RESEARCH ARTICLE 3099
Development 136, 3099-3107 (2009) doi:10.1242/dev.033324
Abi plays an opposing role to Abl in Drosophila
axonogenesis and synaptogenesis
Tzu-Yang Lin1,*, Chiu-Hui Huang1,2,*, Hsiu-Hua Kao2, Gan-Guang Liou1, Shih-Rung Yeh2, Chih-Ming Cheng1,
Mei-Hsin Chen1, Rong-Long Pan2 and Jyh-Lyh Juang1,†
Abl tyrosine kinase (Abl) regulates axon guidance by modulating actin dynamics. Abelson interacting protein (Abi), originally
identified as a kinase substrate of Abl, also plays a key role in actin dynamics, yet its role with respect to Abl in the developing
nervous system remains unclear. Here we show that mutations in abi disrupt axonal patterning in the developing Drosophila central
nervous system (CNS). However, reducing abi gene dosage by half substantially rescues Abl mutant phenotypes in pupal lethality,
axonal guidance defects and locomotion deficits. Moreover, we show that mutations in Abl increase synaptic growth and
spontaneous synaptic transmission frequency at the neuromuscular junction. Double heterozygosity for abi and enabled (ena) also
suppresses the synaptic overgrowth phenotypes of Abl mutants, suggesting that Abi acts cooperatively with Ena to antagonize Abl
function in synaptogenesis. Intriguingly, overexpressing Abi or Ena alone in cultured cells dramatically redistributed peripheral
F-actin to the cytoplasm, with aggregates colocalizing with Abi and/or Ena, and resulted in a reduction in neurite extension.
However, co-expressing Abl with Abi or Ena redistributed cytoplasmic F-actin back to the cell periphery and restored bipolar cell
morphology. These data suggest that abi and Abl have an antagonistic interaction in Drosophila axonogenesis and synaptogenesis,
which possibly occurs through the modulation of F-actin reorganization.
KEY WORDS: Abi, Abl, Drosophila, Genetic suppressor, Axon guidance, Synaptogenesis
1
Division of Molecular and Genomic Medicine, National Health Research Institutes,
Zhunan, Miaoli 35053, Taiwan. 2Department of Life Science, National Tsing Hua
University, Hsinchu 30043, Taiwan.
*These authors contributed equally to this work
†Author for correspondence ([email protected])
Accepted 2 July 2009
proteins, including Wasp (Bogdan et al., 2005) and Ena (Juang and
Hoffmann, 1999; Tani et al., 2003), are involved in CNS
development (Gertler et al., 1995; Ben-Yaacov et al., 2001). Genetic
and immunohistochemical studies in mammals have also
demonstrated that the Abi family proteins play a role in synaptic
development (Grove et al., 2004; Proepper et al., 2007). Together,
these reports suggest that Abl and Abi have similar roles in
axonogenesis and synaptogenesis. However, no study to date has
explored whether abi interacts genetically with Abl during nervous
system development.
In this study we used a multidisciplinary approach, including
genetics,
immunohistochemistry,
electron
microscopy,
electrophysiology and behavioral analysis, to characterize the
functional connection between Abl and Abi in the Drosophila
nervous system. Our results support a model in which Abl and Abi
play opposing roles in regulating Drosophila axonogenesis and
synaptogenesis through actin cytoskeleton reorganization.
MATERIALS AND METHODS
Fly strains
The wild-type strain used in this study was w1118. The abi alleles we used
were two P-element insertion lines, GE23319 (abiP1) and GE24211 (abiP2)
(GenExel), one deficiency line, Df(3R)JY19 (hereafter referred to as abiDf),
uncovering the abi locus (Breen and Harte, 1991), and a gene-targeting
(knockout, KO) line. The KO allele was created by end-out homologous
recombination. Briefly, we excised hsp70-white 2.8 kb cDNA from pBS70w and subcloned it into a filled-in blunting XhoI site (+262 base pairs
relative to translation start site) on abi exon 1. The abi genomic DNA plus
hsp70-white was subcloned into the NotI site of pP{EndsOut2} to the create
targeting plasmid (see Fig. S1A in the supplementary material). pBS-70w
and pP{EndsOut2} plasmids were gifts from Dr Jeff Sekelsky (University
of North Carolina, Chapel Hill, NC, USA). The fly transformation and
targeting crosses were carried out as described previously (Gong and Golic,
2003). The Abl stocks used were Abl1, Abl4, AblDf(3L)st-j7 [from Dr Eric Libel
(Liebl et al., 2003)], and AblEP3101 (Bloomington Stock Center). To
minimize the potential effects of the genetic background, abi and Abl
mutations (abiP1, abiP2, abiKO, Abl4 and AblEP) were outcrossed with w1118
DEVELOPMENT
INTRODUCTION
Two key cellular processes are needed to establish neuronal
connections: axon guidance and synapse formation. These processes
involve extracellular cues and intracellular signaling (Dent and
Gertler, 2003; Zhou and Cohan, 2004). For intracellular signaling,
many actin regulators, including proteins functioning in actin
nucleation, elongation and monomer binding, are required for
axonal connection in the developing central nervous system (CNS)
(Sanchez-Soriano et al., 2007). One actin-binding protein, Abl
tyrosine kinase, regulates axonal guidance in the developing
Drosophila and mammalian CNS (Hoffmann, 1991; Lanier and
Gertler, 2000; Moresco and Koleske, 2003). Many actin regulators,
including ena, chickadee (also known as profilin) and capulet, also
genetically interact with Abl in this process (Gertler et al., 1995;
Wills et al., 1999; Wills et al., 2002). Mammalian Abl (known as
Abl1) also functions in dendrite maintenance and synaptic plasticity
(Moresco and Koleske, 2003; Moresco et al., 2005), although it is
unclear how well these functions are conserved in Drosophila.
