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First published online 14 November 2007
doi: 10.1242/dev.012872
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Department of Neuroscience, University of Pennsylvania School of Medicine, 1113 BRB2/3, 421 Curie Blvd., Philadelphia, PA 19104, USA.
* Author for correspondence (e-mail: gbashaw{at}mail.med.upenn.edu)
Accepted 13 September 2007
 |
SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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C) results in dose-dependent defects in commissural axon
guidance. These findings represent the first systematic dissection of the
cytoplasmic domains required for Fra-mediated axon attraction in the context
of full-length receptors in an intact organism and provide important insights
into attractive axon guidance at the midline.
Key words: Axon guidance, Midline, Attraction, Netrin, DCC, Frazzled, Signaling
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Yu et al., 2002). Growth cones
translate these cues using signaling mechanisms downstream of conserved
guidance receptors and ultimately steer an axon to its target. Several
conserved families of guidance cues and receptors have been discovered
including the Netrin-family of ligands and their DCC family of receptors
including Frazzled (Fra) and Unc-40
(Kennedy, 2000).
The DCC family of Netrin receptors, including UNC-40 in C.
elegans, Fra in Drosophila, and DCC in vertebrates contain
extracellular domains consisting of six immunoglobulin (Ig) repeats and four
fibronectin type III (FNIII) repeats and cytoplasmic domains consisting of
three conserved sequence motifs, P1, P2 and P3
(Kolodziej, 1997). Previous
studies have shown that DCC family members bind to Netrin and mediate axon
outgrowth and growth cone attraction (Chan
et al., 1996; de la Torre et
al., 1997; Keino-Masu et al.,
1996; Kolodziej et al.,
1996; Stein et al.,
2001). For example, in C. elegans, during circumferential
axon guidance, the UNC-40 receptor is required in ventrally migrating cells
that respond to Netrin (Chan et al.,
1996). Genetic analysis in Drosophila also illustrates a
role for Netrin-Frazzled signaling in axon attraction. Netrin and
fra mutants exhibit a decrease in the number of commissural axons
crossing the ventral midline suggesting that attraction is compromised
(Harris et al., 1996;
Kolodziej et al., 1996;
Mitchell et al., 1996). In
addition, using the Xenopus spinal axon turning assay, it was shown
that axons are attracted to an exogenous source of Netrin, and this response
depends on DCC (de la Torre et al.,
1997). Together, these data establish that the DCC/Frazzled/UNC-40
family of receptors responds to Netrin to mediate axon outgrowth and
attraction.
Although the biological relevance of the Netrin-DCC pathway is becoming
clear, the receptor motif requirements and signaling mechanisms downstream of
the receptor are not well understood. Recent studies from vertebrate systems
and C. elegans have investigated which of the cytoplasmic motifs of
DCC/UNC-40 play a role in signaling. For example, in vertebrates it was
reported that the cytoplasmic P3 sequence motif is necessary for receptor
multimerization and Netrin-induced attractive turning of stage 22
Xenopus neurons (Stein et al.,
2001). Versions of DCC lacking the P3 motif are not able to
mediate axon attraction or stimulate outgrowth. However, replacing the P3
motif with the SAM self-association domain (from the EphB receptor) is
sufficient to restore function, suggesting the major role of P3 is to mediate
self-association (Stein et al.,
2001). More recently, several independent reports demonstrated
that one of the major functions of P3 is to recruit focal adhesion kinase
(Fak). This interaction is important for tyrosine phosphorylation of DCC and
for the ability of DCC to elicit axon outgrowth and turning
(Li et al., 2004;
Liu et al., 2004;
Meriane et al., 2004;
Ren et al., 2004).
Specifically, the LD-like motif within P3 of DCC was critical for FAK binding;
however, another P3-independent binding site was also proposed
(Ren et al., 2004).
