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First published online June 6, 2008
doi: 10.1242/10.1242/dev.017319
Laboratory of Developmental Neurobiology, Department of Molecular and Cell Biology, Leiden University Medical Center, P.O. Box 9600, 2300 RC, Leiden, The Netherlands.
* Authors for correspondence (e-mails: J.N.Noordermeer{at}lumc.nl; L.G.Fradkin{at}lumc.nl)
Accepted 5 May 2008
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: Axon guidance, RYK, Src family kinase, Wnt, Signal transduction
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). The ventral
midline of the Drosophila embryonic nervous system has proven to be
an excellent model system in which to identify the molecules that control axon
guidance (Araujo and Tear,
2003). A number of these guidance proteins are encoded by highly
conserved gene families; for example, slit, roundabout and netrin genes have
been shown to play remarkably similar roles to their Drosophila
orthologs at the mammalian floorplate
(Garbe and Bashaw, 2004).
Whereas considerable knowledge has accumulated about the mechanisms
controlling initial midline crossing, less is known about those controlling
routing at intermediate choice points, where extending axons may take
alternative routes. An example of such a decision is commissure choice.
Drosophila contralateral axons project stereotypically through one of
the two major axon tracts in each hemisegment, the anterior (AC) or the
posterior (PC) commissure. An axon's projection through the AC is, at least in
part, dictated by its repulsion away from the PC by the Wnt family member WNT5
acting through the Derailed (DRL) RYK axonal receptor
(Bonkowsky et al., 1999;
Callahan et al., 1995;
Yoshikawa et al., 2003).
Wnt5 (Fradkin et al.,
2004) and drl
(Callahan et al., 1995) mutants
also display altered axon fasciculation, which might reflect changes in
inter-axonal adhesion. Moreover, mutation of drl (also known as
linotte) results in memory deficits
(Dura et al., 1993), likely to
be caused by axon guidance defects in the larval brain
(Moreau-Fauvarque et al.,
1998; Simon et al.,
1998).
Wnt family proteins signal through alternative receptors with distinct
downstream pathways that sometimes have members in common. In many tissues,
Wnts signal by binding to the Frizzled (Fz) family of receptors in conjunction
with LRP co-receptors (Cadigan and Nusse,
1997). Fzs can transduce Wnt signaling via a canonical
Armadillo/β-catenin pathway culminating in the regulation of
TCF/LEF-dependent transcription or via non-canonical pathways
(Widelitz, 2005), some
involving the heterotrimeric GTPases
(Katanaev et al., 2005;
Katanaev and Tomlinson, 2006;
Liu et al., 2001). Recently,
the mammalian WNT5A protein was shown to interact with the receptor tyrosine
kinase (RTK) ROR, resulting in the repression of canonical Wnt signaling via
an as yet uncharacterized mechanism
(Mikels and Nusse, 2006). In
these studies, WNT5A was also shown to activate the canonical Wnt signaling
pathway via interaction with a Fz family member, suggesting that pathway
specificity might sometimes be determined by the Wnt receptor engaged and not
solely by the specific Wnt itself.
In addition to their involvement in a number of diverse developmental
processes (Logan and Nusse,
2004), Wnts play roles in various aspects of nervous system
development, such as cell fate determination, synapse formation, axon guidance
and neurite outgrowth (Ciani and Salinas,
2005; Fradkin et al.,
2005; Zou, 2004).
Wnt-RYK interactions (reviewed by Bovolenta
et al., 2006; Keeble and
Cooper, 2006) underlie the anterior-posterior guidance of subsets
of axons in the mammalian spinal cord (Liu
et al., 2005), cortical axon guidance across the corpus callosum
(Keeble et al., 2006),
establishment of the vertebrate retinotectal topographic map
(Schmitt et al., 2005) and
neurite outgrowth in vivo and in cultured primary cells
(Lu et al., 2004).