Accumulated studies have found that Abelson interacting protein
(Abi), a target of Abl family tyrosine kinases, forms a pentameric
complex with SCAR/WAVE, Sra-1, Kette/Hem and HSPC300 when
regulating actin dynamics (Eden et al., 2002; Innocenti et al., 2004;
Kunda et al., 2003; Rogers et al., 2003). Moreover, all members of
the complex, except Abi, have been reported to be involved in
axonogenesis in Drosophila (Bogdan et al., 2004; Bogdan and
Klambt, 2003; Hummel et al., 2000; Qurashi et al., 2007; Schenck
et al., 2003; Schenck et al., 2004). In addition, other Abi-binding
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Development 136 (18)
for at least five generations. The UAS-GFP-Abi transgenic flies were
generated by subcloning the GFP-Abi cDNA from pAc5-GFP-Abi (Huang
et al., 2007) into pUAST. The following lines were obtained from the
Bloomington Stock Center: UAS-Abl (8567), act5c-GAL4 (ubiquitous
expression; 3954), elavC155-GAL4 (pan-neuronal expression; 458) and
enaGC5 (8570). The 24B-GAL4 (muscle expression) line was provided by Dr
Cheng-Ting Chien (Institute of Molecular Biology, Academia Sinica, Taipei,
Taiwan). The scarΔ37 and ketteC3-20 flies were obtained from Dr Eyal D.
Schejer (Zallen et al., 2002) and Dr Christian Klämbt (Hummel et al., 2000),
respectively.
Immunohistochemistry and image quantification
Embryos were immunostained according to standard procedures (Patel et
al., 1987). To study synapse morphology, third instar larvae were dissected
for immunolabeling as previously described (Bellen and Budnik, 2000).
Bouton numbers on neuromuscular junctions (NMJs) were scored by
counting distinct sphere-shaped synaptic terminals. Antibodies used were:
rabbit anti-Abi (1:100) (Huang et al., 2007); mouse anti-BP102 (1:50),
mouse anti-FasII (ID4, 1:50), mouse anti-Csp (1:40), mouse anti-Dlg
(1:100) and mouse anti-Brp (nc82, 1:10) [Developmental Studies
Hybridoma Bank, Iowa City, USA (DSHB)]; FITC-conjugated anti-Hrp
(1:100, Jackson Laboratories); and rabbit anti-β-galactosidase (1:500,
Chemicon). S2 and BG2-c2 cells were immunostained as previously
described (Huang et al., 2007). Antibodies used were mouse anti-Ena (5G2,
1:50) (DSHB) and mouse anti-HA (1:1000, Babco). Rhodamine and Cy5labeled donkey secondary antibodies were purchased from Jackson
Laboratories. Confocal images were obtained using Leica TCS SP5 or TCS
NT confocal microscopes.
Electrophysiological recordings were performed as previously described
(Tsai et al., 2008).
Electron microscopy
Larvae were filleted and processed for transmission electron microscopy as
described previously (Bellen and Budnik, 2000). A total of six control (w1118)
and seven Abl1/4 boutons were measured. Synaptic vesicles in the clustered
pool were defined as those <250 nm of the AZ T-bar. Measurements were
quantified using ImageJ (NIH).
Cell culture and transient transfection
The culture of Drosophila S2 and BG2-c2 cells and transient transfection of
plasmids into the cells were performed as previously described (Huang et
al., 2007; Lin et al., 2007).
RESULTS
Generation of abi mutants
To date, no abi mutant strain has been reported. To determine the
function of endogenous Abi, we generated a loss-of-function (LOF)
mutant of abi by homologous recombination (see Materials and
methods and Fig. S1A in the supplementary material). The isolated
allele (abiKO) is lethal when homozygous. We also identified two Pelement insertion mutations (abiP1 and abiP2) in the abi gene locus.
All four abi mutants analyzed (abiP1, abiP2, abiKO and abiDf) showed
diminished Abi protein levels in homozygous embryos (Fig. 1A).
Moreover, transheterozygotes with different combinations of the
four alleles are lethal at different developmental stages. Specifically,
the transheterozygotes abiKO/Df, abiP2/Df and abiKO/P2 showed pupal
lethality, suggesting that abiKO and abiP2 are strong LOF or null
alleles. By contrast, over 60% of abiP1/Df and abiP1/KO animals
survived to adulthood, suggesting that abiP1 behaves genetically as
a hypomorphic allele (Fig. 1B). The expression of an act5c-GAL4driven GFP-Abi transgene could efficiently rescue the lethal
phenotypes of abi mutants (see Fig. S1B in the supplementary
material). Moreover, a line with a precise P-element excision from
abiP2 was found to have wild-type viability (data not shown). Our
Fig. 1. Generation and characterization of abi mutants.
(A) Western blotting of Abi from wild-type and abi–/– Drosophila
embryos (abi alleles are indicated) at 14-17 hours after eggs were laid.
(B) Lethal-phase analysis of abi mutant alleles. abi mutant animals were
generated by crossing different abi/TM6 (with TM6 containing the
markers B, Hu, e and Tb). The lethal phase of the prepupal/pupal stage
was determined by counting pupae lacking the Tubby-marked balancer.
