in development remains to be elucidated.
  • Wingless encodes a protein of the Wnt family and acts as a critical regulator in many developmental processes both during embryonic and larval development (reviewed by Siegfried and Perrimon, 1994). Genetic studies in Drosophila, in combination with biochemical studies, have led to the identification of a number of downstream molecules required for Wg signaling (for reviews, see Cadigan and Nusse, 1997; Cox and Peifer, 1998; Dale, 1998). According to a current model, Wg protein bind to the seven transmembrane receptor, Drosophila Frizzed 2 (Dfz2), and transduces its signaling to several downstream components resulting in the accumulation of cytoplasmic Armadillo (Arm).
  • Dfz2 Drosophila Frizzed 2
  • Arm subsequently translocates into the nucleus to form a complex with LEF/TCF transcription factors. Together Arm and TCF regulate the expression of many downstream target genes such as engrailed (en) in the embryonic epidermis, distalless (dll) and genes of the achaete scute complex (acs) in the wing.
  • Wg can exert both short-range and long- range effects during the development of the embryo and imaginal discs (Bejsovec and Martinez Arias, 1991; Struhl and Basler, 1993; Hoppler and Bienz, 1995; Lawrence et al., 1996).
  • wg is expressed in a narrow strip of four to five cells wide straddling the dorso-ventral (D/V) boundary, and Wg proteins form a gradient over a distance of up to 25 cell diameters (Zecca et al., 1996, Neumann and Cohen, 1997; Cadigan et al., 1998).
  • Wg acts as a short-range organizer to activate the expression of several genes adjacent to the wing margin, including members of the acs complex. Wg also functions as a mo ⁇ hogen to directly activate the transcription of several target genes such as dll in a Wg-concentration dependent manner (Zecca et al, 1996, Neumann and Cohen, 1997). However, what factors govern the shape of the Wg mo ⁇ hogen gradient and how the activity of Wg is regulated is not clear. Recent experiments have proposed that the expression of the Wg receptor Dfz2, which is negatively regulated by Wg signaling, plays a role in shaping the gradient of Wg activity. Overexpression of Dfz2 in wing discs was found to increase Wg range, possibly as a result of the stabilization of Wg when it is associated to Dfz2 (Cadigan et al., 1998).
  • Wg and its mouse homolog Wnt-1 are high-affinity Heparin-binding proteins (Bradley and Brown, 1990; Reichsman et al., 1996). Both Wg and mouse Wnt-1 are poorly secreted and usually tightly associated to the cell surface as well as the ECM (Papkoff and Schryver, 1990; Bradley and Brown, 1990; Gonzalez et al, 1991; van den Heuvel et al, 1993; Reichsman et al, 1996). Further, the tightly associated Wg proteins on the cell surface can be released by addition of exogenous Heparin (Bradley and Brown, 1990; Reichsman et al., 1996). These characteristics imply that HSPGs might be important factors in Wg function.
  • Wg signaling can be inhibited by removal of HS with Heparinase or by treatment of cells with sodium perchlorate, a competitive inhibitor that blocks the sulfation of proteoglycans (Reichsman et al., 1996).
  • Wg signaling is defective (Binari et al., 1997; Haecker et al., 1997; Haerry et al., 1997).
  • This enzyme is required for the formation of Glucuronic acid (GlcA). Because GlcA is required for the formation of HS, CS and dermatan sulfate (DS), it is not known what class of proteoglycans are involved for Wg signaling.
  • Sfl is involved in Wg signaling during embryonic development, sfl was identified in a genetic screen to characterize the maternal effects of zygotic lethal mutations (Perrimon et al., 1996; and Experimental Procedures for details). Homozygous sfl mutant animals derived from heterozygous mothers die at the third instar larval or early pupal stages. However, homozygous sfl mutant embryos derived from females lacking germline sfl activity (referred to as sfl null embryos throughout the text) die with a segment polarity phenotype. The sfl maternal effect is completely paternally rescuable indicating that sfl is expressed at least both during oogenesis and early embryonic development.
