1 The Ohio State University, Department of Molecular Genetics and Center for
Molecular Neurobiology, Columbus, OH 43210, USA
2 Denison University, Department of Biology, Granville, OH 43023, USA
3 Vanderbilt University Medical Center, Department of Cell and Developmental
Biology, Center for Molecular Neuroscience, MCN C2210, Nashville, TN
37232-2175, USA
Author for correspondence (e-mail:
seeger.9{at}osu.edu)
Accepted 2 February 2005
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SUMMARY |
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Key words: Drosophila, CNS midline, Axon guidance, Abelson tyrosine kinase, Abl, Trio, Guanine nucleotide-exchange factor, Enabled, Frazzled, Netrin, Actin cytoskeleton, Growth cone attraction
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Introduction |
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An emerging theme in axon guidance is that growth cone receptors recruit
cytoplasmic effectors to modulate reorganization of the actin cytoskeleton
(Huber et al., 2003;
Patel and Van Vactor, 2002
).
Relatively little is known about how Frazzled and its homologs DCC and UNC-40
signal to the cytoskeleton during axon guidance and outgrowth. A screen in
C. elegans for genetic suppressors of an UNC-40 gain-of-function
phenotype identified molecules that may function with UNC-40 and Netrin/UNC-6
to regulate actin dynamics (Gitai et al.,
2003
). These include the actin-binding protein AbLIM/UNC-115,
Enabled (Ena)/UNC-34, and the Rho-family guanosine triphosphatase (GTPase)
Rac/CED-10. AbLIM/UNC-115 behaves genetically as an effector of signaling by
the Rac-2 GTPase (Struckhoff and
Lundquist, 2003
). The vertebrate orthologs of Ena/UNC-34, Mena,
vasodilator-stimulated protein (VASP) and Ena/VASP-like (EVL), antagonize
F-actin capping and allow F-actin filament elongation
(Bear et al., 2002
;
Gitai et al., 2003
). Netrin
stimulation of cultured mouse neurons results in Ena/VASP-dependent filopodia
formation and Mena phosphorylation at a protein kinase A regulatory site
(Lebrand et al., 2004
). In
cultured vertebrate cells, the adaptor Nck1 and the GTPases Cdc42 and Rac1
affect Netrin- and DCC-dependent neurite outgrowth, cell spreading and
filopodia extension (Li et al.,
2002a
; Li et al.,
2002b
; Shekarabi and Kennedy,
2002
). Nck1 binds DCC in vitro, and can regulate actin nucleation
in concert with Rac through WAVE1, an Arp2/3 complex activator; however, it is
not known whether the WAVE complex is activated in response to Netrin-DCC
signaling (Eden et al., 2002
;
Li et al., 2002a
). Similarly,
although Netrin stimulation of DCC-expressing non-neuronal cells leads to
activation of Cdc42 and Rac1, the mechanisms by which DCC regulates small
GTPase activity have not been elucidated
(Li et al., 2002b
;
Shekarabi and Kennedy,
2002
).
Other pathways from DCC to the F-actin cytoskeleton are likely to involve
cytoplasmic tyrosine kinases. DCC interacts with focal adhesion kinase (FAK),
Src and Fyn, and DCC is tyrosine phosphorylated in cells expressing increased
levels of these kinases or upon Netrin stimulation; furthermore,
phosphorylation of DCC is required for attractive axon turning in cultured
neurons and Rac1 activation in non-neuronal cells
(Li et al., 2004;
Liu et al., 2004
;
Meriane et al., 2004
;
Ren et al., 2004
). Tyrosine
phosphorylation of UNC-40 has also been observed, and genetic interactions
indicate that UNC-40 signaling is regulated by the receptor protein tyrosine
phosphatase (RPTP) CLR-1 (Chang et al.,
2004
; Tong et al.,
2001
).
In Drosophila, signaling by Fra and the Netrins is even less
understood. Genetic interactions with fra suggest that Gef64C,
weniger, Arf6-Gef/Schizo, Myosin Light Chain Kinase
(Stretchin-Mlck FlyBase) and the G-protein
Gq (G
q49B FlyBase)
promote commissure formation (Bashaw et
al., 2001
; Hummel et al.,
1999a
; Hummel et al.,
1999b
; Kim et al.,
2002
; Onel et al.,
2004
; Ratnaparkhi et al.,
2002
). However, none of the molecules encoded by these genes nor
any others have been linked biochemically to Fra signaling.
The Drosophila Abelson cytoplasmic tyrosine kinase (Abl), the Trio
Rac/Rho guanosine-exchange factor (GEF) and Ena are expressed in the nervous
system and interact genetically and/or biochemically with receptors known to
regulate nervous system development
(Awasaki et al., 2000;
Bashaw et al., 2000
;
Bateman et al., 2000
;
Crowner et al., 2003
;
Gertler et al., 1989
;
Gertler et al., 1995
;
Liebl et al., 2003
;
Wills et al., 1999
). These
molecules and their homologs in other organisms regulate cytoskeletal dynamics
during diverse developmental processes
(Bateman and Van Vactor, 2001
;
Hakeda-Suzuki et al., 2002
;
Kwiatkowski et al., 2003
;
Lanier and Gertler, 2000
;
Moresco and Koleske, 2003
;
Van Etten, 1999
;
Woodring et al., 2003
). In
cultured cells, these molecules regulate cell migration, neurite extension and
leading edge actin dynamics (Bateman and
Van Vactor, 2001
; Estrach et
al., 2002
; Kwiatkowski et al.,
2003
; Moresco and Koleske,
2003
).