Bars indicate the percentage of survivability in different abi
transheterozygotes at three different developmental stages (blue,
prepupal; red, pupal; green, adult). A minimum of 250 animals was
counted in each cross. (C,C⬘) Abi was enriched in commissural and
longitudinal connectives and slightly enriched in midline (arrowhead in
C⬘). Stage 17 embryos immunolabeled with anti-Abi antibodies,
anterior to the left. C, lateral view; C⬘, ventral view. (D) Abi (green) was
enriched in the third-instar larval brain and nerve cord. Abi was found
in the synaptic neuropil and visual system, including the optic lobe (OL),
optic stalk (os) and eye disc (ED). Anti-Hrp antibody (red) marked the
neuronal membrane. (E) abiKO/P2 showed no Abi immunoreactivity.
(F-H) Anti-FasII staining of wild-type (F), abi heterozygous (G) and abi
maternal hypomorph, zygotic mutant (H) stage 17 embryos, with
anterior-posterior axis from top to bottom. Arrows indicate single axon
bundles that cross the midline. (I) The percentage of embryos with at
least one midline crossover. Genotypes are indicated on the y-axis. The
total number of embryos examined is shown on the right. VNC, ventral
nerve cord. Scale bars: 30 μm.
western blotting analysis confirmed the ectopic expression of GFPAbi and the presence of no or only a low level of endogenous Abi
protein in abiKO/KO, abiKO/P2 and abiKO/Df adults (see Fig. S1C in the
supplementary material). We concluded that abiKO and abiP2 are
strong LOF alleles, and that abiP1 is a hypomorphic allele.
Abi is essential for embryonic CNS development
Given that mammalian Abi family proteins are expressed at high
levels in the developing nervous system (Proepper et al., 2007;
Courtney et al., 2000; Grove et al., 2004), we expected Drosophila
Abi to also be expressed in the nervous system. In our
immunohistochemical study, we found that Abi is expressed primarily
DEVELOPMENT
Electrophysiology
in the brain and ventral nerve cord (VNC) of the embryonic CNS (Fig.
1C,C⬘). Abi is specifically concentrated at the commissural and
longitudinal connectives of the main axonal tracts. There was also
some slight staining of Abi along the CNS midline (Fig. 1C⬘,
arrowhead). At the larval stage, Abi is expressed in diverse regions of
the CNS, including the neuropil area of the brain, axon fascicles in the
VNC, photoreceptor cells in the eye disc and photoreceptor axons
projecting through the optic stalk into the optic lobe, in contrast to the
absence of immunoreactivity found in the abiKO/P2 CNS (Fig. 1D,E).
These results suggest that Abi plays a role in axonogenesis.
Therefore, we asked whether abi–/– displays axonal patterning
defects. Unexpectedly, we found no gross defects in BP102-positive
axonal processes in the abiKO/P2 embryonic CNS (see Fig. S2B in the
supplementary material), raising the possibility that the maternal Abi
protein might play a crucial role in CNS development. Thus, we
generated abi germline clones (Chou and Perrimon, 1996), and found
that embryos lacking both maternal and zygotic Abi (abiMZ) displayed
embryonic lethality and a catastrophic collapse of axonal patterning
(see Fig. S2D in the supplementary material). Since the severe defects
of the abiMZ rendered the interpretation difficult, we used the maternal
hypomorphic transheterozygous combination (abiP1/Df) with the
zygotic allele abiP2 (referred to hereafter as maternal hypomorph,
zygotic mutants) to assess the CNS defect. The embryos displayed
deranged longitudinal connectives and commissural tracts (see Fig.
S2C in the supplementary material). Together, these results clearly
implicate maternal Abi in axonogenesis.
Previous genetic studies have found that Drosophila Abl is
required for the inhibition of axon crossover at the CNS midline
(Wills et al., 2002; Hsouna et al., 2003; Forsthoefel et al., 2005). This
prompted us to investigate whether Abi is also involved in the
regulation of midline crossing. We used anti-FasII antibody to label
specific subsets of longitudinal axons. At embryonic stage 17, 9.6%
and 31.25% of abiKO/P2 and abiKO/Df zygotic null embryos,
respectively, exhibited midline crossing defects in ipsilateral axon
fascicles (Fig. 1G,I). A higher penetrance (57.1%) of this phenotype
was noted in the maternal hypomorph, zygotic mutants (Fig. 1H,I),
suggesting that Abi might play a role in restricting specific
longitudinal axons from crossing the midline.
Heterozygous abi suppresses Abl mutant
phenotypes in lethality and axonal guidance
defects
Although there is a known biochemical interaction between Abl and
Abi, no previous study has defined the genetic interaction between
these two genes. The gene-dosage-sensitive genetic interaction
analysis has been found to be effective in characterizing genes
involved in Abl signal transduction networks (Lanier and Gertler,
2000). We investigated whether Abl–/– lethality could be affected by
reducing the abi gene dosage. Surprisingly, the heterozygous abi
genotype resulted in a dramatic increase in the survival of Abl1/4
progeny to adulthood (Fig. 2A). We found similar results in another
Abl mutant (Abl4/Df(3L)st-j7) (data not shown), suggesting that abi
acts as a genetic antagonist of Abl in the development of Drosophila.
Because both Abi and Abl contribute to CNS development, and
the reduction of abi dosage suppresses Abl mutant lethality, we
investigated the genetic interaction between abi and Abl in
axonogenesis. Consistent with our survival analysis, abi
heterozygosity markedly suppressed abnormal midline crossover in
Abl1/4, from 37% of Abl1/4 embryos having abnormal midline
crossing to 18% of abiP2/+; Abl1/4 embryos (Fig. 2B). This finding,
which is similar to the findings previously reported for ena in the
suppression of Abl axonogenesis phenotypes (Gertler et al., 1995),
RESEARCH ARTICLE 3101
Fig. 2. abi suppresses lethality and axon defects of Abl mutants.