Alternatively, a study from C. elegans showed that the P1 and P2
motifs, but not P3, are required to promote an Unc-40
gain-of-function outgrowth response (Gitai
et al., 2003), suggesting P1 and P2 function in endogenous UNC-40
signaling. It should be noted that the potential function of P3 in full-length
Unc-40 receptor signaling in the context of a normal in vivo attractive
decision has not been examined. Further genetic analysis from this report
argues that unc-34/ena and the Rac GTPase, ced-10, likely
contribute to signaling via the P1 and P2 cytoplasmic domains, respectively
(Gitai et al., 2003). In
Drosophila, ena also genetically interacts with fra further
supporting a role for Ena during attractive midline guidance
(Forsthoefel et al., 2005);
however, in this case, the specific sequence motifs required were not
investigated.
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C) is normally localized to CNS axons and is able to disrupt
commissural axon guidance in a dose-dependent fashion. Expression of this
truncated receptor in a fra heterozygous background causes a more
severe phenotype, whereas expression in fra homozygous mutants leads
to a complete commissureless phenotype suggesting the exciting possibility
that, in addition to acting as a dominant-negative for Frazzled signaling,
FraC also interferes with additional guidance pathways. |
MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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P1-Myc#2; fra3/CyWglacZ,
(11) fra3,
UAS-FraP1-Myc#1.1/CyWglacZ, (12)
UAS-FraP2-Myc#1.1;
fra3/CyWglacZ, (13) fra3/CyWglacZ;
UAS-FraP2-Myc#2.1, (14) fra3,
UAS-FraP3-Myc#2/CyWglacZ, (15)
fra3,
UAS-FraP3-Myc#1.1B/CyWglacZ, (16)
fra3/CyWg; UAS-Myr-Fra-Myc#1.1, (17)
fra3/CyWg; UAS-Myr-Fra-Myc#2.1, (18)
fra3/CyWglacZ;
UAS-FraP3-SAM-Myc#4, (19)
fra3/CyWglacZ;
UAS-FraP3-SAM-Myc#5.2, (20)
fra3, UAS-FraC-HA#4/CyWglacZ,
(21) fra3/CyWglacZ;
UAS-FraC-HA#2, (22)
fra3/CyWglacZ; UAS-FraC-HA#8,
(23) fra3/CyWglacZ;
UAS-FraC-HA#9, (24)
fra4/CyWglacZ; elavGal4, (25)
fra3/CyWglacZ;
UAS-FraCRobo67Myc, (26)
elavGal43A, (27)
UAS-FraC-HA#4/CyWglacZ; elavGal4/TM2, (28)
roboGa285/CyWglacZ, (29) roboGa285/CyWg;
elavGal4, (30) fra3, robo/CyWglacZ, (31)
fra4, robo/CyWglacZ, (32) fra3, robo,
UAS-FraC-HA#4/CyWglacZ, (33)
fra4, robo/CyWglacZ; elavGal4, (34) fra3,
slit2/CyWglacZ, (35) fra4,
slit2/CyWglacZ, (36) fra3,
slit2/CyWglacZ, (37) fra4, slit2,
UAS-FraC-HA#4/CyWglacZ, (38)
fra3, slit2/CyWglacZ; elavGal4.
Molecular biology
All constructs were generated using standard molecular biology techniques.
P1 (aa 1123-1140), P2 (aa 1270-1302), and P3 (aa 1349-1372) motifs are based
on those described by Kolodziej et al.
(Kolodziej et al., 1996) and
precise deletions were made using a `sewing PCR' strategy. Details provided
upon request. For detail of the constructs used, see
Fig. 3 and Fig. S1 in the
supplementary material.
Immunofluorescence
Embryos were collected, fixed and stained as previously described
(Kidd et al., 1998a). The
following primary antibodies were used: (1) Ms-anti-1D4/FasII [(Developmental
Studies Hybridoma Bank (DSHB); 1:100], (2) Ms-mAb BP102 (DSHB; 1:100), (3)
Rb-anti-Myc (Sigma-Aldrich, 1:500), (4) Ms-anti-Sex Lethal (DSHB; M18,
1:1000), (5) Ms-anti-β-gal (DSHB; 40-1a, 1:250), (6) Rb-anti-GFP
(Molecular Probes; 1:500), (7) Rb-anti-HA (Covance; 1:2000). The following
secondary antibodies were used: (1) Alexa Fluor 488 gt-anti-Rb (Molecular
Probes; 1:500), (2) Cy3 gt-anti-Ms (Jackson Laboratories; 1:1000). Stacks of
images were obtained using a Leica DMIRE2 confocal and a 63x oil
immersion objective. After de-speckling the stacks, a maximum projection was
generated with NIH Image/ImageJ software.