  • the cuticle phenotype of sfl null embryos is reminiscent of the phenotypes exhibited by either mutations in wg or hh.
  • the expression patterns of wg and en in these embryos are reminiscent of those observed in either wg or hh null mutants (DiNardo et al., 1988; van den Heuvel et al, 1993; Yoffe et al., 1995; Manoukian et al., 1995; Alcedo et al, 1996), and thus are consistent with a role for sfl in either Wg and/or Hh signaling.
  • Wg is nearly completely absent when En begin to fade during late stage 9 in sfl. This result suggests that in sfl null embryos Wg proteins may have diffused in the extracellular space or have been degraded upon their secretion.
  • Wg is required for D/V patterning and acts as a short-range inducer to activate the expression of several genes such as neuralized (neu) at the wing margin (Phillips and Whittle, 1993; Couso et al., 1994). Wg also functions as a mo ⁇ hogen to directly activate the transcription of several target genes including dll in a concentration dependent manner (Zecca et al., 1996, Neumann and Cohen, 1997). sfl homozygous mutant animals derived from heterozygous mothers die as late larvae to early pupae.
  • sfl encodes heparan sulfate N-deacetylase/N-sulfotransferase
  • HS GAGs are attached to various protein cores to form different HSPGs, we searched for candidate genes that could encode the protein component of the HSPG.
  • dally transcripts are expressed both maternally and zygotically. At early stages dally transcripts are uniformally expressed, however, at stage 8 they are enriched in a segmentally repeated pattern. In stage 8 embryos, dally transcripts are expressed in three to four cells anterior to wg-expressing cells. Interestingly, the stripes of dally expression overlap with those of the Wg receptor Dfz2 (data not shown) (Bhanot et al., 1996 ) suggesting a role for dally in Wg signaling.
  • dsRNA double-stranded RNA
  • dsRNAs corresponding to the entire coding region of dally into wild type embryos We injected dsRNAs corresponding to the entire coding region of dally into wild type embryos. Embryos injected with the dally dsRNAs exhibit a severe segment polarity cuticle defects, similar to those injected with wg or fz + Dfz2 dsRNAs (Kennerdell and Carthew, 1998). No significant defects were observed in a control experiment injected with buffer. This result, together with the genetic interaction observed between sfl and wg strongly argues that dally is a new segmentation polarity gene and that it is required at least for Wg signaling in the embryo.
  • Dally is required for both short and long-range signaling ofWg in wing imaginal discs.
  • the enhancement of the wing defects of dally mutant by a reduction in Wg activity suggests that Dally plays a role in Wg signaling at the wing margin.
  • Dfz2 is involved in shaping the gradient of Wg distribution and determine the response of cells to Wg.
  • Uniform Dfz2 overexpression in the wing pouch leads to ectopic bristles formation in the wing blade, most likely reflecting the activation of Wg signaling above its normal level (Cadigan et al., 1998).
  • ectopic expression of Dfz2 driven by the Gal4 line 69B resulted in wings with ectopic bristles.
  • the formation of ectopic bristles was drastically reduced suggesting that a mutation in dally blocks the activity of Dfz2.
  • Wg distribution in wing imaginal discs overexpressing Dfz2 and mutant for dally, would be affected.
  • Wg distribution was compared in UAS- Dfz2/69B-Gal4 and UAS-Dfz2 dallyP2/69B-Gal4 dallyP2 wing discs.
  • a significant reduction in Wg protein distribution was observed in a dally mutant background, suggesting that a reduction in dally activity destabilizes Wg/Dfz2 complexes.
  • An alternative model to explain the effect of dally on Wg distribution, is that Dally is involved in the transport of Wg proteins.
  • Dally expression is similar to Dfz2 and inhibited by Wg signaling
  • sfl segmentation polarity gene
  • ⁇ S is a ubiquitous GAG that is closely related to Heparin, a GAG expressed in vivo solely as a proteoglycan within the granules of mast cells and basophils (reviewed by Bernfield et al., 1992; Kjellen and Lindahl, 1991).