(A) Heterozygous abi decreased Abl1/4 lethality. Graph depicting survival
to adulthood. A minimum of 300 animals was analyzed for each cross
(n>5 vials/genotype). Error bars represent standard deviation of the mean.
(B,C) Quantification of FasII axonal guidance defects. Genotypes are
indicated on the y-axis. The number of examined embryos is shown on
the right. (B) abi heterozygosity reduced the severity of aberrant midline
crossing defects in Abl embryos. (C) Abl heterozygosity also reduced the
midline crossing defects in abi–/– (abiKO/Df and abiKO/P2) embryos.
further suggests the possibility that Abi plays an opposing role to
Abl in axonogenesis. Therefore, we investigated whether Abl
mutations also modify abi mutant phenotypes in axonogenesis.
Similarly, the presence of a single copy of the Abl gene suppressed
the abiKO/Df and abiKO/P2 midline-crossing phenotypes by
approximately 53% and 69%, respectively (Fig. 2C). Together, these
results suggest that abi and Abl seem to act against or oppose each
other in the axonogenesis of embryonic CNS.
abi mutations suppress locomotion defects of Abl
We noticed inactivity and a lack of coordination in abi and Abl flies.
Performing negative geotaxis assays, we found significant reductions
in locomotor activity in both abi and Abl mutants (see Fig. S3 in the
supplementary material). Because the heterozygosity of abi
suppressed axonal defects of Abl mutants, we tested whether abi
mutations would also modulate the above-mentioned locomotion
defects of Abl mutants. As expected, the presence of abi–/+ increased
the locomotor activity score of flies with Abl hypomorph mutations
by more than 2-fold (see Fig. S3B in the supplementary material).
However, caution should be used when interpreting these results, as
locomotor abnormalities can be caused by a broad spectrum of
developmental and/or functional defects, making it difficult to
systematically examine the molecular mechanisms underlying this
interesting phenotype of abi and Abl flies.
Bouton number at the NMJ is increased in Abl but
not in abi mutants
Because previous studies of mice have suggested that Abi and Abl
proteins play a role in synaptic function (Moresco and Koleske, 2003;
Grove et al., 2004; Proepper et al., 2007), we were prompted to study
whether abi and Abl would genetically interact during synapse
DEVELOPMENT
Abi suppresses Abl in Drosophila
3102 RESEARCH ARTICLE
Development 136 (18)
Fig. 3. Abl mutants displayed bouton overgrowth at the NMJ. (A-D) Confocal images of the NMJ at muscle 6/7 in abdominal segment A2 in
wild-type (A) and Abl mutants (B-D). Anti-Hrp (red) and anti-Csp (green) labeling of neuronal membrane and synaptic boutons, respectively. Bouton
numbers on the NMJ were scored by counting distinct sphere-shaped synaptic terminals. (E) Graph quantifying bouton numbers per NMJ at muscle
6/7. Abl4/EP, Abl1/4, Abl1/Df and Abl4/Df exhibited a significant increase in bouton number compared with controls. Note that the increased bouton
number phenotype in Abl4/Df could be significantly rescued by expressing UAS-Abl under the control of a neuron-specific elav-GAL4 driver, but not
under the control of a muscle-specific 24B-GAL4 driver. Triple asterisks denote statistical significance (***, P<0.005, two-sample Student’s t-test).
Error bars represent standard deviation of the mean. Raw data are shown in Table S1 in the supplementary material.
Abl mutations increase the release of
spontaneous neurotransmitters
To test whether the synaptic overgrowth altered synaptic
transmission in Abl1/4 mutants, we performed intracellular
recordings from muscle 6 at segment A3 at the larval NMJs. The
amplitude of both evoked excitatory junctional potentials (EJPs) and
spontaneous miniature excitatory junctional potentials (mEJPs)
remained unaffected in Abl–/– larvae (Fig. 4A-D), but the frequency
of mEJPs increased by 57% compared with the controls (Fig. 4B,E).
To investigate whether the altered synaptic transmission was
accompanied by a synaptic structural abnormality, we stained NMJs
with an antibody that recognizes Brp, which labels neurotransmitterrelease sites at the active zone (AZ). We found no obvious
morphological differences between Abl1/4 and controls (Fig. 5A,B).
There was also no significant difference in the number of putative
AZs per bouton area in Abl1/4 compared with controls (P=0.13) (Fig.
5G). In our examinations of other pre- and post-synaptic markers,
including Csp, Futsch, FasII, GluRIIA and Dlg, we found no
difference in immunoreactivity between the mutant and control
NMJs (data not shown). These results suggest that the overall
integrity of the synapse structure in the NMJ is not noticeably
compromised in Abl mutants.
Fig. 4. Abl mutations increase the release of spontaneous
neurotransmitters. (A) Representative traces of evoked responses at
larval NMJs showing no difference in peak amplitude between Abl1/4
and controls. (B) Representative traces of mEJPs in wild-type controls
and Abl1/4. (C-E) Bar graphs representing mean values for evoked EJP
amplitude (C), mEJP amplitude (D) and mEJP frequency (E) in control
and Abl1/4 NMJs. There were no significant changes in the amplitude of
the EJPs and mEJPs for Abl1/4 compared with the controls (C,D). Note
that the frequency of mEJPs was significantly elevated (E; P<0.05, twosample Student’s t-test). Error bars represent standard error of the
mean. n.s., not significant.
DEVELOPMENT
formation. We chose to use the Drosophila larval NMJ to study the
genetic interactions during synapse formation (Collins and
DiAntonio, 2007). We chose to study muscle 6/7 in abdominal
segment A2 because no axonal targeting defects have been found in
this region for either abi (our unpublished observations) or Abl
mutants (Kraut et al., 2001). We first examined the NMJ morphology
and electrophysiology function of abi null mutants and found no
obvious phenotype for abi mutants. Both the number of synaptic
boutons and synaptic transmission appeared to be unaffected by the
mutations (see Fig. S4 in the supplementary material).