Biochemistry
For co-ip, S2R+ cells were transfected using the Effectene reagent (Qiagen)
along with 0.5 µg pMT-Gal4, then induced on day 2 with (0.5 mM)
CuSO4. 40 ng of plasmid was transfected per reaction for Myr
constructs and 200 ng for full-length constructs. Cells were lysed in 1 ml
lysis buffer [50 mM Tris-HCl pH 7.4, 500 mM NaCl, 1% NP40, 1 mM EDTA, 0.25%
sodium deoxycholate, 1 mM sodium orthovanadate, 1x complete protease
inhibitor cocktail (Roche) and 1 mM PMSF] on day 3, followed by overnight
incubation with 2 µl of either rabbit anti-Myc antibody (Sigma) or rabbit
anti-HA antibody (Covance). 30 µL of protein G-Sepharose (Zymed) beads were
used followed by three washes in lysis buffer. For immunoblots, mouse anti-Myc
(9E10 1:1000) and mouse anti-HA (Covance 1:1000) antibodies were used.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Higashijima et al., 1996).
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; Higashijima et al.,
1996). Wild-type axons from both the EG and EW clusters cross the
midline in all segments (Fig.
1A and see Fig. S1 in the supplementary material). Conversely, in
approximately three-quarters of fra mutant segments, the EW axons
display defects in midline crossing [not crossing (51%) or significantly
thinner (16%) than wild type]; however the trajectories of the EG axons are
unaffected (Fig. 1C and see
Fig. S1 in the supplementary material). Importantly, these phenotypes are
similar although somewhat stronger than those reported for Netrin
mutants (Fig. 1B)
(Brankatschk and Dickson,
2006). Our own quantification of the defects in the Netrin-A
Netrin-B (NetAB) double mutants reveals that the EW axons fail
to cross the posterior commissure in 34.2% of segments (n=27 embryos,
216 segments), whereas the EG axons fail to cross in only 1% of segments
(n=27 embryos, 214 segments).
The Fra receptor cell autonomously guides commissural axons across the midline
Since it has been reported that Fra has non cell-autonomous functions
(Gong et al., 1999;
Hiramoto et al., 2000), we
next asked if the EW cluster requires Fra exclusively in neurons for proper
guidance. Expressing a wild-type full-length Fra receptor (UAS-Fra-Myc or
UAS-Fra-HA) in the Egl neurons under the control of eagleGal4
(Brand and Perrimon, 1993)
cell-autonomously rescues EW guidance defects
(Fig. 2B,
Fig. 3B and see Fig. S1 in the
supplementary material). By contrast, a myristolated form of Fra lacking the
entire extracellular domain (UAS-MyrFra-Myc) is unable to rescue the mutant
phenotypes, presumably because this truncated protein is unable to bind Netrin
and mediate a directional response. Unlike observations in C. elegans
where expression of a myristolated UNC-40 leads to ectopic neurite outgrowth
(Gitai et al., 2003), our
Myr-Fra construct did not cause similar gain-of-function phenotypes when
expressed in all neurons or within a defined subset of neurons (data not
shown). Together these results are supportive of previous findings
demonstrating that Fra plays an important role in attracting certain classes
of neurons towards and across the midline in response to Netrin
(Kolodziej et al., 1996).
Since the fra defects can be rescued cell autonomously within an
easily quantifiable subset of neurons, this system allows a thorough
investigation of the domains necessary for mediating Netrin attraction.
The P3 motif of Fra is required for midline attraction
To determine the domain requirements for Fra-mediated signaling, we
generated mutant transgenes containing various deletions within the receptor
(Fig. 2A,B and see Fig. S1 in
the supplementary material; see Materials and methods) and tested whether
these mutated receptors could rescue the fra defects in EW neurons.