  • Biosynthesis of HS and Heparin are similar in vertebrates and invertebrates, and are initiated by formation of a polysaccharide chain consisting of multimers of D-glucuronic acid bl,4 - N-acetyl-D- glucosamine al,4 (GlcA-GlcNac) disaccharides.
  • HS N-deacetylase/N-sulfotransferase catalyzes N-deacetylation and N- sulfation, which is a coupled reaction and is the first and key step to initiate further modification reactions of HS/Heparin.
  • Sfl protein is strikingly homologous with all three isoforms of vertebrate NDST.
  • Syndecans and Glypicans differ in many ways. Syndecans bear both CS and HS GAGs while Glypicans are attached exclusively to HS GAGs. Further, Glypicans share several unique features that include a characteristic pattern of 14 highly conserved cysteine residues, 2 to 3 Ser-Gly HS GAG attachment sequences near the C-terminus, and a C-terminal sequence involved in the formation of a GPI-linkage to the membrane (Veugelers and David, 1998), suggesting that they have different functions from Syndecans. Finally, we find that in the embryo Dsyndecan is highly expressed in the mesoderm and trachea suggesting that it may be involved in FGF signaling (unpublished results).
  • Glypicans may also play a role in mammalian Wnt signaling pathways. Consistent with this model, most vertebrate glypicans are expressed in tissues, for example brain and kidney, where Wnt family members are expressed. It has been demonstrated that HSPG is required for the maintenance of mouse Wnt- .77 expression in the ureter tips of the developing kidney. Further, treatment of cultured kidney rudiments with either chlorate or Heparinase III resulted in the reduction of Wnt 11 expression, suggesting that HSPG is required for autocrine signaling activity of Wnt 11.
  • mouse K-glypican is expressed in a very similar pattern to Wnt 11 in a developing kidney (Watanabe et al., 1995).
  • mutation in human Glypican-3 are responsible for the Simpson-Golabi- Behmel Syndrome, a disease associated with prenatal and postnatal overgrowth and a high incidence of neuroblastomas and Wilm's tumors (Pilia et al., 1996). Since many Wnts are required for cell differentiation and growth control in brains and kidney, it is possible that this disease is associated with a defect in Wnt signaling.
  • Dpp activity in the wing is controlled by Hh signaling pathway which itself requires HSPGs (The et al, submitted), raising the possibility that the interaction between Dally and Dpp reflects a role for Dally in Hh signaling.
  • the alternative model is that Dally plays a direct role in Dpp reception.
  • the function of Dally would be tissue specific since we have no evidence for a function of HSPGs in the early function of Dpp in the establishment of D/V embryonic polarity. Tissue specific effects of Dally could be generated either through tissue specific expression of dally during development or tissue specific modification of the HS GAG chains linked to the Dally protein core.
  • Drosophila gene pipe which is involved in D/V patterning in the embryo, encodes a HS 2-0 sulfofransferase (Sen et al., 1998) that is expressed in ventral follicle cells. Future studies will be required to clarify the function of Dally in Dpp signaling. The role of Dally in Wg signaling
  • the co-receptor model for the role of Dally in Wg signaling is similar to the proposed role of HSPGs in other growth factor signaling pathways such as FGF.
  • the binding of growth factors to abundant but low affinity HS GAGs on the cell surface limits the free diffusion of the ligand from three to two dimensions, thereby increasing its local concentration and the probability that it will interact with less abundant, high affinity signaling receptor (Schlessinger et al., 1995).
  • the HS GAG chains of Dally trap secreted Wg.
  • the nature of the GPI anchor may facilitate lateral movement of Wg/Dally complexes such that Wg molecules can encounter less abundant Dfz2 receptor molecules.
  • Dally/Wg may further stabilize it or form an active ligand/receptor complex.
  • Dfz2 are expressed and regulated in a very similar manner.
  • the gradient of Dfz2 expression which is at its highest in cells that receive little Wg, is important for shaping the Wg mo ⁇ hogen gradient.