For the Abl mutants, however, the total synaptic bouton number
(Fig. 3B) and satellite bouton formation (Fig. 5B, arrows) were
significantly increased compared with w1118 controls (Fig. 3A; Fig.
5A). There was a 42% and 67% increase in the normalized bouton
number for Abl hypomorphic (Abl4/EP) and null (Abl4/Df(3L)st-j7)
larvae, respectively (Fig. 3E). To investigate whether a pre- or postsynaptic function of Abl is required for the synaptogenesis, we used
the GAL4/UAS system (Brand and Perrimon, 1993) to ectopically
express a neuronal or muscle Abl transgene in an Abl4/Df(3L)st-j7
mutant background. The presynaptic function of Abl appeared to be
essential for synaptogenesis, because the selective expression of
neuronal Abl (with an elav-GAL4 driver), instead of muscle Abl
(with a 24B-GAL4 driver), fully rescued the Abl4/Df(3L)st-j7 NMJ
phenotypes (Fig. 3C-E). These results suggest that the presynaptic
contribution of Abl is crucial for controlling the overgrowth of
synapses in the NMJ. We also found that the Abl–/– locomotion
defect was alleviated by the re-expression of neuronal, but not
muscle, Abl (data not shown). We concluded that the presynaptic
function of Abl is crucial for synaptogenesis in Drosophila.
Abi suppresses Abl in Drosophila
RESEARCH ARTICLE 3103
Fig. 6. Abl NMJ phenotypes can be suppressed by heterozygous
abi, ena or scar. Quantification of mean bouton number per muscle
area for segment A2, muscle 6/7. Genotypes are indicated in the y-axis.
***, P<0.005, Student’s t-test. n.s., not significant. (A) There was a
significant reduction in synaptic bouton number in abiKO/+, Abl1/4 or
enaGC5/+; abiKO/+, Abl1/4 compared with Abl1/4 mutants.
(B) Heterozygosity of scar (scarΔ37/+), but not of kette (ketteC3-20/+),
suppressed Abl (Abl1/4) NMJ phenotypes. Raw data are shown in Table
S1 in the supplementary material.
Ultrastructural abnormalities at synapses of Abl
mutants
We conducted a transmission electron microscopy study to search
for ultrastructural defects in Abl–/–. However, general features of
synapse structure, including presynaptic bouton morphology, the
electron-dense AZs with T-bars and the structure of the subsynaptic
reticulum (SSR), appeared to be unaffected in Abl–/– synapses (Fig.
5D). Morphometric quantification of the number of synaptic
vesicles clustered at the AZ, the number of AZs per section (Fig. 5CG), the length of AZs and the overall size of synaptic vesicles (data
not shown) also showed no significant differences between Abl–/–
and control synapses. Nevertheless, we found that the average
density of the total synaptic vesicles was decreased by ~50% in an
Abl–/– bouton compared with controls, although the vesicle numbers
in the clustered pool were not notably affected (Fig. 5G). In addition,
we frequently observed a number of enlarged but electron-clear
vesicles near the T-bar in Abl mutants (Fig. 5F, arrow). As we did
not observe an obvious increase in mEJP amplitude, it is likely that
these large electron-clear vesicles contained fewer neurotransmitters
than the electron-dense vesicles. Together, the decrease in overall
synaptic vesicle numbers in the bouton areas might offset the
increase in bouton numbers, in such a way that the EJP amplitude
remains unchanged in Abl–/– synapses.
Double heterozygosity for abi and ena suppresses
Abl NMJ phenotypes
Since the data above suggested that Abl and Abi might contribute to
a common regulatory pathway in the developing nervous system, we
investigated whether abi and Abl genetically interacted in NMJ
development. Similar to the effects observed in axon guidance,
removing one copy of abi moderately, but significantly, suppressed
the Abl–/– NMJ phenotypes (Fig. 6A), suggesting that Abi activity is
required for synaptic overgrowth in Abl.
We and others have previously reported an association between Abi
and Abl, and showed that this association promotes Abl-mediated
phosphorylation of Ena in cell culture systems (Juang and Hoffmann,
1999; Tani et al., 2003). It is intriguing to note that ena also acts as a
genetic antagonist of Abl in axonogenesis (Gertler et al., 1995) and its
protein localizes to motor-axon terminals (Martin et al., 2005). To
investigate whether ena acts cooperatively with abi to functionally
antagonize Abl–/– phenotypes, we examined whether double
heterozygosity for abi and ena further suppressed Abl–/– NMJ
phenotypes. We found an additional suppression of NMJ overgrowth
phenotypes in ena–/+; Abl–/–, abi–/+ larvae (Fig. 6A). Together, these
data lend support to the conclusion that Abi and Ena act in concert to
antagonize the role of Abl in the development of the NMJ.
Previous studies have demonstrated that mutations of other
SCAR/WAVE complex components (containing Scar, Abi, Kette,
Sra-1 and HSPC300) also result in similar axonal abnormalities to
those of abi mutants (Bogdan and Klambt, 2003; Qurashi et al.,
2007; Schenck et al., 2003; Schenck et al., 2004). The similarity in
the phenotypes prompted us to investigate whether the mutation of
other components of SCAR/WAVE complex would also have a
similar effect on inhibiting Abl synaptic phenotypes. However, the
results are fairly divergent with regards to the inhibitory effect on
DEVELOPMENT
Fig. 5. Abl mutants display a decrease in the total number of
synaptic vesicles per bouton. (A,B) Confocal microscopy of synaptic
boutons in muscle 4 labeled with anti-Brp (green) and anti-Hrp (red)
antibodies revealed the presynaptic active zone (AZ) and neuron
membrane, respectively. Arrows indicate the increased satellite boutons
in Abl1/4. (C,D) Transmission electron micrographs of synaptic boutons.