In this study, we expand the understanding of the signaling networks in which Abl, Trio and Ena function by uncovering genetic and biochemical interactions between these molecules and the Netrin receptor Fra. Our results indicate that Abl, Trio and Ena probably function as effectors of Fra signaling in commissural axons, in addition to roles downstream of other growth cone receptors. Furthermore, our observations suggest potential mechanisms by which Fra and other receptors might coordinate actin cytoskeletal dynamics through these molecules.
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Materials and methods |
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All flies were maintained in standard cornmeal-yeast medium at room
temperature. Embryos were fixed in 4% paraformaldehyde/1 xPBS, and the
CNS was visualized using mAb BP102 (1:20, Developmental Studies Hybridoma
Bank, University of Iowa), anti-ß-galactosidase (1:500, Promega), goat
anti-mouse-HRP (1:500, Jackson), and standard immunohistochemical procedures
(Patel et al., 1987). All
alleles were maintained over lacZ-expressing balancers to distinguish
the genotype of embryos. Stage 14-16 embryos were filleted and scored at 400
x magnification.
Constructs
pMET Abl-Myc, pMET Trio-Myc, pMET TrioSPR-Myc, pMET
Fra-Myc, pMET Fra-HA, pMET Fra
CYTO-HA [deleted for amino
acids P1123-C1375 (GenBank Accession Number U71001)], pBSK Abl-Myc, pBKS
Trio-Myc, pBKS Trio
SPR-Myc [deleted for amino acids
L285-D1199 (GenBank Accession Number AF216663)], pBSK Ena-Myc,
pGEX2T-FraCYTO [amino acids C1098-C1375 (GenBank Accession Number
U71001)], pGEX2T-AblSH3 [amino acids E202-K268 (GenBank Accession Number
AH001049)], and pGEX2T-TrioSH3 [amino acids E1177-L1840, deleted for GEF1
(A1281-P1596) (GenBank Accession Number AF216663)] were all constructed using
standard molecular techniques; details are available upon request. pPAC Ena
was provided by A. Comer (Comer et al.,
1998
). pMET Fra constructs were generated using the short isoform
that rescues fra mutant phenotypes
(Kolodziej et al., 1996
). Myc
and HA tags were added C terminally.
Protein-protein interactions and phosphorylation assays
GST and GST-Fracyto were generated in E. coli (BL21),
as described in Amersham Pharmacia's Gene Fusion System Guide. GST pulldowns
of in vitro-translated proteins were performed essentially as described
(Bashaw et al., 2000), except
that non-radiolabeled, epitope-tagged proteins were generated in vitro using
TnT T7-coupled rabbit reticulocyte lysate system (Promega), and Abl, Trio and
Ena constructs cloned into pBluescript (Stratagene). An aliquot from each
reaction (15-25 µl) was added to
10 µg fusion protein bound to
beads suspended in 200 µl binding buffer. Binding was overnight, and, after
washing,
20% of total protein was separated by SDS-PAGE. For GST
pulldowns from S2 cell extracts, 2x107 S2 cells were
transiently transfected with pMET Abl-Myc, pMET Trio-Myc, or pPAC Ena with
CellFectin Reagent (Invitrogen). Twenty-four hours after induction, cells were
lysed in 1 ml IP buffer (Comer et al.,
1998
), and lysates were pre-cleared with 100 µl of Glutathione
Sepharose4B beads prior to GST pulldowns.
For co-immunoprecipitations, 2x107 S2 cells were
transiently transfected with the relevant constructs, and 24 hours after
induction, cells were rinsed once in 1xPBS then lysed in 50 mM Tris (pH
8), 100 mM NaCl, 1 mM MgCl2, 1% NP-40, 10 mM NaF, 2 mM
Na3VO4, and 5 µg/ml each of Aprotinin and Leupeptin
(Roche). Cell extracts were cleared by centrifugation, lysates were
pre-cleared with 40 µl Protein G Sepharose beads (Sigma) for 30 minutes,
and protein complexes were immunoprecipitated with 1 µg rabbit anti-Myc
(Santa Cruz), anti-Ena [5G2, Developmental Studies Hybridoma Bank
(Bashaw et al., 2000)] diluted
1:20, or anti-HA (HA.11, Covance) diluted 1:150, for 60 minutes. Immune
complexes were recovered on 40 µl Protein G beads for 60 minutes, washed
three to four times in lysis buffer containing 1 µg/ml Aprotinin and
Leupeptin, and boiled in 6x sample buffer.