We found that whereas expression of a mutant receptor lacking either P1 or P2
(or both; UAS-FraP1-Myc, UAS-FraP2-Myc or
UAS-FraP1P2-Myc) efficiently rescues the guidance errors of EW
axons, a version of Fra missing P3 (UAS-FraP3-Myc) does not
(Fig. 2A,B and
Fig. 3C-E). Furthermore, a
mutant construct missing only the last 12 amino acids of P3
(UAS-FraP3.5) also does not rescue the attractive guidance defects,
suggesting a critical requirement for these 12 residues during Fra-mediated
attraction at the Drosophila midline
(Fig. 2A,B and
Fig. 3E). Importantly
UAS-FraP3.5 still contains the LD-like motif, the presumed FAK binding
motif identified in vertebrate DCC (Ren et
al., 2004). In addition, individually mutating each of the PXXP
motifs (Fig. 2) did not result
in decreased Fra function; all of these mutant receptors rescued the EW
neurons as well as a wild-type transgene (data not shown). We note that one of
the FraP3 and FraP3.5 lines as well as both FraP1, P2, P3
lines were significantly different from fra mutants alone, suggesting
that these receptors may still provide some attractive function, albeit at
much lower levels than the other mutant Fra receptors. We tested at least two
independent transgenic inserts for each construct
(Fig. 3B and see Fig. S1 in the
supplementary material) and confirmed that they are expressed at similar
levels to wild-type transgenes (Fig.
3 and data not shown).
|
). To test this in an in vivo system, we assessed whether
replacement of the P3 motif of Fra with the SAM self-association domain of the
EphB receptor could rescue function of this mutated receptor when expressed in
Egl neurons in fra mutants. In contrast to observations in vertebrate
systems, substitution of the P3 motif with a SAM multimerization domain could
not rescue EW crossing defects (data not shown), suggesting that
multimerization (at least not P3-dependent multimerization) is not sufficient
for normal Frazzled guidance responses.
To determine whether the inability of FraP3-SAM to rescue crossing
defects in EW neurons is not simply a failure of receptor multimerization, we
sought to confirm that P3 directs receptor self-association in
Drosophila through expression of Fra constructs in cultured
Drosophila S2R+ cells. It has been previously demonstrated that
myristolated DCC cytoplasmic domains exhibit Netrin-independent constitutive
multimerization (Stein et al.,
2001). Interestingly, a myristolated c-terminal version of Fra
(Myr-Fra-Myc) bound to both a myristolated cytoplasmic construct (Myr-Fra-HA)
as well as a full-length construct lacking a P3 domain (Fra-P3-HA),
indicating that multimerization of Fra can occur independently of the P3
domain (Fig. 4A, lanes 1 and
3). Additionally, association was observed between Myc and HA-tagged
constructs in which both P3 domains were deleted, eliminating the possibility
of P3-dependent interaction with a distinct motif
(Fig. 4B). Deletion of P1 and
P2 motifs also had no effect on self-association (data not shown). It will be
interesting to determine which cytoplasmic motif(s) mediate this interaction
in Fra and whether multimerization is indeed required at the
Drosophila midline; however, these results suggest that whatever role
Fra multimerization plays in mediating Fra function, it is not sufficient to
direct axon attraction in the absence of the P3 motif.
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). Our
Myc-tagged mutant transgenes presented the opportunity to establish whether
any of the conserved sequence motifs play a role in receptor localization.
Expression of our singly deleted P1, P2 or P3 mutant
transgenes in all neurons using elavGal4 did not lead to a dramatic
alteration in Fra receptor distribution when compared to a wild-type transgene
(Fig. 3, and data not shown).
Additionally, mutating all three motifs in combination
(UAS-FraP1P2P3-Myc) does not disrupt Fra localization
(data not shown). In fact, overexpressing a Fra receptor missing almost the
entire cytoplasmic domain (UAS-FraC-HA; amino acids 1109-1372 were
deleted; see Fig. 2) still does
not cause a substantial difference in the localization pattern when compared
to a wild-type transgene (Fig.