  • Our data with Dfz2 are consistent with Dally also playing a role in the stabilization of Wg protein at the cell surface because when Dally levels are reduced, Wg distribution stabilized by Dfz2 is also reduced.
  • the second model propose a role for Dally in Wg movement through field of cells rather than playing a direct role in activation of Dfz2.
  • the mechanism underlying the movement of Wg/Wnt molecules through tissues is not understood, and it is not clear whether the gradients of activities that these proteins trigger are established through diffusion of the secreted factors in the extracellular space or through transport mechanisms that involve for example vesicle-like structures which are endocytosed and/or transcytosed.
  • Wnts can be secreted in the extracellular space as well as transported through cells. Wnt proteins are poorly secreted in the extracellular space and bind the extracellular matrix tightly [Bradley, 1990 #39] [Gonzalez, 1991 #29] [Reichsman, 1996 #38].
  • dally alleles dally , dally p , dally are hypomo ⁇ hic alleles and were a gift from S. Selleck ( Nakato et al. 1995). dally is a new hypomo ⁇ hic allele generated by excision of the P-element associated with dally pl (REF? who made this.?). In an attempt to generate stronger loss of function dally allele, we generated following P-element excisions, a number of new dally alleles. However, none were stronger than the original ones described by Nakato et al. (1995).
  • UAS-Dfz2 and UAS-GPI-Dfz2 lines were obtained from R. Nusse (Cadigan et al., 1998).
  • a strong third chromosome UAS-Dfz2 line was used for all experiments.
  • UAS-Dfz2N was obtained from R. Carthew (Zhang and Carthew, 1998).
  • a strong second chromosome UAS-Dfz2N line N33 was used for all the experiment.
  • the UAS-arm act line SlO and the UAS-dTCF line DN4 expressing a deletion from of Arm (Pai et al.1997) or dTCF (van de Wetering et al, 1997) were obtained from M.Peifer.
  • Gal4 lines used were 69B-Gal4 (Brand and Perrimon, 1993), Ptc-Gal4 (Johnson et al.,1995).
  • C96 Gal4 was identified from a collection of PGawB insertions generated by K. Kaiser, wg is a wg null allele (Bejsovec and Wieschaus, 1993).
  • A101 neutralzZ is described in Usui and Kimura (1992).
  • Females with germline clones were generated using the autosomal "FLP-DFS" technique (Chou and Perrimon, 1996).
  • sfl FRT 2A /TM3, Sb females were mated with males of the genotype v w FLP 2 2/+, FRT 2A PfovcP ⁇ f/TM3, Sb.
  • the resulting progeny were heat shocked at 37°C for 2 hrs during the larval stages, and y w FLP 22 /+;
  • sfl FRT 2A /FRT 2A P[ovcpl] females carrying sfl homozygous germline clones were selected.
  • Females with germline clones of dally were generated using a dally FRT 2A recombinant chromosome using the same procedure as for sfl.
  • somatic wing clones For the generation of adult somatic clones in the wing, y w AsFLP122; sfl l(3)03844
  • FRT 2A /TM3, Sb were crossed with males of y w, Pfy+f FRT 2A /TM3, Sb. Larvae from this cross were heat-shock for 2 hours at first or second instar. Adult wings were mounted in Euparal for observation.
  • Anti-Wg serum was a gift of S. Cumberledge and used at 1:500 dilution.
  • Anti-En MAb4D9 was used at 1:300 dilution and obtained from Developmental Studies Hybridoma Bank (Patel et al. 1989).
  • Anti-DU serum was obtained from I. Duncan (Duncan et al., 1998) and used at 1:500.
  • Antibody against the Crumbs protein was used at 1:50 and obtained from E. Knust (Tepass and Knust 1993).
  • RNA probe (Lehmann and Tautz 1994).
  • wg digoxigenin-labeled DNA probe was prepared from a wg cDNA subcloned in the p s p65 plasmid.
  • dally cDNA was obtained from S.
  • Dfz2 cDNA was obtained from R. Nusse (Bhanot et al, 1996).