The subsynaptic reticulum (SSR), mitochondrion (m), AZs (asterisks) and
AZs with T-bars (arrows) are indicated in w1118 (C) and Abl1/4 (D)
micrographs. (E,F) Higher magnification of an AZ showed no obvious
difference in clustered vesicle numbers (<250 nm radius from T-bar)
between control (E) and Abl1/4 (F) synapses. Large electron-clear vesicles
near the AZ in Abl1/4 are indicated with arrows. (G) Quantitative analysis
of AZ number, clustered vesicle number and average of total vesicle
density. For each parameter, n>7; for each genotype, n=3. ***,
P<0.005. Scale bars: 10 μm in B; 500 nm in C; 200 nm in E.
3104 RESEARCH ARTICLE
Development 136 (18)
Abl synaptic phenotypes. Similar to the findings in our study of
abiKO/+, scar heterozygosity (scarΔ37/+) resulted in a significant
decrease in NMJ bouton number in Abl larvae. However, a mutation
in kette (ketteC3-20/+) appeared to be incapable of repressing the same
phenotypes (Fig. 6B). These results might suggest that some, but not
all, of the components of the pentameric SCAR/WAVE complex are
involved in antagonizing the Abl-induced synaptic overgrowth.
Abl regulates Abi and Ena for actin dynamics and
neurite extension
Our results so far suggest that abi ena double heterozygosity can
restrain the synaptic overgrowth of Abl mutants. Since both Abi and
Ena proteins have been implicated in actin binding and
reorganization (Krause et al., 2003; Innocenti et al., 2004), we asked
whether F-actin structure was modulated by the overexpression of
Abi or Ena in neuronal cells. The overexpresssion of either Abi or
Ena alone in BG2-c2 neuronal cells redistributed peripheral F-actin
to cytoplasmic aggregates wherever Abi or Ena had accumulated
(Fig. 7A). However, Ena appeared to be more potent than Abi in
redistributing F-actin to the cytoplasm (compare Fig. 7Ad-f with
7Aj-l). The cytoplasmic F-actin punta were efficiently dispersed and
redistributed back to the cell periphery when Abl was co-expressed
with either Abi or Ena (Fig. 7A). However, in cells co-expressing
both Ena with Abl, Ena proteins were not completely relocalized to
the cell periphery, but rather appeared to exist as cytoplasmic
aggregates (Fig. 7Am-o), suggesting that Abl might affect the
localization of Abi and Ena in neuronal cells differently. Further
biochemical assays found Abi to be crucial for the association of Abl
with Ena (data not shown). Immunocytochemical analysis also
supported the co-existence of Abl-Abi-Ena complexes in cells (Fig.
7B; see Fig. S5 in the supplementary material). Together, these
results suggest that Abl, through binding and modulating Abi and
Ena activity, controls F-actin localization to cell periphery.
Because neurite outgrowth is regulated by the reorganization of
peripheral actin (Schaefer et al., 2008), we investigated whether the
Abl-Abi-Ena interaction affects neuritogenesis. We found that
overexpression of Abi or Ena alone triggered the transformation of the
extended biopolar neuronal cells into retracted cells (Fig. 7Ad-f and
7Aj-l). We also found that the retracted morphology of neuronal cells
could be efficiently reversed by the co-expression of Abl with either
Abi or Ena (Fig. 7Ag-i and 7Am-o). The striking morphological
changes observed in neuronal cells prompted us to measure the
changes in neurite outgrowth in cells overexpressing Abl, Abi or Ena,
either singly or in pairs. In general, over 60% of the BG2-c2 cells
displayed bipolar, neurite-like structures 4 days after plating onto a
coverslip (Fig. 8A). We found a 3- to 60-fold decrease in the
percentage of cells with bipolar morphology when overexpressing Abi
or Ena alone (Fig. 8B). As expected, co-expressing Abl with Abi or
Ena restored bipolar morphology (Fig. 8B), suggesting that Abl plays
an opposing role to that of Abi and Ena in neuritogenesis. Since both
Abi and Ena are physically associated with and functionally
modulated by Abl, we speculated that Abi and Ena might act
cooperatively to regulate the activity of Abl in neuritogenesis. If so,
the co-expression of Abi and Ena might further suppress Abl function
DEVELOPMENT
Fig. 7. Abl regulates Abi and Ena to affect actin
dynamics. (A) Confocal microscopy showing F-actin,
Abl, Abi and Ena distribution in BG2-c2 neuronal cells.
(a-c) The normal localization of Abl and F-actin.
Overexpressed Abi distributed some F-actin to cytosolic
aggregates of Abi (panels d-f). Overexpressed Ena
distributed most F-actin to cytosolic aggregates of Ena
and led to a dramatic retraction of neurites (panels j-l).
Co-expressing Abl with Abi or Ena relocalized cytosolic
aggregates of Abi and Ena, as well as F-actin, to the cell
periphery (g-i,m-o). In cells co-expressing Ena and Abl,
large cytosolic aggregates of Ena were dispersed,
although a punctate pattern of Ena was still observed at
the cell periphery (m). (B) Co-expressing Ena with Abl
dispersed the large cytosolic aggregates of Ena into the
cytosolic puncta of BG2-c2 cells. Abl distributed
uniformly in the cytosol (upper left panel). When Abi,
Ena and Abl were co-expressed, all three proteins
completely colocalized in the cytosolic puncta (right).
