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First published online June 6, 2008
doi: 10.1242/10.1242/dev.017319

Development 135, 2277-2287 (2008)
Published by The Company of Biologists 2008
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Src family kinases are required for WNT5 signaling through the Derailed/RYK receptor in the Drosophila embryonic central nervous system

Rene R. Wouda, Monique R. K. S. Bansraj, Anja W. M. de Jong, Jasprina N. Noordermeer* and Lee G. Fradkin*

Laboratory of Developmental Neurobiology, Department of Molecular and Cell Biology, Leiden University Medical Center, P.O. Box 9600, 2300 RC, Leiden, The Netherlands.


Figure 1
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  		Fig. 1. SFKs play redundant roles during formation of the embryonic CNS commissures. Stage 16 Drosophila embryos of the indicated genotypes were stained with mAb BP102 to label all central axons. Anterior is up. (A) Wild type, (B) Wnt5400, (C) Src42AE1, (D) Src64BKO, (E) Src42AE1/+; Src64BKO/+, (F) Src42AE1; Src64BKO/+, (G) Src42AE1/+; Src64BKO, (H) Src42AE1; Src64BKO, (I) ELAV-GAL4/UAS-RNAi-Src64B and (J) Src42AE1; ELAV-GAL4/UAS-RNAi-Src64B are shown. Defects similar to those seen in Wnt5400, namely `fuzzy' commissures and breaks in the longitudinal pathways, are observed in individuals homozygous for one of the SFK mutants and heterozygous for the other and also in individuals of a Src42A mutant background when Src64B expression is reduced in the nervous system by RNA interference. The commissures are completely fused (white arrow in H) in the double homozygotes. See Table 1 for quantitation. AC, anterior commissure; PC, posterior commissure.

 

Figure 2
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  		Fig. 2. Src64B is required for Wnt5/drl-mediated axon repulsion. (A) Ectopic WNT5 expression in the midline glia (SIM-GAL4, UAS-WNT5/+) results in frequent thinning or complete loss (arrow) of the AC. (B) Heterozygosity for Src64B suppresses the thinning/loss of the AC (SIM-GAL4, UAS-WNT5/+; Src64BPl/+). (C) EG+ neurons crossing in the AC and PC in a Drosophila embryo with one copy of UAS-DRL-MYC and one copy of UAS-NLS-β-Gal visualized by anti-MYC staining (UAS-DRL-MYC/+; UAS-NLS-β-Gal/+; EG-GAL4/+). (D) Overexpression of SRC64B in EG+ neurons does not cause the PC axons to switch commissures (UAS-mCD8-GFP/UAS-SRC64B; EG-GAL4/+). (E) Simultaneous expression of DRL-MYC and SRC64B in the EG+ axons significantly increases the number of the PC-crossing lineages to switch to the AC (arrow) (UAS-DRL-MYC/+; UAS-SRC64B/+; EG-GAL4/+). Quantitation is presented in Tables 2 and 3. Stage 16 embryos are shown, anterior is up.

 

Figure 3
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  		Fig. 3. Src64B mRNA expression overlaps with drl mRNA expression and SRC64B protein is present in axons. (A) Wild-type Drosophila embryo labeled with a Src64B antisense RNA probe shows Src64B expression throughout the ventral nerve cord (arrow) and in the gut. (B) Double RNA in situ staining for endogenous Src64B mRNA (green) and drl mRNA (red) shows that drl and Src64B overlap in the ventral nerve cord in the anterior portion of each segment. (C) Double RNA in situ staining for endogenous Wnt5 mRNA (green) and drl mRNA (red) shows that Wnt5 is predominantly expressed in PC-associated neuronal cell bodies that do not express drl. (D) SRC64B protein is expressed in the wild-type longitudinal and commissural axons. (E) Axons of a homozygous Src64BPI mutant embryo are not stained by anti-SRC64B. (F) Fluorescent double-antibody labeling of a third instar larval neuropile ectopically expressing SRC64B (red) and GFP (green) in motoneurons (OK6-GAL4>UAS-GFP). The arrow indicates fasciculated SRC64B-expressing motoneuron axons.