Members of the RYK family of `dead' or `fractured' RTKs have been found in
all metazoans examined (Halford and
Stacker, 2001). The extracellular domain of RYK members contains a
Wnt-binding WIF domain (Patthy,
2000). RYKs bear substitutions in highly conserved amino acid
residues required for phosphotransfer that are likely to render them inactive
as kinases. Although apparently lacking kinase activity, a human TRKA
(NTRK1)-RYK fusion protein was shown to activate the MAPK pathway when bound
by NGF (Katso et al., 1999),
suggesting that RYKs transduce extracellular signals to downstream targets
within the cell. Recent studies
(Grillenzoni et al., 2007;
Yao et al., 2007) have shown
that DRL can also act to antagonize WNT5 function in the Drosophila
post-embryonic CNS. These functions require the WIF domain, but not the
intracellular region of DRL. Therefore, DRL alone is probably not transducing
a signal in these contexts; instead, it apparently sequesters WNT5, preventing
it from interacting with other as yet unidentified receptors.
Like DRL (Yoshikawa et al.,
2003), human RYK has been shown to bind Wnt proteins
(Lu et al., 2004). Human RYK
acts in a ternary complex with the Fz and Wnt proteins, signaling through the
adaptor protein Dishevelled to increase TCF/LEF-dependent transcription in
transfected cells, suggesting that mammalian RYK induces canonical Wnt pathway
target genes (Lu et al.,
2004). Little is known about the targets of
Wnt5/drl-mediated signaling; however, Wnt5 transcription
increases in embryonic AC neurons in the absence of drl
(Fradkin et al., 2004) and
WNT5 protein is ectopically displayed at the pupal brain midline in
drl mutants (Yao et al.,
2007), indicating that the Wnt5 gene itself is a pathway
target.
Although DRL function in the Drosophila embryonic CNS does not
apparently involve intrinsic tyrosine kinase activity
(Yoshikawa et al., 2001), the
cytoplasmic domain of DRL is required for axon repulsion
(Yoshikawa et al., 2003) and
plays a regulatory role in DRL function during brain development
(Taillebourg et al., 2005). In
addition, mammalian RYK lacking its cytoplasmic domain acts as a
dominant-negative protein (Schmitt et al.,
2005). Thus, the kinase-deficient DRL/RYK receptors probably
interact with other proteins that transduce their signal.
Here, we show that the highly conserved Drosophila Src family non-receptor tyrosine kinases (SFKs), SRC64B and SRC42A, play roles in WNT5/DRL signaling. Src64B and Src42A double-mutant animals display commissural phenotypes similar to Wnt5 and drl mutants, suggesting that the SFKs play at least partially redundant roles. SFK gain- and loss-of-function alleles enhance and suppress, respectively, phenotypes dependent on WNT5/DRL signaling. Furthermore, the SFKs and DRL physically interact resulting in Src activation and DRL tyrosine phosphorylation. Mammalian SFK and RYK orthologs also co-immunoprecipitate from transfected tissue culture cell lysates indicating that RYK-SFK interactions are evolutionarily conserved.
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MATERIALS AND METHODS |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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-MYC,
UAS-NLS-β-Gal, UAS-GFP, UAS-mCD8GFP, DA-GAL4 and EG-GAL4. β-Gal or
GFP balancer chromosomes were used to identify the appropriate progeny.
Transgenic lines were generated using standard techniques in our laboratory
and at BestGene. Two SEMA2B-GAL4 transgenic fly lines were generated (gift of
B. Dickson, Research Institute of Molecular Pathology, Vienna, Austria).
Expression of GAL4 is first detected at embryonic stage 13 in the dorsal
vessel and gut, and from stage 15 onwards in the peripheral nervous system
(chordotonal organs) and the CNS (subsets of AC neurons, see Fig. S1B in the
supplementary material).
Immunohistochemistry and RNA in situ hybridization
Antibody labeling, RNA in situ hybridization and staging of embryos were
performed as described (Fradkin et al.,
1995; Fradkin et al.,
2004). The following antibodies were used on formaldehyde-fixed
embryos: mouse monoclonal antibody (mAb) BP102 (gift of C. Goodman, University
of California, Berkeley, CA), rabbit anti-MYC (Upstate), rabbit anti-GFP
(Invitrogen), mouse mAb anti-Wrapper
(Noordermeer et al., 1998),
mouse mAb anti-REPO (Alfonso and Jones,
2002) and rabbit anti-SRC64B (gift of T. Xu, Yale University, New
Haven, CT). Rabbit anti-SRC64B peptide antibody
(Muda et al., 2002) was used
to stain the larval neuropiles expressing SRC64B in motoneurons.
