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First published online 26 November 2003
doi: 10.1242/dev.00913

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Three Drosophila EXT genes shape morphogen gradients through synthesis of heparan sulfate proteoglycans

Institute of Molecular and Cellular Biosciences, University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113-0032, Japan

View larger version (77K): [in a new window]   Fig. 1. sotv and botv are novel Drosophila EXT genes. All clones are marked by shavenoid– in Drosophila adult wing blade. (A) Wild-type wing. Longitudinal veins 1 to 5 (L1 to L5) are shown. (B) ttv mutant clone made between L1 and L2. The tissues within the clone are deleted and wing blade becomes narrower in A/P orientation. (C) sotv mutant clones. Note that veins L3 and L4 are abnormally close to each other. The region between L3 and L4 is patterned by Hh, whereas the rest of the wing blade is patterned by Dpp. sotv phenotype is relatively weaker than other EXT mutants ttv and botv. (D,E) botv mutant clones located between L2 and L3 or behind L4. The tissues within the clone are deleted. (F) Structures of Drosophila EXTs, Ttv, Sotv and Botv deduced from genome sequence. These proteins have three domains as indicated: light green, transmembrane region; light blue, putative catalytic region; blue, DXD motif. Amino acid sequence alterations in Ttv, Sotv and Botv caused by EMS mutation are indicated by arrows. (G) Glycosaminoglycan synthesis in Drosophila (from Toyoda et al., with modification) (Toyoda et al., 2000). (H) Sequence alignment of Ttv, Sotv and Botv proteins. Black boxes indicate amino acids that are identical in these proteins. The overall similarity between them is 26-32%. The homology is especially high in their C-terminal regions. Conserved DXD motif is indicated in red. Gal, galactose; GlcA, glucuronic acid; GlcNAc, N-acetylglucosamine; Xyl, xylose.

View larger version (92K): [in a new window]   Fig. 2. All three EXT genes are involved in HSPG biosynthesis in vivo. HSPG levels at the lateral surface of cells in the wild-type and mutant wing imaginal discs. (A) In the wild-type disc, uniform staining is detected (gray on the left panel). (B-D) Clones of cells mutant for the EXT genes are marked by the absence of GFP (green on the right panel). HSPG levels are severely reduced in cells mutant for any of the EXT genes. Here, and on all Figures in this report, imaginal discs are oriented anterior to the left and dorsal to the bottom.

View larger version (93K): [in a new window]   Fig. 3. EXT genes are required for Hh signaling. dpp-lacZ expression (gray on the left panel) and Ptc protein level (gray on the middle panel) in the wild-type and mutant wing discs. Some of mutant clones are marked by light-blue lines for clarification. (A) In the wild-type wing discs, dpp expression is induced by Hh that emanates from the posterior compartment and is detected as far as 15 cells away from the anterior-posterior boundary, and Ptc protein can be detected as far as five cells away. A schematic diagram is shown on the right panel. (B) In the botv423 clone (marked by the absence of GFP on the right panel), dpp (red in the right panel) and Ptc (blue in the right panel) levels are reduced (arrows), and cells located at the posterior part of the clone still respond to Hh signal. The reduction in dpp expression extends to the wild-type cells anterior to the mutant clone (arrowhead). (C) In the sotv326 mutant clone, Ptc protein and dpp expression levels are reduced slightly (arrow), indicating that Hh signaling is not severely impaired. (D,E) Loss of botv (D) or sotv (E) activity in the posterior compartment does not affect dpp level in anterior cells (arrow). However, Ptc protein level was sometimes slightly decreased in the anterior cells along the AP boundary (D,E).

View larger version (110K): [in a new window]   Fig. 6. Morphogen distribution in the EXT (ttv524botv510) mutant clones. Hh, Dpp and Wg proteins are significantly reduced in the mutant clones, both in the morphogen-expressing region (A-C) and in the receiving region (D-F). (A) In ttv524botv510 mutant clones (marked by the absence of GFP), Hh protein is severely reduced, whereas hh-lacZ expression was not downregulated. (B) Both Dpp protein and dpp expression levels are decreased in the ttv524botv510 clones. (C) Both Wg protein and wg expression levels are decreased in the ttv524botv510 clones. In the morphogen-receiving region, each of Hh, Dpp-GFP and Wg protein was significantly decreased in the mutant clones (arrowheads; D-F).

View larger version (162K): [in a new window]   Fig. 4. EXT genes are required for Dpp signaling. Levels of Sal and p-Mad in the wild-type and mutant discs. (A) The Sal and p-Mad levels are normally high in the central region of the wild-type wing disc. In A cells close to the A-P boundary, the Sal and p-Mad levels are much lower than those in the adjacent P cells. A schematic diagram is shown on the right panel. (B-D) In botv423 (B), sotv326 (C) and ttv524 mutant cells (marked by the absence of GFP), Sal and p-Mad levels are decreased (red and blue on the right panel, respectively). The A-P boundary is indicated by white arrowheads in B-D, right panel.

View larger version (126K): [in a new window]   Fig. 5. EXT genes are required for Wg signaling. Dll protein levels in the wild-type and mutant wing discs. (A) Wg is expressed at the wing margin and regulates the Dll expression (gray in the left panel) in a concentration-dependent manner. Schematic diagram is shown on the right panel. (B-D) In botv423 (B), sotv326 (C) and ttv524 (D) mutant cells, Dll expression is slightly decreased (arrows). The reduction in Dll protein level is extended to the wild-type cells located on the far side of the mutant clones (C,D).

View larger version (89K): [in a new window]   Fig. 7. Morphogens are accumulated in front of the mutant clones. (A-I) Hh diffusion patterns in the wild-type disc (A-C) and disc with ttv524botv510 clone (D-I). The staining intensity of Hh in the selected area (white boxes in B,E,H) was integrated along the A-P axis, plotted using NIH Image software and presented schematically (C,F,I). (A-C) In the wild-type disc, Hh flows into the anterior compartment with a moderate gradient starting from the middle of the posterior compartment. The distribution pattern of Hh in the ventral region (upper graph in C) is similar to that in the dorsal region (lower graph in C). (D-F) When the mutant clone was located in the anterior-ventral region along the A-P boundary, Hh was accumulated in the posterior-ventral cells. Compare the levels of Hh at the A-P boundary in the ventral region with that in the dorsal region (graphs in F). (G-I) Likewise, when the EXT clone was in the anterior-dorsal compartment, Hh accumulated in the posterior-dorsal cells (graphs in I). (J,K) Dpp-GFP accumulated in front of the EXT mutant clone. In the ventral compartment, Dpp-GFP diffuses with moderate gradient (upper bracket in J). Dpp-GFP accumulated on the cell surface of the wild-type cells adjacent to the mutant clone (lower bracket in J). Compare the levels of Dpp-GFP in the region with mutant clone (lower arrowhead in J) with that in the region without clone (upper arrowhead in J). (L,M) Wg accumulated in front of the EXT mutant clone. Compare the levels of Wg in the region with mutant clone (left bracket and arrowhead in L) with that in the region without clone (right bracket and arrowhead in L). For Hh and Wg, the same results were obtained in cells mutant singly for any of the EXT genes. Single mutation was not tested for the distribution of Dpp-GFP.

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