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First published online 3 March 2004
doi: 10.1242/dev.01051

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

Chun Han^1,2,*, Tatyana Y. Belenkaya^1,*, Marat Khodoun¹, Miyuki Tauchi^1,3, Xinda Lin¹ and Xinhua Lin^1,2,3,

¹ Division of Developmental Biology, Cincinnati Children's Hospital Medical Center, Cincinnati, OH 45229, USA
² Graduate Program in Molecular and Developmental Biology, University of Cincinnati College of Medicine, Cincinnati, OH 45229, USA
³ Graduate Program in Neuroscience, University of Cincinnati College of Medicine, Cincinnati, OH 45229, USA

View larger version (142K): [in a new window]   Fig. 3. The Drosophila EXT proteins are required for the biosynthesis of HS GAG chains in vivo. Third instar larval wing imaginal discs carrying mutant clones of ttv63 (A,A'), sotv44 (B,B') and botv103 (C,C') were fixed, digested with bacterial heparinase III and then stained with mAb 3G10, which recognizes the epitope generated by heparinase III digestion. The mutant clones are marked by the absence of GFP and are outlined with dots. 3G10 staining is absent in mutant clones of ttv63, sotv44 and botv103.

View larger version (78K): [in a new window]   Fig. 4. Involvement of sotv and botv in Hh signalling. All discs are oriented anterior left, dorsal up. (A-C') Ci staining (red) in a wild-type wing disc (A), and in discs carrying mutant clones of sotv44 (B,B') and botv103 (C,C') in the anterior compartment. The mutant clones are marked by the absence of GFP and are outlined with dots. Within the large clones of sotv44 and botv103, accumulated Ci is seen only in a narrow stripe of cells abutting the AP boundary. (D-F') Hh staining in a wild-type wing disc (D), and in discs carrying mutant clones of sotv44 (E,E') or botv103 (F,F'). The AP boundaries are determined by Ci staining (data not shown) and are marked by lines. Clone boundaries are marked by dotted lines. Hh staining is absent in the clones of sotv44 and botv103, except at a residual level in the posterior-most row of cells adjacent to AP boundary.

View larger version (107K): [in a new window]   Fig. 5. Involvement of the Drosophila EXT genes in Dpp signalling. All discs are oriented anterior left, dorsal up. (A-D') p-Mad staining in a wild-type wing disc (A), and in discs carrying mutant clones of ttv63 (B,B'), sotv44 (C,C') and botv103 (D,D'). The mutant clones are marked by the absence of GFP and are outlined with dots. (E-H') UAS-GFP-dpp under the control of dppGal4 in an otherwise wild-type wing disc (E) and in discs carrying mutant clones of ttv63 (F,F'), sotv44 (G,G') and botv103 (H,H'). The mutant clones are marked by the absence of DsRed and are outlined with dots. The distribution of GFP-Dpp outside the Dpp expression domain in the wild-type background appears to be a gradient extending towards the A and P compartments. Within the mutant clones of ttv63, sotv44 and botv103, the ranges of the GFP-Dpp gradient are greatly reduced.

View larger version (95K): [in a new window]   Fig. 6. Involvement of the Drosophila EXT genes in Wg signalling. In all discs carrying mutant clones, the clones are marked by the absence of GFP and are outlined with dots. (A-D'') Dll and Sens staining in a wild-type wing disc (A-A''), and in wing discs carrying mutant clones of ttv63 (B-B''), sotv44 (C-C'') and botv103 (D-D''). Dll expression appears to be a gradient with the peak at the DV boundary. The ranges of the Dll gradient in the mutant clones of ttv63 (B), sotv44 (C) and botv103 (D) are reduced. Note that the level of Dll expression close to the DV boundary within the mutant clones of ttv63 and sotv44 is comparable with that of wild-type cells; however, it is significantly reduced in the botv103 mutant clone. Wg-dependent Sens expression in the wing disc is in two narrow stripes abutting Wg expressing cells. This expression is unaffected in the mutant clones of ttv63 (B') and sotv44 (C'), but is diminished in the botv103 mutant clone (D'). Note that the Wg-independent expression of Sens in botv103 mutant clone is still maintained. (E-H') Extracellular Wg distribution is shown in a wild-type wing disc (E), and in discs carrying mutant clones of ttv63 (F,F'), sotv44 (G,G') and botv103 (H,H'). Extracellular Wg is almost completely lost in the botv103 clone except at the surface of Wg expressing cells, whereas it is maintained at some level around the DV boundary in the mutant clones of ttv63 and sotv44. (I-J') Sens staining in discs bearing clones of ttv63-sotv44 double mutant cells. The Wg-dependent Sens expression is diminished in the double mutant clones.