For Trio and Fra phosphorylation experiments, 2x107 S2 cells were transiently transfected with pMET Trio-Myc, pMET Fra-Myc, pMET Abl, and/or empty pMET vector (to control for transfection efficiency in experiments receiving less than 5 µg pMET Abl). For pervanadate treatment, cells [in Schneider's Media (BRL/Invitrogen), plus 10% FBS] were treated with 2 mM Na3VO4 and 3 mM H2O2 for 30 minutes at room temperature. In all experiments, cells were rinsed once in PBS and then lysed in 50 mM Tris (pH 8), 300 mM NaCl, 5 mM EDTA, 1% Triton X-100, 10 mM NaF, 2 mM Na3VO4, and 5 µg/ml each of Aprotinin and Leupeptin. After immunoprecipitation (IP), beads were washed four to six times in lysis buffer containing 1 µg/ml Aprotinin and Leupeptin, and boiled in 2 x Sample Buffer.
Phosphotyrosine was detected using 4G10 mouse anti-phosphotyrosine (Upstate) at 1:10,000 and goat anti-mouse-HRP (Jackson) at 1:10,000 in low-salt TBST (25 mM NaCl)/5% BSA. Mouse anti-Myc (Roche), rabbit anti-Myc (Santa Cruz), mouse anti-HA, and rabbit anti-Abl (kindly provided by A. Comer) were used at a dilution of 1:2000 in 5% milk/TBST. Mouse anti-Ena 5G2 was used at 1:200 in 5% milk/TBST. Proteins were visualized using ECL (for phosphotyrosine detection), ECL PLUS (Amersham Pharmacia, for protein-protein interactions), or NBT/BCIP detection (for loading controls in co-IP and phosphorylation experiments). Prior to the re-probing of co-IP and phosphotyrosine westerns, blots were stripped in 50 mM Tris-HCl (pH 6.8), 2% SDS and 100 mM ß-mercaptoethanol overnight at 65°C.
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Results |
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In homozygous fra, heterozygous Abl (fra4/Df(2R)vg135; Abl4/+) embryos, the percentage of segments with defective commissures increased to 30%, and this increase was due solely to an increase in the number of thin or missing commissures (24%, Fig. 1G, Table 1). The independently generated Abl1 allele also dominantly enhanced the fra phenotype, demonstrating that lesions in Abl were responsible for the genetic interaction with fra, and not accessory mutations on any of the chromosomes tested (Table 1).
Heterozygosity for trio also enhanced the CNS phenotype in
fra mutant embryos. For example, in fra4/Df(2R)vg135;
trioIMP159.4/+ embryos, 53% of segments had defective
commissures (Fig. 1H,
Table 2). Forty-two percent of
segments in fra4/Df(2R)vg135;trioIMP159.4/+
embryos had thin or missing commissures (versus 13% in
fra4/Df(2R)vg135 animals), and 11% of segments had
commissural pathfinding errors (versus 8% in
fra4/Df(2R)vg135 embryos)
(Fig. 1H,
Table 2). Milder dominant
enhancement of the fra phenotype was observed for a number of other
trio alleles, including the deficiency Df(3L)FpaI, the
hypomorphic P-element insertion allele trioP0368/10, and
the trioM89 allele (which encodes a point mutation in the
GEF1 domain of Trio) originally identified as a dominant enhancer of the
Abl semilethality phenotype
(Liebl et al., 2000)
(Table 2). It is not clear why
trioIMP159.4 (an imprecise excision allele generated by
mobilizing the P element on the trioP0368/10 chromosome)
enhances the fra phenotype so strongly. As the deficiency
Df(3L)FpaI (which completely removes the trio gene) behaves
similarly to the other trio alleles we tested, it is likely that
trioIMP159.4 is not a null or hypomorphic allele, but
rather encodes a Trio protein with unusual properties. It is also not clear
why fra4 homozygotes have more disrupted commissures than
fra4/Df(2R)vg135 animals (this fra allele has not
been characterized), although fra4 homozygous animals are
not immunoreactive with the polyclonal anti-fra serum generated by
Kolodziej et al. (Kolodziej et al.,
1996
).
Although in most fra mutant combinations tested the posterior
commissure (PC) was affected more often than the anterior commissure (AC),
heterozygosity for Abl or trio increased the frequency of
defects in both commissures. For example, in
fra4/fra4 embryos 20% (n=212) of PCs
scored were thin or missing, versus 31% (n=215) in
fra4/fra4;Abl1/+ mutants and 39%
(n=262) in
fra4/fra4;trioM89/+ embryos.
Similarly, 5% (n=212) of ACs scored were thin or missing in
fra4/fra4 embryos, versus 14% (n=215)
in the fra4/fra4;Abl1/+ background
and 18% (n=262) in
fra4/fra4;trioM89/+ embryos. Thus,
although mutations in fra seem to affect posterior commissural axons
preferentially (Kolodziej et al.,
1996), the genetic interactions of fra with Abl
and trio are consistent with a significant role for all of these
genes in anterior commissure formation as well.