Constructs, transfection, immunoblotting and immunoprecipitation
Tagged actin promoter-driven or UAS promoter-driven wild-type and mutant
DRL (HA) and SRC64B or SRC42A (MYC) and wild-type untagged WG and WNT5
expression plasmids were constructed by ORF PCR, oligonucleotide-mediated
mutagenesis and Gateway-mediated recombination (Invitrogen) into appropriate
destination vectors (provided by T. Murphey;
http://www.ciwemb.edu/labs/murphy/Gateway%20vectors.html).
Y. Zou and K-L. Guan, respectively, provided HA-tagged mouse RYK
(Liu et al., 2005) and human
c-SRC (Li et al., 2004)
expression plasmids. The UAS-WRAPPER construct
(Noordermeer et al., 1998) was
co-transfected with pAc-GAL4 to express Wrapper. To generate an
Src64B-specific RNA interference transgene, gene-specific inverted
repeats (bp 1363 to 1963 of accession number NM_080195) were cloned into a
pUAST derivative bearing an intervening intron. Decreases in Src64B
mRNA levels were determined by semi-quantitative reverse transcribed (RT)-PCR
of first strand embryonic cDNA as described (see Fig. S1 in the supplementary
material). Similar reductions in Src64B expression were observed with
two different inserts. All plasmids were verified by sequencing.
S2 and Kc cell transfections were performed using Effectene (Qiagen) and
293T cell transfections with Fugene (Roche). Lysates were prepared using a
high-stringency SDS-containing RIPA buffer
(Muda et al., 2002) containing
a cocktail of protease inhibitors (Roche). Drosophila cell lysate
immunoprecipitations were performed using rabbit anti-MYC (Upstate) or rabbit
anti-DRL or anti-Wrapper mAb. Immunoblots were incubated with mouse 9E10
anti-MYC mAb or rabbit anti-MYC (Upstate) and mouse anti-HA (Sigma) or rabbit
anti-HA (AbCam) for the tagged SRC64B and DRL species, respectively, with the
4G10 or PY20 anti-phosphotyrosine mAbs (Upstate and Sigma, respectively) or
anti-PY434SRC64B affinity-purified antiserum. Bound multiple-label grade HRP-
(Jackson ImmunoResearch) or 800CW (Li-Cor)-conjugated secondary antibodies
were visualized with enhanced ECL (Roche) or an Odyssey two-color laser
scanner (Li-Cor), respectively. The immunoprecipitation resin in the
ExactaCruz Kit (Santa Cruz Biochemicals) or a mouse anti-rabbit light chain
mAb (Jackson ImmunoResearch) was used to reduce the recognition of the rabbit
anti-DRL antibodies used in immunoprecipitation on blots probed with rabbit
antisera. For the double immunoprecipitation of DRL to assess its
phosphotyrosine content, lysates were first precipitated with anti-DRL, washed
immune complexes boiled in 1% SDS, diluted 1:10 into buffer containing 1%
Triton X-100 and then DRL-HA immunoprecipitated with reagents from the
Profound anti-HA Kit (Pierce).
Rabbit anti-DRL was as described (Yao
et al., 2007). Anti-PY434SRC64B antiserum was commercially
generated and purified (Eurogentec) against the SRC64B peptide RVIADDEYCPKQG
and its phosphorylated version (RVIADDEpYCPKQG) as described
(O'Reilly et al., 2006) and
verified as phosphopeptide-specific by ELISA and by its lack of staining on
Src64BKO mutant embryos (data not shown).
293T cell lysate immunoprecipitations were performed using anti-c-SRC mAb
(Upstate) and immunoblots were probed with anti-c-SRC or anti-HA to detect
HA-tagged RYK. Luciferase assays were performed using the Super8XTop/FopFlash
plasmids [(Veeman et al.,
2003); a kind gift from R. Moon]. Lysates were prepared and
assayed using the Dual Luciferase Reporter Assay System (Promega) with
normalization to internal Renilla controls. The Checkmate Mammalian Two-hybrid
System (Promega) was used to assay SRC64B-DRL interaction in vivo. The
cytoplasmic domain of DRL was cloned in frame with the GAL4 DNA-binding domain
in the pBind vector and the full-length wild-type or kinase-deficient SRC64B
ORF was cloned in frame with the VP16 activation domain in the pACT vector.
