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First published online 31 March 2004
doi: 10.1242/dev.01061

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

¹ Developmental and Cell Biology, University of California Irvine, Irvine, CA 92697, USA
² Howard Hughes Medical Institute, Department of Cellular and Molecular Medicine, University of California, San Diego, La Jolla, CA 92093, USA
³ Department of Molecular and Cellular Biology, University of Arizona, Tucson, AZ 85721, USA
⁴ Departments of Pediatrics and Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN 55455, USA

View larger version (33K): [in a new window]   Fig. 8. HS-derived disaccharides from sotv and ttv mutants. HPLC profiles of GAGs from wild-type, sotv and ttv larvae are shown (A). Peaks 1-6 represent the following disaccharides: UA-GlcNAc, UA-GlcNS, UA-GlcNAc6S, UA-GlcNS6S, UA-2S-GlcNS and UA2S-GlcNs6S, respectively. (B) A representative HPLC trace from sotv18.2/Df(2R)Jp8 larvae at a six times more sensitive scale than in A. All six disaccharide species found in wild type can be detected in these animals, albeit at greatly reduced levels. (C) Disaccharides from sotv/Df(2R)Jp8 larvae are shown as a percentage of wild-type controls. The sotv alleles are ordered according to the levels of HS produced, which correlates with the predicted lengths of the mutant proteins (see Fig. 1). The results for ttv mutants are from two different experiments. In one case ttvK06619 was derived maternally and in the other ttv02055 was contributed maternally. Different colored bars indicate individual HS disaccharides.

View larger version (23K): [in a new window]   Fig. 9. HPLC profiles showing CS-derived disaccharides (A). The largest peak, migrating at 4 minutes retention time, represents unsulfated Di-0S, followed by the Di-4S disaccharide. Each tracing represents material from 40 larvae. (B) CS-derived disaccharides from sotv and ttv mutants, shown as a percentage of wild-type controls (fraction of ng disaccharide/mg dry tissue, Ore R control). Unsulfated (Di-0S) and monosulfated disaccharides (Di-4S) across the sotv allelic series show changes in total CS levels as well as the degree of sulfation.

View larger version (62K): [in a new window]   Fig. 1. Clustal alignment detects 47% sequence identity between Sotv and human EXT2. Identical residues are bold and boxed in dark gray, while similarities are boxed in light gray. A strongly hydrophobic region likely to represent a transmembrane domain is indicated by a double-headed arrow. Vertical arrows indicate positions of nonsense mutations in the five characterized sotv alleles. The dumbbell highlights a DXD motif conserved in UDP-sugar-dependent glycosyltransferases.

View larger version (92K): [in a new window]   Fig. 2. Cuticle preparations of ttv and sotv germline clone embryos resemble hh and wg mutants. The ventral surface of wild-type embryos is marked by bands of denticles separated by naked cuticle (A). Zygotic removal of either wg (B) or hh (C) results in loss of naked cuticle and a lawn of ventral denticles. Embryos lacking maternal and zygotic sotv (D), ttv (E), or both ttv and sotv (F) also show a reduction in naked cuticle. The phenotypes in D-F are of similar severity, but less severe than hh or wg nulls.

View larger version (169K): [in a new window]   Fig. 3. Wings mosaic for homozygous patches of ttv (A,D), sotv (B,E), and ttv, sotv (C,F) mutant cells show notching (A-C) and ectopic bristles (D-F) in the vicinity of the wing margin. The insets in D-F are higher magnification views. Clones are unmarked in these examples, but no notching or ectopic bristles were observed in non-mosaic control siblings.

View larger version (143K): [in a new window]   Fig. 4. Formation of the Wg gradient is severely affected in cells that are homozygous mutant for ttv, sotv and ttv, sotv. In this and subsequent figures, antibody staining is in red. The boundaries of informative clones are outlined in white. Staining for extracellular Wg reveals a shallow gradient in the wing pouch that is strongly reduced in clones of ttv (A,D), sotv (B,E) and ttv, sotv (C,F) that cross the Wg expression domain. Arrows draw attention to regions near the prospective wing margin, where a sharp decrease in extracellular wingless levels is visible across the clone boundary. Mutant clones of ttv (G), sotv (H) and ttv, sotv (I) that cross the Wg stripe cause a less dramatic reduction in cytoplasmic Wg levels.

View larger version (32K): [in a new window]   Fig. 5. Expression of the Wg target gene ac is altered in clones lacking HS polymerase activity. In wild type (A), Ac is expressed in the anterior compartment in cells on either side of the Wg expression domain. Ac expression is reduced or lost in ttv (B), sotv (C) or both ttv, sotv (D) mutant clones (see arrows). Occasionally, ectopic Ac is observed in the vicinity of the mutant clone (B, arrowhead), which correlates with the location of ectopic margin bristles in wings from mosaic adults (see Fig. 3D-F).

View larger version (68K): [in a new window]   Fig. 6. Stabilization of the Hh target Ci is restricted, and Hh levels are reduced in mutant clones. Hh dependent inhibition of Ci proteolysis appears as a band of intense staining 8-10 cells wide anterior to Hh-expressing cells in the posterior compartment. The remaining cells in the anterior compartment show low levels of Ci staining. In ttv (A), sotv (B) or double mutant clones (C), the domain of Ci stabilization is reduced to 1-2 cells in width. Hh is uniformly distributed in the posterior compartment except in clones lacking ttv (E), sotv (F) or ttv and sotv (G). Reduced ligand levels are apparent in clones located along the AP boundary (arrows) as well as deep within the posterior compartment (arrowheads). (D,H) By contrast, Hh transcription, visualized using a Hh-lacZ reporter (H), is unaffected in posterior clones mutant for sotv (arrowhead). Mutant clones were visualized by the absence of GFP expression (D).

View larger version (91K): [in a new window]   Fig. 7. Dpp signal transduction and Sal expression are inhibited in cells lacking HS polymerase activity. Staining with PS1 antisera allows visualization of pMad generated by Dpp signaling activity. Cells mutant for ttv (A), sotv (B) or ttv and sotv (C), show reduced pMad levels (arrows in A and C or arrowhead in B) regardless of whether the clones are situated in the anterior or posterior compartments, suggesting that Dpp signaling is compromised independently of Hh. Low levels of pMad can be detected within clones in the vicinity of the clone boundaries, suggesting that Dpp signaling can still occur in mutant cells, although with reduced effectiveness or range. Sal responds to a high threshold of Dpp signaling and is expressed in the wing pouch centered on the AP boundary. However, in ttv (D), sotv (E) and ttv, sotv (F) mutant cells, Sal expression is reduced independently of whether clones lie in the anterior or posterior compartment (arrows). Loss of Sal in anterior clones is a direct result of loss of Dpp signaling rather than an indirect consequence of compromised Hh signaling, since it occurs in a domain beyond the effective range of Hh. Sal staining persists in ttv and sotv clones that overlap the AP boundary (arrowheads), suggesting that Dpp can signal in an autocrine or paracrine fashion, even in mutant cells.