We next asked whether heterozygosity for Abl or trio
modifies the CNS phenotype in embryos mutant for genes encoding the Frazzled
ligands Netrin A and Netrin B. NetA and NetB are both
removed by a deficiency on the X chromosome, Df(1)NP5
(Mitchell et al., 1996). Like
fra mutant embryos, Netrin mutant embryos have fewer
commissural axons, with those in the posterior commissure being most affected
(Mitchell et al., 1996
). As
observed for fra, both Abl and trio dominantly
enhanced the Netrin deficiency phenotypes and increased the frequency
of defects in both commissures (Fig.
1P-R, and data not shown). In
Df(1)NP5/Y;Abl4/+ embryos, 33% of segments had
thin or missing commissures, versus only 24% in Df(1)NP5/Y
hemizygotes; in Df(1)NP5/Y;trioIMP159.4/+
embryos, 56% of segments had thin or missing commissures (Tables
1,
2;
Fig. 1P-R). Genetic
interactions of Abl and trio with the Netrin genes
further support the idea that Abl and trio are required to
attract growth cones to the CNS midline.
Although mutations in Abl and trio dominantly enhanced
the loss-of-commissure phenotype in fra and Netrin mutants,
we did not observe other dose-sensitive interactions between fra, Abl
and trio. For example, mutations in fra did not reciprocally
enhance the loss-of-commissure phenotype in Abl or trio
mutants, although the percentage of segments with axon pathfinding errors
increased when one dose of fra was removed in the Abl mutant
background (Tables 1,
2;
Fig. 1I,J). We also did not
observe transheterozygous interactions between Abl, trio and
fra in single, double and triple transheterozygous mutant
combinations. In all of these cases, no more than 5% of segments had disrupted
commissures (Table 3,
Fig. 1K). Furthermore, even in
embryos that were homozygous mutant for one of these three genes,
heterozygosity for the two remaining genes did not lead to additive or
synergistic increases in commissure defects
(Table 3). Because in other
experiments, Abl and trio behaved genetically as
fra effectors (see below), the inability of fra to
dominantly enhance Abl or trio loss-of-commissure defects,
and the lack of transheterozygous interactions between fra, Abl and
trio, may be due to the presence of maternally-contributed
Abl and trio in the embryo
(Bennett and Hoffmann, 1992;
Liebl et al., 2000
;
Wadsworth et al., 1985
).
Another possibility is that heterozygosity for fra simply does not
reduce the effective dose enough to enhance Abl or trio
loss-of-commissure phenotypes.
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Additionally, although there were occasional breaks (>75% of axons missing) in longitudinal connectives in fra homozygotes [10% (n=519) in fra4/Df(2R)vg135 embryos], in fra;Abl and fra;trio double mutants this type of defect did not increase considerably. For example, only 11% (n=387) of connectives had breaks in fra4/Df(2R)vg135;Abl4/Abl1 embryos, and 16% (n=368) in fra4/Df(2R)vg135;trioIMP159.4/trioM89 embryos. However, analysis of subsets of longitudinally-projecting axons in stage 17 embryos using the monoclonal antibody 1D4 (anti-Fasciclin II) revealed a significant disorganization of these pathways, especially in fra;Abl double mutants (see Fig. S1 and Table S1 in the supplementary material). In fra;Abl and fra;trio double mutants, Fas2-positive longitudinal pathways wandered medially or laterally, often seeming to intertwine so that individual bundles were indistinguishable. In addition, although breaks in all three longitudinal bundles between segments were rare, often one to two (usually lateral) 1D4-positive bundles were discontinuous between segments. Similar defects were observed at a lower frequency in individual fra, Netrin, Abl and trio mutants (see Fig. S1 and Table S1 in the supplementary material). Thus, although BP102 immunohistochemistry did not reveal consistent defects in longitudinal pathways, the disorganization of Fas2-positive axon bundles suggests that Fra, Abl and Trio function during axon pathfinding in longitudinal pathways in addition to their roles during commissure formation.
Mutations in enabled suppress frazzled, Netrin and trio,Abl CNS phenotypes
In the CNS, ena interacts genetically with the repulsive receptor
robo, leading to inappropriate crossing of the midline by
longitudinal axons (Bashaw et al.,
2000). In Abl, trio and fra mutant combinations,
numerous axons fail to cross the midline
(Liebl et al., 2000
) (Tables
1,
2). We explored this apparently
antagonistic relationship further by analyzing genetic interactions among
trio, Abl, fra, the Netrin genes and ena in the
CNS.