 

Figure 4
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  		Fig. 4. SRC64B and DRL and their mammalian orthologs physically associate. (A) Drosophila Kc cells were transfected with the indicated expression constructs, lysates were immunoprecipitated (IP) with antibodies specific to DRL (anti-HA) or SRC64B (anti-MYC) and then immunoblotted (WB) with the reciprocal antibody to detect co-immunoprecipitation. The expression of DRL and SRC64B was confirmed by immunoblotting of the whole-cell extract (WCE). DRL and SRC64B specifically co-immunoprecipitate in the presence or absence of WNT5 protein. (B) The mammalian orthologs of DRL and SRC64B, RYK and c-SRC, physically interact as assayed by their co-immunoprecipitation from transfected human 293T cell lysates. The expression of RYK-HA and untagged c-SRC was confirmed by WCE immunoblot. RYK derived from c-SRC-overexpressing cells migrates faster than control RYK species, presumably owing to altered post-translational processing. Endogenous c-SRC protein is visible in lanes 1 and 2 of the lowermost blot.

 

Figure 5
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  		Fig. 5. SRC64B tyrosine kinase activity is required for the formation or stability of the DRL-SRC64B complex. (A) Herbimycin A treatment leads to significantly decreased amounts of SRC64B co-immunoprecipitating with DRL. Aliquots of Kc cells co-transfected with DRL-HA and SRC64B-MYC expression constructs were treated for 24 hours with increasing concentrations of herbimycin A or equivalent volumes of the DMSO carrier, WCEs prepared, DRL-SRC64B complexes immunoprecipitated with anti-DRL and then immunoblotted with anti-MYC (SRC64B) antibody (top panels). Lanes 1 and 5, untreated samples; lanes 2-4, herbimycin A-treated at 2.5, 5 and 10 µM final concentration, respectively; lanes 6-8, DMSO controls. Equal amounts of WCEs were evaluated for SRC64B-MYC expression (middle panels) and overall tyrosine phosphorylation levels (lower panels). (B) Wild-type (WT), but not kinase-dead (KD), SRC64B interacts with the intracellular domain of DRL (DRL-intra) in a mammalian two-hybrid assay. The indicated fusion protein constructs were transfected into SFK-deficient cells and luciferase activity was measured 48 hours post-transfection and plotted, normalized to an internal control. Immunoblotting for DRL and SRC64B species (inset) indicates equivalent expression of the test plasmids. An irrelevant lane between lanes 5 and 6 was removed in preparing the panel.

 

Figure 6
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  		Fig. 6. DRL is tyrosine phosphorylated in a SRC64B-dependent manner and DRL coexpression activates SRC64B. (A) DRL and SRC64B co-transfected cell lysates contain a predominant protein species with increased tyrosine phosphorylation (asterisk). Drosophila S2 cells were transfected with the indicated plasmids and WCEs analyzed by immunoblotting with an anti-phosphotyrosine mAb. (B) DRL is phosphorylated in a SRC64B-dependent manner. Lysates of S2 cells transiently transfected with the indicated constructs were immunoprecipitated with anti-DRL antiserum, complexes washed, disrupted by boiling and DRL reprecipitated with anti-HA antiserum and analyzed by anti-phosphotyrosine immunoblotting. V, vector alone. (C) Coexpression of DRL and SRC64B results in a WIF and cytoplasmic domain-dependent activation of SRC64B. S2 cells were transfected as follows: lane 1, SRC64B[WT] only; lane 2, DRL[WT] + SRC64B[WT]; lane 3, DRL[{Delta}Cyto] + SRC64B[WT]; lane 4, DRL[{Delta}WIF] + SRC64B[WT]; lane 5, DRL[WT] + SRC64B[KD]; lane 6, DRL[{Delta}TBC] + SRC64B[WT]; and lane 7, DRL[{Delta}PDZ] + SRC64B[WT]. WCEs were immunoblotted to detect active SRC64B (anti-PY434SRC64B), pan-SRC64B (anti-MYC) and DRL (anti-HA). All transfections contained SRC64B-MYC except lane 5. Quantitation of a similar experiment performed in triplicate is shown in Fig. S5 in the supplementary material. (D) DRL-associated SRC64B is, at least in part, catalytically active. Lysates from cells transfected as indicated were immunoprecipitated with anti-DRL and analyzed by anti-PY434SRC64B immunoblotting. Control anti-MYC (SRC64B) and anti-HA (DRL) blots are shown.

 

Figure 7
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  		Fig. 7. The expression of WNT5 and DRL neither activates nor represses canonical TCF/LEF-dependent transcription. Drosophila S2 cells were transfected in triplicate with the expression plasmids indicated and either the TCF/LEF-dependent transcription reporter Super8XTopFlash (black bars) or the control Super8XFopFlash (white bars). Luciferase expression levels were determined, normalized to internal Renilla controls and plotted.

 

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© The Company of Biologists Ltd 2008