SFK-deficient SYF cells (Klinghoffer et
al., 1999) were obtained from LGC Promochem-ATCC and transfected
using Fugene.
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RESULTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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), was found to interact with the DRL intracellular domain
bait fusion protein (data not shown). In order to evaluate possible roles of
Src64B in Wnt5/drl-mediated signaling, we examined
the ventral nerve cord commissures in embryos lacking Src64B, Src42A,
or both. Homozygosity for null alleles of either Src64B or
Src42A or simultaneous heterozygosity for both Src64B and
Src42A resulted in only mild aberrations in commissural projections
(Fig. 1C-E). However, embryos
entirely lacking one of the SFK orthologs and heterozygous for a null allele
of the other (Fig. 1F,G)
displayed `fuzzy' commissures, longitudinal breaks and apparent axon stalling
in the longitudinal pathways, as previously reported for Wnt5-null
mutant embryos (Fradkin et al.,
2004) (Fig. 1B).
Embryos entirely lacking both Src64B and Src42A displayed a
highly penetrant, severe commissural phenotype
(Fig. 1H). Embryos lacking
SRC42A and pan-neuronally expressing Src64B-targeting double-stranded
(ds) RNA (Fig. 1J,
Table 1) displayed qualitative
and quantitative phenotypes similar to those of Src42A;
Src64BKO/+ and Src42A/+;
Src64BKO mutant embryos. This result confirms a
requirement for wild-type Src64B expression levels in
SRC42A-deficient neurons for commissure formation. Altered commissure
formation in the partial SFK mutants is unlikely, therefore, to be due to
secondary effects of the loss of Src64B in other tissues. The failure
of these animals to exhibit the full SFK-null commissural phenotype probably
reflects incomplete knockdown of Src64B expression levels (see also
below). Quantitation of these phenotypes is presented in
Table 1. These results and the
similar commissural phenotypes observed in a previous study examining SFK
roles during Drosophila embryonic development
(Takahashi et al., 2005)
suggest that the Drosophila SFKs play partially redundant roles in
the formation of the embryonic commissures.
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).
Sema2b is expressed in a small subset of segmentally reiterated
neurons that project their axons to the contralateral side through the AC
(Rajagopalan et al., 2000).
Src64B mRNA levels in dsRNA-expressing animals were evaluated by
semi-quantitative RT-PCR and found to be 
10% of wild-type levels (see
Fig. S1A in the supplementary material). Sema2b+ axons
with decreased levels of Src64B misprojected or apparently stalled in
a number of hemisegments (25%, n=383) (see Fig. S1B in the
supplementary material). No apparent changes in Sema2b+
cell fate or cell body position were observed. These data provide further
support for SFK roles in commissure formation.
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) and anti-REPO (Alonso and Jones, 2002) antibodies. The
lateral glial cells appeared in their wild-type positions in all mutant
combinations except the Src42A/+; Src64B and double-null
mutants, whereas the midline glia appeared wild-type in all combinations
except the double-null SFK mutant (see Fig. S2 in the supplementary material).
The intermediate commissural phenotypes seen in the `partial' SFK mutants are
therefore unlikely to be caused by major CNS patterning and fate changes.
Wild-type Src64B expression levels are required for Wnt5/drl-mediated axon repulsion
Next we examined whether Src64B genetically interacts with the
Wnt5/drl-mediated signaling pathway during embryonic nervous
system development. We first evaluated whether wild-type Src64B
levels were required for a previously reported Wnt5 dominant
gain-of-function phenotype (Fradkin et
al., 2004; Yoshikawa et al.,
2003). When Wnt5 was ectopically expressed from a single
transgene in the midline glia using the SIM-GAL4 driver, 16% of
hemisegments displayed the absence or thinning of the AC
(Fig. 2A,
Table 2), owing to the
repulsion of the DRL+ AC axons by WNT5 produced by the midline
glia. This assay was previously used to establish that wild-type expression
levels of the O-acyltransferase porcupine (porc;
por - FlyBase) (Fradkin et al.,
2004) and of drl
(Yoshikawa et al., 2003) are
required for the loss of the AC, thus confirming that these genes are members
of a WNT5 signaling pathway. The removal of only a single copy of
Src64B from this genetic background resulted in a greater than 5-fold
reduction in the loss of the AC (Fig.