Mutations in ena dominantly reduced the severity of the CNS phenotype in trio,Abl mutants. For example, in ena heterozygous, trio,Abl homozygous (enaGC10/+;trioM89,Abl1/Df(3L)FpaI,Abl4) embryos, only 78% of segments had defective commissures, versus 100% in the trio,Abl (trioM89,Abl1/Df(3L)FpaI,Abl4) double mutant (Table 4; and compare Fig. 1D,E with 1T). Overall, there was a 20% reduction in the number of segments with thin or missing commissures (Table 4). However, analysis of individual commissures revealed a dramatic increase in the number of axons which crossed the midline when compared with the trio,Abl double mutant, especially in the anterior commissure. For example, in ena heterozygous, trio,Abl homozygous (enaGC10/+;trioM89,Abl1/Df(3L)FpaI,Abl4) embryos, only 24% of anterior commissures and 59% of posterior commissures were thin or missing (n=206 segments), compared with 64% of anterior commissures and 84% of posterior commissures in trio,Abl embryos (n=160 segments) (Fig. 1D,E,T). In another mutant combination, the increase in the number of axons crossing the midline was even more striking. In Df(3L)FpaI,Abl4/trioIMP159.4,Abl1 embryos, 65% of segments had thin or missing commissures, whereas in enaGC5/+;Df(3L)FpaI,Abl4/trioIMP159.4,Abl1 embryos only 9% of segment commissures were thin or missing (Table 4). Removing one dose of ena in the trio,Abl homozygous mutant background also decreased the number of breaks in longitudinal connectives (Fig. 1D,E,T; and see Table S1 in the supplementary material).
Mutations in ena also dominantly suppressed CNS defects in fra and Netrin mutants. For example, in Df(1)NP5/Y;enaGC10/+ embryos, only 7% of segments had thin/missing commissures, versus 24% in Df(1)NP5/Y embryos (Fig. 1P,S, and Table 4). In fra4,enaGC10/Df(2R)vg135 embryos, 10% of segments had thin or missing commissures, versus 13% in fra4/Df(2R)vg135 animals (Table 4). Heterozygosity for ena also suppressed enhancement of the fra CNS phenotype by Abl or trio. For example, in fra4,enaGC10/fra4;Abl4/+ embryos 14% of segments had thin/missing commissures, versus 33% in fra4/fra4;Abl4/+ mutants, and 23% in fra4/fra4 embryos. Similarly, in fra4/fra4;trioIMP159.4/+ embryos, 80% of segments had thin or missing commissures, compared with only 15% in fra4,enaGC10/fra4;trioIMP159.4/+ embryos (Table 4).
Heterozygosity for Abl, trio and ena suppresses inappropriate midline crossing by axons expressing the chimeric Robo-Fra receptor
The genetic interactions described support roles for Abl, Trio and Ena
during commissure formation, but these types of interactions (dominant
enhancement/suppression and synergistic double mutant genetic interactions)
are usually interpreted to mean that gene products function in parallel
pathways; it is likely, for example, that Abl and Trio are instructed by at
least one other receptor that positively regulates commissure formation. To
test genetically whether Abl, Trio and/or Ena might function as Fra
effector(s) in vivo, we took advantage of the fact that neuronal expression of
a chimeric receptor composed of the extracellular and transmembrane domains of
the repulsive Robo receptor and the intracellular domain of Fra causes CNS
axons to inappropriately sense the midline-secreted repellent Slit as an
attractant and cross the midline boundary inappropriately
(Bashaw and Goodman, 1999). If
Abl, Trio and/or Ena function as effectors of Fra signaling in axons that
cross the midline, then reducing the dose of these molecules genetically would
be expected to reduce the severity of the chimeric Robo-Fra receptor
phenotype. We chose this strategy because, in the Drosophila embryo,
overexpressing full-length Fra in CNS neurons does not cause robust ectopic
midline crossing by CNS axons (D.J.F. and P.A.K., unpublished)
(Kim et al., 2002
).
In wild-type stage 17 embryos, Fas2-positive axons project longitudinally in three bundles on either side of the midline, but these axons never cross the midline boundary (Fig. 2A). In embryos expressing UAS-Robo-Fra in all neurons, numerous Fas2-positive axon bundles crossed the midline inappropriately (Table 5, Fig. 2B). Reducing the gene dose of Abl in embryos expressing the chimeric receptor led to a moderate reduction in the number of these ectopic crossovers (Table 5). Similarly, in three out of four alleles tested, heterozygosity for trio also reduced the severity of the Robo-Fra phenotype (Table 5). In these experiments, the deficiency Df(3L)FpaI suppressed the Robo-Fra receptor phenotype most strongly, and not the imprecise excision allele trioIMP159.4, which acted as the strongest dominant enhancer of the fra loss-of-function phenotype (Table 2). It is possible that other, unidentified genes removed by the FpaI deficiency also function as Fra effectors, that the other trio alleles encode Trio proteins that retain partial function downstream of Fra signaling, or that trioIMP159.4 disrupts signaling by other receptor(s) that mediate commissure formation more strongly than this allele interferes with Fra signaling. We also discovered that if the Df(3L)FpaI chromosome was contributed to progeny by the male parent, rather than the female, genetic suppression of the Robo-Fra phenotype was less severe (Table 5), suggesting that maternal contribution of trio (or another gene removed by this deficiency) plays a role.