Abl was labeled with anti-HA antibody, Ena with antiEna antibody and F-actin with rhodamine-phalloidin.
Fig. 8. Neurite extension is modulated by Abl-Abi-Ena
interaction. (A) BG2-c2 neuronal cells plated onto a coverslip showed
extended bipolar (labeled blue), intermediate (labeled red) or spherical
(labeled green) cell shapes, as marked by rhodamine-phalloidin. (B) Abl,
Abi and Ena expression levels are crucial for neuritogenesis. Transient
expression of eGFP, Abl, Abi or Ena in BG2-c2 cells is indicated on the xaxis. Histograms are the mean percentages of the three categories
shown in A in the total number of cells. Error bars are standard
deviation of the mean percentage. At least 100 cells were analyzed in
three independent experiments.
in neuritogenesis. We found that co-expressing both Abi and Ena with
Abl could diminish neurite extension more than by overexpressing
Abl with either Abi or Ena alone (Fig. 8B). To rule out the possibility
that the changes in neurite extension were related to the levels of Abi,
Ena and Abl protein expression, we conducted western blot analysis.
We found comparable protein expressions in our experiments with
different combinations of gene expression (see Fig. S6 in the
supplementary material).
DISCUSSION
The in vivo role of Abi with respect to Abl has remained enigmatic.
Abi was first identified as an Abl kinase substrate, functioning in
modulating the transformation activity of oncogenic Abl in human
cancers (Dai and Pendergast, 1995; Shi et al., 1995; Wang et al., 2007;
Yu et al., 2008). Intriguingly, we and others have shown that Abi also
functions as an activator of Abl kinase activity (Juang and Hoffmann,
1999; Tani et al., 2003; Lin et al., 2004; Leng et al., 2005; Maruoka et
al., 2005). Moreover, the interaction of Abl and Abi can trigger an
array of biochemical and functional changes in Abi, including protein
phosphorylation, stability and subcellular localization (Huang et al.,
2007), which might ultimately lead to the control of a particular
biological process in vivo. Although both Abi and Abl proteins are
highly expressed in the mammalian and Drosophila nervous systems,
the role of Abi in modulating the function of Abl in developing
nervous systems has remained unclear. In this investigation, we
conducted genetic and functional studies to advance our
understanding of how abi and Abl interact in vivo. To do this, we
generated and characterized abi loss-of-function alleles for genetic
and functional studies in Drosophila. Immunohistochemical analysis
revealed that Abi is primarily expressed in the developing CNS.
Consistent with this finding, our phenotypic analysis suggested that
mutations in abi resulted in axonal guidance defects in the CNS. In an
analysis of Abl mutants, we found Abl to be crucial for restricting
synaptic overgrowth in the larval NMJ. Importantly, our further
studies of the genetic interaction found a functional link between abi
RESEARCH ARTICLE 3105
and Abl in axonogenesis and synaptogenesis. Moreover, Abi and Ena
were found to cooperate in modulating the function of Abl in NMJ
growth. Finally, based on additional cellular biology studies, we
propose that the functional interactions between Abi, Ena and Abl
might be mediated through the modulation of actin cytoskeleton
reorganization.
Accumulated evidence suggests that the highly conserved actinregulatory pathways are essential for synaptogenesis and synaptic
plasticity (Coyle et al., 2004; O’Connor-Giles et al., 2008; Pawson
et al., 2008; Rodal et al., 2008; Stewart et al., 2002; Stewart et al.,
2005). Abi, Ena and Abl proteins are all involved in actin dynamics
(Stradal and Scita, 2006; Lanier and Gertler, 2000). Using the
Drosophila NMJ as a model system, we propose that the abi-enaAbl interaction in synaptogenesis might be associated with actin
cytoskeleton reorganization. In fact, several actin regulatory
molecules associated with both Abl and Abi have been implicated
in synaptic growth. For example, Wiskott-Aldrich Syndrome protein
(Wasp) is a kinase substrate of Abl (Burton et al., 2005) and also a
binding partner for Abi (Bogdan et al., 2005; Innocenti et al., 2005).
The mutations in wasp result in phenotypes very similar to those
present in Abl mutants, with synaptic overgrowth and
hyperbranching at the NMJ (Coyle et al., 2004). Another example
is that of Diaphanous (Dia), which also interacts with both Abl and
Abi, and has recently been found to modulate synaptic growth of the
Drosophila NMJ (Pawson et al., 2008). dia mutant heterozygotes
have been found to be able to enhance the cellularization phenotype
of an Abl maternal-null mutant (Grevengoed et al., 2003), suggesting
that Dia might be involved in the regulation of Abl signaling for
actin reorganization. Consistent with this idea, the interaction of Dia
with Abi protein has been found to be important in regulating the
formation and stability of cell-cell junctions in mammalian cells
(Ryu et al., 2009). Future studies investigating whether and how
Wasp and/or Dia can participate in Abl-Abi signaling for the
regulation of Drosophila synaptogenesis could be interesting.