2, compare A and B; Table
2). The extent of suppression observed in Src64B
heterozygotes was similar to that seen in animals heterozygous for
drl (Table 2).
Heterozygosity for Src42A, by contrast, did not suppress the
WNT5-dependent midline overexpression phenotype
(Table 2). These data indicate
that wild-type Src64B expression levels are required for this
dominant gain-of-function phenotype and that Src64B is therefore
likely to be a member of the Wnt5/drl signaling pathway.
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;
Yoshikawa et al., 2001;
Yoshikawa et al., 2003). We
found that increased expression of Src64B driven by Eagle-GAL4
(EG-GAL4) did not cause PC EG-GAL4+ axons to switch to the AC
(Fig. 2D). To determine whether
wild-type drl expression levels might be limiting our ability to
observe the effect of increased Src64B expression, we used a
UAS-DRL-MYC transgene insert, which did not elicit substantial
EG-GAL4-dependent switching in single copy, but did when present in two copies
(Table 3). Increased expression
of wild-type Src64B in EG-GAL4+ axons in the sensitized
background (single copy of UAS-DRL-MYC) resulted in significant enhancement in
the number of switched axons (1% and 34% switched axons for 1x
UAS-DRL-MYC; 1x UAS-NLS β-Gal and 1x UAS-DRL-MYC; 1x
UAS-SRC64B, respectively; Fig.
2E, Table 3).
Enhancement of switching was also observed when Src42A expression
levels were increased, but to a lesser extent than with overexpression of
SRC64B (8% versus 34%, respectively; Table
3). Expression of a kinase-deficient Src64B transgene
(gift of Willis Li, University of Rochester, Rochester, NY) in this sensitized
background did not increase switching, indicating that the kinase activity of
SRC64B is required for its interaction with DRL
(Table 3). These data support
the hypothesis that the SFKs are members of the Wnt5/drl-signaling
pathway.
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;
Callahan et al., 1995;
Yoshikawa et al., 2003),
whereas Wnt5 mRNA is most predominantly found in neuronal cell bodies
associated with the PC (Fig.
3C) (Fradkin et al.,
2004). SRC64B protein was found in most, if not all, longitudinal
and commissural axonal projections in the CNS
(Fig. 3D) and was not readily
detectable in the Src64BP1 mutant
(Fig. 3E). To confirm that
SRC64B was transported out along axons, a double labeling of third instar
larval neuropiles coexpressing SRC64B and GFP in motoneurons was performed.
SRC64B, like the mammalian (Maness et al.,
1988) and C. elegans
(Itoh et al., 2005) Srcs, was
found to be efficiently transported within the axons
(Fig. 3F). A similar
localization of SRC42A has been reported previously
(Takahashi et al., 2005) and
DRL is also present in AC axonal projections
(Bonkowsky et al., 1999;
Callahan et al., 1995). SRC64B,
SRC42A and DRL are therefore expressed in AC axonal projections, supporting
our observations that they functionally interact there.
SRC64B and DRL and their mammalian orthologs physically interact
We then evaluated whether SRC64B and DRL physically interact by
ascertaining their ability to co-immunoprecipitate from transiently
transfected tissue culture cell lysates. We expressed epitope-tagged DRL
(DRL-HA) and SRC64B (SRC64B-MYC) proteins in Drosophila Kc cells,
which express little, if any, Wnt5 mRNA as assayed by quantitative
RT-PCR and gene expression microarray analyses (our unpublished data and M.
Fornerod, personal communication). Proteins were immunoprecipitated from cell
lysates using antibodies specific for either DRL or the SRC64B fusion protein
and immunoblots of the immunoprecipitated proteins were probed with antibodies
recognizing the reciprocal protein. SRC64B, as with its mammalian orthologs,
is myristoylated and membrane-associated, so immunoprecipitations were
performed under highly stringent conditions to disrupt membrane-protein
interactions (Materials and methods). DRL and SRC64B were found to
reciprocally co-immunoprecipitate both in the presence and absence of WNT5
(Fig. 4A). Similar results were
obtained using Drosophila S2 cells (data not shown). SRC42A also
co-immunoprecipitated with DRL, but at only very low levels, despite its
presumptive membrane localization (see Fig. S3 in the supplementary material).