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|
Heterozygosity for three different alleles of ena also reduced the
severity of the Robo-Fra phenotype (Table
5). Furthermore, reducing the gene dose of both Abl and
ena also led to a synergistic reduction in the number of ectopic
midline crossovers by Fas2-positive axons, similar to the genetic interaction
between Abl and trio in embryos expressing Robo-Fra. For
example, in ELAV-GAL4; UAS-Robo-Fra/ena23;
Abl4/+ embryos, there were only 0.23 crossovers per segment,
compared with 0.47 crossovers/segment in Robo-Fra-expressing embryos
heterozygous for ena23 only
(Table 5,
Fig. 2D). While these data were
initially surprising because heterozygosity for ena led to an
increase in the number of axons that crossed the midline in fra, Netrin,
Abl and trio loss-of-function mutants
(Table 4), they are consistent
with the idea that Ena may be functioning positively as an effector of
signaling via the Fra cytoplasmic domain, similar to orthologs of Ena in other
organisms (see Discussion) (see also Gitai
et al., 2003; Lebrand et al.,
2004
).
Physical interactions between Fra, Abl and Trio
To test whether Fra could physically interact with Abl, Trio or Ena, we
first asked whether glutathione-S-transferase (GST) fusions with the
cytoplasmic domain of Fra (generated in E. coli) could bind in vitro
translated Abl, Trio or Ena. In these experiments, Abl and Trio, but not Ena,
specifically bound GST-FraCYTO, but not GST or glutathione beads
alone (Fig. 3A and data not
shown), indicating that the cytoplasmic domain of Fra can interact directly
with Abl and Trio. In parallel experiments, we also found that GST-Fra could
interact with Abl-Myc and Trio-Myc in extracts from Drosophila
Schneider-2 (S2) cells that had been engineered to express each protein (not
shown).
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Next, we investigated whether full-length Fra could interact with full-length Abl, Trio and/or Ena in S2 cells. Hemagglutinin (HA)-tagged Fra was transiently co-expressed with either Abl-Myc, Trio-Myc or Ena, and extracts were subjected to immunoprecipitation. Fra-HA specifically co-immunoprecipitated in the presence of Abl-Myc or Trio-Myc, but not in their absence (Fig. 3D,E, lanes 1 and 2). This interaction is mediated by the cytoplasmic domain of Fra, as a Fra protein deleted for this domain did not co-immunoprecipitate with either Abl or Trio (Fig. 3D,E, lane 3). We did not observe a similar association between Ena and Fra-HA in S2 cells, using either anti-Ena or anti-HA antibodies for co-immunoprecipitation (data not shown).
Fra and Trio are tyrosine phosphorylated in S2 cells
The genetic and physical interactions that we observed among Fra, Trio and
Abl raised the possibility that Fra and Trio might be substrates for the Abl
tyrosine kinase. To determine whether or not phosphotyrosine could be detected
on Fra or Trio, we transiently expressed full-length, epitope-tagged versions
of Trio and Fra in S2 cells, and treated the cells with pervanadate, a potent
phosphotyrosine phosphatase inhibitor that has been used previously to sustain
tyrosine phosphorylation of proteins in Drosophila cells
(Fashena and Zinn, 1997;
Muda et al., 2002
). In control
S2 cells, Trio tyrosine phosphorylation was not detected, whereas Fra was
moderately tyrosine phosphorylated (Fig.
4A,B, lane 1). After a 30-minute pervanadate treatment, both
molecules were robustly tyrosine phosphorylated, indicating that Trio and Fra
are substrates of tyrosine kinases and phosphatases expressed endogenously in
S2 cells (Fig. 4A,B, lane
2).
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Discussion |
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We found that mutations in Abl and trio dominantly enhance the CNS phenotype in fra and Netrin mutant embryos, and that fra;Abl and fra;trio double mutants have a severe CNS phenotype in which a majority of commissures are thin or missing, similar to the trio,Abl double mutant phenotype. Mutations in Abl and trio reduce the number of axons that cross the midline inappropriately in embryos expressing the chimeric Robo-Fra receptor. Abl and Trio interact physically with the cytoplasmic domain of Fra, and increasing Abl kinase expression in cells increases tyrosine phosphorylation of Fra and Trio. Interpreting these data together, we conclude (1) that Abl, Trio and Fra function together during commissure formation, (2) that the severe double mutant phenotypes reflect the disruption of multiple signaling pathways or networks in the growth cones of commissural axons (i.e. Abl and Trio function downstream of at least one other receptor that positively regulates commissure formation), and (3) that the lack of other dose-sensitive interactions between fra, Abl and trio is a result of redundancy (other receptors/effectors mediating commissure formation), or the presence of maternally contributed proteins.
The interactions of Abl with Fra are intriguing, as they suggest that in
Drosophila, as in other organisms, this evolutionarily conserved
guidance receptor is regulated by tyrosine phosphorylation, and also that Fra
may regulate Abl substrates. Recently, others have demonstrated
Netrin-dependent tyrosine phosphorylation of DCC, Netrin/DCC-dependent
activation of the tyrosine kinases FAK, Src and Fyn, and the requirement of
DCC tyrosine phosphorylation for Netrin-dependent Rac1 activation and growth
cone turning (Li et al., 2004;
Liu et al., 2004
;
Meriane et al., 2004
;
Ren et al., 2004
).