5D,E). For the purpose of this study, we remade UAS-FraC-HA
(Fig. 6F) because previous
experiments (Hiramoto et al.,
2000) were done using a FraC receptor that contained the
transmembrane domain and 67 juxtamembrane cytoplasmic amino acids of the Robo
receptor (FraCRobo67-Myc). We suspect that this exogenous
sequence might interfere with normal Fra distribution. Given that our newly
generated construct localizes similarly to wild-type and differently from the
original FraCRobo67-Myc protein
(Fig. 5, compare C' or
D' to E'), we consider this a reasonable hypothesis. At least two
independent transgenic lines were tested in our localization studies. Our data
suggest that the cytoplasmic domain of Fra is not required for robust axonal
localization. However, it is important to point out that these overexpression
experiments may not reflect the true localization of endogenous proteins with
similar deletions.
FraC interferes with normal axon guidance and depends on Netrin
In the course of investigating the localization pattern of UAS-FraC,
we noticed that overexpression of this receptor in a wild-type background
causes commissures to become thin, suggesting some commissural axons fail to
cross the midline (Fig. 6A,B).
The severity of this phenotype increases when one copy of fra is
removed (Fig. 6D) suggesting
that FraC could be acting as a dominant negative for Fra. When
FraC is expressed at high levels in a wild-type background, most axons
do not cross the midline (Fig.
6C) demonstrating that the effect is dose dependent. Strikingly,
homozygous fra mutants overexpressing only one copy of
UAS-FraC exhibit a near complete loss of axon commissures
(Fig. 6E), a phenotype that
closely resembles that of commissureless (comm)
loss-of-function mutant embryos (Seeger et
al., 1993). We observe this strong commissureless ectopic
expression phenotype in four independent transgenic lines; however, one line,
which is expressed at lower levels (Fig.
5 and data not shown), gives only a mild overexpression phenotype.
The overexpression phenotype cannot be the result of the exogenous HA tag as a
full-length Fra-HA receptor does not show this phenotype and in fact, can
rescue the fra mutant defects in the EW neurons
(Fig. 3B). This suggests that
in addition to acting as a dominant negative for wild-type Fra function,
FraC also affects additional guidance signals including the exciting
possibility that it is inhibiting novel attractive pathways.
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C in a subset
of commissural neurons; once again we turned to the Egl neurons. Expression of
FraC in Egl neurons of wild-type embryos causes a decrease in EW
attraction towards the midline (Fig.
6G), though in many segments the EW neurons cross normally. When
FraC is expressed in fra homozygous embryos, 100% of the EW
axons fail to cross the midline (Fig.
6H). In some segments, we also observe defects in EG axon guidance
(Fig. 6H), a phenotype never
observed in fra mutants. These data support the hypothesis that in
addition to acting as a dominant negative for Fra function, FraC is
also interfering with an additional attractive pathway (one required for EG
guidance) at the Drosophila midline. An alternative although not
mutually exclusive possibility is that FraC might somehow be increasing
axon repulsion thereby preventing axons from approaching the midline (see
below).
To test whether Netrin is required for the commissureless phenotype caused
by FraC expression, we expressed UAS-FraC in a Netrin
mutant background. Two different strategies were used to eliminate Netrin
function. First, we used a X-chromosomal deletion (NP5) known to completely
remove both Netrin genes
(Mitchell et al., 1996), and
second we used a specific NetAB double mutant stock generated by
homologous recombination (Brankatschk and
Dickson, 2006). Expression of FraC did not cause a
commissureless phenotype in either mutant background
(Fig. 7A,B and data not shown)
suggesting that this truncated receptor depends on Netrin to produce the
phenotype.