The inefficient co-immunoprecipitation of SRC64B with the membrane protein
Wrapper (Noordermeer et al.,
1998) from cells coexpressing these proteins further indicates the
specificity of the DRL-SRC64B co-immunoprecipitation (see Fig. S4 in the
supplementary material).
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Formation of the DRL-SRC64B complex results in DRL phosphorylation and increased SRC64B activation and requires SRC64B kinase activity
We then evaluated whether the kinase activity of SRC64B was required in the
formation or stabilization of the DRL-SRC64B complex. The physical association
of DRL and SRC64B was dependent on the kinase activity of SRC64B or an
associated tyrosine kinase: treatment of the co-transfected cells with
herbimycin A, a tyrosine kinase-specific inhibitor, resulted in their reduced
co-immunoprecipitation (Fig.
5A). To further assess the role of SFK kinase activity, we used a
mammalian two-hybrid assay in which plasmids expressing a SRC64B and DRL
intracellular domain fusion protein were transfected into SFK-deficient cells
(Klinghoffer et al., 1999) to
eliminate possible interference by the highly conserved endogenous mammalian
SFKs. Coexpression of wild-type SRC64B and DRL intracellular domain fusion
proteins led to significant increases in luciferase expression above that of
the controls, indicating that these proteins physically interact
(Fig. 5B). No significant
expression of luciferase was observed when catalytically inactive SRC64B (KD)
was coexpressed with DRL (Fig.
5B).
The requirement for tyrosine kinase activity in the formation or stability
of the SRC64B-DRL complex raised the question as to whether either DRL or
SRC64B displayed increased tyrosine phosphorylation upon coexpression.
Evaluation of tyrosine phosphorylation of whole-cell extract proteins derived
from cells transiently transfected with DRL, SRC64B, or both expression
constructs revealed a dramatic increase in the phosphorylation of a 75 kDa
protein(s) in the doubly transfected cells
(Fig. 6A). The tagged DRL and
SRC64B proteins both displayed apparent molecular weights of 75 kDa on
denaturing gels. Therefore, to investigate whether this species includes DRL,
we initially immunoprecipitated the DRL-containing complex with anti-DRL,
dissociated it by boiling and immunoprecipitated DRL with anti-HA (DRL).
Anti-phosphotyrosine immunoblots revealed that DRL tyrosine phosphorylation is
increased upon its coexpression with SRC64B
(Fig. 6B).
SFKs are known to be differentially phosphorylated at specific tyrosine
residues depending on their state of activation (reviewed by
Roskoski, 2005). We therefore
evaluated the degree of phosphorylation of the SRC64B tyrosine at position
434, which is phosphorylated in catalytically active SRC64B
(O'Reilly et al., 2006).
Anti-PY434SRC64B immunoblot analysis of whole-cell lysates derived from
transfected cells revealed DRL-dependent activation of SRC64B
(Fig. 6C). The intracellular
and WIF domains of DRL, but not its putative tetrabasic cleavage (TBC) site or
N-terminal PDZ-binding domain, are required for SRC64B activation
(Fig. 6C and see Fig. S5 in the
supplementary material). Co-transfection of a plasmid expressing WNT5 did not
increase the amount of SRC64B phosphorylation (data not shown), suggesting
that the activation of SRC64B by DRL is independent of WNT5 under these
conditions. Finally, we assessed whether or not the SRC64B that was associated
with DRL included catalytically active molecules. Anti-DRL immunoprecipitation
of lysates from cells co-transfected with SRC64B and DRL expression plasmids
and immunoblotting with anti-PY434SRC64B revealed that at least some of the
SRC64B protein bound to DRL is catalytically active
(Fig. 6D).
|
), we
evaluated whether or not their Drosophila orthologs also signal
through this pathway. Drosophila S2 tissue culture cells were
transfected with the indicated combinations of expression plasmids and a
TCF/LEF-responsive luciferase reporter gene [Super8XTopFlash
(Veeman et al., 2003] or a
control reporter (Super8XFopFlash) and luciferase levels were measured 48
hours post-transfection. Transfection of a construct encoding a canonical Wnt
protein, Wingless (WG), resulted in high TCF/LEF-dependent levels of
luciferase expression. S2 cells express both fz and fz2 as
assayed by quantitative RT-PCR (data not shown), precluding the necessity of
transfecting expression plasmids for either of the canonical Fz receptors.