  • Genomic DNAs flanking the sfl 4 P-element insertion was obtained by plasmid rescue in E. coli (Cooley et al. 1988) .
  • To isolate sfl cDNAs we screened a 0-4 hr embryonic cDNA library (Brown and Kafatos 1988) with random primed probes generated from genomic DNA fragments from plasmid rescue.
  • One full-length 4.89 kb sfl cDNA was isolated. DNAs were sequenced by Taq-polymerase cycle sequencing and an automatic sequencer.
  • rescued plasmids from sfl flies were also sequenced using a primer derived from the P-element.
  • Northern blots of total RNA were carried out by standard procedures (Sambrook et al. 1989). Probes used are as following: 2.5 kb Xho-EcoRI fragment of sfl cDNA and sgl cDNA was used as a control (Haecker et al., 1997). Sequence alignment was produced using "DNA star" software.
  • RNA injection rescue was done as described ( Heacker et al., 1997).
  • the sfl mutation was recombined with a trachealess (trh) mutation (Wilk et al, 1996; Isaac and Andrew 1996) located at 61 Cl-2.
  • trh mutants are missing tracheas and exhibit defective posterior spiracles which are easily scorable by looking at embryonic cuticles.
  • Females that carry trh sfl homozygous germline clones were generated and mated with sfl/trh transheterozygous males, respectively.
  • RNA synthesis and injection were as described (Kennerdell and Carthew, 1998).
  • the dally full length cDNA cloned in pBluescript was used as a template used for RNA synthesis.
  • Both sense and antisense RNA of dally were synthesized using T7 and T3 polymerase by MEGAscript kit from Ambion company. A mixture of equal amount of sense and antisense RNA were heated in boiling water for 1 minute and then allowed to cool to room temperature for overnight. A concentration of I ⁇ g/ ⁇ l of dally dsRNA was injected in the posterior domain extending from 50% to 75% egg length at syncitial blastoderm stage. In a control experiment, the injection buffer was used.

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Abstract

Cette invention a trait à la découverte d'une nouvelle famille de protéines interactives hérisson, dénommées dans le descriptif "protéines interactives hérisson" ou "Exts". Il y est démontré que ces protéines se fixent à des polypeptides hérisson et ce, avec une affinité élevée. Ces protéines sont nécessaires à la diffusion des polypeptides hérisson et, à ce titre, à leur régulation.
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US6511987B1 (en) 1999-11-12 2003-01-28 Neurogen Corporation Bicyclic and tricyclic heteroaromatic compounds
EP1627640A1 (fr) * 2003-05-19 2006-02-22 Seikagaku Corporation Inhibiteur de transferase de groupe sulfate
US7682607B2 (en) 2001-05-01 2010-03-23 The Regents Of The University Of California Wnt and frizzled receptors as targets for immunotherapy in head and neck squamous cell carcinomas

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AU5443394A (en) * 1992-10-15 1994-05-09 New York University Medical Center A new class of rptpases: their structural domains and ligands
WO1998030576A1 (fr) * 1996-10-07 1998-07-16 The Johns Hopkins University School Of Medicine Nouveaux polypeptides derives d'une proteine herisson
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US6511987B1 (en) 1999-11-12 2003-01-28 Neurogen Corporation Bicyclic and tricyclic heteroaromatic compounds
US7326709B2 (en) 1999-11-12 2008-02-05 Neurogen Corporation Bicyclic and tricyclic heteroaromatic compounds
US7682607B2 (en) 2001-05-01 2010-03-23 The Regents Of The University Of California Wnt and frizzled receptors as targets for immunotherapy in head and neck squamous cell carcinomas
EP1627640A1 (fr) * 2003-05-19 2006-02-22 Seikagaku Corporation Inhibiteur de transferase de groupe sulfate
EP1627640A4 (fr) * 2003-05-19 2007-05-23 Seikagaku Kogyo Co Ltd Inhibiteur de transferase de groupe sulfate
US7396818B2 (en) 2003-05-19 2008-07-08 Seikagaku Corporation Sulfotransferase inhibitor

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