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First published online November 21, 2006
doi: 10.1242/10.1242/dev.02711

Development 133, 4969-4979 (2006)
Published by The Company of Biologists 2006
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The metalloprotease Tolloid-related and its TGF-ß-like substrate Dawdle regulate Drosophila motoneuron axon guidance

Mihaela Serpe1,2 and Michael B. O'Connor1,2,*

1 Department of Genetics, Cell Biology and Development and the Developmental Biology Center, University of Minnesota, Minneapolis, MN 55455, USA.
2 Howard Hughes Medical Institute, Minneapolis, MN 55455, USA.


Figure 1
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  		Fig. 1. Tlr expression pattern. (A) Expression of tlr transcript in the muscles of a stage 13 embryo by in situ hybridization. (B) Same stage embryo stained with an anti-Tlr antibody and rolled to show the Tlr protein expression in muscles. (C) tlr transcript is present in the imaginal discs (arrows) and in a distinct pattern in the CNS of the late embryos. (D) Tlr protein expression in the CNS (upper view) and in the corpus allatum portion of the ring gland (arrow in lower view) in a stage 17 embryo. (E-E'') Tlr protein (E', red in E'') is expressed in the corpus allatum of an embryo whereas phantom>GFP is in the prothoracic portion of the ring gland (E, green in E'').

 

Figure 2
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  		Fig. 2. tlr loss-of-function phenotype in the embryonic CNS. (A) tlrex[2-41]/tlrE1 embryos overall have a normal CNS appearance when stained with mAb BP102. Staining with anti-Fas2 antibody reveals defects in the CNS (B-D) and motoneuron arbors (F-L). Red arrows indicate defects; black arrows indicate normal projections. The CNS fasciculation defects (red arrows) vary from mild (C) to severe (B,D). (E) Schematic of the wild-type motoneuron projections, adapted from Landgraf et al. (Landgraf et al., 1997Go). (F-L) tlrex[2-41]/tlrE1 embryos showed a variety of guidance defects (red arrows; black arrows indicate wild-type-like projections): (F) delay in ISN growth and (G) defective ISN terminal arbor morphology; (H) SNa stalling and (I) additional branching; (L) SNb failure to reach ventral muscle targets. Schematic diagrams illustrate SNa and SNb stalling (J) and misguidance (K) in tlr mutants. (M-S) Innervation on ventral muscles in third-instar larvae visualized with anti-Fas2 antibody (M-O) or anti-csp antibody (P-S). Dorsal is up and anterior is left in all except B which is a view from ventral side. Scale bars are 50 µm in B,G,I,L; 100 µm in O,S.

 

Figure 3
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  		Fig. 3. Tlr is a circulatory enzyme. Western blots showing Tlr and Tlr(HA)3 (A) or ß-Gal (B). The serum protein serves as a loading control. The immunoblots were simultaneously probed for non-tagged/endogeneous Tlr (anti-Tlr) and tagged/overexpressed Tlr(HA)3 (12CA5) or mouse anti-ß-Gal. S2, transfected S2 cells; H, hemolymph from third-instar larvae of the indicated genotype; C, third-instar carcasses; *, non-specific bands.

 

Figure 4
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  		Fig. 4. Tlr and Tld process the pro-peptides of several TGF-ß ligands in Drosophila. (A) Alignment of predicted BMP-1/Tolloid cleavage sites in Act, Daw, Myo and Mav pro-peptides. *, residues that were changed to alanine in the mutated forms of the ligands shown in F; red arrow, cleavage site. (B) Schematic of tagged pro-peptide and pro-peptide fragments visible by western analysis. (C-F) Conditioned media from S2* cells co-transfected with ligands and enzymes as indicated were analyzed by western blotting using anti-V5 antibody for detection of N-tagged pro-peptides and cleaved fragments. Tm designates the corresponding mutant in which the BMP-1/Tolloid site was destroyed.

 

Figure 5
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  		Fig. 5. Pro-peptide processing activates Alp in a cell-based signaling assay. (A) Signaling activities of various ligands -/+ Tld were tested using S2 cells expressing Flag-Mad (left panels) or Flag-Smad2 (right panels) and western analysis of the cell extracts. The signals were analyzed using primary antibodies against Mad1-P and Smad2-P (P-Mad1 and P-Smad2, respectively) and an anti-Flag antibody, and anti-rabbit IRDye 800 and anti-mouse IRDye 700 secondary antibodies, followed by scanning and quantification by the Odyssey Infrared Imaging System. (B) Ligands +/-enzymes were analyzed using anti-V5 antibody for Daw pro-peptide detection (arrow) and anti-HA for enzymes detection (parenthesis). (C,E) Dose-dependence of Daw activities +/-Tld or Tlr in signaling assays. S2 cells transfected with Flag-Smad2 were incubated with 0, 25 µl, 50 µl, 100 µl and 150 µl of the conditioned supernatants shown in B, lanes 1, 2 and 4 (C), or 5, 6 and 8 (E). (D,F) Quantification of Tld and Tlr effects on Daw activities. The signals are normalized as the relative ratio Smad2-P/Flag and represented as a function of ligand amount.

 

Figure 6
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  		Fig. 6. daw expression pattern. (A-G) Expression of daw transcript in Drosophila embryos at blastoderm stage (A), stage 7 (B,C), stage 13 (D) and stage 17 (E-G). A,B,D are lateral views; C,E show ventral sides; F,G are ventral and dorsal views, respectively, of dissected CNS. (H-J) Expression of daw transcript in third-instar larval tissues: (H) brain lobes with ring gland and ventral ganglion (insert shows detail of the brain lobe surface); (I) ventral muscle field; (J) eye imaginal disc. (K) Schematic representation of the position of the transposable element (blue arrowhead) and the breakpoints of the imprecise dawex11 excision relative to the daw gene organization.

 

Figure 7
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  		Fig. 7. Axon guidance defects associated with Daw signaling pathway components. (A) Representative axon guidance defects in a ventral muscle field of a stage 17 dawex11/dawex11 embryo. Defects are indicated with red arrows; black arrows indicate wild-type-like projections. (B) CNS and (C) SNa defects in dawex11/dawex11 embryos. (D) 12/13 synapse in dawex11/dawex11 third-instar larvae. (E) SNa and (F) SNb in stage 17 babo52/babo32 embryos derived from babo52 germline clones. (G) Ventral muscle field in 17 put heteroallelic embryos reared at 23.5°C. (H) SNa and (I) SNb in CNS of stage 17 Smad2 embryos derived from germline clones. (J) Quantification of loss-of-function defects for Daw signaling pathway components. Dorsal is up and anterior is left in all images. Scale bars: 100 µm in D; 50 µm in all others.

 

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© The Company of Biologists Ltd 2006