Besides Wasp and Dia, other actin regulators might also contribute
to Abl-Abi signaling during nervous system development, for, as
another study has suggested, Abl may be a key regulator in
modulating different types and sites of actin polymerization within the
cells (Grevengoed et al., 2003). Abi has been shown to play a key role
in the activation of the SCAR/WAVE complex, which relays signaling
from Rac1 to the Arp2/3 complex for actin cytoskeleton remodeling
(Innocenti et al., 2004). Our genetic studies showed that the
heterozygosity of scar, but not of kette, suppressed Abl NMJ
phenotypes. A current model suggests that eliminating any component
from the SCAR/WAVE complex induces the breakdown of other
complex components and subsequently results in abnormal
lamellipodia formation (Stradal and Scita, 2006). Our genetic study
using the Drosophila NMJ as a model does not appear to fully support
this idea. Our results suggested that only a subcomplex of
SCAR/WAVE might be involved in synaptogenesis. In fact, recent
studies have demonstrated that some components of the
SCAR/WAVE complex might work outside the complex to regulate
various biological processes, including neutrophil chemotaxis, cell
motility and adhesion, and the formation of cell-cell junctions (Weiner
et al., 2006; Pollitt and Insall, 2008; Ibarra et al., 2006; Ryu et al.,
2009). Thus, it is possible that Kette is not in a complex with Abi and
Scar to modulate the function of Abl in NMJ growth. To explore this
hypothesis, it will be important to examine the genetic interactions
between abi and scar or kette in NMJ morphogenesis.
This study found strong in vivo evidence for an antagonistic
relationship between Abl and Abi in axonogenesis and
synaptogenesis. Supporting this model, one very recent study has
DEVELOPMENT
Abi suppresses Abl in Drosophila
demonstrated that Abl can inhibit the role of Abi in the
engulfment of apoptotic cells in C. elegans (Hurwitz et al., 2009).
Given that Abi is the Abl kinase substrate and that it also
functions as an adaptor protein for Abl in regulating other
downstream effectors (Juang and Hoffmann, 1999; Tani et al.,
2003; Lin et al., 2004; Leng et al., 2005; Maruoka et al., 2005), it
is feasible that Abi might act downstream of Abl in modulating
NMJ growth. If so, the removal of both copies of abi could
conceivably further suppress Abl–/– NMJ phenotypes. Our
preliminary morphological and functional data both suggest that
the minor NMJ defects of Abl–/– abi+/– are further rescued in Abl–/–
abi–/– mutants (data not shown). However, Abl–/– abi–/– double
mutants showed early lethality and defects in axonal innervations,
rendering the finding inconclusive. Further epistasis analysis
combining abi and abl gain-of-function and loss-of-function
mutations are needed to test this hypothesis.
Since our data suggest an antagonistic interaction between abi and
Abl for the CNS and NMJ phenotypes (Fig. 2), we speculated that
Abl heterozygosity would suppress the semilethal phenotype of abi
mutants. Surprisingly, our preliminary data showed that the lethality
of abi hypomorphic mutants (abiP1/KO and abiP1/Df) was further
increased by Abl+/– (data not shown). This result does not seem to
support a general bidirectional antagonistic relationship between Abl
and abi for the biological processes involved during development.
Thus, a complex genetic interaction network between Abl and abi
might be present in development processes.
Another interesting issue is that the abi mutants did not display
obvious defects in synaptic bouton number or synaptic
transmission, although they exhibited midline crossing defects in
the embryonic CNS. Because other members of SCAR complex,
including Scar, Kette, Sra-1 and HSPC300, exhibit both CNS and
NMJ phenotypes (Bogdan et al., 2004; Hummel et al., 2000;
Qurashi et al., 2007; Schenck et al., 2003; Schenck et al., 2004),
it is still possible that abi mutants might show minor
morphological or functional abnormalities if different phenotypic
characteristics are studied. Detailed morphological assays are
required to investigate other phenotypic traits of the NMJ in larval
or later developmental stages. Alternatively, one could reason that
the roles of Abi in synaptic growth and axonal guidance are not
exactly identical. Results similar to this finding have been
observed for the loss of spastin, a gene enriched in axons and
synaptic connections, as spastin mutants only exhibit NMJ but not
CNS defects (Sherwood et al., 2004).
Our work also suggested that the synaptic overgrowth phenotypes
in Abl mutants could be completely rescued by expressing Abl in the
presynaptic nerve cells but not in the postsynaptic muscles,
suggesting that the presynaptic Abl is more crucial than the
postsynaptic population for Drosophila larval NMJ formation.
However, studies in mammalian Abl and Arg (also known as Abl2)
have shown that both proteins localize to the presynaptic terminals
and dendritic spines of synapses in the hippocampal CA1 area
(Moresco and Koleske, 2003). Abl and Arg have also been shown
to be essential for the agrin-induced clustering of acetylcholine
receptors (AChRs) on the postsynaptic membrane of the mammalian
NMJ, suggesting that Abl function is required in the postsynaptic
region of the mammalian NMJ (Finn et al., 2003). However, these
reports do not exclude the possibility that Drosophila Abl might also
function in postsynaptic regions of the developing brain. The reason
for this speculation is that the mammalian NMJ uses acetylcholine
as the neurotransmitter, unlike the Drosophila NMJ, which uses
glutamate as a transmitter. Since acetylcholine receptors also
function in the developing brain of Drosophila (Yasuyama et al.,
Development 136 (18)
1995), it would be important to investigate whether Drosophila Abl
also plays a role in the postsynaptic region of the neurons, where
acetylcholine receptors are expressed.
In conclusion, our genetic studies in Drosophila suggest that Abi
and Abl play opposing roles in axonogenesis and synaptogenesis.
This conclusion is further supported by a series of biochemical,
immunocytochemical and morphological studies in cultured cells.
These findings offer new insights into the functional interaction
between Abl, Abi and Ena in nervous system development.
Acknowledgements
We thank Drs Jui-Chou Hsu, Henry Sun, Cheng-Ting Chien and Eric C. Liebl for
helpful comments; Mr Pei-I Tsai for reagents; and NHRI Optical Biology Core
for microscopy assistance. This work was supported by the NHRI and the NSC
(NSC95-2311-B-400-001-MY3).
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/136/18/3099/DC1
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DEVELOPMENT
Abi suppresses Abl in Drosophila