Interestingly, 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 (Li et al., 2004
;
Meriane et al., 2004
).
Tyrosine phosphorylation of UNC-40 has also been observed, and although the
kinase(s) responsible has not been identified, genetic interactions suggest
that UNC-40 signaling is regulated by the RPTP CLR-1, supporting the idea that
regulation of tyrosine phosphorylation is a consequence of UNC-6/Netrin
signaling in C. elegans as well
(Chang et al., 2004
;
Tong et al., 2001
). In this
study, we observed more robust tyrosine phosphorylation of Fra in cells with
pervanadate stimulation than with Abl overexpression alone, raising the
possibility that additional kinase(s) may function during Fra signaling.
Further investigation will be needed to address this issue and to determine
how Abl-mediated phosphorylation of Fra modulates commissural growth cone
guidance.
Abl is thought to control actin dynamics in part through its ability to
regulate other proteins through tyrosine phosphorylation
(Lanier and Gertler, 2000;
Woodring et al., 2003
). Thus,
in addition to potential regulation of Fra, Fra may recruit Abl to regulate
other Abl substrates. Abl interacts genetically with trio
(Liebl et al., 2000
), and in
this study, we have found that Trio physically interacts with Abl in vitro and
that Trio tyrosine phosphorylation increases dramatically with co-expression
of Abl. Phosphorylation of Trio may affect its activity, as observed for other
GEFs. For example, Abl regulates phosphorylation and Rac-GEF activity of Sos1,
and Lck, Fyn, Hck and Syk kinases tyrosine phosphorylate Vav GEF and stimulate
its activity (Sini et al.,
2004
; Turner and Billadeau,
2002
).
Trio physically interacts with Fra in vitro and in S2 cells, suggesting
that Fra can recruit Trio directly. In addition, heterozygosity for
trio dominantly modifies the Robo-Fra chimeric receptor phenotype,
consistent with a positive role for Trio as a downstream effector of Fra
signaling in vivo. As a Rac/Rho GEF, Trio may link Netrin-Fra signaling to the
regulation of Rho-family GTPases in commissural axons. Rho-family GTPases have
been rigorously studied with regard to their role in the regulation of
cytoskeletal dynamics and axon guidance, outgrowth and branching
(Dickson, 2001;
Luo, 2000
). Although positive
roles for GTPases in commissure formation in the Drosophila embryo
have not been directly demonstrated, trio (in this study) and
GEF64C, a Rho GEF (Bashaw et al.,
2001
), interact genetically with fra leading to the
dramatic disruption of commissures. Additionally, expression of constitutively
active or dominantly negative isoforms of both Rac and Rho, as well as
constitutively active Cdc42, causes axons to cross the CNS midline
inappropriately (Fan et al.,
2003
; Fritz and VanBerkum,
2002
; Matsuura et al.,
2004
). Recent studies have implicated Cdc42 and Rac1/CED-10 as
effectors of DCC and UNC-40 signaling, but the biochemical mechanisms by which
GTPases are regulated have been elusive
(Gitai et al., 2003
;
Li et al., 2002a
;
Li et al., 2002b
;
Shekarabi and Kennedy, 2002
).
Future experiments must determine whether Netrin-Fra signaling modulates the
GEF activity of Trio, and how this occurs.
In this study, we found that reducing the genetic dose of ena
causes either more or fewer axons to cross the CNS midline, depending on the
genetic background, suggesting that the role of Ena in the growth cone is
complex. Heterozygosity for ena in embryos expressing the Robo-Fra
chimeric receptor reduces the number of axon bundles that inappropriately
cross the CNS midline, consistent with a role for Ena as a positive effector
of Fra signaling. Ena/UNC-34 has been identified genetically as an effector of
DCC/UNC-40 in C. elegans (Gitai
et al., 2003). In cultured mouse neurons, Ena/VASP proteins are
required for Netrin-DCC-dependent filopodia formation, and Mena is
phosphorylated at a PKA regulatory site in response to Netrin stimulation
(Lebrand et al., 2004
). In
migrating fibroblasts, increasing Ena/VASP proteins at the leading edge leads
to unstable lamellae and decreased motility; by contrast, increasing Ena/VASP
levels at the leading edge in growth cones causes filopodia formation,
possibly due to differences in the distribution of actin bundling or branching
proteins (Bear et al., 2000
;
Bear et al., 2002
;
Lebrand et al., 2004
).
Although the role of Ena in actin reorganization in Drosophila has
not been rigorously studied, Ena localizes to filopodia tips in cultured
Drosophila cells, suggesting that the role of Ena in filopodia
formation may be conserved (Biyasheva et
al., 2004
).
We have not observed a direct biochemical interaction between Fra and Ena.