The FraC phenotype is independent of Unc-5 signaling and partially dependent on Slit-Robo signaling
Since Slit and the Robo family of receptors are essential for midline
repulsion (Brose et al., 1999;
Kidd et al., 1999;
Kidd et al., 1998a;
Long et al., 2004), we
reasoned if FraC is causing an increase in repulsion, then Slit-Robo
might be required. To test this possibility, we expressed this mutant
construct, in a fra homozygous background (the background in which we
see a strong commissureless phenotype) in combination with mutations in either
slit or robo. In the Drosophila CNS, axons collapse
at the midline in fra, slit double mutant embryos
(Garbe and Bashaw, 2007); this
phenotype is quite strong (Fig.
7G). Panneural expression of FraC in fra, slit
embryos still prevents many axons from crossing the midline
(Fig. 7H) suggesting that
FraC can prevent axon collapse in the absence of all midline repulsion.
Consistent with this result, expression of FraC in a fra, robo
double mutant background dramatically repels axons, though the defect is less
severe than that seen a fra single mutant background
(Fig. 7E,F; compare with
Fig. 6E). These data suggest
that Slit-Robo signaling is partially required to generate the strongest
FraC-induced misexpression phenotype. Similarly, expressing FraC
in all neurons leads to a partial suppression of the robo single mutant
phenotype (Fig. 7C,D). Taken
together, the above data suggests that repulsion via the Slit-Robo pathway is
not strictly required for FraC to elicit a phenotype. In other words,
in the absence of either the repulsive ligand Slit or its receptor Robo, axons
still avoid the midline when FraC is overexpressed suggesting that some
other mechanism generates the phenotype.
Unc-5 is another guidance receptor known to promote axonal repulsion in
many organisms (Hamelin et al.,
1993; Hedgecock et al.,
1990; Hong et al.,
1999; Keleman and Dickson,
2001), and in Drosophila, ectopic expression of Unc-5
causes a Netrin-dependent phenotype similar to that seen in comm
mutants (Keleman and Dickson,
2001). Therefore, we tested whether repulsion via Unc-5 is
required for the FraC overexpression phenotype. This does not appear to
be the case; panneural overexpression of FraC in a fra, unc-5
double mutant background still causes a commissureless phenotype indicating
that the Unc-5 receptor is not required to prevent axons from approaching the
midline (Fig. 7I,J; compare
with Fig. 6E).
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Although many molecules have been implicated in
mediating guidance decisions, how these factors function together to regulate
axon guidance in vivo is poorly understood. Additionally, our knowledge of how
non-catalytic receptors (for example DCC/Fra) elicit a response and what
domains are required for signaling is not complete. In this study, using a
specific subset of commissural neurons in vivo, we confirm the observation of
in vitro experiments that the P3 sequence motif of Fra/DCC is required at the
Drosophila midline for an attractive response. Additionally, our data
show that neither P1 nor P2 are required for attractive guidance; neurons are
properly guided by receptors missing both of these domains. Our work also
demonstrates that the cytoplasmic domain is not required for normal Fra
distribution in axons as was previously reported. Furthermore, while
investigating these truncated receptors, we noticed that expression of a
receptor lacking the entire cytoplasmic domain causes defects in midline
guidance and produces a completely commissureless phenotype in fra
homozygous embryos. This phenotype is dependent on Netrin, is partially
dependent on Slit-Robo signaling, and is independent of the Unc5 receptor
suggesting that this misexpression phenotype is not a consequence of repulsion
via Unc5 and cannot be completely explained by an increase in Slit-Robo
signaling. Therefore, the emergence of a commissureless phenotype is quite
exciting as it implies that our truncated Fra receptor is interfering with
additional pathways, perhaps those playing a role in axon attraction at the
Drosophila midline.
Domain requirements for Fra-mediated attraction
At first glance, the domain requirements for DCC/Fra/Unc-40 signaling might
appear to differ among species. For example, in vertebrates it has been
demonstrated that the P3 conserved sequence motif is essential for the
outgrowth and turning of cultured Xenopus spinal neurons. Whereas in
C. elegans, P3 is not required to generate a gain-of-function
phenotype associated with expression of MyrUnc-40; however, P1 and P2 are
essential for this response (Gitai et al.,
2003). Can the difference in motif requirements between
vertebrates and C. elegans simply be attributed to a divergence in
conserved functions for these domains throughout evolution? Given the high
degree of sequence similarity of these motifs, this seems unlikely. Here it
should be noted that the potential function of the conserved P1-P3 motifs in
full-length Unc-40 receptor signaling in the context of an in vivo attractive
decision have not been examined. Therefore, perhaps the apparent divergence in
function could simply be attributed to the phenotypic readout of each assay in
the individual systems.
