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First published online November 21, 2006
doi: 10.1242/10.1242/dev.02673

Development 133, 4981-4991 (2006)
Published by The Company of Biologists 2006
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The divergent TGF-ß ligand Dawdle utilizes an activin pathway to influence axon guidance in Drosophila

Louise Parker1, Jeremy E. Ellis1, Minh Q. Nguyen2 and Kavita Arora1,3,*

1 Department of Developmental and Cell Biology, University of California Irvine, Irvine, CA 92697-2300, USA.
2 NIH/NIDCR Bethesda, MD 20892, USA.
3 Developmental Biology Center, University of California Irvine, Irvine, CA 92697-2300, USA.


Figure 1
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  		Fig. 1. Molecular characterization of daw. (A) Genomic organization of the daw region. The synaptotagmin (syt) gene is located 5.5 kb centromere distal of daw and CG2964 is located 300 bp proximal of daw. Alternate isoforms daw-A and daw-B, which share common coding exons, are shown. The P{EP}EP1039 insertion site, genomic rescue fragment Daw-XmnI and deletions daw{Delta}1 and daw{Delta}2 are indicated. (B) Sequence and conceptual translation of daw (Genbank accession no. AY051485). Italics indicate signal sequence, boxes are proteolytic cleavage sites, and circles mark cysteine residues conserved in TGF-ß and activin ligands. Lesions in daw3 (deletion of CA) and daw4 (G->T substitution) are highlighted in gray. (C) Clustal-W alignment of ligand domains of human TGF-ß3, activinC, BMP3, and Drosophila TGF-ß superfamily members. Identities are in dark gray, similarities in light gray, and asterisks mark conserved cysteines.

 

Figure 2
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  		Fig. 2. Expression profile of daw. (A-N) Embryos, larval imaginal discs and brains were hybridized with daw probe. (A) Stage-5 embryos showing uniform maternal transcript. Sites of zygotic expression include: mesoderm at stage 7 (B) and stage 11 (C); visceral and somatic mesoderm (D) and oenocyte (magnified view E) at stage 13; VNC (optical sections F,G), fat body (arrowhead in G), posterior spiracles, hindgut and the maxillary segment (H), median, intermediate and lateral clusters of glial cells in the VNC (magnified views I,I'), at stage 17. (J) VNC of gcm- embryos. Larval expression is in the optic lobe and central brain (K), wing (L) and leg (M) imaginal discs, and bodywall muscles (N). A-C are lateral views; D,E,H are dorsal views; F,G,I,I',J are ventral views; A-J, anterior is to left.

 

Figure 3
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  		Fig. 3. Aberrant pathfinding in daw mutants. (A-C) Late stage 16/17 daw3 embryos labeled with (A) BP102, (B) 1D4 or (C) {alpha}-Repo antibodies. Arrowheads mark rare CNS defects. (D,E) Schematic representation of motoneuron pathfinding in wild-type and daw- embryos. Anterior is left, dorsal is up. Axon trajectories are depicted on the left and muscle targets are included on the right. (D) Five main nerve branches are shown that innervate correspondingly colored muscles. ISNb axons defasciculate to form three branches that innervate the muscle 6/7 cleft, muscle 13 and muscle 12. SNa forms a dorsal branch that innervates muscles 21-24 and a lateral branch that innervates muscles 5 and 8. For detailed description see Landgraf et al. (Landgraf et al., 1997Go). (E) In daw- mutants the ISNb and SNa extend into their correct target fields, but terminate prematurely (black and white arrowheads, respectively). (F-I) Motoneuron projections in late stage 16/17 filleted embryos stained with 1D4. (F) Wild-type ISNb synapses on muscles 6/7, 13 and 12. (G) In daw3 embryos, ISNb stalls at muscle 13 (black and white arrowheads), or reaches muscle 12 but fails to form a synapse (arrow). (H) Wild-type SNa dorsal (d) and lateral (l) branches. (I) daw3 embryos displaying failure of SNa defasciculation and loss of the lateral branch (arrow). Arrowhead marks stalled axons. (J,K) Incidence of ISNb and SNa pathfinding defects in daw- and Oregon R embryos. ISNb defects in J were scored as: stalling at muscles 6/7, muscle 13, or muscle 12 and failure to form a synapse. SNa defects in K were scored as: failure to reach target (short), or defasciculate at branchpoint (bifurcation). Hemisegments scored: daw{Delta}1 n=161/115, daw{Delta}2 n=300/233, daw3 n=222/221, daw4 n=238/200, daw maternal and zygotic nulls (MN) n=93/93, Oregon R n=155/178 for ISNb/SNa pathfinding.

 

Figure 4
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  		Fig. 4. Daw induces Smad2 phosphorylation via activin pathway receptors. S2 cells were transiently transfected with Smad2 alone or with wild-type and dominant-negative receptors as marked. (A) Response to Babo; (B) response to Put. Cells were exposed to control or Daw-conditioned media and western blots probed with {alpha}-PS2 to detect phosphorylated Smad2. Smad2 expression was monitored using {alpha}-FLAG. Smad2 phosphorylation was detected only in the presence of receptor (lanes 2, 8), or ligand (lanes 4, 10), but not in their absence (lanes 1, 7). Exposure to Daw enhanced Smad2 phosphorylation in Babo- and Put-expressing cells (lanes 5, 11). Coexpression of dominantnegative receptors (lanes 6, 12) reduced phosphorylation levels in response to ligand, demonstrating receptor specificity.

 

Figure 5
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  		Fig. 5. Activin signaling pathway components have similar expression and mutant phenotypes. (A,B) babo mRNA is enriched in the VNC and brain in stage 13 (A, lateral view), and stage 15 (B, ventral view) embryos. (C) Incidence of total pathfinding defects in babo, put and Smad2 mutants, as compared with daw- and Oregon R. MN refers to maternal/zygotic nulls. Hemisegments scored: babo32/babo32 n=187/120, babo MN n=211/116, put88/put88 n=208/174, Smad2388/Y n=137/138. (D-F) ISNb and (G-I) SNa guidance defects. Motoneuron projections in late stage 16/17 filleted embryos stained with 1D4. Arrowheads in D-G indicate stalled axons. Arrow in D marks ISNb axons that extend to muscle 12 but fail to form a synapse. In G-I, arrows indicate defasciculation failures and loss of SNa branches.

 

Figure 6
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  		Fig. 6. Disruption of Babo and Put signaling induces pathfinding defects. UAS-babo{Delta}I, UAS-put{Delta}I or UAS-tkv{Delta}I were expressed using different Gal4 drivers at 29°C. Motoneuron projections in late stage 16/17 filleted embryos stained with 1D4. OK6-Gal4 driven expression of Babo{Delta}I (A) and Put{Delta}I (B) results in stalling of ISNb axons and thickening of axon bundles (arrowheads).

 

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© The Company of Biologists Ltd 2006