View larger version (77K): [in a new window]   Fig. 1. Identification of botv and sotv as two novel segment-polarity genes. (A-D) Wings are oriented proximal to the left, anterior up. (A) A wild-type wing. (B-D) Wings bearing somatic clones of ttv63 (B), botv103 (C) and sotv44 (D). Adult wings with clones of these mutations exhibit a variety of phenotypes, including vein loss, vein fusion, blister, narrowed wing and wing notching (data not shown). (E-I) Cuticle preparations of a wild-type embryo (E), a hhIJ35 homozygous embryo (F), and embryos derived from mutant germ-line clones of botv103 (G) and sotv44 (H,I). All embryos are oriented anterior to the left. Embryos lacking both maternal and zygotic activities of botv or sotv exhibit typical segment-polarity phenotypes; however, the defects of sotv mutants can be relatively weak (I). (J-L) En staining of a stage 10 wild-type embryo (J), and embryos derived from mutant germ-line clones of botv103 (K) and sotv44 (L). (M-O) Wg staining of a stage 10 wild-type embryo (M), and embryos derived from mutant germ-line clones of botv103 (N) and sotv44 (O).

View larger version (78K): [in a new window]   Fig. 2. Sotv and Botv are members of the Drosophila EXT family of tumor suppressors. (A) Structures and mutations of Sotv and Botv. Sotv and Botv are putative type II transmembrane proteins. Three mutations of botv and sotv are shown in green bars (except sotv44, which harbors a G to A transition at the start codon). (B) Phylogenetic tree of Drosophila, mouse (m), and human (h) EXT family proteins. Ttv, Sotv and Botv are the Drosophila EXT1, EXT2 and EXTL3 proteins, respectively. (C) Sequence comparison of Ttv, Sotv and Botv. Identical residues are highlighted (light red) and consensus residues are boxed. A conserved nucleotide sugar-binding motif DXD is found at the C-terminal portion of all three proteins and is boxed (blue). The sequence alignment and phylogenetic tree were generated using Lasergene software with the Jotun Hein method.

View larger version (50K): [in a new window]   Fig. 7. Ttv and Sotv form a complex and are co-localized in vivo. (A-C) Co-immunoprecipitation of Ttv with Sotv (A), of Ttv with Botv (B), and of Sotv with Botv (C). Drosophila S2 cells were transfected with plasmids to express Myc-tagged Ttv, V5-tagged Sotv and HA-tagged Botv in various combinations. Cell lysates were immunoprecipitated and then analyzed by western blotting with the antibodies indicated. IP, immunoprecipitation; IB, immunoblot. The `m' in the first lane of the left panel (A) indicates that ttv-myc and sotv-V5 were individually transfected and the cell lysates mixed in vitro prior to immunoprecipitation. Ttv-Myc and Sotv-V5 can easily precipitate each other (A), whereas Botv-HA cannot precipitate Ttv-myc or Sotv-V5, or vice versa (B,C). (D-D'') Double staining of Ttv-Myc (green) and a Golgi marker (red) in Drosophila S2 cells expressing Ttv-Myc. Ttv-Myc is concentrated in the Golgi complex. (E,E') Double staining of Ttv-Myc (green) and the ER marker Calnexin (red) in Drosophila S2 cells expressing Ttv-Myc. Ttv-Myc is co-localized with Calnexin. (F-F''') Co-staining of Ttv-myc (green), Sotv-V5 (blue) and Botv-HA (red) in S2 cells transfected with all three expression constructs. Although the majority of all three proteins are co-localized, Ttv-myc and Sotv-V5 appear to be more precisely co-localized and concentrated in certain compartments, whereas Botv-HA seems more uniformly distributed. (G-J) All embryos are oriented anterior left, dorsal up. All panels show embryos derived from females with corresponding mutant germline clones. (G) A ttv63 null embryo. (H-J) Ectopic expression of ttv-myc driven by hairyGal4 (IJ3) in null embryos of ttv63 (H), sotv44 (I) and botv (J). X-gal staining was used to identify sotv (I) or botv (J) null embryos expressing UAS-ttv-myc by hairyGal4 (I,J). Embryos stained blue were selected for cuticle preparation. The genotypes shown are ttv63/ttv63UAS-nlacZ; IJ3 UAS-ttv-myc/+ (H), sotv44/sotv44 UAS-nlacZ; IJ3 UAS-ttv-myc/+ (I) and botv103/botv103 UAS-nlacZ; IJ3 UAS-ttv-myc/+ (J). Although ectopic expression of ttv-myc can rescue ttv63 null with full penetrance, it cannot rescue sotv44 and botv103 mutant embryos.