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Involvement of the P3 sequence motif during attractive guidance
In vertebrate systems, P3 has been implicated in mediating two distinct
functions. Initially, it was reported that the conserved cytoplasmic P3
sequence motif is necessary and sufficient for ligand-gated receptor
multimerization and Netrin-induced attractive turning in cultured
Xenopus spinal neurons (Stein et
al., 2001). Versions of DCC lacking the P3 motif cannot
self-associate and neurons expressing this form of DCC are no longer able to
respond to Netrin. Replacing the P3 motif with the SAM multimerization domain
from Eph tyrosine kinase receptors
(Bruckner and Klein, 1998;
Thanos et al., 1999) is
sufficient to restore an appropriate Netrin response, suggesting that the
major function of the P3 domain is to mediate self-association
(Stein et al., 2001).
Surprisingly, we have found that P3 does not appear to mediate
self-association of the Drosophila Fra receptor, as mutants lacking
P3 show equivalent biochemical interactions. It seems clear from our data that
P3 function in the context of midline attraction is through a mechanism that
is independent of mediating receptor multimerization. Although it remains an
open question whether the receptor-receptor interactions that we observe in
vitro are necessary for attractive guidance, it is clear that in the absence
of an intact P3 domain they are not sufficient.
Another set of studies proposed that P3 recruits Fak and this recruitment
leads to tyrosine phosphorylation of DCC by Src family non-receptor tyrosine
kinases (Li et al., 2004;
Liu et al., 2004;
Meriane et al., 2004;
Ren et al., 2004). Both Fak
recruitment and tyrosine phosphorylation are important in mediating
Netrin-induced outgrowth and attractive turning in cultured neurons in vitro
(Li et al., 2004;
Liu et al., 2004;
Ren et al., 2004).
Specifically, the LD-like motif within P3 was shown to play a critical role in
FAK association, although a P3-independent binding site was also suggested
(Ren et al., 2004).
Interestingly, we have found that mutant Fra receptors in which the LD motif
is intact, but the rest of P3 is disrupted are still unable to mediate Fra
attraction (Fig. 3), suggesting
that Fak binding may not be important for Fra function in Drosophila.
This is also consistent with the observation that fak mutants do not
have a disrupted CNS phenotype (D.S.G. and G.J.B., unpublished). Nevertheless,
future studies will test for genetic interactions between mutations in
fra and mutations in fak and/or src in the context
of the Drosophila midline. Here it is worth noting that the tyrosine
residue in DCC identified as the principal target of Fyn/Src kinases is not
conserved in Drosophila Fra or C. elegans UNC-40, suggesting
that the precise mechanisms by which Fra/DCC/UNC-40 signaling is regulated by
tyrosine kinases may differ between organisms. However, the facts that, (1)
tyrosine phosphorylation of UNC-40 has been observed in C. elegans
(though the responsible kinase has not been identified) and (2) UNC-40
signaling appears to be negatively regulated by a receptor tyrosine
phosphatase is consistent with an evolutionarily conserved role for tyrosine
phosphorylation of the DCC/Fra/UNC-40 family of receptors
(Chang et al., 2004;
Tong et al., 2001).
Furthermore, the Abl kinase has also been implicated in Netrin-Fra signaling
in Drosophila (Forsthoefel et
al., 2005) suggesting that additional phosphorylation events may
be important for DCC/Fra/UNC-40 output.
Requirements for Fra localization
Previous data determined that the cytoplasmic domain of Fra is necessary
and sufficient for normal distribution of the receptor
(Hiramoto et al., 2000).