Transfection of a WNT5-expressing plasmid alone or in combination with a
DRL-expressing plasmid did not result in expression of luciferase above
control levels (Fig. 7),
suggesting that Wnt5/drl-mediated signaling does not
increase TCF/LEF activity.
As the mammalian WNT5A protein acting through the ROR receptor has been
shown to inhibit canonical Wnt signaling
(Mikels and Nusse, 2006), we
evaluated whether or not WNT5 interaction with DRL might similarly block
canonical signaling. Coexpression of WG, WNT5 and DRL resulted in luciferase
expression levels similar to those seen with WG alone
(Fig. 7), suggesting that
Wnt5/drl-mediated interactions do not apparently inhibit
contemporaneous canonical Wnt signaling.
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DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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); data not shown], are mislocalized in the double-null SFK
animals.
Second, we observe that wild-type levels of Src64B are required
for a dominant gain-of-function phenotype of Wnt5; heterozygosity for
a Src64B-null mutant resulted in a dramatic reduction in the loss of
the AC caused by midline glial overexpression of Wnt5. Similar levels
of suppression are observed in embryos heterozygous for drl or
porc, established members of the Wnt5/drl signaling
pathway. The other Drosophila SFK, SRC42A, which is also expressed in
the embryonic CNS (Takahashi et al.,
2005), plays partially redundant roles to those of SRC64B in other
tissues (Takahashi et al.,
2005; Tateno et al.,
2000; Harris and Beckendorf,
2007). Heterozygosity for Src42A did not suppress the
Wnt5 midline glial expression phenotype, possibly because SRC42A is
not limiting under these conditions.
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) and
drl (Yoshikawa et al.,
2003) mutants. The aberrant pathfinding of Src64B
dsRNA-expressing Sema2b+ neurons, which normally project
through the AC, indicates that wild-type expression levels of Src64B
are required for their correct routing. The incomplete penetrance of this
phenotype possibly reflects the presence of wild-type levels of SRC42A, which
we have shown is at least partially redundant with SRC64B. Alternatively,
expression of Src64B-targeted dsRNA might not adequately reduce
SRC64B expression levels to effect full penetrance.
Fourth, we demonstrate that drl and
Src64B/Src42A interact synergistically in an axon-switching
assay. Increased SFK neuronal expression levels alone could not force axons
that normally traverse the PC to cross in the AC. Use of a DRL-expressing
transgene facilitating only moderate switching showed that elevated
Src64B or Src42A expression levels significantly increased
switching. SFK catalytic activity is required to enhance DRL-dependent
switching, as kinase activity-deficient SRC64B did not increase switching.
Thus, catalytically active SFKs can synergize with limiting levels of DRL to
induce commissure switching, and presumably act to control the wild-type
trajectories of the AC axons. Furthermore, Src64B and drl
have previously been reported to interact genetically during pupal brain
development (Nicolai et al.,
2003) and it was also recently reported that drl
genetically interacts with the SFK genes to control salivary gland migration
(Harris and Beckendorf,
2007).
Supporting these previous observations and the genetic data presented here, we found that SRC64B and DRL physically interact, as assayed by co-immunoprecipitation. The formation or stability of this complex is apparently dependent upon SFK kinase activity as shown by the failure of the proteins to co-immunoprecipitate from lysates derived from cells treated with herbimycin A, a tyrosine kinase inhibitor. Further support for this involvement of SFK catalytic activity was provided by our observation that wild-type, but not catalytically inactive, SRC64B physically interacts with the DRL cytoplasmic domain as assayed in two-hybrid experiments. RYK and c-SRC, like their Drosophila orthologs, co-immunoprecipitate from transfected cell lysates, suggesting that these interactions are evolutionarily conserved.