However, Abl binds and phosphorylates Ena, and heterozygosity for both
Abl and ena further suppresses the Robo-Fra phenotype,
suggesting that Fra may recruit Abl to regulate filopodial extension through
Ena (Comer et al., 1998;
Gertler et al., 1995
).
Alternatively, Fra may regulate Ena through other molecule(s), and the
synergistic suppression of the Robo-Fra phenotype by Abl and
ena is a result of the compromise of parallel pathway(s) regulated by
Fra. It is important to note that the functional consequences of biochemical
interactions between Abl and Ena are not understood
(Comer et al., 1998
;
Grevengoed et al., 2003
;
Krause et al., 2003
).
Therefore it will be of particular interest to determine whether Ena is
tyrosine phosphorylated in response to Netrin-Fra signaling, and if Ena
phosphorylation regulates its activity during filopodial extension.
In addition to suppressing the Robo-Fra chimeric receptor phenotype,
mutations in ena also suppress the loss-of-commissure phenotype in
fra, Netrin, trio and Abl mutant combinations. In
Drosophila (as well as in C. elegans), Ena interacts
genetically and biochemically with the repulsive receptor Robo, indicating
that Ena may restrict axon crossing at the midline
(Bashaw et al., 2000;
Yu et al., 2002
). Thus, the
fact that mutations in ena dominantly suppress fra, Netrin,
trio and Abl CNS phenotypes could simply reflect the compromise
of a parallel, opposing signaling pathway. Consistent with this idea, some
axons that cross the midline in ena heterozygous, trio,Abl
homozygous embryos are Fas2 positive (D.J.F., unpublished), indicating a
partial reduction in repulsive signaling. However, ena also
dominantly suppresses fra and Netrin commissural pathfinding
defects, without causing longitudinal Fas2-positive axons to cross the midline
(D.J.F., unpublished). Reductions in Robo signaling therefore may not fully
explain the ability of ena to suppress defects in fra, Netrin,
Abl and trio mutants.
Based on the fact that mutations in ena suppress a number of
Abl mutant phenotypes, it has been proposed that Abl antagonizes Ena
function (Grevengoed et al.,
2003; Grevengoed et al.,
2001
; Lanier and Gertler,
2000
). In Abl mutant embryos, Ena and actin mislocalize
during dorsal closure and cellularization, and apical microvilli are
abnormally elongated, indicating that Abl regulates the localization of Ena
(Grevengoed et al., 2003
;
Grevengoed et al., 2001
). In
migrating fibroblasts, increasing Ena/VASP levels at the leading edge results
in long, unbranched actin filaments, unstable lamellae, and decreased motility
due to increased antagonism of capping protein
(Bear et al., 2000
;
Bear et al., 2002
).
Interestingly, mutations in the gene encoding Drosophila capping
protein ß enhance CNS axon pathfinding defects in Abl mutants,
including commissure formation (Grevengoed
et al., 2003
). Therefore, if Fra and/or Abl regulate Ena
localization in commissural axons, then in fra, Netrin or
Abl mutants, Ena may be mislocalized in the growth cone, leading to
inappropriate inhibition of capping protein and excessive F-actin filament
elongation. Additionally, reducing regulation of Ena by Fra or Abl may also
allow greater Ena regulation by Slit-Robo signaling. In either case, reducing
the gene dose of ena in fra, Netrin and trio,Abl
mutant embryos would partially relieve these effects, allowing axons to
respond more efficiently to other cues and cross the midline, as we observed.
Consistent with this idea, Lebrand et al.
(Lebrand et al., 2004
) found
that either increasing or decreasing Ena/VASP proteins at the leading edge
impaired the elaboration of growth cone filopodia in response to Netrin-DCC
signaling, suggesting that Ena/VASP levels must be tightly regulated in order
for the growth cone to respond optimally to extracellular signals.
The role of Abl in the growth cone is also likely to be complex. Our
observations implicate Abl as an effector of attractive Fra signaling. In
addition, tyrosine phosphorylation of Robo by Abl is thought to negatively
regulate repulsive signaling by Robo
(Bashaw et al., 2000).
Paradoxically though, loss-of-function mutations in Abl, robo and
slit interact genetically, resulting in inappropriate axon crossing
at the midline, and indicating that Abl may also promote repulsion in
longitudinally migrating growth cones
(Hsouna et al., 2003
;
Wills et al., 2002
).
Obviously, much remains to be understood about the molecular basis for genetic
interactions of Abl, particularly how Abl and its various substrates
cooperate with different growth cone receptors to yield specific cytoskeletal
outputs.
In summary, we have observed genetic and biochemical interactions indicating that Abl, Trio and Ena are integrated into a complex signaling network with Fra and the Netrins during commissure formation. These observations identify another receptor that acts through these effectors, and provide a framework for further investigation of signaling by this key, evolutionarily conserved guidance receptor.
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ACKNOWLEDGMENTS |
---|
![]() |
Footnotes |
---|
Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/132/8/1983/DC1
* We celebrate the life of our friend and colleague Peter Kolodziej who
passed away 3 March 2005. Peter was an inquisitive and insightful scientist
who will be missed.
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