Therefore, we were surprised to find that our newly generated FraC-HA
localized similarly to the wild-type protein. The original experiments from
Hiramoto et al. (Hiramoto et al.,
2000) were performed using a truncated Fra receptor
(Bashaw and Goodman, 1999) that
contained the transmembrane domain and 67 juxtamembrane cytoplasmic amino
acids of the Robo receptor (FraCRobo67-Myc). We hypothesized
that this exongenous protein sequence could be interfering with proper
localization of the Fra receptor. Indeed this seems to be the case; whereas
the FraC-HA construct mimics wild-type receptor localization when
expressed in all neurons by elavGal4, the original
FraCRobo67-Myc does not
(Fig. 5F'). Therefore,
these data suggest that normal Fra localization does not require the
cytoplasmic domain. Additional experiments will help determine the specific
domains of Fra that are sufficient to control receptor distribution. Finally,
all of the above observations are based on overexpression studies and
therefore may not completely reflect the localization of endogenous proteins
with similar deletions.
How does FraC disrupt guidance?
Intriguingly, expression of FraC in a fra mutant background
results in a complete commissureless phenotype, suggesting the possibility
that it is capable of inhibiting Fra-independent axon attraction. Although
this is one of the simplest interpretations, other hypotheses exist. For
example, double mutants between fra null alleles and either
abl or trio also exhibit a near commissureless phenotype
(Forsthoefel et al., 2005).
These data are consistent with Abl and Trio participating in other pathways
that are important for guidance toward and across the Drosophila
midline. Accordingly, one possibility could be that FraC is interfering
with an independent Abl and/or Trio signaling pathway.
Previous results demonstrated that panneural overexpression of Netrin leads
to a phenotype where few axons cross the midline
(Kolodziej et al., 1996;
Mitchell et al., 1996). It was
suggested that wild-type Netrin distribution provides a directional cue that
attracts axons across the midline and when Netrin is misexpressed in all
neurons, axons become confused and are no longer able to decipher the
appropriate path. Along these lines, perhaps the extracellular domain of
FraC is binding Netrin and `presenting' it everywhere thereby confusing
the axons. Indeed, the Fra receptor has been shown to redistribute Netrin
laterally away from its midline source
(Hiramoto et al., 2000).
However, this theory would have to assume that this specific truncation is
somehow misregulated upon ligand binding (for example, perhaps it is not
efficiently internalized) since our other wild-type and deletion receptors -
which presumably bind to and relocalize Netrin similarly to FraC - do
not produce this effect when expressed panneurally. Since we only see a
commissureless phenotype when FraC is overexpressed in a fra
background, we would also have to argue that another receptor elicits the
response to this un-internalized and redistributed Netrin.
Finally, FraC expression may cause increased midline repulsion,
thereby preventing axons from crossing the midline. Accordingly, the
FraC misexpression phenotype shows a striking similarity to embryos
deficient for the gene comm. Comm is a single-pass transmembrane
protein that acts to downregulate Robo expression
(Keleman et al., 2002;
Keleman et al., 2005) and
comm, robo double mutants resemble robo single mutants
indicating that comm acts upstream of the Robo receptor
(Kidd et al., 1998b).
Therefore, if FraC misregulates Comm, then perhaps Robo levels are
increased and axons are repelled away from the midline. This argument would
imply that overexpressing FraC in a robo mutant background
should produce robo-like mutants (similar to the comm, robo
double mutants). Contrary to this hypothesis, we observe that FraC
expression can partially suppress a robo loss-of-function phenotype
suggesting that FraC is not simply misregulating Comm. However, since
FraC cannot prevent all axons from approaching the midline in fra,
slit double mutants, and given that the FraC overexpression
phenotype in a fra, robo double mutant background is weaker than that
seen in fra single mutants, we cannot rule out the possibility that
signaling through Robo is partially required and/or that the upregulation of
another Robo family member, for example Robo2, prevents axons from approaching
the midline when FraC is expressed in all neurons. Whatever the
mechanism by which FraC exerts its influence on commissural axon
guidance, it may provide an important route to a further dissection of the
missing factors that function in addition to Netrin and Fra to guide axons
across the midline.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/24/4325/DC1
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ACKNOWLEDGMENTS |
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REFERENCES |
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