|
Unlike the in vivo assays implicating SFKs in the WNT5/DRL pathway
presented here, neither the physical association of DRL and SRC64B in
Drosophila or mammalian tissue culture cells, nor the activation of
SRC64B upon its coexpression with DRL, displayed WNT5-dependence under the
conditions examined. It is possible, however, that endogenous expression
levels of WNT5 or of another Wnt capable of interacting with DRL are already
saturating for DRL-dependent SRC64B activation. Alternatively, higher than
physiological expression levels of SFK/DRL might bypass WNT5 binding-dependent
recruitment of the SFKs by DRL. Our data, however, indicate that DRL-SRC64B
interactions might be constitutive. Ligand-independent association of
signal-transducing kinases with cell surface receptors is not, however,
unprecedented. The mammalian Janus kinases are constitutively and stably
associated with the GP130 cytokine receptor
(Giese et al., 2003;
O'Shea et al., 2002).
Furthermore, the mammalian RYK protein and its Fz co-receptor have also been
shown to interact in the absence of Wnt protein
(Lu et al., 2004). RYKs might
therefore possibly recruit at least a subset of their co-receptors and
downstream effectors in a Wnt-independent manner. WNT5 binding to DRL might
result in subtle conformational changes to pre-existing DRL-SRC64B complexes,
which were not detected in the assays employed here. Such changes might lead
to alterations in tyrosine kinase target specificity, such as those
demonstrated for the Src-interacting Na+/K+-ATPase
(Tian et al., 2005).
Binding of Wnt protein to RYK stimulates TCF/LEF-dependent transcription
via the Dishevelled adaptor protein in transfected cells, suggesting that the
RYK pathway overlaps with the canonical Wnt pathway
(Lu et al., 2004). Our data
indicate, however, that the Drosophila WNT5/DRL signaling pathway
does not regulate TCF/LEF-dependent transcription. Transfection of
Drosophila S2 tissue culture cells, which respond to the canonical WG
ligand, with DRL and WNT5 expression constructs does not increase
TCF/LEF-dependent reporter gene expression. Furthermore, unlike the recently
reported WNT5A-ROR interaction (Mikels and
Nusse, 2006), Wnt5/drl-mediated signaling does
not apparently block contemporaneous canonical Wnt signaling. The
Drosophila WNT5 protein, however, can also signal via Fz family
receptors to activate a non-canonical Wnt pathway
(Srahna et al., 2006).
Although our data render it unlikely that SRC64B is a member of the canonical
Wnt signaling pathway, it might act in a pathway parallel to canonical Wnt
signaling as has been reported for SFKs during convergent extension cell
movement in zebrafish (Jopling and den
Hertog, 2005) and during cell fate specification and cleavage
orientation in C. elegans (Bei et
al., 2002).
It is presently unclear whether the SFKs relay a WNT5/DRL signal, and if
so, to what downstream pathway members and by what mechanisms. Our
observations that increased Src64B and Src42A expression
levels enhance drl-mediated commissure switching of axons in a
sensitized background and that the SFKs and DRL physically interact, suggest
the possibility that DRL dictates the target specificities of the bound SFKs
by co-localizing them with potential targets. This hypothesis is attractive
because the SFKs are widely expressed throughout the ventral nerve cord and
are also likely to act downstream of other axonal receptors, the mammalian
orthologs of which [e.g. the TRKB (NTRK2), ephrin A and netrin receptors] are
known to interact with the SFKs (Iwasaki
et al., 1998; Knoll and
Drescher, 2004; Liu et al.,
2004; Meriane et al.,
2004; Ren et al.,
2004). Furthermore, DRL-dependent asymmetric localization or
regulation of SFKs within the growth cone might mediate axon steering. Such
localized changes in growth cone SFK activity effect axon turning in cultured
Xenopus primary neurons (Robles
et al., 2005).
Although the identification of the relevant SFK targets and of other members of the WNT5/DRL-mediated signaling pathway lies ahead, the data presented here indicate that the catalytically active SFKs are required for WNT5-mediated axon repulsion via the catalytically inactive DRL receptor. Identification of other pathway members, including potential SFK targets, through a combination of genetic and biochemical approaches should further reveal the mechanisms by which Wnt proteins signal through the RYKs.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/135/13/2277/DC1
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Cyto] + SRC64B[WT]; lane 4, DRL[